Patent Application
20220242812
The present invention is directed to various polycyclic compounds and methods of using these compounds to treat a variety of diseases associated with abnormal SWELL1 signaling including metabolic diseases such as obesity, diabetes, nonalcoholic fatty liver disease; cardiovascular diseases such as hypertension and stroke; neurological diseases; male infertility, muscular disorders, and immune deficiencies.
Obesity-induced diabetes (Type 2 diabetes, T2D) is reaching epidemic proportions with more than one in three Americans obese (36%), >29 million with diabetes and ˜86 million with pre-diabetes in the US alone (in 2014, CDC). The economic consequences of obesity and diabetes in the US alone are close to $500 billion. Globally, this is an even more significant problem, where the incidence of Type 2 diabetes is estimated at 422 million in 2014 and the projected numbers are expected to reach over 700 million within the next decade. Non-alcoholic fatty liver disease (NAFLD), is highly associated with T2D, and has a prevalence of 24% in both the US and globally. NAFLD often progresses to advanced liver disease, cirrhosis and hepatocellular carcinoma, and is currently the second most common indication for liver transplantation in the US, after hepatitis C.
While there are currently several commercially available drugs to treat Type 2 diabetes, physicians remain challenged with effectively treating this disease, as a significant percentage of patients continue to have poorly controlled blood glucose, despite optimal medical therapy. Failure of medical therapy relates to a number of factors, including a narrow mechanism of action (insulin sensitizer vs. secretagogue vs. other), medication non-compliance (particularly for drugs with frequent dosing regimens) and achieving euglycemia while avoiding life-threatening hypoglycemia. Moreover, several current therapies suffer from unwanted and dangerous side effects such as congestive heart failure, weight gain and edema including TZDs that are also used for NAFLD.
Volume regulated anion channels (VRAC) are considered cell swelling-induced anion channels. They modulate vital functions in a variety of organ systems and have been implicated in pathology associated with diabetes, obesity, non-alcoholic fatty liver disease, stroke, hypertension and other conditions. The leucine-rich repeat-containing protein 8A (LRRC8A) which is also known as SWELL1, along with its four other associated homologs (LRRC8B-E) form heteromeric VRACs.
SWELL1 (LRRC8a) is a required component of a volume-sensitive ion channel molecular complex that is activated in the setting of adipocyte hypertrophy and regulates adipocyte size, insulin signaling and systemic glycaemia via a novel SWELL1-PI3K-AKT2-GLUT4 signaling axis. Adipocyte-specific SWELL1 ablation disrupts insulin-PI3K-AKT2 signaling, inducing insulin resistance and glucose intolerance in vivo. As such, SWELL1 is identified as a positive regulator of adipocyte insulin signaling and glucose homeostasis, particularly in the setting of obesity.
In addition to impaired insulin sensitivity, Type 2 diabetes is also characterized by a relative loss of insulin-secretion from the pancreatic β-cell. Regulation of β-cell excitability is a dominant mechanism controlling insulin secretion and systemic glycaemia. Indeed, a cornerstone of current diabetes pharmacotherapy, the sulfonylurea receptor inhibitors (i.e., glibenclamide), are aimed at antagonizing the well-characterized, inhibitory, hyperpolarizing current IK,ATP to facilitate β-cell depolarization, activate voltage-gated calcium channels (VGCC) and thereby trigger insulin secretion. However, in order for such agents to be effective, an excitatory current must exist to allow for membrane depolarization. SWELL1 is required for a prominent swell-activated chloride current in β-cells. SWELL1-mediated VRAC is activated by glucose-mediated β-cell swelling, providing an essential depolarizing current required for β-cell depolarization, glucose-stimulated Ca2+ signaling and insulin secretion.
Normal SWELL1 function is required for normal human immune system development. In one example, expression of a truncated SWELL1 protein caused by a translocation in one allele of SWELL1 inhibits normal β-cell development, causing agammaglobulinemia 5 (AGMS) (Sawada, A., et al. Journal of Clinical Investigation 2003; Kubota, K. et al., FEBS Lett 2004). Because different types of immune system cells (e.g., B-lymphocytes and T-lymphocytes) use similar intracellular signaling pathways, it is likely that the development and/or function of other immune system cells (e.g., T-lymphocytes, macrophages, and/or NK cells) would also be affected in adequate SWELL1 function.
Currently, the molecular causes of male infertility are only partially understood. In mice lacking SWELL1 late spermatids fail to reduce their cytoplasm during development into spermatozoa and have disorganized mitochondrial sheaths with angulated flagella, resulting in reduced sperm motility. This demonstrates that SWELL1 is also required for normal spermatid development and male fertility (Luck, J. C., Journal of Biological Chemistry 2018).
SWELL1 and associated VRAC signaling is also linked to stroke induced neurotoxicity and cardiovascular disease.
There is evidence that a variety of conditions may be treated by inhibiting or otherwise modulating SWELL1 using compounds that directly bind to it. One such compound is DCPIB (4-[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid) (herein referred to as Smod1) described in WO2018/027175, which has affinity for LRRC8A. However, there exists a need for compounds that have improved affinity and metabolic profiles and that target a larger variety of LRRC8 homologs. Such compounds can be useful for improved therapies for diabetes, obesity, non-alcoholic fatty liver disease, stroke, hypertension, immune deficiencies, male infertility, and other conditions.
Various aspects of the present invention are directed to compounds of Formula (I), and salts thereof:
wherein:
R1 and R2 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl;
R3 is —Y—C(O)R4, —Z—N(R5)(R6), or —Z-A;
R4 is hydrogen, substituted or unsubstituted alkyl, —OR7, or —N(R8)(R9);
X1 and X2 are each independently substituted or unsubstituted alkyl, halo, —OR10, or —N(R11)(R12);
R5, R6, R7, R8, R9, R10, R11 and R12 are each independently hydrogen or substituted or unsubstituted alkyl;
Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least 2 carbon atoms;
A is a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least one nitrogen heteroatom, boronic acid or
and
n is 1 or 2.
Further aspects are directed to various methods using the compound of Formula (I) to treat various conditions in a subject in need thereof including insulin sensitivity, obesity, diabetes, nonalcoholic fatty liver disease, metabolic diseases, hypertension, stroke, vascular tone, systemic arterial and/or pulmonary arterial blood pressure, blood flow, male infertility, muscular disorders, and/or immune deficiencies. In general, the method comprises administering to the subject a therapeutically effective amount of a compound of Formula (I).
Other objects and features will be in part apparent and in part pointed out hereinafter.
Statistical significance between the indicated group were calculated with one-way Anova, Tukey's multiple comparisons test. Error bars represent mean±s.e.m. *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001. n=3, independent experiments.
The present invention is directed to various polycyclic compounds and various methods using these compounds to treat a variety of conditions in a subject in need thereof including insulin sensitivity, obesity, diabetes, nonalcoholic fatty liver disease, metabolic diseases, hypertension, stroke, vascular tone, and systemic arterial and/or pulmonary arterial blood pressure and/or blood flow. Various neurological diseases, infertility problems, muscular disorders, and immune deficiencies can also be treated with these compounds.
In various embodiments, compounds of the present invention include those of Formula (I) and salts thereof:
wherein
R1 and R2 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl;
R3 is —Y—C(O)R4, —Z—N(R5)(R6), or —Z-A;
R4 is hydrogen, substituted or unsubstituted alkyl, —OR7, or —N(R8)(R9);
X1 and X2 are each independently hydrogen, substituted or unsubstituted alkyl, halo, —OR10, or —N(R11)(R12);
R5, R6, R7, R8, R9, R10, R11 and R12 are each independently hydrogen or substituted or unsubstituted alkyl;
Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least 2 carbon atoms;
A is a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least one nitrogen heteroatom, boronic acid, or
and
n is 1 or 2.
In various embodiments, at least one of R1 or R2 is a substituted or unsubstituted linear or branched alkyl having at least 2 carbon atoms. In further embodiments, R1 is hydrogen or a C1 to C6 alkyl. For example, in some embodiments, R1 is butyl. In various embodiments, R2 is cycloalkyl (e.g., cyclopentyl).
In various embodiments, R1 and R2 are selected from the group consisting of:
In various embodiments, R3 is —Y—C(O)R4. In some embodiments, R3 is —Z—N(R5)(R6). In further embodiments, R3 is —Z-A.
As noted above, A can be a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least one nitrogen heteroatom. In some embodiments, A is a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least two, three, or four nitrogen heteroatoms. In some embodiments, A is a substituted or unsubstituted 5- or 6-membered heterocyclic ring having at least one nitrogen heteroatom and at least one other heteroatom selected from oxygen or sulfur. In various embodiments, A can be boronic acid or
In various embodiments, A is:
In certain embodiments, A is
In certain embodiments, R3 is selected from the group consisting of:
In various embodiments, R4 is —OW or —N(R8)(R9).
In various embodiments, X1 and X2 are each independently hydrogen, substituted or unsubstituted C1 to C6 alkyl or halo. In some embodiments, X1 and X2 are each independently C1 to C6 alkyl, fluoro, chloro, bromo, or iodo. In certain embodiments, X1 and X2 are each independently methyl, fluoro, or chloro.
In various embodiments, R5, R6, R7, R8, R9, R10, R11 and R12 are each independently hydrogen or alkyl. For example, in some embodiments, R5, R6, R7, R8, R9, R10, R11, and R12 are each independently hydrogen or a C1 to C3 alkyl.
In various embodiments, Y and Z are each independently substituted or unsubstituted alkylene having 2 to 10 carbons, substituted or unsubstituted alkenylene having from 2 to 10 carbons, or substituted or unsubstituted arylene. In some embodiments, Y and Z are each independently alkylene having 2 to 10 carbons, alkenylene having from 2 to 10 carbons, or phenylene. Y and Z can also each independently be cycloalkylene having 4 to 10 carbons. In certain embodiments, Y is an alkylene or an alkenylene having 3 to 8 carbons or 3 to 7 carbons. For example, Y can be an alkylene or any alkenylene having 4 carbons. In further embodiments, Z is an alkylene having 2 to 4 carbons. For example, Z can be an alkylene having 3 or 4 carbons.
In various embodiments, Y or Z can be selected from the group consisting of
In various embodiments, when Y is an alkylene having 2 to 3 carbons then both X1 and X2 are each fluoro or each substituted or unsubstituted alkyl (e.g., methyl or ethyl). In some embodiments, Y is not an alkylene having 3 carbons. In certain embodiments, R7 is not hydrogen or a C1 to C6 alkyl. In some embodiments, X1 and/or X2 are not halo. In certain embodiments, X1 and/or X2 are not chloro. In some embodiments, R1 and/or R2 are not alkyl.
In accordance with the embodiments described herein, the compound of Formula (I) may be selected from the group consisting of:
Various compounds of Formula (I) advantageously can modulate or inhibit a SWELL1 channel. In certain embodiments, the compound of Formula (I) has a higher potency at modulating or inhibiting a SWELL1 channel than an equivalent amount of DCPIB (4-[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid). Therefore, they can be used to treat conditions and diseases associated with impaired SWELL1 activity.
Various aspects of the invention include methods for increasing insulin sensitivity and/or treating obesity, diabetes (e.g., Type I or Type II diabetes), nonalcoholic fatty liver disease, a metabolic disease, hypertension, stroke, vascular tone, and systemic arterial and/or pulmonary arterial blood pressure and/or blood flow in a subject in need thereof. Various aspects of the invention also include methods for treating an immune deficiency or infertility caused by insufficient or inappropriate SWELL1 activity in a subject in need thereof. In various aspects, the immune deficiency can include agammaglobulinemia. In further aspects, the infertility can be a male infertility caused by, for example, abnormal sperm development due to insufficient or inappropriate SWELL1 activity. Various aspects of the invention also include methods for treating or restoring exercise capacity and/or improving muscle endurance. In further aspects, methods are provided for treating a muscular disorder in a subject need thereof. The muscular disorder can include skeletal muscle atrophy. As the SWELL1-LRRC8 complex also regulates myogenesis, methods are also provide for regulating myogenic differentiation and insulin-P13K-AKT-AS160, ERK1/2 and mTOR signaling in myotubules. In general, these methods comprise administering to the subject a therapeutically effective amount of the compound of Formula (I).
In the various methods described herein, the administration of the compound is sufficient to upregulate the expression of SWELL1 or alter expression of a SWELL1-associated protein. In some embodiments, the administration of the compound is sufficient to stabilize SWELL1-LRRC8 channel complexes or a SWELL1-associated protein. In further embodiments, the administration of the compound is sufficient to promote membrane trafficking and activity of SWELL1-LRRC8 channel complexes or a SWELL1-associated protein. In some embodiments, the SWELL1-associated protein is selected from the group consisting of LRRC8, GRB2, Cav1, IRS1, or IRS2. In various methods described herein, the administration of the compound is sufficient to augment SWELL1 mediated signaling.
In accordance with the various methods of the present invention, a pharmaceutical composition comprising a compound of Formula (I) is administered to the subject in need thereof. The pharmaceutical composition can be administered by a routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. In various embodiments, administration is selected from the group consisting of oral, intranasal, intraperitoneal, intravenous, intramuscular, rectal, and transdermal.
The determination of a therapeutically effective dose for any one or more of the compounds described herein is within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which provides the desired result. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
Typically, the normal dosage amount of the compound can vary from about 0.05 to about 100 mg per kg body weight depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. It will generally be administered so that a daily oral dose in the range, for example, from about 0.1 mg to about 75 mg, from about 0.5 mg to about 50 mg, or from about 1 mg to about 25 mg per kg body weight is given. The active ingredient can be administered in a single dose per day, or alternatively, in divided doses (e.g., twice per day, three time a day, four times a day, etc.). In general, lower doses can be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, from about 0.05 mg to about 30 mg, from about 0.1 mg to about 25 mg, or from about 0.1 mg to about 20 mg per kg body weight can be used.
A pharmaceutical composition for oral administration can be formulated using pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the subject. In certain embodiments, the composition is formulated for parenteral administration. Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pa., which is incorporated herein by reference). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
In addition to the active ingredients (e.g., the compound of Formula (I)), the pharmaceutical composition can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil; and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF), and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator based on the desired route of administration.
Unless otherwise indicated, the alkyl, alkenyl, and alkynyl groups described herein preferably contains from 1 to 20 carbon atoms in the principal chain. They may be straight or branched chain or cyclic (e.g., cycloalkyls). Alkenyl groups can contain saturated or unsaturated carbon chains so long as at least one carbon-carbon double bond is present. Alkynyl groups can contain saturated or unsaturated carbon chains so long as at least one carbon-carbon triple bond is present. Unless otherwise indicated, the alkoxy groups described herein contain saturated or unsaturated, branched or unbranched carbon chains having from 1 to 20 carbon atoms in the principal chain.
Unless otherwise indicated herein, the term “aryl” refers to monocyclic, bicyclic or tricyclic aromatic groups containing from 6 to 14 ring carbon atoms and including, for example, phenyl. The term “heteroaryl” refers to monocyclic, bicyclic or tricyclic aromatic groups having 5 to 14 ring atoms and containing carbon atoms and at least 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms.
Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.
The following non-limiting examples are provided to further illustrate the present invention.
A series of compounds (Smod compounds) were synthesized to evaluate the role of a butyrate side chain and aryl substituents on activity (see
SWELL1 expression in vivo by channel-inactive Snot1 was compared to channel-active Smod3 and Smod5. Both Smod3 and Smod5 induce SWELL1 protein in 3T3-F442A adipocytes compared to vehicle, while Snot1 is ineffective (
The recent Cryo-EM structure of Smod1 bound with a SWELL1 homohexamer22 was used to generate binding models in an effort to explain activity profiles for the Smod compounds described in Example 1. As shown in
Human islets and adipocytes were obtained and cultured as described previously (Kang et al., 2018; Zhang et al., 2017). The patients involved in the study were anonymous and information such as gender, age, HbA1c, glucose levels and BMI only were available to the research team.
All C57BL/6 mice involved in study were purchased from Charles River Labs. Both KK.Cg-Ay/J (KKN) and KK.Cg-Aa/J (KKAa) mice involved in study were gender and age-matched mice obtained from Jackson Labs (Stock No: 002468) and bred up for experiments. The mice were fed ad libitum with either regular chow (RC) or high-fat diet (Research Diets, Inc., 60 kcal % fat) with free access to water and housed in a light-, temperature- and humidity-controlled room. For high-fat diet (HFD) studies, only male mice were used and were started on HFD regimen at the age of 6-9 weeks. For all experiments involving KKN and KKAa mice, both males and females were used at approximately 50/50 ratio. In all experiments involving mice, investigators were kept blinded both during the experiments and subsequent analysis.
3T3-F442A (Sigma-Aldrich) cells were maintained in 90% DMEM (25 mM D-Glucose and 4 mM L-Glutamine) containing 10% fetal bovine serum (FBS) and 100 IU penicillin and 100 μg/ml streptomycin.
HEK-293 (ATCC® CRL-1573™) cells were maintained in 90% DMEM (25 mM D-Glucose and 4 mM L-Glutamine) containing 10% fetal bovine serum (FBS) and 100 IU penicillin and 100 μg/ml streptomycin. Overexpression of plasmid DNA in HEK-293 cells were carried out using Lipofectamine 2000 (Invitrogen) reagent.
All compounds were dissolved in Kolliphor® EL (Sigma, #C5135). Either vehicle (Kolliphor® EL), SN-401 (DCPIB, 5 mg/kg of body weight/day, Tocris, D1540), SN-403, SN-406, SN-407 or SN071 were administered i.p. as indicated using 1 cc syringe/26G×½ inch needle daily for 4-10 days, and in one experiment, SN-401 was administered daily for 8 weeks. SN-401, formulated as above, was also administered by oral gavage at 5 mg/kg/day for 5 days using a 20G×1.5 inch reusable metal gavage needle.
Adenovirus type 5 with Ad5-RIP2-GFP (4.1×1010 PFU/ml) and Ad5-CAG-LoxP-stop-LoxP-3×Flag-SWELL 1 (1×1010 PFU/ml) were obtained from Vector Biolabs. Adenovirus type 5 with Ad5-CMV-Cre-wt-1RES-eGFP (8×1010 PFU/ml) was obtained from the University of Iowa Viral Vector Core.
Wildtype (WT) and SWELL1 knockout (KO) 3T3-F442A (Sigma-Aldrich) cells were cultured and differentiated as described previously (Zhang et al., 2017). Preadipocytes were maintained in 90% DMEM (25 mM D-Glucose and 4 mM L-Glutamine) containing 10% fetal bovine serum (FBS) and 100 IU penicillin and 100 μg/ml streptomycin on collagen-coated (rat tail type-I collagen, Corning) plates. Upon reaching confluency, the cells were differentiated in the above-mentioned media supplemented with 5 μg/ml insulin (Cell Applications) and replenished every other day with the differentiation media. For insulin signaling studies on WT and KO adipocytes with or without SWELL1 overexpression (O/E), the cells were differentiated for 10 days and transduced with Ad5-CAG-LoxP-stop-LoxP-SWELL1-3×Flag virus (MOI 12) on day 11 in 2% FBS containing differentiation medium. To induce the overexpression, Ad5-CMV-Cre-wt-lRES-eGFP (MOI 12) was added on day 13 in 2% FBS containing differentiation medium. The cells were then switched to 10% FBS containing differentiation medium from day 15 to 17. On day 18, the cells were starved in serum free media for 6 h and stimulated with 0 and 10 nM insulin for either 5 or 15 min Either Ad5-CAG-LoxP-stop-LoxP-SWELL1-3×Flag or Ad5-CMV-Cre-wt-1RES-eGFP virus transduced cells alone were used as controls. Based on GFP fluorescence, viral transduction efficiency was ˜90%.
For SN-401 treatment and insulin signaling studies in 3T3-F442A preadipocytes, the cells were incubated with either vehicle (DMSO) or 10 μM SN-401 for 96 h. The cells were serum starved for 6 h (+DMSO or SN-401) and washed with PBS three times and stimulated with 0, 3 and 10 nM insulin containing media for 15 mins prior to collecting lysates. In the case of 3T3-F442A adipocytes, the WT and KO cells were treated with either vehicle (DMSO), 1 or 10 μM SN-40X following 7-11 days of differentiation for 96 hand then stimulated with 0 and 10 nM insulin/serum containing media (+DMSO or SN-40X) for 15-30 min for SWELL1 detection. For AKT and AS160 signaling, the serum starved cells in the presence of compounds were washed twice in hypotonic buffer (240 mOsm) and then incubated at 37° C. in hypotonic buffer for 10 min followed by stimulation with insulin/serum containing media. To simulate gluco-lipotoxicity, sodium palmitate was dissolved in 18.4% fatty-acid free BSA at 37° C. in DMEM medium with 25 mM glucose to obtain a conjugation ratio of 1:3 palmitate:BSA (Busch et al., 2002). As described above, the 3T3-F442A adipocytes were incubated with vehicle or SN-401, SN-406, SN072 at 10 μM for 96 h and treated with 1 mM palmitate for additional 16 hand lysates were collected and further processed.
SN-401 and its analogs were docked into the expanded state structure of a LRRCBA-SN-401 homo-hexamer in MSP1E3D1 nanodisc (PDB ID: 6NZZ) using Molecular Operating Environment (MOE) 2016.08 software package [Chemical Computing Group (Montreal, Canada)]. The 3D structure obtained from PDB (PDB ID: 6NZZ) was prepared for docking by first generating the missing loops using the loop generation functionality in Yasara software package followed by sequentially adding hydrogens, adjusting the 3D protonation state and performing energy minimization using Amber10 force-field in MOE. The ligand structures to be docked were prepared by adjusting partial charges followed by energy minimization using Amber10 force-field. The site for docking was defined by selecting the protein residues within 5A from co-crystallized ligand (SN-401). Docking parameters were set as Placement: Triangle matcher; Scoring function: London dG; Retain Poses: 30; Refinement: Rigid Receptor; Re-scoring function: GBVI/WSA dG; Retain poses: 5. Binding poses for the compounds were predicted using the above validated docking algorithm.
Patch-clamp recordings of β-cells and mature adipocytes were performed as described previously (Kang et al., 2018; Zhang et al., 2017). 3T3-F442A WT and KO preadipocytes were prepared as described in the Cell culture section above. For SWELL1 overexpression recordings, preadipocytes were first transduced with Ad5-CAG-LoxP-stop-LoxP-3×Flag-SWELL1 (MOI 12) in 2% FBS culture medium for two days and then overexpression induced by adding Ad5-CMV-Cre-wt-lRES-eGFP (MOI 10-12) in 2% FBS culture medium for two more days and changed to 10% FBS containing culture media and were selected based on GFP expression (˜2-3 days). For cell recordings, islets were transduced with Ad-RIP2-GFP and then dispersed after 48-72 hours for patch-clamp experiments. GFP+ cells marked β-cells selected for patch-clamp recordings. For measuring ICl,SWELL inhibition by SN-401 congeners after activation of ICl,SWELL, HEK-293 cells were perfused with hypotonic solution (Hypo, 210 mOsm) described below and then SN-401 congeners+ Hypo applied at 10 and 7 μM to assess for % ICl,SWELL inhibition. To assess for ICl,SWELL inhibition upon application of SN-401 congeners to the closed SWELL1-LRRC8 channel, HEK-293 cells were preincubated with vehicle (or SN-401, SN-406, SN071 and SN072) for 30 mins prior to hypotonic stimulation and then stimulated with hypotonic solution+SN-401 congeners. Recordings were measured using Axopatch 2008 amplifier paired to a Digidata 1550 digitizer using pClamp 10.4 software. The extracellular buffer composition for hypotonic stimulation contains 90 mM NaCl, 2 mM CsCl, 1 mM MgCl, 1 mM CaCb, 10 mM HEPES, 10 mM Mannitol, pH 7.4 with NaOH (210 mOsm/kg). The extracellular isotonic buffer composition is same as above except for Mannitol concentration of 110 mM (300 mOsm/kg). The composition of intracellular buffer is 120 mM L-aspartic acid, 20 mM CsCl, 1 mM MgCl, 5 mM EGTA, 10 mM HEPES, 5 mM MgATP, 120 mM CsOH, 0.1 mM GTP, pH 7.2 with CsOH. All recordings were carried out at room temperature (RT) with HEK-293 cells, β-cells and 3T3-F442A cells performed in whole-cell configuration and human adipocytes in perforated-patch configuration, as previously described (Kang et al., 2018; Zhang et al., 2017).
Cells were washed twice in ice-cold phosphate buffer saline and lysed in RIPA buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.4) with proteinase/phosphatase inhibitors (Roche). The cell lysate was further sonicated in 10 sec cycle intervals for 2-3 times and centrifuged at 14000 rpm for 20 min at 4° C. The supernatant was collected and further estimated for protein concentration using DC protein assay kit (Bio-Rad). Fat tissues were homogenized and suspended in RIPA buffer with inhibitors in similar fashion as described above. Protein samples were further prepared by boiling in 4× laemmli buffer. Approximately 10-20 μg of total protein was loaded in 4-15% gradient gel (Bio-Rad) for separation and protein transfer was carried out onto the PVDF membranes (Bio-Rad). Membranes were blocked in 5% BSA (or 5% milk for SWELL1) in TBST buffer (0.2 M Tris, 1.37 M NaCl, 0.2% Tween-20, pH 7.4) for 1 hand incubated with appropriate primary antibodies (5% BSA or milk) overnight at 4° C. The membranes were further washed in TBST buffer before adding secondary antibody (Bio-Rad, Goat-anti-rabbit, #170-6515) in 1% BSA (or 1% milk for SWELL1) in TBST buffer for 1 h at RT. The signals were developed by chemiluminescence (Pierce) and visualized using a Chemidoc imaging system (Biorad). The images were further analyzed for band intensities using ImageJ software. Following primary antibodies were used: anti-phospho-AKT2 (#8599s), anti-AKT2 (#3063s), anti-phospho-AS160 (#4288s), anti-AS160 (#2670s) anti-GAPDH (#D16H11) and anti-β-actin (#8457s) from Cell Signaling; Rabbit polyclonal anti-SWELL1 antibody was generated against the epitope QRTKSRIEQGIVDRSE (SEQ ID NO: 13) (Pacific Antibodies).
3T3-F442A preadipocytes (WT, KO) and differentiated adipocytes without or with SWELL1 overexpression (WT+SWELL1 O/E, KO+SWELL1 O/E) were prepared as described in the Cell culture section on collagen coated coverslips. In the case of SWELL1 membrane trafficking, the 3T3-F442A preadipocytes were incubated in the presence of vehicle (or SN-401, SN-406 and SN071) at either 1 or 10 μM for 48 h and further processed. The cells were fixed in ice-cold acetone for 15 min at −20° C. and then washed four times with 1×PBS and permeabilized with 0.1% Triton X-100 in 1×PBS for 5 min at RT and subsequently blocked with 5% normal goat serum for 1 h at RT. Either anti-SWELL1 (1:400) or anti-Flag (1:1500, Sigma #F3165) antibody were added to the cells and incubated overnight at 4° C. The cells were then washed three times (1×PBS) prior and post to the addition of 1:1000 Alexa Flour 488/568 secondary antibody (anti-rabbit, #A11034 or anti-mouse, #A11004) for 1 hour at RT. Cells were counterstained with nuclear TO-PRO-3 (Life Technologies, #T3605) or DAPI (Invitrogen, #D1306) staining (1 μM) for 20 min followed by three washes with 1×PBS. Coverslips were further mounted on slides with Prolong Diamond anti-fading media. All images were captured using Zeiss LSM700/LSM510 confocal microscope with 63× objective (NA 1.4). SWELL1 membrane localization was quantified by stacking all the z-images and converting it into a binary image where the cytoplasmic intensity per unit area was subtracted from the total cell intensity per unit area using ImageJ software.
Mice were fasted for 6 h prior to glucose tolerance tests (GTT). Baseline glucose levels at 0 min timepoint (fasting glucose, FG) were measured from blood sample collected from tail snipping using glucometer (Bayer Healthcare LLC). Either 1 g or 0.75 g D-Glucose/kg body weight were injected (i.p.) for lean or HFD mice, respectively and glucose levels were measured at 7, 15, 30, 60, 90 and 120 min timepoints after injection. For insulin tolerance tests (ITTs), the mice were fasted for 4 h. Similar to GTTs, the baseline blood glucose levels were measured at 0 min timepoint and 15, 30, 60, 90 and 120 min timepoints post-injection (i.p.) of insulin (HumulinR, 1 U/kg body weight for lean mice or 1.25 U/kg body weight for HFD mice). GTTs or ITTs with vehicle (or SN-401, SN-403, SN-406, SN-407 and SN071) treated groups were performed approximately 24 hours after the last injection. For insulin secretion assay, the vehicle (or SN-401, SN-406 and SN071) treated HFD mice were fasted for 6 hand injected (i.p.) with 0.75 g D-Glucose/kg body weight and blood samples were collected at 0, 7, 15 and 30 min time points in microvette capillary tubes (SARSTEDT, #16.444) and centrifuged at 2000×g for 20 min at 4° C. The collected plasma was then measured for insulin content by using Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, #90080). All mice and treatment groups were assessed blindly while performing experiments.
For patch-clamp studies involving primary mouse cells, the mice were anesthesized by injecting Avertin (0.0125 g/ml in H2O) followed by cervical dislocation. HFD or polygenic KKAy mice treated with either vehicle (or (or SN-401, SN-406, SN-407 and SN071) were anesthesized with 1-4% isoflurane followed by cervical dislocation. Islets were further isolated as described previously (Kang et al., 2018). The perifusion of islets were performed using a PER14-02 from Biorep Technologies. For each experiment, around 50 freshly isolated islets (all from the same isolation batch) were handpicked to match size of islets across the samples and loaded into the polycarbonate perifusion chamber between two layers of polyacrylamide-microbead slurry (Bio-Gel P-4, BioRad) by the same experienced operator. Perifusion buffer contained (in mM): 120 NaCl, 24 NaHCO3, 4.8 KCl, 2.5 CaCl, 1.2 MgSO4, 10 HEPES, 2.8 glucose, 27.2 mannitol, 0.25% w/v bovine serum albumin, pH 7.4 with NaOH (300 mOsm/kg). Perifusion buffer kept at 37° C. was circulated at 120 μI/min After 48 min of washing with 2.8 mM glucose solution for stabilization, islets were stimulated with the following sequence: 16 min of 16.7 mM glucose, 40 min of 2.8 mM glucose, 10 min of 30 mM KCl, and 12 min of 2.8 mM glucose. Osmolarity was matched by adjusting mannitol concentration when preparing solution containing 16.7 mM glucose. Serial samples were collected either every 1 or 2 min into 96 wells kept at 4° C. Insulin concentrations were further determined using commercially available ELISA kit (Mercodia). The area under the curve (AUC) for the high-glucose induced insulin release was calculated for time points between 50 to 74/84 min. At the completion of the experiments, islets were further lysed by addition of RIPA buffer and the amount of insulin was detected by ELISA.
The pharmacokinetic studies of SN-401/SN-406 study were performed at Charles River Laboratory as outlined below. Male C57/BL6 mice were used in the study and assessed for a single dose (5 mg/kg) administration. The compounds were prepared in Cremaphor for i.p. and p.o dose routes and in 5% ethanol, 10% Tween-20 and water mix for i.v. route at a final concentration of 1 mg/ml. Terminal blood samples were collected via cardiac venipuncture under anesthesia at timepoints 0.08, 0.5, 2, 8 h post dose for i.v and at timepoints 0.25, 2, 8, 24 h post dose for i.p. and p.o. groups respectively with a sample size of 3 mice per timepoint. The blood samples were collected in tubes with K2 EDTA anticoagulant and further processed to collect plasma by centrifugation at 3500 rpm at 5° C. for 10 min Samples were further processed in LC/MS to determine the concentration of the compounds. Non-compartmental analysis was performed to obtain the PK parameters using the PKPlus software package (Simulation Plus). The area under the plasma concentration-time curve (AUCint) is calculated from time 0 to infinity where the Cmax is the maximal concentration achieved in plasma and t112 is the terminal elimination half-life. Oral bioavailability was calculated as AUCorailAUC1v*100.
In vitro ADMET studies were performed at Charles River Laboratory as outlined below. For the Caco-2 permeability assay, the cells were cultured (DMEM, 10% FBS, 1% L-Glutamax and 1% PenStrep) for 21 days. HBSS was used as the transport buffer and the TEER measurements were taken before the start of the assay. Compounds were added apical side to determine apical to basolateral transport (A-B) and basal side to determine basolateral to apical transport (B-A). Samples (10 μL) were collected at time 0 and 2 h and diluted (5×) with transport buffer. After the quenching reaction, the samples were further diluted in MilliQ water for bioanalysis. The TEER measurements were carried out at the end of the assay and wells with significant decrease in post-assay TEER values were not included in the data and repeated again. The analyte levels (peak area ratios) were measured on apical (A) and basolateral (B) sides at To and T2h-A-B and B-A fluxes were calculated averaging 3 individual measurements. Apparent permeability (Papp, cm/sec) was calculated as dQ (flux)/(dt*area*concentration). The efflux ratio was calculated by Papp(B-A)/Papp(B-A). For the microsomal metabolic stability assay, the microsomes were diluted in potassium phosphate buffer to maintain at a final concentration of 0.5 mg/ml in the assay procedure. The compounds were diluted 10-fold in acetonitrile and incubated with the microsomes at 37° C. with gentle shaking. Samples were collected at different timepoints and quenched. The samples were mixed by vortexing for 10 min and centrifuged at 3100 rpm for 10 min at 4° C. The supernatant was diluted in water and further analyzed in LC/MS autosampler. Half-life (T1/2) was calculated by the formula 0.692/slope where slope is ln(% remaining relative to Tzero vs time). Intrinsic clearance was calculated using the (CLn1)=T112*1/initial concentration*mg prep/g liver*g liver/kg body weight. For the cytochrome P450 inhibition assay, the cofactors and substrate were mixed in Potassium phosphate buffer. A stock concentration of 10 mM compounds (in DMSO) were diluted 5-fold in acetonitrile and mixed with cofactor/substrate mixture (2×). Human liver microsomes were diluted in Potassium phosphate buffer for a final concentration (2×) of 0.2 mg/ml and the reaction was initiated by mixing the microsomes with the compound/cofactor/substrate mixture at 37° C. with gentle shaking. Samples were collected at To and T30 min timepoints and quenched. The samples were then centrifuged at 3100 rpm for 5 min at 5-10° C. and the supernatant was diluted in water and further analyzed in LC/MS autosampler. % inhibition was calculated (using peak area ratios) relative to zero inhibition (full activity) and no activity (full inhibition). In silica prediction of properties and drug likeness of SN-401 and SN-406 drugs were performed using the FAF-Drugs4 and preADMET software packages.
Sterile silicone catheters (Dow-Corning) were placed into the jugular vein of mice under isoflurane anesthesia. Placed catheter was flushed with 200 U/ml heparin in saline and the free end of the catheter was directed subcutaneously via a blunted 14-gauge sterile needle and connected to a small tubing device that exited through the back of the animal. Mice were allowed to recover from surgery for 3 days, then received IP injections of vehicle or SN-401 (5 mg/kg) for 4 days. Hyperinsulinemic euglycemic clamps were performed on day 8 post-surgery on unrestrained, conscious mice as described elsewhere (Ayala et al., 2011; Kim et al., 2000), with some modifications. Mice were fasted for 6 h at which time insulin and glucose infusion were initiated (time 0). At 80 min prior to time 0 basal sampling was conducted, where whole-body glucose flux was traced by infusion of 0.05 μCi/min D-[3-3H]-glucose (Perkin Elmer), after a priming 5 μCi bolus for 1 minute. After the basal period, starting at time 0 D-[3-3H]-glucose was continuously infused at the 0.2 μCi/min rate and the infusion of insulin (Humulin, Eli Lilly) was initiated with a bolus of 80 mU/kg/min then followed by continuous infusion of insulin at the dose of 8 mU/kg/min throughout the assay. Fifty percent dextrose (Hospira) was infused at a variable rates (GIR) starting at the same time as the initiation of insulin infusion to maintain euglycemia at the targeted level of 150 mg/dl (8.1 mM). Blood glucose (BG) measurements were taken every ten minutes via tail vein sampling using Contour glucometer (Bayer). After mouse reached stable BG and GIR (typically, after 75 minutes since starting the insulin infusion; for some mice, a longer time was required to achieve steady state) a single bolus of 12 μCi of [1-14C]-2-deoxy-D-glucose (Perkin Elmer) in 96 μl of saline was administered. Plasma samples (collected from centrifuged blood) for determination of tracers enrichment, glucose level and insulin concentration were obtained at times −80, −20, −10, 0, and every 10 min starting at 80 min post-insulin (5 min after [1-14C]-2-deoxy-D-glucose bolus was administered) until the conclusion of the assay at 140 min. Tissue samples were then collected from mice under isofluorane anesthesia from organs of interest (e.g., liver, heart, kidney, white adipose tissue, brown adipose tissue, gastrocnemius, soleus etc.) for determination of 1-14C1-2-deoxy-D-glucose tracer uptake. Plasma and tissue samples were processed as described previously (Ayala et al., 2011). Briefly, plasma samples were deproteinized with Ba(OH)2 and ZnSO4 and dried to eliminate tritiated water. The glucose turnover rate (mg/kg-min) was calculated as the rate of tracer infusion (dpm/min) divided by the corrected plasma glucose specific activity (dpm/mg) per kg body weight of the mouse. Fluctuations from steady state were accounted for by use of Steele's model. Plasma glucose was measured using Analox GMD9 system (Analox Technologies).
Tissue samples (˜30 mg each) were homogenized in 750 μl of 0.5% perchloric acid, neutralized with 10 M KOH and centrifuged. The supernatant was then used for first measuring the abundance of total [1-14C] signal (derived from both 1-14C-2-deoxy-D-glucose, 1-14C-2-deoxy-D-glucose 6 phosphate) and, following a precipitation step with 0.3 N Ba(OH)3 and 0.3 N ZnSO4, for the measuring of non-phosphorylated 1-14C-2-deoxy-D-glucose. Glycogen was isolated by ethanol precipitation from 30% KOH tissue lysates, as described (Shiota, 2012). Insulin level in plasma at T0 and T140 were measured using a Stellux ELISA rodent insulin kit (Alpco).
3T3-F442A preadipocytes cells treated with either vehicle (DMSO) or 10 μM SN-401 for 96 h were solubilized in TRIzol and the total RNA was isolated using Purelink RNA kit (Life Technologies). The cDNA synthesis, qRT-PCR reaction and quantification were carried out as described previously (Zhang et al., 2017).
HFD mice treated with either vehicle or SN-401 were anesthetized with 1-4% isoflurane followed by cervical dislocation. Gross liver weights were measured and identical sections from right medial lobe of liver were dissected for further examinations. Total triglyceride content was determined by homogenizing 10-50 mg of tissue in 1.5 ml of chloroform:methanol (2:1 v/v) and centrifuged at 12000 rpm for 10 mins at 4° C. An aliquot, 20 ul, was evaporated in a 1.5 ml microcentrifuge tube for 30 mins. Triglyceride content was determined by adding 100 μl of Infinity Triglyceride Reagent (Fisher Scientific) to the dried sample followed by 30 min incubation at RT. The samples were then transferred to a 96 well plate along with standards (0-2000 mg/di) and absorbance was measured at 540 nm and the final concentration was determined by normalizing to tissue weight. For histological examination, liver sections were fixed in 10% zinc formalin and paraffin embedded for sectioning. Hematoxylin and eosin (H&E) stained sections were then assessed for steatosis grade, lobular inflammation and hepatocyte ballooning for non-alcoholic fatty liver disease (NAFLD) scoring as described (Kleiner et al., 2005; Liang et al., 2014; Rauckhorst et al., 2017).
Standard unpaired or paired two-tailed Student's t-test were performed while comparing two groups. One-way Anova was used for multiple group comparison. For GTTs and ITTs, 2-way analysis of variance (Anova) was used. A p-value less than 0.05 was considered statistically significant. *, ** and *** represents a p-value less than 0.05, 0.01 and 0.001 respectively. All data are represented as mean±SEM. All statistical details and analysis are indicated in the brief descriptions of the figures.
General Information: All commercially available reagents and solvents were used directly without further purification unless otherwise noted. Reactions were monitored either by thin-layer chromatography (carried out on silica plates, silica gel 60 F2s4, Merck) and visualized under UV light. Flash chromatography was performed using silica gel 60 as stationary phase performed under positive air pressure. 1H NMR spectra were recorded in CDCb on a Bruker Avance spectrometer operating at 300 MHz at ambient temperature unless otherwise noted. All peaks are reported in ppm on a scale downfield from TMS and using the residual solvent peak in CDCb (H 5=7.26) or TMS (5=0.0) as an internal standard. Data for 1H NMR are reported as follows: chemical shift (ppm, scale), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet and/or multiplet resonances, dd=double of doublets, dt=double of triplets, br=broad), coupling constant (Hz), and integration. All high-resolution mass spectra (HRMS) were measured on Waters Q-Tof Premier mass spectrometer using electrospray ionization (ESI) time-of-flight (TOF).
2-cyclopentyl-1-(2,3-dichloro-4-methoxyphenyl)ethan-1-one (3) was prepared according to Scheme 1 (
To a stirring solution of aluminum chloride (13.64 g, 102 mmol, 1.1 equiv.) in dichloromethane (250 ml) at 0° C. was added cyclopentyl acetyl chloride (15 g, 102 mmol, 1.1 equiv.) and the resulting solution was allowed to stir at 0° C. under nitrogen atmosphere for 10 minutes. To this was added a solution of 2,3-dicholoro anisole (16.46 g, 92.9 mmol, 1 equiv.) in dichloromethane (50 ml) at 0° C. and the resulting solution was allowed to warm to room temperature and stirred for 16 hours. Once complete, the reaction was added to cold concentrated hydrochloric acid (100 ml) followed by extraction in dichloromethane (150 ml×3). The organic fractions were pooled, concentrated and purified by silica gel chromatography using 0-15% ethyl acetate in hexanes as eluent to furnish compound 3 as white solid (22.41 g, 84%). NMR (300 MHz, CDCl3) δ 7.39 (d, J=8.7 Hz, 1H), 6.89 (d, J=8.7 Hz, 1H), 3.96 (s, 3H), 2.96 (d, J=7.2 Hz, 2H), 2.38-2.21 (m, 1H), 1.92-1.75 (m, 2H), 1.69-1.46 (m, 4H), 1.28-1.05 (m, 2H). HRMS (ESI), m/z calcd for C14H17Cl2O2 [M+H]+ 287.0605, found 287.0603.
6,7-dichloro-2-cyclopentyl-5-methoxy-2,3-dihydro-1H-inden-1-one (4) was prepared according to Scheme 1 (
To 2-cyclopentyl-1-(2,3-dichloro-4-methoxyphenyl)ethan-1-one (3) (21.5 g, 74.8 mmol, 1 equiv.) in a round bottom flask was added paraformaldehyde (6.74 g, 224.5 mmol, 3 equiv.), dimethylamine hydrochloride (30.52 g, 374 mmol, 5 equiv.) and acetic acid (2.15 ml) and the resulting mixture was allowed to stir at 85° C. for 16 hours. To the reaction was then added dimethylformamide (92 ml) and the resulting solution was allowed to stir at 85° C. for 4 hours. Once complete, the reaction was diluted with ethyl acetate and then washed with 1N hydrochloric acid. The organic fractions were collected and concentrated under vacuum and used for next step without purification. To the concentrated product in a round bottom flask was added cold concentrated sulfuric acid (120 ml) at 0° C. and the resulting solution was allowed to stir at room temperature for 18 hours. Once complete, the reaction was diluted with cold water and extracted thrice with ethyl acetate (100 ml). The organic fractions were pooled, concentrated and purified by silica gel chromatography using 0-15% ethyl acetate in hexanes as eluent to furnish compound 4 as beige solid (18.36 g, 82%). NMR (300 MHz, CDCl3) δ 6.88 (s, 1H), 4.00 (s, 3H), 3.16 (dd, J=18.1, 8.7 Hz, 1H), 2.80 (d, J=14.4 Hz, 2H), 2.43-2.22 (m, 1H), 1.96 (s, 1H), 1.73-1.48 (m, 5H), 1.46-1.33 (m, 1H), 1.17-1.00 (m, 1H). LRMS (ESI), m/z calcd for C15H17Cl2O2 [M+H]+ 299.0605, found 299.0614.
2-butyl-6,7-dichloro-2-cyclopentyl-5-methoxy-2,3-dihydro-1H-inden-1-one (5) was prepared according to Scheme 1 (
A stirring suspension of 4 (23 gm, 76.8 mmol, 1 equiv.) in anhydrous tert-butanol (220 ml) was allowed to reflux at 95° C. for 30 minutes. To the resulting solution was added potassium tert-butanol (1M in tert-butanol) (84 ml, 84.5 mmol, 1.1 equiv.) and the resulting solution was refluxed for 30 minutes. The reaction was then cooled to room temperature followed by addition of iodobutane (44.2 ml, 384 mmol, 5 equiv.) and the reaction was then allowed to reflux for additional 60 minutes. The reaction was allowed to cool, concentrated and purified by silica gel chromatography using 0-10% ethyl acetate in hexanes as eluent to furnish compound 5 as clear oil (17.75 g, 65%). NMR (300 MHz, CDCl3) δ 6.89 (s, 1H), 4.09-3.90 (m, 3H), 2.98-2.70 (m, 2H), 2.36-2.18 (m, 1H), 1.89-1.71 (m, 2H), 1.58-1.42 (m, 5H), 1.33-1.09 (m, 4H), 1.09-0.94 (m, 2H), 0.93-0.73 (m, 4H). HRMS (ESI), m/z calcd for C19H25Cl2O2 [M+H]+ 355.1231, found 355.1231.
2-butyl-6,7-dichloro-2-cyclopentyl-5-hydroxy-2,3-dihydro-1H-inden-1-one (6) was prepared according to Scheme 1 (
To 5 (3.14 g, 8.87 mmol, 1 equiv.) was added aluminum chloride (2.36 g, 17 mmol, 2 equiv.) and sodium iodide (2.7 g, 17 mmol, 2 equiv.) and the resulting solid mixture was triturated and allowed to stir at 70° C. for 60 minutes. Once complete, the reaction was diluted with dichloromethane and washed with aqueous saturated sodium thiosulfate solution. The organic fractions were collected and concentrated to give a beige solid which was then washed multiple times with hexanes to provide compound 6 as white solid (2.87 g, 95%). NMR (300 MHz, CDCl3) δ 7.03 (s, 1H), 6.32 (s, 1H), 2.97-2.73 (m, 2H), 2.36-2.17 (m, 1H), 1.88-1.68 (m, 2H), 1.62-1.39 (m, 6H), 1.31-1.11 (m, 3H), 1.08-0.97 (m, 2H), 0.97-0.87 (m, 1H), 0.83 (t, J=7.3 Hz, 3H). HRMS (ESI), m/z calcd for C18H23Cl2O2 [M+H]+ 341.1075, found 341.1089.
2-((2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)acetic acid (7) (SN071) was prepared according to Scheme 1 (
To a stirring solution of 5 (170 mg, 0.50 mmol, 1 equiv.) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (76 mg, 0.56 mmol, 1.1 equiv.) and ethyl 2-bromoacetate (61 μl, 0.56 mmol, 1.1 equiv.) and the reaction was allowed to stir at 60° C. for 2 hours. Once complete, to the reaction was added 4 N NaOH (1 ml) and the reaction was allowed to stir at 100° C. for 60 minutes. Once complete, reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as eluent to provide SN071 as a clear solid (173 mg, 87%). NMR (300 MHz, CDCl3) δ 6.80 (s, 1H), 5.88 (s, 1H), 4.88 (s, 2H), 2.87 (q, J=17.9 Hz, 2H), 2.34-2.20 (m, 1H), 1.91-1.69 (m, 2H), 1.66-1.39 (m, 6H), 1.32-1.13 (m, 3H), 1.10-0.95 (m, 2H), 0.94-0.86 (m, 1H), 0.83 (t, J=7.3 Hz, 3H). HRMS (ESI), m/z calcd for C20H25Cl2O4 [M+H]+ 399.1130, found 399.1132.
4-((2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)butanoic acid (8) (SN-401) was prepared according to Scheme 1 (
To a stirring solution of 5 (100 mg, 0.29 mmol, 1 equiv.) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv.) and ethyl 4-bromobutyrate (46 μl, 0.32 mmol, 1.1 equiv.) and the reaction was allowed to stir at 60° C. for 2 hours. Once complete, to the reaction was added 4 N NaOH (1 ml) and the reaction was allowed to stir at 100° C. for 60 minutes. Once complete, reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as eluent to provide SN-401 as a clear solid (111 mg, 89%). NMR (300 MHz, CDCl3) δ 10.77 (s, 1H), 6.86 (s, 1H), 4.21 (t, J=5.9 Hz, 2H), 2.88 (t, J=14.4 Hz, 2H), 2.69 (t, J=7.0 Hz, 2H), 2.26 (dd, J=12.6, 6.1 Hz, 3H), 1.87-1.73 (m, 2H), 1.64-1.44 (m, 6H), 1.35-1.10 (m, 4H), 1.08-0.95 (m, J=15.0, 7.7 Hz, 2H), 0.82 (t, J=7.3 Hz, 3H). HRMS (ESI), m/z calcd for C22H29C1204 [M+H]+427.1443, found 427.1446.
5-((2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)pentanoic acid (9) (SN-403) was prepared according to Scheme 1 (
To a stirring solution of 5 (100 mg, 0.29 mmol, 1 equiv.) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv.) and ethyl 6-bromovalerate (51 μl, 0.32 mmol, 1.1 equiv.) and the reaction was allowed to stir at 60° C. for 2 hours. Once complete, to the reaction was added 4 N NaOH (1 ml) and the reaction was allowed to stir at 100° C. for 60 minutes. Once complete, reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as eluent to provide SN-403 as a clear solid (114 mg, 88%). NMR (300 MHz, CDCl3) δ 10.95 (s, 1H), 6.85 (brs, 1H), 4.16 (t, J=5.7 Hz, 2H), 2.96-2.75 (m, 2H), 2.61-2.44 (m, 2H), 2.35-2.17 (m, 1H), 2.10-1.87 (m, 4H), 1.86-1.70 (m, 2H), 1.66-1.38 (m, 6H), 1.32-1.13 (m, 3H), 1.08-0.96 (m, 2H), 0.94-0.86 (m, 1H), 0.86-0.73 (m, 3H). HRMS (ESI), m/z calcd for C23H31Cl2O4 [M+H]+ 441.1599, found 441.1601.
To a stirring solution of 5 (100 mg, 0.29 mmol, 1 equiv.) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv.) and ethyl 6-bromohexanoate (58 μl, 0.32 mmol, 1.1 equiv.) and the reaction was allowed to stir at 60° C. for 2 hours. Once complete, to the reaction was added 4 N NaOH (1 ml) and the reaction was allowed to stir at 100° C. for 60 minutes. Once complete, reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as eluent to provide SN-406 as a clear solid (115 mg, 86%). 1H NMR (300 MHz, CDCl3) δ 11.70 (s, 1H), 6.85 (s, 1H), 4.13 (t, J=6.2 Hz, 2H), 2.93-2.74 (m, 2H), 2.43 (t, J=7.3 Hz, 2H), 2.32-2.17 (m, 1H), 1.98-1.87 (m, 2H), 1.85-1.68 (m, 4H), 1.66-1.40 (m, 8H), 1.28-1.12 (m, 3H), 1.07-0.93 (m, 2H), 0.91-0.70 (m, 4H). HRMS (ESI), m/z calcd for C24H33Cl2O4 [M+H]+ 455.1756, found 455.1756.
7-((2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)heptanoic acid (11) (SN-407) was prepared according to Scheme 1 (
To a stirring solution of 5 (100 mg, 0.29 mmol, 1 equiv.) in anhydrous dimethylformamide (1 ml) was added potassium carbonate (45 mg, 0.32 mmol, 1.1 equiv.) and ethyl 7-bromoheptanoate (63 μl, 0.32 mmol, 1.1 equiv.) and the reaction was allowed to stir at 60° C. for 2 hours. Once complete, to the reaction was added 4 N NaOH (1 ml) and the reaction was allowed to stir at 100° C. for 60 minutes. Once complete, reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as eluent to provide SN-407 as a clear solid (122 mg, 89%). NMR (300 MHz, CDCl3) δ 11.52 (s, 1H), 6.85 (s, 1H), 4.12 (t, J=6.3 Hz, 2H), 2.84 (q, J=18.2 Hz, 2H), 2.47-2.32 (m, 2H), 2.32-2.18 (m, 1H), 1.96-1.84 (m, 2H), 1.83-1.64 (m, 4H), 1.62-1.39 (m, 10H), 1.28-1.14 (m, 3H), 1.08-0.94 (m, 2H), 0.91 (d, J=8.5 Hz, 1H), 0.81 (t, J=7.3 Hz, 3H). HRMS (ESI), m/z calcd for C25H35Cl2O4 [M+H]+ 469.1912, found 469.1896.
4-((6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)butanoic acid (12) (SN072) was synthesized according to Scheme 2 (
To 4 (100 mg, 0.36 mmol, 1 equiv.) was added aluminum chloride (89 mg, 0.67 mmol, 2 equiv.) and sodium iodide (101 mg, 0.67 mmol, 2 equiv.) and the resulting solid mixture was triturated and allowed to stir at 70° C. for 60 minutes. Once complete, the reaction was diluted with dichloromethane and washed with aqueous saturated sodium thiosulfate solution. The organic fractions were collected and concentrated to give a beige solid which was then washed multiple times with hexanes to provide compound 6 as white solid which was used for the next step. To a stirring solution of the product form the first step in anhydrous dimethylformamide (1 ml) was added potassium carbonate (53 mg, 0.39 mmol, 1.1 equiv.) and ethyl 4-bromobutyrate (55 μl, 0.39 mmol, 1.1 equiv.) and the reaction was allowed to stir at 60° C. for 2 hours. Once complete, to the reaction was added 4 N NaOH (1 ml) and the reaction was allowed to stir at 100° C. for 60 minutes. Once complete, reaction was concentrated and purified by column chromatography using 0-10% methanol in dichloromethane as eluent to provide SN072 as a clear solid (107 mg, 86%). 1H NMR (300 MHz, CDCl3) δ 6.87 (s, 1H), 4.21 (t, J=5.9 Hz, 2H), 3.26-3.02 (m, 1H), 2.94-2.56 (m, 4H), 2.40-2.19 (m, 3H), 2.03-1.90 (m, 1H), 1.74-1.50 (m, 5H), 1.47-1.32 (m, 1H), 1.19-1.00 (m, 1H). HRMS (ESI), m/z calcd for C18H21Cl2O4 [M+H]+ 371.0817, found 371.0808.
Enantiomerically enriched SN-401 isomers were synthesized following literature reported procedure (Cragoe et al., 1982) and as depicted in scheme 3,
SWELL1/LRRC8a ablation impairs insulin signaling in target tissues and insulin secretion from the pancreatic β3-cell, inducing a pre-diabetic state of glucose intolerance. These recent findings show that reductions in SWELL1 may contribute to Type 2 diabetes (T2D). To determine if SWELL1-mediated currents are altered in T2D we measured ICl,SWELL in pancreatic β-cells freshly isolated from T2D mice raised on HFD for 5-7 months (
To test whether SWELL1 regulates insulin signaling we overexpressed Flag-tagged SWELL1 (SWELL1 O/E) in both WT and SWELL1 KO 3T3-F442A adipocytes and measured insulin-stimulated phosphorylated AKT2 (pAKT2) as a readout of insulin-sensitivity (
The small molecule 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB,
To confirm the SN-401-induced increases in SWELL1 protein were mediated by direct binding to the SWELL1-LRRC8 channel complex, as opposed to off-target effects, we designed and synthesized novel SN-401 congeners with subtle structural changes that either maintained or enhanced (SN-403, SN-406, SN-407;
To characterize the structural features of SN-401 responsible for binding to SWELL1-LRRC8, we performed molecular docking simulations of SN-401 and its analogs into the SWELL1 homo-hexamer (PDB: 6NZZ), and identified two molecular determinants predicted to be critical for SN-401-SWELL1-LRRC8 binding (
Additional binding interactions for congeners SN-406 and SN-407 are also predicted along the channel, due to the longer carbon chains affording additional hydrophobic interactions with side chain carbons of the R103 residues (
Collectively, these functional and molecular docking experiments indicate SN-401 and SWELL1-active congeners (SN-403/406/407) bind to SWELL1-LRRC8 hexamers at both R103 (via carboxylate end) and at the interface between LRRC8 monomers (via hydrophobic end), to stabilize the closed state of the channel, thereby inhibiting ICl,SWELL activity. Guided by docking studies and binding models that reveal the SN-401 carboxylate group interacting with R103 residues of multiple LRRC8 monomers within the hexameric channel, along with SN-401 cyclopentyl group binding within hydrophobic clefts between adjacent monomers, we hypothesized that these SN-40X compounds function as molecular tethers to stabilize assembly of the SWELL1-LRRC8 hexamer. This reduces SWELL1-LRRC8 complex disassembly, and subsequent proteasomal degradation, thereby augmenting translocation from ER to plasma membrane signaling domains, functioning as a pharmacological chaperone.
To test this hypothesis, we applied SWELL1-active SN-401 and SN-406 compounds to differentiated 3T3-F442A adipocytes under basal culture conditions for 4 days and then measured SWELL1 protein after 6 h of serum starving. At both 1 and 10 μM, SN-401 and SN-406 markedly augment SWELL1 protein to levels 1.5-2.3-fold to those in vehicle-treated controls, while inactive congeners SN071 and SN072 do not significantly increase SWELL1 protein levels. (
We next asked whether endoplasmic reticulum (ER) stress associated with glucolipotoxicity in metabolic syndrome may impair SWELL1-LRRC8 assembly and trafficking, to promote SWELL1 protein degradation, and thereby reduce ICl,SWELL and SWELL1 protein in T2D (
To determine if SN-401 improves insulin signaling and glucose homeostasis in vivo we treated two T2D mouse models: obese, HFD-fed mice and the polygenic T2D KKN mouse model with SN-401 (5 mg/kg i.p. for 4-10 days). In vivo, SN-401 augments SWELL1 expression 2.3-fold in adipose tissue of HFD-fed T2D mice (
SN-401 has in silica, in vitro, and in vivo characteristics that show it may be an effective oral therapy for T2D. First, several algorithms designed to identify candidate compounds with oral drug-like physicochemical properties (Lipinski (Lipinski et al., 2001), Veber (Veber et al., 2002), Egan (Egan et al., 2000), MDDR (Oprea, 2000)) indicate that SN-401 had oral drug-like properties as compared to current approved oral T2D drugs (Table 7, below).
Second, in vitro studies show SN-401 has good Caco-2 cell monolayer permeability and minimal cytochrome p450 isoenzyme inhibition (Table 8, below). Third, SN-401 has no effect on hERG, human Kv and delayed rectifier channels, and is selective for ICl,SWELL in guinea-pig atrial cells at channel inhibitory concentrations {˜5-10 μM), which is consistent with in silica ADMET predictions (Table 7), and indicates a low likelihood of cardiac QT prolongation and arrhythmia Fourth, in vivo PK studies in mice demonstrate that SN-401 has high oral bioavailability (AUCp.o./AUCi.v.=79%,
To examine the possible contribution of SN-401-mediated enhancements in insulin secretion from pancreatic β-cells, we next measured glucose-stimulated insulin secretion (GSIS) in SN-401 treated mice subjected to 21 weeks of HFD. We find that the impairments GSIS classically observed with long-term HFD (21 weeks HFD) are significantly improved in SN-401-treated HFD mice based on serum insulin measurements (
To more rigorously evaluate SN-401 effects on insulin sensitization and glucose metabolism in T2D mice we compared euglycemic hyperinsulinemic clamps traced with 3H-glucose and 14C-deoxyglucose in T2D KKN mice treated with SN-401 or vehicle. SN-401 treated T2D KKN mice require a higher glucose-infusion rate (GIR) to maintain euglycemia compared to vehicle, consistent with enhanced systemic insulin-sensitivity (
As the SN-401-mediated increase in SWELL1 is expected to enhance insulin-pAKT2-pAS160 signaling, GLUT4 plasma membrane translocation, and tissue glucose uptake, we next measured the effect of SN-401 on glucose uptake in adipose, myocardium and skeletal muscle using 2-deoxyglucose (2-DG). SN-401 enhanced insulin-stimulated 2-DG uptake into inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), and myocardium (
Nonalcoholic fatty liver disease (NAFLD), like T2D, is associated with insulin resistance. NASH is an advanced form of nonalcoholic liver disease defined by three histological features: hepatic steatosis, hepatic lobular inflammation, hepatocyte damage (ballooning) and can be present without or without fibrosis. NAFLD and T2D likely share at least some pathophysiologic mechanisms because more than one-third of patients (37%) with T2D have NASH and almost one-half of patients with NASH (44%) have T2D. (To evaluate the effect of SN-401 on the genesis of NAFLD, mice were raised on HFD for 16 weeks followed by intermittent dosing with SN-401 over the course of 5 weeks (
To determine if the effects of SN-401 observed in vivo in T2D mice are attributable to SWELL1-LRRC8 binding, as opposed to off-target effects, we next measured fasting blood glucose and glucose tolerance in HFD T2D mice treated with either SWELL1-active SN-403 or SN-406 as compared to SWELL1-inactive SN071 (all at 5 mg/kg/day×4 days). In mice treated with HFD for 8 weeks, SN-403 significantly reduced fasting blood glucose and improved glucose tolerance compared to SN071 (
Our current working model is that the transition from compensated obesity (pre-diabetes, normoglycemia) to decompensated obesity (T2D, hyperglycemia) reflects, among other things, a relative reduction in SWELL1 protein expression and signaling in peripheral insulin-sensitive tissues) and in pancreatic β-cells)-metabolically pheno-copying SWELL1-loss-of-function models. This contributes to the combined insulin resistance and impaired insulin-secretion associated with poorly-controlled T2D and hyperglycemia. SWELL1 forms a macromolecular signaling complex that includes heterohexamers of SWELL1 and LRRC8b-e, with stoichiometries that likely vary from tissue to tissue. We propose that SWELL1-LRRC8 signaling complexes are inherently unstable, and thus a proportion of complexes succumb to disassembly and degradation. Glucolipotoxicity and ensuing ER stress associated with T2D states provide an unfavorable environment for SWELL1-LRRC8 complex assembly, contributing to SWELL1 degradation and reductions in SWELL1 protein and SWELL1-mediated ICl,SWELL observed in T2D. Small molecules SN-401 and SN-401 congeners with preserved SWELL1 binding activity serve as pharmacological chaperones to stabilize formation of the SWELL1-LRRC8 complex. This reduces SWELL1 degradation, and enhances the passage of SWELL1-LRRC8 heteromers through the ER and Golgi apparatus to the plasma membrane—thereby rectifying the SWELL1-deficient state in multiple metabolically important tissues in the setting of T2D and metabolic syndrome to improve overall systemic glycemia via both insulin sensitization and secretion mechanisms. Indeed, the concept of small molecule inhibitors acting as therapeutic molecular chaperones to support the folding, assembly and trafficking of proteins (including ion channels) has been demonstrated for Niemann-Pick C disease and congenital hyperinsulinism (SUR1-KATP channel mutants). Also, this therapeutic mechanism is analogous to small molecule correctors for another chloride channel, CFTR (VX-659/VX-445, Vertex Pharmaceuticals), which is proving to be a breakthrough therapeutic approach for cystic fibrosis.
Through structure activity relationship (SAR) and in silica molecular docking studies, we identified hotspots on opposing ends of the SN-401 molecule that interact with separate regions of the SWELL1-LRRC8 complex: the carboxylate group with R103 in multiple LRRC8 subunits at a constriction in the pore, and the cyclopentyl group within the hydrophobic cleft formed by adjacent LRRC8 monomers; functioning like a molecular staple or tether to bind and stabilize loosely associated SWELL1 homomers (especially in the setting of T2D) into a more rigid hexameric structure. Indeed, the cryo-EM structure obtained in lipid nanodiscs required DCPIB/SN-401 binding in order to obtain images of sufficient spatial resolution (Kern et al., 2019), which supports the concept that SN-401 stabilizes the SWELL1 homomer. Another advantage provided by SAR studies was identification and synthesis of SN-401 congeners that removed (SN071/SN072) or enhanced (SN-403/406/407) SWELL1-binding, as these provided powerful tools to query SWELL1-on target activity directly in vitro and in vivo, and also validated the proof-of-concept for developing novel SN-401 congeners with enhanced efficacy.
SWELL1-LRRC8 complexes are broadly expressed in multiple tissues, and consist of unknown combinations of SWELL1, LRRC8b, LRRC8c, LRRC8d and LRRC8e, indicating that SWELL1 complexes will be enormously heterogenous. However, SWELL1-LRRC8 stabilizers like SN-401 may be designed to target many, if not all, possible channel complexes since all will contain the elements necessary for SN-401 binding: at least one R103 (from the requisite SWELL1 monomer: carboxyl group binding site), and the nature of the hydrophobic cleft (cyclopentyl binding site), which is conserved among all LRRC8 monomers. Indeed, traced glucose clamps did reveal insulin sensitization effects in multiple tissues, including adipose, skeletal muscle, liver and heart. The increased glucose-uptake in heart is particularly interesting, since this may provide salutary effects on cardiac energetics that could favorably impact both systolic (HFrEF) and diastolic (HFpEF) function in diabetic cardiomyopathy, and thereby potentially improve cardiac outcomes in T2D, as observed with SGLT2 inhibitors.
The current study provides an initial proof-of-concept for pharmacological induction of SWELL1 signaling using SWELL1 modulators (SN-40X congeners) to treat metabolic diseases at multiple homeostatic nodes, including adipose, liver, and pancreatic β-cell. Hence, SN-401 may represent a tool compound from which a novel drug class may be derived to treat T2D, NASH, and other metabolic diseases.
Animals. All the mice were housed in temperature, humidity, and light-controlled room and allowed free access to water and food. Both male and female SWELL1fl/fl (WT), Myl1Cre;SWELL1fl/fl (Myl1 KO), Myf5Cre;SWELL1fl/fl (skeletal muscle targeted SWELL1 KO), were generated and used in these studies. Myl1Cre (JAX #24713) and Myf5Cre (JAX #007893) mice were purchased from Jackson labs. For high-fat diet (HFD) studies, we used Research Diets Inc. (Cat #D12492) (60 kcal % fat) regimen starting at 14 weeks of age.
Generation of CRISPR/Cas9-mediated SWELL1 floxed (SWELL1fl/fl) mice. SWELL1fl/fl mice were generated as previously described (Zhang et al., 2017). Briefly, SWELL1 intronic sequences were obtained from Ensembl Transcript ID ENSMUST00000139454. All CRISPR/Cas9 sites were identified using ZiFit Targeter Version 4.2. Pairs of oligonucleotides corresponding to the chosen CRISPR-Cas9 target sites were designed, synthesized, annealed, and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 construct (Addgene plasmid #42230), following the protocol detailed in Cong et al., 2013. CRISPR-Cas9 reagents and ssODNs were injected into the pronuclei of F1 mixed C57/129 mouse strain embryos at an injection solution concentration of 5 ng/μl and 75-100 ng/μl, respectively. Correctly targeted mice were screened by PCR across the predicted loxP insertion sites on either side of Exon 3. These mice were then backcrossed >8 generations into a C57BL/6 background.
Antibodies: Rabbit polyclonal anti-SWELL1 antibody was generated against the epitope QRTKSRIEQGIVDRSE (SEQ ID NO: 13) (Pacific Antibodies). All other primary antibodies were purchased from Cells Signaling: anti-β-actin (#8457s), p-AKT1 (#9018s), Akt1 (#2938s), pAKT2 (#8599s), Akt2 (#3063s), p-AS160 (#4288s), AS160 (#2670s), AMPKα (#5831s), pAMPKα (#2535s), FoxO1(#2880s) and pFoxO1(#9464s), p70 S6 Kinase (#9202s), p-p70 S6 Kinase (#9205s), pS6 Ribosomal (#5364s), GAPDH (#5174s), pErk1/2 (#9101s), Total Erk1/2 (#9102s). Purified mouse anti-Grb2 was purchased from BD (610111s). Purified anti-flag mouse antibody was purchased from sigma. Rabbit IgG Santa Cruz (sc-2027). All primary antibodies were used at 1:1000 dilution, except for anti-flag at 1:2000 dilution. All secondary antibody (anti-rabbit-HRP and anti-mouse-HRP) were used at 1:10000 dilution.
Adenovirus. Adenovirus type 5 with Ad5-CMV-mCherry (1×1010 PFU/ml), Ad5-CMV-Cre-mCherry (3×1010 PFU/ml) were obtained from the University of Iowa viral vector core facility. Ad5-CAG-LoxP-stop-LoxP-3×Flag-SWELL1 (1×1010 PFU/ml) were obtained from Vector Biolabs. Ad5-U6-shGRB2-GFP (1×109 PFU/ml) and Ad5-U6-shSCR-GFP (1×1010 PFU/ml) were obtained from Vector Biolabs.
Electrophysiology. All recordings were performed in the whole-cell configuration at room temperature, as previously described (Zhang et al., 2017 and Kang et al., 2018). Briefly, currents were measured with either an Axopatch 200B amplifier or a MultiClamp 700B amplifier (Molecular Devices) paired to a Digidata 1550 digitizer, using pClamp 10.4 software. The intracellular solution contained (in mM): 120 L-aspartic acid, 20 CsCl, 1 MgCl2, 5 EGTA, 10 HEPES, 5 MgATP, 120 CsOH, 0.1 GTP, pH 7.2 with CsOH. The extracellular solution for hypotonic stimulation contained (in mM): 90 NaCl, 2 CsCl, 1 MgCl2, 1 CaCl2), 10 HEPES, 5 glucose, 5 mannitol, pH 7.4 with NaOH (210 mOsm/kg). The isotonic extracellular solution contained the same composition as above except for mannitol concentration of 105 (300 mOsm/kg). The osmolarity was checked by a vapor pressure osmometer 5500 (Wescor). Currents were filtered at 10 kHz and sampled at 100 μs interval. The patch pipettes were pulled from borosilicate glass capillary tubes (WPI) using a P-87 micropipette puller (Sutter Instruments). The pipette resistance was ˜4-6 Mil when the patch pipette was filled with intracellular solution. The holding potential was 0 mV. Voltage ramps from ˜100 to +100 mV (at 0.4 mV/ms) were applied every 4 s.
Primary muscle satellite cell isolation: Satellite cell isolation and differentiation were performed as described previously with minor modifications (Hindi et al., 2017). Briefly, gastrocnemius and quadriceps muscles were excised from SWELL1flfl mice (8-10 weeks old) and washed twice with 1×PBS supplemented with 1% penicillin-streptomycin and fungizone (300 μl/100 ml). Muscle tissue was incubated in DMEM-F12 media supplemented with collagenase II (2 mg/ml), 1% penicillin-streptomycin and fungizone (300 μl/100 ml) and incubated at shaker for 90 minutes at 37° C. Tissue was washed with 1×PBS and incubated again with DMEM-F12 media supplemented with collagenase II (1 mg/ml), dispase (0.5 mg/ml), 1% penicillin-streptomycin and fungizone (300 ul/100 ml) in a shaker for 30 minutes at 37° C. Subsequently, the tissue was minced and passed through a cell strainer (70 μm), and after centrifugation; satellite cells were plated on BD Matrigel-coated dishes. Cells were stimulated to differentiate into myoblasts in DMEM-F12, 20% fetal bovine serum (FBS), 40 ng/ml basic fibroblast growth factor (bfgf, R&D Systems, 233-FB/CF), 1× non-essential amino acids, 0.14 mM β-mercaptoethanol, 1× penicillin/streptomycin, and Fungizone. Myoblasts were maintained with 10 ng/ml bfgf and then differentiated in DMEM-F12, 2% FBS, 1× insulin-transferrin-selenium, when 80% confluency was reached.
Cell culture: WT C2C12 and SWELL1 KO C2C12 cell line were cultured at 37° C., 5% CO2 Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum (FBS; Atlanta Bio selected) and antibiotics 1% penicillin-streptomycin (Gibco, USA). Cells were grown to 80% confluency and then transferred to differentiation media DMEM supplemented with antibiotics and 2% horse serum (HS; GIBCO) to induce differentiation. The differentiation media was changed every two days. Cells were allowed to differentiate into myotubes for up to 6 days. Subsequently, myotube images were taken for quantification of myotube surface area and fusion index.
Myotube morphology, surface area and fusion index quantification: After differentiation (Day 7), cells were imaged with Olympus IX73 microscope (10× objective, Olympus, Japan). For each experimental condition, 5-6 bright field images were captured randomly from 6 well plate. Myotube surface area was quantified manually with ImageJ software. The morphometric quantification was carried out by an independent observer who was blinded to the experimental conditions. For fusion index, differentiated myotube growing on coverslip were washed with 1×PBS and fixed with 2% PFA. After washing with 1×PBS 3 times, cells were permeabilized with 0.1% TritonX100 for 5 minutes at room temperature and subsequently blocking was done with 5% goat serum for 30 minutes. Cells were stained with DAPI (1 μM) for 15 minutes and after washing with 1×PBS, coverslip were mounted on slides with ProLong Diamond anti-fading agent. Cells were imaged with Olympus IX73 microscope (10× objective, Olympus, Japan) with bright field and DAPI filter. Fusion index (number of nuclei incorporated within the myotube/total number of nuclei present in that view field) were analyzed by ImageJ.
RNA sequencing: RNA quality was assessed by Agilent BioAnalyzer 2100 by the University of Iowa Institute of Human Genetics, Genomics Division. RNA integrity numbers greater than 8 were accepted for RNAseq library preparation. RNA libraries of 150 bp PolyA-enriched RNA were generated, and sequencing was performed on a HiSeq 4000 genome sequencing platform (Illumina). Sequencing results were uploaded and analyzed with BaseSpace (Illumina). Sequences were trimmed to 125 bp using FASTQ Toolkit (Version 2.2.0) and aligned to Mus musculus mmp10 genome using RNA-Seq Alignment (Version 1.1.0). Transcripts were assembled and differential gene expression was determined using Cufflinks Assembly and DE (Version 2.1.0). Ingenuity Pathway Analysis (QIAGEN) was used to analyze significantly regulated genes which were filtered using cutoffs of >1.5 fragments per kilobase per million reads, >1.5 fold changes in gene expression, and a false discovery rate of <0.05. Heatmaps were generated to visualize significantly regulated genes.
Myotube signaling studies: For insulin stimulation, differentiated C2C12 myotubes were incubated in serum free media for 6 h and stimulated with 0 and 10 nM insulin for 15 min; while differentiated primary myotubes were incubated in serum free media for 4 h and stimulated with 0 and 10 nM insulin for 2 h. To examine intracellular signaling upon SWELL1 overexpression (SWELL1 O/E), we overexpressed SWELL1-3×Flag by transducing C2C12 myotubes with Ad5-CAG-LoxP-stop-LoxP-SWELL1-3×Flag (MOI 50-60) and Ad5-CMV-Cre-mCherry (MOI 50-60) and polybrene (4 μg/ml) in DMEM (2% FBS and 1% penicillin-streptomycin) for 36 h. Ad5-CMV-Cre-mCherry alone with polybrene (4 μg/ml) (MOI 50-60) was transduced in WT C2C12 or SWELL1 KO C2C12 as controls. Viral transduction efficiency (60-70%) was confirmed by mCherry fluorescence. Cells were allowed to differentiate further in differentiation media up to 6 days. Myotube images were taken before collecting lysates for further signaling studies. GRB2 knock-down was achieved by transducing myotubes with Ad5-U6-shSCR-GFP (Control, MOI 50-60) or Ad5-U6-shSWELL1-GFP (GRB2 KD, MOI 50-60) in DMEM (2% FBS and 1% penicillin-streptomycin) supplemented with polybrene (4 μg/ml) for 24 hour. Cells were allowed to differentiate further in differentiation media up to 6 days. Differentiated myotube images were taken for myotube surface area quantification before collecting the cells for RNA isolation.
Stretch stimulation: C2C12 myotubes were plated in each well of a 6 well BioFlex culture plate. Cells were allowed to differentiate up to 6 days in differentiation media, and then placed into a Flexcell Jr. Tension System (FX-6000T) and incubated at 37° C. with 5% CO2. C2C12 myotubes on flexible membrane were subjected to either no tension or to static stretch of 5% for 15 minutes. Cells were lysed and protein isolated for subsequent Western blots.
Western blot: Cells were washed with ice cold 1×PBS and lysed in ice-cold lysis buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.5) with added proteinase/phosphatase inhibitor (Roche). The cell lysate was further sonicated (20% pulse frequency for 20 sec) and centrifuged at 14000 rpm for 20 min at 4° C. The supernatant was collected and estimated for protein concentration using DC protein assay kit (Bio-Rad). For immunoblotting, an appropriate volume of 4× Laemmli (Bio-rad) sample loading buffer was added to the sample (10-20 μg of protein), then heated at 90° C. for 5 min before loading onto 4-20% gel (Bio-Rad). Proteins were separated using running buffer (Bio-Rad) for 2 h at 110V. Proteins were transferred to PVDF membrane (Bio-Rad) and membrane blocked in 5% (w/v) BSA or 5% (w/v) milk in TBST buffer (0.2 M Tris, 1.37 M NaCl, 0.2% Tween-20, pH 7.4) at room temperature for 1 hour. Blots were incubated with primary antibodies at 4° C. overnight, followed by secondary antibody (Bio-Rad, Goat-anti-mouse #170-5047, Goat-anti-rabbit #170-6515, all used at 1:10000) at room temperature for one hour. Membranes were washed 3 times and imaged by chemiluminescence (Pierce) by using a Chemidoc imaging system (BioRad). The images were further analyzed for band intensities using ImageJ software. β-Actin or GAPDH levels were quantified for equal protein loading.
Immunoprecipitation: C2C12 myotubes were plated on 10 cm dishes in complete media and grown to 80% confluency. For SWELL1-3×Flag overexpression, Ad5-CAG-LoxP-stop-LoxP-3×Flag-SWELL1 (MOI 50-60) and Ad5-CMV-Cre-mCherry (MOI 50-60) along with polybrene (4 ug/ml) were added to cells in DMEM media (2% FBS and 1% penicillin-streptomycin) allowed to grow for 36 hours. Cells were then switched to differentiation media for up to 6 days. After that myotubes were harvested in ice-cold lysis buffer (150 mM NaCL, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.5) with added protease/phosphatase inhibitor (Roche) and kept on ice with gentle agitation for 15 minutes to allow complete lysis. Lysates were incubated with anti-Flag antibody (Sigma #F3165) or control rabbit IgG (Santa Cruz sc-2027) rotating end over end overnight at 4° C. Protein G sepharose beads (GE) were added for 4 h and then samples were centrifuged at 10,000 g for 3 minutes and washed three times with RIPA buffer and re-suspended in laemmli buffer (Bio-Rad), boiled for 5 minutes, separated by SDS-PAGE gel followed by the western blot protocol.
RNA isolation and quantitative RT-PCR: Differentiated cells were solubilized in TRIzol and the total RNA was isolated using PureLink RNA kit (Life Technologies) and column DNase digestion kit (Life Technologies). The cDNA synthesis, qRT-PCR reaction and quantification were carried out as described previously (Zhang et al., 2017). All experiment was performed in triplicate and GAPDH were used as internal standard to normalize the data. All primers used for qRT-PCR are listed in Table 10, below.
Muscle tissue homogenization: Mice were euthanized and gastrocnemius muscle excised and washed with 1×PBS. Muscles tissue were minced with surgical blade and kept in 8 volume of ice cold homogenization buffer (20 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10% (w/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, pH 7.8) supplemented with protease/phosphatase inhibitor (Roche). Tissues were homogenized on ice with a Dounce homogenizer (40-50 passes) and incubated for overnight at 4° C. with continuous rotation. Tissue lysate was further sonicated in 20 sec cycle intervals for 2-3 times and centrifuged at 14000 rpm for 20 min at 4° C. The supernatant was collected for protein concentration estimation using DC protein assay kit (Bio-Rad). Due to the high content of contractile protein in this preparation, Coomassie gel staining was performed to demonstrate equal protein loading, and for quantification normalization of Western blots.
Tissue histology: Mice were anesthetized with isoflurane followed by cervical dislocation. Tibialis anterior (TA) muscle was carefully excised and gently immersed into the tissue-tek O.C.T medium placed on wooden cork. Orientation of the tissue maintained while embedding in the medium. Subsequently, wooden cork with tissue gently immersed into the liquid N2 pre-chilled isopentane bath for 10-14 sec and store at −80° C. Tissue sectioning (10 μm) were done with Leica cryostat and all sections collected on positively charged microscope slide for H&E staining as described earlier (Bonetto et al., 2015). Briefly, TA sectioned slides were stained for 2 minutes in hematoxylin, 1 minute in eosin and then dehydrated with ethanol and xylenes. Subsequently, slides were mounted with coverslip and image were taken with EVOS cell imaging microscope (10×objective). For quantification of fiber cross-sectional area, images were processed using ImageJ software to enhance contrast and smooth/sharpen cell boundaries and clearly demarcate muscle fiber cross sectional area. All measurement was performed with an independent observer who was blinded to the identity of the slides.
Exercise tolerance test and inversion testing: Mouse treadmill exercise protocols were adapted from Dougherty et al., 2016. Briefly, mice were first acclimated with the motorized treadmill (Columbus Instruments Exer3/6 Treadmill (Columbus, Ohio) for 3 days by running 10-15 minutes (with 3 minutes interval) for 3 consecutive days at 7 m/min, with the electric shocking grid (frequency 1 Hz) installed in each lane. During the treadmill testing, mice ran with a gradual increase in speed (5.5 m/minute to 22 m/minute) and inclination (0°-15°) at time intervals of 3 minutes each. The total running distance for each mouse was recorded at the end of the experiment. The predefined criteria for removing the mouse from the treadmill and recording the distance travelled was: continuous shock for 5 sec or receiving 5-6 shocks within a time interval of 15 seconds. These mice were promptly removed from the treadmill and total duration and distance were recorded for further analysis. Mouse inversion test was performed using a wire-grid screen apparatus elevated to 50 cm. Mice were stabilized on the screen inclined at 60°, with the mouse head facing towards the base of the screen. The screen was slowly pivoted to 0° (horizontal), such that the mouse was fully inverted and hanging upside down from the screen. Soft bedding was placed underneath the screen to protect mouse from any injury, were they to fall. The inversion test for each mouse was repeated 2 times with an interval of 45 minutes (resting period). The hang time for each mouse was repeated 3 times with an interval of 5-minute. The maximum hanging time limit for each mouse was set for 3 minutes.
Isolated muscle contractile assessment: Soleus muscle was carefully dissected and transferred to a specialized muscle stimulation system (1500A, Aurora Scientific, Aurora, ON, Canada) where physiology tests were run in a blinded fashion. Muscle was immersed in a Ringer solution (in mM) (NaCl 137, KCl 5, CaCl2) 2, NaH2PO4 1, NaHCO3 24, MgSO4 1, glucose 11 and curare 0.015) maintained at 37° C. The distal tendon was secured with silk suture to the arm of a dual mode ergometer (300C-LR, Aurora Scientific, Aurora, ON, Canada) and the proximal tendon secured to a stationary post. Muscles were stimulated with an electrical stimulator (701C, Aurora Scientific, Aurora, ON, Canada) using parallel platinum plate electrodes extending along the muscle. Muscle slack length was set by increasing muscle length until passive force was detectable above the noise of the transducer and fiber length was measured through a micrometer reticule in the eyepiece of a dissecting microscope. Optimal muscle length was then determined by incrementally increasing the length of the muscle by 10% of slack fiber length until the isometric tetanic force plateaued. At this optimum length, force was recorded during a twitch contraction and isometric tetanic contraction (300 ms train of 0.3 ms pulses at 225 Hz). The muscle was then fatigued with a bout of repeated tetanic contractions every 10 seconds until force dropped below 50% of peak. At this point, the muscle was cut from the sutures and weighed. This weight, along with peak fiber length and muscle density (1.056 g/cm3), was used to calculate the physiological cross-sectional area (PCSA) and convert to specific force (tension). The experimental data were analyzed and quantified using Matlab (Mathworks), and presented as peak tetanic tension (Tetanic Tension)—peak of the force recording during the tetanic contraction, normalized to PCSA; Time to fatigue (TTF)—time for the tetanic tension to fall below 50% of the peak value during the fatigue test; Half relaxation time (HRT)—half the time between force peak and return to baseline during the twitch contraction.
XF-24 Seahorse assay: Cellular respiration was quantified in primary myotubes using the XF24 extracellular flux (XF) bioanalyzer (Agilent Technologies/Seahorse Bioscience, North Billerica, Mass., USA). Primary skeletal muscle cells isolated from SWELL1flfl mice were plated on BD Matrigel-coated plate at a density of 20×103 per well. After 24 hours, cells were incubated in Ad5-CMV-mCherry or Ad5-CMV-Cre-mCherry (MOI 90-100) in DMEM-F12 media (2% FBS and 1% penicillin-streptomycin) for 24 hours. Cells were then switched to differentiation media for another 3 days. For insulin-stimulation, cells were incubated in serum free media for 4 h and stimulated with 0 and 10 nM insulin for 2 h. Subsequently, medium was changed to XF-DMEM, and kept in a non-CO2 incubator for 60 minutes. The basal oxygen consumption rate (OCR) was measured in XF-DMEM. Subsequently, oxygen consumption was measured after addition of each of the following compounds: oligomycin (1 μg/ml) (ATP-Linked OCR), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 μM) (Maximal Capacity OCR) and antimycin A (10 μM; Spare Capacity OCR) For the glycolysis stress test, prior to experimentation, cells were switched to glucose-free XF-DMEM and kept in a non-CO2 incubator for 60 min. Extracellular acidification rate (ECAR) was determined in XF-DMEM followed by these additional conditions: glucose (10 mM), oligomycin (1 μM), and 2-DG (100 mM). Data for Seahorse experiments (normalized to protein) reflect the results of one Seahorse run/condition with 6 replicates.
Metabolic phenotyping: Mouse body composition (fat and lean mass) was measured by nuclear magnetic resonance (NMR); Echo-MRI 3-in-1 analyzer, EchoMRI, LLC). For glucose tolerance test (GTT), mice were fasted for 6 hours and intraperitoneal injection of glucose (lg/kg body weight for lean mice and 0.75 g/kg of body weight for HFD mice) administered. Glucose level was monitored from tail-tip blood using a glucometer (Bayer Healthcare LLC) at the indicated times. For insulin tolerance test (ITT), mice were fasted for 4 hours and after an intra-peritoneal injection of insulin (HumulinR, 1 U/kg for lean mice and 1.25 U/kg for HFD mice) glucose level was measured by glucometer at the indicated times.
Statistics. Data are represented as mean±s.e.m. Two-tail paired or unpaired Student's t-tests were used for comparison between two groups. For three or more groups, data were analyzed by one-way analysis of variance and Tukey's post hoc test. For GTTs and ITTs, 2-way analysis of variance (Anova) was used. A p-value <0.05 was considered statistically significant. *, ** and *** represents a p-value less than 0.05, 0.01 and 0.001 respectively.
SWELL1 (LRRC8a) is the essential component of a hexameric ion channel signaling complex that encodes ICl,SWELL, or the volume-regulated anion current (VRAC). While the SWELL1-LRRC8 complex has been shown to regulate cellular volume in response to application of non-physiological hypotonic extracellular solutions, the physiological function(s) of this ubiquitously expressed ion channel signaling complex remain unknown. To determine the function of the SWELL1-LRRC8 channel complex in skeletal muscle, we genetically deleted SWELL1 from C2C12 mouse myoblasts using CRISPR/cas9 mediated gene editing as described previously (Zhang et al., 2017 and Kim et al., 2000), and from primary skeletal muscle cells isolated from SWELL1flfl mice transduced with adenoviral Cre-mCherry (KO) or mCherry alone (WT control) (Zhang et al., 2017). SWELL1 protein Western blots confirmed robust SWELL1 ablation in both SWELL1 KO C2C212 myotubes and SWELL1 KO primary skeletal myotubes (
In order to further characterize the observed SWELL1 dependent impairment in myotube formation in C2C12 and primary muscle cells we performed genome-wide RNA sequencing (RNA-seq) of SWELL1 KO C2C12 relative to control WT C2C12 myotubes. These transcriptomic data revealed clear differences in the global transcriptional profile between WT and SWELL1 KO C2C12 myotubes (
Guided by the results of the pathway analysis, and the fact that skeletal myogenesis and maturation is regulated by insulin-PI3K-AKT-mTOR-MAPK we directly examined a number of insulin-stimulated pathways in WT and SWELL1 KO C2C12 myotubes, including insulin-stimulated AKT2-AS160, FOXO1 and AMPK signaling. Indeed, insulin-stimulated pAKT2, pAS160, pFOXO1 and pAMPK are abrogated in SWELL1 KO myotubes compared to WT C2C12 myotubes (
To further validate SWELL1-mediated effects on muscle differentiation and signaling we re-expressed SWELL1 in SWELL1 KO C2C12 myoblasts (SWELL1 O/E) and then examined myotube differentiation and basal activity of multiple intracellular signaling pathways by Western blot, including pAKT1, pAKT2, pAS160, p-p70S6K, pS6K and pERK1/2 as compared to WT and SWELL1 KO C2C12 myotubes. SWELL1 O/E to 2.12-fold WT levels fully rescues myotube development in SWELL1 KO myotubes (
In a cellular context, there are numerous reports that VRAC and the SWELL1-LRRC8 complex that functionally encodes it is mechano-responsive. It is well established that mechanical stretch is an important regulator of skeletal muscle proliferation, differentiation and skeletal muscle hypertrophy and may be mediated by PI3K-AKT-MAPK signaling and integrin signaling pathways. To determine if SWELL1 is also required for stretch-mediated AKT and MAP kinase signaling in skeletal myotubes we subjected WT and SWELL1 KO C2C12 myotubes to 0% or 5% equiaxial stretch using the FlexCell stretch system. Mechanical stretch (5%) is sufficient to stimulate PI3K-AKT2/AKT1-pAS160-MAPK (ERK1/2) signaling in WT C2C12 in a SWELL1-dependent manner (
It has been reported earlier in both lymphocyte and adipocytes that the SWELL1-LRRC8 complex interacts with Growth factor Receptor-Bound 2 (GRB2) and regulates PI3K-AKT signaling, whereby GRB2 binds with IRS1/2 and negatively regulates insulin signaling. Indeed, GRB2 knock-down augments insulin-PI3K-MAPK signaling and induces myogenesis and myogenic differentiation genes. To determine if SWELL1 and GRB2 interact in C2C12 myotubes, we overexpressed C-terminal 3×Flag tagged SWELL1 in C2C12 cells followed by immunoprecipitation (IP) with Flag antibody. We observed significant GRB2 enrichment upon Flag IP from lysates of SWELL1-3×Flag expressing C2C12 myotubes, consistent with a GRB2-SWELL1 interaction (
To examine the physiological consequences of SWELL1 ablation in vivo, we generated skeletal muscle specific SWELL1 KO mice using Cre-LoxP technology by crossing Myf5-Cre mice with SWELL1fl/fl mice (Myf5 KO;
Since insulin signaling is an important regulator of skeletal muscle oxidative capacity and endurance, we next examined exercise tolerance on treadmill testing in SWELL1fl/fl (WT) compared to Myf5 KO mice. Myf5 KO mice exhibit a 14% reduced exercise capacity, compared to age and gender matched WT controls (
To determine whether these SWELL1 dependent differences in muscle endurance and force were due to impaired oxidative capacity, we next measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in WT and SWELL1 KO primary skeletal muscle cells, under basal and insulin-stimulated conditions (
Guided by evidence of impaired insulin-PI3K-AKT-AS160-GLUT4 signaling observed in SWELL1 KO C2C12 and primary myotubes we next examined systemic glucose homeostasis and insulin sensitivity in WT and Myf5 KO mice by measuring glucose and insulin tolerance. On a regular chow diet, there are no differences in either glucose tolerance or insulin tolerance (
Since Myf5 is also expressed in brown fat, it is possible that these metabolic phenotypes arise from SWELL1-mediated effects in brown fat and consequent changes in systemic metabolism. To rule out this possibility, we repeated a subset of the above experiments in a skeletal muscle targeted KO mouse generated by crossing the Myl1-Cre and SWELL1fl/fl mice (Myl1-Cre;SWELL1fl/fl) or Myl1 KO (
Our data reveal that the SWELL1-LRRC8 channel complex regulates insulin/stretch-mediated AKT-AS160-GLUT4, MAP kinase and mTOR signaling in differentiated myoblast cultures, with consequent effects on myogenic differentiation, insulin-stimulated glucose metabolism and oxygen consumption. In vivo, skeletal muscle targeted SWELL1 KO mice have smaller skeletal muscle cells, impaired muscle endurance, and force generation, and are predisposed to adiposity, glucose intolerance and insulin resistance. Insulin/stretch-mediated PI3K-AKT, mTOR signaling are well known to be important regulators of myogenic differentiation, metabolism and muscle function showing impaired SWELL1-AKT-mTOR signaling may underlie the defect in myogenic differentiation. Indeed, consistent with our previous findings and proposed model in adipocytes, in which SWELL1 mediates the interaction of GRB2 with IRS1 to regulate insulin-AKT signaling, SWELL1 also associates with GRB2 in skeletal myotubes, and GRB2 knock-down rescues impaired myogenic differentiation in SWELL1 KO muscle cells. Thus, our working model for SWELL1 mediated regulation of insulin-PI3K-AKT and downstream signaling in adipocytes appears to be conserved in skeletal myotubes. The in vitro phenotype that we observe in CRISPR/cas9 mediated SWELL1 KO C2C12 myotubes and in SWELL1 KO primary myotubes is consistent with the observation of Chen et al., 2019 that used siRNA mediated SWELL1 knock-down to demonstrate that the SWELL1-LRRC8 channel complex is required for myogenic differentiation. However, the ability of both GRB2 KD and SWELL1 O/E to rescue myogenic differentiation and augment insulin-AKT, MAP kinase and mTOR signaling in SWELL1 KO myotubes implicates non-canonical, non-conductive signaling mechanisms. Based on our work and also previous studies, SWELL1 O/E does not increase ICl,SWELL/VRAC to supranormal levels, although pAKT, pERK1/2 and mTOR levels are augmented by 2-fold to 3-fold above endogenous levels, upon 2-fold SWELL1 O/E in C2C12 myotubes. These data show that alternative/non-canonical signaling mechanisms underlie SWELL1-LRRC8 signaling, as opposed to canonical/conductive signaling mechanisms.
It is also notable that the profound myogenic differentiation block observed upon SWELL1 ablation in both C2C12 myotubes and primary myotubes in vitro is significantly milder in vivo, where only a 30% reduction in skeletal myocyte cross-sectional area is observed, with no change in total muscle mass, or lean content, in Myf5 KO mice. This discordance in phenotype may reflect fundamental differences in the biology of skeletal muscle differentiation in vitro versus the in vivo milieu.
Although overall muscle development is grossly intact in both Myl1 KO and Myf5 KO mice, there is a consistent reduction in exercise capacity, muscle endurance and force generation, and a propensity for increased adiposity over time compared to age and gender matched controls. The observed impairments in exercise capacity in skeletal muscle SWELL1 KO mice are consistent with some level of insulin resistance, as in db/db mice and in humans, and may be due to impaired skeletal muscle glycolysis and oxygen consumption in SWELL1 depleted skeletal muscle. Furthermore, the increased gonadal adiposity, with preserved glucose and insulin tolerance, observed in Myl1 KO and Myf5 KO mice phenocopy both skeletal muscle specific insulin receptor KO mice (MIRKO) and transgenic mice expressing a skeletal muscle dominant-negative insulin receptor mutant, wherein skeletal muscle specific insulin resistance drives re-distribution of glucose from skeletal muscle to adipose tissue, to promote adiposity. In the case of Myf5 KO mice, overnutrition and HFD feeding unmasks this underlying mild insulin resistance and glucose intolerance. Recent findings from skeletal muscle specific AKT1/AKT2 double KO mice indicate that these effects may not attributable to solely to muscle AKT signaling, but potentially involve other insulin sensitive signaling pathways.
In summary, we show that SWELL1-LRRC8 regulates myogenic differentiation and insulin-PI3K-AKT-AS160, ERK1/2, and mTOR signaling in myotubes via GRB2-mediated signaling. In vivo, SWELL1 is required for maintaining normal exercise capacity, muscle endurance, adiposity under basal conditions, and systemic glycemia in the setting of overnutrition. These findings contribute further to our understanding of SWELL1-LRRC8 channel complexes in the regulation of systemic metabolism.
When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.
As various changes could be made in the above compositions and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/037022 | 6/10/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62982531 | Feb 2020 | US | |
| 62963988 | Jan 2020 | US | |
| 62859499 | Jun 2019 | US |
