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Fifteen years ago orexins were identified as central regulators of energy homeostasis. Since then, that concept has evolved considerably and orexins are currently considered, besides orexigenic neuropeptides, key modulators of sleep-wake cycle and neuroendocrine function. Little is known, however, about the effect of the neuroendocrine milieu on orexins' effects on energy balance. We therefore investigated whether hypothalamic-pituitary axes have a role in the central orexigenic action of orexin A (OX-A) by centrally injecting hypophysectomized, adrenalectomized, gonadectomized (male and female), hypothyroid, and GH-deficient dwarf rats with OX-A. Our data showed that the orexigenic effect of OX-A is fully maintained in adrenalectomized and gonadectomized (females and males) rats, slightly reduced in hypothyroid rats, and totally abolished in hypophysectomized and dwarf rats when compared with their respective vehicle-treated controls. Of note, loss of the OX-A effect on feeding was associated with a blunted OX-A-induced increase in the expression of either neuropeptide Y or its putative regulator, the transcription factor cAMP response-element binding protein, as well as its phosphorylated form, in the arcuate nucleus of the hypothalamus of hypophysectomized and dwarf rats. Overall, this evidence suggests that the orexigenic action of OX-A depends on an intact GH axis and that this neuroendocrine feedback loop may be of interest in the understanding of orexins action on energy balance and GH deficiency.

Humanin (HN) is a novel 24-amino acid mitochondrial-derived peptide that has demonstrated diverse cytoprotective effects, including an emerging role in diabetes. The purpose of this study was to examine the pharmacokinetics of humanin analogues, which show great potential as therapeutic agents (HNG and the non-IGFBP-3 binding, HNGF6A). 11-week-old male IGFBP-3(-/-) and wild type (WT) mice were divided into 3 groups: WT mice treated with HNG, WT mice treated with HNGF6A, and IGFBP-3(-/-) mice treated with HNG. Plasma was obtained from mice following ip injection with HN analogues, and HN levels were measured with ELISA. WT mice treated with HNGF6A and IGFBP-3(-/-) mice treated with HNG displayed a longer half-life of HN compared with WT mice treated with HNG. Following HNG injection, both IGF-1 and IGFBP-3 levels decreased over time. Adult male Sprague Dawley rats were also ip injected with HNG, and HN levels were measured in various tissues (plasma, liver, heart, and brain) by ELISA. The half-life of HN was found to be longer in rats compared with mice. In rats, HN levels were found to be highest in plasma, present in liver, and undetectable in brain or heart. The current study provides evidence of HN and IGFBP-3 association in the circulation and suggests that native HN may modulate the distribution of IGF-1 and IGFBP-3. The results also demonstrate varying kinetic profiles of HN analogues and interspecies variation in rodents. Sustainable levels of circulating HN measured in plasma underline the potential value of HN analogues as a new therapeutic intervention in the treatment of diabetes.

Orexin-A (ORXA) is an orexigenic neuropeptide produced by the lateral hypothalamus that increases food intake when injected into the brain ventricles or forebrain nuclei. We used a licking microstructure analysis to evaluate hindbrain and forebrain ORXA effects in intact and hindbrain-lesioned rats, to identify the motivational and anatomical bases of ORXA hyperphagia. Intact rats with cannulas in the fourth brain ventricle (4V) received vehicle (artificial cerebrospinal fluid) or ORXA (0.1, 0.4, 1, or 10 nm) injections before 90 min access to 0.1 m sucrose. Meal size and frequency were increased in a double-dissociated manner by the 1 and 10 nm doses, respectively. In experiment 2, 4V 1 nm ORXA was applied to rats offered solutions varied in caloric and gustatory intensity (water and 0.1 and 1 m sucrose). ORXA increased meal frequency for all tastants. ORXA increased meal size only for 0.1 m sucrose, by prolonging the meal without affecting early ingestion rate or lick burst size, suggesting that 4V ORXA influenced inhibitory postingestive feedback rather than taste evaluation. In experiment 3, rats with cannulas in the third ventricle (3V) received dorsal medullary lesions centered on the area postrema (APX group) or sham procedures, and licking for water and 0.1 and 1 m sucrose was evaluated after 1 nm 3V ORXA/artificial cerebrospinal fluid injections. The 3V ORXA increased 0.1 m sucrose meal size and meal frequency for all tastants in the sham group, as observed after 4V ORXA in experiment 2. In the APX group, 3V ORXA injections influenced meal frequency, but they no longer increased meal size. However, the APX rats increased meal size for 0.1 m sucrose after food and water deprivation and after 3V angiotensin II injection. They also showed meal size suppression after 3V injection of the melanocortin-3/4 receptor agonist melanotan II (1 nm). These findings suggest that the area postrema and subjacent nucleus of the solitary tract are necessary for increases in consummatory (meal size) but not appetitive (meal frequency) responses to 3V ORXA. The meal size increases may be due to reduced postingestive feedback inhibition induced by ORXA delivered to either the hindbrain or forebrain ventricles.

The dephosphorylation of glycogen synthase is a key step in the stimulation of glycogen synthesis by insulin. To further investigate the hormonal regulation of glycogen synthase activity, enzymatic localization in 3T3-L1 adipocytes was determined by immunocytochemistry and confocal microscopy. In basal cells, glycogen synthase and the protein phosphatase-1-glycogen-targeting subunit, protein targeting to glycogen (PTG), were diffusely distributed throughout the cell. Insulin treatment had no effect on PTG distribution but resulted in a reorganization of glycogen synthase into punctate clusters. Glycogen synthase aggregation was restricted to discrete cellular sites, presumably where glycogen synthesis occurred. Omission of extracellular glucose or substitution with 2-deoxy-glucose blocked the insulin-induced redistribution of glycogen synthase. Addition of the glycogenolytic agent forskolin after insulin stimulation disrupted the clusters of glycogen synthase protein, restoring the immunostaining pattern to the basal state. Conversely, adenoviral-mediated overexpression of PTG resulted in the insulin-independent dephosphorylation of glycogen synthase and a redistribution of the enzyme from the cytosolic- to glycogen-containing fractions. The effects of PTG on glycogen synthase activity were mediated by multisite dephosphorylation, which was enhanced by insulin and 2-deoxy-glucose, and required a functional glycogen synthase-binding domain on PTG. However, PTG overexpression did not induce distinct glycogen synthase clustering in fixed cells, presumably because cellular glycogen levels were increased more than 7-fold under these conditions, resulting in a diffusion of sites where glycogen elongation occurred. Cumulatively, these data indicate that the hormonal regulation of glycogen synthesis rates in 3T3-L1 adipocytes is mediated in part through changes in the subcellular localization of glycogen synthase.