Melanocortin 4 receptors reciprocally regulate sympathetic and parasympathetic preganglionic neurons.

Melanocortin 4 receptors (MC4Rs) in the central nervous system are key regulators of energy and glucose homeostasis. Notably, obese patients with MC4R mutations are hyperinsulinemic and resistant to obesity-induced hypertension. Although these effects are probably dependent upon the activity of the autonomic nervous system, the cellular effects of MC4Rs on parasympathetic and sympathetic neurons remain undefined. Here, we show that MC4R agonists inhibit parasympathetic preganglionic neurons in the brainstem. In contrast, MC4R agonists activate sympathetic preganglionic neurons in the spinal cord. Deletion of MC4Rs in cholinergic neurons resulted in elevated levels of insulin. Furthermore, re-expression of MC4Rs specifically in cholinergic neurons (including sympathetic preganglionic neurons) restores obesity-associated hypertension in MC4R null mice. These findings provide a cellular correlate of the autonomic side effects associated with MC4R agonists and demonstrate a role for MC4Rs expressed in cholinergic neurons in the regulation of insulin levels and in the development of obesity-induced hypertension.

Cytosolic phosphoenolpyruvate carboxykinase as a cataplerotic pathway in the small intestine.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme that is highly expressed in the liver and kidney but is also expressed at lower levels in a variety of other tissues where it may play adjunct roles in fatty acid esterification, amino acid metabolism, and/or TCA cycle function. PEPCK is expressed in the enterocytes of the small intestine, but it is unclear whether it supports a gluconeogenic rate sufficient to affect glucose homeostasis. To examine potential roles of intestinal PEPCK, we generated an intestinal PEPCK knockout mouse. Deletion of intestinal PEPCK ablated ex vivo gluconeogenesis but did not significantly affect glycemia in chow, high-fat diet, or streptozotocin-treated mice. In contrast, postprandial triglyceride secretion from the intestine was attenuated in vivo, consistent with a role in fatty acid esterification. Intestinal amino acid profiles and 13C tracer appearance into these pools were significantly altered, indicating abnormal amino acid trafficking through the enterocyte. The data suggest that the predominant role of PEPCK in the small intestine of mice is not gluconeogenesis but rather to support nutrient processing, particularly with regard to lipids and amino acids. NEW & NOTEWORTHY The small intestine expresses gluconeogenic enzymes for unknown reasons. In addition to glucose synthesis, the nascent steps of this pathway can be used to support amino acid and lipid metabolisms. When phosphoenolpyruvate carboxykinase, an essential gluconeogenic enzyme, is knocked out of the small intestine of mice, glycemia is unaffected, but mice inefficiently absorb dietary lipid, have abnormal amino acid profiles, and inefficiently catabolize glutamine. Therefore, the initial steps of intestinal gluconeogenesis are used for processing dietary triglycerides and metabolizing amino acids but are not essential for maintaining blood glucose levels.

Hyperglycemia in rodent models of type 2 diabetes requires insulin-resistant alpha cells.

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet-induced obese (DIO) and leptin receptor-defective (LepR(-/-)) rodents with and without glucagon receptors (GcgRs). DIO and LepR(-/-),GcgR(+/+) mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR(+/+) mice developed mild T2D, whereas LepR(-/-),GcgR(+/+) mice developed severe T2D. High-fat-fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR(-/-) to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR(-/-),GcgR(-/-) did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR(-/-),LepR(-/-) mice caused the severe hyperinsulinemia and hyperglycemia of LepR(-/-) mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade.

Metabolic manifestations of insulin deficiency do not occur without glucagon action.

To determine unambiguously if suppression of glucagon action will eliminate manifestations of diabetes, we expressed glucagon receptors in livers of glucagon receptor-null (GcgR(-/-)) mice before and after ß-cell destruction by high-dose streptozotocin. Wild type (WT) mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR(-/-) mice with similar ß-cell destruction remained clinically normal without hyperglycemia, impaired glucose tolerance, or hepatic glycogen depletion. Restoration of receptor expression using adenovirus containing the GcgR cDNA restored hepatic GcgR, phospho-cAMP response element binding protein (P-CREB), and phosphoenol pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycemia appeared. When GcgR mRNA spontaneously disappeared 7 d later, P-CREB declined and hyperglycemia disappeared. In conclusion, the metabolic manifestations of diabetes cannot occur without glucagon action and, once present, disappear promptly when glucagon action is abolished. Glucagon suppression should be a major therapeutic goal in diabetes.

Weight-independent effects of roux-en-Y gastric bypass on glucose homeostasis via melanocortin-4 receptors in mice and humans.

BACKGROUND & AIMS: Roux-en-Y gastric bypass (RYGB) improves glucose homeostasis independently of changes in body weight by unknown mechanisms. Melanocortin-4 receptors (MC4R) have weight-independent effects on glucose homeostasis, via autonomic neurons, and also might contribute to weight loss after RYGB. We investigated whether MC4Rs mediate effects of RYGB, such as its weight-independent effects on glucose homeostasis, in mice and humans. METHODS: We studied C57BL/6 mice with diet-induced obesity, MC4R-deficient mice, and mice that re-express MC4R specifically in autonomic neurons after RYGB or sham surgeries. We also sequenced the MC4R locus in patients undergoing RYGB to investigate diabetes resolution in carriers of rare MC4R variants. RESULTS: MC4Rs in autonomic brainstem neurons (including the parasympathetic dorsal motor vagus) mediated improved glucose homeostasis independent of changes in body weight. In contrast, MC4Rs in cholinergic preganglionic motor neurons (sympathetic and parasympathetic) mediated RYGB-induced increased energy expenditure and weight loss. Increased energy expenditure after RYGB is the predominant mechanism of weight loss and confers resistance to weight gain from a high-fat diet, the effects of which are MC4R-dependent. MC4R-dependent effects of RYGB still occurred in mice with Mc4r haplosufficiency, and early stage diabetes resolved at a similar rate in patients with rare variants of MC4R and noncarriers. However, carriers of MC4R (I251L), a rare variant associated with increased weight loss after RYGB and increased basal activity in vitro, were more likely to have early and weight-independent resolution of diabetes than noncarriers, indicating a role for MC4Rs in the effects of RYGB. CONCLUSIONS: MC4Rs in autonomic neurons mediate beneficial effects of RYGB, including weight-independent improved glucose homeostasis, in mice and humans.

Hepatic malonyl-CoA synthesis restrains gluconeogenesis by suppressing fat oxidation, pyruvate carboxylation, and amino acid availability.

Acetyl-CoA carboxylase (ACC) promotes prandial liver metabolism by producing malonyl-CoA, a substrate for de novo lipogenesis and an inhibitor of CPT-1-mediated fat oxidation. We report that inhibition of ACC also produces unexpected secondary effects on metabolism. Liver-specific double ACC1/2 knockout (LDKO) or pharmacologic inhibition of ACC increased anaplerosis, tricarboxylic acid (TCA) cycle intermediates, and gluconeogenesis by activating hepatic CPT-1 and pyruvate carboxylase flux in the fed state. Fasting should have marginalized the role of ACC, but LDKO mice maintained elevated TCA cycle intermediates and preserved glycemia during fasting. These effects were accompanied by a compensatory induction of proteolysis and increased amino acid supply for gluconeogenesis, which was offset by increased protein synthesis during feeding. Such adaptations may be related to Nrf2 activity, which was induced by ACC inhibition and correlated with fasting amino acids. The findings reveal unexpected roles for malonyl-CoA synthesis in liver and provide insight into the broader effects of pharmacologic ACC inhibition.

Ghrelin cell-expressed insulin receptors mediate meal- and obesity-induced declines in plasma ghrelin.

Mechanisms underlying postprandial and obesity-associated plasma ghrelin reductions are incompletely understood. Here, using ghrelin cell-selective insulin receptor-KO (GhIRKO) mice, we tested the impact of insulin, acting via ghrelin cell-expressed insulin receptors (IRs), to suppress ghrelin secretion. Insulin reduced ghrelin secretion from cultured gastric mucosal cells of control mice but not from those of GhIRKO mice. Acute insulin challenge and insulin infusion during both hyperinsulinemic-hypoglycemic clamps and hyperinsulinemic-euglycemic clamps lowered plasma ghrelin in control mice but not GhIRKO mice. Thus, ghrelin cell-expressed IRs are required for insulin-mediated reductions in plasma ghrelin. Furthermore, interventions that naturally raise insulin (glucose gavage, refeeding following fasting, and chronic high-fat diet) also lowered plasma ghrelin only in control mice - not GhIRKO mice. Thus, meal- and obesity-associated increases in insulin, acting via ghrelin cell-expressed IRs, represent a major, direct negative modulator of ghrelin secretion in vivo, as opposed to ingested or metabolized macronutrients. Refed GhIRKO mice exhibited reduced plasma insulin, highlighting ghrelin's actions to inhibit insulin release via a feedback loop. Moreover, GhIRKO mice required reduced glucose infusion rates during hyperinsulinemic-hypoglycemic clamps, suggesting that suppressed ghrelin release resulting from direct insulin action on ghrelin cells usually limits ghrelin's full potential to protect against insulin-induced hypoglycemia.

Gαi/o-coupled Htr2c in the paraventricular nucleus of the hypothalamus antagonizes the anorectic effect of serotonin agents.

The anorexigenic effect of serotonergic compounds has largely been attributed to activation of serotonin 2C receptors (Htr2cs). Using mouse genetic models in which Htr2c can be selectively deleted or restored (in Htr2c-null mice), we investigate the role of Htr2c in forebrain Sim1 neurons. Unexpectedly, we find that Htr2c acts in these neurons to promote food intake and counteract the anorectic effect of serotonergic appetite suppressants. Furthermore, Htr2c marks a subset of Sim1 neurons in the paraventricular nucleus of the hypothalamus (PVH). Chemogenetic activation of these neurons in adult mice suppresses hunger, whereas their silencing promotes feeding. In support of an orexigenic role of PVH Htr2c, whole-cell patch-clamp experiments demonstrate that activation of Htr2c inhibits PVH neurons. Intriguingly, this inhibition is due to Gαi/o-dependent activation of ATP-sensitive K+ conductance, a mechanism of action not identified previously in the mammalian nervous system.

Glucagon and lipid interactions in the regulation of hepatic AMPK signaling and expression of PPARalpha and FGF21 transcripts in vivo.

Hepatic glucagon action increases in response to accelerated metabolic demands and is associated with increased whole body substrate availability, including circulating lipids. The hypothesis that increases in hepatic glucagon action stimulate AMP-activated protein kinase (AMPK) signaling and peroxisome proliferator-activated receptor-α (PPARα) and fibroblast growth factor 21 (FGF21) expression in a manner modulated by fatty acids was tested in vivo. Wild-type (gcgr(+/+)) and glucagon receptor-null (gcgr(-/-)) littermate mice were studied using an 18-h fast, exercise, and hyperglucagonemic-euglycemic clamps plus or minus increased circulating lipids. Fasting and exercise in gcgr(+/+), but not gcgr(-/-) mice, increased hepatic phosphorylated AMPKα at threonine 172 (p-AMPK(Thr(172))) and PPARα and FGF21 mRNA. Clamp results in gcgr(+/+) mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, p-AMPK(Thr(172)), or PPARα and FGF21 mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPARα and FGF21 expression. All effects were absent in gcgr(-/-) mice. These findings demonstrate that glucagon exerts a critical regulatory role in liver to stimulate pathways linked to lipid metabolism in vivo and shows for the first time that effects of glucagon on PPARα and FGF21 expression are amplified by a physiological increase in circulating lipids.

Ghrelin Protects Against Insulin-Induced Hypoglycemia in a Mouse Model of Type 1 Diabetes Mellitus.

Insulin-induced hypoglycemia is a major limiting factor in maintaining optimal blood glucose in patients with type 1 diabetes and advanced type 2 diabetes. Luckily, a counterregulatory response (1) system exists to help minimize and reverse hypoglycemia, although more studies are needed to better characterize its components. Recently, we showed that the hormone ghrelin is permissive for the normal CRR to insulin-induced hypoglycemia when assessed in mice without diabetes. Here, we tested the hypothesis that ghrelin also is protective against insulin-induced hypoglycemia in the streptozotocin (2) mouse model of type 1 diabetes. STZ-treated ghrelin-knockout (KO) (3) mice as well as STZ-treated wild-type (WT) littermates were subjected to a low-dose hyperinsulinemic-hypoglycemic clamp procedure. The STZ-treated ghrelin-KO mice required a much higher glucose infusion rate than the STZ-treated WT mice. Also, the STZ-treated ghrelin-KO mice exhibited attenuated plasma epinephrine and norepinephrine responses to the insulin-induced hypoglycemia. Taken together, our data suggest that without ghrelin, STZ-treated mice modeling type 1 diabetes are unable to mount the usual CRR to insulin-induced hypoglycemia.

Acyl-ghrelin Is Permissive for the Normal Counterregulatory Response to Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia leads to far-ranging negative consequences in patients with diabetes. Components of the counterregulatory response (CRR) system that help minimize and reverse hypoglycemia and coordination between those components are well studied but not yet fully characterized. Here, we tested the hypothesis that acyl-ghrelin, a hormone that defends against hypoglycemia in a preclinical starvation model, is permissive for the normal CRR to insulin-induced hypoglycemia. Ghrelin knockout (KO) mice and wild-type (WT) littermates underwent an insulin bolus-induced hypoglycemia test and a low-dose hyperinsulinemic-hypoglycemic clamp procedure. Clamps also were performed in ghrelin-KO mice and C57BL/6N mice administered the growth hormone secretagogue receptor agonist HM01 or vehicle. Results show that hypoglycemia, as induced by an insulin bolus, was more pronounced and prolonged in ghrelin-KO mice, supporting previous studies suggesting increased insulin sensitivity upon ghrelin deletion. Furthermore, during hyperinsulinemic-hypoglycemic clamps, ghrelin-KO mice required a 10-fold higher glucose infusion rate (GIR) and exhibited less robust corticosterone and growth hormone responses. Conversely, HM01 administration, which reduced the GIR required by ghrelin-KO mice during the clamps, increased plasma corticosterone and growth hormone. Thus, our data suggest that endogenously produced acyl-ghrelin not only influences insulin sensitivity but also is permissive for the normal CRR to insulin-induced hypoglycemia.

Estrogen receptor inhibition enhances cold-induced adipocyte beiging and glucose tolerance.

BACKGROUND:  Low estrogen states, exemplified by postmenopausal women, are associated with increased adiposity and metabolic dysfunction. We recently reported a paradox, in which a conditional estrogen receptor-alpha (ERα) mutant mouse shows a hyper-metabolic phenotype with enhanced brown/beige cell formation ("browning/beiging"). HYPOTHESIS:  These observations led us to consider that although systemic deficiency of estrogen or ERα in mice results in obesity and glucose intolerance at room temperature, cold exposure might induce enhanced browning/beiging and improve glucose metabolism. METHODS AND RESULTS:  Remarkably, studying cold-exposure in mouse models of inhibited estrogen signaling - ERαKO mice, ovariectomy, and treatment with the ERα antagonist Fulvestrant - supported this notion. ERα/estrogen-deficient mice demonstrated enhanced cold-induced beiging, reduced adiposity and improved glucose tolerance. Fulvestrant was also effective in diet-induced obesity settings. Mechanistically, ERα inhibition sensitized cell-autonomous beige cell differentiation and stimulation, including ß3-adrenoreceptor-dependent adipocyte beiging. CONCLUSION:  Taken together, our findings highlight a therapeutic potential for obese/diabetic postmenopausal patients; cold exposure is therefore predicted to metabolically benefit those patients.

Celastrol Reduces Obesity in MC4R Deficiency and Stimulates Sympathetic Nerve Activity Affecting Metabolic and Cardiovascular Functions.

Leptin resistance is a hallmark of obesity with unclear etiology. Celastrol, a compound found in the roots of the Tripterygium wilfordii and known to reduce endoplasmic reticulum (ER) stress, has recently emerged as a promising candidate to treat obesity by improving leptin sensitivity. However, the underlying neural mechanisms by which celastrol reduces obesity remain unclear. Using three different mouse models of obesity-diet-induced obesity (DIO), leptin receptor (LepR)-null, and melanocortin 4 receptor (MC4R)-null mice-in this study, we show that systemic celastrol administration substantially reduces food intake and body weight in MC4R-null comparable to DIO, proving the MC4R-independent antiobesity effect of celastrol. Body weight reduction was due to decreases in both fat and lean mass, and modest but significant body weight reduction was also observed in nonobese wild-type and LepR-null mice. Unexpectedly, celastrol upregulated proinflammatory cytokines without affecting genes involved in ER stress. Importantly, celastrol steadily increased sympathetic nerve activity to the brown fat and kidney with concordant increases of resting metabolic rate and arterial pressure. Our results suggest a previously unappreciated mechanism of action of celastrol in the regulation of energy homeostasis and highlight the need for careful consideration of its development as a safe antiobesity medication.

Pyruvate-Carboxylase-Mediated Anaplerosis Promotes Antioxidant Capacity by Sustaining TCA Cycle and Redox Metabolism in Liver.

The hepatic TCA cycle supports oxidative and biosynthetic metabolism. This dual responsibility requires anaplerotic pathways, such as pyruvate carboxylase (PC), to generate TCA cycle intermediates necessary for biosynthesis without disrupting oxidative metabolism. Liver-specific PC knockout (LPCKO) mice were created to test the role of anaplerotic flux in liver metabolism. LPCKO mice have impaired hepatic anaplerosis, diminution of TCA cycle intermediates, suppressed gluconeogenesis, reduced TCA cycle flux, and a compensatory increase in ketogenesis and renal gluconeogenesis. Loss of PC depleted aspartate and compromised urea cycle function, causing elevated urea cycle intermediates and hyperammonemia. Loss of PC prevented diet-induced hyperglycemia and insulin resistance but depleted NADPH and glutathione, which exacerbated oxidative stress and correlated with elevated liver inflammation. Thus, despite catalyzing the synthesis of intermediates also produced by other anaplerotic pathways, PC is specifically necessary for maintaining oxidation, biosynthesis, and pathways distal to the TCA cycle, such as antioxidant defenses.

Hypoglycemic Effect of Combined Ghrelin and Glucagon Receptor Blockade.

Glucagon receptor (GcgR) blockade has been proposed as an alternative to insulin monotherapy for treating type 1 diabetes since deletion or inhibition of GcgRs corrects hyperglycemia in models of diabetes. The factors regulating glycemia in a setting devoid of insulin and glucagon function remain unclear but may include the hormone ghrelin. Not only is ghrelin release controlled by glucose but also ghrelin has many actions that can raise or reduce falls in blood glucose level. Here, we tested the hypothesis that ghrelin rises to prevent hypoglycemia in the absence of glucagon function. Both GcgR knockout (Gcgr-/-) mice and db/db mice that were administered GcgR monoclonal antibody displayed lower blood glucose levels accompanied by elevated plasma ghrelin levels. Although treatment with the pancreatic ß-cell toxin streptozotocin induced hyperglycemia and raised plasma ghrelin levels in wild-type mice, hyperglycemia was averted in similarly treated Gcgr-/- mice and the plasma ghrelin level was further increased. Notably, administration of a ghrelin receptor antagonist further reduced blood glucose levels into the markedly hypoglycemic range in overnight-fasted, streptozotocin-treated Gcgr-/- mice. A lowered blood glucose level also was observed in overnight-fasted, streptozotocin-treated ghrelin receptor-null mice that were administered GcgR monoclonal antibody. These data suggest that when glucagon activity is blocked in the setting of type 1 diabetes, the plasma ghrelin level rises, preventing hypoglycemia.

Cellular Aging Contributes to Failure of Cold-Induced Beige Adipocyte Formation in Old Mice and Humans.

Cold temperatures induce progenitor cells within white adipose tissue to form beige adipocytes that burn energy and generate heat; this is a potential anti-diabesity therapy. However, the potential to form cold-induced beige adipocytes declines with age. This creates a clinical roadblock to potential therapeutic use in older individuals, who constitute a large percentage of the obesity epidemic. Here we show that aging murine and human beige progenitor cells display a cellular aging, senescence-like phenotype that accounts for their age-dependent failure. Activating the senescence pathway, either genetically or pharmacologically, in young beige progenitors induces premature cellular senescence and blocks their potential to form cold-induced beige adipocytes. Conversely, genetically or pharmacologically reversing cellular aging by targeting the p38/MAPK-p16Ink4a pathway in aged mouse or human beige progenitor cells rejuvenates cold-induced beiging. This in turn increases glucose sensitivity. Collectively, these data indicate that anti-aging or senescence modalities could be a strategy to induce beiging, thereby improving metabolic health in aging humans.

Ire1α in Pomc Neurons Is Required for Thermogenesis and Glycemia.

Whether neuronal inositol-requiring enzyme 1 (Ire1) is required for the proper regulation of energy balance and glucose homeostasis is unclear. We found that pro-opiomelanocortin (Pomc)-specific deficiency of Ire1α accelerated diet-induced obesity concomitant with a decrease in energy expenditure. This hypometabolic phenotype included deficits in thermogenic responses to diet and cold exposure as well as "beiging" of white adipose tissue. We also demonstrate that loss of Ire1α in Pomc neurons impaired whole-body glucose and insulin tolerance as well as hepatic insulin sensitivity. At the cellular level, deletion of Ire1α in Pomc neurons elevated hypothalamic endoplasmic reticulum (ER) stress and predisposed Pomc neurons to leptin and insulin resistance. Together, the current studies extend and confirm conclusions that Ire1α-Xbp1s and associated molecular targets link ER stress in arcuate Pomc neurons to aspects of normal energy and glucose homeostasis.

Skeletal muscle salt inducible kinase 1 promotes insulin resistance in obesity.

OBJECTIVE: Insulin resistance causes type 2 diabetes mellitus and hyperglycemia due to excessive hepatic glucose production and inadequate peripheral glucose uptake. Our objectives were to test the hypothesis that the proposed CREB/CRTC2 inhibitor salt inducible kinase 1 (SIK1) contributes to whole body glucose homeostasis in vivo by regulating hepatic transcription of gluconeogenic genes and also to identify novel SIK1 actions on glucose metabolism. METHODS: We created conditional (floxed) SIK1-knockout mice and studied glucose metabolism in animals with global, liver, adipose or skeletal muscle Sik1 deletion. We examined cAMP-dependent regulation of SIK1 and the consequences of SIK1 depletion on primary mouse hepatocytes. We probed metabolic phenotypes in tissue-specific SIK1 knockout mice fed high fat diet through hyperinsulinemic-euglycemic clamps and biochemical analysis of insulin signaling. RESULTS: SIK1 knockout mice are viable and largely normoglycemic on chow diet. On high fat diet, global SIK1 knockout animals are strikingly protected from glucose intolerance, with both increased plasma insulin and enhanced peripheral insulin sensitivity. Surprisingly, liver SIK1 is not required for regulation of CRTC2 and gluconeogenesis, despite contributions of SIK1 to hepatocyte CRTC2 and gluconeogenesis regulation ex vivo. Sik1 mRNA accumulates in skeletal muscle of obese high fat diet-fed mice, and knockout of SIK1 in skeletal muscle, but not liver or adipose tissue, improves insulin sensitivity and muscle glucose uptake on high fat diet. CONCLUSIONS: SIK1 is dispensable for glycemic control on chow diet. SIK1 promotes insulin resistance on high fat diet by a cell-autonomous mechanism in skeletal muscle. Our study establishes SIK1 as a promising therapeutic target to improve skeletal muscle insulin sensitivity in obese individuals without deleterious effects on hepatic glucose production.

Enhanced insulin sensitivity in skeletal muscle and liver by physiological overexpression of SIRT6.

OBJECTIVE: Available treatment for obesity and type 2 diabetes mellitus (T2DM) is suboptimal. Thus, identifying novel molecular target(s) exerting protective effects against these metabolic imbalances is of enormous medical significance. Sirt6 loss- and gain-of-function studies have generated confounding data regarding the role of this sirtuin on energy and glucose homeostasis, leaving unclear whether activation or inhibition of SIRT6 may be beneficial for the treatment of obesity and/or T2DM. METHODS: To address these issues, we developed and studied a novel mouse model designed to produce eutopic and physiological overexpression of SIRT6 (Sirt6BAC mice). These mutants and their controls underwent several metabolic analyses. These include whole-blood reverse phase high-performance liquid chromatography assay, glucose and pyruvate tolerance tests, hyperinsulinemic-euglycemic clamp assays, and assessment of basal and insulin-induced level of phosphorylated AKT (p-AKT)/AKT in gastrocnemius muscle. RESULTS: Sirt6BAC mice physiologically overexpress functionally competent SIRT6 protein. While Sirt6BAC mice have normal body weight and adiposity, they are protected from developing high-caloric-diet (HCD)-induced hyperglycemia and glucose intolerance. Also, Sirt6BAC mice display increased circulating level of the polyamine spermidine. The ability of insulin to suppress endogenous glucose production was significantly enhanced in Sirt6BAC mice compared to wild-type controls. Insulin-stimulated glucose uptake was increased in Sirt6BAC mice in both gastrocnemius and soleus muscle, but not in brain, interscapular brown adipose, or epididymal adipose tissue. Insulin-induced p-AKT/AKT ratio was increased in gastrocnemius muscle of Sirt6BAC mice compared to wild-type controls. CONCLUSIONS: Our data indicate that moderate, physiological overexpression of SIRT6 enhances insulin sensitivity in skeletal muscle and liver, engendering protective actions against diet-induced T2DM. Hence, the present study provides support for the anti-T2DM effect of SIRT6 and suggests SIRT6 as a putative molecular target for anti-T2DM treatment.

Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin.

The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreER(T2) transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.