Abhiram Sahu

Leptin signaling in the hypothalamus: emphasis on energy homeostasis and leptin resistance

Leptin, the long-sought satiety factor of adipocytes origin, has emerged as one of the major signals that relay the status of fat stores to the hypothalamus and plays a significant role in energy homeostasis. Understanding the mechanisms of leptin signaling in the hypothalamus during normal and pathological conditions, such as obesity, has been the subject of intensive research during the last decade. It is now established that leptin action in the hypothalamus in regulation of food intake and body weight is mediated by a neural circuitry comprising of orexigenic and anorectic signals, including NPY, MCH, galanin, orexin, GALP, alpha-MSH, NT, and CRH. In addition to the conventional JAK2-STAT3 pathway, it has become evident that PI3K-PDE3B-cAMP pathway plays a critical role in leptin signaling in the hypothalamus. It is now established that central leptin resistance contributes to the development of diet-induced obesity and ageing associated obesity. Central leptin resistance also occurs due to hyperleptinimia produced by exogenous leptin infusion. A defective nutritional regulation of leptin receptor gene expression and reduced STAT3 signaling may be involved in the development of leptin resistance in DIO. However, leptin resistance in the hypothalamic neurons may occur despite an intact JAK2-STAT3 pathway of leptin signaling. Thus, in addition to defective JAK2-STAT3 pathway, defects in other leptin signaling pathways may be involved in leptin resistance. We hypothesize that defective regulation of PI3K-PDE3B-cAMP pathway may be one of the mechanisms behind the development of central leptin resistance seen in obesity.

Resistance to the Satiety Action of Leptin Following Chronic Central Leptin Infusion is Associated with the Development of Leptin Resistance in Neuropeptide Y Neurones

Leptin regulates food intake and body weight by acting primarily in the hypothalamus. In humans and rodents, obesity is associated with hyperleptinaemia, suggesting a possible state of leptin resistance. Thus, to begin to examine the mechanisms of leptin resistance, we developed a rat model in which chronic central leptin infusion results in the development of resistance to leptins satiety action. Adult male rats were infused chronically into the lateral cerebroventricle with leptin (160 ng/h) or phosphate‐buffered saline via Alzet pumps for 28 days, followed by artificial cerebrospinal fluid infusion for 3 weeks. After the initial decrease in food intake, rats developed resistance to the satiety action of leptin, and withdrawal of the chronic leptin infusion resulted in hyperphagia. During leptin infusion, body weight was gradually decreased to reach a nadir on day 12, and thereafter, body weight was sustained at a reduced level throughout the entire 28‐day infusion, despite normalization in food intake. Body weight was mostly normalized by day 22 postleptin. Since neuropeptide Y (NPY) neurones are one of the targets of leptin signalling in the hypothalamus, we next examined whether the development of resistance to the satiety action of leptin was due to altered NPY gene expression. On day 3–4 of infusion, hypothalamic NPY mRNA levels, as determined by RNAse protection assay (RPA), were significantly decreased in leptin treated rats compared to controls. By contrast, on day 16 of infusion, NPY mRNA levels in the leptin treated group had returned to control levels. In situ hybridization study confirmed the results obtained with RPA and showed further that the effect of chronic leptin infusion on NPY mRNA levels was restricted to the rostral and middle parts of the arcuate nucleus. Overall, the finding that the action of continuous leptin exposure on NPY neurones was not sustained suggests that NPY neurones may be involved in the development of leptin resistance to the satiety action of leptin in the hypothalamus.

Evidence that neurotensin mediates the central effect of leptin on food intake in rat

Recent evidence suggests that leptins action on food intake and body weight regulation is mediated by a number of orexigenic and anorectic neuronal systems in the hypothalamus. Our previous demonstration that central injections of leptin induce hypothalamic neurotensin (NT) gene expression in association with a reduced food intake and decreased body weight in rats indicates that NT, an anorectic peptide, is involved in mediating leptins action on feeding and body weight regulation. To begin to examine the relative role of NT in this regard we evaluated the effects of NT antiserum (NT-AS) or NT receptor antagonist, SR 48692, on the satiety action of leptin in rats. In the first experiment, 3rd cerebroventricular (i.c.v.) administration of either 1 or 5 microl of NT-AS, 30 min prior to leptin (4 microg) injection, completely blocked the effects of leptin on food deprivation (FD)-induced feeding. In the second experiment, intraperitoneal (i.p.) administration of SR 48692 (40 microg/kg) also completely prevented leptins satiety action on FD-induced feeding. These results showing the reversal of leptins satiety action by either NT immunoneutralization or NT-receptor antagonism support our hypothesis that NT is involved in mediating leptins action on feeding and further suggest that this neuropeptide is a quantitatively important component of the leptin sensitive neural circuitry.

Intracellular Leptin-Signaling Pathways in Hypothalamic Neurons: The Emerging Role of Phosphatidylinositol-3 Kinase- Phosphodiesterase-3B-cAMP Pathway

Leptin is secreted primarily by fat cells and acts centrally, particularly in the hypothalamus, to reduce food intake and body weight. Besides the classical JAK2 (Janus kinase-2)-STAT3 (signal transducer and activator of transcription-3) pathway, several non-STAT3 pathways play an important role in mediating leptin signaling in the hypothalamus. We have demonstrated that leptin action in the hypothalamus is mediated by an insulin-like signaling pathway involving stimulation of PI3K (phosphatidylinositol-3 kinase) and PDE3B (phosphodiesterase-3B), and reduction in cAMP levels, and that a PI3K-PDE3B-cAMP pathway interacting with the JAK2-STAT3 pathway constitutes a critical component of leptin signaling in the hypothalamus. It appears that defective regulation of multiple signaling pathways in the hypothalamus causes central leptin resistance, a major cause of obesity. In this regard, we have shown that leptin resistance in hypothalamic neurons following chronic central infusion of this hormone is associated with a defect in the PI3K-PDE3B-cAMP, and not due to compromised signaling in the JAK2-STAT3 pathway. Similarly, the PI3K, but not the STAT3, pathway is impaired in the hypothalamus during the development of diet-induced obesity. Additionally, our recent work suggests that suppressor of cytokine signaling-3 negatively regulates the PI3K pathway of leptin signaling in the hypothalamus, a mechanism expected to play a significant role in diet-induced obesity. Together, the PI3K-PDE3B-cAMP pathway appears to emerge as a major mechanism of leptin signaling in the hypothalamus in regulating energy balance.

Effects of Orchidectomy on Levels of the mRNAs Encoding Gonadotropin‐Releasing Hormone and Other Hypothalamic Peptides in the Adult Male Rhesus Monkey (Macaca mulatta)1

The testicular regulation of luteinizing hormone (LH) secretion in the adult rhesus monkey is mediated by an indirect action of testosterone to decelerate pulsatile gonadotrophin releasing hormone (GnRH) release. Whether this negative feedback action of testosterone involves regulation of GnRH gene expression is unknown. Therefore, the effect of bilateral orchidectomy on hypothalamic levels of the mRNA encoding this hypophysiotropic factor was examined. The feedback action of testosterone is generally considered to be mediated through non‐GnRH cells, and the present experiment provided the opportunity to also examine testicular influences on mRNAs encoding putative hypothalamic factors implicated in the testicular regulation of LH secretion. Adult male rhesus monkeys were orchidectomized (n=5) or sham‐orchidectomized (n=5) and killed 6 weeks later, after a castration‐induced hypersecretion of LH was established. Separate preoptic and mediobasal hypothalamus containing areas were collected, and levels of GnRH mRNA, as well as those of mRNAs encoding pro‐opiomelanocortin (POMC), the γ‐aminobutyric acid (GABA) synthesizing enzymes (glutamic acid decarboxylase 65 and 67; GAD65 and GAD67, respectively), neuropeptide Y, galanin and transforming growth factor (TGF)α, were quantified using RNase protection assay. Values were expressed in terms of optical density relative to that of cyclophilin mRNA levels. Bilateral orchidectomy produced a significant increase in GnRH mRNA levels that was restricted to the mediobasal hypothalamus and that was associated with a significant decrease in POMC, GAD65 and GAD67 mRNA levels in this region of the hypothalamus. In contrast, neuropeptide Y, galanin and TGFα mRNA levels were not affected by castration. These results indicate that, in the monkey, the deceleration of pulsatile GnRH release that is imposed by the testis, and presumably mediated by testosterone, is associated with a concomitant down regulation of GnRH gene expression in the mediobasal hypothalamus. They also support the notion that this hypothalamic feedback action may be mediated by POMC‐and GABA‐producing neurones in the mediobasal hypothalamus.

Hypothalamic phosphatidylinositol 3-kinase-phosphodiesterase 3B-cyclic AMP pathway of leptin signalling is impaired following chronic central leptin infusion.

Leptin signalling in the hypothalamus is critical for the maintenance of normal body weight. Although hyperleptinaemia in obese people suggests a state of leptin resistance, and diet‐induced obesity in rodents is associated with central leptin resistance, the underlying mechanisms remain unclear. Recent evidence suggests that, in addition to the signal transducer and activator of the transcription‐3 (STAT3) pathway, leptin action is critical for energy homeostasis through an insulin‐like signalling pathway involving an increase in phosphatidylinositol 3‐kinase (PI3K) and phosphodiesterase 3B (PDE3B) activities and reduction in cyclic AMP (cAMP) levels in the hypothalamus. Here, we show that chronic central leptin (160 ng/h) infusion, which resulted in the development of resistance to the satiety action of leptin, impaired the PI3K‐PDE3B‐cAMP pathway of leptin signalling in the hypothalamus in that PI3K and PDE3B activities were increased and cAMP levels were decreased in the hypothalamus on day 2 of leptin infusion but remained unchanged on day 16. Additionally, induction of tyrosyl phosphorylation of insulin receptor substrate‐1 observed on day 2 was not evident on day 16 of leptin infusion. By contrast, signalling through the STAT3‐pathway remained activated in the hypothalamus throughout 16 days of leptin infusion. These findings show a differential response in PI3K‐PDE3B‐cAMP (impaired) and STAT3 (up‐regulated) pathways to chronic central leptin infusion, and suggest a selective resistance in the PI3K‐PDE3B‐cAMP pathway of leptin signalling following a chronic increase in hypothalamic leptin tone attained by central infusion of this peptide hormone.

Interactions of neuropeptide Y, hypocretin-I (orexin A) and melanin-concentrating hormone on feeding in rats

Amongst various neuropeptidergic systems, neuropeptide Y (NPY), hypocretin-1 and melanin-concentrating hormone (MCH) producing neurons have been shown to play an important role in the regulation of food intake and body weight. All of these neuropeptides are orexigenic signals and recent evidence suggests the existence of morphological connections between these neuronal systems in the hypothalamus. However, the functional interactions between these neuronal systems are not clearly understood. Therefore, in the present study, we examined whether there is a cooperative action on food intake between these neuropeptides after third intracerebroventricular (icv) injection in the rat. The icv administration of NPY (0.118, 0.588, 1.176 nmol), hypocretin-1 (1, 3 nmol) and MCH (0.42, 1.048, 2.096 nmol) stimulated food intake in a dose dependent manner. Coinjection with 0.118 nmol of NPY and hypocretin-1 (1, 3 nmol) or MCH (0.42, 1.048, 2.096 nmol) had no additive effect on food intake as compared to that of NPY alone. However, coinjection with lower dose of NPY (0.023 nmol) and hypocretin-1 (0.25 nmol), that did not have any effect alone, significantly induced food intake. In contrast, combination of a lower dose of NPY (0.023 nmol) or hypocretin-1 (0.25 nmol) with lower stimulatory dose (0.21, 0.42 nmol) of MCH did not result in further increase in food intake as compared to that of MCH alone. Also, combination of 0.25 nmol hypocretin-1 and a non-stimulatory dose of MCH (0.105 nmol) was ineffective in stimulating food intake. Finally, coinjection with of 0.023 nmol NPY and 0.105 nmol MCH significantly induced food intake as compared to saline control group but not as compared to NPY or MCH treated groups. In total, these results show (1) a synergistic action between NPY and hypocretin-1, (2) no interaction between hypocretin-1 and MCH and (3) very little interaction, if any, between NPY and MCH in inducing food intake. In conclusion, these results provide a physiological concomitant to the previous demonstration of morphological contacts between NPY and hypocretin producing neurons by suggesting an interaction between these two orexigenic signals in control of food intake, and further suggest that MCHs action on feeding may be independent of NPY and hypocretin-1 action.

Effect of Estrogen on Hypothalamic Transforming Growth Factor Alpha and Gonadotropin-Releasing Hormone Gene Expression in the Female Rhesus Monkey

In order to study whether hypothalamic transforming growth factor alpha (TGFα) gene expression in the monkey is estrogen-sensitive, long-term ovariectomized rhesus macaques were implanted subcutaneously with either estradiol-containing (n = 3) or blank (n = 3) Silastic capsules. Blood samples were collected every other day while the animals were lightly sedated with ketamine hydrochloride to monitor circulating LH and estradiol concentrations. Animals were killed with a lethal dose of pentobarbital sodium after a marked suppression of LH secretion was confirmed (81 days of estradiol treatment); the preoptic area (POA), mediobasal hypothalamus (MBH) and samples of cerebral cortex were dissected out, snap-frozen in liquid nitrogen and processed for the determination of TGFα messenger RNA (mRNA) by ribonuclease protection assay using a cRNA probe. The opportunity was also taken to study the action of estrogen on hypothalamic GnRH mRNA levels. Although circulating estradiol concentrations of 50–150 pg/ml achieved in the steroid-treated group produced a decrease in hypothalamic GnRH mRNA levels, which was significant in the MBH, TGFα mRNA levels in this hypothalamic region and in the POA were not influenced by estrogen treatment. These findings indicate that TGFα is probably not involved in mediating the inhibitory action of estradiol on GnRH neurons. Additionally, the relevance of our results to the understanding of the neurobiological mechanisms underlying the initiation of puberty in primates is discussed.

Central Nervous System Receptors Involved in Mediating the Inhibitory Action of Neuropeptide Y on Luteinizing Hormone Secretion in the Male Rhesus Monkey (Macaca mulatta)

An earlier finding that gonadotropin‐releasing hormone (GnRH) secretion may be triggered prematurely in the juvenile male monkey by central administration of 1229U91, a Y1 receptor antagonist, contributed to our current hypothesis that neuropeptide Y (NPY) is a major component of the brake that holds pulsatile GnRH release in check during prepubertal development in primates. However, 1229U91 is also a Y4 receptor agonist, and the present study was conducted to further examine the role of the Y1 receptor in mediating the putative inhibitory action of NPY on GnRH release. Agonadal juvenile and postpubertal male monkeys were implanted with i.v. and i.c.v. cannulae to gain continuous access to the venous and cerebroventricular circulations without sedation. Luteinizing hormone (LH) secretion was measured to provide an indirect index of GnRH release. The specific Y1 antagonists, VD‐11 (476 µg; n = 4) and isopropyl 3‐chloro‐5‐[1‐({6‐[2‐(5‐ethyl‐4‐methyl‐1,3‐thiazol‐2‐yl)ethyl]‐4‐morpholin‐4‐ylpyridin‐2‐yl}amino)ethyl]phenylcarbamate (Compound A, 300 µg; n = 4), did not mimic the stimulatory action of 1229U91 on GnRH secretion in the juvenile male monkey. Additionally, neither NPY (200 µg; n = 2), a general Y receptor agonist, nor rPP (100 µg; n = 4), a Y4 agonist, mimicked the action of 1229U91 in stimulating GnRH release. Moreover, previous exposure of the hypothalamus of juvenile monkeys (n = 5) to NPY (660 µg) failed to block 1229U91‐induced (200 µg) GnRH release. However, the action of NPY (364 µg) in inhibiting GnRH release postpubertally was attenuated by 1229U91 (300 µg). We conclude that, although the action of exogenous NPY to suppress GnRH release from the postpubertal hypothalamus appears to be mediated, at least in part, by the Y1 receptor, the existence of a Y1 receptor pathway inhibitory to GnRH release in the prepubertal hypothalamus remains to be substantiated.

Ovarian Regulation of Kisspeptin Neurones in the Arcuate Nucleus of the Rhesus Monkey (Macaca mulatta)

Tonic gonadotrophin secretion throughout the menstrual cycle is regulated by the negative‐feedback actions of ovarian oestradiol (E2) and progesterone. Although kisspeptin neurones in the arcuate nucleus (ARC) of the hypothalamus appear to play a major role in mediating these feedback actions of the steroids in nonprimate species, this issue has been less well studied in the monkey. In the present study, we used immunohistochemistry and in situ hybridisation to examine kisspeptin and KISS1 expression, respectively, in the mediobasal hypothalamus (MBH) of adult ovariectomised (OVX) rhesus monkeys. We also examined kisspeptin expression in the MBH of ovarian intact females, and the effect of E2, progesterone and E2 + progesterone replacement on KISS1 expression in OVX animals. Kisspeptin or KISS1 expressing neurones and pronounced kisspeptin fibres were readily identified throughout the ARC of ovariectomised monkeys but, on the other hand, in intact animals, kisspeptin cell bodies were small in size and number and only fine fibres were observed. Replacement of OVX monkeys with physiological levels of E2, either alone or with luteal phase levels of progesterone, abolished KISS1 expression in the ARC. Interestingly, progesterone replacement alone for 14 days also resulted in a significant down‐regulation of KISS1 expression. These findings support the view that, in primates, as in rodents and sheep, kisspeptin signalling in ARC neurones appears to play an important role in mediating the negative‐feedback action of E2 on gonadotrophin secretion, and also indicate the need to study further their regulation by progesterone.