Patent Application
20230129745
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
This invention is directed to compounds, compositions, and methods to treat metabolic diseases. For example, the invention is drawn to compounds identified in extracts from Artemisia scoparia for the treatment of metabolic disease.
Adipose tissue (AT) is a critical player in metabolic regulation (Kusminski, Bickel, and Scherer 2016). Obesity, the main disorder involving AT, is arguably the greatest health issue currently affecting the Western world, as it is the major driver of the high rates of cardiovascular disease and insulin resistance in developed nations (Bhupathiraju and Hu 2016; Ranasinghe et al. 2017; Saklayen 2018). In obese states, the normal functions of adipose tissue are disrupted, contributing to whole-body metabolic dysfunction (Vidal-Puig 2013). Although the inhibition of fat cell development may intuitively sound like a beneficial strategy to reduce obesity and associated disorders, obesity is associated with impaired adipocyte differentiation, along with ectopic lipid deposition and metabolic dysfunction in muscle and liver (Danforth 2000; Gustafson et al. 2015; Kim et al. 2007; Smith and Kahn 2016).
The present invention provides a botanical composition comprising an extract isolated from Artemisa scoparia. In embodiments, the extract comprises a compound of
In embodiments, the botanical extract comprises a polar solvent or a nonpolar solvent. For example, the polar solvent comprises ethyl alcohol (ethanol), ethyl acetate, butyl alcohol (butanol), methyl alcohol (methanol), n-propanol, and water. For example, the non-polar solvent comprises isooctane, hexane, diethyl ether, or chloroform.
In embodiments, the botanical composition comprises an isomer of Formula (V).
In embodiments, the botanical composition comprises a compound of
or any combination thereof.
In embodiments, the botanical composition comprises a compound that is an isomer of Formula (I), Formula (II), Formula (III), or Formula (IV).
Aspects of the invention are also directed towards a pharmaceutical composition. For example, such pharmaceutical composition can comprise a therapeutically effective amount of a botanical extract of Artemisa scoparia.
In embodiments, the pharmaceutical composition comprises a compound of
wherein bonds can be cis or trans; R1 comprises H, OH, and OAc, R2 comprises H, OH, and OAc; R3 comprises H, OH, and OAc; R4 comprises H, OH, and OAc, or any combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
In embodiments, the pharmaceutical extract can comprise a botanical extract described herein, wherein the botanical extract comprises a polar solvent or a non-polar solvent. For example, the polar solvent comprises ethyl alcohol (ethanol), ethyl acetate, butyl alcohol (butanol), methyl alcohol (methanol), n-propanol, and water. For example, the non-polar solvent comprises isooctane, hexane, diethyl ether, or chloroform.
In embodiments, the pharmaceutical composition comprises an isomer of Formula (V).
In embodiments, the pharmaceutical composition comprises a compound of:
or any combination thereof.
In embodiments, the pharmaceutical composition comprises a compound that is an isomer of Formula (I), Formula (II), Formula (III), or Formula (IV).
In embodiments, the pharmaceutical composition can further comprise one or more additional active agents. For example, such one or more additional active agents can synergize with a compound of Formula (V).
Aspects of the invention are further drawn to a method of treating or preventing a metabolic disease, for example obesity, diabetes, or metabolic syndrome. For example, the method can comprise administering to a subject in need thereof a therapeutically effective amount of the botanical extract as described herein or a pharmaceutical composition as described herein. For example, the therapeutically effective amount can comprise about 0.1 μg/kg to about 1000 mg/kg.
Aspects of the invention are also drawn to a method of treating or preventing a drug-induced metabolic disturbance. For example, the method can comprise administering to a subject in need thereof a therapeutically effective amount of the botanical extract as described herein or a pharmaceutical composition as described herein. For example, the therapeutically effective amount can comprise about 0.1 μg/kg to about 1000 mg/kg.
Aspects of the invention are still further drawn to a method of extending the lifespan of a subject. For example, the method can comprise administering to a subject in need thereof a therapeutically effective amount of the botanical extract as described herein or a pharmaceutical composition as described herein. For example, the therapeutically effective amount can comprise about 0.1 μg/kg to about 1000 mg/kg.
Also, aspects of the invention are drawn to a method of preparing a botanical extract, such as a botanical extract isolated from Artemisa scoparia. In embodiments, the method comprises: obtaining fresh plant material; grinding the plant material to a powder; combining the powder with a liquid comprising at least one of water and ethanol to concentrate the solution to obtain an alcohol extract of the plant materials; extracting plant material by liquid to form a liquid extract; and separating the liquid extract from the plant material.
In embodiments, the liquid extract comprises a compound of
wherein bonds can be cis or trans; R1 comprises H, OH, and OAc, R2 comprises H, OH, and OAc; R3 comprises H, OH, and OAc; R4 comprises H, OH, and OAc, or any combination thereof.
For example, the liquid extract comprises a compound of:
or any combination thereof.
Aspects of the invention are further drawn to a therapeutic composition prepared by the methods as described herein.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
Throughout human history, botanicals have been used as therapeutics, and many pharmaceuticals in use today have been derived, directly or indirectly, from plant compounds. A primary challenge in botanical research is that plant extracts are comprised of hundreds of compounds, and it is difficult to identify which of these compounds confer biological activity. Also, the isolation of specific compounds from plant extracts can be challenging.
Artemisa scoparia is a Eurasian species in the genus Artemisia, in the sunflower family. Dozens of extract fractions were screened for their ability to promote adipocyte development, as has been shown for parent SCO extracts isolated from Artemisia scoparia (SCO). As described herein, the inventors have identified the structures of at least three botanically-derived compounds that are active ingredients within SCO extracts, all of which are prenylated coumaric acid derivatives (PCAs). Two of these compounds, capillartemisin A and capillartemisin B, have been previously reported in A. scoparia (Kitagawa et al. 1983), but until now their use to treat metabolic disease remained unknown. The third compound, for example, the compound of Formula I or Formula II, was previously unknown. As described herein, pharmaceutical compositions comprising these botanically-derived compounds can promote adipocyte differentiation and, without wishing to be bound by theory, improve overall metabolic health based on studies of parent SCO extract in diabetic mice.
Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
The singular forms “a”, “an” and “the” include plural reference unless the context dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can refer to “one,” but it is also consistent with “one or more,” “at least one,” and “one or more than one.”
Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.
The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.
The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” refers to the process including at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.
As used herein the term “about” is used herein to refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. For example, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
As described herein, aspects of the invention refer to compositions and methods comprising botanically-derived extracts and compounds.
As used herein, “botanical” can refer to a material that is or may be tree-, plant-, weed- or herb-derived. As used herein, “botanically derived” can refer to a material capable of having been derived from a botanical, as by isolation or extraction; however, “botanically derived” is not limited in this application to materials which actually are isolated or extracted from a botanical, but also includes materials obtained commercially or synthetically. As used herein,
Aspects of the invention are drawn to botanically-derived compounds isolated from Artemisa scoparia. For example, the botanically-derived compounds can include, but are not limited to:
Embodiments can further comprise “isomers” of the botanically-derived compounds. The term “isomer” can refer to compounds that have the same composition and molecular weight, but differ in physical and/or chemical properties. Such substances have the same number and kind of atoms but differ in structure. The structural difference may be in constitution (geometric isomers) or in an ability to rotate the plane of polarized light (stereoisomers). See, for example, Formula (I) and Formula (II), which each have the chemical formula of C21H26O6, exact mass of 374.1729, and molecular weight of 374.4330.
The term “botanically-derived extract”, “plant extract”, or “botanical extract” can refer to any extract obtainable from a plant or any portion thereof. For example, the plant extract can comprise the active ingredient/s thereof. Thus, the plant extract can be obtained from the fruit, the skin or rind of the fruit, the seeds, the bark, the leaves, the roots, the rhizome, the root bark or the stem of a plant, or a combination of same. In embodiments, “extracted” or an “extraction” can refer to the process and product, respectively, of separating a substance from a matrix wherein the matrix can be solid or liquid. In embodiments, the matrix is a plant, a plant product, or extract thereof. As used herein the term “botanical composition” can refer to a composition that is comprises material derived from a plant, a part of a plant, or a combination thereof.
The botanically-derived extract can be obtained from Artemisa scoparia. According to an embodiment of the invention, the plant extract is an aqueous extract, a hydrophilic extract, a non-polar extract or a polar extract. For example, the plant extract is an ethanolic extract. A representative extraction procedure includes the one disclosed in Boudreau, Anik, et al. “Distinct fractions of an Artemisia scoparia extract contain compounds with new adipogenic bioactivity.” Frontiers in nutrition 6 (2019): 18, which is incorporated by reference herein in ites entirety. Plant extracts can be divided into polar and non-polar extracts, and hydrophilic and hydrophobic extracts.
Thus, the plant extracts can be purified by the use of a polar solvent (i.e. polar extract) such as, without being limited to, ethyl alcohol (ethanol), butyl alcohol (butanol), methanol, water, acetic acid, tetrahydrofuran, N,N-dimethylformamide, dichloromethane, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide, acetone, or n-propanol. As used herein, the term “solvent” can refer to a substance capable of dissolving or dispersing one or more substances. As used herein, the term “polar solvent” can refer to a solvent that comprises dipole moments. For example, a polar solvent can be miscible with water and polar solvents. For example, a polar solvent can comprise chemical species in which the distribution of electrons between covalently bonded atoms is not even. For example, the polarity of solvents can be assessed by measuring any parameter known to those of skill in the art, including dielectric constant, polarity index, and dipole moment (see, e.g., Przybytec (1980) “High Purity Solvent Guide,” Burdick and Jackson Laboratories, Inc.). The polar extracts of the invention can comprise any percentage of polar solvent including, but not limited to, for example 1-10% polar solvent, 10-20% polar solvent, 20-30% polar solvent, 30-40% polar solvent, 40-50% polar solvent, 50-60% polar solvent, 70-80% polar solvent, 80-90% polar solvent and 90-100% polar solvent.
In other embodiments, the plant extracts can be purified by the use of a non-polar solvent (i.e. non-polar extract) such as, without being limited to, isooctane, hexane, pentane, benzene, chrloroform, diethyl ether, hydrocarbons, cyclohexane, toluene, or 1,4-dioxane. As used herein, “nonpolar” and “non-polar” can be used interchangabely. As used herein, the term “nonpolar solvent” can refer to a solvent comprising molecules that do not have an overall dipole. For example, the solvent comprises molecules comprising bonds between atoms with similar electrogenativities (e.g. a carbon-hydrogen bond). For example, the nonpolar molecule comprises equal sharing of electrons between atoms or the arrangement of polar bonds leads to overall no net molecular dipole moment. The non-polar extracts of the invention can comprise any percentage of non-polar solvent, including but not limited to, for example, 1-10% non-polar solvent, 10-20% non-polar solvent, 20-30% non-polar solvent, 30-40% non-polar solvent, 40-50% non-polar solvent, 50-60% non-polar solvent, 70-80% non-polar solvent, 80-90% non-polar solvent, and 90-100% non-polar solvent.
Hydrophobic molecules can be non-polar and thus can interact with (e.g. associate, aggregate, etc.) other neutral molecules and non-polar solvents. For example, nonpolar or hydrophobic molecules can interact through non-covalent interactions. For example, the non-covalent interaction is a van der Waals interaction. For example, the van der Waals interaction are London forces. Hydrophilic molecules can be polar and dissolve by water and other polar substances.
Thus, the plant extracts of the invention can be produced by any method known in the art including a polar extract such as a water (aqueous) extract or an alcohol extract (e.g., butanol, ethanol, methanol, hydroalcoholic, see for example Swanson R L et al., 2004, Biol. Bull. 206: 161-72) or a non-polar extract (e.g., hexane or isooctane, see for example, Ng L K and Hupe M. 2003, J. Chromatogr A. 1011: 213-9; Diwanay S, et al., 2004, J. Ethnopharmacol. 90: 49-55.
Regardless of the exact solvent employed, plant extracts can be made by placing a plant sample (e.g., leaves, seeds) in a mortar along with a small quantity of liquid (e.g., 10 ml of water, alcohol or an organic solvent for every 2 grams of plant sample) and grinding the sample thoroughly using a pestle. When the plant sample is completely ground, the plant extract is separated from the ground plant material, such as by centrifugation, filtering, cation-exchange chromatography, and the like, and the collected liquid can be further processed if need be (such as by a concentrating column and the like), active ingredients can be separated from this extract via affinity chromatography, mass chromatography and the like.
Extraction methods are known to the skilled artisan. See, for example, Boudreau, Anik, et al. “Distinct fractions of an Artemisia scoparia extract contain compounds with new adipogenic bioactivity.” Frontiers in nutrition 6 (2019): 18, which is incorporated herein by reference in its entirety.
For example, ethanolic extracts can be prepared from greenhouse-grown plants as described in the literature. The ethanolic extract of Artemisia scoparia (SCO, 10 g) can be dissolved in water (200 ml) and partitioned 3 times with hexane (3×200 ml). Hexane partitions can be combined and dried by rotary evaporation to produce the H crude fraction. The water portion can be further partitioned 3 times with ethyl acetate (3×200 ml). The ethyl acetate (EA) partitions can be combined and dried by rotary evaporation to produce the EA crude fraction. Remaining water can be dried of residual solvents by rotary evaporation and by freeze drying to produce the W crude fraction.
The SCO EA crude fraction can be fractionated using a semipreparatory high-performance liquid chromatography (HPLC) system consisting of Waters™ Alliance e2695 Separations Module and 2998 Photodiode Array Detector with a Phenonmenex Synergi 4 μm 80 Å Hydro-RP column 250×21.2 mm. The mobile phases consisted of two components: Solvent A (0.5% ACS grade acetic acid in double distilled de-ionized H2O), and Solvent B (acetonitrile). Separation can be completed using a gradient run of 25% B in A to 95% B over 35 min at a flow rate of 8 mL/min. Five fractions, H1-H5, can be obtained.
The SCO EA crude fraction can also be subjected to fast centrifugal partition chromatography (FCPC) fractionation using countercurrent chromatography separation (CCS) with a biphasic solvent system (HEMWat+3) composed of Hexanes/Ethyl-Acetate/Methanol/Water (6:4:6:4 v/v) on a Kromaton (Annonay, France) bench-scale fast centrifugal partition chromatography system FCPC1000, v 1.0, equipped with a 1,000 ml volume rotor. For fractionation, the column can be first filled with the lower phase 40 ml/min using a Chrom Tech® PR-Pump while rotating at 300 rpm. The system can be then equilibrated with upper phase at a flow rate of 10 ml/min and 750 rpm. A 2 g sample of SCO can be suspended in 10 ml of each phase of the solvent system used for separation, sonicated and filtered (Millipore filter type NY11). The sample can be injected with the upper phase flow at 10 ml/min and rotor rotation of 750 rpm. UV detection can be performed at 254 nm using a Visacon VUV-24 detector, and fractions can be collected manually. Ten fractions, F1-F10, can be obtained.
F2 of the above FCPC fractions can be further fractionated using a semipreparatory HPLC system consisting of Waters™ 600 Controller and 486 Tunable Absorbance Detector with a Phenonmenex Synergi 10 μm 80 Å MAX-RP column 250×21.2 mm. The mobile phases consisted of two components: Solvent A (0.10% ACS grade acetic acid in double distilled deionized H2O), and Solvent B (100% acetonitrile). For the initial separation, a gradient run of 40% B in A to 70% B over 35 min was used at a flow rate of 15 ml/min. SCO fraction 2-2 can be further fractionated using the same semipreparatory HPLC system with a Phenomenex Kinetex 5 μm C18 100 Å LC column 250×10.0 mm. The mobile phases consisted of two components: Solvent A (0.1% ACS grade formic acid in double distilled de-ionized H2O), and Solvent B (100% acetonitrile). For the secondary separation, a gradient run of 40% B in A to 80% B over 80 min was used at a flow rate of 5 ml/min. Fractions can be collected manually as defined by UV (254 nm) peak designations for both separations.
The plant extracts can be further treated to purify those active ingredients such as those having biological activities described herein. Active ingredients can be purified from plant extracts or used synthetically. In embodiments, active ingredients can refer to an ingredient that is biologically active. In embodiments, biological activity can refer to the in vivo activities of a compound, such as physiological responses that result upon in vivo administration of a compound, composition comprising the compound, or other mixtures.
For example, the active ingredients present in the plant extracts of the invention can include, but are not limited to:
wherein bonds can be cis or trans; R1 comprises H, OH, and OAc, R2 comprises H, OH, and OAc; R3 comprises H, OH, and OAc; R4 comprises H, OH, and OAc, or any combination thereof.
For example, the active ingredients present in the plant extracts of the invention can include, but are not limited to:
Aspects of the invention are drawn to pharmaceutical compositions, such as those useful for preventing or treating a metabolic disorder. In embodiments, the compositions of the disclosure can comprise a single botanically-derived extract or can comprise several botanically derived extracts. In other embodiments, the compositions of the disclosure can comprise a single botanically-derived compound or can comprise several botanically-derived compounds. Thus, according to one embodiment of the disclosure, the composition comprises an Artemisia scoparia plant extract or compounds isolated therefrom (i.e., Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V)).
The concentration of the plant extract or compounds therefrom within the composition can vary, such as dependent on the therapeutic effect desired and/or the extraction method utilized. For example, a concentration of each of the plant extract or compounds therefrom within the composition can be in a range of about 0.01 to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.
The compositions of the disclosure can be administered to the subject per se, or in a pharmaceutical composition. As used herein a “pharmaceutical composition” can refer to a preparation of one or more active ingredient(s) described herein, such as Formula I, Formula II, Formula III, Formula IV, or Formula V, with other chemical components such as physiologically suitable carriers and excipients. The purpose of the composition is to facilitate administration of the active ingredients (e.g., botanically-derived extract) to the subject.
As used herein the term “active ingredient” can refer to the botanically-derived extract, compositions isolated therefrom, or a synthetic composition derived therefrom that is biologically active. For example, one or more active ingredients can be accountable for the intended biological effect (i.e., for treatment or prevention of a metabolic disease).
Embodiments herein can further comprise one or more one or more additional active agents. The phrase “additional active agent” can refer to an agent, other than a compound(s) of the inventive composition, that exerts a pharmacological, or any other beneficial activity. Such additional active agents include, but are not limited to, an antidiabetic agent, a corticosteroid, or an anti-obesity agent. In embodiments, the additional active agent can synergize with one or more of the botanically-derived composition. For the example, the additional active agent can comprise one or more botanically-derived compound(s) or composition(s), such as from a fraction of a SCO extract. For example, the additional active agent can comprise a synthetic compound, for example a synthetic compound based on or derived from a compound of a botanical extract. In embodiments, the additional active agent can comprise a pharmaceutical or a drug, such as but not limiting to a glucocorticoid.
The phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which can be interchangeably used can refer to a carrier, excipient, or diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered active ingredients. An adjuvant is included under these phrases.
The term “excipient” can refer to an inert substance added to the composition (pharmaceutical composition) to further facilitate administration of an active ingredient of the present invention.
Techniques for formulation and administration of drugs can be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
The pharmaceutical compositions can be formulated for numerous types of administrations. For example, suitable routes of administration can, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
One can administer the pharmaceutical composition in a local rather than systemic manner, for example, by injecting the composition including the active ingredient (e.g., plant extract or compounds isolated therefrom) and a physiologically acceptable carrier directly into a tissue region of a patient.
In one embodiment, the pharmaceutical composition is formulated for oral administration. As used herein the phrase “oral administration” can refer to administration of the composition of the disclosure by mouth e.g. in the form of a liquid, a solution, a tablet, a capsule, a tincture, a gummy, or an elixir.
In another embodiment, the pharmaceutical composition is formulated for administration by injection.
Compositions of the disclosure can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Compositions for use in accordance with embodiments of the invention thus can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the composition can be formulated in aqueous solutions, such as in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition can be sterile and can be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In embodiments, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions can be prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
For oral administration, the composition can be formulated readily by combining the active compounds with carriers (e.g. pharmaceutically acceptable carriers) known in the art. Such carriers enable the composition to be formulated as tablets, pills, dragees, capsules, liquids, tinctures, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients can be fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). For example, disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used which can contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.
The composition described herein can be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients can be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions can contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
In embodiments, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.
The composition of embodiments can also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
The composition of embodiments can also be formulated in inhalable compositions. For example, the inhalable composition is formulated to for use in a nebulizer.
Compositions suitable for use in context of embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose, such as to prevent or treat a metabolic disease.
As used herein the phrase “therapeutically effective amount” can refer to an amount of an active ingredient (i.e. botanically-derived extract or an active ingredient or compound thereof, as described above) effective in preventing or treating a metabolic disease. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
In embodiments, the therapeutically effective amount is at least about 0.1 mg/kg body weight, at least about 0.25 mg/kg body weight, at least about 0.5 mg/kg body weight, at least about 0.75 mg/kg body weight, at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, at least about 100 mg/kg body weight, at least about 200 mg/kg body weight, at least about 250 mg/kg body weight, at least about 300 mg/kg body weight, at least about 3500 mg/kg body weight, at least about 400 mg/kg body weight, at least about 450 mg/kg body weight, at least about 500 mg/kg body weight, at least about 550 mg/kg body weight, at least about 600 mg/kg body weight, at least about 650 mg/kg body weight, at least about 700 mg/kg body weight, at least about 750 mg/kg body weight, at least about 800 mg/kg body weight, at least about 900 mg/kg body weight, or at least about 1000 mg/kg body weight.
The dosage can vary depending upon a number of factors known to those of ordinary skill in the art, such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the subject; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl, E. et al. (1975), “The Pharmacological Basis of Therapeutics,” Ch. 1, p. 1.)
Depending on the severity of the condition and the responsiveness of the subject to treatment, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, several months or several years, or until cure is effected or diminution of the disease state is achieved. Alternatively, the compositions are administered in order to prevent occurrence of a metabolic disorder in a subject at risk of developing a metabolic disorder. The compositions can be administered for prolonged periods of time (e.g. several days, several weeks, several months or several years) as to prevent occurrence of a metabolic disorder.
According to an embodiment of the disclosure, the compositions can be administered at least once a day. For example, the compositions can be administered daily. According to another embodiment, the compositions can be administered twice a day, three times a day or more. In embodiments, the compositions can be administered weekly, such as about once a week or about twice a week. In embodiments, the compositions can be administered monthly, such as about once a month or about twice a month.
According to an embodiment of the disclosure, the composition can be administered to a subject chronically. Such is the case, for example, when treating a subject afflicted with a chronic disease or condition. In other embodiments, the composition can be administered to a subject as long as the subject is at risk of a disease or condition, or as long as symptoms of a disease or condition persists. For example, embodiments of the invention can be administered to a subject for at least 7 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, at least 21 days, at least 24 days, at least 27 days, at least 30 days, at least 60 days, at least 90 days, or longer than 90 days.
The amount of a composition to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like. The exact amount of a composition to be administered can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl, E. et al. (1975), “The Pharmacological Basis of Therapeutics,” Ch. 1, p. 1.)
The compositions of the disclosure can be formulated as a unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredients such as for a single administration. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, an ampule, a dispenser, an adhesive bandage, a non-adhesive bandage, a wipe, a baby wipe, a gauze, a pad and a sanitary pad.
The quantity of active compound in a unit dose of preparation can be varied or adjusted according to the particular application.
Compositions of the disclosure can be presented in a pack or dispenser device, such as a kit, which can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The pack or dispenser device can also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, can include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a pharmaceutically acceptable carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as detailed herein.
Since the compositions of the disclosure are utilized in vivo, the compositions can be of high purity and substantially free of harmful contaminants, e.g., at least National Food (NF) grade. Compositions as described herein can be at least analytical grade. Compositions as described herein can be at least pharmaceutical grade. To the extent that a given compound must be synthesized prior to use, such synthesis or subsequent purification can result in a product that is substantially free of any contaminating toxic agents that can have been used during the synthesis or purification procedures.
Aspects of the invention are further drawn to methods of treating or preventing a metabolic disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein comprising a botanically-derived extract or compound, thereby treating and/or preventing the metabolic disease in the subject.
The term “treating” can refer to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
The term “preventing” can refer to keeping a disease, disorder or condition from occurring in a subject. In some cases, the subject can be at risk for developing the disease but has not yet been diagnosed as having the disease.
Subjects that can be treated according to this aspect of the invention include mammals such as human beings or domesticated animals including, but not limited to, horses (i.e. equine), cattle, goat, sheep, pig, dog, cat, camel, alpaca, llama and yak, male or female, at any age that is in need of treatment or prevention of a metabolic disease.
As used herein, the term “subject in need thereof” can refer to a subject that would benefit biologically, medically, or in a quality of life from the treatment, a subject exhibiting one or more symptoms or indications described herein, and/or a subject at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or ameloriation with the compositions or methods described herein. For example, the disease or indication comprises metabolic disease.
The term “metabolism” can refer to, for example, the sum of the processes by which a particular substance is handled in the living body, and/or the sum of the metabolic activities taking place. For example, metabolism can refer to the chemical changes in living cells by which energy is provided for vital processes and activities and new material is assimilated.
The term “metabolic disease” can refer to a group of identified disorders in which errors of metabolism, imbalances in metabolism, or sub-optimal metabolism occur. The metabolic diseases as described herein also include diseases that can be treated through the modulation of metabolism, although the disease itself may or may not be caused by a specific metabolic defect. Non-limiting examples of metabolic diseases that can be treated or prevented by embodiments herein include Type 2 diabetes, obesity, obesity-related conditions, insulin resistance and metabolic syndrome.
The term “metabolic health” can refer to the presence or absence of metabolic disease. For example, metabolic health can refer to having less than ideal or ideal levels of blood sugar, triglycerides, high-density lipoprotein (HDL) cholesterol, blood pressure, and/or waist circumference. For example, a subject with poor metabolic health can be afflicted with or at risk of a metabolic disease. Such subject can have less than ideal levels of blood sugar, triglycerides, high-density lipoprotein (HDL) cholesterol, blood pressure, and/or waist circumference.
In embodiments, the compositions and methods described herein can be used to treat or prevent metabolic disturbances induced by therapeutics or drugs. As used herein, the phrase “drug-induced metabolic disturbances” can refer to a metabolic disturbance induced by any agent or ingredient whose administration to a subject can result in metabolic disturbances. As used herein the term “metabolic disturbance” can refer to any metabolic disease, perturbation, or disorder. For example, the metabolic disturbances can comprise elevated glucocorticoids, obesity, overproduction of cortisol, insulin resistance, and nonalcoholic fatter liver disease.
“Diabetes” can refer to a heterogeneous group of disorders that share impaired glucose tolerance in common. Its diagnosis and characterization, including pre-diabetes, type I and type II diabetes, and a variety of syndromes characterized by impaired glucose tolerance, impaired fasting glucose, and abnormal glycosylated hemoglobin, are well known in the art. It can be characterized by hyperglycemia, glycosuria, ketoacidosis, neuropathy or nephropathy, increased hepatic glucose production, insulin resistance in various tissues, insufficient insulin secretion and enhanced or poorly controlled glucagon secretion from the pancreas.
The term “obesity” as used herein is defined in the WHO classifications of weight. Underweight is less than 18.5 BMI (thin); healthy is 18.5-24.9 BMI (normal); grade 1 overweight is 25.0-29.9 BMI (overweight); grade 2 overweight is 30.0-39.0 BMI (obesity); grade 3 overweight is greater than or equal to 40.0 BMI. BMI is body mass index (morbid obesity) and is kg/m2. Waist circumference can also be used to indicate a risk of metabolic complications. Waist circumference can be measured (in cm) at midpoint between the lower border of ribs and the upper border of the pelvis. Other measures of obesity include, but are not limited to, skinfold thickness and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution.
The term “obesity-related condition” can refer to any disease or condition that is caused by or associated with (e.g., by biochemical or molecular association) obesity or that is caused by or associated with weight gain and/or related biological processes that precede clinical obesity. Examples of obesity-related conditions include, but are not limited to, diabetes (e.g., type 1 diabetes, type 2 diabetes, and gestational diabetes), metabolic syndrome, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, insulin resistance, hypercholesterolemia, atherosclerosis, coronary artery disease, peripheral vascular disease, and hypertension. For example, “metabolic syndrome” can refer to a syndrome marked by the presence of a combination of factors (e.g. high blood pressure, abdominal obesity, high triglyceride levels, low HDL levels, and high fasting levels of blood sugar) that are linked to an increased risk of cardiovascular disease and type 2 diabetes. For example, metabolic syndrome can be marked by the presence of three or more of such factors.
Regardless of the indication, a therapeutically effective amount of a composition comprising the botanically-derived extract or compound, or a unit dosage form which comprises the same, is administered to the subject. The term “administration” can refer to introducing a substance, such as the botanically-derived compound or extract, into a subject. Any route of administration can be utilized including, such as those described herein.
The term “subject” or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Subjects to which compounds of the disclosure can be administered include mammals, such as primates, for example humans. For veterinary applications, a wide variety of subjects are be suitable, for example, livestock such as cattle, sheep, goats, cows, swine, and the like; poultry, such as chickens, ducks, geese, turkeys, and the like; and domesticated animals, such as pets, for example dogs and cats. For diagnostic or research applications, a wide variety of mammals are suitable subjects, including rodents (for example, mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted above or another organism that is alive. The term “living subject” can also refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.
Kits
Aspects of the invention are directed towards kits.
The term “kit” can refer to a set of articles that facilitates the process, method, assay, analysis, or manipulation of a sample. The kit can include instructions for using the kit (eg, instructions for the method of the invention), materials, solutions, components, reagents, chemicals, or enzymes required for the method, and other optional components.
For example, the compositions as described herein can be provided in a kit. In one embodiment, the kit includes (a) a container that contains the compositions described herein or components thereof (e.g., solvents, buffers, extracts, botanical compounds), and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that encompasses the methods described herein and/or the use of the agents for therapeutic benefit. In an embodiment, the kit also includes a second agent, such as at least one additional active agent.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material encompasses methods of manufacturing one or more botanical compositions, and/or methods of administering botanical compositions to a subject, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject). The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material.
The composition in the kit can include other ingredients, such as a solvent or buffer, culture media, a stabilizer, or a preservative. The compositions of the kit thereof can be provided in any form, e.g., liquid, dried or lyophilized form, and can be substantially pure and/or sterile. When the compositions are provided in a liquid solution, the liquid solution can be an aqueous solution or an alcohol solution. When the compositions or components thereof are provided as a dried form, reconstitution, for example, is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit. The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes the botanical composition and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be air-tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.
Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Botanicals have been used as therapeutics, and some pharmaceuticals in use today have been derived, directly or indirectly, from plant compounds. One such drug is metformin, originally derived from French lilac (Galega officinalis) and is now used as a first line therapeutic for the treatment of type 2 diabetes mellitus. Our screening of over 400 compounds for the ability to enhance lipid accumulation and promote adipocyte differentiation of 3T3-L1 adipocytes led to the identification of an ethanolic extract of Artemisia scoparia (SCO) as promoting adipocyte differentiation in vivo. Studies in diet-induced obese mice showed the SCO supplementation in the high-fat diet had beneficial effects on the adipose tissue, increasing adiponectin expression and insulin signaling and decreasing markers of inflammation. Additionally, SCO improved whole-body insulin sensitivity and reduced circulating triglycerides, glycerol, and free fatty acids.
Adipose tissue (AT) is a critical player in metabolic regulation (Kusminski, Bickel, and Scherer 2016). Obesity, the main disorder involving AT, is arguably the greatest health issue currently affecting the Western world, as it is the major driver of the high rates of cardiovascular disease and insulin resistance in developed nations (Bhupathiraju and Hu 2016; Ranasinghe et al. 2017; Saklayen 2018). In obese states, the normal functions of adipose tissue are disrupted, contributing to whole-body metabolic dysfunction (Vidal-Puig 2013). Although the inhibition of fat cell development can sound like a beneficial strategy to reduce obesity and associated disorders, obesity is associated with impaired adipocyte differentiation, along with ectopic lipid deposition and metabolic dysfunction in muscle and liver (Danforth 2000; Gustafson et al. 2015; Kim et al. 2007; Smith and Kahn 2016). The insulin-sensitizing thiazolidinediones (TZDs) improve metabolic health through activation of peroxisome proliferator-activated receptor-γ (PPARγ) and promotion of adipogenesis (Hammarstedt et al. 2005; Soccio, Chen, and Lazar 2014). Concerns over side effects and safety have led to a decline in clinical use of TZDs in recent years, however selective PPARγ modulators (SPPARMs) that can enhance adipocyte differentiation are a subject of study as therapeutics for obesity-related metabolic disease (Chigurupati, Dhanaraj, and Balakumar 2015; Dunn et al. 2011; Feldman, Lambert, and Henke 2008; Higgins and Depaoli 2010).
Botanicals have been used as therapeutics, and some pharmaceuticals in use today have been derived, directly or indirectly, from plant compounds. One such drug is metformin, originally derived from French lilac (Galega officinalis) and is now used as a first line therapeutic for the treatment of type 2 diabetes mellitus (T2DM)(Thomas and Gregg 2017; Witters 2001). To identify proadipogenic/anti-diabetic compounds, we performed a blinded screen of over 400 botanical extracts for their ability to enhance lipid accumulation and promote adipocyte differentiation of 3T3-L1 adipocytes. These efforts led to the identification of an ethanolic extract of Artemisia scoparia (SCO) as promoting adipocyte differentiation in vitro (Allison J Richard, Fuller, et al. 2014; Richard, Burris, et al. 2014).
Following our discovery that SCO could improve adipocyte differentiation in vitro, we performed studies in diet-induced obese (DIO) mice to determine if SCO could have metabolically favorable effects in vivo. SCO supplementation in the high-fat diet (HFD) had beneficial effects on the adipose tissue, increasing adiponectin expression and insulin signaling, and decreasing markers of inflammation. In addition, SCO improved whole-body insulin sensitivity and reduced circulating triglycerides, glycerol, and free fatty acids (Richard, Burris, et al. 2014; Allison J. Richard, Fuller, et al. 2014). For these animal studies, the botanical was either administered by gavage or incorporated into the mouse food. Other studies examined the effects of SCO and these studies have observed a variety of effects for SCO. In one study, SCO was shown to increase in the ratio of Bacteroidetes to Firmicutes bacterial populations in the mucosal layer of the ileum in a mouse model of diet-induced obesity (Wicks et al. 2014).
A role of adipocytes/adipose tissue is to store and release lipid stores as needed. In fasted conditions, adrenergic input to the adipose tissue promotes the release of fatty acids to the circulation to be used as an energy source by other tissues, a process known as lipolysis. In contrast, in fed conditions, low adrenergic input and high insulin levels favor lipogenesis and energy storage in adipocytes. Insulin resistant states are associated with elevated rates of basal lipolysis in the fed state, which in turn lead to further metabolic dysfunction (Morigny et al. 2016). The pro-inflammatory conditions present in obesity and insulin resistance are known to contribute to this increased basal lipolysis. A major mediator of AT inflammation, tumor necrosis factor-alpha (TNFα) can induce lipolysis in adipocytes in the absence of adrenergic stimulation (Frahbeck et al. 2014; Kawakami et al. 1987; Sharma and Puri 2016). Gene expression studies from the Stephens labs indicate that SCO could reduce inflammatory markers in AT. We also observed that SCO reduced circulating FFAs and glycerol in mice (Boudreau, Richard, Jasmine A. Burrell, et al. 2018). Hence, we examined anti-lipolytic effects of SCO in vitro in cultured adipocytes. We observed that SCO could inhibit TNFα-induced lipolysis in cultured 3T3-L1 adipocytes and this was accompanied by changes in the levels and phosphorylation of two proteins involved in lipolysis, perilipin and hormone-sensitive lipase (Boudreau, Richard, Jasmine A. Burrell, et al. 2018). SCO reduced glycerol and free fatty acid release induced by TNFα, but not by isoproterenol (a β-adrenergic agonist), supporting a role for SCO in limiting inflammation-driven lipolysis in AT.
Like obesity, chronically elevated glucocorticoid levels promote metabolic disruptions such as insulin resistance, hypertension, and hepatic lipid accumulation reviewed here (Strohmayer 2011). Although high cortisol levels caused by naturally occurring Cushing's Syndrome are rare, synthetic glucocorticoids can be prescribed for chronic conditions such as asthma, autoimmune disorders, cancer and other inflammatory conditions (Benard-Laribiere et al. 2017; Fardet, Petersen, and Nazareth 2011; Overman, Yeh, and Deal 2013). The Cushingoid phenotype has been successfully recapitulated and studied in a variety of rodent models incorporating synthetic glucocorticoid administration and genetic manipulations. In both humans and rodents, high glucocorticoid levels have been found to increase adipose tissue lipolysis and to induce insulin resistance (Divertie, Jensen, and Miles 1991; Harvey et al. 2018; Hochberg et al. 2015; Shen et al. 2017). Given the clinical need for synthetic glucocorticoids and their widespread use, adjuvant therapies to combat the metabolic disturbances induced by these drugs would be beneficial. Since we have observed reductions in TNFα-induced lipolysis and improvements in insulin sensitivity with SCO treatment (Boudreau, Richard, Jasmine A Burrell, et al. 2018), we tested SCO's ability to regulate glucocorticoid-induced lipolysis in cultured adipocytes, in the presence or absence of the glucocorticoid receptor (GR) antagonist RU486. We found that SCO attenuated lipolysis to the same extent as RU486, and that the effects of RU486 and SCO on lipolysis were additive. These results indicate that SCO does not act directly on GR signaling and, therefore, without wishing to be bound by theory, can attenuate the lipolytic effects of glucocorticoids without inhibiting their therapeutic anti-inflammatory activity.
Inflammation in adipose tissue contributes to whole-body metabolic dysfunction (Engin 2017; Kawakami et al. 1987; Ohmura et al. 2007; Stephens, Lee, and Pilch 1997; Stephens and Pekala 1992). This process involves interactions between adipocytes and AT immune cells, for example, macrophages (Burhans et al. 2018; Olefsky and Glass 2010). To further characterize the effects of SCO in the context of AT inflammation, we induced activation of macrophages with lipopolysaccharide (LPS). We observed that SCO mitigated LPS-induced gene expression of interleukin 1 beta (Il-1b) and inducible nitric oxide synthase (iNOS), but not that of TNFa(Tnf). In TNFα-treated adipocytes, we have observed that SCO pretreatment mitigates induction of the inflammatory genes interleukin 6 (I16), C—C motif chemokine ligand 2 (Ccl2), and lipocalin 2 (Lcn2). Overall, these results show that, without wishing to be bound by theory, that SCO can potently reduce inflammatory gene expression in macrophages and adipocytes.
A challenge in botanical research is that plant extracts are comprised of hundreds of compounds, and it is difficult to identify which of these compounds confer biological activity. Activity-guided fractionation studies of SCO extracts have been performed for over a decade. There were a lot of complications (technical, mechanical, and personnel) along the way. We have screened dozens of extract fractions for their ability to promote adipocyte development as has been shown for the parent SCO extracts. This process revealed that SCO contains non-overlapping active fractions, indicating the presence of several compounds that can promote adipogenesis. See, for example, Boudreau et al. 2019, which is incorporated by reference herein in its entirety. We have recently elucidated the structures of three of these compounds, all of which are prenylated coumaric acid derivatives (PCAs). These compounds were purified by and the used for NMR structural and chemical compositions analysis. Two of these compounds, capillartemisin A and capillartemisin B, have been previously reported in A. scoparia (Kitagawa et al. 1983).The third compound, however, is new. For example, another prenylated coumaric acid, Artepillin C, has been shown to activate PPARγ and enhance adipogenesis (Choi et al. 2011).
Described herein is, for example, the discovery that three compounds isolated from SCO can promote adipocyte differentiation and, without wishing to be bound by theory, improve overall metabolic health based on studies of parent SCO extract in diabetic mice.
Botanicals have been used as therapeutics, and many pharmaceuticals in use today have been derived, directly or indirectly, from plant compounds. One such drug is metformin, originally derived from French lilac (Galega officinalis) and now used as a first line therapeutic for Type 2 diabetes mellitus. To study the effects of botanicals on adipocyte development, we screened over 400 botanical extracts from plants across the globe for the ability to modulate adipocyte differentiation in vitro. Less than 5% of the extracts we screened regulated adipogenesis, and only one of them could promote adipocyte development—an ethanolic extract of the botanical Artemisia scoparia (SCO). In addition to promoting adipogenesis in vitro, we have demonstrated that SCO decreases lipolysis in mice as well as in cultured adipocytes in a cell-autonomous manner, and that SCO supplementation in vivo did not induce weight loss but did protect mice from some of the negative effects of high-fat feeding. SCO can also attenuate some of the negative effects of glucocorticoids (GC) and tumor necrosis factor alpha (TNFα) on adipocytes. In another screening study, we observed that SCO significantly enhanced longevity in the nematode C. elegans. Collectively, our data on SCO demonstrate its capabilities to improve multiple aspects of systemic metabolic health.
We purified and identified three compounds in SCO that can promote lipid accumulation and adipogenic marker gene expression in murine preadipocytes. One of these compounds, which can be referred to as “scoprenyl” (SP), is a prenylated coumaric acid that we have shown can also promote adipogenesis in human preadipocytes. Without wishing to be bound by theory, at least some of the in vivo effects of SCO can be mediated by scoprenyl. The aims are to investigate the pharmacokinetics, in vivo bioactivity, and toxicity of scoprenyl.
Project Narrative: Obesity and Type 2 diabetes are global epidemics that negatively impact the quality of life. To combat these epidemics, new approaches are required that are safe, widely available and inexpensive. Many therapeutics are developed from compounds obtained from plants. Extracts of Artemisia scoparia (SCO) have anti-diabetic effects in vivo. Our in vivo studies will assess efficacy, pharmokinetics, and toxicity of scoprenyl, a component we purified from SCO.
Aims:
Previously, our lab screened over 400 botanical extracts from plants across the globe to examine their ability to modulate adipocyte differentiation in vitro. Less than 5% of the extracts we screened regulated adipogenesis, and only one of them could promote adipocyte development—an ethanolic extract of the botanical Artemisia scoparia (SCO). In addition to its ability to promote adipogenesis in vitro, we have also observed that SCO decreases lipolysis in vitro and in vivo. Our studies in mice revealed that SCO supplementation did not induce weight loss, but did protect mice from some of the negative effects of high-fat feeding, including fatty liver and elevated insulin levels. We also observed improvements in insulin signaling in adipose tissue adipose tissue with SCO supplementation. Moreover, our recent studies demonstrate that SCO can extend life span in C. elegans. Although it has taken many years, we have recently identified three bioactive compounds in the SCO extract that promote adipocyte development in vitro. NMR data confirm that one of these compounds is a prenylated coumaric acid, which can be referred to as “scoprenyl” (SP). Based on the activity of the SCO parent extract, SP can also promote metabolic health by enhancing adipogenesis and reducing ectopic lipid accumulation. Our aims will consist of validation of SP. The studies described herein will validate that scoprenyl is a viable therapeutic for drug development and treatment of metabolic disease states.
Aim 1: To assess pharmacological properties of scoprenyl (SP). Our studies have established that SCO extract can enhance adipocyte differentiation in vitro, and mitigate metabolic disruptions associated with obesity in mice. Recently, we identified SP as a constituent compound of SCO that can recapitulate the effects of SCO on adipogenesis in vitro, and we now will study SP as a therapeutic for high-fat diet-induced metabolic dysfunction. This will require validation of SP's pharmacological properties. We will assess SP's solubility and stability in vitro, as well as pharmacokinetics, bioavailability, and tissue distribution in vivo. Since SP can behave differently when assayed as a pure compound versus within a whole SCO extract, all in vivo parameters will be assessed using both SP alone and the SCO extract at a dose which we know to be effective in improving insulin sensitivity in HFD-fed mice. Data obtained in this aim will not only validate SP as a therapeutic, but will also validate the optimal SP dose for use, as we will be able to compare circulating levels of SP in mice gavaged with SP only versus the SCO extract.
Aim 2: SP's enhance glucose tolerance in diet-induced obese mice and assess in vivo metabolism of SP in a chronic feeding study. Our studies have shown beneficial effects of parent SCO extract in the C57Bl/6J mice, a well characterized mouse model of obesity and insulin resistance. We will validate that SP can also reduce some of the negative consequences of high-fat feeding in male and female mice. We will incorporate SP in the diet of the mice for six weeks; SP dose will be determined based on data obtained in Aim 1. We will also perform insulin and glucose tolerance tests, as well as measures of adipose tissue function and lipid metabolism. These studies will allow us to validate that SP improves metabolic health in the context of diet-induced metabolic dysfunction. In addition, because diet incorporation can alter pharmacological parameters of SP, we will collect serum and tissue samples from this study for analysis of SP content. These data will be compared to those obtained from gavaged animals in Aim 1.
Aim 3: To examine the toxicity of scoprenyl using the C. elegans model. Our data demonstrate that SCO parent extract extends life span in C. elegans (
It is known that genetic, dietary, pathological, or aging-related disruptions of adipose tissue function are among the underlying causes of the global epidemic of metabolic diseases. Adipocytes not only store and release lipid, but are insulin sensitive and are the sole source of some endocrine hormones; disruption of any one of these adipocyte functions can lead to systemic metabolic dysfunction1-3. In addition, inhibition of adipocyte differentiation impairs adipose tissue expansion and has negative metabolic consequences. Adipocyte dysfunction is associated with ectopic lipid accumulation, inflammation in adipose tissue, and altered adipokine expression. Hence, the identification of specific compounds that improve or maintain adipocyte function and systemic metabolic health has merit.
We have been studying an extract of Artemisia scoparia (SCO) that improves adipocyte function in vitro and in vivo47. Throughout human history, botanicals have been used as therapeutics, and many pharmaceuticals in use today have been derived, directly or indirectly, from plant compounds. One such drug is metformin, originally derived from French lilac (Galega officinalis) and now used as a first line therapeutic for Type 2 diabetes mellitus (T2DM)8,9. To study the effects of botanicals on adipocyte development, we screened over 400 botanical extracts from plants across the globe for the ability to modulate adipocyte differentiation in vitro. Less than 5% of the extracts we screened regulated adipogenesis, and only one of them promoted adipocyte development—an ethanolic extract of the botanical Artemisia scoparia (SCO). In addition to promoting adipogenesis in vitro, we have demonstrated that SCO decreases lipolysis in mice as well as in cultured adipocytes in a cell-autonomous manner, and that SCO supplementation in vivo did not induce weight loss, but did protect mice from some of the negative effects of high-fat feeding. Our studies indicate that SCO can also attenuate some of the negative effects of both glucocorticoids (GC) and tumor necrosis factor alpha (TNFα) on adipocytes. In another screening study, SCO was also shown to significantly enhance longevity in the nematode C. elegans. Our efforts have identified bioactive compounds in SCO (
Artemisia: There are over 1600 genera in Asteraceae and at least 68 different Artemisia species in this genus, many of which have a history of medicinal use10. Artemisia annua, a plant widely used in traditional Chinese medicine, is the source of the anti-malarial compound artemisinin, whose discovery was awarded the Nobel Prize in Physiology or Medicine in 201511. Artemisinin-based combination therapies are recommended treatments for malaria, and artemisinin is being studied for a range of therapeutic effects related to cancer and other diseases12-14. Interestingly, SCO also contains artemisinin, and without wishing to be bound by theory, can be an alternative source for the therapeutic compound15. Although certain beneficial effects of Artemisia extracts are known16-20, they have not been supported by rigorous basic or clinical research. One exception is the well documented effectiveness of Artemisia dracunculus L., to regulate insulin action20,21,30,31,22-29. However, the involvement of adipose tissue in the whole-body effects of Artemisia species has not been evaluated.
Artemisia scoparia (SCO): SCO has been investigated in various diseases. In a spontaneously hypertensive rat model of Alzheimer's disease, rats fed diets containing 2% (w/w) of SCO for 6 weeks had various improvements, including lower levels of amyloid R and phosphorylated tau proteins, compared to controls32. SCO extracts have several beneficial effects on carbon tetrachloride induced oxidative stress in rat kidneys, including reduction of DNA damage in renal nuclei33, indicating a therapeutic role of SCO in renal oxidative stress-related disorders. Hepatoprotective, antitumor, antiviral, antihypertensive, hemostatic, and free radical-scavenging effects of SCO have also been reported34-41. Anti-inflammatory properties of SCO have been observed in several models42-44. Flavonoids from SCO reduce intracellular oxidative stress and inflammatory cytokine production in macrophages, and suppress inflammatory responses in a model of acute lung injury. In these studies, and without wishing to be bound by their, repression of NF-κB and MAP kinase signaling pathways can be the mechanism for these effects45. In a mouse model of atopic dermatitis, SCO and some of its components can reduce clinical symptoms of skin lesions and levels of various inflammatory mediators in both the lesions and the serum. DEQA (3,5-dicaffeoyl-epi-quinic acid), a major component of a butanol-extracted SCO fraction in these studies, can reduce caspase 1 activity46. Another study in mast cells also showed anti-inflammatory effects of both SCO and DEQA, including reduced cytokine expression and caspase activity47. Although we have detected DEQA in our SCO extracts, these compounds do not promote adipogenesis4. Ethnobotanical studies in various regions of Pakistan document the medicinal and common use of SCO as well as a high-fidelity level for SCO and diabetes48-50. This ethnobotanical research underscores the importance of traditional herbal preparations in remote regions, where they are often the only available form of medication, and highlights the wealth of traditional knowledge in such areas.
Innovation:
Significance: SCO enhances the development of both murine and human adipocytes in vitro, reduces lipolysis in mice and in adipocytes in a cell-autonomous manner, and mitigates metabolic dysfunction in vivo in a mouse model of diet-induced obesity (5-7,51 and
DATA:
Published data from our laboratory have shown that SCO promotes differentiation of 3T3-L1 adipocytes67. Activity-guided fractionation efforts have determined that non-overlapping fractions of SCO could recapitulate the effects of the SCO parent extract on adipogenesis, indicating the presence of several bioactive compounds4. Recently, we purified and identified three distinct compounds in the SCO parent extract that could independently promote lipid accumulation and adipogenic marker gene expression in 3T3-L1 cells (
Human subcutaneous preadipocytes from two lean donors were purchased from Zenbio, Inc. As shown in
Collaborators screened a nuclear receptor ligand-binding domain (LBD) library for activation by SCO in HEK293 cells, using vectors containing the LBDs of all 48 human nuclear receptors fused to the Gal4 DNA-binding domain, and a reporter construct encoding the Gal4 upstream activator sequence to drive luciferase expression. Of the 48 nuclear receptors, only peroxisome proliferator activated receptor gamma (PPARγ) was significantly activated by SCO in this model system6,7. We have recently examined the ability of SCO to modulate transcriptional activity of PPARγ in NIH-3T3 fibroblasts using a consensus PPAR response element (PPRE) from the acyl CoA oxidase promoter element linked to a luciferase reporter. In these studies, we observed that ROSI activates PPARγ transcription activity in NIH-3T3 cells transfected with ectopic PPARγ. To our surprise, however, we did not observe an induction of PPARγ transcription activity with SCO (
A class of PPARγ agonist with a dibenzoazepine scaffold has been identified55, but these compounds are also distinct from our identified bioactives (
Lipolysis is the release of fatty acids and glycerol from adipocytes. Obesity and insulin resistance are associated with both AT inflammation and elevated basal lipolysis57. Also, enhancement of lipolysis by the inflammatory cytokine TNFα, contributes to metabolic dysfunction58-60. Like TNFα, glucocorticoids (GCs) can also cause insulin resistance in vivo and promote lipolysis in cultured adipocytes61-64. We have shown that SCO supplementation of a high-fat diet (HFD) reduces circulating levels of free fatty acids (C18:0, C18:1, C16:0, and C16:1) and glycerol5. SCO inhibits TNFα-induced lipolysis in 3T3-L1 adipocytes, but has no significant effect on isoproterenol-induced lipolysis, which is mediated by adrenergic signaling5. SCO can also attenuate GC (dexamethasone)-induced lipolysis in adipocytes (
Cellular communication between immune cells and adipocytes plays an important role in insulin resistance, and it is well established that macrophage-derived TNFα levels in AT are elevated in conditions of obesity and insulin resistance. TNFα acts in a paracrine manner on preadipocytes to inhibit adipogenesis, and on adipocytes to induce production of inflammatory mediators and promote insulin resistance. Hence, we examined the ability of SCO to modulate TNFα-induced inflammatory cytokine gene expression in fat cells. SCO pretreatment significantly inhibited TNFα-induced expression of lipocalin-2 (Lcn2), interleukin 6 (Il6), and C—C motif chemokine ligand 2 (Ccl2), also known as monocyte chemoattractant protein 1 (Mcp1) in adipocytes (
NF-kB signaling is induced by TNFα in adipocytes, and contributes to metabolic dysfunction60,65-67. In addition, inflammatory cytokines induced by TNFα are known to be transcriptional targets of NF-kB (www.nf-kb.org). We have observed that a chronic pretreatment with SCO significantly attenuates TNFα-induced p65 nuclear translocation in mature 3T3-L1 adipocytes, indicating a role for NF-kB signaling in SCO's anti-inflammatory effects.
Notably, the TZD pioglitazone does not attenuate the ability of TNFα to activate NF-kB in 3T3-L1 adipocytes, despite inhibiting insulin signaling at these same time points68. In another study, the TZD troglitazone suppressed the expression of some NF-kB target genes, but did not inhibit IkBa phosphorylation, or NF-kB activation or DNA-binding activity in response to TNFα in 3T3-L1 adipocytes69. These studies also provided evidence that the p65 subunit of NF-kB and PPARγ could antagonize each other's transcriptional activities69. Based on our observations with SCO, SCO can regulate a functional antagonism between p65 and PPARγ, and act on multiple integrated signals that impact adipocyte gene expression and function. We will continue our current work to validate the mechanisms involved in the ability of SCO and SP to affect adipocyte function. We note that SCO does not modulate NF-kB in a manner shared by two other PPARγ activators, indicating that SP and/or other SCO bioactives will be very distinct from drugs that target PPARγ.
We have validated the effects of the SCO parent extract in animal studies. For example,
Aim 1: To Assess Pharmacological Properties of SP.
Rationale: While we have established the ability of SCO diet supplementation to improve measures of metabolic health in a DIO mouse model (
Preparation of SCO extracts and purified compounds: Ethanolic extracts will be prepared from greenhouse-grown plants as described previously5,7. We observed that hexane and ethyl acetate, but not water, partitions of SCO contain components that promote adipocyte development4. However, starting from the ethyl acetate fraction, we have performed two successive and distinct semi-preparatory HPLC protocols to obtain the three bioactives shown in
In vitro solubility and stability assays of SP: Assessments of aqueous solubility and chemical and metabolic stability can provide estimations of in vivo compound availability and metabolic clearance, respectively. These are considered critical steps in early drug discovery stages. We will perform solubility studies in phosphate buffered saline (PBS) at pH 7.4 and pH 4.0 (to mimic acidic conditions in the stomach). We will also validate intrinsic clearance (CLint) of SP in murine liver S9 fractions that are enriched in microsomes and cytosol containing a wide variety of drug metabolizing enzymes. A half-life approach will be used to validate metabolic stability. Such parameters, for example, can be used to design the in vivo PK/bioavailability studies.
In vivo pharmacokinetic, bioavailability, and tissue distribution studies of SCO and SP: We will also conduct the in vivo portion of this aim. A single low dose of SP (1 mg/kg) or SCO (500 mg/kg, a dose shown to have efficacy on metabolic parameters in vivo) will be administered to mice either intravenously or by oral gavage. Blood will be collected after 0.25, 0.5, 1, 2, 4, 8, and 24 hours in both groups, with an additional collection after 5 minutes for the IV group only. Samples will be analyzed for SP levels. The initial 1 mg/kg SP dose was calculated based on the estimated percent of SP within the total SCO extract (less than 1%) and dose of SCO with demonstrated efficacy. If circulating SP is not readily detected at this low dose, we will test three to five higher doses. Various pharmacokinetic parameters will be calculated, and descriptive statistics will be generated using Phoenix WinNonlin software. At the 24-hour time point of the low-dose study, mice will be sacrificed, and liver, adipose tissue, lungs, kidneys, and brain will be collected to analyze tissue distribution of SP. For adipose tissue, we will focus analyses on gonadal and inguinal depots, but will collect other fat pads, such as mesenteric and retroperitoneal, and have them in storage should data indicate the need to analyze them. Of course, SP levels in fat tissue will be of interest to us, since, without being bound by theory, we consider adipose tissue to be the primary mediator of SCO's in vivo effects. We will validate whether SP accumulates in fat tissue, as we have demonstrated that SCO and SP have cell-autonomous effects on adipocytes and preadipocytes.
To conduct in vivo assessments of SP's bioavailability or pharmacokinetics, we sent six-year-old archived serum samples from our last SCO feeding study in high fat-fed mice for analysis. These samples were collected at the time of sacrifice after a 4-hour fast, in conditions that were not designed for detection of SCO compounds in the circulation. As indicated in
Aim 2: SP Enhances Glucose Tolerance in Diet-Induced Obese Mice, and Validate In Vivo Metabolism of SP in a Chronic Feeding Study.
Rationale: SCO promotes adipogenesis in cultured 3T3-L1 adipocytes, and, without wishing to be bound by theory, these adipogenic effects are responsible for SCO's ability to enhance insulin sensitivity in mice fed a HFD. Our studies have revealed that SP can recapitulate the effects of SCO on adipogenesis in murine and human cells (
Without wishing to be bound by theory, Scoprenyl can improve metabolic dysfunction during diet-induced obesity in mice.
Validate SP's ability to improve metabolic function in DIO mice. The overall study design is shown in
Validate the metabolism of SP in vivo, in mice fed HFD supplemented with SCO or SP. PK and bioavailability studies will rely on oral gavage of SCO and SP, while our efficacy study will use diet supplementation. After 1 day, and 1, 3, and 5 weeks on supplemented diets, we will collect blood from 3 mice per group in rotating cohorts to assess levels of SP in circulation. This will allow us to validate whether there are substantial differences in PK and bioavailability between oral gavage and diet incorporation of SP and to compare the levels of circulating SP in diet-fed versus gavage conditions. It will also allow us to validate when steady state levels are reached and whether there is any accumulation in circulation with chronic administration. Tissues collected at the end of the study, adipose tissue (gonad and inguinal depots which can accumulate lipophilic compounds), liver, kidneys, and lung (highly perfused tissues), and brain (to determine CNS penetration) will be collected from 3 mice per SCO and SP treatment groups to be analyzed for SP content. This analysis will be important to validate tissue distribution and bioavailability of SP and if there is any bioaccumulation. Analysis of SP content in plasma and tissue samples will be performed at Inotiv. These analyses will also validate if there are any differences in PK and bioavailability of SP between males and females.
Aim 3: To Validate the Toxicity of Scoprenyl Using the C. elegans Model.
Assessing toxicity of candidate drug molecules in animals is an important step in validating therapeutic applicability. The nematode C. elegans provides an alternative animal model for assessing toxicity75,78, which has consistently proven reliable and predictive of toxicity in mammalian species79-83. Greater than 80% of genes in C. elegans have known human homologs84, and characterized drug molecules show similar modes of action in worms and mammalian systems78.
Compared to other animals, C. elegans has a fast generation time; it grows from embryo to reproductive adult in roughly three days85. Upon reaching adulthood, C. elegans hermaphrodites begin to self-fertilize, each producing hundreds of offspring until sperm are depleted86. Thus, it is easy and relatively inexpensive to establish large, synchronous populations of worms, which can be used to analyze the toxicity of compounds at a wide range of concentrations. Importantly, toxicity screening in C. elegans allows one to assess responses at an organismal level, which cannot be done when toxicity is assessed in vitro using mammalian cell lines78. Further, wild-type C. elegans live only two to three weeks86. This provides a unique opportunity to validate safety and/or toxic effects on whole, intact animals at different life stages.
Worms can respond to SCO active compounds, and that this treatment is non-toxic at the tested concentration (500 μg/ml). However, the toxicity of the new SP compound (
Without wishing to be bound by theory, Scoprenyl is non-toxic to both young and old animals.
Analyze acute toxicity and DART (developmental and reproductive toxicity): Toxicity in humans is best validated when multiple measures of toxicity are assessed in animal models. Recently, a platform for simultaneously scoring acute toxicity and DART in C. elegans was described87. We will apply this approach to validate SP's safety and/or toxicity. Analysis will be performed using 24-well plates. Each well will contain NGM solid medium seeded with E. coli OP50 as a food source88. DMSO (control) or varying concentrations of SP (nM to mM range) will be added to the NGM in the different wells. Five wild-type N2 hermaphrodites will be transferred to each well at the L1 larval stage, and plates will be maintained at 20° C.87. Pictures of the wells will be taken on each subsequent day. At non-toxic concentrations, wild-type Lis should develop into reproductive adults and begin producing progeny by the third day. The expanding populations should begin to starve by the fifth or sixth day as all the food is consumed. We will score acute toxicity by determining concentrations at which the PO animals die. We will score developmental and reproductive delay (i.e., DART) by quantifying differences in the time to starvation between experimental populations and controls87. For comparison, we will analyze SP alongside chemicals that are known to cause acute toxicity and/or DART at a wide range of concentrations in mammals and worms87. Examples include boric acid, warfarin, aldicarb, fenoxycarb, spirotetramat, and piperazine (ToxCast database, https://www.epa.gov/chemicalresearch/toxcast-chemicals; Pesticide database, http://pesticideinfo.org/). We will also validate SP safety and/or toxicity in bus (bacterially unswollen) mutants89, which lack a protective cuticle90 that, when intact, could interfere with an accurate assessment of toxic effects87. Without wishing to be bound by theory, SP will be less toxic than known toxic compounds.
Determine LC50 at different time points during aging: Chemical toxicity can be exacerbated by aging; some chemicals can have more toxic effects in older animals than in younger ones91. This is an important consideration, because metabolic disease is most common among older populations92, and viable therapeutic leads would need to be non-toxic to older individuals. We can easily validate chemical safety and/or toxicity during aging using C. elegans because of its short lifespan. We will administer varying concentrations of SP to C. elegans at different time points of adulthood (day 1, day 5, day 10, and day 15). Worms will initially be raised on standard NGM agar with OP50 and then moved to SP- or DMSO-supplemented plates on the relevant day of adulthood. We will score acute toxicity after treatment to determine the 24-hour, 48-hour, and 96-hour LC50 for SP for the different age groups. To maintain adult populations that are separate from progeny, worms will be transferred to new plates every two days.
Statistical analysis: At least five replicates will be performed per concentration for the acute toxicity and DART analyses that start with L1 animals. Statistical analysis of delays in food consumption will be carried out using ANOVA tests. To validate 24-hour, 48-hour, and 96-hour LC50 using worms of different ages, at least 100 worms will be scored for each concentration at each time point of adulthood. Each experiment will be repeated at least three times, and mean LC50s at the different ages will be compared by ANOVA tests.
Summary: The first aim includes in vitro and in vivo assessments of SP's pharmacological properties. These will serve to demonstrate the presence of SP in animal sera and validate the appropriate SP dose and route of delivery for achieving doses comparable to those obtained with a SCO dose known to be effective in improving insulin sensitivity.
Once we have identified the best dose of SP to use, we will perform mouse studies in the second aim to validate that SP can reduce some of the negative consequences of diet-induced obesity. Further, we can also validate whether other bioactives that we have identified (
These studies will significantly improve upon or differentiate from existing clinical care. We know that SP can promote adipogenesis in vitro. There is evidence that promotion of adipogenesis in vivo is metabolically healthy, and the importance of adipogenesis and adipose tissue expansion has been recognized for nearly twenty years93. As described herein, we will characterize SP and validate its suitability as a therapeutic compound.
We can also synthesize derivatives of these compounds and explore their structure-activity relationships by validating their ability to promote adipogenesis in vitro and, ultimately, their efficacy in vivo.
Mouse PK Experiments: Male C57BL/6J mice (8-10 weeks of age) will be used. Animals arrive and acclimate at SLU vivarium space for 3-7 days. Following acclimation, the animals will be given a single dose of scoprenyl or the parent SCO extract, and euthanized 24 hours after dosing. Mice will be administered a single dose of each compound/extract intravenously (IV) or by oral (PO) gavage in nine mice per compound/extract per route of administration (ROA) (n=3 per bleeding time point). Volume for a single IV bolus or PO administration is limited to 20 mL/kg. A full PK curve (7-8 time points) will be obtained by sparse sampling with rotating cohorts of 3 mice per time point per compound/extract per ROA. Two or three timed blood samples will be obtained from each mouse. The first two samples will be obtained via peripheral blood collections using the submandibular procedure with retro-orbital route blood collections as a secondary option. The third and final sample will be obtained via cardiac puncture following euthanasia.
Mouse Efficacy Experiments: Male and female C57BL/6J mice fed low-fat diet (10% kcal fat) or high fat diet (45% kcal fat) for 12 weeks beginning at 6 weeks of age will be used. Following a 7-day quarantine and acclimation period within the PBRC CBC vivarium, mice (purchased from commercial vendor) will be fed diet supplemented with test compound/extract for 6 additional weeks. Blood for will be collected via submandibular vein of each mouse for baseline measures of adipokine, insulin, glucose, glycerol, and NEFA and for 3 mice (in rotating groups) at day 1 and weeks 1, 3, and 6 for pharmacokinetic measures. Mice will undergo the following procedures: measurement of body weight and body composition (via NMR), oral glucose tolerance tests (OGTT) and intraperitoneal insulin tolerance tests (IP-ITT). Mice will also be subjected to 4 hours of fasting prior to GTT or ITT and euthanasia. During GTTs and IP-ITTs, blood will be collected via tail snip at baseline and 5-6 designated time points between 0 and 120 min following glucose or insulin administration. For OGTTs, glucose will be delivered via oral gavage using a flexible, plastic gavage needle, and this procedure will be performed by a well-trained technician. Terminally, blood will be collected via cardiac puncture under deep anesthesia by isoflurane gas inhalation. Mice will be euthanized by isoflurane overdose or carbon dioxide inhalation followed by cervical dislocation, and tissues will be removed for RNA, protein analyses, and histology. Male and female mice will be used for all animal studies.
Survival blood collections for both PK and efficacy experiments are limited to a total volume of 10 mL/kg/animal over the course of 14 days.
Aims:
Nearly a decade ago, our lab screened over 400 botanical extracts from plants across the globe for the ability to modulate adipocyte differentiation in vitro. Less than 5% of the extracts we screened regulated adipogenesis, and only one of them could promote adipocyte development—an ethanolic extract of the botanical Artemisia scoparia (SCO). In addition to promoting adipogenesis in vitro, we have demonstrated that SCO decreases lipolysis in mice as well as in cultured adipocytes in a cell-autonomous manner, and that SCO supplementation in vivo did not induce weight loss but did protect mice from some of the negative effects of high-fat feeding.
Our more recent studies demonstrate that SCO can also attenuate some of the negative effects of glucocorticoids (GC) and tumor necrosis factor-alpha (TNFα) on adipocytes. SCO was also shown to significantly enhance longevity in C. elegans. In addition, we have recently identified three bioactive compounds in SCO that promote adipocyte development in vitro. Without wishing to be bound by theory, SCO promotes metabolic health and longevity by enhancing adipocyte development and reducing inflammation.
We will use a variety of in vitro and in vivo model systems to perform the validation studies. Overall, these aims will leverage our published and preliminary data to examine the metabolic mechanisms involved in SCO's action on adipocytes and metabolic health. Similar to metformin, SCO and its bioactives may work by affecting several pathways that impact metabolic diseases.
Aim 1: To validate the ability of SCO to promote adipogenesis in vivo and to validate that the effects of SCO are dependent on PPARγ expression. SCO promotes adipogenesis in both murine and human adipocytes. Our studies have shown that SCO enhances insulin sensitivity in high fat-fed mice. Without wishing to be bound by theory, the ability of SCO to increase adipogenesis during HFD feeding confers at least part of its insulin sensitizing effects. We will use the AdipoChaser mice, a state-of-the-art mouse model, to validate SCO's ability to promote adipocyte development in vivo. We will use another animal model (adipocyte-specific inducible PPARγ knockouts) to validate the ability of SCO to promote metabolic health is dependent on adipocyte PPARγ expression in adult male and female mice.
Aim 2: Underlying mechanisms involved in SCO to attenuate the metabolic dysfunction induced by glucocorticoids. Our data demonstrate that SCO can reduce GC-induced lipolysis. This observation is relevant because elevated lipolysis is associated with metabolic dysfunction. Our studies in cultured adipocytes also revealed that SCO attenuates the ability of GCs to induce the expression of a subset of GC-regulated genes. We will validate the effects of SCO on GC action in adipocytes and to determine if SCO can attenuate GC-induced insulin resistance in vivo.
Aim 3: To validate the mechanisms involved in the ability of SCO and its bioactives to inhibit TNFα action in adipocytes. SCO promotes adipogenesis, but we have also observed other effects of this botanical extract, including anti-inflammatory properties. SCO can reduce some actions of the proinflammatory cytokine TNFα. This aim will include in vitro experiments in primary mouse adipocytes to assess the anti-inflammatory abilities of the SCO bioactives, as well as mechanistic experiments to validate how SCO modulates NF-κB activity in adipocytes.
Significance: These studies will assess metabolic mechanisms and pathways involved in the ability of SCO to modulate adipocyte function and systemic metabolic health. We will employ innovative mouse models to study adipogenesis in vivo and to validate the role of PPARγ in mediating SCO's effects. Additional experiments will also validate that SCO can mitigate GC-induced metabolic dysfunction. We will use mechanistic studies to validate the pathways responsible for SCO's anti-inflammatory effects in adipocytes and to evaluate the ability of individual bioactive compounds from SCO to mediate these effects. Overall, these aims will provide mechanistic information on SCO's significant effects on adipocyte function.
Background
Adipocytes: Genetic, dietary, pathological, or aging-related disruptions of adipose tissue (AT) function are among the underlying causes of the global epidemic of metabolic diseases. Adipocytes not only store and release lipids, but are insulin sensitive and are the sole source of some endocrine hormones; disruption of any one of these adipocyte functions can lead to systemic metabolic dysfunction1-3. In addition, inhibition of adipocyte differentiation impairs AT expansion and has negative metabolic consequences. Adipocyte dysfunction is associated with ectopic lipid accumulation, inflammation in AT, and altered adipokine expression. Hence, the identification of plant extracts and specific compounds that improve or maintain adipocyte function and systemic metabolic health has merit.
For the past decade, we have been studying an extract of Artemisia scoparia (SCO) that improves adipocyte function in vitro and in vivo4-7.
Artemisia: There are >1600 genera in Asteraceae and at least 68 different Artemisia species in this genus, many of which have a history of medicinal use8. Artemisia annua, a plant widely used in traditional Chinese medicine, is the source of the anti-malarial compound artemisinin, whose discovery was awarded the Nobel prize in 20158. Artemisinin-based combination therapies are a recommended treatment for malaria and artemisinin is being studied for a range of therapeutic effects related to cancer and other diseases9-11. Interestingly, SCO also contains artemisinin, and can be an alternative source for the therapeutic compound12. Although certain beneficial effects of Artemisia extracts are known13-17, they have not been supported by rigorous basic or clinical research. One exception is the well documented effectiveness of Artemisia dracunculus L., to regulate insulin action 17,18,27,28,19-26. However, the involvement of AT in the whole-body effects of Artemisia species has not been evaluated.
Artemisia scoparia (SCO): SCO has been investigated in various disease states, including a spontaneously hypertensive rat model of Alzheimer's disease, oxidative stress-related renal disorders, cancer, hypertension, viral infection, hepatotoxicity, and hemostatic disorders29-37. Anti-inflammatory properties of SCO have been observed in models such as acute lung injury, atopic dermatitis, and mast cell activation38-43. Quantitative ethnobotanical studies in various regions of Pakistan document the medicinal and common use of SCO, with a high-fidelity level for SCO and diabetes44-46. This type of research underscores the importance of traditional herbal preparations in remote regions, where they are often the only available form of medication.
Innovation:
SCO promotes adipocyte development in mouse and human adipocytes.
Three SCO bioactives promote adipogenesis.
SCO reduces lipolysis in vitro and in vivo.
SCO has anti-inflammatory effects on adipocytes in vitro.
SCO reduces some of the metabolically unhealthy effects of glucocorticoids on adipocytes.
SCO significantly increases longevity in C. elegans.
SCO enhances the development of both murine and human adipocytes in vitro and mitigates metabolic dysfunction in vivo in mouse models of diet-induced obesity. Our studies will validate metabolic mechanisms and pathways involved in the ability of SCO to modulate adipocyte function and promote metabolic health. We will employ innovative mouse models to study adipogenesis in vivo and to validate the role of the transcription factor, peroxisome proliferator-activated receptor, gamma (PPARγ), in mediating SCO's effects. We will use rigorous mechanistic studies to validate the pathways responsible for SCO's anti-inflammatory effects in adipocytes and evaluate the ability of individual bioactive compounds from SCO to mediate these effects. We will use state-of-the-art methodologies to validate genes that mediate the ability of SCO to increase longevity in worms. These studies, as well as experiments in glucocorticoidinduced insulin resistant mice, are of great translational relevance and will also reveal pathways and SCO compounds that mediate the anti-inflammatory effects of SCO.
Data:
We have shown that SCO promotes differentiation of 3T3-L1 adipocytes4,5. Activity-guided fractionation efforts have determined that non-overlapping fractions of SCO could recapitulate the effects of the parent extract on adipogenesis, indicating the presence of several active compounds6. Recently, we identified three distinct compounds in SCO that could independently promote lipid accumulation and adipogenic gene expression in 3T3-L1 cells (
Human subcutaneous preadipocytes from two lean donors were purchased from Zenbio, Inc. As shown in
We also validated the ability of SCO to modulate transcriptional activity of PPARγ in NIH-3T3 fibroblasts using a consensus PPAR response element (PPRE) from the acyl CoA oxidase promoter element linked to a luciferase reporter. In these studies, we observed that ROSI activates PPARγ transcription activity in NIH-3T3 cells transfected with ectopic PPARγ. To our surprise, however, we did not observe an induction of PPARγ transcription activity with SCO (
PPARγ protein levels are significantly reduced in adipocytes in response to specific ligands such as thiazolidinediones48. Ligand-induced activation of PPARγ triggers its polyubiquitylation and proteasomal degradation, and is therefore associated with increased protein turnover49. To further examine the effects of SCO on PPARγ, we treated mature adipocytes with the protein synthesis inhibitor, cycloheximide. As shown in
Lipolysis is the release of fatty acids and glycerol from adipocytes. Obesity and insulin resistance are associated with both AT inflammation and elevated basal lipolysis50. Also, enhancement of lipolysis by the inflammatory cytokine, tumor necrosis factor, alpha (TNFα), contributes to metabolic dysfunction51-53. Like TNFα, glucocorticoids (GCs) can cause insulin resistance in vivo and promote lipolysis in cultured adipocytes54-57. We have shown that SCO supplementation of a high-fat diet (HFD) reduces circulating levels of free fatty acids (C18:0, C18:1, C16:0, and C16:1) and glycerol7. SCO inhibits TNFα-induced lipolysis in 3T3-L1 adipocytes, but has no significant effect on isoproterenol-induced lipolysis, which is mediated by adrenergic signaling7. SCO can also attenuate GC (dexamethasone) induced lipolysis in adipocytes (
Both products of lipolysis, glycerol and non-esterified fatty acids (NEFA), were assayed. Cellular communication between immune cells and adipocytes plays an important role in insulin resistance, and in conditions of obesity and insulin resistance, there is an increase in macrophage-derived TNFα levels in AT. TNFα acts in a paracrine manner on preadipocytes to inhibit adipogenesis and on adipocytes to induce production of inflammatory mediators and promote insulin resistance. Hence, we examined the ability of SCO to modulate TNFα-induced inflammatory cytokine gene expression in fat cells. SCO pretreatment significantly inhibited TNFα-induced expression of lipocalin-2 (Lcn2), interleukin 6 (I16), and C-C motif chemokine ligand 2 (Ccl2), also known as monocyte chemoattractant protein 1 (Mcp1) in adipocytes (
NF-κB signaling is induced by TNFα in adipocytes and contributes to metabolic dysfunction53,58-60. In addition, inflammatory cytokines induced by TNFα are known to be transcriptional targets of NF-κB (www.nf-kb.org). We have observed that a chronic pretreatment with SCO significantly attenuates TNFα-induced p65 nuclear translocation in mature 3T3-L1 adipocytes, indicating a role for NF-κB signaling in SCO's anti-inflammatory effects.
Aim 1: To Validate the Ability of SCO to Promote Adipogenesis In Vivo and to Determine if the Effects of SCO are Dependent on PPARγ Expression in Mature Adipocytes.
Rationale: Our data demonstrate that SCO promotes adipogenesis in murine and human adipocytes in vitro, and specifically activates the ligand binding domain (LBD) of PPARγ fused to the Gal4 DNA-binding domain (DBD). Since PPARγ agonists such as TZDs promote adipogenesis and improve metabolic health62-65, without wishing to be bound by theory, the ability of SCO to increase adipogenesis during HFD feeding can confer at least part of its insulin sensitizing effects. Assessing SCO's effects on adipogenesis in vivo will be critical to understanding SCO's metabolic actions. The inability of SCO to induce PPARγ transcriptional activity in NIH-3T3 cells stably transfected with PPARγ, or in 3T3-L1 adipocytes, when a PPRE-luciferase reporter construct was used (
Clinical use of insulin-sensitizing full agonists of PPARγ, such as ROSI, has declined sharply in recent years, due to negative side effects including weight gain, fluid retention, bone loss, and heart problems71. However, so-called selective PPARγ modulators (SPPARMs) are the subject of intense study as therapeutics for obesity-related metabolic disease72-75. Therefore, SCO's partial PPARγ agonism could make it a therapeutic.
In our previous animal studies, we did not observe increased body weight or adiposity when mice were SCO treated from 2 to 12 weeks4,5. Moreover, mice consuming SCO-supplemented HFD for 12 weeks did not have decreased hematocrit levels (
Without wishing to be bound by theory, SCO promotes adipogenesis in vivo. Use the AdipoChaser mouse model to determine if SCO promotes the formation of new adipocytes in vivo. Adipocytes are long-lived cells, and technical limitations have long precluded in vivo assessment of adipocyte formation. However, the AdipoChaser mouse, an inducible adipocyte-tagging system established over five years ago77, has provided important data on the development of AT and its modulation by various physiological challenges78. We will use this mouse model to validate that SCO modulates adipogenesis in subcutaneous and visceral AT depots in male and female mice under low- and high-fat feeding conditions. It is known that inhibiting AT expansion results in ectopic lipid accumulation and metabolic dysfunction79-82. Hence, without wishing to be bound by theory, SCO enhances metabolic health by promoting adipocyte expansion. To validate this, we will perform the experiment outlined in the
To create the AdipoChaser mouse, we will cross three transgenic lines: the adiponectin promoter driven tetracycline-on (Teton) transcription factor rtTA (adiponectinP-rtTA)83 line, a Tet-responsive Cre (TRE-cre) line that can be activated by rtTA in the presence of doxycycline (DOXY)84, and another transgenic line carrying the Rosa26 promoter-driven loxP-stop-loxP-O-galactosidase (Rosa26-loxP-stop-loxP-lacZ)77,85.
The triple transgenic AdipoChaser mice express the transcription factor rtTA in all of their mature adiponectin expressing adipocytes. DOXY administration activates Cre expression via the TRE promoter, and the Cre protein specifically excises the floxed transcriptional stop cassette and turns on LacZ expression, resulting in permanent LacZ expression in these adipocytes, even after removal of the DOXY. LacZ-expressing cells stain blue when histological AT sections are exposed to an appropriate P-galactosidase substrate.
Starting at 8 weeks of age (WOA), mice will be fed either low-fat diet (LFD) or HFD for 10 weeks, followed by 7 days of DOXY supplementation (600 mg/kg diet) and a 3-day DOXY washout period on their respective diets to label mature adipocytes. Based on data, this protocol labels 100% of the mature adipocytes and allows for complete washout of residual DOXY prior to additional treatment or intervention. This will be followed by LFD or HFD feeding with or without SCO or ROSI supplementation for 6 weeks. We have used this experimental design successfully to establish insulin resistance in HFD-fed mice and have observed positive effects of SCO.
Research Strategy
A total of 15 animals per treatment group will be used. At study's end, we will anesthetize and perfuse 6 mice from each group with 0.2% glutaraldehyde prior to collecting gonadal and inguinal white adipose tissue (gWAT and iWAT) for histology and 3-gal staining as described in 77. It will be critical to determine whether metabolic outcomes correlate with changes in adipocyte formation in this experiment. To that end, 6 animals per group will be subjected to insulin tolerance tests (ITT) one week prior to sacrifice. At the endpoint, these mice will be euthanized, and iWAT, gWAT, mesenteric WAT (mWAT), liver, and skeletal muscle will be excised and snap frozen. Markers of proliferation, inflammation, neurons, vasculature, and adipocyte function will be assessed in whole-tissue extracts. Finally, three animals per group will be used for ex vivo lipolysis assays, where basal, induced (adrenergic, TNFα), and suppressed (insulin) lipolysis will be assessed in the iWAT and gWAT explants, as measured by glycerol and NEFA release into incubation medium. Blood samples will be collected from all non-perfused mice (n=9/condition) at time of sacrifice, after a 4-hour fast, and assayed for glycerol and NEFA levels as indicators of in vivo lipolysis, and for circulating insulin, glucose, lipids, and adipokines. The HFD used for these experiments will be 45 kcal % fat (D12451 from Research Diets), and the LFD (D12450H) will have 10 kcal % fat with matching amount of sucrose.
Without wishing to be bound by theory, ROSI and SCO supplementation will both enhance adipogenesis in subcutaneous AT in both male and female mice, in both LFD and HFD conditions, and that ROSI will have a greater effect than SCO.
Without wishing to be bound by theory, SCO will improve glucose tolerance and reduce the hepatic lipid accumulation that occurs with high-fat feeding. Since female C57BL/6J mice are less metabolically compromised by HFD than male mice, we may not observe profound effects of SCO on metabolic outcomes (GTTs, fasting insulin levels, circulating lipids, adipogenesis) in females.
Without wishing to be bound by theory, SCO won't alter lipolysis in LFD-fed mice, but will lower circulating glycerol and NEFA levels in HFD mice. Ex vivo lipolysis will allow us to assess insulin's ability to suppress lipolysis in AT explants, which might be improved by SCO in HFD-fed mice.
Without wishing to be bound by theory, the ability of SCO to improve HFD-induced glucose intolerance is not dependent on PPARγ in mature adipocytes. Use an inducible adipocyte-specific PPARγ knockout mouse to determine if the metabolic effects of SCO in vivo are dependent on PPARγ expression in mature adipocytes. We have established that SCO exerts profound effects on adipocyte development and function in vitro, and on whole-body metabolism in vivo, and that it acts as a type of agonist for PPARγ. Although PPARγ is crucial in regulating the metabolic functions of AT, including lipolysis, inflammation, and adipokine secretion, studies have shown that the PPARγ agonist ROSI can improve glucose tolerance in mice that lack PPARγ only in mature adipocytes of adult mice89, indicating that PPARγ in mature adipocytes is not necessary for the metabolic benefits of ROSI. Our understanding of metabolic mechanisms of SCO will be enhanced if we can validate that its effects on metabolic health in vivo require the presence of PPARγ in mature adipocytes.
We will create triple transgenic inducible adipocyte-specific PPARγ knockout mice (Adn-PPARγ−/−) by crossing the adiponectinPrtTA 83 and TRE-cre lines84 described herein for the AdipoChaser experiments with the PPARγflox/flox line from Jackson Laboratories (stock #: 004584) and treat with DOXY as described89. Cre expression will be selectively turned on in adipocytes following DOXY treatment, and the Cre protein will excise the floxed PPARγ locus to knock out PPARγ expression selectively in mature adipocytes in adult mice. We will use PPARγflox/flox mice as controls, and all mice will receive DOXY in the diet.
As shown in
Studies characterizing the inducible adipocyte PPARγ knockout model demonstrated, quite surprisingly, that ROSI could improve insulin sensitivity independently of PPARγ expression in mature adipocytes89. Without wishing to be bound by theory, this study will repeat this observation. Without wishing to be bound by theory, since SCO behaves as a partial PPARγ agonist, it can recapitulate some but not all of ROSI's actions. Should SCO improve glucose tolerance in the knockout, this would add to the similarities we have observed between ROSI and SCO. If SCO fails to improve glucose metabolism in the absence of adipocyte PPARγ, this can represent a difference between SCO and ROSI and demonstrate PPARγ independent effects of SCO. Without wishing to be bound by theory, PPARγ dependence of SCO's metabolic effects would indicate that SCO can have a distinct mode of action from TZDs.
Aim 2: Mechanisms Involved in SCO to Attenuate the Metabolic Dysfunction Induced by Glucocorticoids (GCs).
GCs are prescribed for a variety of serious and chronic medical conditions91-93, but their use often results in a metabolic disease state94. Therefore, it is paramount to identify ways to attenuate the harmful effects of these drugs. GC signaling in AT plays a role in the development of insulin resistance and fatty liver disease associated with excess GCs57,95,96. Data has shown improvements in obesity-associated insulin resistance and a reduction in hepatic lipid accumulation following SCO treatment in HFD-fed mice4,5,97. However, until recently, we had not examined the effects of SCO on glucocorticoid-induced metabolic dysfunction. We therefore performed experiments to validate whether SCO could attenuate some of the negative effects of GCs on murine adipocytes, and found that SCO suppresses GC-induced lipolysis in 3T3-L1 adipocytes (
We validated the effects of dexamethasone (DEX), a synthetic glucocorticoid, on gene expression in 3T3-L1 adipocytes, with or without SCO pretreatment, and observed that SCO reduced the ability of DEX to induce serum/glucocorticoid regulated kinase 1 (Sgk1) and serine (or cysteine) peptidase inhibitor, clade A, member 3n (Serpina3n) mRNA expression, but not that of other known GC-regulated genes such as dual specificity phosphatase 1 (Dusp1) or lipin 1 (
Without wishing to be bound by theory, SCO attenuates effects of glucocorticoids on adipocytes in a cell-autonomous manner. Characterize SCO's effects on DEX-induced changes in SGK1 and SERPINA3N protein levels, glucocorticoid receptor (GR) translocation, and GR binding to Sgk1 and Serpina3n promoters. Our data have identified two glucocorticoid-induced genes, Sgk1 and Serpina3n, whose DEXmediated induction is attenuated by SCO in adipocytes. To identify mechanisms involved in this regulation, we will validate that alterations in Sgk1 and Serpina3n mRNA levels are accompanied by changes in protein levels. We will also validate that SCO reduces the nuclear translocation of the glucocorticoid receptor (GR) in adipocytes, and examine the binding of GR to the promoters of these two genes in the presence and absence of SCO, using Chromatin Immunoprecipitation (ChIP) analysis. A GRE (GC response element) has been characterized in the human Sgk1 promoter112. We will identify the GRE(s) in the mouse Sgk1 promoter and determine if there is a GRE in the Serpina3n promoter. These experiments will be conducted in mature primary murine adipocytes after treatment with SCO or vehicle control for 72 hours, an experimental paradigm in which SCO reduces DEX-induced lipolysis (
Validate that regulation of Serpina3n or Sgk1 is required for SCO's effects on lipolysis and glucose uptake in glucocorticoid-treated adipocytes. DEX increases adipocyte lipolysis and regulates the mRNA and protein expression of several lipolytic enzymes and beta-adrenergic signaling proteins55,113-116 and SCO reduces DEX-induced lipolysis in 3T3-L1 adipocytes (
Without wishing to be bound by theory, SCO will reduce the DEX-mediated induction of Sgk1 and Serpina3n protein expression, however these effects can or cannot be mediated by alterations in GR trafficking to the nucleus or in GR promoter binding. We have substantial experience performing ChIP122, and have previously studied GR translocation in adipocytes123. Experiments will be conducted in murine adipocytes, and repeated in human adipocytes purchased from Zenbio, Inc. We have purchased and successfully used both human preadipocytes (
Without wishing to be bound by theory, SCO can attenuate glucocorticoid-induced insulin resistance in vivo. SCO reduces some of the metabolic dysfunction associated with HFD in mice4,5, and a one-week treatment of corticosterone (CORT), the active endogenous GC in rodents, induces insulin resistance in mice124. Without wishing to be bound by theory, we will validate that SCO can mitigate CORT-induced insulin resistance in vivo. Since there are sex differences in obesity, diabetes, and GCs in various mouse models, such as C57BL/6 mice125, both male and female C57BL/6J mice will be used. Mice will be ordered from the Jackson Laboratory at 5 WOA and will have 3 weeks to acclimate to their new surroundings, then body weight, body composition, and blood glucose will be used to determine randomization into groups. At 8 WOA, mice will be fed a LFD diet with or without 1% SCO supplementation for two weeks. Mice will then be treated with 100 mg/mL CORT, or 1% ethanol (vehicle), in their drinking water for an additional week while remaining on their respective diets. An ITT will be performed via intraperitoneal injection of insulin (1U/kg of lean body mass) following 5 days of CORT treatment (
Blood will be collected prior to diet switch, prior to CORT treatment, and prior to euthanasia, to assess various metabolic outcomes, including circulating glucose and insulin. Mice will be euthanized after one week of CORT treatment. At this time, liver and AT will be excised and either snap frozen or placed in formalin to examine GCregulated gene and protein expression or histology, respectively. Additionally, small samples of AT will be removed to assess ex vivo lipolysis.
Without wishing to be bound by theory, the GC treatment described above will induce insulin resistance as judged by an ITT 124, and that SCO supplementation can reduce this effect. SCO can improve GC-induced insulin resistance, this approach will help validate this effect. If SCO can ameliorate GC-induced metabolic dysfunction, this could be a very high-impact observation given the widespread use of GCs and the ongoing search for therapies to mitigate its unfavorable actions. In our cultured adipocyte experiments, not all DEXinduced genes were affected by the SCO supplementation (
Aim 3—to Validate the Mechanisms Involved in the Ability of SCO and its Bioactives to Inhibit TNFα Action in Adipocytes.
TNFα acts via several signaling pathways including the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB)/NF-κB and MAP kinase pathways. NF-κB induces expression of many inflammatory cytokines through nuclear translocation of its p65 subunit, which then binds to its target gene promoters (reviewed in 53,58 & www.nf-kb.org). Our data show that SCO significantly inhibits the expression of the inflammatory genes Lcn2, I16, and Ccl2(Mcp1) in TNFα-treated 3T3-L1 adipocytes (
These observations are of physiological importance, since circulating levels of LCN2 are elevated in obese and insulin resistant states127-129. We have also shown that SCO inhibits nuclear translocation of NF-κB p65 in TNFα-treated adipocytes (
Without wishing to be bound by theory, SCO attenuates TNFα-induced inflammatory cytokine expression through inhibition of NF-κB signaling in adipocytes.
We will validate that SCO can reduce TNFα-induced activation of an NF-κB response element. SCO substantially reduces the TNFα-induced expression of some inflammatory genes (
We will validate that SCO can reduce p65 binding to promoters of specific genes induced by TNFα in adipocytes. NF-κB is required for TNFα-induced induction of Lcn2 in 3T3-L1 adipocytes 126 and 116 and Mcp1 are known to be induced by NF-κB in various cell types. To validate that SCO can inhibit TNFα-induced binding of p65 to these specific gene promoters in adipocytes, chromatin immunoprecipitation (ChIP) assays will be conducted in which we will assess binding of the p65 subunit of NF-κB to the promoters of Lcn2, Ccl2, and 116 in primary mouse adipocytes treated with TNFα.
We will validate that SCO affects TNFα-induced changes in phosphorylation of NF-κB p65 or degradation of IκB proteins in adipocytes. Induction of the NF-κB signaling pathway by TNFα involves the phosphorylation of IκB, which targets it for proteasomal degradation13. Nuclear translocation and transcriptional activity of the p65 subunit of NF-κB are also modulated by phosphorylation at several sites131. We will treat mouse primary adipocytes with TNFα and determine whether pretreatment with SCO can limit degradation of IκB or alter phosphorylation of p65 at Ser276 and Ser536, the principal sites involved in TNFα-induced gene activation131,132.
Without wishing to be bound by theory, dicaffeoylquinic acids (DCQA) and/or prenylated coumaric acids (PCA) present in SCO can inhibit TNFα-induced inflammatory cytokine expression. We have purified three different prenylated coumaric acids (PCA) from SCO, each of which can enhance adipogenesis in 3T3-L1 preadipocytes (
Since SCO inhibits TNFα-induced nuclear translocation of p65, without wishing to be bound by theory, SCO will modulate the transcriptional activity of p65, as measured by direct activation of the NF-κB response element. Also, SCO will reduce binding of p65 to the promoters of our genes of interest (I16, Ccl2, and Lcn2), whose expression levels are inhibited by SCO. However, the observed SCO effects on inflammatory cytokine expression may not require changes in p65 activity or DNA binding. Indeed, data from our laboratory show that transcription of Lcn2 induced by TNFα is suppressed by ERK inhibition, in the absence of any effects on binding of p65 to the Lcn2 promoter59. Also, the effects of SCO on DNA binding may be gene-specific, which would be an intriguing finding. Also, three MAP kinase pathways are known to be activated by TNFα in adipocytes (ERK 1/2, p38, and Jnk) 53; any one of them could be impacted by SCO in a way that interferes with NF-κB signaling and target gene expression. SCO can also alter the phosphorylation of specific sites in p65 and/or the degradation of IκB proteins known to be triggered by TNFα. Although SCO's effects on gene expression can occur without such changes, answering these questions will allow us to validate which signaling elements are modulated by SCO.
Vertebrate Animals:
1. Description of Procedures: We will isolate preadipocytes from inguinal adipose tissue of 5 to 6-week-old C57BL/6J, Adn-PPARgfl/fl, and PPARgfl/fl male and female mice; these cells will be differentiated to establish mouse primary adipocyte cultures as described in Aims 2 and 3. We will also breed AdipoQ-rtTA and TRE-Cre mice to either Rosa26-loxP-stop-loxP-lacZ or PPARgfl/fl mice to generate the AdipoChaser or doxycycline-inducible PPARg adipocyte knockout (Adn-PPARg−/−) in adult mice. All of the mice are on a C57BL/6J background. All mice will be genotyped between 2-3 weeks of age using a tail snip method. In Aim 1, male and female AdipoChaser mice, Adn-PPARgfl/fl, and control PPARgfl/fl mice will be fed doxycycline-supplemented LFD or HFD for 4-7 days. These mice will then be fed LFD or HFD plus or minus doxycycline and SCO or Rosi or appropriate control LFD or HFD for an additional 7 days-6 weeks. The HFD used for these experiments will be 45 kcal % fat, and the LFD will have 10 kcal % fat with matching amount of sucrose. Terminal assessment will be performed at 21 or 26 weeks of age. For adipocyte-specific inducible PPARg KO experiments, doxycycline-fed floxed littermates will be used as controls. In Aim 2, C57BL/6J will be fed LFD diet with or without SCO supplementation for 3 weeks with additional corticosterone or ethanol vehicle administration via their drinking water during the final week prior to euthanasia and blood and tissue collection at 11 weeks of age. Mice will undergo the following procedures: measurement of body weight and body composition (via NMR), OGTTs, IPGTT, and IP-ITT. Mice will also be subjected to 4 hours of fasting prior to GTT or ITT and euthanasia. During GTTs and IP-ITTs, blood will be collected via tail snip at baseline and 15, 30, 60, 90, and 120 min following glucose or insulin administration. For OGTTs, glucose will be delivered via oral gavage using a flexible, plastic gavage needle, and this procedure will be performed by a well-trained technician. IP-GTTs will be performed by injecting 2-[3H]-DG for 120 min prior to euthanasia. Blood for adipokine, insulin, glucose, glycerol, and NEFA analyses may also be collected via submandibular vein. Terminally, mice will undergo glutaraldehyde perfusion (Aim 1, Adipochaser mice) or blood will be collected via cardiac puncture (Aims 1 and 2) under deep anesthesia by isoflurane gas inhalation. Mice will be euthanized by isoflurane overdose or carbon dioxide inhalation followed by cervical dislocation, and tissues will be removed for RNA, protein analyses, and histology. Male and female mice will be used for all animal studies.
References cited in this example:
Prenylated Coumaric Acids from Artemisia scoparia Beneficially Modulate Adipogenesis
Abstract
Two new di-prenylated coumaric acid isomers (1a/b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR, LC-MS, and their purity assessed by 1D 1H pure shift qNMR analysis. The bioactivity of 1a/b-3 was validated by enhanced accumulation of lipid, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin, in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the extract, 1a/b-3 showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This indicates that additional bioactives can contribute to metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.
Introduction
Artemisia is one of the largest and most diverse genera of the family Asteraceae and comprised of over 500 species, which contain a diverse array of phytochemicals that are used as traditional medicines and sources of pharmaceutical agents. Artemisia scoparia Waldst. & Kit is an annual herb distributed from Central Europe to Western Asia that has been studied for the biological activity of its essential oil as well as for other phytochemical preparations. We identified adipogenic activity in an EtOH extract of A. scoparia (SCO), as hundreds of screening results of plants for bioactivity related to metabolic syndrome.1 Several bioactivities of SCO have been identified, including the promotion of adipogenesis in vitro, as measured by both lipid accumulation and the expression of adipogenic gene), as well as the enhancement of insulin sensitivity in high-fat diet-induced obese mice.2,3 SCO also showed distinct inhibitory effects on lipolysis (the release of lipids) in both cultured murine adipocytes and in mice fed a high-fat diet supplemented with SCO.4,5 In an effort to demonstrate how the constituents of SCO contribute to the observed adipogenic bioactivity, we conducted a series of the bioactivity-guided fractionation studies. This previously led to the putative identification of the principal bioactive constituents in SCO as prenylated coumaric acids, coumarin monoterpene ethers, 6-demethoxycapillarisin, and two polymethoxyflavones.5
A study by Yahagi et al. identified a collection of compounds isolated from an aqueous EtOH extract of A. scoparia that inhibited triglyceride accumulation in adipocytes.6 Indeed, the inhibition of lipid accumulation and adipocyte differentiation was once considered an appropriate strategy for combating obesity-related metabolic dysfunction. However, it is well established that impairments in adipogenesis and adipose tissue expansion can promote the metabolic dysregulation associated with insulin resistance.7-10 This is underscored by the potent insulin-sensitizing effects of the thiazolidinedione (TZD) drugs, which act by enhancing adipocyte differentiation.11 The compounds identified as inhibitors of triglyceride accumulation were primarily chromane derivatives that were active at higher concentrations than those tested.6 In our present studies, compounds 1-3 were purified from SCO using bioassay-guidance, structurally characterized by spectroscopic methods including NMR, and validated as bioactive markers for SCO by a panel of in vitro assays.
Results and Discussion
An extract of A. scoparia, SCO, was previously shown to have beneficial adipogenic properties in vitro and in vivo, which were maintained through the process of the bioactivity-guided fractionation and present in distinct fractions comprised of prenylated coumarins and related compounds.5 Further separation of the fractions led to the isolation of six compounds that were subsequently structurally characterized and subject to bioassays. Three of those compounds retained some, i.e., a statistically significant portion, but not all the biological activity of their parent fractions. The structures of compounds 1-3 are shown in
Bioactivity-guided fractionation is a method used to identify the active constituents of complex mixtures of compounds and relies on the use of appropriate in vitro assays where small amounts of separated materials can be tested. While it can lead to bioactive markers, the inability to assess “synergistic” bioactivity is its major and widely recognized limitation. Compared to the previously published process,5 the separation and extraction process was optimized for this study in terms of compound yield and minimization of samples to be tested. EtOAc extraction of the dried A. scoparia plants replaced the broader polarity EtOH extraction and its subsequent fractionation by CPC. HPLC separation of the EtOAc extract produced three fractions that contained the bioactive compounds previously described. Individual HPLC methods utilizing a chiral column were then used to produce nearly pure compounds (see discussion herein about purity) from each of those fractions.5 Compounds 1-3 were obtained from fractions III and V. While fraction IV was also separated further, no resulting component exhibited adipogenic activity and, thus, no structural studies were performed on these components.
Structure Elucidation of Di-prenylated Coumaric Acid Derivatives. The crude ethyl acetate partition of the aerial parts of A. scoparia was purified by HPLC column chromatography (CC), which led to two di-prenylated coumaric acids, 1a and 1b, isolated as a mixture, along with two known compounds, 2 and 3, which were characterized as capillartemisins A (2) and B (3) by 1D and 2D NMR spectroscopic data and comparison with reported spectroscopic data.12-15
Compound 1a was isolated as an amorphous solid and as a mixture with 1b. The HRESIMS (m/z 397.1616 [M+Na]+, calcd for C21H26O6Na+, 397.1622) indicated a molecular formula of C21H26O6 associated with an LC retention time of 5.8 min (
The molecular formula of 1b (C21H26O6), which was the second component in the mixture along with 1a, was also deduced from the HRESIMS (m/z 397.1627 [M+Na]+), exhibiting the same molecular formula of C21H26O6 ([M+Na]+, calcd for C21H26O6Na+, 397.1622) as 1a, but exhibited at an LC retention time of 6.0 min. The 1H and 13C NMR data of 1b were almost identical to those of 1. The key difference of 1b arose from a cis double bond of coumaric acid with olefinic hydrogen resonating at δH 5.81 ppm and exhibiting a characteristic J coupling [d, J=12.6 Hz, H-7)/6c 122.6 (C-7) and δH 6.44 (d, J=12.6 Hz, H-8)/6c 136.2 (C-8)]. The di-prenylated group could be located via the HMBC correlation from H-1′ to C-2 and C-3 (δC 130.5 and 128.3 ppm, esp.), and from H-1″ to C-5 and C-6 (δC 128.5 and 130.5, resp.). The HMBC correlation (
Cis-trans Isomerism of Di-prenylated Coumaric Acid. Photochemical mechanisms are known to generate cis-trans isomerization of olefins. Other mechanisms are induced thermally, by acid or base catalysis, or by reaction with molecules that contain an odd number of electrons.16,17 Among these isomerism factors, photoisomerization can occur spontaneously in the plant due to the exposure to sunlight. As the di-prenylated coumaric acids possess an α,β-unsaturation, absorption of longer wavelengths can promote trans-to-cis isomerization in these compounds.18
Without wishing to be bound by theory, the trans (E) isomer tends to be more stable than the cis (Z) isomer.19,20 Notably, the di-prenylated coumaric acid has three α,β-unsaturated olefinic hydrogens, each can undergo cis-trans isomerization. Thus, besides the presence of the diastereomeric species 1a and 1b, additional cis-trans isomers exist in the plant, the mother fraction that contained 1a and 1b, and purified 1a/1b samples. To validate this, the sample containing the mixture of 1a/1b was evaluated using pure shift NMR spectroscopy confirmed the presence of the isomeric compounds which are shown as the singlet peaks at the characteristic chemical shift of di-prenylated proton region, aided by the extraction of additional ion traces from the total ion LC-MS chromatogram (
Spectroscopic Detection of Isomeric Patterns. Representing a versatile structural tool, NMR is widely utilized to elucidate the structures of isolated (“pure”) natural products. While the classical 1D 1H NMR spectrum contributes essential information (number of hydrogens, their chemical shift and spin-spin coupling constants), the spectra of pure compounds are often already (over) crowded, which explains why spectra of materials that contain other, near-identical structures as mixtures often challenge NMR data interpretation. One solution to the signal overlap challenge is the pure shift NMR spectrum,21 which removes spin-spin coupling and, thereby, reduces the resonance of each hydrogen to a singlet. This improves the signal resolution and simplifies spectral analysis and interpretation.22 In the given case of a mixture of closely related, diastereomeric congeners, the ability to distinguish molecular species is a valuable addition to the structural information obtained from coupling patterns to determine the cis-trans isomerization.
The pure shift 1H NMR spectrum of 1a and 1b was, thus, obtained to suppress the spin-spin coupling patterns in the crowded spectral regions between δH 1.70 and 5.70 ppm (
Purity Analysis Using 100% PF-qNMR Method. The pure shift NMR data confirmed the presence of other isomeric di-prenylated coumaric acids, which gave rise to additional minor NMR signals resonating close-by those of 1a and 1b. The pure shift experiments also revealed that the presence of minor isomers were hidden under the “shoulders” of the main resonances of 1a and 1b. Applying the peak-fitting (PF) approach using peak deconvolution, PF-qNMR enabled the disentangling of the overlapped resonances for the purpose of their relative quantitation.23 Subsequent application of the 100% qNMR method led to the measurement of the relative ratio of 1a and 1b to be calculated as 71.6% 5.70 and 28.4%±1.86, respectively (
Compounds from A. scoparia promote adipogenesis. Bioactivity-guided fractionation studies were previously used to identify several fractions from SCO that can promote adipocyte differentiation, as assessed by enhanced accumulation of lipid and increased expression of adipogenic genes in differentiating 3T3-L1 adipocytes.4,5 Six distinct sub-fractions were isolated from these fractions as “single” compounds in order to assign the bioactivity to individual compounds (
Compounds 1a/b-3 also increased the expression of adipogenic genes for adiponectin (AdipoQ), fatty acid binding protein 4 (Fabp4), and peroxisome proliferation activated-receptor gamma (PPARγ) in 3T3-L1 adipocytes relative to the negative controls (
Compounds from A. scoparia inhibit TNFα-induced lipolysis. The sub-fractions were also tested in 3T3-L1 adipocytes relative to SCO for their ability to inhibit TNFα-induced lipolysis. SCO was previously shown to reduce lipolysis in vitro (TNFα-induced lipolysis in cultured adipocytes) and in vivo (mice fed a high-fat diet with SCO supplementation) bioassays.4 In order to validate that the SCO compounds with pro-adipogenic activity in differentiating adipocytes also have anti-lipolytic effects in mature adipocytes, mature 3T3-L1 adipocytes were pretreated with each of the six sub-fractions and then with TNFα to induce lipolysis. Lipolysis was measured on the basis of glycerol release, which is enhanced by insulin resistance and TNFα treatment of cell cultures. SCO was able to reduce TNFα-induced lipolysis to basal levels as previously demonstrated, but only the highest concentration of 1a/b showed any statistically significant inhibitory activity (
In conclusion, this study led to the characterization of bioactive marker compounds that are associated with the adipogenic bioactivity observed from an extract of A. scoparia (SCO). One of the compounds was identified as a new set prenylated cinnamic acid isomers, cis-scopa-trans-coumaricin and cis-scopa-cis-coumaricin (1a/b). The others were prenylated coumaric acid isomers previously indicated as capillartemisin A (2) and B (3). These compounds share the cinnamic acid core and di-prenyl substitution in both meta positions, which indicates this motif being the core of the pharmacophore. The adipogenic bioactivities used for the characterization of the extract could be replicated for the isolates.
Plant Material. Artemisia scoparia Waldst. & Kit herb was grown in a greenhouse facility in New Brunswick, N.J. (40° 28′41.9″ N 74° 26′15.7″ W) and harvested the whole aerial parts of the plant at the flowering stage for the production of extract. Some plants were left to produce seed to maintain the seed source for future cultivation. Voucher specimens are retained under the guidance of a taxonomist.
Extraction and Isolation. A. scoparia extract (SCO) was prepared from greenhouse-grown plants as described previously.4 Briefly, the herb was freeze-dried and extracted in 80% EtOH (1:20 w/v). The preparation of distinct fractions from SCO was previously described in detail. Each of the fractions consisted of compounds that were not purified for structural characterization but maintained adipogenic activities were originally characterized from SCO. The fractions were created using multiple chromatographic techniques including solvent partitioning, CPC and HPLC.5
The chromatographic response of the active compounds obtained from the enriched fractions of SCO was used to enhance the purification process. Here, the A. scoparia plants were freeze-dried, ground into a powder and extracted directly with EtOAc (1:20 w/v), sonicated for one hour, and incubated at 22° C. for 24 h. Solids were removed by filtration, and the extract was dried by rotary evaporation. The yield of this crude extract was 4.6% from the dried plant.
The freeze-dried extract was dissolved in 90% EtOH (25 mg/mL) for initial HPLC separation. The sample was separated on a semi-preparatory HPLC system consisting of Waters™ Alliance e2695 Separations Module and 2998 Photodiode Array Detector with a Phenomenex Synergi 4p 80A Hydro-RP column 250×21.2 mm. The mobile phases consisted of two components: Solvent A (0.1% ACS grade acetic acid in double-distilled de-ionized H2O) and Solvent B (CH3CN). The separation was completed using a linear gradient run of 40% B in A to 60% B over 20 min at a flow rate of 15 mL/min, followed by reconditioning of the column with 100% B for 5 min and return to initial conditions. Fractions III, IV and V were manually collected at 14-16 min, 16-17 min and 17-18 min, respectively. The yields of fractions III and V were 9.3% and 5.3%, respectively, from the total extract injected. Each fraction was dried by rotary evaporation.
Fraction III was separated and purified using the same HPLC system described above with a Phenomenex Lux® 5 μM Cellulose-2 chiral column 250×10 mm. The mobile phases consisted: Solvent A (0.1% ACS grade formic acid in double-distilled de-ionized H2O), and Solvent B (CH3CN). The separation was completed using a linear gradient run of 35% B in A to 65% B over 60 min at a flow rate of 5 mL/min, followed by reconditioning of the column with 100% B for 10 min and return to initial conditions. Fractions III-1 and III-2 were collected at 14-18 min and 21-25 min, respectively. Fractions III-1 and III-2 were analyzed by LC-MS for exact mass and identified using NMR.
Fraction IV was separated using the same HPLC system as fraction III. However, the separation was completed using a different linear gradient as 45% B in A to 60% B over 30 min, then to 100% B at 35 min at a flow rate of 5 mL/min, followed by reconditioning of the column with 100% B for 10 min and return to initial conditions. Fractions IV-1 and IV-2 were collected at 36-39 min and 39-41 min, respectively.
Fraction V was separated using the same HPLC system as fraction III with a linear gradient 40% B in A to 50% B over 20 min at a flow rate of 5 m/min, followed by reconditioning of the column with 100% B for 10 min and return to initial conditions. Fractions V-1 and V-2 were collected at 12-14 min and 29-30 min.
General NMR and MS Procedures. The samples were dissolved in 200 μL of methanol-d4, then transferred into a 3 mm NMR tube. 1D and 2D NMR spectra were acquired at 298 K using Bruker AVANCE I 900/225 NMR spectrometer. The spectrometer was equipped with a 5-mm Bruker TCI triple resonance, inverse-detection cryoprobe with a z-axis pulse field gradient. The pure shift spectroscopic data were acquired at 297.9 K using an AVANCE III NMR spectrometer equipped with an inverse-detection C/H Cryoprobe Bruker 600 MHz. Processing was accomplished using the Mnova software package (v.14.1.1, Mestrelab Research S.L., A Coruña, Spain). 1H spectra were processed with the following parameters: GM (LB−0.3 Hz, GB 0.5), zero filling to SI=256 k and with the application of automatic phasing, the pure shift spectra were processed with the following parameter: GM (LB−0.3 Hz, GB 0.5, zero fillings to SI=64k with a linear prediction of MIST method application of automatic phasing, using Mnova software. The integral values were obtained after using a fifth order polynomial fit baseline correction.
Mass spectrometry analyses were carried out using a Bruker Impact II, quadrupole time-of-flight (Bremen, Germany) coupled to a Shimadzu Nexera X2 UHPLC system (Kyoto, Japan). Data analyses were performed on the Compass Data Analysis software (Bruker Version 4.4). The machine is equipped with an electrospray source. And the analysis was made in positive mode a capillary voltage at 4.5 kV, nebulizer and drying gas (N2) at 3.0 bar and 12.0 L/min, respectively, dry temperature of 225° C., and mass scan range set from m/z 200 to 800, along with the negative electrospray ionization mode using a capillary voltage at −2.5 kV, nebulizer and drying gas (N2) at 4.0 bar and 12.0 L/min, respectively, dry temperature of 225° C., and mass scan range set from m/z 200 to 800. The separation was performed on a CORTECS C18 (100×3.0 mm, 2.7 μm) UHPLC column.
UPLC/MS Analysis of A. scoparia fractions: Compounds in samples were separated and analyzed during the process of fractionation by a UPLC/MS system (
Compound 1a and 1b: amorphous solid; UV (MeOH) Amax (log ε) action; 1H NMR (900 MHz, methanol-d4) and 13C NMR (225 MHz, methanol-d4) data, see
Cell Culture and Treatments. 3T3-L1 preadipocytes (murine) were cultured as previously described,3 and induced to differentiate two days after reaching confluence. For experiments using mature adipocytes, cells were induced with a standard methylxanthine-dexamethasone-insulin (MDI) cocktail, which contains 0.5 mM isobutylmethylxanthine (IBMX), 1 μM dexamethasone (DEX), and 1.72 μM insulin in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). IBMX, DEX, insulin, and DMEM were obtained from Sigma-Aldrich (St. Louis, Mo.), and FBS from Hyclone (GE Healthcare Life Sciences, Logan, Utah). For differentiation experiments, cells were induced with half-strength MDI cocktail containing. In both cases, cells were fed with high-glucose DMEM+10% FBS with 0.43 μM insulin three days following MDI induction. Test compounds were dissolved in dimethylsulfoxide (DMSO) as 1000× stocks. For differentiation experiments, cells were treated with the compounds (or DMSO vehicle as a control) at the time of induction and at the first feeding there after (3 days post-MDI). For lipolysis experiments on mature adipocytes, cells were fed with high-glucose DMEM+10% FBS 6 days after MDI and treated with test compounds or DMSO vehicle for three days. On the third day cells were also treated overnight with 0.75 nM tumor necrosis factor alpha (TNFα) (Life Technologies, Carlsbad, Calif.) or its vehicle [0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS)]. The following morning, the culture medium was replaced with lipolysis incubation medium (low-glucose DMEM+2% BSA) and 0.75 nM TNFα(or vehicle). After 4 hours, conditioned medium and cell lysates were collected.
Lipid Accumulation Assay. Five days after MDI induction, cell monolayers were fixed in 10% neutral buffered formalin (ThermoFisher, Waltham, Mass.) and stained with the neutral lipid stain, Oil Red O (ORO). Plates were scanned to generate images of staining. The ORO was then eluted in isopropyl alcohol, and absorbance of eluates was measured at 520 nm for quantitation.
RNA Purification and Gene Expression. Four days after MDI induction, cells were harvested and RNA purified using the RNeasy Mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, Calif.), according to the manufacturer's protocol. Gene expression assays using primers from Integrated DNA Technologies (Skokie, Ill.). Primer sequences are shown. SYBR Premix (Takara Bio, Mountain View, Calif.) was used for quantitative polymerase chain reaction (qPCR), performed on the Applied Biosystems 7900HT system. Data analysis used SDS 2.3 software. Target gene data were normalized to the reference gene, non-POU-domain-containing, octamer binding protein (Nono). All primer sequences are shown in
1H NMR spectrum of compounds 1a and 1b in CD3OD at 900 MHz; DEPTQ-135 spectrum of compounds 1a and 1b in CD3OD at 900 MHz; COSY spectrum of compounds 1a and 1b in CD3OD at 900 MHz; HSQC spectrum of compounds 1a and 1b in CD3OD at 900 MHz; HMBC spectrum of compounds 1a and 1b in CD3OD at 900 MHz, LC-MS chromatogram of compounds 1a and 1b; LC-MS chromatogram of capillartemisin A; LC-MS chromatogram of capillartemisin B; table of a rational nomenclature for the prospective naming scheme of di-prenylated coumaric acids; deconvolution of pure shift spectroscopic data of compounds 1a and 1b.
Mechansims of Artemisia scoparia's Anti-Inflammatory Activity in Cultured Adipocytes, Macrophages, and Pancreatic β-Cells
Abstract
Objective: An ethanolic extract of Artemisia scoparia (SCO) improves adipose tissue function and reduces negative metabolic consequences of high-fat feeding. A. scoparia has a long history of medicinal use across Asia and has anti-inflammatory effects in various cell types and disease models. The objective of the current study was to validate SCO's effects on inflammation in cells relevant to metabolic health.
Methods: Inflammatory responses were assayed in cultured adipocytes, macrophages, and insulinoma cells, by quantitative PCR, immunoblotting, and NF-κB promoter reporter assays.
Results: In TNFα-treated adipocytes, SCO mitigated ERK and NF-κB signaling, as well as transcriptional responses, but had no effect on fatty acid-binding protein 4 (FABP4) secretion. SCO also reduced levels of deleted in breast cancer 1 (DBC1) protein in adipocytes, and inhibited inflammatory gene expression in stimulated macrophages. Finally, in pancreatic β-cells, SCO decreased NF-κB-responsive promoter activity induced by IL-1β treatment.
Conclusions: SCO's ability to promote adipocyte development and function can mediate its insulin-sensitizing actions in vivo. Our findings that SCO inhibits inflammatory responses through at least two distinct signaling pathways (ERK and NF-κB) in three cell types known to contribute to metabolic disease, reveal that SCO can act to improve metabolic health.
Study Questions
Introduction
The obesity epidemic and its associated metabolic disorders are among the major health challenges of our time (1). Adipose tissue plays a role in these metabolic disease states, and disruptions in adipocyte development and function negatively impact whole-body insulin sensitivity and systemic metabolic health (2). Adipocyte differentiation and adipose tissue expansion are often impaired in obese states, leading to insulin resistance and metabolic dysregulation (3, 4). In addition, obesity and insulin resistance are associated with enhanced basal lipolysis rates, which exacerbate metabolic disease (reviewed in (5)). Inflammatory processes in adipose tissue are also a feature of obesity and the metabolic syndrome, and there is crosstalk between inflammation, adipogenesis, endocrine function, and lipid metabolism in adipose tissue (5, 6). Because of its role in obesity and diabetes, adipose tissue is considered a target for therapeutic intervention (2).
Botanicals have a history of medicinal use across cultures the world over, and plants have been a source of pharmaceutical compounds. Metformin, the first-line drug in treating type 2 diabetes mellitus (T2DM), was derived from galegine, a bioactive first isolated from Galega officinalis, also known as goat's rue or French lilac (7). Screening efforts in our laboratory identified an extract of Artemisia scoparia (SCO) as a potent enhancer of adipocyte differentiation in vitro. Subsequent studies in a diet-induced obesity (DIO) mouse model have shown that SCO also has metabolically beneficial effects on adipose tissue in vivo (8, 9). In addition, SCO supplementation improves whole-body insulin sensitivity, and reduces circulating levels of fatty acids and triglycerides (8-10). More recently, we have demonstrated that SCO can act on adipocytes in vitro to reduce lipolysis induced by the inflammatory cytokine, TNFα(11).
A. scoparia has a history of medicinal use (12, 13), and it has been shown to have a range of effects in disease models related to Alzheimer's, renal oxidative stress, hepatotoxicity, hypertension, and others (14-17). Anti-inflammatory effects of A. scoparia have been described in a range of cell types and contexts (18-20). In adipose tissue, TNFα secreted from resident macrophages is a mediator of obesity-associated inflammation (6, 21, 22). Given the important role of adipose tissue inflammation in obesity-related metabolic dysfunction, we examined the ability of SCO to regulate TNFα action and inflammatory gene expression in adipocytes. We observed that SCO reduced TNFα's effects on inflammatory cytokine expression and NF-κB activation. SCO also reduced nuclear levels of deleted in breast cancer 1 (DBC1, also known as cell cycle and apoptosis regulator 2 or CCAR2), a protein associated with impaired metabolic function (23-26). Although SCO reduced lipolysis induced by TNFα, it had no effect on TNFα-induced secretion of FABP4, which has been shown to be enhanced by lipolytic stimuli and reduced by interventions that inhibit lipolysis. (27, 28). Finally, we observed anti-inflammatory effects of SCO in two other cell types critical in metabolic disease states: macrophages and pancreatic β-cells. Data presented herein validate SCO as a therapeutic in the treatment of metabolic disease.
Materials and Methods
Botanical extract source and preparation. Artemisia scoparia plants were grown and harvested, and ethanolic extracts were prepared as previously reported (8, 11). SCO extracts were dissolved in DMSO at 1000× final concentration (10 mg/ml and 50 mg/ml respectively for 10 or 50 μg/ml treatments).
Adipocyte cell culture and treatments. 3T3-L1 preadipocytes were grown and differentiated as previously described (8). Briefly, cells were grown in high-glucose DMEM supplemented with 10% bovine calf serum. Cells were induced to differentiate two days after reaching confluence, in DMEM plus 10% fetal bovine serum (FBS) containing 0.5 mM IBMX, 1 μM dexamethasone, and 1.7 μM insulin. Cells were fed 48-72 hours later with DMEM plus 10% FBS and 0.425 μM insulin, then every 48-72 hours with DMEM plus 10% FBS. DMEM, IBMX, dexamethasone, and insulin were obtained from Sigma-Aldrich (St. Louis, Mo.); calf serum and FBS were purchased from Hyclone (GE Life Sciences, Logan, Utah). Treatments with SCO were initiated between 6 and 13 days after induction of differentiation. Murine TNFα was purchased from Life Technologies (Carlsbad, Calif.) and dissolved at 0.5 μM in PBS containing 0.1% bovine serum albumin BSA and added to cell culture media at the final concentrations indicated in figure legends.
Macrophage cell culture and treatments. RAW 264.7 macrophages were cultured in high-glucose DMEM supplemented with 10% FBS. Cells were pretreated for 2 hours with SCO, then lipopolysaccharide from E. coli 0111:B4 strain (LPS) (InvivoGen, San Diego, Calif.) was added for an additional 5.5-hour incubation before harvest.
Insulinoma cell culture and treatments. 832/13 rat insulinoma cells were cultured as described previously (29) and transfected with an adenoviral luciferase reporter construct under the control of 5 copies of a consensus NF-κB element (vector obtained from Vector Biolabs, Malvern, Pa.: catalog number 1740). 12 hours after transfection, cells were treated overnight with 5 or 10 μg/ml of A. scoparia (SCO) or A. santolinaefolia, then for 4 hours with 1 ng/ml IL-10. Luciferase reporter activity was assessed by luminometry and normalized to total protein content.
RNA isolation and gene expression analysis. Treated cells were harvested in RLT cell lysis buffer (Qiagen, Hilden, Germany). Lysates were stored at −80° C. prior to RNA extraction using the RNeasy Mini kit (Qiagen). The High-Capacity Reverse Transcription kit (Applied Biosystems, Foster City, Calif.) was used to reverse transcribe the RNA samples, and gene expression was assayed by qPCR with Takara SYBR premix (Takara Bio USA, Mountain View, Calif.) on the Applied Biosystems 7900HT system (cycling conditions: 2 min @ 50° C.; 10 min @ 95° C.; 40 cycles of 15s @ 95° C. and 1 min @ 60° C.; dissociation stage: 15 s @ 95° C., 15 s @ 60° C., and 15 s @ 95° C. with end step ramp rate of 2%). Data were analyzed using SDS 2.3 or 2.4 software. Target gene data were normalized to the reference gene non-POU-domain-containing, octamer-binding protein (Nono). Primers were obtained from Integrated DNA Technologies (IDT, Skokie, Ill.), and sequences were as follows: Nono forward 5′-CATCATCAGCATCACCACCA-3′ (SEQ ID NO: [ ]), reverse 5′-TCTTCAGGTCAATAGTCAAGCC-3′(SEQ ID NO: [ ]); Ccl2 (Mcp1) forward 5′-GCAGAGAGCCAGACGGGAGGA-3′(SEQ ID NO: [ ]), reverse 5′-TGGGGCGTTAACTGCATCTGG-3′(SEQ ID NO: [ ]); I16 forward 5′-TCCTCTCTGCAAGAGACTTCCATCC-3′(SEQ ID NO: [ ]), reverse 5′-AAGCCTCCGACTTGTGAAGTGGT-3′(SEQ ID NO: [ ]); Lcn2 forward 5′-TGCAAGTGGCCACCACGGAC-3′(SEQ ID NO: [ ]), reverse 5′-GCATTGGTCGGTGGGGACAGAGA-3′(SEQ ID NO: [ ]), Nos2 (iNos) forward 5′-CCCTCCTGATCTTGTGTTGGA-3′(SEQ ID NO: [ ]), reverse 5′-TCAACCCGAGCTCCTGGAA-3′(SEQ ID NO: [ ]); Tnfa forward 5′-AGACCCTCACACTCAGATCA-3′(SEQ ID NO: [ ]), reverse 5′-TCTTTGAGATCCATGCCGTTG-3′(SEQ ID NO: [ ]).
Protein samples and immunoblotting. For whole-cell extract preparation, adipocyte monolayers were harvested in radioimmunoprecipitation assay (RIPA) buffer (30) containing the following protease and phosphatase inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 μM leupeptin, 1 mM 10-phenanthroline, 0.2 mM sodium orthovanadate, and 100 μM sodium fluoride. Lysates were stored at −80° C., then thawed, passed through a 20G needle three times and clarified by centrifugation at 13000 g. Supernatants were recovered, and protein concentrations were determined by bicinchoninic acid (BCA) assay (Sigma-Aldrich). Equal amounts of protein from each sample were loaded onto polyacrylamide gels, subjected to electrophoresis, and transferred to nitrocellulose. Standard immunoblotting techniques were applied to probe for target proteins. Primary antibodies were purchased from Cell Signaling Technologies (Danvers, Mass.) for ERK 1/2, CCAR2 (DBC1), and NF-κB p65; from R&D Systems (Bio-Techne, Minneapolis, Minn.) for lipocalin 2 (LCN2), Promega (Madison, Wis.) for phosphorylated ERK 1/2 (active MAPK), and Abcam (Cambridge, Mass.) for FABP4. Detection was performed with horseradish peroxidase-conjugated antibodies from Jackson Immunoresearch (West Grove, Pa.). Autoradiography films were scanned, and densitometry analysis performed using ImageStudio software from Li-Cor Biosciences (Lincoln, Nebr.).
Subcellularfractionation. After treatment, adipocytes were harvested in nuclear homogenization buffer (NHB) (20 mM Tris pH 7.4, 10 mM NaCl, and 3 mM MgCl2). IGEPAL CA-630 (Sigma-Aldrich) was added to the cell suspension at a final concentration of 0.15% prior to Dounce homogenization on ice. The nuclear fraction was pelleted by centrifugation at 517 g, washed in NHB, and resuspended in IP buffer. NHB and IP buffer were supplemented with protease and phosphatase inhibitors. Protein concentrations were determined by BCA assay.
FABP4 secretion and lipolysis assays. Cells pretreated for three days with SCO or DMSO vehicle, were treated overnight with or without 0.75 nM TNFα. The following morning, culture medium was replaced with lipolysis incubation medium (low-glucose DMEM+2% BSA). Cells treated with TNFα overnight were treated with the same concentration of TNFα for the lipolysis/FABP4 secretion analysis. The remaining cells were treated with either vehicle (controls), or 2 nM or 10 μM of isoproterenol. Conditioned media samples were collected after four hours and analyzed for FAPB4 content by immunoblotting. Lipolysis was also assessed by measuring glycerol concentrations in the samples, using free glycerol reagent from Sigma-Aldrich.
Results
SCO Inhibits TNFα-Induced Inflammatory Gene Expression in 3T3-L1 Adipocytes
Our previous studies have demonstrated that SCO could reduce protein levels of the inflammatory cytokine C-C motif chemokine ligand 2 (CCL2) (also known as monocyte chemoattractant protein 1, MCP-1) in the adipose tissue of high-fat diet-fed mice, and inflammatory gene expression in cultured adipocytes (9). We have sought to characterize the cell-autonomous anti-inflammatory effects of SCO in 3T3-L1 adipocytes treated with TNFα, a predominant mediator of adipose tissue inflammation that increases the expression of several inflammatory genes in adipocytes (21). As shown in
SCO Inhibits TNFα-Induced Lipocalin 2 Secretion in 3T3-L1 Adipocytes
Lipocalin 2 (LCN2) is a pro-inflammatory mediator secreted by adipocytes (reviewed in (32)). We have previously shown that LCN2 expression and secretion are induced by TNFα in 3T3-L1 adipocytes (33). As shown in
SCO Reduces Total and Phosphorylated EFK Levels
In adipocytes, TNFα signaling mediates transcriptional regulation of numerous target genes involved in adipocyte function (inflammation, insulin signaling, lipolysis, endocrine function, stress responses), through the activation of several signaling pathways (reviewed in (21)), including the MAP kinase, ERK. To further characterize the effects of SCO in the context of TNFα action in adipocytes, we examined ERK activation in the presence or absence of SCO. As shown in
SCO inhibits TNFα-induced nuclear translocation of NF-κB and reduces nuclear levels of Deleted in breast cancer 1 (DBC1) in 3T3-L1 adipocytes.
Another major signaling pathway engaged by TNFα in adipocytes is the IKK/NF-κB pathway. TNFα causes phosphorylation of the IKK complex, as well as phosphorylation and degradation of its target, inhibitor of nuclear factor-KB (IκB), resulting in the translocation of NF-κB to the nucleus. In order to determine whether SCO can modulate the TNFα activation of NF-κB in fat cells, we treated 3T3-L1 adipocytes with TNFα with or without a 3-day SCO pretreatment. The cytosolic and nuclear compartments were analyzed by immunoblotting. As shown in
The nuclear protein, Deleted in breast cancer 1 (DBC1), also known as cell cycle and apoptosis regulator 2 (CCAR2), has been implicated in several processes related to inflammation, adipocyte biology, and insulin resistance (23-25). Work in our laboratory has described a role for DBC1 in regulating TNFα-induced lipolysis in 3T3-L1 adipocytes (26). We examined DBC1 protein levels in cytosolic and nuclear fractions in TNFα-treated cells in the presence and absence of SCO (
SCO does not Reduce TNFα- or Isoproterenol-Induced FABP4 Secretion
In addition to inducing expression of inflammatory mediators in adipocytes, TNFα also stimulates lipolysis, thereby increasing circulating fatty acid levels and promoting further metabolic dysregulation in obese and insulin-resistant states (5). We have shown that SCO inhibits TNFα-induced, but not adrenergic-stimulated lipolysis in adipocytes (11). Fatty acid-binding protein 4 (FABP4) is secreted from adipocytes and adipose tissue in response to lipolytic conditions, including exposure to forskolin, cyclic AMP, or isoproterenol (27, 28). To our knowledge, the effects of TNFα on FABP4 secretion have not been reported. Therefore, we induced lipolysis in adipocytes using TNFα or isoproterenol, with or without SCO pretreatment, and measured FABP4 levels in the conditioned medium to assess whether TNFα could induce secretion of FABP4 in 3T3-L1 adipocytes under prolipolytic conditions. Consistent with previous studies, both doses of isoproterenol tested stimulated FABP4 secretion (
SCO Reduces LPS-Induced Expression of IIIb and Nos2 (iNos), but not Tnfa in Macrophages
Adipose tissue macrophages promote inflammation and contribute to adipocyte dysfunction in obese and insulin resistant states (6, 22). Hence, we validate that SCO can modulate inflammatory gene expression in RAW 264.7 cells treated with LPS. As shown in
SCO Reduces IL-1P-Induced NF-κB Activation in Cultured Pancreatic Beta Cells.
NF-κB signaling is involved in mediating the inflammatory transcriptional responses which contribute to pancreatic R-cell dysfunction in diabetes (34-38). To determine whether SCO could regulate NF-κB activation in β-cells, we transduced 823/13 rat insulinoma cells with a NF-κB luciferase reporter, then treated cells with SCO at 5 or 10 μg/ml, or vehicle overnight before stimulating cells with IL-1β for 4 hours. IL-1β treatment induced NF-κB promoter activation 28.6-fold over untreated controls. As shown in
Discussion
The impacts of obesity, metabolic syndrome, and T2DM justify the search for therapeutic approaches, and adipose tissue is studied as a target for such interventions. Obesity is considered a pro-inflammatory state, and the importance of adipose tissue inflammation in the progression of insulin resistance is documented. In obesity, infiltration and activation of pro-inflammatory immune cells (such as macrophages) in adipose tissue contribute to impaired adipocyte differentiation and function, as well as to systemic insulin resistance, mediated at least in part by the paracrine actions of macrophage-derived TNFα on adipocytes (6, 21, 22). The study demonstrates that SCO can impede inflammatory processes through at least two different signaling pathways (MAP kinase and NF-κB), and in three cell types relevant to metabolic health (adipocytes, macrophages, and pancreatic β-cells).
In adipocytes, we discovered gene-specific temporal patterns of induction by TNFα and inhibition by SCO. For example, three of the four genes we examined showed a steady increase over an 8-hour TNFα treatment, while the fourth gene, Il6, showed a biphasic response (
Activation of the MAP kinase ERK by phosphorylation mediates some of TNFα's effects in adipocytes (21). Although phosphorylated:total ERK1/2 ratios were not significantly altered by SCO pretreatment, TNFα-treated cells had lower absolute levels of active phosphorylated ERK 1/2 when pretreated with SCO (
The NF-κB pathway is also characterized and known to mediate TNFα's effects on inflammatory cytokine gene transcription through nuclear translocation of the p65 (RelA) subunit of NF-κB, and NF-κB signaling events are involved in inflammation-related metabolic disease (reviewed in (40)). A study in LPS-stimulated macrophages has shown that the total flavonoid fraction of A. scoparia can inhibit inflammatory signaling through MAP kinases and NF-κB (19). Our studies in adipocytes reveal that SCO caused substantial inhibition of both pathways in adipocytes, and provide further evidence that SCO impedes inflammatory signaling pathways in fat cells.
Without wishing to be bound by theory, DBC1 is a multi-functional protein at the interface between aging, cancer, and metabolism (41). Loss of DBC1 in adipocytes inhibits TNFα-induced lipolysis (26), and DBC1 knockout mice are protected from several metabolic effects of DIO (23). In addition, DBC1 knockdown in adipocytes promotes adipogenesis and reduces inflammatory cytokine expression (24, 25), while in B cells, DBC1 has been shown to interact with proteins of the IKK complex, which directly regulates NF-kB signaling (42). These studies link DBC1 inhibition with favorable metabolic effects. We have shown that SCO reduces DBC1 protein levels in adipocytes (
Elevated plasma levels of FABP4 are associated with obesity and insulin resistance in both mice and humans, and inhibition of plasma FABP4 activity improves glucose homeostasis in DIO mice (43). Acute activation of cAMP- or cGMP-dependent protein kinases (PKA and PKG) by increased intracellular cAMP or cGMP levels, respectively, stimulates lipolysis, and has been shown to promote FABP4 secretion from adipocytes. (28, 44). The mechanisms involved in the lipolytic effects of TNFα are distinct from those induced by fasting or adrenergic stimulation (5, 21). We report here that induction of lipolysis by TNFα is associated with increased FABP4 secretion (
In LPS-treated RAW 264.7 macrophages, SCO significantly inhibited Il6 and Nos2 expression, but had no effect on Tnfa expression (
Interestingly, in a similar study in macrophages using a preparation of total flavonoids from A. scoparia, LPS-induced levels of 116 and Tnfa were both inhibited (19). Although the reason for this discrepancy cannot be ascertained, LPS treatment in this study was longer than ours (20 versus 5 hours). Under these more chronic conditions, regulation of Tnfa expression can be NF-κB dependent and thus susceptible to inhibition by SCO. Alternatively, differences in the composition of both extracts could explain these results. Analysis of our SCO extract by chromatography and mass spectrometry has revealed a complex mixture of compounds, many of which are not flavonoids (50). In addition, plants grown in different locations and conditions yield extracts with distinct chemical constituents and bioactivities. While SCO did not inhibit Tnfa gene expression in macrophages (
We have shown that SCO reduces NF-κB-responsive promoter activity in pancreatic β-cells. Recruitment of immune cells and activation of inflammatory pathways are contributors to the pancreatic-cell dysfunction that occurs with obesity and insulin resistance, and NF-κB is known to mediate these processes (36). The finding that SCO attenuates a measure of NF-κB activation in cultured-cells (
Our laboratory has studied the effects of SCO on adipocyte differentiation and function, as well as in a DIO mouse model, for several years. We have now shown that SCO significantly impairs inflammatory signaling and transcriptional responses in three cell types that play roles in obesity, diabetes, and metabolic syndrome. In addition, we have described effects of SCO on adipocyte levels of DBC1, a protein that has garnered attention in recent years for its involvement in adipogenesis, inflammation, and metabolic dysfunction. We have also discovered that FABP4 secretion, known to be induced by various lipolytic agents, is stimulated by TNFα, although SCO did not modulate this response. Our findings reveal that SCO interferes with inflammatory signaling to attenuate responses that promote metabolic dysfunction, and demonstrate, for the first time, that SCO has favorable effects in pancreatic β-cells. These results support further investigation of SCO as a nutritional supplement to promote metabolic health in the context of obesity and insulin resistance.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.
This invention was made with government support under P50 AT002776 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/17055 | 2/8/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62971054 | Feb 2020 | US |
