FNDC5 expression and circulating irisin levels are modified by diet and hormonal conditions in hypothalamus, adipose tissue and muscle.

Irisin is processed from fibronectin type III domain-containing protein 5 (FNDC5). However, a controversy exists concerning irisin origin, regulation and function. To elucidate the relationship between serum irisin and FNDC5 mRNA expression levels, we evaluated plasma irisin levels and FNDC5 gene expression in the hypothalamus, gastrocnemius muscle and different depots of adipose tissue in models of altered metabolism. In normal rats, blood irisin levels diminished after 48-h fast and with leptin, insulin and alloxan treatments, and serum irisin concentrations increased in diabetic rats after insulin treatment and acute treatments of irisin increased blood insulin levels. No changes were observed during long-term experiments with different diets. We suggested that levels of circulating irisin are the result of the sum of the irisin produced by different depots of adipose tissue and skeletal muscle. This study shows for the first time that there are differences in FNDC5 expression depending on white adipose tissue depots. Moreover, a considerable decrease in visceral and epididymal adipose tissue depots correlated with increased FNDC5 mRNA expression levels, probably in an attempt to compensate the decrease that occurs in their mass. Hypothalamic FNDC5 expression did not change for any of the tested diets but increased with leptin, insulin and metformin treatments suggesting that the regulation of central and peripheral FNDC5/irisin expression and functions are different.

Changes in hypothalamic expression of the Lin28/let-7 system and related microRNAs during postnatal maturation and after experimental manipulations of puberty.

Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.

Influence of ghrelin and growth hormone deficiency on AMP-activated protein kinase and hypothalamic lipid metabolism.

Current evidence demonstrates that the stomach-derived hormone ghrelin, a potent growth hormone (GH) secretagogue, promotes feeding through a mechanism involving the short-term activation of hypothalamic AMP-activated protein kinase (AMPK), which in turn results in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. Despite this evidence, no data have been reported about the effect of chronic, central ghrelin administration on hypothalamic fatty acid metabolism. In the present study, we examined the differences in hypothalamic fatty acid metabolism in the presence and absence of GH, by using a model for the study of GH-deficiency, namely the spontaneous dwarf rat and the effect of long-term central ghrelin treatment and starvation on hypothalamic fatty acid metabolism in this animal model. Our data showed that GH-deficiency induces reductions in both de novo lipogenesis and beta-oxidation pathways in the hypothalamus. Thus, dwarf rats display reductions in fatty acid synthase (FAS) mRNA expression both in the ventromedial nucleus of the hypothalamus (VMH) and whole hypothalamus, as well as in FAS protein and activity. CPT1 activity was also reduced. In addition, in the present study, we show that chronic ghrelin treatment does not promote AMPK-induced changes in the overall fluxes of hypothalamic fatty acid metabolism in normal rats and that this effect is independent of GH status. By contrast, we demonstrated that both chronic ghrelin and fasting decreased FAS mRNA expression in the VMH of normal rats but not dwarf rats, suggesting GH status dependency. Overall, these results suggest that ghrelin plays a dual time-dependent role in modulating hypothalamic lipid metabolism. Understanding the molecular mechanism underlying the interplay between GH and ghrelin on hypothalamic lipid metabolism will allow new strategies for the design and development of suitable drugs for the treatment of GH-deficiency, obesity and its comorbidities.

Regulation of growth hormone secretion by the growth hormone releasing hexapeptide (GHRP-6).

Growth hormone (GH) secretion is regulated by a complex system of central and peripheral signals. Recently, a new GH-releasing hexapeptide (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) called GHRP-6 which specifically releases GH has been studied. In the present work the mechanism of action of GHRP-6 has been addressed in experimental animal models as well as in obese subjects. GHRP-6 releases GH independently of the hypothalamic factors GHRH and somatostatin and is a powerful GH releaser in obesity.

Effect of central cholinergic neurotransmission enhancement by pyridostigmine on the growth hormone secretion elicited by clonidine, arginine, or hypoglycemia in normal and obese subjects.

Obesity is associated with an impairment of the GH secretion elicited by all stimuli known to date, but the basic mechanism of this alteration is unknown. To determine whether obesity is associated with a chronic state of tonic somatostatin secretion, several tests with GH stimuli with or without pyridostigmine were undertaken in both obese subjects and matched controls. Pyridostigmine reduces somatostatin release from the hypothalamus by increasing central cholinergic neurotransmission. The administration of clonidine (300 micrograms, orally) to obese subjects did not modify basal GH values (1.9 +/- 0.7 micrograms/L at 90 min), while in control subjects the clonidine-induced GH peak was 13.1 +/- 1.6 micrograms/L. Pretreatment with pyridostigmine (120 mg, orally) notably increased clonidine-stimulated GH secretion in both the obese (6.9 +/- 1.8 micrograms/L) and control (17.6 +/- 2.7 micrograms/L) subjects. Since clonidine acts by releasing endogenous GHRH, similar studies were undertaken employing arginine, which presumably enhances GH release by reducing somatostatin discharge. Arginine administration in obese subjects induced an increase in GH levels of 5 +/- 2.3 micrograms/L, which was significantly smaller than that in the matched control subjects (13.3 +/- 2.4 micrograms/L). Pretreatment with pyridostigmine increased the arginine action toward a GH peak of 12.2 +/- 2.2 micrograms/L in the obese and 21.6 +/- 2.5 micrograms/L in control subjects. As a third hypothalamic stimulus of GH secretion, trials of insulin-induced hypoglycemia were carried out. Hypoglycemia induced an increase in GH levels in obese subjects of 12.2 +/- 1.8 micrograms/L, which was higher than that produced by any other stimulus, but lower than that in control subjects (28.4 +/- 5.5 micrograms/L). In contrast with the previous two GH stimuli, pretreatment with pyridostigmine did not modify the hypoglycemia-induced GH release in either obese or normal subjects. Our results lend support to the view that clonidine acts through GH-releasing hormone release and arginine by reducing somatostatin discharge from the hypothalamus. In addition, they seem to indicate that hypoglycemia acts by a combination of both mechanisms, mainly through a reduction in somatostatin release. These findings support the idea that obesity is associated with a state of chronic somatostatin hypersecretion as the basis for the derangements in GH secretion.

Cholinergic receptor activation by pyridostigmine restores growth hormone (GH) responsiveness to GH-releasing hormone administration in obese subjects: evidence for hypothalamic somatostatinergic participation in the blunted GH release of obesity.

GH secretion in response to provocative stimuli is decreased in obese individuals. However, the precise mechanism causing this decrease is unknown. In an attempt to determine if reduced cholinergic stimulation accounts for the decreased GH secretion, we studied the effect of enhanced cholinergic tone induced by pyridostigmine on GHRH-stimulated GH secretion in a group of seven obese and seven normal subjects. When GHRH (100 micrograms, iv) was administered after placebo in the obese group, mean plasma GH rose from 0.5 +/- (0.1 (+/- SE) to 3.6 +/- 1.5 micrograms/L at 30 min. When the same obese subjects were given GHRH 60 min after pyridostigmine administration (120 mg, orally), the mean plasma GH level rose from 1.8 +/- 0.6 to 21.0 +/- 7.5 micrograms/L at 30 min. The responses to placebo and pyridostigmine were significantly different at 15, 30, 45, 60, and 90 min. In the normal subjects, a similar dose of GHRH induced a GH peak of 24.3 +/- 7.1 micrograms/L, and the GHRH-stimulated peak was significantly higher (56.2 +/- 16.8 micrograms/L) after pyridostigmine administration. To study the effect of pyridostigmine alone six other obese and six other normal subjects were tested with pyridostigmine or placebo on different days. In the normal subjects the mean peak plasma GH level after pyridostigmine was 12.5 +/- 3.1 micrograms/L, and in the obese subjects it was 4.6 +/- 1.3 micrograms/L. Thus, pyridostigmine potentiated the action of GHRH, rather than merely being additive. We conclude that pyridostigmine stimulates GH secretion in obese as well as normal subjects, although the response was less in the former group. Pyridostigmine potentiates the response to GHRH in both groups, but again, the response was less in the obese subjects. These results suggest that the impaired somatotroph responsiveness in obese subjects may be due to chronically decreased hypothalamic cholinergic tone, resulting in enhanced somatostatinergic tone.