S100A9 exerts insulin-independent antidiabetic and anti-inflammatory effects.

Type 1 diabetes mellitus (T1DM) is characterized by insulin deficiency leading to hyperglycemia and several metabolic defects. Insulin therapy remains the cornerstone of T1DM management, yet it increases the risk of life-threatening hypoglycemia and the development of major comorbidities. Here, we report an insulin signaling-independent pathway able to improve glycemic control in T1DM rodents. Co-treatment with recombinant S100 calcium-binding protein A9 (S100A9) enabled increased adherence to glycemic targets with half as much insulin and without causing hypoglycemia. Mechanistically, we demonstrate that the hyperglycemia-suppressing action of S100A9 is due to a Toll-like receptor 4-dependent increase in glucose uptake in specific skeletal muscles (i.e., soleus and diaphragm). In addition, we found that T1DM mice have abnormal systemic inflammation, which is resolved by S100A9 therapy alone (or in combination with low insulin), hence uncovering a potent anti-inflammatory action of S100A9 in T1DM. In summary, our findings reveal the S100A9-TLR4 skeletal muscle axis as a promising therapeutic target for improving T1DM treatment.

Hepatic non-parenchymal S100A9-TLR4-mTORC1 axis normalizes diabetic ketogenesis.

Unrestrained ketogenesis leads to life-threatening ketoacidosis whose incidence is high in patients with diabetes. While insulin therapy reduces ketogenesis this approach is sub-optimal. Here, we report an insulin-independent pathway able to normalize diabetic ketogenesis. By generating insulin deficient male mice lacking or re-expressing Toll-Like Receptor 4 (TLR4) only in liver or hepatocytes, we demonstrate that hepatic TLR4 in non-parenchymal cells mediates the ketogenesis-suppressing action of S100A9. Mechanistically, S100A9 acts extracellularly to activate the mechanistic target of rapamycin complex 1 (mTORC1) in a TLR4-dependent manner. Accordingly, hepatic-restricted but not hepatocyte-restricted loss of Tuberous Sclerosis Complex 1 (TSC1, an mTORC1 inhibitor) corrects insulin-deficiency-induced hyperketonemia. Therapeutically, recombinant S100A9 administration restrains ketogenesis and improves hyperglycemia without causing hypoglycemia in diabetic mice. Also, circulating S100A9 in patients with ketoacidosis is only marginally increased hence unveiling a window of opportunity to pharmacologically augment S100A9 for preventing unrestrained ketogenesis. In summary, our findings reveal the hepatic S100A9-TLR4-mTORC1 axis in non-parenchymal cells as a promising therapeutic target for restraining diabetic ketogenesis.

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.

Combined Loss of Ghrelin Receptor and Cannabinoid CB1 Receptor in Mice Decreases Survival but does not Additively Reduce Body Weight or Eating.

Ghrelin administration increases food intake, body weight (BW), adiposity, and blood glucose. In contrast, although mouse models lacking ghrelin or its receptor (Growth Hormone Secretagogue Receptor (GHSR)) exhibit life-threatening hypoglycemia in starvation-like states, they do not exhibit appreciable reductions in food intake, BW, adiposity, blood glucose, or survival when food availability is unrestricted. This suggests the existence of a parallel neuromodulatory system that can compensate for disruptions in the ghrelin system in certain settings. Here, we hypothesized that the cannabinoid CB1 receptor (CB1R) may encode this putative redundancy, and as such, that genetic deletion of both GHSR and CB1R would exaggerate the metabolic deficits associated with deletion of GHSR alone. To test this hypothesis, we assessed food intake, BW, blood glucose, survival, and plasma acyl-ghrelin in ad libitum-fed male wild-type mice and those that genetically lack GHSR (GHSR-nulls), CB1R (CB1R-nulls), or both GHSR and CB1R (double-nulls). BW, fat mass, and lean mass were similar in GHSR-nulls and wild-types, lower in CB1R-nulls, but not further reduced in double-nulls. Food intake, plasma acyl-ghrelin, and blood glucose were similar among genotypes. Deletion of either GHSR or CB1R alone did not have a statistically-significant effect on survival, but double-nulls demonstrated a statistical trend towards decreased survival (p = 0.07). We conclude that CB1R is not responsible for the normal BW, adiposity, food intake, and blood glucose observed in GHSR-null mice in the setting of unrestricted food availability. Nor is CB1R required for plasma acyl-ghrelin secretion in that setting. However, GHSR may be protective against exaggerated mortality associated with CB1R deletion.

Hypothalamic POMC neurons promote cannabinoid-induced feeding.

Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide ß-endorphin. CB1R activation selectively increases ß-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

Hepatocyte Toll-like receptor 4 regulates obesity-induced inflammation and insulin resistance.

Chronic low-grade inflammation is a hallmark of obesity and thought to contribute to the development of obesity-related insulin resistance. Toll-like receptor 4 (Tlr4) is a key mediator of pro-inflammatory responses. Mice lacking Tlr4s are protected from diet-induced insulin resistance and inflammation; however, which Tlr4-expressing cells mediate this effect is unknown. Here we show that mice deficient in hepatocyte Tlr4 (Tlr4LKO) exhibit improved glucose tolerance, enhanced insulin sensitivity and ameliorated hepatic steatosis despite the development of obesity after a high-fat diet (HFD) challenge. Furthermore, Tlr4LKO mice have reduced macrophage content in white adipose tissue, as well as decreased tissue and circulating inflammatory markers. In contrast, the loss of Tlr4 activity in myeloid cells has little effect on insulin sensitivity. Collectively, these data indicate that the activation of Tlr4 on hepatocytes contributes to obesity-associated inflammation and insulin resistance, and suggest that targeting hepatocyte Tlr4 might be a useful therapeutic strategy for the treatment of type 2 diabetes.

Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis.

Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking serotonin 2C receptors (Htr2c) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of serotonin 2C receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2c loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that serotonin 2C receptor-expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of serotonin 2C receptor agonists on weight-loss induction and improved glycemic control.

Leptin engages a hypothalamic neurocircuitry to permit survival in the absence of insulin.

The dogma that life without insulin is incompatible has recently been challenged by results showing the viability of insulin-deficient rodents undergoing leptin monotherapy. Yet, the mechanisms underlying these actions of leptin are unknown. Here, the metabolic outcomes of intracerebroventricular (i.c.v.) administration of leptin in mice devoid of insulin and lacking or re-expressing leptin receptors (LEPRs) only in selected neuronal groups were assessed. Our results demonstrate that concomitant re-expression of LEPRs only in hypothalamic γ-aminobutyric acid (GABA) and pro-opiomelanocortin (POMC) neurons is sufficient to fully mediate the lifesaving and antidiabetic actions of leptin in insulin deficiency. Our analyses indicate that enhanced glucose uptake by brown adipose tissue and soleus muscle, as well as improved hepatic metabolism, underlies these effects of leptin. Collectively, our data elucidate a hypothalamic-dependent pathway enabling life without insulin and hence pave the way for developing better treatments for diseases of insulin deficiency.

Cannabinoid receptor 1 in the vagus nerve is dispensable for body weight homeostasis but required for normal gastrointestinal motility.

The cannabinoid receptor 1 (CB(1)R) is required for body weight homeostasis and normal gastrointestinal motility. However, the specific cell types expressing CB(1)R that regulate these physiological functions are unknown. CB(1)R is widely expressed, including in neurons of the parasympathetic branches of the autonomic nervous system. The vagus nerve has been implicated in the regulation of several aspects of metabolism and energy balance (e.g., food intake and glucose balance), and gastrointestinal functions including motility. To directly test the relevance of CB(1)R in neurons of the vagus nerve on metabolic homeostasis and gastrointestinal motility, we generated and characterized mice lacking CB(1)R in afferent and efferent branches of the vagus nerve (Cnr1(flox/flox); Phox2b-Cre mice). On a chow or on a high-fat diet, Cnr1(flox/flox); Phox2b-Cre mice have similar body weight, food intake, energy expenditure, and glycemia compared with Cnr1(flox/flox) control mice. Also, fasting-induced hyperphagia and after acute or chronic pharmacological treatment with SR141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide] (CB(1)R inverse agonist) paradigms, mutants display normal body weight and food intake. Interestingly, Cnr1(flox/flox); Phox2b-Cre mice have increased gastrointestinal motility compared with controls. These results unveil CB(1)R in the vagus nerve as a key component underlying normal gastrointestinal motility.

Direct leptin action on POMC neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice.

Leptin action on its receptor (LEPR) stimulates energy expenditure and reduces food intake, thereby lowering body weight. One leptin-sensitive target cell mediating these effects on energy balance is the proopiomelano-cortin (POMC) neuron. Recent evidence suggests that the action of leptin on POMC neurons regulates glucose homeostasis independently of its effects on energy balance. Here, we have dissected the physiological impact of direct leptin action on POMC neurons using a mouse model in which endogenous LEPR expression was prevented by a LoxP-flanked transcription blocker (loxTB), but could be reactivated by Cre recombinase. Mice homozygous for the Lepr(loxTB) allele were obese and exhibited defects characteristic of LEPR deficiency. Reexpression of LEPR only in POMC neurons in the arcuate nucleus of the hypothalamus did not reduce food intake, but partially normalized energy expenditure and modestly reduced body weight. Despite the moderate effects on energy balance and independent of changes in body weight, restoring LEPR in POMC neurons normalized blood glucose and ameliorated hepatic insulin resistance, hyperglucagonemia, and dyslipidemia. Collectively, these results demonstrate that direct leptin action on POMC neurons does not reduce food intake, but is sufficient to normalize glucose and glucagon levels in mice otherwise lacking LEPR.

SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance.

Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

A treasure trove of hypothalamic neurocircuitries governing body weight homeostasis.

Changes in physical activities and feeding habits have transformed the historically rare disease of obesity into a modern metabolic pandemic. Obesity occurs when energy intake exceeds energy expenditure over time. This energy imbalance significantly increases the risk for cardiovascular disease and type 2 diabetes mellitus and as such represents an enormous socioeconomic burden and health threat. To combat obesity, a better understanding of the molecular mechanisms and neurocircuitries underlying normal body weight homeostasis is required. In the 1940s, pioneering lesion experiments unveiled the importance of medial and lateral hypothalamic structures. In the 1980s and 1990s, several neuropeptides and peripheral hormones critical for appropriate feeding behavior, energy expenditure, and hence body weight homeostasis were identified. In the 2000s, results from metabolic analyses of genetically engineered mice bearing mutations only in selected neuronal groups greatly advanced our knowledge of the peripheral/brain feedback-loop modalities by which central neurons control energy balance. In this review, we will summarize these recent progresses with particular emphasis on the biochemical identities of hypothalamic neurons and molecular components underlying normal appetite, energy expenditure, and body weight homeostasis. We will also parse which of those neurons and molecules are critical components of homeostatic adaptive pathways against obesity induced by hypercaloric feeding.

Leptin induces phosphorylation of neuronal nitric oxide synthase in defined hypothalamic neurons.

Studies have indicated that the neurotransmitter nitric oxide (NO) mediates leptin's effects in the neuroendocrine reproductive axis. However, the neurons involved in these effects and their regulation by leptin is still unknown. We aimed to determine whether NO neurons are direct targets of leptin and by which mechanisms leptin may influence neuronal NO synthase (nNOS) activity. Nicotinamide adenine dinucleotide phosphate diaphorase activity and leptin-induced phosphorylation of signal transducer and activator of transcription-3 immunoreactivity were coexpressed in subsets of neurons of the medial preoptic area, the paraventricular nucleus of the thalamus, the arcuate nucleus (Arc), the dorsomedial nucleus of the hypothalamus (DMH), the posterior hypothalamic area, the ventral premammillary nucleus (PMV), the parabrachial nucleus, and the dorsal motor nucleus of the vagus nerve. Fasting blunted nNOS mRNA expression in the medial preoptic area, Arc, DMH, PMV, and posterior hypothalamic area, and this effect was not restored by acute leptin administration. No difference in the number of neurons expressing nNOS immunoreactivity was noticed comparing hypothalamic sections of fed (wild type and ob/ob), fasted, and fasted leptin-treated mice. However, we found that in states of low leptin levels, as in fasting, or lack of leptin, as in ob/ob mice, the number of neurons expressing the phosphorylated form of nNOS is decreased in the Arc, DMH, and PMV. Notably, acute leptin administration to fasted wild-type mice restored the number of phosphorylated form of nNOS neurons to that observed in fed wild-type mice. Herein we identified the first-order neurons potentially involved in NO-mediated effects of leptin and demonstrate that leptin regulates nNOS activity predominantly through posttranslational mechanisms.

SIRT1 deacetylase in POMC neurons is required for homeostatic defenses against diet-induced obesity.

Feeding on high-calorie (HC) diets induces serious metabolic imbalances, including obesity. Understanding the mechanisms against excessive body weight gain is critical for developing effective antiobesity strategies. Here we show that lack of nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1 in pro-opiomelanocortin (POMC) neurons causes hypersensitivity to diet-induced obesity due to reduced energy expenditure. The ability of leptin to properly engage the phosphoinositide 3-kinase (PI3K) signaling in POMC neurons and elicit remodeling of perigonadal white adipose tissue (WAT) is severely compromised in mutant mice. Also, electrophysiological and histomorphomolecular analyses indicate a selective reduction in sympathetic nerve activity and brown-fat-like characteristics in perigonadal WAT of mutant mice, suggesting a physiologically important role for POMC neurons in controlling this visceral fat depot. In summary, our results provide direct genetic evidence that SIRT1 in POMC neurons is required for normal autonomic adaptations against diet-induced obesity.

Central administration of resveratrol improves diet-induced diabetes.

Resveratrol is a natural polyphenolic compound that activates nicotinamide adenosine dinucleotide-dependent deacetylase SIRT1. Resveratrol has recently been shown to exert potent antidiabetic actions when orally delivered to animal models of type 2 diabetes. However, the tissue(s) mediating these beneficial effects is unknown. Because SIRT1 is expressed in central nervous system (CNS) neurons known to control glucose and insulin homeostasis, we hypothesized that resveratrol antidiabetic effects are mediated by the brain. Here, we report that long-term intracerebroventricular infusion of resveratrol normalizes hyperglycemia and greatly improves hyperinsulinemia in diet-induced obese and diabetic mice. It is noteworthy that these effects are independent of changes in body weight, food intake, and circulating leptin levels. In addition, CNS resveratrol delivery improves hypothalamic nuclear factor-kappaB inflammatory signaling by reducing acetylated-RelA/p65 and total RelA/p65 protein contents, and inhibitor of nuclear factor-kappaB alpha and IkappaB kinase beta mRNA levels. Furthermore, this treatment leads to reduced hepatic phosphoenolpyruvate carboxykinase 1 mRNA and protein levels and ameliorates pyruvate-induced hyperglycemia in this mouse model of type 2 diabetes. Collectively, our results unveiled a previously unrecognized key role for the CNS in mediating the antidiabetic actions of resveratrol.

Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity.

A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.

Hypomorphic mutation of PGC-1beta causes mitochondrial dysfunction and liver insulin resistance.

PGC-1beta is a transcriptional coactivator that potently stimulates mitochondrial biogenesis and respiration of cells. Here, we have generated mice lacking exons 3 to 4 of the Pgc-1beta gene (Pgc-1beta(E3,4-/E3,4-) mice). These mice express a mutant protein that has reduced coactivation activity on a subset of transcription factors, including ERRalpha, a major target of PGC-1beta in the induction of mitochondrial gene expression. The mutant mice have reduced expression of OXPHOS genes and mitochondrial dysfunction in liver and skeletal muscle as well as elevated liver triglycerides. Euglycemic-hyperinsulinemic clamp and insulin signaling studies show that PGC-1beta mutant mice have normal skeletal muscle response to insulin but have hepatic insulin resistance. These results demonstrate that PGC-1beta is required for normal expression of OXPHOS genes and mitochondrial function in liver and skeletal muscle. Importantly, these abnormalities do not cause insulin resistance in skeletal muscle but cause substantially reduced insulin action in the liver.

Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice.

PGC-1alpha is a coactivator of nuclear receptors and other transcription factors that regulates several metabolic processes, including mitochondrial biogenesis and respiration, hepatic gluconeogenesis, and muscle fiber-type switching. We show here that, while hepatocytes lacking PGC-1alpha are defective in the program of hormone-stimulated gluconeogenesis, the mice have constitutively activated gluconeogenic gene expression that is completely insensitive to normal feeding controls. C/EBPbeta is elevated in the livers of these mice and activates the gluconeogenic genes in a PGC-1alpha-independent manner. Despite having reduced mitochondrial function, PGC-1alpha null mice are paradoxically lean and resistant to diet-induced obesity. This is largely due to a profound hyperactivity displayed by the null animals and is associated with lesions in the striatal region of the brain that controls movement. These data illustrate a central role for PGC-1alpha in the control of energy metabolism but also reveal novel systemic compensatory mechanisms and pathogenic effects of impaired energy homeostasis.

Adaptive thermogenesis in hummingbirds.

The occurrence of non-shivering thermogenesis in birds has long been a controversial issue. Although birds are endothermic vertebrates, sharing with mammals (placental mammals and marsupials) a common ancestor, they do not possess brown adipose tissue or a similar type of tissue, unlike their mammalian counterparts. Some bird species are, however, able to withstand very low ambient temperatures (-70 degrees C) or undergo periods of heterothermia, and there is now good experimental evidence showing that non-shivering thermogenesis may indeed occur in birds under such conditions. The skeletal muscles of birds, particularly the flight muscles, occupy a significant fraction (approximately 30 %) of the total body mass, and recent results have shown that they are likely to be the main sites for non-shivering thermogenesis. The precise mechanisms involved in adaptive thermogenesis in birds are still not fully understood. The translocation of Ca(2+) between intracellular compartments and the cystosol mediated by the sarcoplasmic reticulum Ca(2+)-ATPase, uncoupled from ATP synthesis, is one mechanism whereby chemi-osmotic energy can be converted into heat, and it has been proposed as one of the possible mechanisms underlying non-shivering thermogenesis in birds on the basis of data obtained mainly from ducklings acclimatized to cold conditions. The recent characterization of an uncoupling protein homolog in avian skeletal muscle and the expression of its mRNA at different stages of the torpor/rewarming cycle of hummingbirds indicate that it has the potential to function as an uncoupling protein and could play a thermogenic role during rewarming in these birds.