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WO2021203033A2 - Procédés et compositions pour modifier la libération de gaba hépatique pour traiter des problèmes de santé liés à l'obésité - Google Patents

Procédés et compositions pour modifier la libération de gaba hépatique pour traiter des problèmes de santé liés à l'obésité Download PDF

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Publication number
WO2021203033A2
WO2021203033A2 PCT/US2021/025629 US2021025629W WO2021203033A2 WO 2021203033 A2 WO2021203033 A2 WO 2021203033A2 US 2021025629 W US2021025629 W US 2021025629W WO 2021203033 A2 WO2021203033 A2 WO 2021203033A2
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composition
gaba
hepatic
expression
activity
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PCT/US2021/025629
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English (en)
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WO2021203033A3 (fr
Inventor
Caroline E. GEISLER
Benjamin J RENQUIST
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Arizona Board Of Regents On Behalf Of The University Of Arizona
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Priority claimed from PCT/US2020/052571 external-priority patent/WO2021062048A2/fr
Application filed by Arizona Board Of Regents On Behalf Of The University Of Arizona filed Critical Arizona Board Of Regents On Behalf Of The University Of Arizona
Publication of WO2021203033A2 publication Critical patent/WO2021203033A2/fr
Publication of WO2021203033A3 publication Critical patent/WO2021203033A3/fr
Priority to US17/937,604 priority Critical patent/US20230128194A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]

Definitions

  • the present invention relates to methods and compositions for treating obesity-related conditions such as type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperphagia, and obesity-related conditions. More particularly, the present invention describes but is not limited to methods and compositions for treating conditions caused by altered hepatic GABA production and release, including obesity, hyperinsulinemia and insulin resistance, wherein manipulating the expression and/or activity of specific GABA transporters (e.g., increasing expression of SLC6A12 and/or SLC6A13 genes or increasing activity of the proteins for which they encode, BGT1 and/or GAT2; or decreasing expression of SLC6A6 and SLC6A8 genes or increasing the activity of the proteins for which they encode, TauT and/or CRT) can increase hepatic GABA re-uptake or decrease hepatic GABA release to improve insulin sensitivity and prevent hypertension.
  • the present invention also describes methods and systems for modulating hepatic GABA production and release or hepatic vagal afferent nerve signaling to alter
  • T2D Type II diabetes
  • diabetes affects 30 million Americans, while an additional 81 million Americans have pre-diabetes.
  • the high prevalence, mortality, and economic burden of T2D underscores a critical need for the development of additional therapeutics to treat diabetes.
  • hepatic lipid accumulation is directly associated with increased energy intake.
  • NAFLD non-alcoholic fatty liver disease
  • Hepatic lipid accumulation is a hallmark of T2D and is associated with obesity-induced hyperinsulinemia, insulin resistance, and hyperphagia.
  • GABA-T GABA-transaminase
  • the liver produces and releases hepatokines into circulation in response to acute and chronic nutrient status.
  • FGF21 and ANGPTL4 are secreted from hepatocytes in response to liver nutrient flux and can act in an endocrine fashion to impact whole body metabolism.
  • the present invention establishes that obesity-induced hepatic lipid accumulation increases hepatocyte production and release of the inhibitory neurotransmitter, GABA, in mice that acts in a paracrine fashion to decrease the firing activity of the hepatic vagal afferent nerve (HVAN), resulting in increased insulin secretion and decreased skeletal muscle glucose clearance.
  • GABA e.g., hepatokine
  • the literature suggests that the hepatic vagal nerve communicates with the central nervous system to affect pancreatic insulin release and peripheral tissue insulin sensitivity.
  • the HVAN regulates parasympathetic efferent nerve activity at the pancreas to alter insulin secretion.
  • a decrease in HVAN firing frequency stimulates insulin secretion, whereas an increase in HVAN firing frequency decreases insulin secretion.
  • the HVAN is also involved in regulating whole-body insulin sensitivity. Hepatic vagotomy diminishes insulin sensitivity (assessed as insulin-stimulated glucose uptake) in insulin sensitive rats, while improving insulin sensitivity and glucose tolerance in insulin resistant mice. Therefore, the firing frequency of the HVAN is integral to controlling insulin secretion and sensitivity.
  • liver is also a key endocrine organ which produces a significant number of hepatokines that are altered by obesity, NAFLD, and exercise and signal to change metabolic function in other tissues.
  • HVAN histone deacetylase
  • hepatocellular lipid accumulation depolarizes hepatocytes.
  • the present invention shows that hepatic steatosis dysregulates glucose and insulin homeostasis. Obesity-induced hepatocellular lipid accumulation results in hepatocyte depolarization. The present invention shows that hepatocyte depolarization depresses hepatic afferent vagal nerve firing, increases GABA release from liver slices, and causes hyperinsulinemia.
  • Preventing hepatic GABA release or eliminating the ability of the liver to communicate to the hepatic vagal nerve ameliorates the hyperinsulinemia and insulin resistance associated with diet-induced obesity.
  • hepatic expression of GABA transporters is associated with basal serum insulin, hepatic insulin sensitivity index, and glucose infusion and disposal rates during a hyperinsulinemic euglycemic clamp.
  • Single nucleotide polymorphisms in hepatic GABA re-uptake transporters are associated with an increased incidence of D2M.
  • the present invention features a new use of GABA as a novel hepatokine that is dysregulated in obesity and whose release can be manipulated to mute or exacerbate the glucoregulatory dysfunction common to obesity.
  • the present invention describes the use of four GABA transporters whose activity can be manipulated to alter hepatic slice GABA release. Hepatic expression of SLC6A12 and SLC6A13 is positively correlated with insulin sensitivity. The present invention shows that inhibiting these two transporters increases liver slice media GABA concentrations by preventing re-uptake. Hepatic expression of SLC6A6 and SLC6A8 is negatively associated with insulin sensitivity, proposing that these two transporters export hepatic GABA.
  • the present invention utilizes pharmacological agents that increase expression or activity of SLC6A12 and/or SLC6A13 or pharmacological agents that inhibit expression or activity of SLC6A6 and/or SLC6A8 to improve insulin sensitivity and prevent hypertension.
  • the present invention provides compositions and methods for decreasing hepatic GABA release or increasing hepatic GABA re-uptake to treat obesity and obesity-related conditions (e.g., hyperphagia, hypertension, insulin resistance, and hyperinsulinemia) as specified in the independent claims.
  • obesity and obesity-related conditions e.g., hyperphagia, hypertension, insulin resistance, and hyperinsulinemia
  • Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.
  • the present invention features a method of treating obesity and/or obesity-related conditions in a subject in need thereof.
  • the method comprises administering to the subject a therapeutic amount of a composition for altering GABA release by increasing hepatic expression or activity of GABA transporters encoded for by SLC6A12 and/or SLC6A13, or by inhibiting hepatic expression or activity of GABA transporters encoded for by SLC6A6 and/or SLC6A8.
  • This altering of activity of specific hepatic GABA transporters decreases GABA release.
  • the methods herein may help improve insulin sensitivity, decrease body mass, cause weight loss, decrease food intake, and/or prevent hypertension.
  • a non-limiting example of the present invention describes 1) increasing the liver expression of SLC6A12 and/or SLC6A13 or activity of their proteins (BGT1 and GAT2, respectively) improves GABA re-uptake and/or 2) decreasing the liver expression of SLC6A6 and/or SLC6A8 or activity of their proteins (TauT and CRT, respectively) prevents GABA release. Either improving GABA re-uptake or preventing GABA release will help to prevent obesity, insulin resistance, hypertension, hyperinsulinemia, and/or hyperphagia.
  • the present invention also features a method for improving insulin sensitivity in a subject in need thereof.
  • the method comprises: administering to the subject a therapeutic amount of a composition for altering GABA release by increasing hepatic expression or activity of GABA transporters, SLC6A12 and/or SLC6A13, or by inhibiting hepatic expression or activity of GABA transporters, SLC6A6 and/or SLC6A8.
  • This altering of activity of specific hepatic GABA transporters alters GABA re-uptake and release to improve insulin sensitivity, decrease body mass, cause weight loss, decrease food intake, and/or prevent hypertension.
  • the present invention also features a pharmaceutical composition, for example for use in the methods described herein such as but not limited to methods for treating an obesity-related condition.
  • the composition comprises an activator or stimulator of hepatic expression of GABA transporters, SLC6A12 and/or SLC6A13, or activity of their proteins, BGT1 and GAT2, to improve GABA re-uptake, or an inhibitor of GABA transporters, SLC6A6 and/or SLC6A8 or activity of their proteins, TauT and/or CRT to prevent GABA release.
  • the composition may be effective for decreasing blood glucose, decreasing blood insulin, improving insulin sensitivity, increasing glucose tolerance, and decreasing/normalizing blood pressure or a combination thereof.
  • the present invention also features a method of causing a subject in need thereof to lose weight.
  • the method comprises administering to the patient a therapeutic amount of a composition for altering hepatic activity and/or expression of GABA transporters.
  • the composition is for increasing hepatic expression of genes encoding GABA transporters, SLC6A12 and/or SLC6A13, or for decreasing hepatic expression of genes encoding GABA transporters, SLC6A6 and/or SLC6A8.
  • the composition is for increasing expression or activity of the GABA transporters, BGT1 and/or GAT2, or decreasing expression of activity of the GABA transporters, TauT and/or CRT, decreasing hepatic GABA synthesis or hepatic GABA release or increasing hepatic GABA re-uptake.
  • altering hepatic activity and/or expression of GABA transporters, SLC6A12, SLC6A13, SLC6A6, SLC6A8 causes a decrease in food intake so that the subject loses weight and/or adiposity.
  • the present invention features altering food intake by regulating GABA release.
  • the present invention features methods and compositions for inducing weight loss (reducing food intake) by depressing hepatic GABA release.
  • the present invention also features methods and compositions for increasing weight gain by increasing food intake through enhancing hepatic GABA production or release.
  • the present invention features methods and compositions for treating obesity-related conditions by regulating glucose homeostasis.
  • the methods and compositions herein may feature limiting hepatic mitochondrial uncoupling, decreasing hepatic GABA release, hyperpolarizing the hepatocyte, and preventing obesity induced depolarization of the hepatocyte membrane potential.
  • the methods may feature inhibitors for GABA synthesis and/or inhibitors for GABA release, e.g., inhibitors for GABA-T, TauT (GABA transporter), or CRT (GABA transporter).
  • the methods and compositions herein may be used for a variety of purposes including but not limited to treating obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, hypertension, and/or hyperphagia.
  • the present invention may be used for altering food intake by regulating GABA production or GABA release.
  • the present invention features methods and compositions for losing weight (reducing food intake) by depressing hepatic GABA production or release.
  • the present invention also features methods and compositions for gaining weight by increasing food intake, a result of enhanced hepatic GABA production or release.
  • the composition normalizes blood pressure, reduces blood glucose, improves glucose homeostasis in obesity, improves obesity-induced metabolic dysfunction, decreases body mass and fat mass, and/or reduces food intake in obesity.
  • the present invention features methods of reducing food intake in a monogastric animal (e.g., pig, chicken, dog, cat, horse, rodent, e.g., mouse, rat, etc.) or a human.
  • the method comprises administering to the monogastric animal or human an effective amount of a composition that depresses hepatic GABA production or release, wherein depressing hepatic GABA production or release causes the monogastric animal or human to reduce its food intake as compared to its food intake prior to being administered the composition.
  • the method may be applied for weight loss purposes.
  • the composition inhibits GABA signaling on the hepatic vagal afferent nerve.
  • the composition inhibits expression of or activity of GABA transaminase (GABA T) or inhibits GABA production in certain embodiments, the composition that inhibits expression of or activity of GABA transaminase, or inhibits GABA production comprises valproic acid, vigabatrin, phenylethylidenehydrazine (PEH), ethanolamine-O-sulfate (EOS), L-cycloserine, aminooxyacetic acid, gabaculine, phenelzine, rosmarinic acid, branched chain fatty acid, 2-methyl, 2-ethylcaproic acid, 2,2-dimethylvaleric acid, S-vigabatrin, [3-(aminomethyl)phenyl]acetic acid, [2-(aminomethyl)phenyl]acetic acid, ursolic acid, succinic semialdehyde, succinate, Sr2+, SH-group reagent, pyruvate, propionic acid,
  • 6-Azathymine 5-thiouracil, 5-nitrouracil, DL-3-amino-1-cyclopentene-1-carboxyiic acid,
  • the composition that inhibits expression of or activity of GABA transaminase or inhibits GABA production is an AMPK activator.
  • the composition inhibits GABA release. In certain embodiments, the composition inhibits expression or activity of GABA transporters that export hepatic GABA. In certain embodiments, the composition that inhibits GABA release inhibits mRNA or protein expression of the Solute Carrier Family 6 Member 6 ( SLC6A6 ) gene or the Solute Carrier Family 6 Member 8 ( SLC6A8 ) gene. In certain embodiments, the composition inhibits mRNA or protein expression of or activity of TauT, a GABA transporter protein encoded by the SLC6A6 gene. In certain embodiments, the composition inhibits mRNA or protein expression of or activity of creatine transporter (CRT), a GABA transporter protein encoded by SLC6A8 gene.
  • CRT creatine transporter
  • the composition that inhibits mRNA or protein expression or activity of SCL6A6 or TauT is vigabatrin, d-ALA, guvacine, taurine, Beta-alanine, Guanidinoacetate, b-Guanidinopropionate, g-Guanidinobutyrate, Guanidinoethansulfonate, and taurine in certain embodiments, the composition that inhibits expression or activity of SCL6A8 or CRT is an AMPK activator, Guanidinoacetate, b-Guanidinopropionate, g-Guanidinobutyrate, Guanidinoethansulfonate, creatinine, methylguanidine, l-arginine, RGX-202, 2,4-dinitro-1-fluorobenzene, tetraethylammonium, guanidine, creatine, arginine, lysine, DTBM, DNFB, or NEM.
  • the composition improves GABA re-uptake.
  • the composition increases mRNA or protein expression of the Solute Carrier Family 6 Member 12 ( SLC6A12 ) gene or the Solute Carrier Family 6 Member 13 ( SLC6A13 ) gene.
  • the composition increases mRNA or protein expression of or activity of BGT1, a GABA transporter protein encoded by the SLC6A12 gene.
  • the composition increases mRNA or protein expression of or activity of GAT2, a GABA transporter protein encoded by the SLC6A13 gene.
  • the composition that increases expression of SLC6A12 and/or SLC6A 13 is an AMPK activator.
  • the composition inhibits expression or activity of succinate semialdehyde dehydrogenase.
  • the composition that inhibits expression or activity of succinate semialdehyde dehydrogenase comprises 2-methyl, 2-ethylcaproic acid; 2,2-dimethylvaleric acid, 2-oxoglutaric semialdehyde,
  • 4-dimethylaminoazobenzene-4-iodoacetamide 4-hydroxy-trans-2-nonenal, 4-hydroxybenzaldehyde, 4-methoxybenzaldehyde, 4-tolualdehyde, 5,5'-dithiobis(2-nitrobenzoic acid), Acetaldehyde, Acrolein, ADP, AMP, Arsenite, ATP, Benzaldehyde, Ca2+, Cd2+, Chloral hydrate, Cu2+, Disulfiram, Dithionitrobenzoate, Fe3+, Glyoxylate, Hg2+, lodoacetamide, m-hydroxybenzaldehyde, Mg2+, Mn2+, N-ethylmaleimide, N-formylglycine, NAD+, NADH, NEM, Ni2+, o-phthalaldehyde, p-bromobenzaldehyde, p-chlorobenzaldehyde,
  • the AMPK activator is a biguanide, a thiazolidinedione, a ginsenoside, or a polyphenol.
  • the AMPK activator is A-769662, metformin, resveratrol, troglitazone, pioglitazone, rosiglitazone, quercetin, genistein, epigallocatechin gallate, berberine, curcumin, ginsenoside Rb1, alpha-lipoic acid, cryptotanshinone, 5-aminoimidazole-4-carboxaminde ribonucleoside (AICAR), benzimidazole, salicylate, compound-13, PT-1 , MT63-78, and APC.
  • AICAR 5-aminoimidazole-4-carboxaminde ribonucleoside
  • the composition comprises an inhibitor of sodium potassium ATPase. In certain embodiments, the composition reduces hepatic mitochondrial uncoupling.
  • the present invention features methods of increasing food intake in a monogastric animal.
  • the method comprises administering to the monogastric animal an effective amount of a composition that increases hepatic GABA production or release, wherein increasing hepatic GABA production or release causes the monogastric animal to increase its food intake as compared to its food intake prior to being administered the composition.
  • the method may be applied for improving weight gain.
  • the composition is a drug, a compound, or a molecule (such as but not limited to an anti-sense oligonucleotide).
  • the composition activates GABA signaling on the hepatic vagal afferent nerve.
  • the composition increases expression of or activity of GABA transaminase (GABA T) or increases GABA production.
  • GABA T GABA transaminase
  • the composition increases or activates GABA release.
  • the composition increases expression or activity of GABA transporters that export hepatic GABA.
  • the composition that increases GABA release increases expression of the Solute Carrier Family 6 Member 6 (SLC6A6) gene or the Solute Carrier Family 6 Member 8 ( SLC6A8 ) gene.
  • the composition increases expression of or activity of TauT, a GABA transporter protein encoded by the SLC6A6 gene.
  • the composition increases expression of or activity of creatine transporter (CRT), a GABA transporter protein encoded by SLC6A8 gene.
  • CRT creatine transporter
  • the composition decreases GABA re-uptake. In certain embodiments, the composition decreases expression of the Solute Carrier Family 6 Member 12 ( SLC6A12 ) gene or the Solute Carrier Family 6 Member 13 ( SLC6A13 ) gene. In certain embodiments, the composition decreases expression of or activity of BGT1, a GABA transporter protein encoded by the SLC6A12 gene. In certain embodiments, the composition decreases expression of or activity of GAT2, a GABA transporter protein encoded by the SLC6A13 gene.
  • the composition increases expression or activity of succinate semialdehyde dehydrogenase.
  • the composition comprises an activator of sodium potassium ATPase.
  • the composition increases hepatic mitochondrial uncoupling.
  • the methods related to increasing food intake may be directed to animals, e.g., monogastric animals, the present invention is not limited to the application of said method to non-human animals. For example, there may be instances wherein the method is applied to humans in order to help increase food intake and/or gain weight.
  • Non-limiting examples of compounds that may be considered for weight loss and a reduction in food intake include vigabatrin and ethanoiamine-O-sulfate (EOS). This finding was surprising, since those in the field believe that vigabatrin is associated with weight gain (Ben-Menachem, 2007, Epilepsia 48 Suppl 9:42-5; Lambert and Bird, 1997, Seizure 6:233-235).
  • FIGs. 1A-1L show that GABA-Transaminase inhibition improves glucose homeostasis in obesity.
  • HFD-induced obese mice were intraperitoneally injected with GABA-Transaminase inhibitors ethanolamine-O-sulfate (EOS) or vigabatrin (8 mg/day), or phosphate buffered saline (PBS; control) for 5 days.
  • FIG. 1A shows body weight during treatment.
  • FIG. 1B shows basal serum insulin (B)
  • FIG. 1C shows glucose
  • FIG. 1D shows glucose.insulin ratio pre-treatment, on treatment day 4, and after a 2-week washout.
  • FIG. 1E shows Serum glucagon in response to EOS.
  • FIG. 1A shows body weight during treatment.
  • FIG. 1B shows basal serum insulin (B)
  • FIG. 1C shows glucose
  • FIG. 1D shows glucose.insulin ratio pre-treatment, on treatment day 4, and after a 2-week washout.
  • FIG. 1E
  • FIG. 1F shows Oral glucose tolerance (OGTT), and FIG. 1G shows OGTT area under the curve (AUC) on treatment day 4.
  • FIG. 1H shows Glucose stimulated serum insulin pre-treatment, on treatment day 4, and after a 2-week washout.
  • FIG. 11 shows Insulin tolerance (ITT) and FIG. 1J shows ITT AUC on treatment day 4.
  • FIG. 1L shows Tissue specific 3 H-2-deoxy-D-glucose uptake during a glucose tolerance test spiked with 3 H-2-deoxy-D-glucose (10 pCi/mouse) and cGMP content, indicative of vasodilatory signal (L) on treatment day 5.
  • DPM disintegrations per minute
  • NS non-significant.
  • FIGs. 2A-2K show Acute hepatic GABA-Transaminase knockdown improves obesity induced metabolic dysfunction.
  • FIG. 2A shows GABA-T mRNA expression in liver, whole brain, and pancreas after 1 week of injections with a GABA-T targeted or scramble control antisense oligonucleotide (ASO; 12.5 mg/kg IP twice weekly) in high fat diet-induced obese mice.
  • FIG. 2B shows release of GABA (pmol/mg DNA) from hepatic slices.
  • FIG. 2C shows body weight during treatment.
  • FIG. 2D shows basal serum insulin.
  • FIG. 2E shows glucose
  • FIG. 2F shows glucose:insulin ratio.
  • FIG. 2G shows oral glucose tolerance (OGTT)
  • FIG. 2H shows OGTT area under the curve (AUC; H)
  • FIG. 2I shows oral glucose stimulated serum insulin
  • FIG. 2J shows insulin tolerance (ITT)
  • FIGs. 3A-3G show one week of hepatic GABA-Transaminase knockdown improves insulin sensitivity assessed by hyperinsulinemic euglycemic clamp.
  • High fat diet-induced obese mice received 1 week of injections with a GABA-T targeted or scramble control antisense oligonucleotide ⁇ ASO; 12.5mg/kg IP twice weekly) before hyperinsulinemic euglycemic clamps were performed.
  • FIG. 3A shows body weight the day of clamp procedures.
  • FIG. 3B shows blood glucose concentrations and
  • FIG. 3C shows glucose infusion rate during the clamps.
  • FIG. 3D shows serum insulin concentrations before insulin infusion (basal) and during the clamp.
  • FIG. 3E shows endogenous glucose appearance (Ra) and FIG. 3F shows glucose disappearance (Rd) before insulin infusion (basal) and during the clamp.
  • FIGs. 4A-4H show obesity induced hepatic GABA production increases phagic drive.
  • FIGs. 5A-5F show GABA-Transaminase knockdown or inhibition decreases body mass and fat mass.
  • Body composition in antisense oligonucleotide (ASO) treated mice was assessed by Dual-Energy X-ray Absorptiometry (DEXA) at the UC Davis Mouse Metabolic Phenotyping Center.
  • FiG. 5A shows change in body mass (A)
  • FIG. 5B shows fat mass (B)
  • FiG FiG.
  • FIG. 5C shows lean mass after 1 and 4 weeks of GABA-T targeted or scramble control ASO ⁇ 12.5 mg/kg IP twice weekly) relative to pre-treatment body composition.
  • FIG. 5D shows body mass (D)
  • FIG. 5E shows fat mass (E)
  • FIG. 5F shows lean mass (F) on day 0 and 7 of EOS treatment (3 g/L in drinking water). All data are presented as mean ⁇ SEM.
  • FIGs. 6A-6C show associations between hepatic GABA system and glucoregulatory markers in people with obesity.
  • Multivariate regressions including intrahepatic triglyceride % (IHTG%), hepatic ABAT (GABA-T) mRNA, and the hepatic GABA transporter ( SLC6A12 ) mRNA as explanatory variables for variations in serum insulin (FIG. 6A) or hepatic insulin sensitivity index (HISI; FIG. 6B), ABAT and SLC6A12 mRNA (FPKMUQ; fragments per kilobase million reads upper quartile) were quantified by RNA-Seq from liver tissue.
  • Single nucleotide polymorphisms (SNPs) in the ABAT promoter are associated with a decreased risk of type 2 diabetes (T2D; FIG. 6C). All data are presented as mean ⁇ SEM.
  • FIGs. 7A-7H show glucose homeostasis in obese male mice treated with the GABA-Transaminase inhibitor ethanolamine-O-sulfate (EOS; 3 g/L in drinking water).
  • EOS effects on serum insulin (FIG. 7A), glucose (FIG. 7B), and glucose:insulin ratio (FIG. 7C) pre-treatment and after 4 days of treatment.
  • Oral glucose tolerance (OGTT; FIG.7D) OGTT area under the curve (OGTT AUC; E), and oral glucose stimulated insulin (FIG.7F) pre-treatment and after 3 days of treatment.
  • FIGs. 8A-8H show glucose homeostasis in lean male mice treated with GABA-Transaminase inhibitors ethanolamine-O-sulfate (EOS) or vigabatrin (8 mg/day), or phosphate buffered saline (PBS; control).
  • Serum insulin (FIG. 8A), glucose (FIG. 8B), and glucoseiinsulin ratio (FIG. 8C) on treatment day 4.
  • NS non-significant.
  • a b Bars that do not share a common letter differ significantly (P ⁇ 0.05; number below bar denotes n per group). All data are presented as mean ⁇ SEM.
  • FIGs. 9A-9F show chronic hepatic GABA-Transaminase knockdown improves obesity induced metabolic dysfunction.
  • High fat diet-induced obese mice were treated for 4 weeks with a GABA-T targeted or scramble control antisense oligonucleotide (ASO; 12.5 mg/kg IP twice weekly).
  • ASO GABA-T targeted or scramble control antisense oligonucleotide
  • FIG. 9A Body weight during treatment
  • FIG. 9B Basal serum insulin
  • FIG. 9D glucose
  • FOG. 9E glucose:insulin ratio
  • Serum glucagon (FIG. 9F) after 4 weeks of treatment.
  • Number below bar denotes n per group.
  • NS non-significant. All data are presented as mean ⁇ SEM.
  • FIGs. 10A-10K show glucose homeostasis in lean mice treated with the scramble control antisense oligonucleotide (ASO), or 1 of 2 GABA-Transaminase (GABA-T) targeted ASO sequences (GABA-T or GABA-T 2; 12.5 mg/kg IP twice weekly) for 4 weeks.
  • ASO scramble control antisense oligonucleotide
  • GABA-T GABA-Transaminase
  • FIG. 10D glucose (FIG.10E), and glucose:insulin ratio (FIG. 10F) pre-treatment and after 1 , 2, 3, and 4 weeks of treatment.
  • Oral glucose tolerance (OGTT; FIG. 10G) OGTT area under the curve (AUC; FIG.10H), oral glucose stimulated serum insulin (FIG. 101), insulin tolerance (ITT; FIG. 10J), and ITT AUC (FIG. 10K).
  • FIGs. 11A-11K show GABA-Transaminase inhibition improves glucose homeostasis in sham but not vagotomy mice.
  • HFD induced sham operated and hepatic vagotomized mice were treated with the GABA-Transaminase inhibitor ethanolamine-O-sulfate (EOS) (8mg/day) for 5 days.
  • EOS GABA-Transaminase inhibitor ethanolamine-O-sulfate
  • Body weight during treatment (FIG. 11A).
  • Basal serum insulin (FIG. 11 B), glucose (FIG. 11C), and glucoseiinsulin ratio (FIG. 11D) pre-treatment, on treatment day 5, and after a 2-week washout.
  • Oral glucose tolerance in sham mice OGTT; FIG. 11 E), oral glucose tolerance in vagotomized mice (FIG.
  • FIG. 11 F OGTT area under the curve (AUC; FiG. 11G), and glucose stimulated serum insulin (FIG. 11 H) pre-treatment, on treatment day 4, and after a 2-week washout.
  • NS non-significant.
  • a b Bars that do not share a common letter differ significantly within injection treatment (P ⁇ 0.05; number below bar denotes n per group). All data are presented as mean ⁇ SEM.
  • FIGs. 12A-12J show hepatic GABA-Transaminase knockdown mediated improvements in glucose homeostasis are dependent on an intact hepatic vagal nerve.
  • Diet-induced obese hepatic vagotomized and sham operated mice were treated with a GABA-T targeted antisense oligonucleotide (ASO; 12.5 mg/kg IP twice weekly) for 4 weeks.
  • Body weight during treatment (FIG. 12A).
  • Basal serum insulin (FIG. 12B), glucose (FIG. 12C), and glucose.insulin ratio (FIG. 12D) pre-treatment and after 4 weeks of treatment.
  • Serum glucagon (FIG. 12E), oral glucose tolerance (OGTT; FIG.
  • FIGs. 13A-13G show hepatic GABA-Transaminase knockdown does not affect fast induced refeeding or leptin sensitivity.
  • FIGs. 14A-14I show hepatic GABA-T knockdown does not alter energy expenditure in obesity. Energy expenditure, respiratory exchange ratio, and activity level were assessed by Comprehensive Lab Animal Monitoring System (CLAMS) at the UCDavis Mouse Metabolic Phenotyping Center in diet-induced obese mice after 0, 1 , and 4 weeks of GABA-T targeted or scramble control antisense oligonucleotide treatment (ASO; 12.5 mg/kg IP twice weekly). Energy expenditure during the light cycle (FIG. 14A), dark cycle (FIG.14B), and over 24 hours (FIG. 14C). Respiratory exchange ratio (RER) during the light cycle (FIG. 14D) and dark cycle (FIG. 14E). 24 hour water intake (FIG. 14F).
  • CLAMS Comprehensive Lab Animal Monitoring System
  • NS non-significant. All data are presented as mean ⁇ SEM.
  • FIGs 15A-15D show hepatic vagotomy decreases light cycle food intake on HFD, while GABA-Transaminase knockdown normalizes sham mice food intake to vagotomy mice.
  • Cumulative basal light cycle, dark cycle, and daily food intake (FIG. 15A) and cumulative body weight change (FIG.15B).
  • FIGs. 16A-16H show insulin tolerance tests (ITT) presented as raw glucose values.
  • ITT on day 4 of EOS or Vigabatrin (8 mg/day), or PBS treatment in obese mice (FIG. 16A).
  • ITT pre-treatment, on day 4 of oral EOS (3 g/L in drinking water) treatment, and after a 2-week washout period (FIG. 16B).
  • ITT on day 4 of EOS or Vigabatrin (8mg/day), or PBS treatment in lean mice FIG.16C
  • ITT in obese mice after 1 week of control or GABA-T antisense oligonucleotide (ASO) treatment (FIG.16D).
  • ASO GABA-T antisense oligonucleotide
  • FIGs. 17A-17L show hepatic vagotomy protects against diet-induced hyperinsulinemia.
  • Visual operative field for hepatic vagotomy surgeries (FIG. 17 A).
  • Arrow A indicates the hepatic branch of the vagus which was severed to vagotomize mice.
  • Arrow A also indicates the electrode placement to record firing activity of the hepatic vagal afferent nerve (FIG. 17F).
  • Arrow B indicates where the hepatic vagal nerve was cut after securing the electrode to eliminate vagal efferent activity (FIG. 17F).
  • HFD high fat diet
  • FIG. 17B induced weight gain
  • serum insulin FIG. 17C
  • glucose FIG.
  • FIGs. 17C-17E * denotes significance (P ⁇ 0.05) between bars of the same color.
  • FIG. 17F Effect of hepatic vagotomy after 9 weeks of HFD feeding on serum glucagon (FIG. 17G), oral glucose tolerance (OGTT; FIG. 17H), OGTT area under the curve (AUC; FIG. 171), oral glucose stimulated serum insulin (FIG. 17J), insulin tolerance (ITT; FIG. 17K), and ITT AUC (FIG. 17L).
  • NS non-significant. Number below bar denotes n per group. All data are presented as mean ⁇ SEM.
  • FIGs. 18A-18L show acute hepatocyte depolarization depresses hepatic vagal afferent nerve activity and elevates serum insulin.
  • FIGs. 18A-18C 10X magnification
  • GFP green fluorescent protein
  • FIGs. 18E, 18F, 18G, 18H, and 181 show data from albumin-cre and wildtype mice tail-vein injected with an AAV8 encoding liver specific expression of the PSEM89S ligand activated depolarizing channel whose expression is dependent on cre-recombinase.
  • PSEM89S ligand ⁇ 30 mM PSEM89S ligand induced change in hepatocyte membrane potential
  • PSEM89S ligand induced relative change in hepatic vagal afferent nerve activity
  • FIG. 18F Data in FIG. 18F was collected concurrently with data in panel E. Serum insulin (FIG. 18G), glucose (FIG. 18H), and glucosednsulin ratio (FIG. 181) in albumin-cre and wildtype virus injected mice 15 minutes after saline or PSEM89S ligand (30 mg/kg) administration.
  • FIGs. 19A-190 show hepatic hyperpolarization protects against diet-induced metabolic dysfunction.
  • Liver specific expression of the Kir2.1 hyperpolarizing channel in a wildtype mouse ⁇ FIG. 19A; 10X magnification).
  • Barium (BaCI; 50 pM) induced change in hepatocyte membrane potential in Kir2.1 and eGFP (control) expressing mice (FIG. 19B).
  • HFD high fat diet
  • FIG. 19C serum insulin
  • FIG. 19E glucose
  • FIG. 19F glucose.insulin ratio
  • FIG. 19G Regression of body weight and serum insulin concentrations during HFD feeding in Kir2.1 and eGFP mice. Effect of hepatic Kir2.1 expression after 9 weeks of HFD feeding on serum glucagon FIG. 19H), oral glucose tolerance (OGTT; FIG. 19 I), OGTT area under the curve (AUC; FIG. 19J), oral glucose stimulated serum insulin (K; * denotes significance (P ⁇ 0.05) between bars of the same color), insulin tolerance (ITT; FIG. 19L), ITT AUC (FIG. 19M), pyruvate tolerance (PTT; FIG. 19N), and PTT AUC (FIG. 190).
  • NS non-significant. Number below bar denotes n per group. All data are presented as mean ⁇ SEM.
  • FIGs. 20A-20J show hepatic slice GABA Release. Release of GABA (pmol/mg DNA) from hepatic slices (FIG. 20A), hepatic GABA-Transaminase mRNA expression (FIG. 20B), relationship between hepatic GABA release and liver triglyceride concentration (FIG. 20C), hepatic ATP concentration (nmol/g tissue; FIG. 20D), release of GABA in slices incubated with the Na+/K+ ATPase inhibitor, Ouabain (1 mM; FIG.
  • NS non-significant. Number below bar denotes n per group. All data are presented as mean ⁇ SEIVI.
  • FIG. 21 shows the working model of hepatic lipid accumulation induced changes in hepatic metabolism and resulting changes in hepatic vagal nerve signaling to affect insulin secretion and sensitivity.
  • High levels of b-oxidation in the obese liver increase the mitochondrial NADH 2 .NAD + and FADH 2 :FAD + ratios driving succinate to succinate semialdehyde, generating substrate for GABA-Transaminase.
  • GABA-Transaminase produces GABA and a-ketoglutarate, a substrate for aspartate aminotransferase.
  • Increased gluconeogenic flux in obesity drives the mitochondrial export of OAA as malate. The increased GABA release is encouraged by the depolarized membrane in obesity.
  • GABA is co-transported with 3Na + and 1 Cl ⁇ ions, so an increase in intracellular cation concentration (hepatocyte depolarization) encourages GABA export, while a decrease in intracellular cation concentration (hepatocyte hyperpolarization) limits GABA export.
  • Kir2.1 expression induces hepatic K + efflux and hyperpolarization, inhibiting GABA export. Obesity decreases hepatic ATP concentrations, impairing activity of the Na7K + ATPase pump and increasing intracellular Na + concentrations, driving GABA export. This mechanism explains how hepatic lipid accumulation increases hepatic GABA release.
  • OAA oxaloacetate
  • AST aspartate aminotransferase
  • GABA-T GABA-Transaminase
  • a-KG a-ketoglutarate
  • SSADH succinate semialdehyde dehydrogenase.
  • FIGs. 22A-22C show associations between hepatic GABA system and glucoregulatory markers in obese humans. Multivariate regressions including intrahepatic triglyceride % (IHTG%) and the mRNA for the hepatic GABA transporters (Slc6A6, Slc6A8, Slc6A12, and Scl6A12) as explanatory variables for variations in glucose infusion rate during a hyperinsulinemic euglycemic clamp (pMol/kg fat free mass/min; FIG. 22A), and the glucose disposal rate calculated during a hyperinsulinemic-euglycemic clamp (Glucose Rd, % increase; FIG. 22B).
  • RNA-Seq RNA-Seq from liver tissue.
  • SNPs Single nucleotide polymorphisms that cause missense mutations in Slc6A12 or Slc6A13 are associated with an increased incidence of type 2 diabetes (T2D) adjusted for body mass index (BMI). Regression data are presented as mean ⁇ SEM.
  • FIGs. 23A-23J show hepatic Kir2.1 expression alters glucose homeostasis in the lean mouse.
  • NS non-significant. Number below bar denotes n per group. All data are presented as mean ⁇ SEM.
  • FIGs. 24A-24E show glucose homeostasis in Kir2.1 and eGFP control mice at 3 weeks of high fat diet feeding. Effect of hepatic Kir2.1 expression on oral glucose tolerance (OGTT; FIG. 24A), OGTT area under the curve (AUC; FIG. 24B), oral glucose stimulated serum insulin (FIG. 24C), insulin tolerance (ITT; FIG. 24D), and ITT AUC (FIG. 24E).
  • OGTT oral glucose tolerance
  • AUC OGTT area under the curve
  • ITT insulin tolerance
  • FIG. 24E insulin tolerance
  • NS non-significant. Number below bar denotes n per group. All data are presented as mean ⁇ SEM.
  • FIGs. 25A-25D show insulin tolerance tests (ITT) presented as raw glucose values.
  • ITT insulin tolerance tests
  • FIG. 25A ITT in HFD fed sham and vagotomized mice
  • FIG. 25B ITT in Kir2.1 and eGFP control mice on chow diet
  • FIG. 25C ITT in Kir2.1 and eGFP control mice on chow diet
  • FIG. 25C ITT in Kir2.1 and eGFP control mice on chow diet
  • FIG. 25C 9 weeks of HFD feeding
  • ⁇ Denotes the data point is not significantly different from time 0 for that group (P > 0.05). Unless indicated, all other timepoints are significantly different from time 0 within a group of mice.
  • * Denotes significance between groups specified in the panel within a timepoint. All data are presented as mean ⁇ SEM.
  • animal includes but is not limited to a human, mouse, rat, rabbit, dog, cat, pig, chicken, non-human primates, etc.
  • Antisense oligonucleotide refers to a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid. Antisense technology is emerging as an effective means for reducing the expression of specific gene products.
  • Effective Amount refers to a dosage of a compound or a composition effective for eliciting a desired effect. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, mammal, human, etc. Effective amount may vary depending upon body mass of the individual to be treated, the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, depending on the evaluation, and other relevant factors of a medical condition of an individual varies between individuals obtain.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow the development of a disease or condition, such as slow down the development of obesity, or reducing at least one adverse effect or symptom of a condition, disease or disorder, e.g., any disorder characterized by insufficient or undesired function.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced as that term is defined herein.
  • a treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or decrease of markers of the disease, but also a cessation or slowing of progress or worsening of a symptom that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already diagnosed with a condition, as well as those likely to develop a condition due to genetic susceptibility or other factors such as weight, diet and health.
  • the present invention features compositions and methods for altering hepatic GABA reuptake and/or release to treat obesity and obesity-related conditions, including hyperphagia, hypertension, insulin resistance, and hyperinsulinemia.
  • compositions e.g., compounds, drugs, molecules, e.g , siRNA, etc.
  • the present invention features methods for treating obesity-related complications using compositions (e.g., compounds, drugs, molecules, e.g., siRNA, etc.) that inhibit the activity or expression of (or silence) GABA-transaminase, hepatic succinate semialdehyde dehydrogenase, TauT (protein encoded for by SLC6A6), CRT (protein encoded for by SLC6A8), the like, or a combination thereof.
  • they may increase BGT1 (protein encoded for by SLC6A12) and/or GAT2 (protein encoded for by SLC6A13) to encourage GABA re-uptake.
  • the present invention also features methods for treating obesity-related complications by hyperpolarizing liver cells or by preventing obesity induced depolarization of liver cells. This changing membrane potential will alter the activity of the GABA transporters.
  • the compositions of the present invention improve insulin sensitivity and glucose clearance, decrease blood glucose and insulin concentrations, and/or decrease/normalize blood pressure.
  • the present invention is for inhibiting hepatic GABA release; increasing hepatic aspartate release; hyperpolarizing the hepatocyte/preventing the obesity induced depolarization of the hepatocyte and GABA transporter mediated release, while encouraging GABA transporter mediated GABA re-uptake; preventing GABA signaling on the hepatic vagal afferent nerve; increasing Aspartate signaling on the hepatic vagal afferent nerve; blocking muscarinic 3 receptor signaling on the beta and alpha cell; blocking pancreatic parasympathetic efferent signaling; increasing muscarinic receptor signaling on endothelial cells in the vasculature to limit vasoconstriction/encourage vasodilation; enhancing skeletal muscle parasympathetic efferent signaling; and the like.
  • the present invention features methods of treating obesity or an obesity-related condition in a subject in need thereof.
  • the method comprises administering to the subject a therapeutic amount of a composition for increasing hepatic GABA re-uptake and/or decreasing hepatic GABA release, wherein increasing hepatic GABA re-uptake or decreasing hepatic GABA release decreases blood glucose and improves insulin sensitivity.
  • the composition prevents obesity-induced depolarization of hepatocytes.
  • the composition normalizes blood pressure.
  • the composition reduces hepatic mitochondrial uncoupling.
  • the composition comprises an inhibitor of GABA-T. In certain embodiments, the composition comprises an activator of BGT1. In certain embodiments, the composition comprises an activator of GAT2. In certain embodiments, the composition comprises an inhibitor of M3R for inhibiting insulin release. In certain embodiments, the composition comprises an activator of M3R for improving insulin sensitivity and stimulating insulin release. In certain embodiments, the composition comprises an inhibitor of UCP2. In certain embodiments, the composition comprises an inhibitor of hepatic succinate semialdehyde dehydrogenase. In some embodiments, the composition comprises an inhibitor of GHB production. In some embodiments, the composition comprises an inhibitor of GHB conversion to succinate semialdehyde (SSA). In some embodiments, the composition comprises a GHB dehydrogenase inhibitor.
  • the composition is a drug, a compound, or a molecule.
  • the molecule is an anti-sense oligonucleotide.
  • the composition inhibits GABA signaling on the hepatic vagal afferent nerve.
  • the obesity-related condition is diabetes, hyperglycemia, insulin resistance, glucose intolerance, or hypertension.
  • the composition causes a fasting blood glucose of 120 mg/dL or less. In certain embodiments, the composition causes a fasting blood glucose of 110 mg/dL or less. In certain embodiments, the composition causes a fasting blood glucose of 100 mg/dL or less. In certain embodiments, the composition causes a fasting blood glucose of 90 mg/dL or less. In certain embodiments, the composition causes a fasting blood glucose from 90 mg/dL to 100 mg/dL. In certain embodiments, the composition causes a fasting insulin level of 5 mmol/mL or less. In certain embodiments, the composition causes a fasting insulin level of 10 mmol /mL or less. In certain embodiments, the composition causes a fasting insulin level from 2 to 10 mmol/mL.
  • the composition comprises ethanolamine-O-sulfate (EOS). In certain embodiments, the composition comprises vigabatrin. In certain embodiments, the composition does not cross the blood-brain barrier. In certain embodiments, the composition comprises a derivative of vigabatrin or EOS that does not cross the blood-brain barrier.
  • EOS ethanolamine-O-sulfate
  • the present invention also features methods for improving insulin sensitivity in a subject in need thereof.
  • the method comprises administering to the subject a therapeutic amount of a composition for increasing hepatic GABA reuptake or decreasing hepatic GABA release, wherein increasing hepatic GABA reuptake or decreasing hepatic GABA release improves insulin sensitivity.
  • the composition restores insulin sensitivity to that of a non-diabetic individual.
  • the present invention also features methods for improving insulin sensitivity and limiting hyperinsulinemia in a subject in need thereof.
  • the method comprises administering to the subject a therapeutic amount of a composition for decreasing hepatic GABA synthesis or hepatic GABA release, wherein decreasing hepatic GABA synthesis or release improves insulin sensitivity and decreases hyperinsulinemia.
  • the obesity-related condition is diabetes, hyperglycemia, insulin resistance, glucose intolerance, excess body adiposity, excessive food intake, or hypertension.
  • the present invention also features a pharmaceutical composition for treating an obesity-related condition, wherein the composition is effective to decrease blood glucose, decrease blood insulin, improve insulin sensitivity, increase glucose tolerance, and decrease/normalize blood pressure or a combination thereof.
  • the composition comprises an inhibitor of beta-cell M3R for inhibiting insulin release.
  • the composition comprises an activator of endothelial cell M3R for improving insulin sensitivity and stimulating insulin release.
  • the composition comprises an inhibitor of UCP2.
  • the composition comprises an inhibitor of hepatic succinate semialdehyde dehydrogenase.
  • the composition comprises an inhibitor of GHB production.
  • the composition comprises an inhibitor of GHB conversion to succinate semialdehyde (SSA).
  • the composition comprises a GHB dehydrogenase inhibitor.
  • the composition comprises ethanolamine-O-sulfate (EOS). In certain embodiments, the composition comprises vigabatrin. In certain embodiments, the composition does not cross the blood-brain barrier. In certain embodiments, the composition comprises a derivative of vigabatrin or EOS that does not cross the blood-brain barrier.
  • EOS ethanolamine-O-sulfate
  • the hepatic GABA transporters are electrogenic and comprising members of the Na + /CI -dependent neurotransmitter transporter (SLC6) family, wherein the members comprise proteins encoded for by Slc6A12 (Betaine GABA transporter 1, BGT1), Slc6A13 (GABA transporter 2, GAT2), Slc6A6 (Taurine Transporter, TauT), and Slc6A8 (Creatine transporter, CRT) BGT1 and GAT2 both co-transport 3 Na + , 1 Cl and GABA, moving 2 positive charges in the direction of GABA transport.
  • SLC6A12 Betaine GABA transporter 1, BGT1
  • Slc6A13 GABA transporter 2, GAT2
  • Slc6A6 Taurine Transporter, TauT
  • Slc6A8 Creatine transporter, CRT
  • the GABA transporter moves a novel hepatokine, GABA, that is dysregulated in obesity and whose release can be manipulated to mute or exacerbate the glucoregulatory dysfunction common to obesity.
  • composition comprises an inhibitor of GABA-T or ABAT gene expression, an activator of BGT1 activity and/or expression or an activator of SLC6A12 expression, , an activator of GAT2 activity and/or expression or an activator of SLC6A13 expression, an inhibitor of TauT activity and/or expression or an inhibitor of SLC6A6 expression, an inhibitor of CRT activity and/or expression or an inhibitor of SLC6A8 expression, an inhibitor of Beta-cell M3R for inhibiting insulin release or an activator of endothelial cell M3R to improve insulin sensitivity or Beta-cell M3R to stimulate insulin release.
  • mice All animal studies were conducted using male wildtype C57BL/6J mice purchased from Jackson Laboratories or bred in-house (Bar Harbor, ME). Mice were kept on a 14-hour light/10-hour dark schedule and housed 3-5 mice per cage until 1 week prior to study initiation, at which point animals were individually housed. Studies were conducted in lean chow fed mice (7013 NIH-31, Teklad Wl, 3.1 kcal/g, 18% kcal from fat, 59% kcal from carbohydrate, 23% kcal from protein) at 12-16 weeks of age.
  • mice treated with ethanolamine-O-sulfate in their drinking water were treated with GABA-T targeted or scramble control antisense oligonucleotides (ASO) were performed after 8-10 weeks on a high fat diet (TD 06414, Teklad Wl, 5.1 kcal/g, 60.3% kcal from fat, 21.3% kcal from carbohydrate, 18.4% kcal from protein; 20-26 weeks of age).
  • mice were stratified by body weight and assigned to an injection treatment (control or GABA-T).
  • studies in obese vagotomy and sham mice were performed after 9 weeks of high fat diet feeding.
  • mice Unless fasted, mice had ad libitum access to food and water. All studies were approved by The University of Arizona Institutional Animal Care and Use Committee. [0089] Wildtype lean or diet-induced obese mice received twice-weekly intraperitoneal injections (12.5 mg/kg; 0.1 mL/10 g body weight) of murine GABA-Transaminase (GABA-T) targeted antisense oligonucleotides (ASO; iONIS 1160575) or scramble control ASO (lONIS 549144) for 1 or 4 weeks prior to experimentation. The control ASO does not have complementarity to known genes and was employed to demonstrate the specificity of target reduction.
  • GABA-T murine GABA-Transaminase
  • ASO scramble control ASO
  • Wildtype lean and obese mice were randomly divided into treatment groups and dosed daily with 8 mg of ethanolamine-O-sulfate (EOS; Sigma-Aldrich, St. Louis, MO), vigabatrin (United States Pharmacopeia, Rockville, MD) or PBS.
  • EOS ethanolamine-O-sulfate
  • vigabatrin United States Pharmacopeia, Rockville, MD
  • PBS ethanolamine-O-sulfate
  • Obese sham and vagotomy mice were dosed daily with 8 mg of EOS.
  • basal bleeds were taken prior to initiation of an ITT.
  • Lean mice received treatment by oral gavage (0.3 mL/mouse) while obese mice were treated by intraperitoneal injection (0.3 mL/mouse). Pre-treatment studies were conducted in the week immediately prior to beginning drug administration.
  • EOS was provided ad libitum in the water (3 g/L) for 4 days.
  • An OGTT and ITT were performed on days 3 and 4 of treatment, respectively.
  • the water was then removed, and an ITT was performed 2 weeks later to establish the timing of restoration of insulin resistance after drug removal.
  • mice were housed on wood chip bedding (Harlan Laboratories; Cat #7090 Sani-Chips) to limit consumption of nutrients from bedding during the fasting period. Mice were fasted for 16 hours beginning at 5pm and food was returned at 9am. Food and body weight were measured at 10am, 11am, and 1pm to determine 1, 2, and 3-4 hour fast-induced refeeding.
  • mice or diet-induced obese control or GABA-T targeted ASO treated mice received an intraperitoneal injection of phosphate buffered saline (PBS; 0.1 mL/10 g body weight) on day 1 and leptin (2 mg/kg; 0.1 mL/10 g body weight; CAT# 498-OB, R&D Systems, Minneapolis, MN) on day 2 at 6am. Mice were not fasted before injections. Food and body weight were measured every day at 6am and 6pm.
  • PBS phosphate buffered saline
  • Body composition was assessed under isoflurane anesthesia by DEXA after each CLAMS run. Animals were allowed to recover from anesthesia before returning to their home cage except for the final (3rd) DEXA run, after which animals were terminated and no tissues collected. Animals were acclimated to the CLAMS cages for 48 hours and to the light and temperature-controlled chamber for 24 hours prior to testing. Animals were held and calorimetry data was collected for 48 hrs. Analyzed data constitutes data collected from 48 hours of continuous measurement (2 light/2 dark cycles). Oxygen consumption and carbon dioxide production were measured and used to calculate energy expenditure (or heat production, kilocalories (kcal)) and respiratory exchange ratio (RER: VC02/V02).
  • Cage-mounted sensors detected and recorded measurements of physical activity, food intake and water intake.
  • Body composition was measured by dual-energy X-ray absorptiometry under isoflurane anesthesia, using a Lunar PIXImus II Densitometer (GE Medical Systems, Chalfont St. Giles, UK) immediately after completion of the indirect respiration calorimetry measurements.
  • Body composition was assessed in diet-induced obese mice on day 0 and 7 days after continuous provision of EOS in the drinking water ⁇ 3 g/L) using an EchoMRI 900 with A10 insert for mice. Calibration was performed daily and had the water stage set to on.
  • Clamps were performed as previously described 46. Briefly, one week prior to the experiment, diet-induced obese mice underwent surgical catheterization of the jugular vein under isoflurane anesthesia. Mice were given a single post-operative dose of slow release formulated buprenorphine analgesic (1.2 mg/kg slow release, sub-cutaneous) and food and body weight were assessed daily for 1 week. The same day following surgery completion mice received their first injection of either the scramble control or GABA-T targeted ASO (12.5 mg/kg; 0.1 mL/10 g body weight). Four days post-surgery mice received a second ASO injection, and clamps were performed 7 days after catheterization.
  • mice were infused with 3-[3HJ-D-glucose (0.05 pCi/pl in saline) at a rate of 10 pL/min for 2 minutes and then decreased to a rate of 1 pL/min for the remaining 90 minutes. All blood was collected from the tip of the tail in heparinized capillary tubes and immediately spun down to collect plasma for tracer and insulin analysis.
  • mice were given a bolus of 14C-2-deoxyglucose (12 pCi in 48 pL followed by a 100 pL saline flush) after the 120min timepoint. Blood samples were collected 2, 10, and 25 minutes following the bolus.
  • mice were anesthetized with isoflurane using the bell-jar method and the soleus, quadricep, calf, white adipose tissue, and perirenal adipose tissue were collected and immediately frozen in liquid nitrogen for analysis of tissue specific glucose uptake.
  • Plasma samples were deproteinized using barium hydroxide and zinc sulfate and tracer analysis and tracer standards was processed as described 46.
  • Plasma glucose was analyzed by colorimetric assay (Cat. # G7519, Pointe Scientific Inc., Canton Ml) from the samples collected for tracer measurement.
  • Plasma insulin was analyzed by enzyme-linked immunosorbent assay (ELISA; Cat. # 80-INSMSU-E01 , E10, Alpco, Salem, NH).
  • Oral glucose (2.5 g/kg; 0.1 mL/10 g body weight; Chem-lmpex Int’l Inc., Wood Dale, IL) was given to 4 hour fasted individually housed mice. All glucose tolerance tests began at 1 pm and glucose was measured in whole blood, collected from the tail vein, by glucometer (Manufacture # D2ASCCONKIT, Bayer, Leverkusen, Germany) at 0, 15, 30, 60, 90, and 120 minutes after glucose gavage. Blood for serum insulin (oral glucose stimulated insulin secretion; OGSIS) and glucose determination was collected from the tail vein 15 minutes following glucose administration.
  • OGSIS oral glucose stimulated insulin secretion
  • Intraperitoneal insulin (0.5 U/kg; 0.1 mL/10 g body weight; Sigma Aidrich, St. Louis, MO) was given to 4 hour fasted individually housed mice. All insulin tolerance tests began at 1 pm and glucose was measured in whole blood, collected from the tail vein, by glucometer (Manufacture # D2ASCCONKIT, Bayer, Leverkusen, Germany) at 0, 30, 60, 90, and 120 minutes after insulin injection.
  • mice Liver slices from ASO treated mice were incubated ex vivo to measure hepatic GABA release.
  • a peristaltic pump perfusion system was used to deliver warmed KH buffer to the liver through the portal vein. Briefly, mice were anesthetized with an intraperitoneal injection of ketamine (10 mg/mL) and diazepam (0.5 mg/mL). Once mice were unresponsive, an incision in the lower abdomen through the skin and peritoneal membrane was made vertically through the chest along with transverse incisions on both sides to expose the liver. A 30-gauge needle was inserted into the hepatoportal vein to blanch the liver. The inferior vena cava was cut to relieve pressure in the circulatory system and allow blood to drain.
  • liver slices were taken from each mouse. Tissue slices were placed individually into a well on a 12-well plate pre-filled with 1mL of KH buffer that had been sitting in an incubator set to 37°C and gassed with 5% C02. Liver slices were incubated in the initial well for 1 hour to stabilize before being transferred to a fresh well pre-filled with KH buffer. After 1 hour in the second well, tissue and media were collected. Liver slice samples and KH media samples from both wells of each mouse were pooled.
  • Liver slices were snap frozen in liquid nitrogen, while media was centrifuged for 5 minutes at 10,000xg at 4°C to remove tissue debris and both were frozen and stored at -80°C pending analysis.
  • the media was thawed collected from the ex vivo hepatic slice culture on ice. GABA was then measured in the supernatant using a commercially available ELISA (REF# BA E-2500, Labor Diagnostika Nord, Nordhorn, Germany) pmol GABA concentrations were corrected for liver slice DNA concentrations.
  • 3H-2-deoxy-D-glucose 2DG; 10 uCi/mouse; PerkinElmer, Waltham, MA
  • 2DG was given by oral gavage in a solution of glucose (2.5 g/kg) and each mouse received the same dose based off the average body weight for their cohort (0.1 mL/10 g body weight). Mice were anesthetized by isoflurane and sacrificed by cervical dislocation 45 minutes following oral gavage.
  • Liver, soleus, quadriceps femoris, and gonadal white adipose tissue were collected, weighed, and dissolved overnight in 1N NaOH (0.5 mL/50 mg tissue) at 55°C on a shaker plate.
  • 0.5 mL of dissolved tissue was added to 5 mL of scintillation cocktail (Ultima Gold, PerkinElmer, Waltham, MA) and disintegrations per minute (DPM) were measured using a LS 6500 Multipurpose Scintillation Counter (Beckman Coulter, Brea, CA).
  • DPM/g tissue weight was determined for each tissue and normalized based on a correction factor calculated by the average total DPM/g for all tissues divided by the total DPM/g for all tissues of the individual mouse.
  • mice were sacrificed and the quadricep and soleus tissues were collected and frozen on dry ice. Prior to analysis, frozen quadricep were powdered using a liquid nitrogen cooled mortar and pestle to obtain homogenous muscle samples. 15-20 mg of quadricep and the entire soleus tissue (6-12 mg) were homogenized in 200 pL of a 5% trichloroacetic acid solution. Following 15 minutes of centrifugation at 3,000xg at 4°C, supernatant was transferred to a fresh tube for analysis of muscle cGMP by enzyme-linked immunosorbent assay (ELISA; ADI-900-164, Enzo Life Sciences, Farmingdale, NY).
  • ELISA enzyme-linked immunosorbent assay
  • livers Prior to analysis, frozen livers were powdered using a liquid nitrogen cooled mortar and pestle to obtain homogenous liver samples. To measure liver DNA content (ng dsDNA/g tissue), 10-20 mg of powdered liver was sonicated in 500 pL DEPC H20 and dsDNA determined by fluorometric assay (Cat. # P7589, Invitrogen, Waltham, MA). Whole liver and hypothalamic mRNA were isolated from powered liver samples with TRI Reagent® (Life Technologies, Grand Island, NY) and phenol contamination was eliminated by using water-saturated butanol and ether as previously described 47.
  • TRI Reagent® Life Technologies, Grand Island, NY
  • cDNA was synthesized by reverse transcription with Verso cDNA synthesis kit (Thermo Scientific, Inc., Waltham, MA), and qPCR performed using SYBR 2X mastermix (Bio-Rad Laboratories, Hercules, CA) and the Biorad iQTM5 iCycler (Bio-Rad Laboratories, Hercules, CA).
  • GABA-Transaminase ABAT
  • ACT b-actin
  • insulin Ins
  • NPY neuropeptide Y
  • AgRP agouti related peptide
  • POMC pro-opiomelanocortin
  • LinReg PCR analysis software was used to determine the efficiency of amplification from raw output data.
  • AOTb served as the reference gene for liver and brain tissue, and ins served as the reference gene for pancreas tissue for calculating fold change in ABAT gene expression using the efficiency-AACt method.
  • HECP in conjunction with stable isotopically labelled glucose tracer infusion
  • CTL Clinical Translational Research Unit
  • This procedure was performed to determine: i) hepatic insulin sensitivity, which was assessed as the product of the basal endogenous glucose production rate (in pmoi-kg fat-free mass (FFM)-1 -min-1) and fasting plasma insulin concentration (in mU/L).
  • Liver tissue was obtained by needle biopsy during the bariatric surgical procedure, before any intra-operative procedures were performed. Liver tissue was rinsed in sterile saline, immediately frozen in liquid nitrogen, then stored at -80°C until RNA extraction. Total RNA was isolated from frozen hepatic tissue samples by using Trizol reagent (Invitrogen, Carlsbad, CA). Library preparation was performed with total RNA and cDNA fragments were sequenced on an lllumina HiSeq-4000. The fragments per kilobase million reads upper quartile (FPKM-UQ) values were calculated and used for further gene expression analyses. All RNA-seq data used in this study have been deposited into the NCBI GEO database under accession number GSE144414.
  • the main effect was treatment (PBS, Vigabatrin, or EOS).
  • EOS treated sham and vagotomy mice the main effects were surgery (sham or vagotomy) and treatment (PBS or EOS).
  • Pre-, during, and post-treatment measures were taken for basal glucose, insulin, and glucose stimulated insulin, thus a repeated measure analysis including time (pre-, during, or post-treatment) was performed separately within each injection or surgical treatment.
  • Pre-, during, or post-treatment was performed separately within each injection or surgical treatment.
  • analysis of the effect of EOS on 2DG uptake and cGMP analysis was performed separately for each tissue.
  • the main effect was injection treatment (control of GABA-T ASO).
  • the vagotomy analyses the main effect was surgery (sham or vagotomy).
  • Pre-treatment and ASO week 4 measures were taken for basal glucose, insulin, and the glucose:insulin ratio, thus a repeated measure analysis including time (pre-, or week 4-treatment) was performed separately within each injection or surgical treatment.
  • a multivariate regression analysis was performed on data from human clinical patients using IHTG%, ABAT mRNA, and SLC6a12 mRNA as explanatory variables for variations in serum insulin, HOMA-IR, M-Value, and Glucose Rd.
  • Statistics performed by the UC Davis Mouse Metabolic Phenotyping Center are described as follows: data are presented as means ⁇ SEM and a Student t-test was used to test for significant differences between groups.
  • GABA-T inhibitors improved insulin sensitivity assessed by insulin tolerance test (ITT) within 4 days of initiating treatment (FIG. 11-1 J).
  • ITT insulin tolerance test
  • EOS provided in the drinking water (3 g/L) for 4 days.
  • EOS in the drinking water similarly improved measures of glucose homeostasis and the response washed out after 2 weeks without EOS in the drinking water (FIG. 7A-7H).
  • tissue specific 3 H-2-deoxy-D-glucose (2DG) uptake was measured following an oral glucose gavage on day 5 of EOS or saline treatment.
  • EOS treatment increased 2DG uptake by the soleus (22%) but did not affect 2DG clearance by the quadriceps femoris (quad) or gonadal white adipose tissue (WAT; FIG. 1K).
  • cGMP was subsequently measured.
  • cGMP is a key second messenger downstream of nitric oxide (NO) signaling that regulates blood flow.
  • EOS increased cGMP in the soleus (59%) but had no effect in quad (FIG. 1L).
  • an ASO model was next used to specifically knockdown hepatic GABA-T expression.
  • Peripherally administered ASOs do not cross the blood brain barrier. Outside the central nervous system, the liver and pancreas express the most GABA-T.
  • a GABA-T targeted ASO (12.5 mg/kg IP twice weekly) decreased hepatic GABA-T mRNA expression by > 98% within 1 week.
  • this GABA-T targeted ASO did not affect pancreatic or whole-brain GABA-T mRNA expression (FIG. 2A).
  • ex vivo liver slice GABA release was measured.
  • GABA-T knockdown cut obesity induced liver slice GABA release by 61% (FIG. 2B).
  • One week of GABA-T knockdown in obese mice did not affect body weight but decreased basal serum insulin and glucose concentrations and elevated the glucose:insulin ratio (FIGs. 2C-2F).
  • GABA-T targeted ASO injections also improved oral glucose clearance without affecting oral glucose stimulated serum insulin concentrations (FIGs. 2G-2I), and improved insulin sensitivity compared to scramble control ASO injected mice (FIGs. 2J-2K).
  • GABA-T knockdown did not affect the rate of endogenous glucose appearance (Ra) during the basal or clamp periods (FIG. 3E).
  • the basal rate of glucose disappearance (Rd) did not differ between ASO treatments.
  • hyperinsulinemia during the clamp nearly doubled Rd in GABA-T knockdown but not control mice (FIG. 3F).
  • mice received a bolus of 14 C-2-dexyglucose and were sacrificed 30 minutes later to assess tissue specific glucose uptake.
  • GABA-T knockdown improved glucose uptake by the soleus, quadricep, and calf skeletal muscles (FIG. 3G).
  • Hepatic vagotomy prevents the liver from altering afferent signaling to the brain, without affecting normal vagal afferent input originating from the nodose. In turn, vagotomy prevents inhibition of the vagal afferent tone by GABA produced In the liver. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that hepatic GABA mediates the effects that are reported herein by acting on the HVAN to induce hyperinsulinemia and insulin resistance. Accordingly, the effect of EOS treatment was assessed in HFD-induced obese hepatic vagotomized and sham operated mice. Although body weight did not differ between surgical groups during EOS treatment (FIG.
  • EOS the response to EOS was dependent on an intact hepatic vagal nerve.
  • EOS decreased serum insulin and glucose, elevated the glucose.insulin ratio, improved oral glucose tolerance, diminished glucose stimulated serum insulin concentrations, and tended to improve insulin sensitivity as measured by an insulin tolerance test (FIGs. 11B-11K). Washout restored all parameters to pre-treatment measures. Vagotomy eliminated most of the responses to EOS (FIGs. 11B-11K). Since hepatic GABA production supports gluconeogenic flux (FIG. 21), it was expected GABA-T inhibition to decrease gluconeogenesis through direct actions at the liver. In turn, diminished hepatic glucose output explains the vagal nerve independent decrease in serum glucose during EOS treatment in hepatic vagotomized mice (FIG. 11C).
  • GABA-T knockdown (12.5 mg/kg GABA-T targeted ASO IP twice weekly) was assessed in diet-induced obese sham operated and hepatic vagotomized mice. Consistent with previous observations, pre-treatment body weight was lower in obese, vagotomized mice compared to sham operated control mice (FIG. 12A). As shown with pharmacological GABA-T inhibition (EOS), GABA-T knockdown decreased basal serum glucose concentrations independent of the hepatic vagal nerve (FIG. 12C), again likely due to reduced hepatic glucose output.
  • EOS pharmacological GABA-T inhibition
  • GABA-T knockdown decreased basal serum insulin, improved oral glucose tolerance, limited oral glucose stimulated serum insulin, and improved insulin sensitivity in sham, but not in vagotomized mice (FIGs. 12B, 12E-12J).
  • Hepatic vagotomy protects against obesity-induced insulin resistance GABA-T knockdown allowed sham operated animals to achieve similar glucose tolerance, glucose stimulated serum insulin, and insulin sensitivity to that measured in hepatic vagotomized mice.
  • GABA-T knockdown may decrease food intake or increase energy expenditure. Accordingly, daily food intake and body mass were assessed during the light and dark cycle for the first 2 weeks of ASO treatment in lean and obese mice. In lean mice, GABA-T knockdown did not affect cumulative light cycle, dark cycle, or daily food intake (FIG. 4A). Cumulative daily body mass change was also not affected by GABA-T knockdown in lean mice (FIG. 4B). In obese mice, GABA-T knockdown decreased cumulative light cycle, dark cycle, and daily food intake (FIG. 4C).
  • GABA-T knockdown did not alter food intake in response to a 16-hour fast in either lean or obese mice (FIGs. 13A-13B).
  • GABA-T knockdown did not affect fasting mRNA expression of the canonical hypothalamic neuropeptides regulating food intake (NPY, AgRP, and POMC; FIG. 13C). It cannot be ruled out an effect of central GABA-T knockdown as a 14% reduction was observed in hypothalamic GABA-T expression at 7 weeks of GABA-T ASO injections (FIG. 13C).
  • the anorexigenic hormone leptin induces satiety and weight loss, while leptin resistance in obesity contributes to hyperphagia and weight gain.
  • GABA-T knockdown in obesity may have improved leptin sensitivity as a potential was tested as a mechanism to decrease appetite and cause weight loss.
  • a single 6AM injection of leptin (2 mg/kg) did not affect food intake in mice on either ASO treatment at any timepoint, suggesting that mice on both treatments remained leptin resistant (FIG. 13D). Consistent with the decreased food intake previously observed in response to GABA-T knockdown, GABA-T knockdown decreased light cycle, dark cycle, and 24-hour food intake independent of injection (FIG. 13D).
  • Body composition assessed by Dual-Energy X-ray Absorptiometry (DEXA), revealed that 4 weeks of GABA-T knockdown decreased total body mass and fat mass without affecting lean mass (FIGs. 5A-5C).
  • the decreased fat mass with GABA-T knockdown suggests that weight loss induced by limiting hepatocyte GABA production reflects a loss of adiposity.
  • Body composition was additionally assessed on day 0 and 7 of providing obese mice with EOS in their drinking water (3 g/L). It was found that EOS treatment decreased total body mass (10%), fat mass (16.27%), and lean mass (6.15%). Although there was a small loss of lean mass, diminished fat mass contributes the majority of lost body mass with EOS treatment.
  • Hepatic vagotomy decreased weight gain on a high fat diet.
  • hepatic vagotomy decreases 1-week cumulative light cycle food intake by 22% in diet-induced obese mice, resulting in a 5.3% decrease in cumulative 24-hour food intake (FIG. 15A).
  • Daily food intake measurements were continued for the next 2 weeks as all mice were treated with the GABA-T targeted ASO.
  • GABA-T knockdown the previously observed difference in food intake in sham operated and vagotomized mice was eliminated.
  • cumulative light cycle, dark cycle, and daily food intake were similar in sham operated and vagotomized mice (FIG. 15B).
  • the GABA-T ASO resulted in a greater cumulative week 4 body mass loss in sham operated than in vagotomized mice which experienced no net change in body mass (FIG. 15D).
  • the glucoregulatory, phagic, and body mass changes in response to GABA-T knockdown all appear to be dependent on the hepatic vagal afferent nerve.
  • ABAT encodes for GABA-T
  • GABA transporter SLC6A6 , encodes for taurine transporter, TauT
  • SLC6A8 encodes for the creatine transporter, CRT
  • SLC6A12 encodes for the Betaine-GABA Transporter 1 , BGT1, and SLC6A13 encodes for GABA Transporter 2, GAT2
  • IHTG intrahepatic triglyceride
  • MRI magnetic resonance imaging
  • HECP hyperinsulinemic-euglycemic clamp procedure
  • HISI hepatic insulin sensitivity index
  • SNPs Single Nucleotide Polymorphisms in the promoter of the ABAT gene, which encodes for GABA transaminase, are associated with a decreased odds ratio (OR) for type 2 diabetes (T2D; Source: knowledge portal diabetes database). MAP - minor allele frequency.
  • Hepatic GABA production improves insulin sensitivity primarily by increasing skeletal muscle glucose clearance (FIGs. 1K and 3G).
  • the results reported herein propose that some of the improvements in glucose clearance may be mediated by increased blood flow.
  • Vasodilation of the microvasculature accounts for 40% of insulin stimulated muscle glucose uptake.
  • Insulin and acetylcholine signaling at endothelial cells stimulates NO production, which signals to smooth muscle cells inducing cGMP production and vasodilation.
  • the microvascular vasodilatory response is reduced in obesity, directly contributing to systemic insulin resistance.
  • EOS treatment increased soleus muscle cGMP concentrations (FIG. 1L).
  • GABA-T knockdown improves glucose homeostasis independent of the decrease in food intake and body weight.
  • One week of GABA-T ASO treatment does not decrease food intake (FIG. 4E) or body weight (FIGs. 2C and 4F) compared to controls, yet basal serum insulin concentrations, glucose tolerance, and insulin sensitivity are markedly improved (FIGs. 2D, 2G, and 2J).
  • the HVAN has long been implicated in transmitting liver derived signals to the hindbrain to regulate feeding behavior.
  • hepatic portal infusions of glucose, amino acids, and lipids suppress food intake, while more recent studies support that carbohydrate signals originating from the liver regulate feeding behavior through HVAN dependent mechanisms.
  • Peripheral satiation factors including glucagon like peptide-1 (GLP-1), cholecystokinin (CCK), lipids, and leptin all reduce food intake dependent upon increasing gastric and hepatic vagal branch afferent firing, while the orexigenic hormone ghrelin suppresses vagal afferent tone.
  • hepatic vagotomy shifts the diurnal feeding pattern to increase light cycle food intake, potentially mediated by the loss of peripheral light cycle anorexigenic stimuli. Diet-induced obese, hepatic vagotomized mice eat less during the light cycle than sham operated controls, suggesting that hepatic vagotomy protects against the obesity-induced increase in daytime feeding. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that preventing the GABA mediated depression of HVAN activity from reaching the hindbrain not only improves glycemic control but decreases light cycle food intake, explaining the decreased weight gain with HFD feeding in hepatic vagotomized mice. Further supporting a role of hepatic GABA in HFD induced weight gain, it was previously observed that inducing hepatic Kir2.1 expression limited hepatic GABA release and HFD induced weight gain.
  • mice were conducted in lean chow fed mice ⁇ 7013 NIH-31 , Teklad Wl, 3.1 kcal/g, 18% kcal from fat, 59% kcal from carbohydrate, 23% kcal from protein) at 12-16 weeks of age.
  • Studies in diet-induced obese sham and vagotomy mice were performed after 9 weeks on a high fat diet (TD 06414, Teklad Wl, 5.1 kcal/g, 60.3% kcal from fat, 21.3% kcal from carbohydrate, 18.4% kcal from protein; 20-26 weeks of age).
  • the depolarizing channel (PSAM L141FY115F -5HT3HC), originally engineered by Dr. Scott Sternsons group, was made by mutating the acetylcholine binding domain of a chimeric channel that included the binding domain of the a7 nicotinic acetylcholine receptor and the ion pore domain of the serotonin receptor 3a.
  • the ligand binding domain mutations (Leu 141 ® Phe and Tyr 115 Phe) limited the agonist action of acetylcholine and allowed for stimulation by a pharmacologically selective effector molecule PSEM89S.
  • the exogenous ligand PSEM89S opens the serotonin receptor 3a channel allowing Na + , K + , and Ca ++ passage into the cell and membrane depolarization.
  • AAV8 viral vectors were used for plasmid delivery in all the reported studies and were synthesized by the Penn Vector Core. Hepatic specific expression of the depolarizing channel was achieved through two different methods. First, expression of a cre-recombinase dependent depolarizing channel was driven by a globally expressed CAG promoter. LoxP sites limited expression to cre-recombinase expressing tissue, and tail vein injection of 1X10 10 viral genome copies established hepatocyte expression in albumin-cre but not wildtype mice.
  • a separate AAV8 viral vector induced hepatic specific expression of the same depolarizing channel by driving expression using the thyroxine binding globulin (TBG) promoter.
  • TBG thyroxine binding globulin
  • Tail vein injection of 1X10 11 viral genome copies established hepatocyte expression (FiG. 18C).
  • the thyroxine binding globulin promoter also drove hepatic expression of the hyperpolarizing, inward-rectifier K + channel, Kir2.1.
  • Tail vein injection of 1X10 11 viral genome copies established hepatocyte specific expression (FIG. 19A).
  • eGFP enhanced green fluorescent protein
  • mice All studies in virus injected mice were conducted at least 5 days post virus injection to allow for maximal channel expression. Individually housed mice were intraperitoneally injected with the ligand for the depolarizing channel (PSEM89S; 30mg/kg; 0.1ml_/10g body weight) or PBS (0.1mL/10g body weight).
  • PSEM89S the depolarizing channel
  • PBS 0.1mL/10g body weight
  • mice expressing the cre-independent depolarizing channel were expressing the cre-independent depolarizing channel.
  • Mice received an oral glucose gavage (2.5 g/kg) 10 minutes after intraperitoneal injection of the PSEM89S ligand or saline. 15 minutes following glucose administration (25 minutes post treatment injection), blood for serum insulin and glucose determination was collected from the tail vein. All mice received both saline and PSEM89S ligand injection on separate days. These studies were repeated in 2 cohorts.
  • Both nerve and intracellular signals were sent to an A/D converter (Digidata 1322A, Molecular Devices, Sunnyvale, CA), digitized at 20 kHz and viewed on a computer monitor using pCIamp software (version 10.2; Molecular Devices).
  • A/D converter Digidata 1322A, Molecular Devices, Sunnyvale, CA
  • pCIamp software version 10.2; Molecular Devices
  • hepatocyte impalement was determined by an abrupt negative deflection upon penetration of the cell and a stable intracellular potential (-45 to -25 mV for mouse hepatocytes) for at least 2 minutes. If the recording of hepatocyte membrane potential was not stable the electrode was removed and membrane potential was measured on another hepatocyte.
  • a cryostat HM 520 (MICROM International GmbH, Walldorf, Germany) was used to get 10 mM thick slices which were collected onto Superfrost Plus slides. Immunohistochemistry for GFP alone (FIGs. 18A-18C, 24A-24B) was performed as follows: Briefly, slides were washed twice in PBS and twice in PBST (3% Triton in PBS) before being exposed to blocking solution (5% normal goat serum in PBST) for 1 h. Slides were subsequently exposed to a 1:5000 dilution of the primary anti-GFP antibody in blocking solution (Alexa488-conjugated rabbit anti-GFP; Life Technologies, Waltham, MA) for 3 hours at room temperature.
  • the secondary anti-rabbit and anti-goat antibodies were used at a 1 :500 dilution (Alexa56S conjugated donkey anti-rabbit A10042 and alexa488 conjugated donkey anti-goat A32814; Thermo Fisher, Waltham, MA). Images were collected by fluorescent microscopy (Leica DM5500B, Leica Microsystems, Wetzlar, Germany), captured using HCImage Live, and formatted in Image-Pro Premier 9.2. 10X magnification was used to ensure a wide field of vision and accurate assessment of degree of expression. 20X magnification was used to image co-staining for GFP and arginase-1.
  • Oral glucose (2.5g/kg; 0.1mL/10g body weight; Chem-lmpex Int’l Inc., Wood Dale, IL) was given to 4 hour fasted individually housed mice. All glucose tolerance tests began at 1 pm and glucose was measured in whole blood, collected from the tail vein, by glucometer (Manufacture # D2ASCCONKIT, Bayer, Leverkusen, Germany) at 0, 15, 30, 60, 90, and 120 minutes after glucose gavage. Blood for serum insulin (oral glucose stimulated insulin secretion; OGSIS) and glucose determination was collected from the tail vein 15 minutes following glucose administration.
  • OGSIS oral glucose stimulated insulin secretion
  • Intraperitoneal insulin (0.75U/kg; 0 1mL/10g body weight; Sigma Aldrich, St. Louis, MO) was given to 4 hour fasted individually housed mice. All insulin tolerance tests began at 1 pm and glucose was measured in whole blood, collected from the tail vein, by glucometer (Manufacture # D2ASCCONKIT, Bayer, Leverkusen, Germany) at 0, 30, 60, 90, and 120 minutes after insulin injection.
  • Intraperitoneal sodium pyruvate (1.5g/kg; 0.1mL/10g body weight; Alfa Aesar, Ward Hill, MA) was given to 16 hour fasted individually housed mice. Mice were switched to wood chip bedding (Harlan Laboratories; Cat. # 7090 Sani-Chips) at the initiation of the fast. All pyruvate tolerance tests began at 9 am and the rise in glucose was measured in whole blood, collected from the tail vein, by glucometer (Manufacture # D2ASCCONKIT, Bayer, Leverkusen, Germany) at 0, 30, 60, 90, and 120 minutes after pyruvate injection. This is indicative of hepatic gluconeogenic potential from pyruvate.
  • ELISA enzyme-linked immunosorbent assay
  • mice Liver slices from experimental mice were incubated ex vivo to measure release of signaling molecules.
  • a peristaltic pump perfusion system was used to deliver warmed KH buffer to the liver through the portal vein. Briefly, mice were anesthetized with an intraperitoneal injection of ketamine (10mg/mL) and diazepam (0.5mg/mL). Once mice were unresponsive, an incision in the lower abdomen through the skin and peritoneal membrane was made vertically through the chest along with transverse incisions on both sides to expose the liver. A 30-gauge needle was inserted into the hepatoportal vein to blanch the liver. The inferior vena cava was cut to relieve pressure in the circulatory system and allow blood to drain.
  • Liver slices treated with the GABA-T inhibitor EOS were incubated in media containing EOS (5.3 mM) during the second hour of incubation.
  • Liver slices treated with the Na7K + ATPase inhibitor ouabain were incubated in media containing ouabain (1 mM) media was collected after 15 minutes of incubation.
  • Liver slices treated with the GABA transporter inhibitors for BGT1 and GAT2 were incubated in media containing betaine (1 mM) or Nipecotic acid (1 mM) or both during the second hour of incubation.
  • reduced and low NaCI medias respectively, 58 and 103 mM of NaCI were replaced with 116 and 206 mM of mannitol to maintain the osmolarity of the buffer.
  • tissue and media were collected.
  • Liver slice samples and KH media samples from both wells of each mouse were pooled. Liver slices were snap frozen in liquid nitrogen, while media was frozen and stored at -80°C for future analysis.
  • Preliminary media samples were sent to the Mayo Clinic Metabolomics Regional Core for mass spectrophotometry analysis using their neuromodulators panel (Data Table 5).
  • Data Table 5 For all liver slice GABA and aspartate release data, the media collected from the ex vivo hepatic slice culture was thawed on ice and centrifuged for 5 minutes at 10,000xg at 4°C to remove tissue debris. GABA was then measured in the supernatant using a commercially available ELISA (REF# BA E-2500, Labor Diagnostika Nord, Nordhorn, Germany).
  • Aspartate release was measured using liquid chromatography-mass spectrometry. Samples were prepared for analysis by LC-MS/MS using protein precipitation. Twenty mI of each sample and standard curve increment was transferred to 1.5 ml tubes. One hundred eighty pi acetonitrile (ACN) was added to each tube followed by a 5 second vortex. All samples were incubated at 4°C for one hour for precipitation. Samples were then centrifuged at 10,000 RPM for 10 minutes and the supernatant transferred to 300 mI HPLC vials for analysis. The aqueous portion of the mobile phase was buffered using 10 mM ammonium bicarbonate with the pH adjusted to 7.4 using 1M formic acid and ammonium hydroxide.
  • ACN acetonitrile
  • Methanol was used as the organic portion of the mobile phase.
  • the column for separation was a Phenomenex Luna Silica with 5 pm particle diameter and 100 A pore size. Column internal diameter was 4.6 mm and length was 150 mm.
  • a Shimadzu LC10 series HPLC with two dual piston pumps was used for sample injection and solvent delivery. The flow rate was fixed at 300 mI per minute. Aspartate was quantified using an LTQ Velos Pro mass spectrometer. Eluate from the Shimadzu HPLC was ionized using a Thermo ESI source. Source voltage was 6 kV; sheath and auxiliary gas flows were 40 and 20 units respectively. The ion transfer capillary was heated to 300°C.
  • the LTQ Velos Pro was operated in negative SRM mode using two transitions: 132.1 ->115 for quantification and 132.1 ->88.1 as a qualifier. Data integration and quantification were performed using the Thermo Xcalibur software packaged with the LTQ Velos Pro.
  • livers Prior to analysis, frozen livers were powdered using a liquid nitrogen cooled mortar and pestle to obtain homogenous liver samples.
  • liver DNA content ng dsDNA/g tissue
  • 10-20 mg of powdered liver was sonicated in 200pL DEPC H 2 0 and dsDNA determined by fluorometric assay (Cat. # P7589, Invitrogen, Waltham, MA).
  • Fluorometric assay Cat. # P7589, Invitrogen, Waltham, MA.
  • Whole liver mRNA was isolated from powered liver samples with TRI Reagent® (Life Technologies, Grand Island, NY) and phenol contamination was eliminated by using water-saturated butanol and ether as previously described.
  • cDNA was synthesized by reverse transcription with Verso cDNA synthesis kit (Thermo Scientific, Inc., Waltham, MA), and qPCR performed using SYBR 2X mastermix (Bio-Rad Laboratories, Hercules, CA) and the Biorad iQTM5 iCycler (Bio-Rad Laboratories, Hercules, CA). Expression of b-actin (ACTp) and GABA-Transaminase (ABAT) mRNA were measured using primers as described previously (Ramakers et al., 2003). LinReg PCR analysis software was used to determine the efficiency of amplification from raw output data. AOTb served as the reference gene for calculating fold change in gene expression using the efficiency ⁇ 01 method.
  • liver lipids were extracted from powdered liver samples. Briefly, 10-20 mg of sample was sonicated in 100pL PBS. 1 mL of 100% ethanol was added to each sample and vortexed for 10 minutes. Following 5 minutes of centrifugation at 16,000xg at 4°C, supernatant was transferred to a fresh tube for analysis of liver triglycerides (Cat. # T7531, Pointe Scientific Inc., Canton, Ml).
  • Hepatic NADH and NAD were quantified by fluorometric assay (ab176723, Abeam, Cambridge, UK). Hepatic ATP concentrations were assessed as previously described.
  • CTRU Clinical Translational Research Unit
  • Liver tissue was obtained by needle biopsy during the bariatric surgical procedure, before any intra-operative procedures were performed. Liver tissue was rinsed in sterile saline, immediately frozen in liquid nitrogen, then stored at -80°C until RNA extraction. Total RNA was isolated from frozen hepatic tissue samples by using Trizol reagent (Invitrogen, Carlsbad, CA). Library preparation was performed with total RNA and cDNA fragments were sequenced on an lllumina HiSeq-4000. The fragments per kilobase million reads upper quartile (FPKM-UQ) values were calculated and used for further gene expression analyses. All RNA-seq data used in this study have been deposited into the NCBI GEO database under accession number GSE144414.
  • Chronic hepatic vagotomy eliminates the ability of the liver to alter vagal afferent nerve activity.
  • hepatic vagotomy does not prevent otherwise basal signaling of the vagus in the central nervous system.
  • basal signaling at the nucleus of the solitary tract (NTS) is restored within 1 month of surgery.
  • hepatic lipid accumulation drives hyperinsulinemia and insulin resistance by altering HVAN activity. It was examined whether or not obesity-induced insulin dysregulation is dependent on an intact hepatic vagal nerve. It was expected that hepatic vagotomy would mute obesity-induced hyperinsulinemia and insulin resistance. Hepatic vagotomy or sham surgery was performed in lean mice and then provided them a 60% high fat diet (HFD; Teklad, TD 06414) for 9 weeks. The operative field is visualized in FIG. 17A, with arrow A indicating the hepatic vagal branch which was severed to hepatic vagotomize mice (FIG. 17A).
  • HFD 60% high fat diet
  • Vagotomy suppressed weight gain on a HFD starting at week 6 (FIG. 17B). Hepatic vagotomy lowered serum insulin and elevated the glucose:insulin ratio at both 0 and at 9 weeks on the HFD, while decreasing serum glucose concentrations after 9 weeks of HFD feeding (FIG. 17C-17E). For the same increase in body weight during HFD feeding, the rise in serum insulin was greater in sham than vagotomized mice (FIG. 17F). Thus, the vagotomy induced protection against obesity-induced hyperinsulinemia is not due to limited weight gain on the HFD. Serum glucagon concentrations in HFD fed mice were not different between surgical treatments (FIG. 17G).
  • Vagotomy improved oral glucose tolerance at 9 weeks on the HFD, while simultaneously decreasing glucose stimulated insulin concentrations (Figs. 17H-17J) Vagotomy also improved insulin sensitivity in obese mice (FIGs. 17K-17L).
  • An adeno-associated virus serotype 8 (AAV8) encoding this PSEM89S ligand-gated depolarizing channel and green fluorescent protein (eGFP) flanked by LoxP sites to wildtype mice or mice expressing cre-recombinase driven by the albumin promoter was intravenously delivered.
  • This channel will only be expressed in hepatocytes of cre-recombinase expressing mice and activated by PSEM89S ligand.
  • Immunohistochemistry was performed against GFP to confirm liver specific expression in albumin-cre expressing mice and no expression in wildtype mice (FIGs. 18A-18B). No GFP expression was observed in skeletal muscle, pancreas, or adipose tissue of albumin-cre mice.
  • hepatocyte membrane potential and HVAN activity was simultaneously measured in the anesthetized mouse.
  • HVAN firing activity the hepatic vagal nerve was gently lifted and placed onto a hook-shaped electrode (FIG. 17A; arrow A). After the electrode placement was secured, the hepatic vagal nerve to the right of the hook near the esophagus (FIG. 17A; arrow B) was cut to eliminate efferent firing activity.
  • FIG. 17C To induce a chronic hyperpolarized state an AAV8 vector, was used encoding TBG promoter driven expression of the K+ channel, Kir2.1, and eGFP (FIG. 18A). Although this channel is inwardly rectifying in neurons, in hepatocytes, with a resting membrane potential that ranges from -20 to -50 mV, Kir2.1 expression supports K+ efflux and hyperpolarization 19.
  • the hyperpolarizing effect of Kir2.1 was confirmed by in vivo intracellular measurement of hepatocyte membrane potential before and after bath application of the Kir2.1 antagonist, Barium (Ba2+; 50 mM) 19. Ba2+ induced a 6.86 ⁇ 1.54 mV depolarization of hepatocytes in Kir2.1 expressing mice but had no effect (-0.62 ⁇ 1.86 mV) in control eGFP expressing mice (FIG. 19B).
  • hepatocyte hyperpolarization decreased basal serum insulin and glucose concentrations (FIG. 23A-25C), improved glucose clearance (FIG. 23D-25F) and enhanced insulin sensitivity (FIGs. 23G-H).
  • Kir2.1 expression did not affect gluconeogenic potential, as assessed by a pyruvate tolerance test (FIGs. 23I-J). This establishes that hepatocyte membrane potential affects systemic glucose homeostasis in non-disease, non-obese conditions.
  • Kir2.1 expression depressed weight gain on a HFD, reaching significance from weeks 6-9 (FIG. 19C). As observed in lean mice, the beneficial response to hepatocyte hyperpolarization was also observed at 3 weeks on a HFD, when body weight remained similar. At 3 weeks on the HFD, Kir2.1 expression improved glucose clearance without altering glucose stimulated serum insulin (FIGs. 24A-C). Kir2.1 expression tended to improve insulin sensitivity at 3 weeks of HFD feeding (P 0.064; FIGs. 24D-E). Kir2.1 expression limited the rise in serum insulin and glucose in response to 3, 6, or 9 weeks of HFD feeding, and increased the glucose:insulin ratio after 9 weeks on the HFD (FIGs. 19D-19F).
  • Kir2.1 expression limited HFD-induced weight gain, the same increase in body weight led to a greater increase in serum insulin concentration in eGFP control than in Kir2.1 expressing mice (FIG. 19G).
  • Kir2.1 expression decreased serum glucagon in diet-induced obese mice (FIG. 19H).
  • Kir2.1 expression improved glucose tolerance and insulin sensitivity (FIGs. 19I-19M), while having no effect on gluconeogenic potential from pyruvate (FIGs. 19N-190).
  • the absence of hyperinsulinemia in obese Kir2.1 expressing mice despite the development of hepatic steatosis supports hepatocyte depolarization as a critical mediator in the relationship between hepatic lipid accumulation and dysregulated glucose homeostasis.
  • liver slice GABA import and export may be mediated by ion dependent transporters.
  • the liver expresses 4 electrogenic GABA transporters that are members of the Na+/CI-dependent neurotransmitter transporter (SLC6) family. These include proteins encoded for by Slc6A12 (Betaine GABA transporter 1, BGT1), Slc6A13 (GABA transporter 2, GAT2), Slc6A6 (Taurine Transporter, TauT), and Slc6A8 (Creatine transporter, CRT).
  • BGT1 and GAT2 both co-transport 3 Na+, 1 Cl- and GABA, moving 2 positive charges in the direction of GABA transport.
  • TauT co-transports 2.5 Na+, 1 Cl- and GABA, moving 1.5 positive charges in the direction of GABA transport.
  • the CRT transporter co-transports 2 Na+, 1 Cl- and GABA (or creatine) moving a single positive charge in the direction of GABA transport.
  • hepatic slice GABA release was encouraged by incubation in media with low NaCI concentrations (FIG. 20F). It is also shown that incubation with the BGT1 and GAT2 inhibitors, betaine (1 mM) and nipecotic acid (NA; 1 mM), respectively, increases media GABA concentrations (FIG.
  • GABA-T is most frequently thought to be an enzyme key to GABA breakdown.
  • early in vitro studies established that the reaction in the direction of GABA synthesis was favored with a Keq 0.04 25. The reason that this reaction most frequently proceeds in the reverse direction is a lack of succinate semialdehyde (SSA), for which SSA dehydrogenase (SSADH) has a nearly 10X lower Km than GABA-T.
  • SSA succinate semialdehyde
  • SSADH SSA dehydrogenase
  • liver slices were treated with the irreversible GABA-T inhibitor, ethanolamine-O-sulphate ex vivo (EOS; 5.3 mM).
  • EOS decreased GABA export from obese control and obese Kir2.1 expressing liver slices, but not liver slices from lean mice (FIG. 20I).
  • GABA production is elevated in obesity and that GABA-T mediated synthesis of GABA is not impaired by Kir2.1 expression.
  • Hepatocytes from obese mice also released less of the excitatory neurotransmitter, aspartate, than hepatocytes from lean mice (FIG. 20J).
  • Vagal afferent innervation in the liver has previously been identified using the vagal sensory immunohistochemical marker calretinin.
  • Calcitonin gene-related peptide (CGRP) has also been proposed as a marker of hepatic vagal afferent innervation and hepatic CGRP staining is eliminated by capsaicin treatment and substantially reduced following bilateral vagotomy.
  • CGRP Calcitonin gene-related peptide
  • GABA transporters SLC6A6 , encodes for taurine transporter, TauT; SLC6A8, encodes for the creatine transporter, CRT; SLC6A12, encodes for the Betaine-GABA Transporter 1, BGT 1 ; and SLC6A13, encodes for GABA Transporter 2, GAT2
  • IHTG intrahepatic triglyceride
  • MRI magnetic resonance imaging
  • HECP hyperinsulinemic-euglycemic clamp procedure
  • IHTG % was negatively associated with both glucose infusion rate during a clamp (FIG. 22A) and the percent increase in glucose rate of disposal from the basal state to the hyperinsulinemic clamp (FIG. 22B and Data Table 7).
  • BGT1 and GAT2 are primarily acting as GABA re-uptake transporters and that Tau-T and CRT are acting to export GABA.
  • This hypothesized role of BGT 1 and GAT2 in hepatic GABA re-uptake is supported by the explant data (Fig 4G).
  • FFM fat free mass
  • Glucose Rd glucose disposal rate.
  • Data Table 7 Regression coefficient estimates showing the association between hepatic mRIMA expression of genes involved in GABA production (ABAT) and GABA transport (Slc6A6, A8, A12, and A13) and glucose infusion rate (pMol/Kg Fat Free Mass/min) and Glucose Rd (rate of disposal; % increase) during a hyperinsulinemic-euglycemic clamp.
  • SNPs Single nucleotide polymorphisms that result in missense mutations in GABA transporters are associated with an increased incidence (OR; odds ratio) of type 2 diabetes (T2D; source: knowledge portal diabetes database). MAF - minor allele frequency.
  • the present invention features a new model that links hepatic lipid accumulation to HVAN activity and the development of hyperinsulinemia and insulin resistance (Fig. 5).
  • Hepatic lipid accumulation increases flux through gluconeogenesis and increases the hepatic FADH:FAD and NADH:NAD ratio (Fig. 4H).
  • the altered hepatic redox state inhibits the conversion of succinate to fumarate in the TCA cycle and instead drives succinate to succinate semialdehyde.
  • Succinate semialdehyde serves as substrate for GABA-T mediated GABA production.
  • the oketoglutarate formed by GABA-T produces oxaloacetate and glutamate, which feeds back into the GABA-T catalyzed reaction.
  • the demand for gluconeogenic substrate and the high NADH:NAD ratio drives the carbons in oxaloacetate to malate and through gluconeogenesis.
  • This gluconeogenic drive increases aspartate metabolism, explaining the decreased aspartate release in liver slices from obese mice (Fig. 4J).
  • gluconeogenic flux and a more reduced mitochondrial redox state direct the flow of intermediate molecules in obesity resulting in elevated hepatic GABA production and increased aspartate utilization.
  • hepatocyte GABA export sensitive to changes in membrane potential. Since GABA transporters are sodium co-transporters, an increase in intracellular sodium ions and hepatocyte depolarization increases GABA export (Fig. 4E). Obesity decreases hepatic ATP content (Fig. 4D) and lowers Na+/K+ ATPase activity 21, providing a mechanism by which obesity depolarizes hepatocytes 12 (Fig. 2D) and encourages GABA export (Fig. 4A). In fact, type II diabetics have lower hepatic ATP concentrations, and both peripheral and hepatic insulin sensitivity is significantly correlated with liver ATP concentrations.
  • hepatic lipid accumulation ultimately increases hepatic GABA signaling through two separate mechanisms.
  • hepatic GABA production is stimulated as a result of increased GABA-T expression (Fig. 4B) and gluconeogenic flux (Fig 5), and second, hepatic GABA release is stimulated by hepatocyte depolarization (Fig. 4E).
  • Fig. 4B hepatic GABA production is stimulated as a result of increased GABA-T expression
  • Fig 5 gluconeogenic flux
  • hepatic GABA release is stimulated by hepatocyte depolarization (Fig. 4E).
  • hepatocyte depolarization in diet-induced metabolic dysfunction.
  • hepatocyte membrane potential is closely regulated by insulin and glucagon. Acutely, insulin depolarizes while glucagon hyperpolarizes hepatocytes 33,34.
  • the hyperpolarizing effect of glucagon is proposed to be mediated by cAMP and is dependent on the Na+/K+ ATPase 33. Accordingly, cAMP or glucagon counteracts insulin stimulated hepatocyte depolarization 35,36
  • High fat diet feeding decreases hepatic cAMP content in mice 37. The decrease in hepatic cAMP along with diminished ATP may contribute to obesity-induced hepatocyte depolarization.
  • Hepatocellular carcinoma is characterized by hepatocyte depolarization and increased GABAergic signaling, while increasing hepatocyte polarization protects against tumor proliferation 38.
  • GABAergic signaling increases hepatocyte polarization
  • Physiological concentrations of insulin and glucagon induce a 5-7 mV change in hepatocyte membrane potential. This is comparable to the 6.86 ⁇ 1.54 mV hyperpolarization induced by Kir2.1 expression (Fig. 3B).
  • the PSEM89S ligand maximally depolarized hepatocytes by 28 ⁇ 5.4 mV (Fig. 2E), which exceeds the depolarization observed in obesity (13 ⁇ 4.7 mV; Fig. 2D), and likely represents a supraphysiological response.
  • hepatocyte depolarization was 17.0 ⁇ 5.4 mV, representing a more physiological change in membrane potential.
  • liver derived signals communicate to the central nervous system via the HVAN is well established in the literature, the degree hepatocyte vagal afferent innervation has remained controversial. Provided herein is evidence of vagal sensory innervation in close proximity to hepatocytes and have established the presence of GABAA receptors on both calretinin and CGRP immunoreactive neurons in the liver (Figs. 6A-D). Once exported, hepatic GABA can act at GABAA receptors on vagal afferents to induce chloride influx and decrease firing rate 41 (Fig. 5), providing a connection between hepatic lipid accumulation and decreased HVAN activity.
  • hepatic vagotomy eliminates the dysfunctional hepatocyte-vagal signaling in obesity, while preserving signaling from the HVAN above the surgical resection.
  • hepatic vagotomy has been used extensively to investigate liver vagal denervation, several limitations must be acknowledged when interpreting this model.
  • the improvements in glucose homeostasis in response to hepatic vagotomy are a result of interrupting non-vagal afferent signaling or partial loss of vagal efferent pancreatic innervation.
  • afferent parasympathetic signals from the liver may affect pancreatic insulin release.
  • Activity of the HVAN is inversely related to parasympathetic efferent nerve activity at the pancreas, which stimulates insulin release 4,5.
  • portal glucose inhibits HVAN activity and increases pancreatic parasympathetic outflow to stimulate b-cell muscarinic 3 receptor (M3R) signaling and insulin release.
  • M3R muscarinic 3 receptor
  • vagotomy reduces glucose stimulated insulin secretion and basal hyperinsulinemia in obese rats by reducing cholinergic action on b-cells 4,46.
  • cholinergic blockade decreases basal serum insulin concentrations in obese but not lean mice, suggesting that elevated basal pancreatic parasympathetic efferent tone underlies obesity-induced hyperinsulinemia.
  • the HVAN also regulates insulin sensitivity. Hepatic vagotomy acutely reduces insulin sensitivity in lean rats, decreasing skeletal muscle glucose clearance by 45%. In contrast, chronic hepatic vagotomy improves insulin sensitivity and glucose clearance in insulin resistant mice 6 (Fig. 1L). Portal glucose delivery decreases HVAN firing activity and skeletal muscle glucose clearance.
  • hepatic GABA production in obesity decreases HVAN activity to limit muscle glucose clearance and drive peripheral insulin resistance.
  • the present invention identified enzymes involved in GABA production and transporters involved in hepatic GABA re-uptake and release as novel therapeutic targets for correcting the inherent metabolic disturbances in T2D.
  • descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of or “consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of or “consisting of is met.

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Abstract

Procédés et compositions pour traiter des états provoqués par la production et la libération de GABA hépatique modifiée, notamment l'hyperinsulinémie, la résistance à l'insuline, le diabète de type II, l'obésité et les problèmes de santé liés à l'obésité. La présente invention décrit le GABA hépatique hépatokine. Les procédés de l'invention comprennent la manipulation de l'expression et/ou de l'activité de transporteurs de GABA spécifiques, par exemple, l'augmentation de l'expression de gènes SLC6A12 et/ou SLC6A13 ou l'augmentation de l'activité des protéines pour lesquelles ils codent, BGT1 et/ou GAT2 ; ou la diminution de l'expression de gènes SLC6A6 et SLC6A8 ou l'augmentation de l'activité des protéines pour lesquelles ils codent, TauT et/ou CRT, ce qui peut augmenter la réabsorption de GABA hépatique ou diminuer la libération de GABA hépatique pour améliorer la sensibilité à l'insuline et prévenir l'hypertension.
PCT/US2021/025629 2017-05-26 2021-04-02 Procédés et compositions pour modifier la libération de gaba hépatique pour traiter des problèmes de santé liés à l'obésité WO2021203033A2 (fr)

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