Central leptin differentially modulates ultradian secretory patterns of insulin, leptin and ghrelin independent of effects on food intake and body weight

We tested the hypothesis that leptin acts centrally to differentially modulate the ultradian communication of leptin, insulin and ghrelin with the hypothalamus. The ultradian fluctuation of these hormones in plasma after central leptin gene therapy was analyzed. Increased leptin transgene expression in the hypothalamus significantly decreased energy intake and body weight concomitant with severe hypoleptinemia and hypoinsulinemia resulting from drastically suppressed peak heights with unchanged frequency discharge of these hormones. Ghrelin secretion was, however, increased solely due to increased pulse amplitude. In pair-fed control rats leptin and ghrelin secretion was unchanged. In conclusion, independent of restraint on caloric intake and weight, leptin acting centrally modulates only the pulse amplitude of ultradian rhythmicity of the three afferent signals involved in the hypothalamic integration of energy balance. Since rhythmic discharge patterns dictate target response of hormones, these findings reveal a novel hypothalamic action of leptin in the pathophysiology of the obesity-dependent metabolic syndrome.     

Leptin TRH and ghrelin: influence on energy homeostasis at rest and during exercise.

The hypothalamus has long been recognized as a major site in the central nervous system (CNS) where a spectrum of internal and external environmental information is integrated for energy homeostasis. The isolation and sequencing of leptin in the mid 90 s, together with the demonstration of leptin administration's ability to correct the obesity syndrome in leptin-deficient ob/ob mice and humans by suppressing food intake and weight gain in laboratory rodents, confirmed the hypothesized existence of a direct humoral signal from adipose tissue to the hypothalamus, thus integrating the energy-related signals. In the 80 s, neuropeptide Y (NPY) was identified as a potent appetite-stimulating neuropeptide produced, released and acting locally within the hypothalamus. This is recognized as a major physiological appetite transducer and central neurochemical substrate receiving, interpreting and processing incoming information on energy status. More recently, ghrelin, produced in the stomach and released into the general circulation, has drawn attention as the other limb of the feedback circuit that stimulates appetite at NPY network level. Prolonged fasting suppresses serum leptin, while suppressing TSH secretion. Intervention with leptin replacement can prevent fasting-induced changes in TSH, suggesting that leptin regulates TSH. Low leptin levels in sportsmen and sportswomen as well as in recreational runners are consistent with reduction in body fat, but are also influenced by the presence of low insulin, hypothyroxemia, and elevated cortisol levels. These metabolic adaptations to chronic energy deficits indicate a role in leptin regulation. A study within the general population found that activity levels and leptin were significantly negatively associated in both sexes. Circulating ghrelin levels, however, do not change during energy expenditure.     

Neuroendocrine actions and regulation of hypothalamic neuropeptide Y during lactation

The expression of neuropeptide Y (NPY) and its co-messenger, agouti-related peptide (AgRP), in arcuate neurons of the hypothalamus is increased during lactation in rats. Our research has been addressing the questions of the physiological actions of these peptides during lactation and the physiological signals associated with lactation that result in increased expression of their genes. Our studies indicate that NPY and AgRP exert pleiotropic actions during lactation that help integrate neuroendocrine regulation of energy balance with controls over anterior and posterior pituitary hormone secretion. Further, reciprocal signaling to the NPY/AgRP system by leptin and ghrelin is responsible for the changes in expression of these hypothalamic peptides in lactating animals, and thus, may contribute to regulation of food intake and the various neuroendocrine adaptations of lactation.     

Orexin A/Hypocretin Modulates Leptin Receptor-Mediated Signaling by Allosteric Modulations Mediated by the Ghrelin GHS-R1A Receptor in Hypothalamic Neurons

The hypothalamus is a key integrator of nutrient-seeking signals in the form of hormones and metabolites originated in both the central nervous system and the periphery. The main autocrine and paracrine target of orexinergic-related hormones such as leptin, orexin/hypocretin, and ghrelin are neuropeptide Y neurons located in the arcuate nucleus of the hypothalamus. The aim of this study was to investigate the expression and the molecular and functional relationships between leptin, orexin/hypocretin and ghrelin receptors. Biophysical studies in a heterologous system showed physical interactions between them, with potential formation of heterotrimeric complexes. Functional assays showed robust allosteric interactions particularly different when the three receptors are expressed together. Further biochemical and pharmacological assays provided evidence of heterotrimer functional expression in primary cultures of hypothalamic neurons. These findings constitute evidence of close relationships in the action of the three hormones already starting at the receptor level in hypothalamic cells.     

Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.     

To subjugate NPY is to improve the quality of life and live longer

The interactive network of neuropeptide Y (NPY) and cohorts is necessary for integrating the hypothalamic regulation of appetite and energy expenditure with the endocrine and neuroendocrine systems on a daily basis. Genetic and environmental factors that produce an insufficiency of leptin restraint on NPY and cognate receptors deregulate the homeostasis to engender various life-threatening risk factors. Recent studies from our laboratory show that neurotherapy consisting of a single central administration of recombinant adeno-associated virus vector encoding the leptin gene can repress the hypothalamic NPY system for the lifetime of rodents. A major benefit of this stable tonic restraint is deceleration of pathophysiologic sequalae that shorten life span. These include suppression of weight gain, fat accumulation, circulating adipokines, amelioration of major symptoms of metabolic syndrome, improved reproduction and bone health. Thus, sustained repression of NPY signaling in the hypothalamus by leptin transgene expression can improve the quality of life and extend longevity.     

A role for beta-melanocyte-stimulating hormone in human body-weight regulation

Pro-opiomelanocortin (POMC) expressing neurons mediate the regulation of orexigenic drive by peripheral hormones such as leptin, cholecystokinin, ghrelin, and insulin. Most research effort has focused on alpha-melanocyte-stimulating hormone (alpha-MSH) as the predominant POMC-derived neuropeptide in the central regulation of human energy balance and body weight. Here we report a missense mutation within the coding region of the POMC-derived peptide beta-MSH (Y5C-beta-MSH) and its association with early-onset human obesity. In vitro and in vivo data as well as postmortem human brain studies indicate that the POMC-derived neuropeptide beta-MSH plays a critical role in the hypothalamic control of body weight in humans.     

Leptin sensitive neurons in the hypothalamus.

A major paradigm in the field of obesity research is the existence of an adipose tissue-brain endocrine axis for the regulation of body weight. Leptin, the peptide mediator of this axis, is secreted by adipose cells. It lowers food intake and body weight by acting in the hypothalamus, a region expressing an abundance of leptin receptors and a variety of neuropeptides that influence food intake and energy balance. Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that co-express the anabolic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and those that express proopiomelanocortin (POMC), the precursor of the catabolic peptide, alphaMSH. These cell types contain mRNA encoding leptin receptors and show changes in neuropeptide gene expression in response to changes in food intake and circulating leptin levels. Decreased leptin signaling in the arcuate nucleus is hypothesized to increase the expression of NPY and AGRP. Levels of leptin receptor mRNA and leptin binding are increased in the arcuate nucleus during fasting, principally in NPY/AGRP neurons. These findings suggest that changes in leptin receptor expression in the arcuate nucleus are inversely associated with changes in leptin signaling, and that the arcuate nucleus is an important target of leptin action in the brain.     

Filling the interstices: ghrelin neurons plug several holes in regulation of energy balance

Of several circulating hormones that act on hypothalamus to affect body energy balance, only ghrelin is also expressed in hypothalamic neurons. From the studies of Horvath and colleagues appearing in this issue of Neuron, it appears that neuronal ghrelin acts presynaptically to stimulate release of the orexigenic peptide, neuropeptide Y, and other neurotransmitters, thus defining a new and subtle modulatory circuit.     

Severe pulmonary hypertension in aging female apolipoprotein E-deficient mice is rescued by estrogen replacement therapy

Corticotropin-releasing factor overexpressing (CRF-OE) male mice showed an inhibited feeding response to a fast, and lower plasma acyl ghrelin and Fos expression in the arcuate nucleus compared to wild-type (WT) mice. We investigated whether hormones and hypothalamic feeding signals are impaired in CRF-OE mice and the influence of sex. Male and female CRF-OE mice and WT littermates (4–6 months old) fed ad libitum or overnight fasted were assessed for body, adrenal glands and perigonadal fat weights, food intake, plasma hormones, blood glucose, and mRNA hypothalamic signals. Under fed conditions, compared to WT, CRF-OE mice have increased adrenal glands and perigonadal fat weight, plasma corticosterone, leptin and insulin, and hypothalamic leptin receptor and decreased plasma acyl ghrelin. Compared to male, female WT mice have lower body and perigonadal fat and plasma leptin but higher adrenal glands weights. CRF-OE mice lost these sex differences except for the adrenals. Male CRF-OE and WT mice did not differ in hypothalamic expression of neuropeptide Y (NPY) and proopiomelanocortin (POMC), while female CRF-OE compared to female WT and male CRF-OE had higher NPY mRNA levels. After fasting, female WT mice lost more body weight and ate more food than male WT, while CRF-OE mice had reduced body weight loss and inhibited food intake without sex difference. In male WT mice, fasting reduced plasma insulin and leptin and increased acyl ghrelin and corticosterone while female WT showed only a rise in corticosterone. In CRF-OE mice, fasting reduced insulin while leptin, acyl ghrelin and corticosterone were unchanged with no sex difference. Fasting blood glucose was higher in CRF-OE with female > male. In WT mice, fasting increased hypothalamic NPY expression in both sexes and decreased POMC only in males, while in CRF-OE mice, NPY did not change, and POMC decreased in males and increased in females. These data indicate that CRF-OE mice have abnormal basal and fasting circulating hormones and hypothalamic feeding-related signals. CRF-OE also abolishes the sex difference in body weight, abdominal fat, and fasting-induced feeding and changes in plasma levels of leptin and acyl ghrelin.     

High‐fat Diet‐induced Ultradian Leptin and Insulin Hypersecretion are Absent in Obesity‐resistant Rats*

Objective: Sprague‐Dawley rats fed a high‐fat diet (HFD) are either obesity prone (OP) or obesity resistant (OR). We tested the hypothesis that differences in the ultradian rhythmic patterns of insulin and ghrelin in OP vs. OR rats promote obesity in OP rats. Research Methods and Procedures: Rats were fed regular chow or an HFD, and ultradian fluctuations in leptin, insulin, and ghrelin were analyzed in blood samples collected at 5‐minute intervals from intrajugular cannulae of freely moving rats. Results: Regular chow feeding resulted in a slow weight gain accompanied by small increases in insulin and leptin and a decrease in ghrelin discharge, with only the pulse amplitude significantly altered. Similar changes were observed in OR rats, despite HFD consumption. In contrast, OP rats exhibited a high rate of weight gain and marked hyperinsulinemia, hyperleptinemia, and hypoghrelinemia; amplitude was altered, but frequency was stable. In a short‐term experiment, HFD elicited similar secretory patterns of smaller magnitude even in the absence of weight gain. Discussion: We showed that three hormonal signals of disparate origin involved in energy homeostasis were secreted in discrete episodes, and only the pulse amplitude component was vulnerable to age and HFD consumption. Increases in insulin and leptin and decreases in ghrelin pulse amplitude caused by HFD were exaggerated in OP rats relative to OR rats and preceded the weight increase. These findings show that a distinct genetic predisposition in the endocrine organs of OR rats confers protection against high‐fat intake‐induced ultradian hypersecretion of obesity‐promoting hormonal signals.     

AMP-activated protein kinase plays a role in the control of food intake

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3-36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs.     

Hypophysectomy Prevents Ghrelin-Induced Adiposity and Increases Gastric Ghrelin Secretion in Rats

Objective: The novel gastric hormone ghrelin has recently been identified as an important modulator of energy homeostasis. Leptin-responsive hypothalamic neuropeptide Y/Agouti-related protein neurons are believed to mediate afferent ghrelin signals. Little is known, however, about ghrelin-induced efferent signals. We therefore investigated if hypothalamic-pituitary axes have a role in transferring ghrelin-induced changes of energy balance to the periphery. Research Methods and Procedures: We subcutaneously injected hypophysectomized, as well as adrenalectomized, thyroidectomized, and sham-operated control rats with GH secretagogues [ghrelin, growth hormone (GH)-releasing peptide] for 1 week. Body weight, food intake, and body composition (chemical carcass analysis) were analyzed and compared with vehicle-treated controls. In addition, we quantified circulating levels of endogenous ghrelin in hypophysectomized and GH–treated normal rats. Results: GH-secretagogue treatment of sham-operated control rats dose-proportionally increased food intake, body weight, and fat mass compared with vehicle-injected controls (p < 0.01). These effects, however, were not observed in ghrelin-treated hypophysectomized, thyroidectomized, or adrenalectomized rats, indicating an essential role for the pituitary axis in ghrelin-induced adiposity. Circulating levels of endogenous ghrelin were reduced by administration of GH in normal rats and were about 3-fold higher in hypophysectomized rats (n = 20, p = 0.001), suggesting a regulatory feedback loop involving the stomach and the pituitary to regulate gastric ghrelin secretion. Discussion: According to these results, the endocrine pituitary is mediating ghrelin-induced changes toward a positive energy balance and is involved in the regulation of ghrelin secretion through a gastro-hypophyseal feedback loop.     

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.     

The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.     

Hormonal regulation of energy-protein homeostasis in hemodialysis patients: an anorexigenic profile that may predispose to adverse cardiovascular outcomes

To assess whether endocrine dysfunction may cause derangement in energy homeostasis in patients undergoing hemodialysis (HD), we profiled hormones, during a 3-day period, from the adipose tissue and the gut and the nervous system around the circadian clock in 10 otherwise healthy HD patients and 8 normal controls. The protocol included a 40-h fast. We also measured energy-protein intake and output and assessed appetite and body composition. We found many hormonal abnormalities in HD patients: 1) leptin levels were elevated, due, in part, to increased production, and nocturnal surge in response to daytime feeding, exaggerated. 2) Peptide YY (PYY), an anorexigenic gut hormone, was markedly elevated and displayed an augmented response to feeding. 3) Acylated ghrelin, an orexigenic gut hormone, was lower and did not exhibit the premeal spike as observed in the controls. 4) neuropeptide Y (NPY), a potent orexigenic peptide, was markedly elevated and did not display any circadian variation. 5) Norepinephrine, marginally elevated, did not exhibit the normal nocturnal dip. By contrast, α-melanocyte-stimulating hormone and glucagon-like peptide-1 were not different between the two groups. Despite these hormonal abnormalities, HD patients maintained a good appetite and had normal body lean and fat mass, and there was no evidence of increased energy expenditure or protein catabolism. We explain the hormonal abnormalities as well as the absence of anorexia on suppression of parasympathetic activity (vagus nerve dysfunction), a phenomenon well documented in dialysis patients. Unexpectedly, we noted that the combination of high leptin, PYY, and NPY with suppressed ghrelin may increase arterial blood pressure, impair vasodilatation, and induce cardiac hypertrophy, and thus could predispose to adverse cardiovascular events that are the major causes of morbidity and mortality in the HD population. This is the first report attempting to link hormonal abnormalities associated with energy homeostasis to adverse cardiovascular outcome in the HD patients.     

Leptin: a possible link between food intake, energy expenditure, and reproductive function

Several regulatory substances participate in the regulation of both food intake/energy metabolism and reproduction in mammals. Most of these neuropeptides originate and act in the central nervous system, mainly at specific hypothalamic areas. Leptin represents a signal integrating all these functions, but originating from the periphery (adipose tissue) and carrying information mainly to central structures. Observations in rodent models of leptin deficiency have suggested that leptin participates in the control of reproduction, in conjunction with that of food intake and energy expenditure. Indeed, leptin administration resulted in the restoration of normal body weight, food intake, and fertility in the ob mouse, lacking circulating leptin. Specific targets of leptin in the hypothalamus are neurons expressing neuropeptide Y, proopiomelanocortin and gonadotropin-releasing hormone, but the presence of leptin receptors in peripheral reproductive structures suggests that leptin might also act at these sites. Human obesity is often associated with reproductive disturbances. The situation in humans is more complex than in the animal models of leptin deficit and the presence of leptin resistance in these subjects is suggested. In conclusion, leptin fits many requirements for a molecule linking the regulation of energy balance and the control of reproduction.     

Peptide signals regulating food intake and energy homeostasis

The adiposity hormone leptin has been shown to decrease food intake and body weight by acting on neuropeptide circuits in the hypothalamus. However, it is not clear how this primary hypothalamic action of leptin is translated into a change in food intake. We hypothesize that the behavioral effect of leptin ultimately involves the integration of neuronal responses in the forebrain with those in the nucleus tractus solitarius in the caudal brainstem, where ingestive behavior signals are received from the gastrointestinal system and the blood. One example is the peptide cholecystokinin, which is released from the gut following ingestion of a meal and acts via vagal afferent nerve fibers to activate medial nucleus tractus solitarius neurons and thereby decrease meal size. While it is established that leptin acts in the arcuate nucleus in the hypothalamus to stimulate anorexigenic neurons that inhibit food intake while simulataneously inhibiting orexigenic neurons that increase food intake, the mechanisms linking these effects with regions of the caudal brainstem that integrate cues related to meal termination are unclear. Based on an increasing body of supportive data, we hypothesize that this integration involves a pathway comprising descending projections from neurons from the paraventricular nucleus to neurons within the nucleus tractus solitarius that are activated by meal-related satiety factors. Leptin's anorexic effect comprises primarily decreased meal size, and at subthreshold doses for eliciting an effect on food intake, leptin intensifies the satiety response to circulating cholecystokinin. The location of neurons subserving the effects of intracerebroventricular administration of leptin and intraperitoneal injection of cholecystokinin on food intake has been identified by analysis of Fos expression. These studies reveal a distribution that includes the paraventricular nucleus and regions within the caudal brainstem, with the medial nucleus tractus solitarius having the most pronounced Fos expression in response to leptin and cholecystokinin, and support the hypothesis that the long-term adiposity signal leptin and the short-term satiety signal cholecystokinin act in concert to maintain body weight homeostasis.     

Effects of insulin, leptin, and glucagon on ghrelin secretion from isolated perfused rat stomach

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally purified from the rat stomach. Although ghrelin has been recognized as an important regulator of energy metabolism, the regulation of the ghrelin secretion is largely unknown. Here, we examined the direct effects of insulin, leptin, and glucagon on the release of ghrelin from the isolated rat stomach. The isolated pancreas-spleen-duodenum deprived preparation of rat stomach was used. After a baseline control infusion into the left gastric artery, insulin, leptin, or glucagon were infused for 15 min at concentrations of 0.1, 1, and 10 nM. The levels of immunoreactive ghrelin in the venous effluents were measured with a radioimmunoassay. Insulin and leptin inhibited ghrelin secretion dose-dependently (total amount of ghrelin release: insulin at 1 nM, 73.5+/-7.3% of the control infusion; leptin at 1 nM, 81.8+/-2.5% of the control infusion; n=5, P<0.05), while glucagon increased it dose-dependently (total amount of ghrelin released at 10 nM was 143.9+/-19.3% of the control infusion; n=5, P<0.01). These results indicate that the ghrelin responses observed in vivo could be due to direct effects of multiple hormonal signals on the stomach.