Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3.

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3' UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3' UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3' UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states.

Perturbation of hypothalamic microRNA expression patterns in male rats after metabolic distress: impact of obesity and conditions of negative energy balance.

The hypothalamus plays a crucial role in body weight homeostasis through an intricate network of neuronal circuits that are under the precise regulation of peripheral hormones and central transmitters. Although deregulated function of such circuits might be a major contributing factor in obesity, the molecular mechanisms responsible for the hypothalamic control of energy balance remain partially unknown. MicroRNAs (miRNAs) have been recognized as key regulators of different biological processes, including insulin sensitivity and glucose metabolism. However, the roles of miRNA pathways in the control of metabolism have been mostly addressed in peripheral tissues, whereas the potential deregulation of miRNA expression in the hypothalamus in conditions of metabolic distress remains as yet unexplored. In this work, we used high-throughput screening to define to what extent the hypothalamic profiles of miRNA expression are perturbed in two extreme conditions of nutritional stress in male rats, namely chronic caloric restriction and high-fat diet-induced obesity. Our analyses allowed the identification of sets of miRNAs, including let-7a, mir-9*, mir-30e, mir-132, mir-145, mir-200a, and mir-218, whose expression patterns in the hypothalamus were jointly altered by caloric restriction and/or a high-fat diet. The predicted targets of these miRNAs include several elements of key inflammatory and metabolic pathways, including insulin and leptin. Our study is the first to disclose the impact of nutritional challenges on the hypothalamic miRNA expression profiles. These data will help to characterize the molecular miRNA signature of the hypothalamus in extreme metabolic conditions and pave the way for targeted mechanistic analyses of the involvement of deregulated central miRNAs pathways in the pathogenesis of obesity and related disorders.

Central resistin regulates hypothalamic and peripheral lipid metabolism in a nutritional-dependent fashion.

Evidence suggests that the adipocyte-derived hormone resistin (RSTN) directly regulates both feeding and peripheral metabolism through, so far, undefined hypothalamic-mediated mechanisms. Here, we demonstrate that the anorectic effect of RSTN is associated with inappropriately decreased mRNA expression of orexigenic (agouti-related protein and neuropeptide Y) and increased mRNA expression of anorexigenic (cocaine and amphetamine-regulated transcript) neuropeptides in the arcuate nucleus of the hypothalamus. Of interest, RSTN also exerts a profound nutrition-dependent inhibitory effect on hypothalamic fatty acid metabolism, as indicated by increased phosphorylation levels of both AMP-activated protein kinase and its downstream target acetyl-coenzyme A carboxylase, associated with decreased expression of fatty acid synthase in the ventromedial nucleus of the hypothalamus. In addition, we also demonstrate that chronic central RSTN infusion results in decreased body weight and major changes in peripheral expression of lipogenic enzymes, in a tissue-specific and nutrition-dependent manner. Thus, in the fed state central RSTN is associated with induced expression of fatty acid synthesis enzymes and proinflammatory cytokines in liver, whereas its administration in the fasted state does so in white adipose tissue. Overall, our results indicate that RSTN controls feeding and peripheral lipid metabolism and suggest that hepatic RSTN-induced insulin resistance may be mediated by central activation of de novo lipogenesis in liver.

Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.

Current evidence suggests that hypothalamic fatty acid metabolism may play a role in regulating food intake; however, confirmation that it is a physiologically relevant regulatory system of feeding is still incomplete. Here, we use pharmacological and genetic approaches to demonstrate that the physiological orexigenic response to ghrelin involves specific inhibition of fatty acid biosynthesis induced by AMP-activated protein kinase (AMPK) resulting in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. In addition, we also demonstrate that fasting downregulates fatty acid synthase (FAS) in a region-specific manner and that this effect is mediated by an AMPK and ghrelin-dependent mechanisms. Thus, decreasing AMPK activity in the ventromedial nucleus of the hypothalamus (VMH) is sufficient to inhibit ghrelin's effects on FAS expression and feeding. Overall, our results indicate that modulation of hypothalamic fatty acid metabolism specifically in the VMH in response to ghrelin is a physiological mechanism that controls feeding.

Osmoregulatory action of 17beta-estradiol in the gilthead sea bream Sparus auratus.

The osmoregulatory action of 17beta-estradiol (E2) was examined in the euryhaline teleost Sparus auratas. In a first set of experiments, fish were injected once with vegetable oil containing E2 (1, 2 and 5 microg/g body weight), transferred 12h after injection from sea water (SW, 38 ppt salinity) to hypersaline water (HSW, 55 ppt) or to brackish water (BW, 5 ppt salinity) and sampled 12h later (i.e. 24 h post-injection). In a second experiment, fish were injected intraperitoneally with coconut oil alone or containing E2 (10 microg/g body weight) and sampled after 5 days. In the same experiment, after 5 days of treatment, fish of each group were transferred to HSW, BW and SW and sampled 4 days later (9 days post-implant). Gill Na+,K+ -ATPase activity, plasma E2 levels, plasma osmolality, and plasma levels of ions (sodium and calcium), glucose, lactate, protein, triglyceride, and hepatosomatic index were examined. Transfer from SW to HSW produced no significant effects on any parameters assessed. E2 treatment did not affect any parameter. Transfer from SW to BW resulted in a significant decrease in plasma osmolality and plasma sodium but did not affect gill Na+,K+ -ATPase activity. A single dose of E2 attenuated the decrease in these parameters after transfer from SW to BW, but was without effect on gill Na+,K+ -ATPase activity. An implant of E2 (10 microg/g body weight) for 5 days significantly increased plasma calcium, hepatosomatic index, plasma metabolic parameters, and gill Na+,K+ -ATPase activity. In coconut oil-implanted (sham) fish, transfer from SW to HSW or BW during 4 days significantly elevated gill Na+,K+ -ATPase. Gill Na+,K+ -ATPase activity remained unaltered after transfer of E2-treated fish to HSW or BW. However, in E2-treated fish transferred from SW to SW (9 days in SW after E2-implant), gill Na+,K+ -ATPase activity decreased with respect to HSW- or BW-transferred fish. Shams transferred to HSW showed increased levels of lactate, protein, and trygliceride in plasma, while those transferred to BW only displayed increased trygliceride levels. E2-treated fish transferred to HSW showed higher protein levels without any change in other plasmatic parameters, while those transferred to BW displayed elevated plasma glucose levels but decreased osmolality and protein levels. These results substantiate a chronic stimulatory action of E2 on gill Na+,K+ -ATPase activity in the euryhaline teleost Sparus auratas.