Sexually different actions of leptin in proopiomelanocortin neurons to regulate glucose homeostasis

Leptin regulates energy balance and glucose homeostasis, at least in part, via activation of receptors in the arcuate nucleus of the hypothalamus located in proopiomelanocortin (POMC) neurons. Females have greater sensitivity to central leptin than males, suggested by a greater anorectic effect of central leptin administration in females. We hypothesized that the regulation of energy balance and peripheral glucose homeostasis of female rodents would be affected to a greater extent than in males if the action of leptin in POMC neurons were disturbed. Male and female mice lacking leptin receptors only in POMC neurons gained significantly more body weight and accumulated more body fat. However, female mice gained disproportionately more visceral adiposity than males, and this appeared to be largely the result of differences in energy expenditure. When maintained on a high-fat diet (HFD), both male and female mutants had higher levels of insulin following exogenous glucose challenges. Chow- and HFD-fed males but not females had abnormal glucose disappearance curves following insulin administrations. Collectively, these data indicate that the action of leptin in POMC neurons is sexually different to influence the regulation of energy balance, fat distribution, and glucose homeostasis.     

Endothelial leptin receptor mutation provides partial resistance to diet-induced obesity

Leptin, a polypeptide hormone produced mainly by adipocytes, has diverse effects in both the brain and peripheral organs, including suppression of feeding. Other than mediating leptin transport across the blood-brain barrier, the role of the endothelial leptin receptor remains unclear. We recently generated a mutant mouse strain lacking endothelial leptin receptor signaling, and showed that there is an increased uptake of leptin by brain parenchyma after its delivery by in situ brain perfusion. Here, we tested the hypothesis that endothelial leptin receptor mutation confers partial resistance to diet-induced obesity. These ELKO mice had similar body weight and percent fat as their wild-type littermates when fed with rodent chow, but blood concentrations of leptin were significantly elevated. In response to a high-fat diet, wild-type mice had a greater gain of body weight and fat than ELKO mice. As shown by metabolic chamber measurement, the ELKO mice had higher oxygen consumption, carbon dioxide production, and heat dissipation, although food intake was similar to that of the wild-type mice and locomotor activity was even reduced. This indicates that the partial resistance to diet-induced obesity was mediated by higher metabolic activity in the ELKO mice. Since neuronal leptin receptor knockout mice show obesity and diabetes, the results suggest that endothelial leptin signaling shows opposite effects from that of neuronal leptin signaling, with a facilitatory role in diet-induced obesity.     

Overexpression of leptin in transgenic mice leads to decreased basal lipolysis, PKA activity, and perilipin levels

Transgenic mice overexpressing leptin (LepTg) exhibit substantial reductions in adipose mass. Since the binding of leptin to its receptor activates the sympathetic nervous system, we reasoned that the lean state of the LepTg mice could be caused by chronic lipolysis. Instead, the LepTg mice exhibited a low basal lipolysis state and their lean phenotype was not dependent on the presence of beta3-adrenergic receptors. In their white adipose tissue, protein levels of protein kinase A, hormone-sensitive lipase, and ADRP were not impaired. However, compared to normal mice, perilipin, perilipin mRNA, and cAMP-stimulated PKA activity were significantly attenuated. Overall, we demonstrate that the lean phenotype of the LepTg mice does not result in a chronically elevated lipolytic state, but instead in a low basal lipolysis state characterized by a decrease in perilipin and PKA activity in white fat.     

Effects of dietary fat types on body fatness,leptin, and ARC leptin receptor,NPY, and AgRP mRNA expression

Some, but not all, fats are obesogenic. The aim of the present studies was to investigate the effects of changing type and amount of dietary fats on energy balance, fat deposition, leptin, and leptin-related neural peptides: leptin receptor, neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC), in C57Bl/6J mice. One week of feeding with a highly saturated fat diet resulted in ~50 and 20% reduction in hypothalamic arcuate NPY and AgRP mRNA levels, respectively, compared with a low-fat or an n-3 or n-6 polyunsaturated high-fat (PUFA) diet without change in energy intake, fat mass, plasma leptin levels, and leptin receptor or POMC mRNA. Similar neuropeptide results were seen at 7 wk, but by then epididymal fat mass and plasma leptin levels were significantly elevated in the saturated fat group compared with low-fat controls. In contrast, fat and leptin levels were reduced in the n-3 PUFA group compared with all other groups. At 7 wk, changing the saturated fat group to n-3 PUFA for 4 wk completely reversed the hyperleptinemia and increased adiposity and neuropeptide changes induced by saturated fat. Changing to a low-fat diet was much less effective. In summary, a highly saturated fat diet induces obesity without hyperphagia. A regulatory reduction in NPY and AgRP mRNA levels is unable to effectively counteract this obesogenic drive. Equally high fat diets emphasizing PUFAs may even protect against obesity.     

Adipose tissue as an endocrine organ regulating growth, puberty, and other physiological functions.

There are reports on some patients with clearly manifested specific features of genotype and phenotype similar to those of ob/ob and db/db mice. Three patients from Turkey were described who had a homozygous mutation in the gene of leptin identical to the mutation in C57BL6J ob/ob mice. This mutation is a C --> T substitution in codon 105 of the amino acid sequence of leptin. In mice this mutation generates a stop-codon; in humans it substitutes Arg-105 with Trp. The mutant human leptin cannot be secreted by the cells and thus has no effect on the hypothalamus. Patients with a homozygous mutation of the leptin receptor resulting in the G --> T substitution in the splice donor site of exon 16 were studied in a family of Kabilian origin. Exon 16 was not included in the mature mRNA molecule, and a truncated leptin receptor was synthesized which lacked the transmembrane and intracellular domains; this receptor was unable to transduce the hormonal signal. Both groups of patients suffered from obesity, delayed linear growth, infertility, increased blood insulin level, and other disorders. Leptin influences lipid metabolism by stimulating the expression of the proopiomelanocortin (POMC) gene in melanocortinergic neurons of the hypothalamus. POMC is the precursor of alpha-melanocyte-stimulating hormone (alpha-MSH), which binds to the melanocortin receptor MC4-R in the brain, decreases appetite, and activates lipid metabolism. Patients with mutations in MC4-R suffered only from obesity, but their growth and puberty were not affected. Thus, leptin apparently stimulates growth and puberty not through its binding to the receptors on melanocortinergic neurons, but through its binding to receptors on other hypothalamic neurons; this effect of leptin is not affected by mutations in the MC4-R gene.     

Method of leptin dosing,strain, and group housing influence leptin sensitivity in high-fat-fed weanling mice

High-fat diets are reported to induce resistance to peripherally administered leptin. In an attempt to develop a model of juvenile diet-induced obesity, mice were weaned onto high-fat diet. Male and female, 35-day-old, C57BL/6J high-fat (45% kcal fat) diet-fed mice housed individually on grid floors did not decrease food intake or body weight in response to intraperitoneal (30 microg), lateral ventricle (5 microg), or third ventricle (0.5 microg) injections of leptin. Body weight and fat were significantly reduced by 13-day intraperitoneal infusions of 10 microg leptin/day, which doubled circulating leptin. Leptin infusion also reduced body fat in weanling, high-fat diet-fed NIH Swiss mice. Group housing mice on bedding prevented loss of fat in high-fat diet-fed male and female NIH Swiss and female C57BL/6J mice. These results indicate that peripherally infused leptin reduces fat in part by increasing thermogenesis and that inhibition of food intake in high-fat diet-fed mice requires either chronic activation of central leptin receptors or is independent of receptors that inhibit feeding in response to an acute central injection of leptin.     

Leptin-Dependent Control of Glucose Balance and Locomotor Activity by POMC Neurons

Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways, leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that, specifically, pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility, we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor-deficient Lepr^db/db mice. Here, we show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Lepr^db/db mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Lepr^db/db mice.     

Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells

Suppressor of cytokine signaling-3 (Socs-3) negatively regulates the action of various cytokines, as well as the metabolic hormones leptin and insulin. Mice with haploinsufficiency of Socs-3, or those with neuronal deletion of Socs-3, are lean and more leptin and insulin sensitive. To examine the role of Socs-3 within specific neurons critical to energy balance, we created mice with selective deletion of Socs-3 within pro-opiomelanocortin (POMC)-expressing cells. These mice had enhanced leptin sensitivity, measured by weight loss and food intake after leptin infusion. On chow diet, glucose homeostasis was improved despite normal weight gain. On a high-fat diet, the rate of weight gain was reduced, due to increased energy expenditure rather than decreased food intake; glucose homeostasis and insulin sensitivity were substantially improved. These studies demonstrate that Socs-3 within POMC neurons regulates leptin sensitivity and glucose homeostasis, and plays a key role in linking high-fat diet to disordered metabolism.     

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β₃ adrenergic receptor (β₃‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β₃‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.     

Serotonin 2C receptor activates a distinct population of arcuate pro-opiomelanocortin neurons via TRPC channels

Serotonin 2C receptors (5-HT(2C)Rs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT(2C)Rs have not yet been identified. In the present study, we found the putative transient receptor potential C (TRPC) channels mediate the activation of a subpopulation of POMC neurons by mCPP (a?5-HT(2C)R agonist). Interestingly, mCPP-activated POMC neurons were found to be a distinct population from those activated by leptin. Together, our data suggest that 5-HT(2C)R and leptin receptors are expressed by distinct subpopulations of arcuate POMC neurons and that both 5-HT and leptin exert their actions in POMC neurons via TRPC channels. VIDEO ABSTRACT:     

Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis

Neuroanatomical and electrophysiological studies have shown that hypothalamic POMC neurons are targets of the adipostatic hormone leptin. However, the physiological relevance of leptin signaling in these neurons has not yet been directly tested. Here, using the Cre/loxP system, we critically test the functional importance of leptin action on POMC neurons by deleting leptin receptors specifically from these cells in mice. Mice lacking leptin signaling in POMC neurons are mildly obese, hyperleptinemic, and have altered expression of hypothalamic neuropeptides. In summary, leptin receptors on POMC neurons are required but not solely responsible for leptin's regulation of body weight homeostasis.     

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb ^+/+ mice and in Leprb ^db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.     

The melanocortin system and energy balance

The melanocortins, a family of peptides produced from the post-translational processing of pro-opiomelanocortin (POMC), regulate ingestive behavior and energy expenditure. Loss of function mutations of genes encoding POMC, or of either of two melanocortin receptors expressed in the central nervous system (MC3R, MC4R), are associated with obesity. The analyses of MC4R knockout mice indicate that activation of this receptor is involved in the regulation of appetite, the adaptive metabolic response to excess caloric consumption, and negative energy balance associated with cachexia induced by cytokines. In contrast, MC3R knockout mice exhibit a normal, or even exaggerated, response to signals that induce a state of negative energy balance. However, loss of the MC3R also results in an increase in adiposity. This article discusses the regulation of energy balance by the melanocortins. Published and newly presented data from studies analyzing of energy balance of MC3R and MC4R knockout mice indicate that increased adiposity observed in both models involves an imbalance in fat intake and oxidation.     

Increased Adiposity in Annexin A1-Deficient Mice

Production of Annexin A1 (ANXA1), a protein that mediates the anti-inflammatory action of glucocorticoids, is altered in obesity, but its role in modulation of adiposity has not yet been investigated. The objective of this study was to investigate modulation of ANXA1 in adipose tissue in murine models of obesity and to study the involvement of ANXA1 in diet-induced obesity in mice. Significant induction of ANXA1 mRNA was observed in adipose tissue of both C57BL6 and Balb/c mice with high fat diet (HFD)-induced obesity versus mice on chow diet. Upregulation of ANXA1 mRNA was independent of leptin or IL-6, as demonstrated by use of leptin-deficient ob/ob mice and IL-6 KO mice. Compared to WT mice, female Balb/c ANXA1 KO mice on HFD had increased adiposity, as indicated by significantly elevated body weight, fat mass, leptin levels, and adipocyte size. Whereas Balb/c WT mice upregulated expression of enzymes involved in the lipolytic pathway in response to HFD, this response was absent in ANXA1 KO mice. A significant increase in fasting glucose and insulin levels as well as development of insulin resistance was observed in ANXA1 KO mice on HFD compared to WT mice. Elevated plasma corticosterone levels and blunted downregulation of 11-beta hydroxysteroid dehydrogenase type 1 in adipose tissue was observed in ANXA1 KO mice compared to diet-matched WT mice. However, no differences between WT and KO mice on either chow or HFD were observed in expression of markers of adipose tissue inflammation.These data indicate that ANXA1 is an important modulator of adiposity in mice, with female ANXA1 KO mice on Balb/c background being more susceptible to weight gain and diet-induced insulin resistance compared to WT mice, without significant changes in inflammation.     

β‐Aminoisobutyric Acid Prevents Diet‐induced Obesity in Mice With Partial Leptin Deficiency

β‐Aminoisobutyric acid (BAIBA), a thymine catabolite, increases fatty acid oxidation (FAO) in liver and reduces the gain of body fat mass in Swiss (lean) mice fed a standard chow. We determined whether BAIBA could prevent obesity and related metabolic disorders in different murine models. To this end, BAIBA (100 or 500 mg/kg/day) was administered for 4 months in mice totally deficient in leptin (ob/ob). BAIBA (100 mg/kg/day) was also given for 4 months in wild‐type (+/+) mice and mice partially deficient in leptin (ob/+) fed a high‐calorie (HC) diet. BAIBA did not limit obesity and hepatic steatosis in ob/ob mice, but reduced liver cytolysis and inflammation. In ob/+ mice fed the HC diet, BAIBA fully prevented, or limited, the gain of body fat, steatosis and necroinflammation, glucose intolerance, and hypertriglyceridemia. Plasma β‐hydroxybutyrate was increased, whereas expression of carnitine palmitoyltransferase‐1 was augmented in liver and white adipose tissue. Acetyl‐CoA carboxylase was more phosphorylated, and de novo lipogenesis was less induced in liver. These favorable effects of BAIBA in ob/+ mice were associated with a restoration of plasma leptin levels. The reduction of body adiposity afforded by BAIBA was less marked in +/+ mice. Finally, BAIBA significantly stimulated the secretion of leptin in isolated ob/+ adipose cells, but not in +/+ cells. Thus, BAIBA could limit triglyceride accretion in tissues through a leptin‐dependent stimulation of FAO. As partial leptin deficiency is not uncommon in the general population, supplementation with BAIBA may help to prevent diet‐induced obesity and related metabolic disorders in low leptin secretors.