Central orchestration of peripheral nutrient partitioning and substrate utilization: Implications for the metabolic syndrome

Energy homoeostasis is maintained through a complex interplay of nutrient intake and energy expenditure. The central nervous system is an essential component of this regulation, as it integrates circulating signals of hunger and satiety to develop adaptive responses at the behavioural and metabolic levels, while the hypothalamus is regarded as a particularly crucial structure in the brain in terms of energy homoeostasis. The arcuate nucleus (ARC) of the hypothalamus contains at least two intermingled neuronal populations: the neurons that produce neuropeptide Y (NPY); and the Agouti-related protein (AgRP) produced by AgRP/NPY neurons situated below the third ventricle in close proximity to proopiomelanocortin (POMC)-producing neurons. POMC neurons exert their catabolic and anorectic actions by releasing α-melanocyte-stimulating hormone (α-MSH), while AgRP neurons oppose this action by exerting tonic GABAergic inhibition of POMC neurons and releasing the melanocortin receptor inverse agonist AgRP. The release of neurotransmitters and neuropeptides by second-order AgRP neurons appears to take place on a multiple time scale, thereby allowing neuromodulation of preganglionic neuronal activity and subsequent control of nutrient partitioning – in other words, the coordinated regulation of conversion, storage and utilization of carbohydrates vs. lipids. This suggests that the function of AgRP neurons extends beyond the strict regulation of feeding to the regulation of efferent organ activity, such that AgRP neurons may now be viewed as an important bridge between central detection of nutrient availability and peripheral nutrient partitioning, thus providing a mechanistic link between obesity and obesity-related disorders.     

Diurnal changes in hypothalamic neuropeptide and SOCS-3 expression: effects of lactation and relationship with serum leptin and food intake

Rats normally eat about 85% of their food at night. Lactation increases food intake 3- to 4-fold, but the diurnal pattern of food intake persists. The mechanisms responsible for the diurnal and lactation-induced changes in food intake are still unresolved, hence we have further investigated the possible roles of serum leptin and hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in rats. Suppressor of cytokine signalling-3 (SOCS-3) acts as a feedback inhibitor of leptin signalling in the hypothalamus, hence changes in expression of SOCS-3 were also investigated. Changes in expression of NPY, AgRP or POMC alone could not account for the diurnal changes in intake and their alteration by lactation. However, there were increased AgRP mRNA:POMC mRNA ratios at night and also during lactation, which were very similar to estimated changes in food intake. Such changes in expression may result in dominance of the orexigenic AgRP peptide over the appetite-suppressing POMC-derived peptides, and so could contribute to the hyperphagia in these states. Diurnal and lactation-related changes in the AgRP mRNA:POMC mRNA ratio and food intake are not due to changes in leptin alone. However, hypoleptinaemia, possibly through increased expression of NPY, may contribute to the hyperphagia of lactation. In the dark, expression of SOCS-3 was decreased in non-lactating rats; lactation decreased SOCS-3 expression in both light and dark phases. However, such changes are likely to enhance the ability of leptin-responsive neurones to transmit the leptin signal, and so are unlikely to contribute to either the nocturnal increase in appetite or the hyperphagia of lactation.     

Differential regulation of agouti-related protein and neuropeptide Y in hypothalamic neurons following a stressful event

Stress affects eating behaviour in rodents and humans, suggesting that the regulation of energy balance and the stress response are coupled physiological processes. Neuropeptide Y (NPY) and agouti-related protein (AgRP) are potent food-stimulating neuropeptides that are highly co-localised in arcuate nucleus neurons of the hypothalamus. Recent studies have shown that NPY and AgRP mRNA levels in these neurons respond similarly to fasting and leptin, indicating functional redundancy of the neuropeptide systems in these orexigenic neurons. However, we have found that NPY and AgRP mRNA expression in arcuate nucleus neurons are dissociated immediately following a stressful event. Two hours following a brief session of inescapable foot shocks, AgRP mRNA levels are down-regulated (P < 0.0001). In contrast, NPY mRNA levels are up-regulated (P < 0.0001). To provide physiological relevance for this acute down-regulation of AgRP, an inverse agonist of melanocortin receptors, we have shown that acute intracerebroventricular injection of a melanocortin receptor agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), caused a significantly stronger activation of the hypothalamus-pituitary-adrenal-cortical (HPA) axis following a stressful event than in controls. Thus, AgRP and NPY mRNA levels in similar arcuate nucleus neurons are differentially regulated following a stressful event. This may contribute to increased sensitivity for alpha-MSH to activate the HPA axis following a repeated stressful experience.     

Leptin regulates neuropeptides associated with food intake and GnRH secretion

The present review focused on the most important effects of leptin on the hypothalamus and on how leptin regulates neuropeptides associated with food intake and GnRH secretion. This review of the literature suggests that a reduction in leptin serum concentrations results from lower body energy reserves or poor energy availability, leading to hypothalamic secretion of neuropeptides such as NPY/AgRP and QRFP to stimulate food intake. Under these negative metabolic conditions, GnRH secretion is reduced, impairing reproductive functions. In contrast, when metabolic status is inversed by an increase in food availability, energy reserves or both, leptin serum concentrations increase to an action threshold reversing the pattern of secretion: i.e., reducing NPY/AgRP and QRFP and increasing POMC and Kisspeptin, and thereby reducing food intake and stimulating GnRH secretion to promote reproductive function.     

Hypothalamic SIRT1 prevents age-associated weight gain by improving leptin sensitivity in mice

Aims/hypothesis

Obesity is associated with ageing and increased energy intake, while restriction of energy intake improves health and longevity in multiple organisms; the NAD⁺-dependent deacetylase sirtuin 1 (SIRT1) is implicated in this process. Pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical for energy balance regulation, and the level of SIRT1 protein decreases with age in the ARC. In the current study we tested whether conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevents age-associated weight gain and diet-induced obesity.

Methods

We targeted Sirt1 cDNA sequence into the Rosa26 locus and generated conditional Sirt1 knock-in mice. These mice were crossed with mice harbouring either Pomc-Cre or Agrp-Cre and the metabolic variables, food intake, energy expenditure and sympathetic activity in adipose tissue of the resultant mice were analysed. We also used a hypothalamic cell line to investigate the molecular mechanism by which Sirt1 overexpression modulates leptin signalling.

Results

Conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevented age-associated weight gain; overexpression in POMC neurons stimulated energy expenditure via increased sympathetic activity in adipose tissue, whereas overexpression in AgRP neurons suppressed food intake. SIRT1 improved leptin sensitivity in hypothalamic neurons in vitro and in vivo by downregulating protein-tyrosine phosphatase 1B, T cell protein-tyrosine phosphatase and suppressor of cytokine signalling 3. However, these phenotypes were absent in mice consuming a high-fat, high-sucrose diet due to decreases in ARC SIRT1 protein and hypothalamic NAD⁺ levels.

Conclusions/interpretation

ARC SIRT1 is a negative regulator of energy balance, and decline in ARC SIRT1 function contributes to disruption of energy homeostasis by ageing and diet-induced obesity.     

Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/Agouti-related protein neurons

The neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus are thought to promote feeding. Here, we demonstrate that feeding state in vivo, through a leptin-dependent process, induces large and persistent changes in the electrophysiological activity of these neurons as measured extracellularly in vitro. Consistent with an orexigenic role, fasting induced a 4-fold increase in the basal action potential frequency of NPY/AgRP neurons. Leptin, when injected into fasted wild-type mice, induced a dose- and time-dependent decrease in spike frequency, which approached fed levels 2-3 h post treatment. In leptin-deficient (lep(ob)/lep(ob)) and leptin receptor-deficient (lepr(db)/lepr(db)) mice, NPY/AgRP spike frequency was not significantly increased by fasting, and even in mutant mice fed ad libitum, spike frequency was at least as high as in fasted wild-type mice. All recordings included GABA(A) and ionotropic glutamate receptor antagonists, suggesting that expression of this modulation is potentially intrinsic and not synaptically dependent. Recorded neurons were unambiguously identified using NPY-Sapphire transgenic mice. This is a remarkably straightforward example of a very robust in vitro electrophysiogical effect produced by a simple behavioral manipulation, food restriction.     

Selective loss of leptin receptors in the ventromedial hypothalamic nucleus results in increased adiposity and a metabolic syndrome

Leptin, an adipocyte-derived hormone, has emerged as a critical regulator of energy homeostasis. The leptin receptor (Lepr) is expressed in discrete regions of the brain; among the sites of highest expression are several mediobasal hypothalamic nuclei known to play a role in energy homeostasis, including the arcuate nucleus, the ventromedial hypothalamic nucleus (VMH), and the dorsomedial hypothalamic nucleus. Although most studies have focused on leptin's actions in the arcuate nucleus, the role of Lepr in these other sites has received less attention. To explore the role of leptin signaling in the VMH, we used bacterial artificial chromosome transgenesis to target Cre recombinase to VMH neurons expressing steroidogenic factor 1, thereby inactivating a conditional Lepr allele specifically in steroidogenic factor 1 neurons of the VMH. These knockout (KO) mice, designated Lepr KO(VMH), exhibited obesity, particularly when challenged with a high-fat diet. On a low-fat diet, Lepr KO(VMH) mice exhibited significantly increased adipose mass even when their weights were comparable to wild-type littermates. Furthermore, these mice exhibited a metabolic syndrome including hepatic steatosis, dyslipidemia, and hyperleptinemia. Lepr KO(VMH) mice were hyperinsulinemic from the age of weaning and eventually developed overt glucose intolerance. These data define nonredundant roles of the Lepr in VMH neurons in energy homeostasis and provide a model system for studying other actions of leptin in the VMH.     

Control of blood pressure, appetite, and glucose by leptin in mice lacking leptin receptors in proopiomelanocortin neurons

Although the central nervous system melanocortin system is an important regulator of energy balance, the role of proopiomelanocortin (POMC) neurons in mediating the chronic effects of leptin on appetite, blood pressure, and glucose regulation is unknown. Using Cre/loxP technology we tested whether leptin receptor deletion in POMC neurons (LepR(flox/flox)/POMC-Cre mice) attenuates the chronic effects of leptin to increase mean arterial pressure (MAP), enhance glucose use and oxygen consumption, and reduce appetite. LepR(flox/flox)/POMC-Cre, wild-type, LepR(flox/flox), and POMC-Cre mice were instrumented for MAP and heart rate measurement by telemetry and venous catheters for infusions. LepR(flox/flox)/POMC-Cre mice were heavier, hyperglycemic, hyperinsulinemic, and hyperleptinemic compared with wild-type, LepR(flox/flox), and POMC-Cre mice. Despite exhibiting features of metabolic syndrome, LepR(flox/flox)/POMC-Cre mice had normal MAP and heart rate compared with LepR(flox/flox) but lower MAP and heart rate compared with wild-type mice. After a 5-day control period, leptin was infused (2 μg/kg per minute, IV) for 7 days. In control mice, leptin increased MAP by ≈5 mm Hg despite decreasing food intake by ≈35%. In contrast, leptin infusion in LepR(flox/flox)/POMC-Cre mice reduced MAP by ≈3 mm Hg and food intake by ≈28%. Leptin significantly decreased insulin and glucose levels in control mice but not in LepR(flox/flox)/POMC-Cre mice. Leptin increased oxygen consumption in LepR(flox/flox)/POMC-Cre and wild-type mice. Activation of POMC neurons is necessary for the chronic effects of leptin to raise MAP and reduce insulin and glucose levels, whereas leptin receptors in other areas of the brain other than POMC neurons appear to play a key role in mediating the chronic effects of leptin on appetite and oxygen consumption.     

Glucose regulates the effects of leptin on hypothalamic POMC neurons

Leptin is believed to exert its potent appetite-suppressing effects via stimulation of hypothalamic anorexigenic proopiomelanocortin (POMC)-containing neurons and inhibition of orexigenic agouti-related protein (AgRP) neurons. We show here that at 11 mM glucose leptin excites POMC cells. At 5 mM glucose, however, leptin hyperpolarizes POMC neurons and suppresses action potential firing, by producing a greater decrease in excitatory synaptic tone than inhibitory tone. These results argue that when glucose is low (5 mM or less) AgRP neurons will be more important for mediating the anorectic effects of leptin than POMC cells. However, at high glucose concentrations (11 mM), activation of POMC cells may contribute to the appetite-suppressing effects of leptin. Our data also suggest the regulation of neuropeptide efficacy as a novel function of hypothalamic glucose sensing.     

Gamma-aminobutyric acid neurons integrate and rapidly transmit permissive and inhibitory metabolic cues to gonadotropin-releasing hormone neurons

Negative energy balance inhibits fertility by decreasing GnRH release; however, the mechanisms are not well understood. GnRH neurons can be excited by activation of gamma-aminobutyric acid (GABA)(A) receptors, and GABAergic neurons provide a major synaptic input. We hypothesized that permissive metabolic signals mediated by leptin and inhibitory signals conveyed by neuropeptide Y (NPY) and opiates rapidly alter GABA(A) receptor-mediated drive to GnRH neurons. In fed and fasted female mice, GABAergic postsynaptic currents (PSCs) were recorded from GnRH neurons before and after in vitro treatment with leptin, NPY, or met-enkephalin. Leptin increased PSC frequency in fed and fasted mice, indicating that it increased presynaptic activity. Leptin also increased PSC size. Inhibiting leptin receptor signaling pathways within GnRH neurons abolished the latter effect, indicating a direct action on these cells. In fed, but not fasted, mice, NPY and met-enkephalin decreased PSC frequency in an antagonist-reversible manner, but did not alter PSC size. NPY-1 receptor antagonists alone increased frequency in fed and fasted mice, as did opiate receptor blockade in fasted animals, suggesting that endogenous NPY and opiates modulate GABAergic drive to GnRH neurons. These data suggest that GABAergic afferents integrate metabolic signals for delivery to GnRH neurons. Decreased sensitivity to NPY and opiates in fasted mice indicate that these peptides send physiologically relevant signals regarding energy balance to GnRH neurons.     

Neuropeptide Y and agouti-related peptide mediate complementary functions of hyperphagia and reduced energy expenditure in leptin receptor deficiency

Neuropeptide Y (NPY) and agouti-related peptide (AGRP) can produce hyperphagia, reduce energy expenditure, and promote triglyceride deposition in adipose depots. As these two neuropeptides are coexpressed within the hypothalamic arcuate nucleus and mediate a major portion of the obesity caused by leptin signaling deficiency, we sought to determine whether the two neuropeptides mediated identical or complementary actions. Because of separate neuropeptide receptors and signal transduction mechanisms, there is a possibility of distinct encoding systems for the feeding and energy expenditure aspects of leptin-regulated metabolism. We have genetically added NPY deficiency and/or AGRP deficiency to LEPR deficiency isolated to AGRP cells. Our results indicate that the obesity of LEPR deficiency in AGRP/NPY neurons can produce obesity with either AGRP or NPY alone with AGRP producing hyperphagia while NPY promotes reduced energy expenditure. The absence of both NPY and AGRP prevents the development of obesity attributable to isolated LEPR deficiency in AGRP/NPY neurons. Operant behavioral testing indicated that there were no alterations in the reward for a food pellet from the AGRP-specific LEPR deficiency.