Hypothalamic malonyl-CoA and CPT1c in the treatment of obesity

Metabolic integration of nutrient sensing in the central nervous system has been shown to be an important regulator of adiposity by affecting food intake and peripheral energy expenditure. Modulation of de novo fatty acid synthetic flux by cytokines and nutrient availability plays an important role in this process. Inhibition of hypothalamic fatty acid synthase by pharmacologic or genetic means leads to an increased malonyl-CoA level and suppression of food intake and adiposity. Conversely, the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus is sufficient to promote feeding and adiposity. Based on these and other findings, metabolic intermediates in fatty acid biogenesis, including malonyl-CoA and long-chain acyl-CoAs, have been implicated as signaling mediators in the central control of body weight. Malonyl-CoA has been hypothesized to mediate its effects in part through an allosteric interaction with an atypical and brain-specific carnitine palmitoyltransferase-1 (CPT1c). CPT1c is expressed in neurons and binds malonyl-CoA, however, it does not perform the same biochemical function as the prototypical CPT1 enzymes. Mouse knockout models of CPT1c exhibit suppressed food intake and smaller body weight, but are highly susceptible to weight gain when fed a high-fat diet. Thus, the brain can directly sense and respond to changes in nutrient availability and composition to affect body weight and adiposity.     

The role of hypothalamic malonyl-CoA in energy homeostasis

Energy balance is monitored by hypothalamic neurons that respond to peripheral hormonal and afferent neural signals that sense energy status. Recent physiologic, pharmacologic, and genetic evidence has implicated malonyl-CoA, an intermediate in fatty acid synthesis, as a regulatory component of this energy-sensing system. The level of malonyl-CoA in the hypothalamus is dynamically regulated by fasting and feeding, which alter subsequent feeding behavior. Fatty acid synthase (FAS) inhibitors, administered systemically or intracerebroventricularly to lean or obese mice, increase hypothalamic malonyl-CoA leading to the suppression of food intake. Conversely, lowering malonyl-CoA with an acetyl-CoA carboxylase (ACC) inhibitor or by the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus increases food intake and reverses inhibition by FAS inhibitors. Physiologically, the level of hypothalamic malonyl-CoA appears to be determined through phosphorylation/dephosphorylation of ACC by AMP kinase in response to changes in the AMP/ATP ratio, an indicator of energy status. Recent evidence suggests that the brain-specific carnitine:palmitoyl-CoA transferase-1 (CPT1c) may be a regulated target of malonyl-CoA that relays the "malonyl-CoA signal" in hypothalamic neurons that express the orexigenic and anorexigenic neuropeptides that regulate food intake and peripheral energy expenditure. Together these findings support a role for malonyl-CoA as an intermediary in the control of energy homeostasis.     

Defective hypothalamic autophagy directs the central pathogenesis of obesity via the IkappaB kinase beta (IKKbeta)/NF-kappaB pathway

Autophagy has been recently demonstrated to control cell and tissue homeostasis, including the functions of various metabolic tissues. However, it remains unclear whether autophagy is critical for the central nervous system and particularly the hypothalamus for exerting metabolic regulation. Using autophagy-related protein 7 (Atg7) as an autophagic marker, this work showed that autophagy was highly active in the mediobasal hypothalamus of normal mice. In contrast, chronic development of dietary obesity was associated with autophagic decline in the mediobasal hypothalamus. To investigate the potential role of autophagy in the hypothalamic control of metabolic physiology, a mouse model was developed with autophagic inhibition in the mediobasal hypothalamus using site-specific delivery of lentiviral shRNA against Atg7. This model revealed that hypothalamic inhibition of autophagy increased energy intake and reduced energy expenditure. These metabolic changes were sufficient to increase body weight gain under normal chow feeding and exacerbate the progression of obesity and whole-body insulin resistance under high-fat diet feeding. To explore the underlying mechanism, this study found that defective hypothalamic autophagy led to hypothalamic inflammation, including the activation of proinflammatory IκB kinase β pathway. Using brain-specific IκB kinase β knockout mice, it was found that the effects of defective hypothalamic autophagy in promoting obesity were reversed by IκB kinase β inhibition in the brain. In conclusion, hypothalamic autophagy is crucial for the central control of feeding, energy, and body weight balance. Conversely, decline of hypothalamic autophagy under conditions of chronic caloric excess promotes hypothalamic inflammation and thus impairs hypothalamic control of energy balance, leading to accelerated development of obesity and comorbidities.     

A role for hypothalamic malonyl-CoA in the control of food intake

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.     

Glial Fatty Acid-Binding Protein 7 (FABP7) Regulates Neuronal Leptin Sensitivity in the Hypothalamic Arcuate Nucleus

The hypothalamus is involved in the regulation of food intake and energy homeostasis. The arcuate nucleus (ARC) and median eminence (ME) are the primary hypothalamic sites that sense leptin and nutrients in the blood, thereby mediating food intake. Recently, studies demonstrating a role for non-neuronal cell types, including astrocytes and tanycytes, in these regulatory processes have begun to emerge. However, the molecular mechanisms involved in these activities remain largely unknown. In this study, we examined in detail the localization of fatty acid-binding protein 7 (FABP7) in the hypothalamic ARC and sought to determine its role in the hypothalamus. We performed a phenotypic analysis of diet-induced FABP7 knockout (KO) obese mice and of FABP7 KO mice treated with a single leptin injection. Immunohistochemistry revealed that FABP7⁺ cells are NG2⁺ or GFAP⁺ in the ARC and ME. In mice fed a high-fat diet, weight gain and food intake were lower in FABP7 KO mice than in wild-type (WT) mice. FABP7 KO mice also had lower food intake and weight gain after a single injection of leptin, and we consistently confirmed that the number of pSTAT3⁺ cells in the ARC indicated that the leptin-induced activation of neurons was significantly more frequent in FABP7 KO mice than in WT mice. In FABP7 KO mice-derived primary astrocyte cultures, the level of ERK phosphorylation was lower after leptin treatment. Collectively, these results indicate that in hypothalamic astrocytes, FABP7 might be involved in sensing neuronal leptin via glia-mediated mechanisms and plays a pivotal role in controlling systemic energy homeostasis.     

Oral Leucine Supplementation Is Sensed by the Brain but neither Reduces Food Intake nor Induces an Anorectic Pattern of Gene Expression in the Hypothalamus

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity.     

Central lactate metabolism suppresses food intake via the hypothalamic AMP kinase/malonyl-CoA signaling pathway

Previous studies showed that centrally administered glucose and fructose exert different effects on food intake - glucose decreasing and fructose increasing food intake. Because of the uncertainty of whether fructose can cross the blood-brain-barrier, the question is raised; can dietary fructose directly enter the CNS? Evidence is presented that fructose administered by intraperitoneal (ip) injection to mice is rapidly (<10 min) converted to lactate in the hypothalamus. Thus, fructose can cross the blood-brain-barrier to enter the CNS/hypothalamus for conversion to lactate without prior (slower) conversion to glucose in the liver. Fructose-derived hypothalamic lactate is not, however, responsible for the orexigenic effect of fructose. Ip lactate administered at a level equivalent to that of fructose generates a higher level of hypothalamic lactate, which rapidly triggers dephosphorylation/inactivation of AMP-kinase. Thereby, ACC — a substrate of AMP-kinase that catalyzes malonyl-CoA formation — is dephosphorylated and activated. Consistent with these findings, ip or centrally (icv) administered lactate rapidly increases (<10 min) hypothalamic malonyl-CoA. Increasing hypothalamic malonyl-CoA suppresses the expression of the orexigenic and increases the expression of the anorexigenic neuropeptides, which decrease food intake. All downstream effects of hypothalamic lactate are blocked by icv administered oxamate, a potent inhibitor of lactate dehydrogenase, thus verifying the central action of lactate.     

Small molecule insulin mimetics reduce food intake and body weight and prevent development of obesity

Obesity and insulin resistance are major risk factors for a number of metabolic disorders, such as type 2 diabetes mellitus. Insulin has been suggested to function as one of the adiposity signals to the brain for modulation of energy balance. Administration of insulin into the brain reduces food intake and body weight, and mice with a genetic deletion of neuronal insulin receptors are hyperphagic and obese. However, insulin is also an anabolic factor; when administered systemically, pharmacological levels of insulin are associated with body weight gain in patients. In this study, we investigated the efficacy and feasibility of small molecule insulin mimetic compounds to regulate key parameters of energy homeostasis. Central intracerebroventricular (i.c.v.) administration of an insulin mimetic resulted in a dose-dependent reduction of food intake and body weight in rats, and altered the expression of hypothalamic genes known to regulate food intake and body weight. Oral administration of a mimetic in a mouse model of high-fat diet-induced obesity reduced body weight gain, adiposity and insulin resistance. Thus, insulin mimetics have a unique advantage over insulin in the control of body weight and hold potential as a novel anti-obesity treatment.     

NPY Neuron-Specific Y2 Receptors Regulate Adipose Tissue and Trabecular Bone but Not Cortical Bone Homeostasis in Mice

Background

Y2 receptor signalling is known to be important in neuropeptide Y (NPY)-mediated effects on energy homeostasis and bone physiology. Y2 receptors are located post-synaptically as well as acting as auto receptors on NPY-expressing neurons, and the different roles of these two populations of Y2 receptors in the regulation of energy homeostasis and body composition are unclear.

Methodology/Principal Findings

We thus generated two conditional knockout mouse models, Y2^lox/lox and NPYCre/+;Y2^lox/lox, in which Y2 receptors can be selectively ablated either in the hypothalamus or specifically in hypothalamic NPY-producing neurons of adult mice. Specific deletion of hypothalamic Y2 receptors increases food intake and body weight compared to controls. Importantly, specific ablation of hypothalamic Y2 receptors on NPY-containing neurons results in a significantly greater adiposity in female but not male mice, accompanied by increased hepatic triglyceride levels, decreased expression of liver cartinine palmitoyltransferase (CPT1) and increased expression of muscle phosphorylated acetyl-CoA carboxylase (ACC). While food intake, body weight, femur length, bone mineral content, density and cortical bone volume and thickness are not significantly altered, trabecular bone volume and number were significantly increased by hypothalamic Y2 deletion on NPY-expressing neurons. Interestingly, in situ hybridisation reveals increased NPY and decreased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of mice with hypothalamus-specific deletion of Y2 receptors in NPY neurons, consistent with a negative feedback mechanism between NPY expression and Y2 receptors on NPY-ergic neurons.

Conclusions/Significance

Taken together these data demonstrate the anti-obesogenic role of Y2 receptors in the brain, notably on NPY-ergic neurons, possibly via inhibition of NPY neurons and concomitant stimulation of POMC-expressing neurons in the arcuate nucleus of the hypothalamus, reducing lipogenic pathways in liver and/or skeletal muscle in females. These data also reveal as an anti-osteogenic effect of Y2 receptors on hypothalamic NPY-expressing neurons on trabecular but not on cortical bone.     

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.     

Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production

The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo beta-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.     

Effects of myeloid sirtuin 1 deficiency on hypothalamic neurogranin in mice fed a high-fat diet

Hypothalamic inflammation has been known as a contributor to high-fat diet (HFD)-induced insulin resistance and obesity. Myeloid-specific sirtuin 1 (SIRT1) deletion aggravates insulin resistance and hypothalamic inflammation in HFD-fed mice. Neurogranin, a calmodulin-binding protein, is expressed in the hypothalamus. However, the effects of myeloid SIRT1 deletion on hypothalamic neurogranin has not been fully clarified. To investigate the effect of myeloid SIRT1 deletion on food intake and hypothalamic neurogranin expression, mice were fed a HFD for 20 weeks. Myeloid SIRT1 knockout (KO) mice exhibited higher food intake, weight gain, and lower expression of anorexigenic proopiomelanocortin in the arcuate nucleus than WT mice. In particular, KO mice had lower ventromedial hypothalamus (VMH)-specific neurogranin expression. However, SIRT1 deletion reduced HFD-induced hypothalamic neurogranin. Furthermore, hypothalamic phosphorylated AMPK and parvalbumin protein levels were also lower in HFD-fed KO mice than in HFD-fed WT mice. Thus, these findings suggest that myeloid SIRT1 deletion affects food intake through VMH-specific neurogranin-mediated AMPK signaling and hypothalamic inflammation in mice fed a HFD.     

Hypothalamic malonyl-coenzyme A and the control of energy balance

An intermediate in the fatty acid biosynthetic pathway, malonyl-coenzyme A (CoA), has emerged as a major regulator of energy homeostasis not only in peripheral metabolic tissues but also in regions of the central nervous system that control satiety and energy expenditure. Fluctuations in hypothalamic malonyl-CoA lead to changes in food intake and peripheral energy expenditure in a manner consistent with an anorexigenic signaling intermediate. Hypothalamic malonyl-CoA is regulated by nutritional and endocrine cues including glucose and leptin, respectively. That malonyl-CoA is an essential component in the energy homeostatic signaling system of the hypothalamus is supported by convergence of physiological, pharmacological, and genetic evidence. This review will focus on evidence implicating malonyl-CoA as a central player in the control of body weight and adiposity as well as clues to the molecular mechanism by which carbon flux through the fatty acid biosynthetic pathway is linked to the neural control of energy balance.     

Hypothalamic lipotoxicity and the metabolic syndrome

Ectopic accumulation of lipids in peripheral tissues, such as pancreatic β cells, liver, heart and skeletal muscle, leads to lipotoxicity, a process that contributes substantially to the pathophysiology of insulin resistance, type 2 diabetes, steatotic liver disease and heart failure. Current evidence has demonstrated that hypothalamic sensing of circulating lipids and modulation of hypothalamic endogenous fatty acid and lipid metabolism are two bona fide mechanisms modulating energy homeostasis at the whole body level. Key enzymes, such as AMP-activated protein kinase (AMPK) and fatty acid synthase (FAS), as well as intermediate metabolites, such as malonyl-CoA and long-chain fatty acids-CoA (LCFAs-CoA), play a major role in this neuronal network, integrating peripheral signals with classical neuropeptide-based mechanisms. However, one key question to be addressed is whether impairment of lipid metabolism and accumulation of specific lipid species in the hypothalamus, leading to lipotoxicity, have deleterious effects on hypothalamic neurons. In this review, we summarize what is known about hypothalamic lipid metabolism with focus on the events associated to lipotoxicity, such as endoplasmic reticulum (ER) stress in the hypothalamus. A better understanding of these molecular mechanisms will help to identify new drug targets for the treatment of obesity and metabolic syndrome.     

Anti-obesity effects of the methanolic extract and chakasaponins from the flower buds of Camellia sinensis in mice

The methanolic extract from the flower buds of Camellia sinensis cultivated in Fujian Province showed inhibitory effects on body weight gain and the weight of visceral fats in high-fat diet-fed mice and/or Tsumura Suzuki Obese Diabetic (TSOD) mice. A suppressive effect of the extract on food intake was suggested to contribute to the anti-obesity effect. The n-butanol (BuOH)-soluble fraction also reduced food intake in normal diet-fed mice. A principal constituent, chakasaponin II, inhibited gastric emptying (GE) as well as food intake. These inhibitory effects were partly reduced by pretreatment with a high dose of capsaicin. The n-BuOH-soluble fraction and chakasaponin II suppressed mRNA levels of neuropeptide Y (NPY), an important regulator of body weight through its effects on food intake and energy expenditure, in the hypothalamus. Furthermore, chakasaponin II enhanced the release of serotonin (5-HT) from the isolated ilea of mice in vitro. These findings suggested that the active saponins suppressed the appetite signals in the hypothalamus through stimulation of the capsaicin-sensitive sensory nerves, probably vagal afferent nerves, or enhancement of 5-HT release from the ilea, leading to reduced food intake and body weight gain.     

Shp2 controls female body weight and energy balance by integrating leptin and estrogen signals

In mammals, leptin regulates food intake and energy balance mainly through the activation of LepRb in the hypothalamus, and estrogen has a leptin-like effect in the hypothalamic control of metabolism. However, it remains to be elucidated how estrogen signaling is intertwined with the leptin pathway. We show here that Shp2, a nonreceptor tyrosine phosphatase, acts to integrate leptin and estrogen signals. The expression of a dominant-active mutant (Shp2(D61A)) in forebrain neurons conferred female, but not male, transgenic mice resistance to high-fat diet (HFD)-induced obesity and liver steatosis, accompanied by improved insulin sensitivity and glucose homeostasis. Fed with either HFD or regular chow food, Shp2(D61A) female mice showed dramatically enhanced leptin sensitivity. Microinjection of Shp2(D61A)-expressing adeno-associated virus into mediobasal hypothalamus elicited a similar antiobese effect in female mice. Biochemical analyses showed a physical association of Shp2 with estrogen receptor alpha, which is necessary for the synergistic and persistent activation of Erk by leptin and estrogen. Together, these results elucidate a mechanism for the direct cross talk of leptin and estrogen signaling and offer one explanation for the propensity of postmenopausal women to develop obesity.     

Cerebral Cell Renewal in Adult Mice Controls the Onset of Obesity

The hypothalamus plays a crucial role in the control of the energy balance and also retains neurogenic potential into adulthood. Recent studies have reported the severe alteration of the cell turn-over in the hypothalamus of obese animals and it has been proposed that a neurogenic deficiency in the hypothalamus could be involved in the development of obesity. To explore this possibility, we examined hypothalamic cell renewal during the homeostatic response to dietary fat in mice, i.e., at the onset of diet-induced obesity. We found that switching to high-fat diet (HFD) accelerated cell renewal in the hypothalamus through a local, rapid and transient increase in cell proliferation, peaking three days after introducing the HFD. Blocking HFD-induced cell proliferation by central delivery of an antimitotic drug prevented the food intake normalization observed after HFD introduction and accelerated the onset of obesity. This result showed that HFD-induced dividing brain cells supported an adaptive anorectic function. In addition, we found that the percentage of newly generated neurons adopting a POMC-phenotype in the arcuate nucleus was increased by HFD. This observation suggested that the maturation of neurons in feeding circuits was nutritionally regulated to adjust future energy intake. Taken together, these results showed that adult cerebral cell renewal was remarkably responsive to nutritional conditions. This constituted a physiological trait required to prevent severe weight gain under HFD. Hence this report highlighted the amazing plasticity of feeding circuits and brought new insights into our understanding of the nutritional regulation of the energy balance.