Patent Application
20240415878
A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference.
The hallmarks of diet and aging associated metabolic syndrome are mitochondrial dysfunction, dysregulation of cellular ion homeostasis, metabolic perturbation, and inadequate nutrient and oxygen supply by microvascular density loss. Emerging evidence highlights the vital role of ion channels as regulators of mitochondrial bioenergetics and cellular metabolism including oxidative phosphorylation (OXPHOS), ATP production, mitochondrial integrity, mitochondrial volume, enzyme activity, signal transduction, proliferation, and apoptosis. Although the progressive changes were either correlated phenomena or circumstantial evidence, the direct cause and effect relationship has not been established. Substantial progress in developing small molecule medicines for metabolic and cardiovascular diseases is becoming increasingly apparent, the limited understanding of energy biology and the interrelationship between bioenergetics and diet influences are therefore obligatory. In multicellular organisms, mitochondrial Ca2+ uptake is fundamentally essential for cellular bioenergetics despite its influence on Ca2+-induced mitochondrial permeability pore activation leading to energetic collapse. Given that mitochondrial dysfunction involves a perturbation of metabolism and intracellular ion homeostasis, the direct link between the mitochondrial divalent ions flux and its buffering capacity that controls bioenergetics needs to be understood.
The impaired divalent cation dynamics at the cellular level is linked to mitochondrial dysfunction. Although mitochondria are the cellular hub for metabolism, any metabolic anomalies have been linked to several metabolic disorders including obesity, diabetes, and cardiovascular disease. The major mitochondrial Ca2+ uptake machinery MCU is essential to shape cytosolic Ca2+ dynamics and promote glucose and fatty acid oxidation dependent mitochondrial respiration. Genetic ablation of MCU in liver, cardiac, and skeletal tissues promote triglyceride accumulation, lowers ketone body production, and increases total body fat. The hepatic and extrahepatic lipid accumulation phenotype observed in the murine and zebrafish model systems suggest a conservation of MCU-regulated cellular metabolism across species. In contrast, several pieces of evidence link intracellular Mg2+ (iMg2+) is an endogenous inactivator of several channels including mitochondrial Ca2+ uniporter channel that is essential to ignite TCA cycle for reducing equivalents production and ATP synthesis. Unlike mitochondrial Ca2+ dynamics, mMg2+ uptake machinery Mrs2 is conserved in both anaerobic and aerobic species. iMg2+ is essential for wide range of cellular processes including membrane integrity, charge neutralization and stabilization of nucleic acids and retention of biological activity of nucleotides, and cofactor for numerous cytosolic and mitochondrial metabolic enzymes, however the molecular signals that rely on mMg2+ dynamics have not been fully elucidated. Remarkably, mammalian glycolytic end-product, L-lactate but not prokaryotic D-lactate acts as an activator that triggers a dynamic transfer of free Mg2+ ions between the ER and mitochondria to shape bioenergetics and cellular metabolism. The molecular switch that links iMg2+ dynamics and cellular metabolism is unknown.
Unlike other organs, the liver plays a dominant role in glucose and lipid metabolism during increased energy intake and starvation. This cyclic process is distinctly controlled by hormones, ligand-gated GPCR signals, and ion channels. In hepatocytes, glucagon, vasopressin, and epinephrine are known to stimulate GPCR-linked iCa2+ mobilization which is a prerequisite for glucose and fatty acid oxidation to generate heat and ATP production. Over decades, several mammalian Mg2+ transporters have been proposed to regulate cellular Mg2+ homeostasis, however its role in balancing carbohydrate and fat metabolism is ambiguous. Since the basal “free” Mg2+ dynamically controls i[Ca2+], any aberration to this signaling cascade may contribute to type II diabetes, obesity manifestation, hepatic NAFLD, progression to NASH, cirrhosis, and hepatocellular carcinoma. Nevertheless, how mammalian Mg2+ channels shape liver and adipose metabolism is unknown. The interrogation of iMg2+ channels influence on long-term Western diet-induced hepatic metabolic signaling events leading to hepatic steatosis and obesity are extremely important.
There is a need for compositions and methods for modulation the iMg2+ dynamics and whole-body metabolism.
The inventor has discovered that limiting mitochondrial Mg2+ uptake (mMg2+) prevents diet-induced obesity. The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-μM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference. Although this phenomenon has been ascribed for several decades, the molecular queues remain unknown. Genetic ablation of mitochondrial Mg2+ channel Mrs2 enhanced fatty-acid transport into the mitochondria that evades adipose tissue expansion and fatty liver phenotype. Mrs2 deficiency restrains citrate efflux from the mitochondria that is a precursor of de novo lipogenesis. Elevated endogenous Mg2+ chelator citrate directly causes HIF-1α destabilization and subsequent biomass accumulation. Unbiased mRNA profiling identified that HIF-1α is linked to its capacity to enhance glycolysis and β-oxidation coupled thermogenesis exclusively in Mrs2 KO mice. Cooperatively, brown adipose tissue markers are markedly elevated in white adipose tissue (WAT) bed and confers weight-gain against long-term Western diet. Mrs2 channel blocker chloropentaamminecobalt (III) chloride (CPACC) has been identified as a treatment that lowers lipid droplet size in hepatocytes and prevents weight gain in obesity mouse model with normal liver function. The inventor contemplates that citrate is a negative regulator of HIF-1α-dependent signaling and is essential for cellular metabolism.
In certain aspects a modulator can be chloropentaammine cobalt(III) chloride (CPACC) or a derivative thereof.
In other embodiments a CPACC derivative can have a chemical structure of Formula II.
wherein X1, X2, X3, X4, and X5 are independently selected from a halogen, amine, nitro, mercapto, hydroxyl, C1 to C4 alky, C1 to C4 heteroalkyl, alkoxy, alkylthio, or alkylamino. In certain aspects X1, X2, X3, X4, and X5 are independently chlorine (Cl), bromine (Br), fluorine (F), or iodine (I). In certain aspects 1, 2, 3, 4, or all 5 of X1, X2, X3, X4, and X5 are a halogen. In certain aspects 1, 2, 3, 4, or all 5 of X1, X2, X3, X4, and X5 are chlorine.
Certain embodiments are directed to methods of ameliorating a metabolic syndrome comprising administering an effective amount of chloropentaammine cobalt (III) chloride (CPACC) or a variant thereof to a subject. The CPACC or a derivative thereof can be administered at a dose of between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, to 500 μg or mg, including all values and ranges there between. The CPACC or a derivative thereof can be formulated as a tablet, a capsule, a concentrate, a powder, a drink, a baked good, chocolate, caramel, and/or snack. In certain aspects the CPACC or derivative thereof is formulated at a nutritional supplement. In certain aspects CPACC or a derivative thereof is administered orally. The CPACC or a derivative thereof can be administered once, twice, three times every day, week, month.
In certain embodiments the CPACC derivative has a chemical structure of Formula II.
wherein X1, X2, X3, X4, and X5 are independently selected from a halogen, amine, nitro, mercapto, hydroxyl, C1 to C4 alky, C1 to C4 heteroalkyl, alkoxy, alkylthio, or alkylamino. In certain aspects X1, X2, X3, X4, and X5 are independently chlorine (Cl), bromine (Br), fluorine (F), or iodine (I). In a particular aspect 1, 2, 3, 4, or 5 of X1, X2, X3, X4, and X5 are chlorine.
Certain embodiments are directed to methods of ameliorating diet-induced metabolic syndrome comprising administering chloropentaammine cobalt chloride (CPACC) or a variant thereof to a subject. The CPACC or a derivative thereof can be administered at a dose of between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, to 500 μg or mg, including all values and ranges there between. The CPACC or a derivative thereof can be formulated as a tablet, a capsule, a concentrate, a powder, a drink, a baked good, chocolate, caramel, and/or snack. In certain aspects the CPACC or derivative thereof is formulated at a nutritional supplement. In certain aspects CPACC or a derivative thereof is administered orally. The CPACC or a derivative thereof can be administered once, twice, three times every day, week, month.
Certain embodiments are directed to methods for treating obesity comprising administering an effective amount of CPACC or a derivative thereof to an obese subject. The subject can have a body mass index (BMI) of 30 or greater. The subject can be diagnosed with pre-diabetes. The CPACC or a derivative thereof can be administered at a dose of between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, to 500 μg or mg, including all values and ranges there between. The CPACC or a derivative thereof can be formulated as a tablet, a capsule, a concentrate, a powder, a drink, a baked good, chocolate, caramel, and/or snack. In certain aspects the CPACC or derivative thereof is formulated at a nutritional supplement. In certain aspects CPACC or a derivative thereof is administered orally. The CPACC or a derivative thereof can be administered once, twice, three times every day, week, month.
Certain embodiments are directed to methods for treating pre-diabetes comprising administering an effective amount of CPACC or a derivative thereof to a pre-diabetic subject. The CPACC or a derivative thereof can be administered at a dose of between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, to 500 μg or mg, including all values and ranges there between. The CPACC or a derivative thereof can be formulated as a tablet, a capsule, a concentrate, a powder, a drink, a baked good, chocolate, caramel, and/or snack. In certain aspects the CPACC or derivative thereof is formulated at a nutritional supplement. In certain aspects CPACC or a derivative thereof is administered orally. The CPACC or a derivative thereof can be administered once, twice, three times every day, week, month.
Certain embodiments are directed to methods for treating diabetes comprising administering an effective amount of CPACC or a derivative thereof to a subject having diabetes. The CPACC or a derivative thereof can be administered at a dose of between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, to 500 μg or mg, including all values and ranges there between. The CPACC or a derivative thereof can be formulated as a tablet, a capsule, a concentrate, a powder, a drink, a baked good, chocolate, caramel, and/or snack. In certain aspects the CPACC or derivative thereof is formulated at a nutritional supplement. In certain aspects CPACC or a derivative thereof is administered orally. The CPACC or a derivative thereof can be administered once, twice, three times every day, week, month.
Certain embodiments are directed to a composition comprising CPACC or a derivative thereof, the CPACC derivative having a chemical structure of Formula II
wherein X1, X2, X3, X4, and X5 are independently selected from a halogen, amine, nitro, mercapto, hydroxyl, C1 to C4 alky, C1 to C4 heteroalkyl, alkoxy, alkylthio, or alkylamino.
Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and/or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps) but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and/or method.
As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consisting of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.
As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a chemical composition and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.
The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be examples of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.
It has been established that the four major bulky cations Na+, K+, Mg2+, and Ca2+ control numerous cellular functions including membrane polarization, bioenergetics, metabolism, transcription, and proliferation. During cellular activation, the highly compartmentalized Ca2+ ions rapidly raise cytosolic concentration to initiate ‘on’ reactions. Consequently, the antagonistic cellular Mg2+ dynamics carry out the offset reactions that are in the in the form of bound or free ionic states. Although this concept was proposed for several decades, iMg2+ dynamics and its intrinsic role remains unknown. Cellular Mg2+ is the intrinsic component of numerous metabolic enzymes, phosphometabolites, and metabolic precursors such as citrate. For instance, GPCR, Nicotinic, and NMDA receptors, L-type Ca2+ channels, and ion pumps activity were modulated by Mg2+ and aberrations led to several prevalent diseases such as hypertension, heart disease, diabetes, ischemia/reperfusion, preeclampsia, cancer, COPD, asthma, migraine, X-linked T-cell immunodeficiency, neurological diseases, Parkinson's disease, and Alzheimer's disease. The precise mechanism of action remains a mystery and is often surrounded by a matter of controversy.
Glycolytic end-product L-lactate acts as an activator of ER Mg2+ release, and the elevated free iMg2+ was subsequently taken up by mitochondria through a highly conserved Mg2+ selective Mrs2 transport machinery. The physiological function of Mrs2-mediated Mg2+ uptake and its effect on bioenergetics and whole-body metabolic consequences thereof are unknown. To recognize the contribution of mMg2+ in physiology and disease context, a mouse model of long-term Western diet induced metabolic disease progression was used, providing various salient findings that were undiscovered. To identify the pathways that facilitate diet-induced metabolic disorders, control and global Mrs2 null mice were fed for over 52 weeks with Western diet. The findings reveal both qualitatively and quantitatively different mechanisms in diet regulated metabolic syndrome that is Mg2+ dependent. The gross anatomical phenotype of Mrs2 KO mice exhibited no body weight gain, near complete ablation of energy storage expansion and prevention of spontaneous hepatocellular carcinoma incidence, retention of sinusoidal microvascular density with normal liver function during the course of Western diet regime. Remarkably, Mrs2 KO mice had consumed more food with the retention of normal circadian rhythm in the form of energy expenditure otherwise this phenomenon is disrupted in the WT WD mice. Having observed such a significant physiological outcome, it is contemplated that the common feature underlying these diverse regulatory mechanisms including thermogenic activation, hyper glucose and fatty oxidation, and oxidative phosphorylation events were under low [Mg2+]m scenario. Histological, biochemical, and global transcriptomic regulation analyses fully support the absence of metabolic syndrome phenotype in Mrs2 KO mice.
It has also been found that a notable reprograming of energy storage organ transcript profile favors thermogenesis, glycolysis, fatty acid oxidation, and oxidative phosphorylation in Mrs2 KO that memory was enhanced upon Western diet supplementation. The data indicate that the retrograde signal that emanates from mitochondria controls transcriptional profiles through Mg2+ dependent manner. A marked suppression of mitochondrial citrate efflux and subsequent stabilization of HIF-1α was identified in Mrs2 KO mice indicating that enhanced HIF-1α-dependent glucose uptake, glycolysis, and vascularization (oxygenation) are essential for oxidative phosphorylation. This new finding sheds light on how intracellular divalent cation dynamics control diet-induced metabolic dysregulation and disease progression. For instance, glucose clearance and oxidation are suppressed in diabetes and other metabolic disease conditions. It has been proven that aerobic or ischemic-mediated HIF-1α stabilization exerts protection against coronary artery disease which is consistent with the finding that lowering mMg2+ prevents Western diet induced obesity in rodents. Aerobic stabilization of HIF-1α transcriptionally controls several monovalent, divalent cation channels, and mitochondria localized SOD2 that prevents oxidative stress and hypertension implying the influence of mMg2+ and HIF-1α-dependent signaling in cellular metabolism.
The findings reveal that blockade of mitochondrial Mg2+ flux by both genetic and pharmacologic interventions diminished hepatic lipid droplet occupancy (
In certain aspects the Magnesium transporter MRS2 homolog has the amino acid sequence
In certain aspects a metabolic modulator can be chloropentaammine cobalt (III) chloride (CPACC) (Formula I) or a derivative thereof.
In other embodiments a CPACC derivative can a chemical structure of Formula II.
wherein X1, X2, X3, X4, and X5 are independently selected from a halogen, amine, nitro, mercapto, hydroxyl, C1 to C4 alky, C1 to C4 heteroalkyl, alkoxy, alkylthio, or alkylamino. In certain aspects X1, X2, X3, X4, and X5 are independently chlorine (Cl), bromine (Br), fluorine (F), or iodine (I). In certain aspects 1, 2, 3, 4, or all 5 of X1, X2, X3, X4, and X5 are a halogen. In certain aspects 1, 2, 3, 4, or all 5 of X1, X2, X3, X4, and X5are chlorine.
As used herein, the term “nitro” means-NO2; the term “halo” or “halogen” designates —F, —Cl, —Br or —I; the term “mercapto” means —SH, and the term “hydroxy” means —OH.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a linear (i.e. unbranched) or branched carbon chain, which may be fully saturated, mono-or polyunsaturated. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Saturated alkyl groups include those having one or more carbon-carbon double bonds (alkenyl) and those having one or more carbon-carbon triple bonds (alkynyl). The groups, —CH. (Me), —CH2CH3(Et), —CH2CH2CH3(n-Pr), —CH(CH3)2 (iso-Pr), —CH2CH2CH2CH3 (n-Bu), —CH(CH3)CH2CH3(sec-butyl), —CH2CH(CH3)2(iso-butyl), —C(CH3)3(tert-butyl), —CH2C(CH3)3(neo-pentyl), are all non-limiting examples of alkyl groups.
The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si. In certain embodiments, the heteroatoms are selected from the group consisting of O and N. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive. The following groups are all non-limiting examples of heteroalkyl groups: trifluoromethyl, trichloromethyl, —CH2F, —CH2Cl, —CH2Br, —CH2OH, —CH2OCH3, —CH2OCH2CF3, —CH2OC(O)CH3, —CH2NH2, —CH2NHCH3, —CH2N(CH3)2, —CH2CH2Cl, —CH2CH2OH, and —CH2CH2OC(O)CH3.
Various groups are described herein as substituted or unsubstituted (i.e., optionally substituted). Optionally substituted groups may include one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, alkylthio, or alkylamino.
The term “alkoxy” means a group having the structure —OR′, where R′is an optionally substituted alkyl or cycloalkyl group. The term “heteroalkoxy” similarly means a group having the structure —OR, where R is a heteroalkyl or heterocyclyl.
The term “amino” means a group having the structure —NR′R″, where R′ and R″ are independently hydrogen or an optionally substituted alkyl, or heteroalkyl. The term “amino” includes primary, secondary, and tertiary amines.
The term “oxo” as used herein means an oxygen that is double bonded to a carbon atom.
Pre-diabetes is the state in which some but not all of the diagnostic criteria for diabetes are met, including impaired fasting glycemia or impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). IFG refers to a condition in which the fasting blood glucose is elevated above what is considered normal levels but is not high enough to be classified as diabetes mellitus. Fasting blood glucose levels are in a continuum within a given population, with higher fasting glucose levels corresponding to a higher risk for complications caused by the high glucose levels. IFG is defined as a fasting glucose that is higher than the upper limit of normal, but not high enough to be classified as diabetes mellitus. Some patients with impaired fasting glucose can also be diagnosed with IGT as described below, but many have normal responses to a glucose tolerance test.
IFG is considered a pre-diabetic state, associated with insulin resistance, increased mortality, and increased risk of cardiovascular pathology, although of lesser risk than IGT (Barr et al. Circulation. 2007, 116(2):151-157). There is a 50% risk over 10 years of progressing to overt diabetes, but many newly identified IFG patients progress to diabetes in less than three years (Nichols et al. Diabetes Care. 2007, 30(2):228-233).
IGT is a pre-diabetic state of dysglycemia, that is associated with insulin resistance and increased risk of cardiovascular pathology. IGT may precede type 2 diabetes mellitus by many years. IGT is also a risk factor for mortality (Nichols et al. Diabetes Care. 2007, 30(2):228-233).
Following the ADA criteria, pre-diabetes can be diagnosed with a blood test with any of the following results: (1) Fasting blood sugar (glucose) level from 100-125 mg/dl (5.6-6.9 mM); (2) A blood sugar level of 140 to 199 mg/dL (7.8 to 11.0 mM) two hours after ingesting the standardized 75 gram glucose solution in the glucose tolerance test; (3) Glycated hemoglobin between 5.7 and 6.4%.
Diabetes can be diagnosed with a blood test with any of the following results: (1) Fasting blood sugar (glucose) level≥126 mg/dl (7.0 mM); (2) A blood sugar level≥200 mg/dL (11.1 mM) two hours after ingesting the standardized 75 gram glucose solution in the glucose tolerance test; (3) Glycated hemoglobin≥6.5%; (4) Symptoms of hyperglycemia and casual plasma glucose≥200 mg/dl (11.1 mM).
Obesity refers to a condition where excessive fat accumulates within the body. In general, when a person's body mass index (BMI) is greater than 30, they are diagnosed as obese. Body mass index (BMI) is a widely used method for estimating body fat mass and is an accurate reflection of body fat percentage in the majority of the adult population. BMI is calculated by dividing the subject's mass by the square of his or her height, typically expressed either in metric or US “Customary” units as kg/m2 or pounds×703/inches2. A person with a BMI of 30.0 or greater is defined as obese, with higher BMI values being further classified as severe obesity (35.0 to 40), morbid obesity (40.0 to 45), and super obese (BMI≥45).
Obesity is caused by an energy imbalance over a long period when an excessive amount of calories are ingested with respect to the amount of energy being expended. Treatment of obesity normally requires behavior therapy as well as a reduction of calories ingested and/or an increase in the amount of calories expended.
Low levels of the protein adiponectin have been associated with a higher risk of developing metabolic syndromes such as obesity. In certain aspects the methods described herein result in the upregulation of adiponectin. Adiponectin (also referred to as GBP-28, apM1, AdipoQ and Acrp30) is a 244 amino acid protein that in humans is encoded by the ADIPOQ gene. It is a protein hormone that modulates a number of metabolic processes, including glucose regulation and fatty acid oxidation. Adiponectin is secreted into the bloodstream from adipose tissue and also from the placenta in pregnancy (Chen et al. Diabetalogica. 2006, 49(6):1292-1302). In the bloodstream, adiponectin accounts for approximately 0.01% of all plasma protein at around 5-10 μg/mL and is very abundant in plasma relative to many hormones. Levels of the hormone are inversely correlated with body fat percentage in adults, while the association in infants and young children is less clear (Ukkola and Santaniemi. J Mol Med. 2002, 80(11):696-702).
Transgenic mice with increased adiponectin show impaired adipocyte differentiation and increased energy expenditure associated with protein uncoupling (Bauche et al. Endocrinology. 148(4):1539-1549). The hormone plays a role in the suppression of the metabolic derangements that may result in type 2 diabetes, obesity, atherosclerosis, non-alcoholic fatty liver disease (NAFLD) and an independent risk factor for metabolic syndrome (Diez and Iglesias. Eur J Endocrinol. 2003, 148(3):293-300; Ukkola and Santaniemi. J Mol Med. 2002, 80(11):696-702; Renaldi et al. Acta Med Indones. 2009, 41(1):20-24). Adiponectin in combination with leptin has been shown to completely reverse insulin resistance in mice (Yamauchi et al. Nat Med. 2001, 7(8):941-946). Levels of adiponectin are reduced in diabetics compared to non-diabetics. Weight reduction significantly increases circulating levels (Coppola et al, Int J Cardiol. 2008 134(3):414-416).
CPACC and derivatives thereof can be administered to a subject either orally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously), intraperitoneally, or locally (for example, powders, ointments or drops). In certain aspects the compounds are provided in a nutritional supplement formulation. A nutritional supplement formulation can be in any form, e.g., liquid, solid, gel, emulsion, powder, tablet, capsule, or gel cap (e.g., soft or hard gel cap). A nutritional supplement formulation typically will include one or more compositions that have been purified, isolated, or extracted (e.g., from plants) or synthesized, which are combined to provide a benefit (e.g., a health benefit in addition to a nutritional benefit) when used to supplement food in a diet.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or may comprise sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, triglycerides, including vegetable oils such as olive oil, or injectable organic esters such as ethyl oleate. 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 dispersions, and/or by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and/or dispersing agents. Prevention of microorganism contamination of the compositions can be accomplished by the addition of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents capable of delaying absorption, for example, aluminum monostearate and/or gelatin.
Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, mannitol, or silicic acid; (b) binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, or acacia; (c) humectants, as for example, glycerol; (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, or sodium carbonate; (e) solution retarders, as for example, paraffin; (f) absorption accelerators, as for example, quaternary ammonium compounds; (g) wetting agents, as for example, cetyl alcohol or glycerol monostearate; (h) adsorbents, as for example, kaolin or bentonite; and/or (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be used as fillers in soft or hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.
Solid dosage forms such as tablets, capsules, and granules can be prepared with coatings or shells, such as enteric coatings and others well known in the art. They may also contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame seed oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.
Suspensions, in addition to the active compound(s), may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol or sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, or tragacanth, or mixtures of these substances, and the like.
Dosage forms for topical administration of ursolic acid and resveratrol include ointments, powders, sprays and inhalants. The compound(s) are admixed with a physiologically acceptable carrier, and any preservatives, buffers, and/or propellants that may be required.
For the compounds of the present invention, alone or as part of a therapeutic or supplement composition, the doses are between about 1, 100, 200, 300, 400, 500, 600 to 500, 600, 700, 800, 900, 1000 μg or mg, preferably between 10 and 500 μg or mg. In certain aspects, a composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day, week or month. In certain aspects, the compounds are administered once every 1, 2, 3, 4, 5, 6, or 7 days.
The term “effective amount” means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
Effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician.
The term “subject” means animals, such as dogs, cats, cows, horses, sheep, geese, and humans. Particularly preferred patients are mammals, including humans of both sexes.
The terms “treating”, “treat” and/or “treatment” include preventative (e.g., prophylactic) and palliative treatment.
The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Targeted Metabolite Screen Identifies Lactate as activator agonist for Mitochondrial Mg2+ Uptake. To search for agonists that promote mitochondrial Mg2+ ([Mg2+]m) uptake, the inventors developed a cell-based confocal fluorescence imaging. As an initial feature, the inventors tested whether GPCR agonist-induced second messengers promote Mg2+ dynamics. To discriminate intracellular Ca2+ and Mg2+ dynamics, MEFs were loaded with cell permeant Ca2+ indicator, Fluo-4-AM or Mg2+ indicator Mag green-AM followed by GPCR agonist, thrombin stimulation. As expected, application of thrombin rapidly elicited cytosolic Ca2+ mobilization, while intracellular Mg2+ dynamics were absent indicating that GPCR-derived second messengers like inositol-trisphosphate (IP3) and diacyl glycerol (DAG) are not agonist/activators of [Mg2+]i dynamics. It is established that intracellular Ca2+ dynamics could be monitored using a wide range of fluorescent dyes or genetically encoded Ca2+ indicators. Conversely, Mg2+ sensitive indicators are very limited due to either lack of agonists or robust dynamic nature. To identify the agonist, primary murine hepatocytes were loaded with [Mg2+]i indicator, cell permeant Mag Green-AM (5 μM). Subsequently, dye loaded hepatocytes were stimulated with major cellular metabolites and mitochondrial oxidative phosphorylation (OXPHOS) inhibitors following baseline recording. Of the metabolites tested, aerobic/anerobic glycolytic end-product lactate consistently exhibited a rapid and robust depletion of ER Mag-Green signal followed by a subsequent elevation in the mitochondria (
Mitochondrial Mg2+ Transporter is Temperature Sensitive. As revealed above, lactate is one of the carbohydrate catabolic end-products that accumulates normally in the range of ˜0.5-1.0 mM but higher levels (5.0-20.0 mM) were detected during intense exercise and several disease states. To assess optimal concentration of lactate required to trigger Mg2+ depletion from ER compartment and mitochondrial Mg2+ uptake, both ER-depletion and mitochondrial Mg2+ uptake was measured in hepatocytes at 37° C. The data clearly shows that lactate concentration≥2 mM promotes ER depletion and partial uptake of Mg2+ into mitochondria (
ER-Dependent Efflux of Free Mg2+ Promotes Mitochondrial Mg2+ Uptake. The results thus far suggest that lactate acts as a ligand/activator of mitochondrial Mg2+ uptake. Since extracellular delivery of lactate rapidly induces ER-depletion and subsequent mitochondrial uptake, it was asked whether rapid influx of lactate regulates this mechanism. Most mammalian cells utilize monocarboxylate transporters (MCT1-4) for plasma membrane lactate entry. To test this, primary hepatocytes were treated with MCT1 or MCT1/2 inhibitors for 60 minutes. Remarkably, both inhibitors abrogated lactate-induced Mg2+ uptake into mitochondria (
Lactate-Induced Mitochondrial Mg2+ uptake Requires Mrs2. Having established that lactate elicits mitochondrial Mg2+ uptake, the inventors tested whether yeast Mrs2p homologue Mrs2 is the bonafide candidate for this process. A global Mrs2 knockout mouse model was generated by CRISPR/Cas9 gene editing strategy. Candidate sgRNAs were selected based on CRISPR/Cas9 nickase design platform, which substantially reduces off-target by ˜50-1500 fold. The selected paired gRNAs targeting exon 8 of Mrs2, the Cas9 nickase mRNA and single-stranded oligodeoxynucleotide (ssODN), for HDR-mediated gene KO were transfected in MEFs. Genotyping was performed by PCR-RFLP analysis with BamH1. The Mrs2p targeted allele was cleaved yielding two fragments (388 and 368 bp) whereas the wild type was not. After validation of gRNAs and ssODN in wild-type MEFs, two gRNAs, Cas9n mRNA and single-stranded oligodeoxynucleotide template (ssODN) were injected into C57BL6n zygotes and then implanted into pseudopregnant female mice. Founder mice were examined for ssODN incorporation by PCR amplification and restriction enzyme digestion (aa changes introduced a BamH1 cut site). After verifying germline transmission and breeding to heterozygosity DNA was isolated and sequenced to confirm the in-frame point mutations.
Lowering mMg2+ mitigates long-term Western diet-induced metabolic syndrome. The highly conserved bacterial CorA homologue, Mrs2, is an inner mitochondrial membrane-localized Mg2+ selective uptake machinery whose biological function is not well understood (Daw et al., 2020). To dissect the physiological significance of mMg2+ dynamics, a CRISPR/Ca9-mediated global Mrs2−/− mouse model was established by introducing a premature stop codon to interrupt exon 8 (Daw et al., 2020). Using this model, it was recently demonstrated that limiting mMg2+ alleviates endotoxin-induced acute multi-organ failure via enhanced mitochondrial bioenergetics (Daw et al., 2020). Here, it was investigated whether long-term WD-induced metabolic syndrome could be prevented by perturbation of mMg2+ dynamics. 14 weeks old male mice were subjected to WD feeding for up to 52 weeks. The Western diet (WD) is composed of 40% (kcal) fat in comparison to 17% in the standard chow diet (CD) (Table 1). This protocol leads to obesity and deterioration of organ function. Wildtype (WT) and Mrs2 knockout (KO, Mrs2−/−) mice were fed normal CD or obesogenic WD and body weights were regularly measured over 30 weeks. Remarkably, consumption of the Western diet caused obesity in WT but not Mrs2−/− mice (
Mice were placed in indirect calorimetry chambers to examine energy expenditure and behavior. In contrast to WT mice, Mrs2−/− mice fed WD maintained body weights close to those of chow fed controls (
Deletion of Mrs2 prevents Western diet-induced liver steatosis and Microvascular rarefaction and spontaneous tumor prevalence. Having observed these gross anatomic and metabolic parameters, liver function was assessed by measuring the plasma levels of ALT (alanine transaminase), a marker of liver damage. Indeed, the WT WD mice, but not the Mrs2−/− WD or the CD controls, had elevated ALT (
HIF-1α pathway as a switchable link in the Western-diet induced obesity. Although lowering [Mg2+]m enhances the mitochondrial oxidative decarboxylation cascade and OXPHOS activity, the physiological and molecular phenotypes of Mrs2−/− mice have never been described in detail (Daw et al., 2020). To identify the causal link between iMg2+ dynamics and metabolic disease progression, we performed global RNA profiling of liver and adipose tissues were performed. The panoramic transcriptomic changes on liver from WT WD and Mrs2−/− WD showed that ≈5500 protein-coding genes were differentially regulated, of which more than 200 are involved in metabolic diseases and cellular processes such as glucose and fatty acid metabolism and mitochondrial bioenergetics, consistent with the antagonistic role of Mg2+ against Ca2+ dependent signaling (Daw et al., 2020; Ramachandran et al., 2022). However, comparison of livers from WT and Mrs2−/− CD revealed that only nine genes were significantly changed and seven were differentially regulated in both dietary conditions. WT WD liver samples exhibited a significant elevation of markers of tumorigenesis, inflammation, and fibrosis when compared with Mrs2−/− WD (
Changes in the transcriptional profiles of inguinal white adipose tissue (iWAT) where assessed. Although gene expression profiles were changed considerably in this tissue, including 74 protein coding genes differentially regulated in both dietary conditions with all of them regulated in the same direction, again the focus was on the major metabolic pathways and HIF1α signaling (Kanehisa, 2019; Kanehisa et al., 2021; Kanehisa and Goto, 2000). Using the ChEA unbiased transcription factor analysis software on iWAT differenentially regulated genes revealed HIF1-α as a strong candidate for controlling the transcriptional program in the inguinal WAT of livers of WD-fed animals. Since a massive elevation of thermogenic candidates was observed under CD as well as WD in the iWAT of Mrs2−/− mice, histologic analysis was performed which revealed a browning of iWAT tissue in Mrs2−/− CD that was amplified when fed with WD in Mrs2−/− as compared the respective WT groups. Next the vascular density in the adipose tissue was measured and was found to be significantly increased in Mrs2−/− WD. Specific markers for brown/beige adipocytes were quantified. As expected, the mt-encoded OXPHOS and ATP synthase subunits were significantly elevated in Mrs2−/− WD vs WT WD. Similarly, the RNA-seq analyses showed upregulation of beige markers and fatty acid catabolismgenes in Mrs2−/− WD mice. Quantification of (Jepl and Lep gene expression in iWAT tissue by RT-qPCR mirrored the RNA-Seq findings. Upon examination of the glycolytic pathway, HIF-1α controlled glycolytic transcripts were significantly upregulated in Mrs2−/− WD mice. How mitochondrial function was changed in the iWAT of Mrs2−/− animals was examined using fresh fat explants and homogenate. Spectacularly, the phenotype of TMRE mitochondrial staining mirrored the RNA-Seq findings. Western diet-fed WT animals had hypertrophic, unilocular adipocytes with minimal mitochondrial network; meanwhile, Mrs2−/− WD had smaller, multilocular, adipocytes with a heavy mitochondrial network (
Western diet induced suppression of mitochondrial fatty acid flux and Ca2+ uptake machinery is prevented in Mrs2 KO mice. To understand the molecular changes that facilitate glucose and lipid oxidation in Mrs2−/− mice in response to WD, hepatocytes were isolated from WT and Mrs2−/− mice fed control or WD for 52-54 weeks. Overnight cultured hepatocytes were co-stained with the lipid indicator BODIPY and the mitochondrial potential indicator TMRE to quantify lipid droplets and mitochondrial ΔΨm using confocal microscopy. The WT WD hepatocytes exhibited more and larger lipid droplets when compared to other groups (
Since MCU-mediated mitochondrial Ca2+ flux is essential for numerous metabolic oxidative enzymes including glucose and fatty acid oxidation, TCA cycle, and electron transport chain complex activities, liver tissue lysates were subjected to Western analysis to determine the MCU and MICU1 protein abundance (Alevriadou et al., 2021). WT WD showed a striking low abundance of MCU and MICU1 while Mrs2−/− WD MCU complex abundance was comparable to WT or Mrs2−/− CD mice (
Efflux of the de novo lipogenesis precursor and endogenous Mg2+ chelator citrate is blunted in Mrs2 KO mice. Production of acetyl-CoA from citrate is a key node in the synthesis of cholesterol and fatty acids, that contribute to hyperlipidemia and deposition of triglyceride species in tissues (Pinkosky et al., 2017). Because citrate is the major precursor of de novo lipogenesis in metazoans, the plasma citrate levels were measured from these four cohorts. Blood samples were drawn from these mice before harvesting organs for RNA-Seq and histological studies. As depicted the plasma citrate concentration was not significantly affected by WD in WT mice (
A fluorometric assay was developed to determine the extramitochondrial citrate accumulation following a supplementation of citrate precursor oxaloacetate. Citrate is the second strongest endogenous chelator of iMg2+ (London, 1991), a property exploited by utilizing the Mg2+ fluorescent indicator Mag-Green (KD˜1 mM) (Daw et al., 2020). Because the binding affinity for citrate and Mg2+ complex (KD˜0.48 mM) (London, 1991) is superior to MagGreen's Mg2+ binding affinity, the rate of Mag-Green intensity loss (quenching) is a function of citrate accumulation in the cytosol. WT or Mrs2−/− hepatocytes were permeabilized and bathed with 5 μM Mag-Green before 1 mM MgCl2 bolus delivery. The extramitochondrial Mg2+ was rapidly taken up by WT but not Mrs2−/− hepatocyte mitochondria (
HIF-1α is stabilized in Mrs2−/− mice. To examine the intrinsic role of Mrs2, Mrs2−/− mice were challenged to LPS-induced inflammatory response. It was noticed that there was a barely detectable HIF-1α stabilization in control but a significant HIF-1α protein accumulation following LPS stimulation in Mrs2−/− hepatocytes (Daw et al., 2020). Having observed the remarkable response against an inflammatory stimulus, the HIF-1α stabilization was compared between genotypes following stimulation with LPS or the generic prolyl hydroxylase (PHD) inhibitor cobalt chloride (CoCl2). Hepatocytes derived from Mrs2−/− mice showed higher accumulation of HIF-1α (
Bacterial CorA blocker selectively inhibits Mrs2 channel activity. Bacterial CorA forms a pentameric complex that selectively drives Mg2+ uptake in an electrogenic manner. CorA is highly selectively inhibited by cobalt and ruthenium derivatives (cobalt(III) hexaammine, ruthenium(III) hexaammine, chloropentaammine cobalt(III) chloride (CPACC; IC50˜100 μM), chloropentaammine ruthenium(III)chloride) with an IC50˜3-100 μM (Kucharski et al., 2000) (
Next, CPACC was assessed to determine if it directly affects the assembly of purified human MRS2 using dynamic light scattering (DLS). DLS has been used to determine whether the complex stability of proteins/channels is altered by molecular chaperones or drugs (Kitamura et al., 2006). Because of the high sensitivity of DLS to changes in complex sizes, full-length Mrs2 assembly was evaluated in the presence Ca2+ (5 mM), Mg2+ (5 mM), Co2+ (5 mM) or the Mrs2 blocker CPACC (0.5 mM) (
CPACC treatment enhances adipose browning and lowers whole body weight. Given the potent mMg2+ uptake inhibiting properties and low toxicity of CPACC, we hypothesized that CPACC could mimic Mrs2−/− cellular phenotype. WT hepatocytes isolated from over 12 months old mice were treated with 50 μM CPACC for 48 hours. Control and CPACC treated hepatocytes were stained with BODIPY and TMRE for intracellular tracing using confocal imaging. CPACC treated hepatocytes displayed a marked reduction of lipid droplet size and appeared to dissipate or smear. Importantly, no negative effect on mitochondrial function was observed (
CPACC Treatment Prevents Western Diet Induced Weight Gain and Promotes Weight Loss. 14-week-old WT mice were started on Western diet (n=8) and regularly monitored for 20 weeks and their body weight recorded. At 20 weeks mice started receiving either vehicle or CPACC (40 mg/kg) intraperitoneal (i.p.) injection (n=4 each group) (
Cell lines. HEK293 (ATCC #CRL-1573), 293T/17 (ATCC #CRL-11268) and COS-7 (ATCC #CRL-1651) cells were grown in high glucose complete growth medium (high glucose-Dulbecco's modified Eagle's medium (DMEM). HepG2 and HepG2-C3A were grown in normal glucose complete growth medium (Dulbecco's modified Eagle's medium (DMEM) These cell lines were supplemented with 10% (v/v) FBS, 1% (v/v) antimycotic-antibacterial cocktail (Gibco). HK-2 (ATCC CRL2190) cells were maintained in specialized serum-free Keratinocyte-SFM media supplemented with required EGF and BGE (Gibco). All cells kept in a 37° C., 5% CO2 incubator. Cells lines were detached using Trypsin-EDTA 0.05%, (HEK293, 293T/17, HK-2) or 0.25% (Cos-7, HeLa, HepG2, HepG2-C3A). All transfected cells were grown in corresponding complete growth media supplemented with puromycin (2 μg/ml) or G418 (500 μg/ml).
Animal models. Wild-type (WT) and Mrs2−/− (KO) C57BL/6J mice were housed and maintained in our animal breeding facility with prior approval and accordance with the Institutional Animal Care and Use Committee (IACUC). Mice were fed a Western Diet (WD; Research Diets D09100310), a standard Chow Diet (CD; Envigo 7012), or a High Fat Diet (HFD; Research Diets D12492). For most experiments, WT and KO mice were maintained on the WD or the CD starting at 12 weeks of age. With termination at 12 months. WT mice maintained on a HFD starting at 6 weeks were administered control (saline) or CPACC (20 mg/kg BW in saline) I.P. every 3 days starting after 6 weeks of maintained diet. Body weight, health, and food were measured regularly for all groups. When needed, blood was collected in K3/EDTA coated tubes and plasma prepared by centrifugation at 1,000×g for 10 min at 4° C.
Isolation and culture of primary murine hepatocytes. Primary adult murine hepatocytes were isolated using portal vein perfusion. After cannulation of the portal vein, the liver is perfused with wash media (35 mM HEPES and 0.75 mM EGTA) followed by digestion media (DMEM, GIBCO, Cat #12320, GIBCO) containing freshly added Collagenase D (380 μg/mL, Worthington) to dissociate extracellular matrix. After perfusion, liver lobes were gently dissected and dissociated in isolation media (DMEM supplemented with 1% (v/v) FBS). The crude hepatocytes were filtered to 100 μm and subjected to three steps of centrifugation-wash cycles (50×g, 4° C., 5 min). After each spin, the pellet was washed in 25 mL of isolation media. The cell's final resuspension is in hepatocyte growth media (Williams E media (Sigma, #W4128) containing 10% (v/v) FBS, 1% (v/v) antibiotic-antimycotic solution, and 200 mM L-glutamine). Finally, cells are counted using trypan-blue exclusion and seeded in hepatocyte growth media according to planned experimental procedures. Hepatocytes are grown in pre-coated collagen culture dishes (Corning BioCoat); or for experiments using confocal microscopy, seeded on in-house collagen coated 25-mm glass coverslips. After 4-8 hours, cell attachment is visually examined, and media is replaced with fresh hepatocyte growth media.
Whole-body respirometry, body composition, and blood chemistry. Mouse metabolic studies were performed at the Penn Diabetes Research Center Rodent Metabolic Phenotyping Core (University of Pennsylvania). A standard 12 h light/dark cycle was maintained throughout the study in a dedicated temperature controlled (20-22° C.) housing room. Mice were acclimated for 5 days before recording. Indirect calorimetry data were recorded using a computer-controlled Promethion Core system (Sable Systems, Las Vegas, NV). Each cage is thermally controlled and equipped with an XYZ beam break array (BXYZ-R, Sable Systems), a voluntary running wheel (WHEEL-M,), and mass measurement modules (2 mg resolution) for food intake and water intake. All animals had ad-libitum access to specified diet and water for 5 days of the study. On the final day, mice were overnight fasted from 1900 to 0900 using a computer-controlled script for automated access to food hopper (Promethion AC-2 Access Control Module) in order to restrict feeding at designated time intervals during the calorimetry run. Respiratory gases are measured with an integrated fuel cell oxygen analyzer, NDIR CO2 analyzer, capacitive water vapor partial pressure analyzer and barometric pressure analyzer (CGF, Sable Systems). Gas sensors were calibrated using 100% N2 as the zero reference. Oxygen consumption (VO2) and carbon dioxide (VCO2) production are measured for each mouse at 5 min intervals for 20 seconds resulting in a 3 min cycle time. Respiratory Exchange Ratio (RER) is calculated as the ratio of VCO2/VO2. Energy expenditure is calculated using the abbreviated Weir equation: Kcal/hr=60*(0.003941*VO2+0.001106*VCO2). Consecutive adjacent infrared beam breaks were counted and converted to distance, with a minimum movement threshold set at 1 cm/s. Voluntary wheel revolutions were also measured continuously as revolutions converted to distance. Data acquisition and instrument control were coordinated by IM-3 software and the obtained raw data were processed using MacroInterpreter v.2.38 (Sable Systems) using an analysis script detailing all aspects of data transformation (One-click macro v.2.45; Sable Systems). EchoMRI nuclear magnetic resonance spectrometer (Echo Medical Systems, Houston, TX) was used to measure whole body lean and fat mass. Blood chemistry parameters were determined as followed: plasma cholesterol with an enzymatic assay (Stanbio), glucose using a ReliON glucometer, an insulin using ELISA.
Histology and quantification. Tissues were fixed in 10% neutral buffered formalin and washed in 70% ethanol overnight before paraffin embedment and generation of unstained slides. Paraffin embedment, mounting, slide preparation, and standard staining was done by the Histology Laboratory in UT Health San Antonio Department of Pathology & Laboratory Medicine with detailed protocols on file. Briefly, tissues are dried, paraffin embedded, sectioned, and placed on slides. For H&E and Masson's Trichome staining, sections were processed according to the Histology Laboratory's established protocols. Unstained liver tissue sections were used to conduct IHC (immunohistochemistry) in-house. Sections were deparaffinized then rehydrated, followed by antigen retrieval (0.01 M citrate buffer). Endogenous peroxidase blocking was done using 3% H2O2 for 10 minutes. Avidin and biotin blocking was conducted using Vector Laboratories Avidin/Biotin Blocking Kit (SP02001). Nonspecific binding was blocked by 1 hour incubation in normal horse serum (NHS, Vector Laboratories, S-2000-20) containing 0.1% Tween-20 in 0.01M PBS and 10% BSA. Unconjugated goat anti-mouse CD-1/PECAM-1 (14 μg/mL; R&D Systems, AF3628) primary was applied overnight at 4° C. in 10% NHS. Sections were incubated with biotinylated ready-to-use horse anti-goat IgG secondary (Vector Laboratories, BP-9500-50) for 1 hour at room temperature. Secondary development was done using the VECTASTAIN Elite ABC-HRP kit (Vector Laboratories PK-6100) and DAB reagent according to manufacturer's recommendations. Developed sections were counterstained with Mayer's Hematoxylin. Thorough washing with PBS was conducted after each step. Slides were imaged using a Olympus light microscope and imaged at 20× magnification. Vascular density was quantified from H&E-stained slides. A digital filter was placed over selected images for vascular clarity and the number of micro vessels observed in each image was recorded. 2-3 slides (different mice) per group were used.
Plasma alanine transaminase activity and citrate measurements. Liver function and damage was determined by measuring alanine transaminase (ALT). The ALT activity in the plasma was determined using the Alanine Transaminase Activity Assay Kit (Abcam ab105134) following manufacturer's protocol. The plasma ALT levels over two different time points were obtained and corresponding ALT activity was determined by reading in a plate reader at 570 nm. The generation of citrate in the TCA cycle and concomitant extrusion from the liver into the blood stream was investigated by estimating the levels of citrate in the plasma. Citrate levels were determined using the Citrate Assay Kit (Abcam ab83396) by reading in a plate reader at 570 nm following the manufacturer's protocol. When required, deproteination was accomplished using a trichloroacetic acid precipitation kit (Abcam ab204708) for tissue and cells, or 10,000 MWCO spin column for plasma.
RNAseq and analysis. RNAseq data generation was contracted with NovoGene. Libraries were constructed using poly-T magnetic beads following by fragmentation, cDNA synthesis, adaptor ligation, and finally PCR. Quality control was conducted by removing low quality reads or those containing the adaptor or poly-N strings. Reads were aligned to the reference genome using Hisat2 v2.0.5. FeatureCounts v1.5.0-p3 was used to count mapped reads and FPKM values were subsequently calculated to correct for gene length and sequencing depth. Consistency between biological replicates was confirmed by correlation analysis. DESeq2 v1.20.0 was used for differential expression analysis; P-values were adjusted using the Benjamini-Hochberg method and statistical significance was considered by an adjusted P-value<0.05. Gene set enrichment analysis (GSEA) was done using the publicly available GSEA analysis tool from Broad Institute (URL www.broadinstitute.org/gsea/index.jsp). The R package ClusterProfile was used to test for significant enrichment of KEGG pathways. Following NovoGene's preliminary analysis, further in-house analysis was conducted. Normalized FPKM values for heatmaps were generated using the STANDARDIZE formula in Microsoft Excel and each gene was normalized individually. Relative mRNA abundance was calculated by normalization from the mean FPKM for the gene and genes were normalized independently. Finally, the Sankey plot was generated using the SankeyMATIC tool (URL sankeymatic.com/).
Immunoblotting. Crushed tissues were homogenized in lysis buffer (ThermoFisher) on ice for one minute (20-30 strokes) using an automatic Dounce homogenizer set to 2000 RPM. Whole cell lysate was prepared in lysis buffer (Abcam) using 3 sets of 3-second sonication (power level 3) intervals on ice. Protein concentration was estimated using Pierce BCA assay kit and samples were prepared and heated for 90° C. for 5 minutes. Equal amounts of protein were loaded and separated on 4%-12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific), transferred to a PVDF membrane, blocked for 1-2 h using 5% fat free skim milk, washed and finally, probed with corresponding antibodies as specified below. Antibodies were from Cell Signaling Technology (HIF-1α and Hydroxy HIF-1α dilution 1:3000, MCU dilution 1.5000, MICU1 dilution 1:3000), Abcam Abcam (CPT-1a, CPT-2, OXPHOS cocktail dilution 1:3000), ZYMED Laboratories (β-actin; dilution 1:1,000), and Amersham (secondary antibodies conjugated with peroxidase). Development was done using X-ray film using a series of timed exposures and Image J was used for densitometric analysis on scanned film images.
Mitochondrial oxygen consumption rate. Primary murine hepatocytes were plated on in-house collagen coated 96-well Seahorse XF Cell Culture Microplates (Agilent) at a density of 4×105 cells/well. Cells were maintained in their normal growth media until 1 hour before assay start time. Hepatocytes were treated with 5, 10 and 25 μM of chloropentammine cobalt(III) chloride (CPACC) for 1 hour at 37° C. Media was changed to Seahorse XF Cell Mito Stress Test Kit (Agilent) assay media supplemented with glucose, glutamine, pyruvate, and HEPESn with concentrations equivalent to that of the growth media 1 hour before the experiment start time. After media change, per manufacturer instructions, cells were placed in a CO2-free incubator for 1 hour. Oxygen consumption rate (OCR) was measured at 37° C. in an XF96 extracellular flux analyzer (Seahorse Bioscience, Agilent) calibrated using Seahorse XF Calibrant solution (Seahorse Bioscience, Agilent) in a CO2-free incubator overnight. Respiratory chain inhibitors (2 μM oligomycin, 5 M FCCP, and a mixture of 1 μM antimycin A and 1 μM rotenone) were added at the indicated time points. Data was collected using Agilent Seahorse Wave 2.6.1 Desktop software and analyzed using GraphPad Prism version 8 (Irrinki et al., 2011; Tomar et al., 2016).
Confocal microscopic examination of cellular lipid localization and maintenance. Primary murine hepatocytes were stained with the lipid indicator BODIPY (ex/em 493/503 nm, 1 μg/ml) and mitochondrial membrane potential indicator tetramethylrhodamine, ethyl ester (TMRE, ex/em 556/610 nm, 100 nM) in serum-free conditions for 30 min (5% CO2) and washed before imaging. (Tomar et al., 2019). For CPACC-treated experiments, hepatocytes were acquired from 12 month-old mice and cells were treated with CPACC (50 μM) for 12 hours. All confocal microscopic images were acquired using a Leica SP8 confocal microscope (Manheim, Germany) coupled with a temperature-controlled environmental chamber. Leica Application Suite X was used to quantify lipid-mitochondrial colocalization using line scan analysis followed by smooth curve fitting. ImageJ was used to calculate Pearson's and Mander's colocalization coefficients. The lipid droplet sizes and mitochondrial membrane potential were quantified using both Leica Application Suite X and ImageJ. All data was analyzed using GraphPad Prism v8.
Evaluation of dose dependent Mrs2 inhibition by confocal live cell imaging system. Primary murine hepatocytes were treated with varying doses (1, 2, 5, 10, 25 μM) of CPACC, or control, for 12 hours. To visualize Mg2+ and the mitochondria, cells were stained with 2.5 mM Magnesium Green-AM (ex/em 488/510 nm) and 1 mM MitoTracker Deep Red FM (ex/em 644/665 nm) for 30 minutes in their normal growth conditions. Using the Leica SP8 confocal microscope, time lapse images were collected every 3 seconds. After 30 seconds of baseline recording, the cells were stimulated with lactate (5 mM). Data was quantified using Leica Application Suite X.
Visualization of citrate flux in live cells using confocal microcopy. Primary murine hepatocytes were grown and seeded as previously described. HepG2 cells were grown on 0.1% gelatin coated glass coverslips. After overnight growth, cells were transiently transfected with 2 μg of genetically encoded biosensors, CMV-Citron or CMV mito-Citron1 (deposited by Robert Campbell) at Addgene, #134303 and #134305; GFP ex/em 488/510 nm) using Lipofectamine 3000 transfection reagent. After 24 hr, transfected hepatocytes were infected with adenoviruses (Vector BioLabs), Ad-RFP and Ad-Mrs2(mut)mRFP-FLAG (MOI 10). After 48 h of infection, the cells were washed and imaged using the Leica SP8 Confocal microscope under 60×oil immersion. After 30 seconds of baseline recording, cells were stimulated with 20 mM of glucose (hepatocytes) or 16.7 mM (HepG2) and the corresponding fluorescence emissions in the cytosol and mitochondria were recorded. The citrate transient-generated fluorescence emission was quantified using Leica Application Suite X and analyzed using GraphPad Prism v8.
Spectrofluorimetric measurement of mitochondrial Mg2+ and Ca2+ dynamics and citrate mediated MagGreen fluorescence quenching. Fluorescence measurements were conducted in a multiwavelength excitation dual wavelength emission spectrofluorometer (Delta RAM, PTI, HORIBA). Cells were washed with Ca2+ and Mg2+ free DPBS, pH 7.4. Following centrifugation (50×g, 4° C., 5 min), approximately 4-5×106 cells were resuspended and permeabilized using 40 μg/mL digitonin in 1.5 mL of intracellular medium (ICM) (mM, 120 KCl, 10 NaCl, 1 KH2PO4, 20 HEPES-Tris, pH 7). Suspension was additionally supplemented with succinate (5 mM), ATP, and a fluorescent dye. Magnesium measurements were performed using K+/ATP (1.5 mM) and Mag Green (0.5 mM); meanwhile, calcium measurements were performed using Mg2+/ATP (1.5 mM) and Fura-2FF (1 μM). Mag-Green has an excitation of 505 nm and emissions of 535 nm and 595 nm based on Mg2+ binding. Fura2-FF has excitations 340 nm and 380 nm based on Ca2+ binding and emits at 510 nm). Changes in extramitochondrial Mg2+ ([Mg2+]out) or Ca2+ ([Ca2+]out) was used as an indicator of mitochondrial uptake of these ions. After a 400 sec background acquisition period, cells were pulsed with either a single bolus of 1 mM Mg2+ or multiple 20 μM Ca2+ pulses followed by the mitochondrial uncoupler, FCCP (2 μM). To measure the inhibition of mMg2+ uptake, permeabilized hepatocytes in ICM were supplemented with different concentrations (0.05, 0.1, 0.5, 1, 2, 5, 10, 25 μM) of CPACC, or control, and pulsed with a bolus of 1 mM Mg2+ at 450 s followed by FCCP (2 μM) at 1000 s. This set of experiments was also conducted using hexaammine Co(III) chloride at the same dosages; additionally, ruthenium ion based inhibition of mMg2+ was investigated using hexaammine Ru(III) chloride (5 μm) and chloropentammine Ru(III) chloride (5 μM). The mMg2+ uptake rate was calculated from the linear portion of the traces immediately after Mg2+ addition. Since citrate acts as an endogenous chelator of magnesium ions, this property can be exploited to visualize citrate production as an inverse function of MagGreen (Mg2+ fluorescent dye) intensity. Hepatocytes were pulsed with 1 mM Mg2+ after a 600 s baseline recording period followed by 5 mM oxaloacetate (OAA) at 800 s. All experiments were done at 37° C. with constant stirring.
Cellular uptake assay. Hepatocytes were treated with varying doses of CPACC for 30 minutes, cells were washed in Ca2+ and Mg2+ free DPBS, and cell pellets flash-frozen. Pellets were suspended in ice cold lysis buffer (0.1% Triton X-100 and 0.2% HNO3 in ultrapure water). The suspension was vortexed for 30 s and incubated on ice for 45 min. The cell lysate was centrifuged, and the supernatant was transferred to a clean tube prior to analysis. The Co3+ content of the lysate was determined using graphite furnace atomic absorption spectroscopy (GFAAS) and was normalized to the protein content of the sample, which was determined using the bicinchoninic acid (BCA) assay kit following manufacturer instructions (ThermoFisher). Results are reported as the average mass ratio of Co to protein (pg/μg) in each sample±SEM.
MRS2 cloning, expression, and purification. The full length human MRS2 (NCBI accession NP_065713.1), identified as MRS258-443 (i.e. residues 58-443) and excluding the mitochondrial targeting sequence, was cloned into pET-28a using Ndel and Xhol restriction sites. The sequence and frame of the MRS258-443 coding insert within the pET-28a vector was confirmed by Sanger DNA sequencing. Transformed BL21 (DE3) codon+Escherichia coli were grown in Luria-Bertani (LB) broth supplemented with 60 mg/mL kanamycin to an optical density (600 nm) of≈0.6-0.8 at 37° C. Expression was induced upon addition 300 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and growth was continued overnight. The protein was purified from the harvested cells under native conditions according to the HisPur nickel-nitriloacetic acid (Ni-NTA) manufacturer guidelines (ThermoFisher). After elution from the Ni-NTA beads using buffer containing 20 mM Tris, 150 mM NaCl, 1 mM DTT, 10 mM CHAPS, 325 mM imidazole, pH 8.0, the 6×His-MRS258-443 was dialyzed in the same buffer without imidazole and thrombin digested (≈1 U/mg protein) to remove the 6×His tag. A final size exclusion chromatography (SEC) purification step was performed through a Superdex 200 10/300 GL column connected to an AKTA Pure FPLC (GE Healthcare). The buffer for the SEC and all experiments was 20 mM Tris, 150 mM NaCl, 1 mM DTT, 10 mM CHAPS, pH 8.0.
Dynamic light scattering (DIS). A Dynapro Nanostar (Wyatt) equilibrated to 20° C. was used for all DLS experiments. Protein samples ≈0.5 mg/mL in 20 mM Tris, 150 mM NaCl, 1 mM DTT, 10 mM CHAPS, pH 8.0 without or with 5 mM CaCl2, 5 mM MgCl2, 5 mM CoCl2 or 0.5 mM CPACC were centrifuged at 12,000×g for 5 min before a 5 mL aliquot of the supernatant was removed and loaded into a JC501 cuvette (Wyatt). The sample was equilibrated for 5 min at 20° C. before 10 autocorrelation function acquisitions of 5 s each were recorded and averaged. Two aliquots (technical replicates) from each sample were averaged, and each experimental condition was performed on three independent/individual protein expression preparations (biological replicates). Autocorrelation functions were deconvoluted using the regularization algorithm to extract polydisperse distributions of hydrodymic radii and cumulants algorithm to extract weight-averaged monodisperse hydrodynamic radii, using the accompanying Dynamics (v7.8.1.3) software (Wyatt). The dependence of hydrodynamic size on CPACC concentration was determined using ≈0.5 mg/mL protein supplemented with 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM or 0.75 mM CPACC, using a similar experimental setup as described above and cumulants analysis.
Statistical Analysis. Two-tailed Student's t test and One Way ANOVA with Tukey's multiple comparisons were used as indicated. GraphPad Prism version 8 was used for statistical testing and regression analysis. Data is represented as mean+/−SEM unless otherwise indicated.
This Application is an International Application claiming priority to U.S. Provisional Application Ser. No. 63/255,077 filed Oct. 13, 2021, which is incorporated herein by reference in its entirety.
This invention was made with government support under R01GM109882 and R01HL086699 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/046631 | 10/13/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63255077 | Oct 2021 | US |
