Neural control of energy balance: translating circuits to therapies.

Recent insights into the neural circuits controlling energy balance and glucose homeostasis have rekindled the hope for development of novel treatments for obesity and diabetes. However, many therapies contribute relatively modest beneficial gains with accompanying side effects, and the mechanisms of action for other interventions remain undefined. This Review summarizes current knowledge linking the neural circuits regulating energy and glucose balance with current and potential pharmacotherapeutic and surgical interventions for the treatment of obesity and diabetes.

Leptin mediates the increase in blood pressure associated with obesity.

Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin's effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

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.

A cardiac microRNA governs systemic energy homeostasis by regulation of MED13.

Obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific overexpression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.

Leptin and brain-adipose crosstalks.

Interactions between the brain and distinct adipose depots have a key role in maintaining energy balance, thereby promoting survival in response to metabolic challenges such as cold exposure and starvation. Recently, there has been renewed interest in the specific central neuronal circuits that regulate adipose depots. Here, we review anatomical, genetic and pharmacological studies on the neural regulation of adipose function, including lipolysis, non-shivering thermogenesis, browning and leptin secretion. In particular, we emphasize the role of leptin-sensitive neurons and the sympathetic nervous system in modulating the activity of brown, white and beige adipose tissues. We provide an overview of advances in the understanding of the heterogeneity of the brain regulation of adipose tissues and offer a perspective on the challenges and paradoxes that the community is facing regarding the actions of leptin on this system.

NURR1 activation in skeletal muscle controls systemic energy homeostasis.

Skeletal muscle plays a central role in the control of metabolism and exercise tolerance. Analysis of muscle enhancers activated after exercise in mice revealed the orphan nuclear receptor NURR1/NR4A2 as a prominent component of exercise-responsive enhancers. We show that exercise enhances the expression of NURR1, and transgenic overexpression of NURR1 in skeletal muscle enhances physical performance in mice. NURR1 expression in skeletal muscle is also sufficient to prevent hyperglycemia and hepatic steatosis, by enhancing muscle glucose uptake and storage as glycogen. Furthermore, treatment of obese mice with putative NURR1 agonists increases energy expenditure, improves glucose tolerance, and confers a lean phenotype, mimicking the effects of exercise. These findings identify a key role for NURR1 in governance of skeletal muscle glucose metabolism, and reveal a transcriptional link between exercise and metabolism. Our findings also identify NURR1 agonists as possible exercise mimetics with the potential to ameliorate obesity and other metabolic abnormalities.

CB1Rs in VMH neurons regulate glucose homeostasis but not body weight.

Cannabinoid 1 receptor (CB1R) inverse agonists reduce body weight and improve several parameters of glucose homeostasis. However, these drugs have also been associated with deleterious side effects. CB1R expression is widespread in the brain and in peripheral tissues, but whether specific sites of expression can mediate the beneficial metabolic effects of CB1R drugs, while avoiding the untoward side effects, remains unclear. Evidence suggests inverse agonists may act on key sites within the central nervous system to improve metabolism. The ventromedial hypothalamus (VMH) is a critical node regulating energy balance and glucose homeostasis. To determine the contributions of CB1Rs expressed in VMH neurons in regulating metabolic homeostasis, we generated mice lacking CB1Rs in the VMH. We found that the deletion of CB1Rs in the VMH did not affect body weight in chow- and high-fat diet-fed male and female mice. We also found that deletion of CB1Rs in the VMH did not alter weight loss responses induced by the CB1R inverse agonist SR141716. However, we did find that CB1Rs of the VMH regulate parameters of glucose homeostasis independent of body weight in diet-induced obese male mice.NEW & NOTEWORTHY Cannabinoid 1 receptors (CB1Rs) regulate metabolic homeostasis, and CB1R inverse agonists reduce body weight and improve parameters of glucose metabolism. However, the cell populations expressing CB1Rs that regulate metabolic homeostasis remain unclear. CB1Rs are highly expressed in the ventromedial hypothalamic nucleus (VMH), which is a crucial node that regulates metabolism. With CRISPR/Cas9, we generated mice lacking CB1Rs specifically in VMH neurons and found that CB1Rs in VMH neurons are essential for the regulation of glucose metabolism independent of body weight regulation.

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.

MC4R-expressing glutamatergic neurons in the paraventricular hypothalamus regulate feeding and are synaptically connected to the parabrachial nucleus.

Activation of melanocortin-4 receptors (MC4Rs) restrains feeding and prevents obesity; however, the identity, location, and axonal projections of the neurons bearing MC4Rs that control feeding remain unknown. Reexpression of MC4Rs on single-minded 1 (SIM1)(+) neurons in mice otherwise lacking MC4Rs is sufficient to abolish hyperphagia. Thus, MC4Rs on SIM1(+) neurons, possibly in the paraventricular hypothalamus (PVH) and/or amygdala, regulate food intake. It is unknown, however, whether they are also necessary, a distinction required for excluding redundant sites of action. Hence, the location and nature of obesity-preventing MC4R-expressing neurons are unknown. Here, by deleting and reexpressing MC4Rs from cre-expressing neurons, establishing both necessity and sufficiency, we demonstrate that the MC4R-expressing neurons regulating feeding are SIM1(+), located in the PVH, glutamatergic and not GABAergic, and do not express oxytocin, corticotropin-releasing hormone, vasopressin, or prodynorphin. Importantly, these excitatory MC4R-expressing PVH neurons are synaptically connected to neurons in the parabrachial nucleus, which relays visceral information to the forebrain. This suggests a basis for the feeding-regulating effects of MC4Rs.

Dnmt3a in Sim1 neurons is necessary for normal energy homeostasis.

Obesity rates continue to rise throughout the world. Recent evidence has suggested that environmental factors contribute to altered energy balance regulation. However, the role of epigenetic modifications to the central control of energy homeostasis remains unknown. To investigate the role of DNA methylation in the regulation of energy balance, we investigated the role of the de novo DNA methyltransferase, Dnmt3a, in Single-minded 1 (Sim1) cells, including neurons in the paraventricular nucleus of the hypothalamus (PVH). Dnmt3a expression levels were decreased in the PVH of high-fat-fed mice. Mice lacking Dnmt3a specifically in the Sim1 neurons, which are expressed in the forebrain, including PVH, became obese with increased amounts of abdominal and subcutaneous fat. The mice were also found to have hyperphagia, decreased energy expenditure, and glucose intolerance with increased serum insulin and leptin. Furthermore, these mice developed hyper-LDL cholesterolemia when fed a high-fat diet. Gene expression profiling and DNA methylation analysis revealed that the expression of tyrosine hydroxylase and galanin were highly upregulated in the PVH of Sim1-specific Dnmt3a deletion mice. DNA methylation levels of the tyrosine hydroxylase promoter were decreased in the PVH of the deletion mice. These results suggest that Dnmt3a in the PVH is necessary for the normal control of body weight and energy homeostasis and that tyrosine hydroxylase is a putative target of Dnmt3a in the PVH. These results provide evidence for a role for Dnmt3a in the PVH to link environmental conditions to altered energy homeostasis.

Shift in Kiss1 cell activity requires estrogen receptor α.

Reproductive function requires timely secretion of gonadotropin-releasing hormone, which is controlled by a complex excitatory/inhibitory network influenced by sex steroids. Kiss1 neurons are fundamental players in this network, but it is currently unclear whether different conditions of circulating sex steroids directly alter Kiss1 neuronal activity. Here, we show that Kiss1 neurons in the anteroventral periventricular and anterior periventricular nuclei (AVPV/PeN) of males and females exhibit a bimodal resting membrane potential (RMP) influenced by K(ATP) channels, suggesting the presence of two neuronal populations defined as type I (irregular firing patterns) and type II (quiescent). Kiss1 neurons in the arcuate nucleus (Arc) are also composed of firing and quiescent cells, but unlike AVPV/PeN neurons, the range of RMPs did not follow a bimodal distribution. Moreover, Kiss1 neuronal activity in the AVPV/PeN, but not in the Arc, is sexually dimorphic. In females, estradiol shifts the firing pattern of AVPV/PeN Kiss1 neurons and alters cell capacitance and spontaneous IPSCs amplitude of AVPV/PeN and Arc Kiss1 populations in an opposite manner. Notably, mice with selective deletion of estrogen receptor α (ERα) from Kiss1 neurons show cellular activity similar to that observed in ovariectomized females, suggesting that estradiol-induced changes in Kiss1 cellular properties require ERα. We also show that female prepubertal Kiss1 neurons are under higher inhibitory influence and all recorded AVPV/PeN Kiss1 neurons were spontaneously active. Collectively, our findings indicate that changes in cellular activity may underlie Kiss1 action in pubertal initiation and female reproduction.

Elevated resistin levels induce central leptin resistance and increased atherosclerotic progression in mice.

AIMS/HYPOTHESIS: Resistin was originally identified as an adipocyte-derived factor upregulated during obesity and as a contributor to obesity-associated insulin resistance. Clinically, resistin has also been implicated in cardiovascular disease in a number of different patient populations. Our aim was to simultaneously address these phenomena. METHODS: We generated mice with modest adipocyte-specific resistin overexpression. These mice were crossed with mice deficient in the LDL receptor (Ldlr (-/-)) to probe the physiological role of resistin. Both metabolic and atherosclerotic assessments were performed. RESULTS: Resistin overexpression led to increased atherosclerotic progression in Ldlr (-/-) mice. This was in part related to elevated serum triacylglycerol levels and a reduced ability to clear triacylglycerol upon a challenge. Additional phenotypic changes, such as increased body weight and reduced glucose clearance, independent of the Ldlr (-/-) background, confirmed increased adiposity associated with a more pronounced insulin resistance. A hallmark of elevated resistin was the disproportionate increase in circulating leptin levels. These mice thus recapitulated both the proposed negative cardiovascular correlation and the insulin resistance. A unifying mechanism for this complex phenotype was a resistin-mediated central leptin resistance, which we demonstrate directly both in vivo and in organotypic brain slices. In line with reduced sympathetic nervous system outflow, we found decreased brown adipose tissue (BAT) activity. The resulting elevated triacylglycerol levels provide a likely explanation for accelerated atherosclerosis. CONCLUSIONS/INTERPRETATION: Resistin overexpression leads to a complex metabolic phenotype driven by resistin-mediated central leptin resistance and reduced BAT activity. Hypothalamic leptin resistance thus provides a unifying mechanism for both resistin-mediated insulin resistance and enhanced atherosclerosis.

Shp2 signaling in POMC neurons is important for leptin's actions on blood pressure, energy balance, and glucose regulation.

Previous studies showed that Src homology-2 tyrosine phosphatase (Shp2) is an important regulator of body weight. In this study, we examined the impact of Shp2 deficiency specifically in proopiomelanocortin (POMC) neurons on metabolic and cardiovascular function and on chronic blood pressure (BP) and metabolic responses to leptin. Mice with Shp2 deleted in POMC neurons (Shp2/Pomc-cre) and control mice (Shp2(flox/flox)) were implanted with telemetry probes and venous catheters for measurement of mean arterial pressure (MAP) and leptin infusion. After at least 5 days of stable control measurements, mice received leptin infusion (2 µg·kg(-1)·day(-1) iv) for 7 days. Compared with Shp2(flox/flox) controls, Shp2/Pomc-cre mice at 22 wk of age were slightly heavier (34 ± 1 vs. 31 ± 1 g) but consumed a similar amount of food (3.9 ± 0.3 vs. 3.8 ± 0.2 g/day). Leptin infusion reduced food intake in Shp2(flox/flox) mice (2.6 ± 0.5 g) and Shp2/Pomc-cre mice (3.2 ± 0.3 g). Despite decreasing food intake, leptin infusion increased MAP in control mice, whereas no significant change in MAP was observed in Shp2/Pomc-cre mice. Leptin infusion also decreased plasma glucose and insulin levels in controls (12 ± 1 to 6 ± 1 µU/ml and 142 ± 12 to 81 ± 8 mg/100 ml) but not in Shp2/Pomc-cre mice. Leptin increased V̇o2 by 16 ± 2% in controls and 7 ± 1% in Shp2/Pomc-cre mice. These results indicate that Shp2 signaling in POMC neurons contributes to the long-term BP and antidiabetic actions of leptin and may play a modest role in normal regulation of body weight.

Adipose tissue biology and cardiomyopathy: translational implications.

It is epidemiologically established that obesity is frequently associated with the metabolic syndrome and poses an increased risk for the development of type 2 diabetes mellitus and cardiovascular disease. The molecular links that connect the phenomenon of obesity, per se, with insulin resistance and cardiovascular disease are still not fully elucidated. It is increasingly apparent that fully functional adipose tissue can be cardioprotective by reducing lipotoxic effects in other peripheral tissues and by maintaining a healthy balance of critical adipokines, thereby allowing the heart to maintain its full metabolic flexibility. The present review highlights both basic and clinical findings that emphasize the complex interplay of adipose tissue physiology and adipokine-mediated effects on the heart exerted by either direct effects on cardiac myocytes or indirect actions via central mechanisms through sympathetic outflow to the heart.

Steroidogenic factor 1 directs programs regulating diet-induced thermogenesis and leptin action in the ventral medial hypothalamic nucleus.

The transcription factor steroidogenic factor 1 (SF-1) is exclusively expressed in the brain in the ventral medial hypothalamic nucleus (VMH) and is required for the development of this nucleus. However, the physiological importance of transcriptional programs regulated by SF-1 in the VMH is not well defined. To delineate the functional significance of SF-1 itself in the brain, we generated pre- and postnatal VMH-specific SF-1 KO mice. Both models of VMH-specific SF-1 KO were susceptible to high fat diet-induced obesity and displayed impaired thermogenesis after acute exposure to high fat diet. Furthermore, VMH-specific SF-1 KO mice showed significantly decreased LepR expression specifically in the VMH, leading to leptin resistance. Collectively, these results indicate that SF-1 directs transcriptional programs in the hypothalamus relevant to coordinated control of energy homeostasis, especially after excess caloric intake.

Orexin receptor 2 expression in the posterior hypothalamus rescues sleepiness in narcoleptic mice.

Narcolepsy is caused by a loss of orexin/hypocretin signaling, resulting in chronic sleepiness, fragmented non-rapid eye movement sleep, and cataplexy. To identify the neuronal circuits underlying narcolepsy, we produced a mouse model in which a loxP-flanked gene cassette disrupts production of the orexin receptor type 2 (OX2R; also known as HCRTR2), but normal OX2R expression can be restored by Cre recombinase. Mice lacking OX2R signaling had poor maintenance of wakefulness indicative of sleepiness and fragmented sleep and lacked any electrophysiological response to orexin-A in the wake-promoting neurons of the tuberomammillary nucleus. These defects were completely recovered by crossing them with mice that express Cre in the female germline, thus globally deleting the transcription-disrupter cassette. Then, by using an adeno-associated viral vector coding for Cre recombinase, we found that focal restoration of OX2R in neurons of the tuberomammillary nucleus and adjacent parts of the posterior hypothalamus completely rescued the sleepiness of these mice, but their fragmented sleep was unimproved. These observations demonstrate that the tuberomammillary region plays an essential role in the wake-promoting effects of orexins, but orexins must stabilize sleep through other targets.

Leptin Reduction as a Required Component for Weight Loss.

Partial leptin reduction can induce significant weight loss, while weight loss contributes to partial leptin reduction. The cause-and-effect relationship between leptin reduction and weight loss remains to be further elucidated. Here, we show that FGF21 and the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide rapidly induced a reduction in leptin. This leptin reduction contributed to the beneficial effects of GLP-1R agonism in metabolic health, as transgenically maintaining leptin levels during treatment partially curtailed the beneficial effects seen with these agonists. Moreover, a higher degree of leptin reduction during treatment, induced by including a leptin neutralizing antibody with either FGF21 or liraglutide, synergistically induced greater weight loss and better glucose tolerance in diet-induced obese mice. Furthermore, upon cessation of either liraglutide or FGF21 treatment, the expected immediate weight regain was observed, associated with a rapid increase in circulating leptin levels. Prevention of this leptin surge with leptin neutralizing antibodies slowed down weight gain and preserved better glucose tolerance. Mechanistically, a significant reduction in leptin induced a higher degree of leptin sensitivity in hypothalamic neurons. Our observations support a model that postulates that a reduction of leptin levels is a necessary prerequisite for substantial weight loss, and partial leptin reduction is a viable strategy to treat obesity and its associated insulin resistance.

Interaction between the gut microbiota and colonic enteroendocrine cells regulates host metabolism.

Nutrient handling is an essential function of the gastrointestinal tract. Hormonal responses of small intestinal enteroendocrine cells (EECs) have been extensively studied but much less is known about the role of colonic EECs in metabolic regulation. To address this core question, we investigated a mouse model deficient in colonic EECs. Here we show that colonic EEC deficiency leads to hyperphagia and obesity. Furthermore, colonic EEC deficiency results in altered microbiota composition and metabolism, which we found through antibiotic treatment, germ-free rederivation and transfer to germ-free recipients, to be both necessary and sufficient for the development of obesity. Moreover, studying stool and blood metabolomes, we show that differential glutamate production by intestinal microbiota corresponds to increased appetite and that colonic glutamate administration can directly increase food intake. These observations shed light on an unanticipated host-microbiota axis in the colon, part of a larger gut-brain axis, that regulates host metabolism and body weight.

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.

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.