RANKL inhibition improves muscle strength and insulin sensitivity and restores bone mass.

Receptor activator of Nfkb ligand (RANKL) activates, while osteoprotegerin (OPG) inhibits, osteoclastogenesis. In turn a neutralizing Ab against RANKL, denosumab improves bone strength in osteoporosis. OPG also improves muscle strength in mouse models of Duchenne's muscular dystrophy (mdx) and denervation-induce atrophy, but its role and mechanisms of action on muscle weakness in other conditions remains to be investigated. We investigated the effects of RANKL inhibitors on muscle in osteoporotic women and mice that either overexpress RANKL (HuRANKL-Tg+), or lack Pparb and concomitantly develop sarcopenia (Pparb-/-). In women, denosumab over 3 years improved appendicular lean mass and handgrip strength compared to no treatment, whereas bisphosphonate did not. HuRANKL-Tg+ mice displayed lower limb force and maximal speed, while their leg muscle mass was diminished, with a lower number of type I and II fibers. Both OPG and denosumab increased limb force proportionally to the increase in muscle mass. They markedly improved muscle insulin sensitivity and glucose uptake, and decrease anti-myogenic and inflammatory gene expression in muscle, such as myostatin and protein tyrosine phosphatase receptor-γ. Similarly, in Pparb-/-, OPG increased muscle volume and force, while also normalizing their insulin signaling and higher expression of inflammatory genes in skeletal muscle. In conclusions, RANKL deteriorates, while its inhibitor improves, muscle strength and insulin sensitivity in osteoporotic mice and humans. Hence denosumab could represent a novel therapeutic approach for sarcopenia.

Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors.

The type 1 cannabinoid receptor (CB1) modulates numerous neurobehavioral processes and is therefore explored as a target for the treatment of several mental and neurological diseases. However, previous studies have investigated CB1 by targeting it globally, regardless of its two main neuronal localizations on glutamatergic and GABAergic neurons. In the context of cocaine addiction this lack of selectivity is critical since glutamatergic and GABAergic neuronal transmission is involved in different aspects of the disease. To determine whether CB1 exerts different control on cocaine seeking according to its two main neuronal localizations, we used mutant mice with deleted CB1 in cortical glutamatergic neurons (Glu-CB1) or in forebrain GABAergic neurons (GABA-CB1). In Glu-CB1, gene deletion concerns the dorsal telencephalon, including neocortex, paleocortex, archicortex, hippocampal formation and the cortical portions of the amygdala. In GABA-CB1, it concerns several cortical and non-cortical areas including the dorsal striatum, nucleus accumbens, thalamic, and hypothalamic nuclei. We tested complementary components of cocaine self-administration, separating the influence of primary and conditioned effects. Mechanisms underlying each phenotype were explored using in vivo microdialysis and ex vivo electrophysiology. We show that CB1 expression in forebrain GABAergic neurons controls mouse sensitivity to cocaine, while CB1 expression in cortical glutamatergic neurons controls associative learning processes. In accordance, in the nucleus accumbens, GABA-CB1 receptors control cocaine-induced dopamine release and Glu-CB1 receptors control AMPAR/NMDAR ratio; a marker of synaptic plasticity. Our findings demonstrate a critical distinction of the altered balance of Glu-CB1 and GABA-CB1 activity that could participate in the vulnerability to cocaine abuse and addiction. Moreover, these novel insights advance our understanding of CB1 neuropathophysiology.

Hepatic PTEN deficiency improves muscle insulin sensitivity and decreases adiposity in mice.

BACKGROUND & AIMS: PTEN is a dual lipid/protein phosphatase, downregulated in steatotic livers with obesity or HCV infection. Liver-specific PTEN knockout (LPTEN KO) mice develop steatosis, inflammation/fibrosis and hepatocellular carcinoma with aging, but surprisingly also enhanced glucose tolerance. This study aimed at understanding the mechanisms by which hepatic PTEN deficiency improves glucose tolerance, while promoting fatty liver diseases. METHODS: Control and LPTEN KO mice underwent glucose/pyruvate tolerance tests and euglycemic-hyperinsulinemic clamps. Body fat distribution was assessed by EchoMRI, CT-scan and dissection analyses. Primary/cultured hepatocytes and insulin-sensitive tissues were analysed ex vivo. RESULTS: PTEN deficiency in hepatocytes led to steatosis through increased fatty acid (FA) uptake and de novo lipogenesis. Although LPTEN KO mice exhibited hepatic steatosis, they displayed increased skeletal muscle insulin sensitivity and glucose uptake, as assessed by euglycemic-hyperinsulinemic clamps. Surprisingly, white adipose tissue (WAT) depots were also drastically reduced. Analyses of key enzymes involved in lipid metabolism further indicated that FA synthesis/esterification was decreased in WAT. In addition, Ucp1 expression and multilocular lipid droplet structures were observed in this tissue, indicating the presence of beige adipocytes. Consistent with a liver to muscle/adipocyte crosstalk, the expression of liver-derived circulating factors, known to impact on muscle insulin sensitivity and WAT homeostasis (e.g. FGF21), was modulated in LPTEN KO mice. CONCLUSIONS: Although steatosis develops in LPTEN KO mice, PTEN deficiency in hepatocytes promotes a crosstalk between liver and muscle, as well as adipose tissue, resulting in enhanced insulin sensitivity, improved glucose tolerance and decreased adiposity.

New automated procedure to assess context recognition memory in mice.

RATIONALE AND OBJECTIVES: Recognition memory is an important aspect of human declarative memory and is one of the routine memory abilities altered in patients with amnestic syndrome and Alzheimer's disease. In rodents, recognition memory has been most widely assessed using the novel object preference paradigm, which exploits the spontaneous preference that animals display for novel objects. Here, we used nose-poke units instead of objects to design a simple automated method for assessing context recognition memory in mice. METHODS: In the acquisition trial, mice are exposed for the first time to an operant chamber with one blinking nose-poke unit. In the choice session, a novel nonblinking nose-poke unit is inserted into an empty spatial location and the number of nose poking dedicated to each set of nose-poke unit is used as an index of recognition memory. RESULTS: We report that recognition performance varies as a function of the length of the acquisition period and the retention delay and is sensitive to conventional amnestic treatments. By manipulating the features of the operant chamber during a brief retrieval episode (3-min long), we further demonstrate that reconsolidation of the original contextual memory depends on the magnitude and the type of environmental changes introduced into the familiar spatial environment. CONCLUSIONS: These results show that the nose-poke recognition task provides a rapid and reliable way for assessing context recognition memory in mice and offers new possibilities for the deciphering of the brain mechanisms governing the reconsolidation process.

PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is dependent on lipid metabolism. Hepatocyte steatosis occurs frequently in HCV infection, but the relationship between steatosis and HCV life cycle is unclear. We showed that HCV induces steatosis via the downregulation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). We here investigated how PTEN may affect HCV production. METHODS: The effect of overexpression or silencing of PTEN on HCV secretion was assessed in genomic-length Jc1 infected HuH7 cells. The role of PTEN protein and lipid phosphatase activities on lipid metabolism and infectious viral particle secretion was investigated using dominant-negative PTEN mutants. The importance of cholesterol metabolism for PTEN-dependent lipid droplet biogenesis and viral particle secretion was examined using statins. RESULTS: PTEN silencing in Jc1 infected HuH7 cells stimulated HCV particle secretion, while PTEN overexpression decreased virus egress. Viral secretion was also increased by overexpression of protein phosphatase-deleted (PTENY138L), but not lipid phosphatase-deleted (PTENG129E), PTEN mutant, thus indicating that the protein phosphatase activity of PTEN controls viral secretion. Similarly, PTENY138L, but not PTENG129E mutant induced the formation of large lipid droplets. PTENY138L mutant did not affect biosynthesis of triglycerides, but promoted the biosynthesis of cholesterol esters. Consistently, statins prevented the increased cholesterol ester production, large lipid droplet formation, and viral secretion in cells expressing the PTENY138L mutant. CONCLUSIONS: Downregulation of PTEN protein phosphatase activity by HCV affects cholesterol metabolism, thereby inducing the appearance of large lipid droplets and increasing virion egress.

Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (<2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

PPARs in Liver Diseases and Cancer: Epigenetic Regulation by MicroRNAs.

Peroxisome-proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors that exert in the liver a transcriptional activity regulating a whole spectrum of physiological functions, including cholesterol and bile acid homeostasis, lipid/glucose metabolism, inflammatory responses, regenerative mechanisms, and cell differentiation/proliferation. Dysregulations of the expression, or activity, of specific PPAR isoforms in the liver are therefore believed to represent critical mechanisms contributing to the development of hepatic metabolic diseases, disorders induced by hepatic viral infections, and hepatocellular adenoma and carcinoma. In this regard, specific PPAR agonists have proven to be useful to treat these metabolic diseases, but for cancer therapies, the use of PPAR agonists is still debated. Interestingly, in addition to previously described mechanisms regulating PPARs expression and activity, microRNAs are emerging as new important regulators of PPAR expression and activity in pathophysiological conditions and therefore may represent future therapeutic targets to treat hepatic metabolic disorders and cancers. Here, we reviewed the current knowledge about the general roles of the different PPAR isoforms in common chronic metabolic and infectious liver diseases, as well as in the development of hepatic cancers. Recent works highlighting the regulation of PPARs by microRNAs in both physiological and pathological situations with a focus on the liver are also discussed.

Prevention of diabetes in db/db mice by dietary soy is independent of isoflavone levels.

Recent evidence points towards the beneficial use of soy proteins and isoflavones to improve glucose control and slow the progression of type 2 diabetes. Here, we used diabetic db/db mice fed a high soy-containing diet (SD) or a casein soy-free diet to investigate the metabolic effects of soy and isoflavones consumption on glucose homeostasis, hepatic glucose production, and pancreatic islet function. Male db/db mice fed with a SD exhibited a robust reduction in hyperglycemia (50%), correlating with a reduction in hepatic glucose production and preserved pancreatic ß-cell function. The rapid decrease in fasting glucose levels resulted from an inhibition of gluconeogenesis and an increase in glycolysis in the liver of db/db mice. Soy consumption also prevented the loss of pancreatic ß-cell mass and thus improved glucose-stimulated insulin secretion (3-fold), which partly accounted for the overall improvements in glucose homeostasis. Comparison of SD effects on hyperglycemia with differing levels of isoflavones or with purified isoflavones indicate that the beneficial physiological effects of soy are not related to differences in their isoflavone content. Overall, these findings suggest that consumption of soy is beneficial for improving glucose homeostasis and delaying the progression of diabetes in the db/db mice but act independently of isoflavone concentration.

Mechanisms of the anti-obesity effects of oxytocin in diet-induced obese rats.

Apart from its role during labor and lactation, oxytocin is involved in several other functions. Interestingly, oxytocin- and oxytocin receptor-deficient mice develop late-onset obesity with normal food intake, suggesting that the hormone might exert a series of beneficial metabolic effects. This was recently confirmed by data showing that central oxytocin infusion causes weight loss in diet-induced obese mice. The aim of the present study was to unravel the mechanisms underlying such beneficial effects of oxytocin. Chronic central oxytocin infusion was carried out in high fat diet-induced obese rats. Its impact on body weight, lipid metabolism and insulin sensitivity was determined. We observed a dose-dependent decrease in body weight gain, increased adipose tissue lipolysis and fatty acid ß-oxidation, as well as reduced glucose intolerance and insulin resistance. The additional observation that plasma oxytocin levels increased upon central infusion suggested that the hormone might affect adipose tissue metabolism by direct action. This was demonstrated using in vitro, ex vivo, as well as in vivo experiments. With regard to its mechanism of action in adipose tissue, oxytocin increased the expression of stearoyl-coenzyme A desaturase 1, as well as the tissue content of the phospholipid precursor, N-oleoyl-phosphatidylethanolamine, the biosynthetic precursor of the oleic acid-derived PPAR-alpha activator, oleoylethanolamide. Because PPAR-alpha regulates fatty acid ß-oxidation, we hypothesized that this transcription factor might mediate the oxytocin effects. This was substantiated by the observation that, in contrast to its effects in wild-type mice, oxytocin infusion failed to induce weight loss and fat oxidation in PPAR-alpha-deficient animals. Altogether, these results suggest that oxytocin administration could represent a promising therapeutic approach for the treatment of human obesity and type 2 diabetes.

Down-regulation of phosphatase and tensin homolog by hepatitis C virus core 3a in hepatocytes triggers the formation of large lipid droplets.

UNLABELLED: Hepatitis C virus (HCV) perturbs the host's lipid metabolism and often results in hepatic steatosis. In nonalcoholic fatty liver disease, the intrahepatic down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a critical mechanism leading to steatosis and its progression toward fibrosis and hepatocellular carcinoma. However, whether an HCV infection triggers the formation of large lipid droplets through PTEN-dependent mechanisms is unknown. We assessed PTEN expression in the livers of patients infected with HCV genotype 1 or 3 with or without steatosis. The role of PTEN in the HCV-induced biogenesis of lipid droplets was further investigated in vitro with hepatoma cells transduced with the HCV core protein of genotype 1b or 3a. Our data indicate that PTEN expression was down-regulated at the posttranscriptional level in steatotic patients infected with genotype 3a. Similarly, the in vitro expression of the HCV genotype 3a core protein (but not 1b), typically leading to the appearance of large lipid droplets, down-regulated PTEN expression by a mechanism involving a microRNA-dependent blockade of PTEN messenger RNA translation. PTEN down-regulation promoted in turn a reduction of insulin receptor substrate 1 (IRS1) expression. Interestingly, either PTEN or IRS1 overexpression prevented the development of large lipid droplets, and this indicates that the down-regulation of both PTEN and IRS1 is required to affect the biogenesis of lipid droplets. However, IRS1 knockdown per se did not alter the morphology of lipid droplets, and this suggests that other PTEN-dependent mechanisms are involved in this process. CONCLUSION: The down-regulation of PTEN and IRS1 is a critical event leading to the HCV genotype 3a-induced formation of large lipid droplets in hepatocytes.

PTEN in liver diseases and cancer.

The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt axis is a key signal transduction node that regulates crucial cellular functions, including insulin and other growth factors signaling, lipid and glucose metabolism, as well as cell survival and apoptosis. In this pathway, PTEN acts as a phosphoinositide phosphatase, which terminates PI3K-propagated signaling by dephosphorylating PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). However, the role of PTEN does not appear to be restricted only to PI3K signaling antagonism, and new functions have been recently discovered for this protein. In addition to the well-established role of PTEN as a tumor suppressor, increasing evidence now suggests that a dysregulated PTEN expression and/or activity is also linked to the development of several hepatic pathologies. Dysregulated PTEN expression/activity is observed with obesity, insulin resistance, diabetes, hepatitis B virus/hepatitis C virus infections, and abusive alcohol consumption, whereas mutations/deletions have also been associated with the occurrence of hepatocellular carcinoma. Thus, it appears that alterations of PTEN expression and activity in hepatocytes are common and recurrent molecular events associated with liver disorders of various etiologies. These recent findings suggest that PTEN might represent a potential common therapeutic target for a number of liver pathologies.

CD4 dimerization requires two cysteines in the cytoplasmic domain of the molecule and occurs in microdomains distinct from lipid rafts.

CD4 is engaged in APC-mediated T-cell activation and serves as the primary receptor for HIV. CD4 dimerization and location in specific microdomains has been previously suggested to control its pathophysiological activity. In this study, we investigated (i) whether the CD4 cytoplasmic domain contributes to its dimerization by evaluating the dimerization of mutants, bearing deletions or point mutations in their cytoplasmic tail, (ii) whether CD4 monomers and dimers segregate in distinct microdomains by subcellular fractionation, and (iii) how CD4 dimerization is affected by T-cell activation or HIV-1 viral proteins. Our results indicated that within the cytoplasmic tail of CD4, two cysteines played a crucial role in the dimers formation, since point mutations or truncation upstream of these residues prevented dimerization. The solubility of CD4 dimers and monomers in various detergents was different and CD4 dimers were poorly associated with lipid rafts, but strongly interacted with the tetraspanin CD81. Neither cytoskeleton-disrupting drugs nor cholesterol-sequestering agents had an effect on the CD4 dimerization indicating that dimers formation was independent of CD4 association with the cytoskeleton or lipid rafts. Finally, whereas T-cell activation poorly impact on CD4 dimerization, HIV-1 gp120 and Nef drastically reduced the ratio of CD4 dimers/monomers. Together, these findings demonstrate that two cysteines within the CD4 cytoplasmic tail are critical for dimerization, that CD4 dimers locate preferentially in microdomains distinct than classical lipid rafts, likely tetraspanin-enriched microdomains, and that CD4 dimers are implicated in the process of HIV infection.

PTEN in non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and cancer.

The tumor suppressor PTEN is a protein/phosphoinositide phosphatase regulating the PI3K/Akt signaling pathway and is mutated or deleted in a variety of human cancers, including hepatocellular carcinoma (HCC). Accumulating evidence indicates that alterations of PTEN expression and activity in hepatocytes are common and recurrent molecular events associated with liver disorders of various etiologies including obesity, the metabolic syndrome, hepatitis B virus/hepatitis C virus infection and abusive alcohol consumption. Genetic and molecular studies, particularly in the context of non-alcoholic fatty liver disease (NAFLD), support a critical role for PTEN in hepatic insulin sensitivity and the development of steatosis, steatohepatitis and fibrosis. PTEN mutations/deletion or low PTEN expression are also associated with diverse liver malignancies, suggesting a critical role for PTEN in hepatic cancers. This review provides an overview of the current knowledge on pathological dysregulations of PTEN expression/activity in the liver with obesity and the metabolic syndrome, and the role of this enzyme in the development of non-alcoholic fatty liver disease and hepatocellular carcinoma.