Gsα deficiency in adipose tissue improves glucose metabolism and insulin sensitivity without an effect on body weight.

Gsα, the G protein that transduces receptor-stimulated cAMP generation, mediates sympathetic nervous system stimulation of brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT), which are both potential targets for treating obesity, as well as lipolysis. We generated a mouse line with Gsα deficiency in mature BAT and WAT adipocytes (Ad-GsKO). Ad-GsKO mice had impaired BAT function, absent browning of WAT, and reduced lipolysis, and were therefore cold-intolerant. Despite the presence of these abnormalities, Ad-GsKO mice maintained normal energy balance on both standard and high-fat diets, associated with decreases in both lipolysis and lipid synthesis. In addition, Ad-GsKO mice maintained at thermoneutrality on a standard diet also had normal energy balance. Ad-GsKO mice had improved insulin sensitivity and glucose metabolism, possibly secondary to the effects of reduced lipolysis and lower circulating fatty acid binding protein 4 levels. Gsα signaling in adipose tissues may therefore affect whole-body glucose metabolism in the absence of an effect on body weight.

Neural innervation of white adipose tissue and the control of lipolysis.

White adipose tissue (WAT) is innervated by the sympathetic nervous system (SNS) and its activation is necessary for lipolysis. WAT parasympathetic innervation is not supported. Fully-executed SNS-norepinephrine (NE)-mediated WAT lipolysis is dependent on ß-adrenoceptor stimulation ultimately hinging on hormone sensitive lipase and perilipin A phosphorylation. WAT sympathetic drive is appropriately measured electrophysiologically and neurochemically (NE turnover) in non-human animals and this drive is fat pad-specific preventing generalizations among WAT depots and non-WAT organs. Leptin-triggered SNS-mediated lipolysis is weakly supported, whereas insulin or adenosine inhibition of SNS/NE-mediated lipolysis is strongly supported. In addition to lipolysis control, increases or decreases in WAT SNS drive/NE inhibit and stimulate white adipocyte proliferation, respectively. WAT sensory nerves are of spinal-origin and sensitive to local leptin and increases in sympathetic drive, the latter implicating lipolysis. Transsynaptic viral tract tracers revealed WAT central sympathetic and sensory circuits including SNS-sensory feedback loops that may control lipolysis.

Gsα deficiency in the dorsomedial hypothalamus leads to obesity, hyperphagia, and reduced thermogenesis associated with impaired leptin signaling.

OBJECTIVE: Gsα couples multiple receptors, including the melanocortin 4 receptor (MC4R), to intracellular cAMP generation. Germline inactivating Gsα mutations lead to obesity in humans and mice. Mice with brain-specific Gsα deficiency also develop obesity with reduced energy expenditure and locomotor activity, and impaired adaptive thermogenesis, but the underlying mechanisms remain unclear. METHODS: We created mice (DMHGsKO) with Gsα deficiency limited to the dorsomedial hypothalamus (DMH) and examined the effects on energy balance and thermogenesis. RESULTS: DMHGsKO mice developed severe, early-onset obesity associated with hyperphagia and reduced energy expenditure and locomotor activity, along with impaired brown adipose tissue thermogenesis. Studies in mice with loss of MC4R in the DMH suggest that defective DMH MC4R/Gsα signaling contributes to abnormal energy balance but not to abnormal locomotor activity or cold-induced thermogenesis. Instead, DMHGsKO mice had impaired leptin signaling along with increased expression of the leptin signaling inhibitor protein tyrosine phosphatase 1B in the DMH, which likely contributes to the observed hyperphagia and reductions in energy expenditure, locomotor activity, and cold-induced thermogenesis. CONCLUSIONS: DMH Gsα signaling is critical for energy balance, thermogenesis, and leptin signaling. This study provides insight into how distinct signaling pathways can interact to regulate energy homeostasis and temperature regulation.

Gsα deficiency in the dorsomedial hypothalamus underlies obesity associated with Gsα mutations.

Gsα, encoded by Gnas, mediates hormone and neurotransmitter receptor-stimulated cAMP generation. Heterozygous Gsα-inactivating mutations lead to obesity in Albright hereditary osteodystrophy (AHO) patients, but only when the mutations occur on the maternal allele. This parent-of-origin effect is due to Gsα imprinting in the CNS, although the relevant CNS regions are unknown. We have now shown that mice with a Gnas gene deletion disrupting Gsα expression on the maternal allele, but not the paternal allele, in the dorsomedial nucleus of the hypothalamus (DMH) developed obesity and reduced energy expenditure without hyperphagia. Although maternal Gnas deletion impaired activation of brown adipose tissue (BAT) in mice, their responses to cold environment remained intact. Similar findings were observed in mice with DMH-specific deficiency of melanocortin MC4R receptors, which are known to activate Gsα. Our results show that Gsα imprinting in the DMH underlies the parent-of-origin metabolic phenotype that results from Gsα mutations and that DMH MC4R/Gsα signaling is important for regulation of energy expenditure and BAT activation, but not the metabolic response to cold.

Role of AgRP on Ghrelin-induced feeding in the hypothalamic paraventricular nucleus.

MT II, agonist for MC3/4-Rs, inhibited Ghrelin's orexigenic effect in the paraventricular nucleus of the hypothalamus (PVN). To further investigate the role of the melanocortin system as mediator of ghrelin's orexigenic actions, we explored the involvement of AgRP in Ghrelin's orexigenic effect by testing the effect on food intake after their co-administration in the PVN, during the light and dark phases of feeding in rats. During both the phases of feeding, co-administration of Ghrelin with either AgRP 50 or AgRP 100 pmol into the PVN did not produce a synergistic effect on the food intake, suggesting that ghrelin induction of feeding occurs by recruiting Agrp as one of the obligatory mediators of its orexigenic effect.

The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis.

Recent research progress indicates a close link between ghrelin, a natural ligand of GH secretagogues receptor (GHS-R), and both the metabolic balance and body composition. To clarify the involvement of ghrelin and GHS-R in the process of adipogenesis, we measured the expression of GHS-R and peroxisome proliferator-activated receptor gamma 2 (PPAR-gamma 2) mRNA in rat adipocytes using semiquantitative RT-PCR methods. The levels of GHS-R mRNA increased by up to 4-fold in adipose tissue from epididymal and parametrial regions as the rat aged from 4-20 wk and were significantly elevated during the differentiation of preadipocytes in vitro. Ghrelin (10(-8) M for 10 d) stimulated the activity of glycerol-3-phosphate dehydrogenase and the differentiation of rat preadipocytes in vitro. Ghrelin treatment also significantly increased the levels of PPAR-gamma 2 mRNA in primary cultured rat differentiated adipocytes. In addition, isoproterenol (10(-8) M, 40 min)-stimulated lipolysis was significantly reduced by simultaneous ghrelin treatment in a dose-dependent manner in vitro. In conclusion, the expression of GHS-R in rat adipocytes increases with the age and during adipogenesis. Ghrelin in vitro stimulates the differentiation of preadipocytes and antagonizes lipolysis. Ghrelin may therefore play an important role in the process of adipogenesis in rats.

Action of MT-II on ghrelin-induced feeding in the paraventricular nucleus of the hypothalamus.

Ghrelin is a 28 amino-acid peptide that has been shown to induce positive energy balance when administered both peripherally and centrally. This effect appears to occur by increasing food intake and by reducing fat utilization. Ghrelin injected into the PVN increases food intake dose-dependently. The NPY receptor has been implicated in the orexigenic effect of ghrelin, but until now, the role of melanocortins on the effect of ghrelin in the PVN has not been reported. Sprague-Dawley rats were stimulated to eat by PVN ghrelin. Pre-injection of 10 pmol of MT II into the PVN caused a significant decrease in ghrelin-induced feeding in both 0-1 h and 0-4 h food intake studies. This finding indicates that MC 3/4-R signaling appears to be recruited by ghrelin, in the PVN, in its role to induce feeding.

Pmch-deficiency in rats is associated with normal adipocyte differentiation and lower sympathetic adipose drive.

The orexigenic neuropeptide melanin-concentrating hormone (MCH), a product of Pmch, is an important mediator of energy homeostasis. Pmch-deficient rodents are lean and smaller, characterized by lower food intake, body-, and fat mass. Pmch is expressed in hypothalamic neurons that ultimately are components in the sympathetic nervous system (SNS) drive to white and interscapular brown adipose tissue (WAT, iBAT, respectively). MCH binds to MCH receptor 1 (MCH1R), which is present on adipocytes. Currently it is unknown if Pmch-ablation changes adipocyte differentiation or sympathetic adipose drive. Using Pmch-deficient and wild-type rats on a standard low-fat diet, we analyzed dorsal subcutaneous and perirenal WAT mass and adipocyte morphology (size and number) throughout development, and indices of sympathetic activation in WAT and iBAT during adulthood. Moreover, using an in vitro approach we investigated the ability of MCH to modulate 3T3-L1 adipocyte differentiation. Pmch-deficiency decreased dorsal subcutaneous and perirenal WAT mass by reducing adipocyte size, but not number. In line with this, in vitro 3T3-L1 adipocyte differentiation was unaffected by MCH. Finally, adult Pmch-deficient rats had lower norepinephrine turnover (an index of sympathetic adipose drive) in WAT and iBAT than wild-type rats. Collectively, our data indicate that MCH/MCH1R-pathway does not modify adipocyte differentiation, whereas Pmch-deficiency in laboratory rats lowers adiposity throughout development and sympathetic adipose drive during adulthood.

Direct effects of nutrients, acetylcholine, CCK, and insulin on ghrelin release from the isolated stomachs of rats.

Ghrelin is a powerful orexigenic peptide predominantly secreted by the stomach. Blood concentration of ghrelin increases before meals and fall postprandial. Its regulation appears to be influenced by the type of macronutrient ingested, the vagus nerve stimulation and by other post-meal stimulated hormonal factors. However, the direct role of nutrients (amino acids or lipids), neuronal (vagal neurotransmitter acetylcholine) and satiety-inducing factor such as CCK are not known. To study this we applied amino acids, lipids, acetylcholine and CCK via vascular perfusion to the isolated stomachs and found that amino acids significantly reduced ghrelin release from the isolated stomach by approximately approximately 30% vs. the control while lipids (10% intralipid) had no affect. Acetylcholine (1 microM) increased ghrelin release from the stomach by approximately 37% whereas insulin (10nM) decreased it by approximately 30% vs. the control. Interestingly, CCK (100 nM) potently increased ghrelin release by approximately 200% vs. the control. Therefore it appears that ghrelin secretion from the stomach is under direct influence of amino acids, neurotransmitter acetylcholine and hormones such as insulin and CCK.

Effect of reducing hypothalamic ghrelin receptor gene expression on energy balance.

Central and peripheral injections of fghrelin potently stimulates food intake via its receptor, GHSR1a expressed in the brain. In this study, we explored the role of GHSR1a in the paraventricular nucleus of the hypothalamus (PVN) by reducing their gene expression using the RNA interference (RNAi). pSUPER plasmids inserted with sh (short hairpin)-GHSR1a were injected into the PVN to reduce its expression. The transfected rats were monitored daily for their food intake and body weight throughout the experimental period lasting 8 days. We found that knockdown of GHSR1a did not affect daily food intake but significantly reduced body weight and blood ghrelin levels. This suggests that the central ghrelin system could selectively regulate body weight without affecting energy intake.