Hepatocyte-specific O-GlcNAc transferase downregulation ameliorates nonalcoholic steatohepatitis by improving mitochondrial function.

OBJECTIVE: O-GlcNAcylation is a post-translational modification that directly couples the processes of nutrient sensing, metabolism, and signal transduction, affecting protein function and localization, since the O-linked N-acetylglucosamine moiety comes directly from the metabolism of glucose, lipids, and amino acids. The addition and removal of O-GlcNAc of target proteins are mediated by two highly conserved enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA), respectively. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, and cardiovascular diseases. The contribution of deregulated O-GlcNAcylation to the progression and pathogenesis of NAFLD remains intriguing, and a better understanding of its roles in this pathophysiological context is required to uncover novel avenues for therapeutic intervention. By using a translational approach, our aim is to describe the role of OGT and O-GlcNAcylation in the pathogenesis of NAFLD. METHODS: We used primary mouse hepatocytes, human hepatic cell lines and in vivo mouse models of steatohepatitis to manipulate O-GlcNAc transferase (OGT). We also studied OGT and O-GlcNAcylation in liver samples from different cohorts of people with NAFLD. RESULTS: O-GlcNAcylation was upregulated in the liver of people and animal models with steatohepatitis. Downregulation of OGT in NAFLD-hepatocytes improved diet-induced liver injury in both in vivo and in vitro models. Proteomics studies revealed that mitochondrial proteins were hyper-O-GlcNAcylated in the liver of mice with steatohepatitis. Inhibition of OGT is able to restore mitochondrial oxidation and decrease hepatic lipid content in in vitro and in vivo models of NAFLD. CONCLUSIONS: These results demonstrate that deregulated hyper-O-GlcNAcylation favors NAFLD progression by reducing mitochondrial oxidation and promoting hepatic lipid accumulation.

Dicer ablation in Kiss1 neurons impairs puberty and fertility preferentially in female mice.

Kiss1 neurons, producing kisspeptins, are essential for puberty and fertility, but their molecular regulatory mechanisms remain unfolded. Here, we report that congenital ablation of the microRNA-synthesizing enzyme, Dicer, in Kiss1 cells, causes late-onset hypogonadotropic hypogonadism in both sexes, but is compatible with pubertal initiation and preserved Kiss1 neuronal populations at the infantile/juvenile period. Yet, failure to complete puberty and attain fertility is observed only in females. Kiss1-specific ablation of Dicer evokes disparate changes of Kiss1-cell numbers and Kiss1/kisspeptin expression between hypothalamic subpopulations during the pubertal-transition, with a predominant decline in arcuate-nucleus Kiss1 levels, linked to enhanced expression of its repressors, Mkrn3, Cbx7 and Eap1. Our data unveil that miRNA-biosynthesis in Kiss1 neurons is essential for pubertal completion and fertility, especially in females, but dispensable for initial reproductive maturation and neuronal survival in both sexes. Our results disclose a predominant miRNA-mediated inhibitory program of repressive signals that is key for precise regulation of Kiss1 expression and, thereby, reproductive function.

Kappa-Opioid Receptor Blockade Ameliorates Obesity Caused by Estrogen Withdrawal via Promotion of Energy Expenditure through mTOR Pathway.

Weight gain is a hallmark of decreased estradiol (E2) levels because of menopause or following surgical ovariectomy (OVX) at younger ages. Of note, this weight gain tends to be around the abdomen, which is frequently associated with impaired metabolic homeostasis and greater cardiovascular risk in both rodents and humans. However, the molecular underpinnings and the neuronal basis for these effects remain to be elucidated. The aim of this study is to elucidate whether the kappa-opioid receptor (k-OR) system is involved in mediating body weight changes associated with E2 withdrawal. Here, we document that body weight gain induced by OVX occurs, at least partially, in a k-OR dependent manner, by modulation of energy expenditure independently of food intake as assessed in Oprk1-/-global KO mice. These effects were also observed following central pharmacological blockade of the k-OR system using the k-OR-selective antagonist PF-04455242 in wild type mice, in which we also observed a decrease in OVX-induced weight gain associated with increased UCP1 positive immunostaining in brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Remarkably, the hypothalamic mTOR pathway plays an important role in regulating weight gain and adiposity in OVX mice. These findings will help to define new therapies to manage metabolic disorders associated with low/null E2 levels based on the modulation of central k-OR signaling.

Connecting nutritional deprivation and pubertal inhibition via GRK2-mediated repression of kisspeptin actions in GnRH neurons.

BACKGROUND: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. METHODS: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, ßARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. RESULTS: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca+2 responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. CONCLUSIONS: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.

Central Ceramide Signaling Mediates Obesity-Induced Precocious Puberty.

Childhood obesity, especially in girls, is frequently bound to earlier puberty, which is linked to higher disease burden later in life. The mechanisms underlying this association remain elusive. Here we show that brain ceramides participate in the control of female puberty and contribute to its alteration in early-onset obesity in rats. Postnatal overweight caused earlier puberty and increased hypothalamic ceramide content, while pharmacological activation of ceramide synthesis mimicked the pubertal advancement caused by obesity, specifically in females. Conversely, central blockade of de novo ceramide synthesis delayed puberty and prevented the effects of the puberty-activating signal, kisspeptin. This phenomenon seemingly involves a circuit encompassing the paraventricular nucleus (PVN) and ovarian sympathetic innervation. Early-onset obesity enhanced PVN expression of SPTLC1, a key enzyme for ceramide synthesis, and advanced the maturation of the ovarian noradrenergic system. In turn, obesity-induced pubertal precocity was reversed by virogenetic suppression of SPTLC1 in the PVN. Our data unveil a pathway, linking kisspeptin, PVN ceramides, and sympathetic ovarian innervation, as key for obesity-induced pubertal precocity.

MCH Regulates SIRT1/FoxO1 and Reduces POMC Neuronal Activity to Induce Hyperphagia, Adiposity, and Glucose Intolerance.

Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

Gonadal hormone-dependent vs. -independent effects of kisspeptin signaling in the control of body weight and metabolic homeostasis.

BACKGROUND: Kisspeptins, encoded by Kiss1, have emerged as essential regulators of puberty and reproduction by primarily acting on GnRH neurons, via their canonical receptor, Gpr54. Mounting, as yet fragmentary, evidence strongly suggests that kisspeptin signaling may also participate in the control of key aspects of body energy and metabolic homeostasis. However, characterization of such metabolic dimension of kisspeptins remains uncomplete, without an unambiguous discrimination between the primary metabolic actions of kisspeptins vs. those derived from their ability to stimulate the secretion of gonadal hormones, which have distinct metabolic actions on their own. In this work, we aimed to tease apart primary vs. secondary effects of kisspeptins in the control of key aspects of metabolic homeostasis using genetic models of impaired kisspeptin signaling and/or gonadal hormone status. METHODS: Body weight (BW) gain and composition, food intake and key metabolic parameters, including glucose tolerance, were comparatively analyzed, in lean and obesogenic conditions, in mice lacking kisspeptin signaling due to global inactivation of Gpr54 (displaying profound hypogonadism; Gpr54-/-) vs. Gpr54 null mice with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54-/-Tg), where kisspeptin signaling elsewhere than in GnRH neurons is ablated but gonadal function is preserved. RESULTS: In male mice, global elimination of kisspeptin signaling resulted in decreased BW, feeding suppression and increased adiposity, without overt changes in glucose tolerance, whereas Gpr54-/- female mice displayed enhanced BW gain at adulthood, increased adiposity and perturbed glucose tolerance, despite reduced food intake. Gpr54-/-Tg rescued mice showed altered postnatal BW gain in males and mildly perturbed glucose tolerance in females, with intermediate phenotypes between control and global KO animals. Yet, body composition and leptin levels were similar to controls in gonadal-rescued mice. Exposure to obesogenic insults, such as high fat diet (HFD), resulted in exaggerated BW gain and adiposity in global Gpr54-/- mice of both sexes, and worsening of glucose tolerance, especially in females. Yet, while rescued Gpr54-/-Tg males displayed intermediate BW gain and feeding profiles and impaired glucose tolerance, rescued Gpr54-/-Tg females behaved as controls, except for a modest deterioration of glucose tolerance after ovariectomy. CONCLUSION: Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. SUMMARY OF TRANSLATIONAL RELEVANCE: Kisspeptins, master regulators of reproduction, may also participate in the control of key aspects of body energy and metabolic homeostasis; yet, the nature of such metabolic actions remains debatable, due in part to the fact that kisspeptins modulate gonadal hormones, which have metabolic actions on their own. By comparing the metabolic profiles of two mouse models with genetic inactivation of kisspeptin signaling but different gonadal status (hypogonadal vs. preserved gonadal function), we provide herein a systematic dissection of gonadal-dependent vs. -independent metabolic actions of kisspeptins. Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. These data pave the way for future analyses addressing the eventual contribution of altered kisspeptin signaling in the development of metabolic alterations, especially in conditions linked to reproductive dysfunction.

Metabolic regulation of female puberty via hypothalamic AMPK-kisspeptin signaling.

Conditions of metabolic distress, from malnutrition to obesity, impact, via as yet ill-defined mechanisms, the timing of puberty, whose alterations can hamper later cardiometabolic health and even life expectancy. AMP-activated protein kinase (AMPK), the master cellular energy sensor activated in conditions of energy insufficiency, has a major central role in whole-body energy homeostasis. However, whether brain AMPK metabolically modulates puberty onset remains unknown. We report here that central AMPK interplays with the puberty-activating gene, Kiss1, to control puberty onset. Pubertal subnutrition, which delayed puberty, enhanced hypothalamic pAMPK levels, while activation of brain AMPK in immature female rats substantially deferred puberty. Virogenetic overexpression of a constitutively active form of AMPK, selectively in the hypothalamic arcuate nucleus (ARC), which holds a key population of Kiss1 neurons, partially delayed puberty onset and reduced luteinizing hormone levels. ARC Kiss1 neurons were found to express pAMPK, and activation of AMPK reduced ARC Kiss1 expression. The physiological relevance of this pathway was attested by conditional ablation of the AMPKα1 subunit in Kiss1 cells, which largely prevented the delay in puberty onset caused by chronic subnutrition. Our data demonstrate that hypothalamic AMPK signaling plays a key role in the metabolic control of puberty, acting via a repressive modulation of ARC Kiss1 neurons in conditions of negative energy balance.

Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways.

Obesity and its comorbidities are reaching epidemic proportions worldwide. Maternal obesity is known to predispose the offspring to metabolic disorders, independently of genetic inheritance. This intergenerational transmission has also been suggested for paternal obesity, with a potential negative impact on the metabolic and, eventually, reproductive health of the offspring, likely via epigenetic changes in spermatozoa. However, the neuroendocrine component of such phenomenon and whether paternal obesity sensitizes the offspring to the disturbances induced by high-fat diet (HFD) remain poorly defined. We report in this work the metabolic and reproductive impact of HFD in the offspring from obese fathers, with attention to potential sex differences and alterations of hypothalamic Kiss1 system. Lean and obese male rats were mated with lean virgin female rats; male and female offspring were fed HFD from weaning onward and analyzed at adulthood. The increases in body weight and leptin levels, but not glucose intolerance, induced by HFD were significantly augmented in the male, but not female, offspring from obese fathers. Paternal obesity caused a decrease in luteinizing hormone (LH) levels and exacerbated the drop in circulating testosterone and gene expression of its key biosynthetic enzymes caused by HFD in the male offspring. LH responses to central kisspeptin-10 administration were also suppressed in HFD males from obese fathers. In contrast, paternal obesity did not significantly alter gonadotropin levels in the female offspring fed HFD, although these females displayed reduced LH responses to kisspeptin-10. Our findings suggest that HFD-induced metabolic and reproductive disturbances are exacerbated by paternal obesity preferentially in males, whereas kisspeptin effects are affected in both sexes.

Direct Actions of Kisspeptins on GnRH Neurons Permit Attainment of Fertility but are Insufficient to Fully Preserve Gonadotropic Axis Activity.

Kisspeptins, ligands of the receptor, Gpr54, are potent stimulators of puberty and fertility. Yet, whether direct kisspeptin actions on GnRH neurons are sufficient for the whole repertoire of their reproductive effects remains debatable. To dissect out direct vs. indirect effects of kisspeptins on GnRH neurons in vivo, we report herein the detailed reproductive/gonadotropic characterization of a Gpr54 null mouse line with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54(-/-)Tg; rescued). Despite preserved fertility, adult rescued mice displayed abnormalities in gonadal microstructure, with signs of precocious ageing in females and elevated LH levels with normal-to-low testosterone secretion in males. Gpr54(-/-)Tg rescued mice showed also altered gonadotropin responses to negative feedback withdrawal, while luteinizing hormone responses to various gonadotropic regulators were variably affected, with partially blunted relative (but not absolute) responses to kisspeptin-10, NMDA and the agonist of tachykinin receptors, NK2R. Our data confirm that direct effects of kisspeptins on GnRH cells are sufficient to attain fertility. Yet, such direct actions appear to be insufficient to completely preserve proper functionality of gonadotropic axis, suggesting a role of kisspeptin signaling outside GnRH cells.