Sex-associated differences in the leptin and ghrelin systems related with the induction of hyperphagia under high-fat diet exposure in rats

Leptin and ghrelin are known to be main hormones involved in the control of food intake, with opposing effects. Here we have explored whether changes in the leptin and ghrelin system are involved in the long-term effects of high-fat (HF) diet feeding in rats and whether sex-associated differences exist. Male and female Wistar rats were fed until the age of 6 months with a normal-fat (NF) or an HF-diet. Food intake and body weight were followed. Gastric and serum levels of leptin and ghrelin, and mRNA levels of leptin (in stomach and adipose tissue), ghrelin (in stomach), and NPY, POMC, and leptin and ghrelin receptors (OB-Rb and GHS-R) (in the hypothalamus) were measured. In both males and females, total caloric intake and body weight were greater under the HF-diet feeding. In females, circulating ghrelin levels and leptin mRNA expression in the stomach were higher under HF-diet. HF-diet feeding also resulted in higher hypothalamic NPY/POMC mRNA levels, more marked in females, and in lower OB-Rb mRNA levels, more marked in males. In addition, in females, serum ghrelin levels correlated positively with hypothalamic NPY mRNA levels, and these with caloric intake. In males, hypothalamic OB-Rb mRNA levels correlated positively with POMC mRNA levels and these correlated negatively with caloric intake and with body weight. These data reflect differences between sexes in the effects of HF-diet feeding on food intake control systems, suggesting an impairment of the anorexigenic leptin-POMC system in males and an over-stimulation of the orexigenic ghrelin-NPY system in females.     

Central mechanisms involved in the orexigenic actions of ghrelin

Ghrelin is a stomach hormone, secreted into the bloodstream, that initiates food intake by activating NPY/AgRP neurons in the hypothalamic acruate nucleus. This review focuses on recent evidence that details the mechanisms through which ghrelin activate receptors on NPY neurons and downstream signaling within NPY neurons. The downstream signaling involves a novel CaMKK-AMPK-CPT1-UCP2 pathway that enhances mitochondrial efficiency and buffers reactive oxygen species in order to maintain an appropriate firing response in NPY. Recent evidence that shows metabolic status affects ghrelin signaling in NPY is also described. In particular, ghrelin does not activate NPY neurons in diet-induced obese mice and ghrelin does not increase food intake. The potential mechanisms and implications of ghrelin resistance are discussed.     

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.     

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 μg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 μg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12 h. Ghrelin and desacyl ghrelin (64 μg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.     

Plasma nesfatin-1 concentrations in restricting-type anorexia nervosa

Restricting-type anorexia nervosa (AN-R) is characterized by chronic food restriction and severe emaciation due to various cognitive biases such as a distorted self-image. In spite of several treatments, AN-R continues to be a refractory disease because of its unknown pathogenesis. Although previous studies have shown that changes in feeding regulatory peptides such as ghrelin are involved in anorexia, few reports have described the relationship between AN-R and nesfatin-1, a recently identified satiety peptide. Therefore, we examined the plasma nesfatin-1 levels in AN-R patients to determine its role in AN-R. A total of 15 women participated in the study; 7 patients with AN-R and 8 age-matched healthy controls (average BMI, 13.02 ± 0.30 vs. 21.57 ± 0.48, respectively). Our results showed that plasma nesfatin-1 levels were significantly lower in AN-R group than in control group (6.23 ± 0.70 ng/ml vs. 8.91 ± 0.85 ng/ml, respectively, P < 0.05). Plasma acyl ghrelin and des-acyl ghrelin levels were significantly higher in AN-R group than in control group (acyl ghrelin: 62.4 ± 10.15 fmol/ml vs. 27.20 ± 5.60 fmol/ml, P < 0.01 and des-acyl ghrelin: 300.17 ± 55.95 fmol/ml vs. 107.34 ± 40.63 fmol/ml, P < 0.05). Although AN-R is associated with emaciation for a prolonged period, our result suggested that nesfatin-1 levels may be regulated by nutrition status and response to starvation.     

Action of anorexigenic peptide injected into the brain: Dissociation of effect on body weight and feeding in the rat

Previous reports on the effect of anorexigenic peptide (AXP) administered systemically in the rodent are inconsistent in terms of the effect of the tri-peptide on food intake and body weight. The purpose of this study was to examine the effect of AXP infused into the brain on these measures. In post-pubescent female rats of the Sprague-Dawley strain, guide cannulae were permanently implanted in the lateral cerebral ventricle for repeated intracerebroventricular (ICV) infusion. Postoperatively, measures of food and water intake and body weight were obtained every day at the same time. After a 7-day base-line period, AXP was infused bilaterally in a total volume of 15 μl and in a dose of either 0.25 μg (n=7) or 1.25 μg (n=5), with artificial CSF vehicle serving as the control solution (n=6). ICV infusions were given once daily for 20 consecutive days, after which the same intake and body weight measures were recorded for another 7-day period. The rats given 0.25 μg AXP showed a significant suppression in weight gain with the overall slope of the growth curve being 0.358. In contrast, the growth slope of the rats given the 1.25 μg dose of AXP was 0.621, whereas those given the CSF was 0.823. Although the trends of intake of food tended to follow the curves of the rats' body weight, the difference between g/kg food intake of rats during ICV infusions of either dose of AXP was not significantly different from that of the CSF controls. Water intake also was unaffected by either dose of AXP. These results demonstrate that this tri-peptide derived from urine of patients afflicted with anorexia nervosa exerts a direct effect on the brain. Since the 0.25 μg dose of AXP infused acutely ICV caused a sustained hyperthermia, its mechanism of action is apparently a metabolic one; that is, the interruption in the gain in body weight of the rat is independent of the amount of food it ingests.     

Overeating of palatable food is associated with blunted leptin and ghrelin responses

Palatable food is rich in fat and/or sucrose. In this study we examined the long-term effects of such diets on food intake, body weight, adiposity and circulating levels of the satiety peptide leptin and the hunger peptide ghrelin. The experiments involved rats and mice and lasted 5 weeks. In rats, we examined the effect of diets rich in fat and/or sucrose and in mice the effect of a high fat diet with or without sucrose in the drinking water. Animals fed with the palatable diets had a larger intake of calories, gained more weight and became more adipose than animals fed standard rat chow. Fasted animals are known to have low serum leptin and high serum ghrelin and to display elevated serum leptin and lowered serum ghrelin postprandially. With time, a sucrose-rich diet was found to raise the fasting level of leptin and to lower the fasting level of ghrelin in rats. A fat-rich diet suppressed serum ghrelin without affecting serum leptin; high sucrose and high fat in combination greatly reduced serum ghrelin and raised serum leptin in the fasted state. The mRNA expression of leptin in the rat stomach was up-regulated by sucrose-rich (but not by fat-rich) diets, whereas the expression of ghrelin seemed not to be affected by the palatable diets. Mice responded to sucrose in the drinking water with elevated serum leptin (fasted state) and to all palatable diets with low serum ghrelin. The expression of both leptin and ghrelin mRNA in the stomach was suppressed in fasted mice that had received a high fat diet for 5 weeks. We conclude that the expression of leptin mRNA in stomach and the concentration of leptin in serum were elevated in response to sucrose-rich rather than fat-rich diets, linking leptin with sucrose metabolism. In contrast, the expression of ghrelin and the serum ghrelin concentration were suppressed by all palatable diets, sucrose and fat alike. In view of the increased body weight and adiposity neither elevated leptin nor suppressed ghrelin were able to control/restrain the overeating that is associated with palatable diets.     

Hypothalamic orexigenic peptides are overexpressed in young Long-Evans rats after early life exposure to fat-rich diets

Nutritional factors have a critical influence during prenatal life on the development and regulation of networks involved in body weight and feeding regulation. To establish the influence of the macronutrient type on feeding regulatory mechanisms and more particularly on stimulatory pathways (galanin and orexins), we fed female rats on either a high-carbohydrate (HC), a high-fat (HF), or a well-balanced control diet during gestation and lactation, and measured peptide expression in the hypothalamus and important hormones (leptin, insulin) in their pups at weaning. HF weanlings were 30% lighter than control and HC pups (P<0.001). They were characterized by reduced plasma glucose and insulin levels (P<0.01 or less). Their galanin and orexin systems were upregulated as shown by the significant augmentation of mRNA expression in the paraventricular nucleus and lateral hypothalamus, respectively. Inhibitory peptides like corticotropin-releasing hormone and neurotensin were not affected by this dietary treatment during early life. There was, therefore, a more intense drive to eat in HF pups, perhaps to compensate for the lower body weight at weaning. HF diets during early life had meanwhile some positive consequences: the lower metabolic profile might be beneficial in precluding the development of obesity and metabolic syndrome later in life. This is however valid only if the orexigenic drive is normalized after weaning.     

AdipoR1 mediates the anorexigenic and insulin/leptin-like actions of adiponectin in the hypothalamus

Adiponectin exerts an insulin-sensitizing effect, improving insulin action in peripheral tissues and restraining insulin resistance. Here, we explore the hypothesis that adiponectin can reproduce some of the actions of insulin/leptin in the hypothalamus. The presence of AdipoR1 and AdipoR2 was mapped to the arcuate and lateral hypothalamic nuclei. Icv adiponectin reduced food intake, which was accompanied by activation/engagement of IRS1/2, ERK, Akt, FOXO1, JAK2 and STAT3. All these actions were dependent on AdipoR1, since inhibition of this receptor, and not of AdipoR2, completely reversed the effects described above. Thus, adiponectin acts in the hypothalamus, activating elements of the canonical insulin and leptin signaling pathways and promoting reduction of food intake.     

Consummatory behavior and metabolic indicators after central ghrelin injections in rats

Ghrelin, an endogenous ligand for the growth-hormone-secretagogue receptor, is a 28-amino acid peptide with a post-translational acyl modification necessary for its activity. It has central nervous system actions that affect appetite, body mass and energy balance. An intracerebroventricular (ICV) injection protocol of sub-nanomolar doses of ghrelin, known to alter the morphology of ACTH and GH producing pituicytes and plasma levels of these hormones, was used to provide an overview of metabolic changes linked to energy metabolism. Variables measured were: food intake (FI), water intake (WI), fecal mass, urine volume, body weight (BW), retroperitoneal (RP) and epididymal (EPI) white adipose tissue (WAT), and changes in serum leptin, insulin, triglycerides, cholesterol, and glucose. Five injections of rat ghrelin or PBS (n = 8 per group) were given ICV every 24 h (1 μg/5 μL PBS) to adult male rats. Ghrelin had a positive and cumulative effect on FI, WI and BW (p < 0.05), but not feces mass or urine volume (p > 0.05). Centrally applied ghrelin clearly increased RP WAT (by 235%, p < 0.001), EPI WAT (by 85%, p < 0.05) and serum insulin levels (by 43%, p < 0.05), and decreased serum leptin levels (by 77%, p < 0.05) without (p > 0.05) evoking changes in blood triglyceride cholesterol, or glucose levels.

These data and the available literature clearly document that exposure of the brain of normal rats, over time, to sub-nanomolar doses of ghrelin results in metabolic dysregulation culminating in increased body mass, consummatory behavior, and lipid stores as well as changes in blood leptin/insulin levels. Thus, modulation of central ghrelin receptors may represent a pharmacological approach for controlling multiple factors involved in energy balance and obesity.     

Voluntary exercise contributed to an amelioration of abnormal feeding behavior,locomotor activity and ghrelin production concomitantly with a weight reduction in high fat diet-induced obese rats

In the present study, effects of voluntary exercise in an obese animal model were investigated in relation to the rhythm of daily activity and ghrelin production. Male Sprague–Dawley rats were fed either a high fat diet (HFD) or a chow diet (CD) from four to 16 weeks old. They were further subdivided into either an exercise group (HFD-Ex, CD-Ex) with a running wheel for three days of every other week or sedentary group (HFD-Se, CD-Se). At 16 weeks old, marked increases in body weight and visceral fat were observed in the HFD-Se group, together with disrupted rhythms of feeding and locomotor activity. The induction of voluntary exercise brought about an effective reduction of weight and fat, and ameliorated abnormal rhythms of activity and feeding in the HFD-Ex rats. Wheel counts as voluntary exercise was greater in HFD-Ex rats than those in CD-Ex rats. The HFD-obese had exhibited a deterioration of ghrelin production, which was restored by the induction of voluntary exercise. These findings demonstrated that abnormal rhythms of feeding and locomotor activity in HFD-obese rats were restored by infrequent voluntary exercise with a concomitant amelioration of the ghrelin production and weight reduction. Because ghrelin is related to food anticipatory activity, it is plausible that ghrelin participates in the circadian rhythm of daily activity including eating behavior. A beneficial effect of voluntary exercise has now been confirmed in terms of the amelioration of the daily rhythms in eating behavior and physical activity in an animal model of obesity.     

Leptins and leptin receptor expression in the goldfish (Carassius auratus). Regulation by food intake and fasting/overfeeding conditions

Leptin is a hormone involved in feeding and body weight regulation in vertebrates, but the relationship between energy status and leptin has not been clearly established in fish. The aim of this study was to investigate in a teleost, the goldfish (Carassius auratus), the tissue expression pattern of two leptins (gLep-aI and gLep-aII) and leptin receptor (gLepR); and the effect of feeding on expression of these genes. Leptin system expression in goldfish was firstly analyzed in fish under overfeeding (2 weeks) or fasting (1 week), and secondly, at different postfeeding times (0, 3, 6, 9 and 12 h). Goldfish has two Lep-a paralog genes, gLep-aI was widely expressed in central and peripheral tissues, whereas gLep-aII was preferentially expressed in brain. This different distribution pattern of leptins suggests that they can play different physiological roles in goldfish. The gLepR mRNA was ubiquitous expressed, with the highest expression in the telencephalon and hypothalamus. No significant differences in the leptin system expression were found among control, overfed and fasting groups, suggesting an apparent lack of correlation between nutritional status and leptin system in goldfish. Hepatic expression of gLep-aI significantly increased 9 h after feeding time, while hypothalamic leptin system expression did not change after feeding. In summary, leptin in goldfish could signal short-term changes in food intake, as postprandial satiety, but seems to be independent of fasting/overfeeding conditions in this teleost. The widespread distribution of leptins and leptin receptor in goldfish strongly supports that this hormone may have pleitropic actions in fish.     

Interface between metabolic balance and reproduction in ruminants: Focus on the hypothalamus and pituitary

This article is part of a Special Issue “Energy Balance”.     

Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long-Evans rats. PEPS at doses of 1 and 10 microg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose (P<0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (-48%; P<0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.     

Gender-specific differences in biomarkers responses to acute coronary syndromes and revascularization procedures

A growing body of gender-related research in coronary artery disease is beginning to gradually elucidate differences between women and men. In patients presenting with acute coronary syndromes (ACS), these sex differences include varying risk factor profiles, accuracy of diagnostic testing, clinical presentations, treatment practices and outcomes. There is also a differential expression of cardiac biomarkers by sex, which remains unexplained. This paper reviews all the available information on the effect of gender on cardiac biomarkers by search of MEDLINE using the terms gender differences, biomarkers, ACS and revascularization procedures. A better understanding of the sex disparities in biomarkers along with all other clinical information is essential to optimal management and patient care in the future.     

Ghrelin concentrations and cardiac vagal tone are decreased after pharmacologic and cognitive-behavioral treatment in patients with bulimia nervosa

Patients with bulimia nervosa (BN) have bulimic and depressive symptoms, which have been associated with abnormalities in the neuroendocrine and vagal systems. Subjects included twenty-four female drug-free outpatients with BN that were selected from patients seeking treatment for eating behavior in our hospital along with twenty-five age-matched healthy females who served as controls. We investigated ghrelin and leptin levels, cardiac vagal tone and sympathovagal balance, frequency of sets of binge-eating and vomiting episodes per week and the Profile of Mood States (POMS) depression scale in BN before and after a 16-week administration of the serotonin selective reuptake inhibitor (SSRI) paroxetine combined with cognitive-behavioral therapy. Compared to controls, the BN group had higher ghrelin levels and resting cardiac vagal tone, and lower leptin levels and resting cardiac sympathovagal balance before treatment, although there was a significant difference between the two groups for the body mass index (BMI). The elevated ghrelin levels (301.7 +/- 18.9 pmol/l, mean +/- SEM vs. 202.8 +/- 15.6 pmol/l, P < 0.01), cardiac vagal tone (2246.4 +/- 335.5 ms(2) vs. 1128.5 +/- 193.3 ms(2), P < 0.01), frequency of sets of binge-eating and purging episodes and T scores for the POMS depression scale were all significantly decreased after treatment despite similar BMI, percent body fat and leptin levels. In close association with cardiac vagal function and ghrelin recoveries, abnormal eating behavior and depressive symptoms improved, indicating the usefulness of these indexes in the assessment of clinical condition and therapeutic efficacy in BN.     

Corticotropin-releasing factor (CRF) is involved in the acute anorexic effect of alpha-melanocyte-stimulating hormone: a study using CRF-deficient mice

Alpha-melanocyte-stimulating hormone (α-MSH) and its receptors are critical and indispensable for maintaining appropriate feeding behavior and energy homeostasis in both mice and humans. Corticotropin-releasing factor (CRF) is a candidate for mediating the anorexic effect of α-MSH. In the present study, we examined whether CRF and its receptors are involved in the anorexic effect of α-MSH, using CRF-deficient (CRFKO) mice and a CRF receptor antagonist. Intracerebroventricular administration of NDP-MSH, a synthetic α-MSH analogue, suppressed food intake in wild-type (WT) mice. This effect was abolished by pretreatment with a non-selective CRF receptor antagonist, astressin, suggesting that the effect of α-MSH-induced anorexia was mediated by a CRF receptor. In CRFKO mice, administration with NDP-MSH did not affect food intake at an early phase (0–4 h). In addition, CRF mRNA levels in the hypothalamus were significantly increased in NDP-MSH-treated mice. Therefore, our findings, using CRFKO, strongly support evidence that CRF is involved in the acute anorexic effect of α-MSH. On the other hand, NDP-MSH administered to CRFKO mice led to suppressed food intake at the late phase (4–12 h), similar to the effect in WT mice. Further, NDP-MSH similarly reduced food intake during the late phase in all types of mice, including WT, CRFKO, and CRFKO with corticosterone replacement. The results would suggest that α-MSH-induced suppression of food intake at late phase was independent of glucocorticoids and CRF.     

Differential inhibition of galanin- and ghrelin-induced food intake by i.c.v. GLP-1(7-36)-amide

Feeding regulation involves both anorectic and orexigenic neuropepetides mainly located in the hypothalamus. To gain further insight into the interaction between these two groups of regulators inhibition of feeding induced by glucagon-like peptide-1 (GLP-1) was examined during stimulation of food intake by equimolar doses of ghrelin and galanin. The experiments were carried out in freely feeding rats. Intracerebroventricular (i.c.v.) injections were accomplished through stereotaxically implanted cannulae aimed at the lateral cerebral ventricle. Food intake of standard rat chow pellets was subsequently recorded for 2 h. Ghrelin and galanin stimulated food intake significantly with no difference between these two peptides. During ghrelin stimulation GLP-1 inhibited feeding in doses between 0.015 and 1.5 nmol. During galanin stimulation of food intake a ten fold higher dose (0.15 nmol) was required to inhibit food intake. In conclusion equimolar doses of i.c.v. ghrelin and galanin are similarly effective stimuli of food intake when given alone. However in combination with an anorectic neuropeptide such as GLP-1 they have substantially different potencies of feeding stimulation. Such interaction could also be of interest for therapeutic strategies involving both regulating groups of neuropeptides.