O-GlcNAcylated p53 in the liver modulates hepatic glucose production.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.

Vav2 catalysis-dependent pathways contribute to skeletal muscle growth and metabolic homeostasis.

Skeletal muscle promotes metabolic balance by regulating glucose uptake and the stimulation of multiple interorgan crosstalk. We show here that the catalytic activity of Vav2, a Rho GTPase activator, modulates the signaling output of the IGF1- and insulin-stimulated phosphatidylinositol 3-kinase pathway in that tissue. Consistent with this, mice bearing a Vav2 protein with decreased catalytic activity exhibit reduced muscle mass, lack of proper insulin responsiveness and, at much later times, a metabolic syndrome-like condition. Conversely, mice expressing a catalytically hyperactive Vav2 develop muscle hypertrophy and increased insulin responsiveness. Of note, while hypoactive Vav2 predisposes to, hyperactive Vav2 protects against high fat diet-induced metabolic imbalance. These data unveil a regulatory layer affecting the signaling output of insulin family factors in muscle.

Beta-Cell-Specific Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 5 Aggravates High-Fat Diet-Induced Impairment of Islet Insulin Secretion in Mice.

Aims: Nicotinamide adenine dinucleotide phosphate oxidases (NOX-es) produce reactive oxygen species and modulate ß-cell insulin secretion. Islets of type 2 diabetic subjects present elevated expression of NOX5. Here, we sought to characterize regulation of NOX5 expression in human islets in vitro and to uncover the relevance of NOX5 in islet function in vivo using a novel mouse model expressing NOX5 in doxycycline-inducible, ß-cell-specific manner (RIP/rtTA/NOX5 mice). Results:In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 messenger ribonucleic acid (mRNA) and protein expressions in human islets. In cultures of dispersed islets, NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8 mM glucose) conditions. Small interfering ribonucleic acid (siRNA)-mediated knockdown of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion (GIIS) in vitro. However, when islets were preincubated in high (16.7 mM) glucose media for 12 h, NOX5 appeared also in insulin-positive (ß) cells. In vivo, mice with ß-cell NOX5 expression developed aggravated impairment of GIIS compared with control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate preincubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared with their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation, suggesting impaired cyclic adenosine monophosphate (cAMP)-mediated signaling due to increased phosphodiesterase activation. Innovation and Conclusions: Our data provide the first insight into the complex regulation and function of NOX5 in islets implying an important role for NOX5 in δ-cell-mediated intraislet crosstalk in physiological circumstances but also identifying it as an aggravating factor in ß-cell failure in diabetic conditions.

Functional Gut Microbiota Remodeling Contributes to the Caloric Restriction-Induced Metabolic Improvements.

Caloric restriction (CR) stimulates development of functional beige fat and extends healthy lifespan. Here we show that compositional and functional changes in the gut microbiota contribute to a number of CR-induced metabolic improvements and promote fat browning. Mechanistically, these effects are linked to a lower expression of the key bacterial enzymes necessary for the lipid A biosynthesis, a critical lipopolysaccharide (LPS) building component. The decreased LPS dictates the tone of the innate immune response during CR, leading to increased eosinophil infiltration and anti-inflammatory macrophage polarization in fat of the CR animals. Genetic and pharmacological suppression of the LPS-TLR4 pathway or transplantation with Tlr4-/- bone-marrow-derived hematopoietic cells increases beige fat development and ameliorates diet-induced fatty liver, while Tlr4-/- or microbiota-depleted mice are resistant to further CR-stimulated metabolic alterations. These data reveal signals critical for our understanding of the microbiota-fat signaling axis during CR and provide potential new anti-obesity therapeutics.

Hepatic protein tyrosine phosphatase receptor gamma links obesity-induced inflammation to insulin resistance.

Obesity-induced inflammation engenders insulin resistance and type 2 diabetes mellitus (T2DM) but the inflammatory effectors linking obesity to insulin resistance are incompletely understood. Here, we show that hepatic expression of Protein Tyrosine Phosphatase Receptor Gamma (PTPR-γ) is stimulated by inflammation in obese/T2DM mice and positively correlates with indices of inflammation and insulin resistance in humans. NF-κB binds to the promoter of Ptprg and is required for inflammation-induced PTPR-γ expression. PTPR-γ loss-of-function lowers glycemia and insulinemia by enhancing insulin-stimulated suppression of endogenous glucose production. These phenotypes are rescued by re-expression of Ptprg only in liver of mice lacking Ptprg globally. Hepatic PTPR-γ overexpression that mimics levels found in obesity is sufficient to cause severe hepatic and systemic insulin resistance. We propose hepatic PTPR-γ as a link between obesity-induced inflammation and insulin resistance and as potential target for treatment of T2DM.

Caloric Restriction Leads to Browning of White Adipose Tissue through Type 2 Immune Signaling.

Caloric restriction (CR) extends lifespan from yeast to mammals, delays onset of age-associated diseases, and improves metabolic health. We show that CR stimulates development of functional beige fat within the subcutaneous and visceral adipose tissue, contributing to decreased white fat and adipocyte size in lean C57BL/6 and BALB/c mice kept at room temperature or at thermoneutrality and in obese leptin-deficient mice. These metabolic changes are mediated by increased eosinophil infiltration, type 2 cytokine signaling, and M2 macrophage polarization in fat of CR animals. Suppression of the type 2 signaling, using Il4ra(-/-), Stat6(-/-), or mice transplanted with Stat6(-/-) bone marrow-derived hematopoietic cells, prevents the CR-induced browning and abrogates the subcutaneous fat loss and the metabolic improvements induced by CR. These results provide insights into the overall energy homeostasis during CR, and they suggest beige fat development as a common feature in conditions of negative energy balance.

Leptin as a Potential Regulator of FGF21.

BACKGROUND/AIMS: Fibroblast growth factor 21 (FGF21), a potent metabolic regulator, has been shown to improve insulin sensitivity in animal models of insulin resistance. Several studies have focused on identifying mediators of FGF21 effects. However, the identification of factors involved in FGF21 regulation is far from complete. As leptin is a potent metabolic modulator as well, we aimed at characterizing whether leptin may regulate FGF21. METHODS: We investigated a potential regulation of FGF21 by leptin in vivo in Wistar rats and in vitro using human derived hepatocarcinoma HepG2 cells. This model was chosen as the liver is considered the main FGF21 expression site. RESULTS: We found that leptin injections increased plasma FGF21 levels in adult Wistar rats. This was confirmed in vitro, as leptin increased FGF21 expression in HepG2 cells. We also showed that the leptin effect on FGF21 expression was mediated by STAT3 activation in HepG2 cells. CONCLUSION: New findings regarding a leptin-STAT3-FGF21 axis were provided in this study, although investigating the exact mechanisms linking leptin and FGF21 are still needed. These results are of great interest in the context of identifying potential new clinical approaches to treat metabolic diseases associated with insulin resistance, such as obesity and type 2 diabetes.

Central melanin-concentrating hormone influences liver and adipose metabolism via specific hypothalamic nuclei and efferent autonomic/JNK1 pathways.

BACKGROUND & AIMS: Specific neuronal circuits modulate autonomic outflow to liver and white adipose tissue. Melanin-concentrating hormone (MCH)-deficient mice are hypophagic, lean, and do not develop hepatosteatosis when fed a high-fat diet. Herein, we sought to investigate the role of MCH, an orexigenic neuropeptide specifically expressed in the lateral hypothalamic area, on hepatic and adipocyte metabolism. METHODS: Chronic central administration of MCH and adenoviral vectors increasing MCH signaling were performed in rats and mice. Vagal denervation was performed to assess its effect on liver metabolism. The peripheral effects on lipid metabolism were assessed by real-time polymerase chain reaction and Western blot. RESULTS: We showed that the activation of MCH receptors promotes nonalcoholic fatty liver disease through the parasympathetic nervous system, whereas it increases fat deposition in white adipose tissue via the suppression of sympathetic traffic. These metabolic actions are independent of parallel changes in food intake and energy expenditure. In the liver, MCH triggers lipid accumulation and lipid uptake, with c-Jun N-terminal kinase being an essential player, whereas in adipocytes MCH induces metabolic pathways that promote lipid storage and decreases lipid mobilization. Genetic activation of MCH receptors or infusion of MCH specifically in the lateral hypothalamic area modulated hepatic lipid metabolism, whereas the specific activation of this receptor in the arcuate nucleus affected adipocyte metabolism. CONCLUSIONS: Our findings show that central MCH directly controls hepatic and adipocyte metabolism through different pathways.

A role for adipose tissue de novo lipogenesis in glucose homeostasis during catch-up growth: a Randle cycle favoring fat storage.

Catch-up growth, a risk factor for type 2 diabetes, is characterized by hyperinsulinemia and accelerated body fat recovery. Using a rat model of semistarvation-refeeding that exhibits catch-up fat, we previously reported that during refeeding on a low-fat diet, glucose tolerance is normal but insulin-dependent glucose utilization is decreased in skeletal muscle and increased in adipose tissue, where de novo lipogenic capacity is concomitantly enhanced. Here we report that isocaloric refeeding on a high-fat (HF) diet blunts the enhanced in vivo insulin-dependent glucose utilization for de novo lipogenesis (DNL) in adipose tissue. These are shown to be early events of catch-up growth that are independent of hyperphagia and precede the development of overt adipocyte hypertrophy, adipose tissue inflammation, or defective insulin signaling. These results suggest a role for enhanced DNL as a glucose sink in regulating glycemia during catch-up growth, which is blunted by exposure to an HF diet, thereby contributing, together with skeletal muscle insulin resistance, to the development of glucose intolerance. Our findings are presented as an extension of the Randle cycle hypothesis, whereby the suppression of DNL constitutes a mechanism by which dietary lipids antagonize glucose utilization for storage as triglycerides in adipose tissue, thereby impairing glucose homeostasis during catch-up growth.

The HPA axis modulates the CNS melanocortin control of liver triacylglyceride metabolism.

The central melanocortin system regulates lipid metabolism in peripheral tissues such as white adipose tissue. Alterations in the activity of sympathetic nerves connecting hypothalamic cells expressing melanocortin 3/4 receptors (MC3/4R) with white adipocytes have been shown to partly mediate these effects. Interestingly, hypothalamic neurons producing corticotropin-releasing hormone and thyrotropin-releasing hormone co-express MC4R. Therefore we hypothesized that regulation of hypothalamo-pituitary adrenal (HPA) and hypothalamo-pituitary thyroid (HPT) axes activity by the central melanocortin system could contribute to its control of peripheral lipid metabolism. To test this hypothesis, we chronically infused rats intracerebroventricularly (i.c.v.) either with an MC3/4R antagonist (SHU9119), an MC3/4R agonist (MTII) or saline. Rats had been previously adrenalectomized (ADX) and supplemented daily with 1mg/kg corticosterone (s.c.), thyroidectomized (TDX) and supplemented daily with 10 µg/kgL-thyroxin (s.c.), or sham operated (SO). Blockade of MC3/4R signaling with SHU9119 increased food intake and body mass, irrespective of gland surgery. The increase in body mass was accompanied by higher epididymal white adipose tissue (eWAT) weight and higher mRNA content of lipogenic enzymes in eWAT. SHU9119 infusion increased triglyceride content in the liver of SO and TDX rats, but not in those of ADX rats. Concomitantly, mRNA expression of lipogenic enzymes in liver was increased in SO and TDX, but not in ADX rats. We conclude that the HPA and HPT axes do not play an essential role in mediating central melanocortinergic effects on white adipose tissue and liver lipid metabolism. However, while basal hepatic lipid metabolism does not depend on a functional HPA axis, the induction of hepatic lipogenesis due to central melanocortin system blockade does require a functional HPA axis.

The Lou/C rat: a model of spontaneous food restriction associated with improved insulin sensitivity and decreased lipid storage in adipose tissue.

The inbred Lou/C rat, originating from the Wistar strain, has been described as a model of resistance to diet-induced obesity, but little is known about its metabolism. Since this knowledge could provide some clues about the etiology of obesity/insulin resistance, this study aimed at characterizing glucose and lipid metabolism in Lou/C vs. Wistar rats. This was achieved by performing glucose and insulin tolerance tests, euglycemic hyperinsulinemic clamps, and characterization of intracellular insulin signaling in skeletal muscle. Substrate-induced insulin secretion was evaluated using perfused pancreas and isolated islets. Finally, body fat composition and the expression of various factors involved in lipid metabolism were determined. Body weight and caloric intake were lower in Lou/C than in Wistar rats, whereas food efficiency was similar. Improved glucose tolerance of Lou/C rats was not related to increased insulin output but was related to improved insulin sensitivity/responsiveness in the liver and in skeletal muscles. In the latter tissue, this was accompanied by improved insulin signaling, as suggested by higher activation of the insulin receptor and of the Akt/protein kinase B pathway. Fat deposition was markedly lower in Lou/C than in Wistar rats, especially in visceral adipose tissue. In the inguinal adipose depot, expression of uncoupling protein-1 was detected in Lou/C but not in Wistar rats, in keeping with a higher expression of peroxisome proliferator-activated receptor-gamma coactivator-1 in these animals. The Lou/C rat is a valuable model of spontaneous food restriction with associated improved insulin sensitivity. Independently from its reduced caloric intake, it also exhibits a preferential channeling of nutrients toward utilization rather than storage.

Unsaturated fatty acids inhibit the expression of tumor suppressor phosphatase and tensin homolog (PTEN) via microRNA-21 up-regulation in hepatocytes.

UNLABELLED: Phosphatase and tensin homolog (PTEN) is a regulator of phosphoinositide 3-kinase signaling and an important tumor suppressor mutated/deleted in human cancers. PTEN deletion in the liver leads to insulin resistance, steatosis, inflammation, and cancer. We recently demonstrated that unsaturated fatty acids trigger steatosis by down-regulating PTEN expression in hepatocytes via activation of a mammalian target of rapamycin (mTOR)/nuclear factor kappa B (NF-kappaB) complex, but the molecular mechanisms implicated in this process are still unknown. Here, we investigated potential genetic and epigenetic mechanisms activated by fatty acids leading to PTEN down-regulation. Our results indicate that unsaturated fatty acids down-regulate PTEN messenger RNA expression in hepatocytes through mechanisms unrelated to methylation of the PTEN promoter, histone deacetylase activities, or repression of the PTEN promoter activity. In contrast, unsaturated fatty acids up-regulate the expression of microRNA-21, which binds to PTEN messenger RNA 3'-untranslated region and induces its degradation. The promoter activity of microRNA-21 was increased by mTOR/NF-kappaB activation. Consistent with these data, microRNA-21 expression was increased in the livers of rats fed high-fat diets and in human liver biopsies of obese patients having diminished PTEN expression and steatosis. CONCLUSION: Unsaturated fatty acids inhibit PTEN expression in hepatocytes by up-regulating microRNA-21 synthesis via an mTOR/NF-kappaB-dependent mechanism. Aberrant up-regulation of microRNA-21 expression by excessive circulating levels of fatty acids exemplify a novel regulatory mechanism by which fatty acids affect PTEN expression and trigger liver disorders.

Effects of a high-glucose environment on the pituitary growth hormone-releasing hormone receptor: type 1 diabetes compared with in vitro glucotoxicity.

The present study investigated the effects of diabetes and high glucose on GHRH receptor (GHRH-R) mRNA and protein levels in the pituitary of diabetic rats 2, 21, and 60 days post-streptozotocin (post-STZ) administration. Two days post-STZ, the 2.5-kb GHRH-R mRNA transcript was increased. Twenty-one days post-STZ, both the 2.5- and 4-kb transcripts and a 72-kDa (125)I-GHRH-GHRH-R complex were elevated. Sixty days post-STZ, the 4-kb transcript remained increased and the 45-kDa (125)I-GHRH-GHRH-R complex (functional receptor) was decreased. Hypothalamic GHRH mRNA and serum total IGF-I levels were reduced at all three time points. To better understand the role of high glucose on GHRH-R regulation, time-course effects of 33 compared with 6 mM d-glucose (DG) were examined in cultured anterior pituitary cells from 2-mo-old healthy rats. Membrane lipoperoxidation was present in 33 mM DG, and GHRH-R mRNA levels were diminished after 24 h, Fluo-GHRH internalization was marginal after 16-24 h, and GHRH-induced cAMP levels were decreased after 24 and 48 h. Altogether, these results indicate that the increase of the 2.5-kb GHRH-R mRNA transcript in vivo could be a consequence of a decrease of hypothalamic GHRH mRNA levels in STZ rats. Since it does not affect primarily functional GHRH-R levels, the initial diminution of circulating IGF-I levels could result from a decreased GHRH-R stimulation by GHRH. Thus, the effect of glucotoxicity would be related to a decrease of functional GHRH-R protein, as observed in rats 60 days post-STZ and in cultured pituitary cells from healthy rats exposed to a high-glucose environment.

PTEN down-regulation by unsaturated fatty acids triggers hepatic steatosis via an NF-kappaBp65/mTOR-dependent mechanism.

BACKGROUND & AIMS: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor and a regulator of insulin sensitivity in peripheral tissues. In the liver, PTEN deletion increases insulin sensitivity, but induces steatosis, steatohepatitis, and hepatocellular carcinoma. Here, we investigated the pathophysiologic mechanisms regulating PTEN expression in the liver and the development of steatosis. METHODS: PTEN expression was evaluated in the liver of rats and human beings having metabolic syndrome. Signaling pathways regulating PTEN expression and lipid accumulation in hepatocytes were examined in vitro. RESULTS: PTEN expression is down-regulated in the liver of rats having steatosis and high plasma levels of fatty acids, as well as in steatotic human livers. Unsaturated fatty acids inhibited PTEN expression in HepG2 cells via activation of a signaling complex formed by the mammalian target of rapamycin (mTOR) and nuclear factor-kappaB (NF-kappaB). Down-regulation of PTEN expression induced steatosis by affecting import, esterification, and extracellular release of fatty acids. CONCLUSIONS: Hepatic steatosis can be mediated by alterations of PTEN expression in hepatocytes exposed to high levels of unsaturated fatty acids. Furthermore, our data revealed interaction between mTOR and NF-kappaB, suggesting cross-talk between these 2 pathways.

Internalization and trafficking of the human and rat growth hormone-releasing hormone receptor.

Internalization and intracellular trafficking of the growth hormone-releasing hormone receptor (GHRH-R) were studied in rat anterior pituitary and human (h) and rat (r) GHRH-R-transfected BHK cells, with the GHRH agonist, [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2), Ala(8), Ala(15), Lys(22)]hGHRH(1-29)NH(2) (Fluo-GHRH). Time- and temperature-dependent internalization of stimulated GHRH-R was blocked by phenyl arsine oxide (PAO) in both cell types. In anterior pituitary and rGHRH-R-transfected BHK cells, only filipin III and cerulenin blocked receptor-mediated internalization of Fluo-GHRH while in hGHRH-R-transfected BHK cells, only hyperosmolar sucrose inhibited this process. These results suggest that hGHRH-R internalization is clathrin-dependent, while fatty acid acylation of rGHRH-R appears to be a prerequisite to caveolin-dependent internalization. Experiments in anterior pituitary using Bodipy-FL-C(5) ganglioside GM1, a specific marker of lipid rafts such as caveolae, confirmed this latter pathway. Co-localization of Fluo-GHRH with LysoTracker indicated that Fluo-GHRH was directed to acidic organelles in both cell types. Finally, studies using cycloheximide and monensin showed that upon stimulation with GHRH, an optimal concentration of functional GHRH-R was maintained at the plasma membrane due to de novo synthesis and recycling in pituitary cells and to de novo synthesis solely in hGHRH-R-transfected BHK cells. This first study on the dynamics of the GHRH/GHRH-R complexes using fluorescence imaging in a native environment compared to cell system models, revealed that both receptor primary structure and concentration at the plasma membrane play important roles in internalization and trafficking of specific G-protein-coupled receptors (GPCR).

Localization and regulation of a functional GHRH receptor in the rat renal medulla.

To provide information about the kidney GHRH receptor (GHRH-R), we assessed its tissue and cellular localization, defined its pattern of expression in developing and aging rats, and studied the effects of GHRH on the regulation of GHRH-R mRNA levels and receptor internalization. In situ hybridization and ribonuclease protection assay demonstrated that GHRH-R mRNA is restricted to the Henle's loop (HL). GHRH-R mRNA levels were low in the medulla from 3- and 12-d-old male rats, increased significantly in that from 30- to 70-d-old rats, and decreased in that from 12- and 18-month-old animals. Compared with the GHRH-R mRNA profile obtained in the pituitary, these data support the concept of a tissue-specific regulation of GHRH-R. In HL cell cultures from 70-d-old rats, a 4-h incubation with 1-100 nM rat GHRH-(1-29)NH(2) reduced GHRH-R mRNA levels significantly. As anti-GHRH-R- (392-404) immunoreactivity was demonstrated in HL cells, internalization of [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2),Ala(8), Ala(15),Lys(22)]hGHRH-(1-29)NH(2) in a time- and temperature-dependent manner and inhibition of this process by phenyl arsine oxide indicate that desensitization to GHRH involves both GHRH-R internalization and down-regulation of GHRH-R mRNA levels. Localization of a functional GHRH-R in HL and its regulation during development and aging suggest roles associated with cellular proliferation, differentiation, and/or water/electrolyte transport.