Manuel Ros

Ageing increases SOCS-3 expression in rat hypothalamus: effects of food restriction.

Aged Wistar rats are characterized by leptin and insulin resistance. The expression of SOCS-3 in hypothalamus increases with ageing. Food restriction during 3 months decreases obesity Lee index in aged rats with respect to their ad libitum aged-mates and brings serum leptin concentrations to values close to those of young rats. Food restriction partially reverts the increases in SOCS-3 mRNA levels associated with ageing. These results suggest that SOCS-3 may be a mediator of hypothalamic leptin resistance in the aged Wistar rat and that the hyperleptinemia associated with ageing is, at least in part, responsible for the increase of SOCS-3 expression in hypothalamus.

Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice

OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action.

ESSENTIAL ROLE OF PROTEIN TYROSINE PHOSPHATASE 1B IN OBESITY-INDUCED INFLAMMATION AND PERIPHERAL INSULIN RESISTANCE DURING AGING

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes (T2DM). In this study, we have evaluated the role of PTP1B in the development of aging‐associated obesity, inflammation, and peripheral insulin resistance by assessing metabolic parameters at 3 and 16 months in PTP1B−/− mice maintained on mixed genetic background (C57Bl/6J × 129Sv/J). Whereas fat mass and adipocyte size were increased in wild‐type control mice at 16 months, these parameters did not change with aging in PTP1B−/− mice. Increased levels of pro‐inflammatory cytokines, crown‐like structures, and hypoxia‐inducible factor (HIF)‐1α were observed only in adipose tissue from 16‐month‐old wild‐type mice. Similarly, islet hyperplasia and hyperinsulinemia were observed in wild‐type mice with aging‐associated obesity, but not in PTP1B−/− animals. Leanness in 16‐month‐old PTP1B−/− mice was associated with increased energy expenditure. Whole‐body insulin sensitivity decreased in 16‐month‐old control mice; however, studies with the hyperinsulinemic–euglycemic clamp revealed that PTP1B deficiency prevented this obesity‐related decreased peripheral insulin sensitivity. At a molecular level, PTP1B expression and enzymatic activity were up‐regulated in liver and muscle of 16‐month‐old wild‐type mice as were the activation of stress kinases and the expression of p53. Conversely, insulin receptor‐mediated Akt/Foxo1 signaling was attenuated in these aged control mice. Collectively, these data implicate PTP1B in the development of inflammation and insulin resistance associated with obesity during aging and suggest that inhibition of this phosphatase by therapeutic strategies might protect against age‐dependent T2DM.

Adenosine A1 receptor in cultured neurons from rat cerebral cortex : Colocalization with Adenosine deaminase

Adenosine A1 receptors (A1Rs) have been characterized in primary cultures of neurons from cerebral cortex. The specific adenosine A1 antagonist 8‐cyclopentyl‐1,3‐[3H]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a KD value of 0.76 nM and a Bmax of 57 fmol/mg of protein were obtained. Competition assays revealed a pharmacological profile characteristic of A1Rs. The presence of this receptor was further confirmed by RT‐PCR analysis. The expression of the receptor showed no significant changes during the period of culture studied, up to 12 days in vitro. A1R agonist inhibited forskolin‐stimulated adenylyl cyclase, showing the functional coupling of these receptors with the effector. αGi1,2 protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlated with an increase in the mRNA level of αGi1 but not αGi2. By immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A1Rs with microtubule‐associated protein 2 and cell surface adenosine deaminase was shown by confocal microscopy. The high degree of colocalization observed between A1Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine‐mediated neuromodulation.

Desensitization of adenosine A1 receptor-mediated inhibition of adenylyl cyclase in cerebellar granule cells.

Agonist-induced desensitization of adenosine A1 receptor-mediated inhibition of adenylyl cyclase has been studied in cerebellar granule cells. Exposure of cells to the adenosine A1 receptor agonist R-phenylisopropyl adenosine (R-PIA) from 2 to 48 h brings about desensitization of this signal transduction pathway. Associated with the desensitization process, a decrease in radioligand binding performed in intact cells with the adenosine A1 receptor agonist [3H]cyclohexyladenosine (CHA) has been detected. Simultaneously, an increase of adenosine A1 radioligand binding has also been detected in microsomes. A decrease in the steady-state level of α-Gi in both, plasma membrane and microsomes also has been detected during the desensitization process. These data may account for the desensitization of the inhibitory pathway of the adenylyl cyclase in cerebellar granule cells described herein. After a transient increase in adenosine A1 receptor mRNA, no changes were observed in this parameter after 12 hr of treatment with the adenosine A1 agonist R-PIA, suggesting a post-transcriptional regulation of this receptor during long-term desensitization.

Ceramide mediates TNF-α-induced insulin resistance on GLUT4 gene expression in brown adipocytes

Abstract Tumour necrosis factor (TNF)-α impaired insulin induction on GLUT4 mRNA in foetal brown adipocytes, as demonstrated by quantitative RT-PCR and Northern blot. We have explored the hypothesis that some effects of TNF-α could be mediated by the generation of ceramide, since TNF-α treatment induced the production of ceramide in these primary cells. A short-chain ceramide analogue, C2-ceramide, precluded insulin-induced GLUT4 mRNA accumulation and GLUT4-chloramphenicol acetyltransferase (CAT) full promoter activation. Moreover, inhibition of the ceramide biosynthesis with fumonisin B, which inhibits ceramide synthase, completely restored insulin-induced GLUT4 mRNA and protein accumulation as well as GLUT4-CAT transactivation in the presence of TNF-α. In consequence, TNF-α-induced insulin resistance on glucose uptake was completely alleviated. In addition, TNF-α down-regulated insulin-induced CCAAT/enhancer binding protein (C/EBP)-alpha gene expression and DNA binding activity, but fumonisin B precludes these effects. Furthermore, co-transfection with a wild-type C/EBP-α construct transactivates GLUT4-CAT construct. Our results indicate that de novo ceramide produced by TNF-α-induced insulin resistance on GLUT4 gene expression in brown adipocytes by interfering C/EBP-α expression, a transcription factor essential for the expression of GLUT4.

Adaptation and failure of pancreatic β cells in murine models with different degrees of metabolic syndrome

SUMMARY The events that contribute to the expansion of β-cell mass and enhanced β-cell function in insulin-resistant states have not been elucidated fully. Recently, we showed that β-cell adaptation failed dramatically in adult, insulin-resistant POKO mice, which contrasts with the appropriate expansion of β cells in their ob/ob littermates. Thus, we hypothesised that characterisation of the islets in these mouse models at an early age should provide a unique opportunity to: (1) identify mechanisms involved in sensing insulin resistance at the level of the β cells, (2) identify molecular effectors that contribute to increasing β-cell mass and function, and (3) distinguish primary events from secondary events that are more likely to be present at more advanced stages of diabetes. Our results define the POKO mouse as a model of early lipotoxicity. At 4 weeks of age, it manifests with inappropriate β-cell function and defects in proliferation markers. Other well-recognised pathogenic effectors that were observed previously in 16-week-old mice, such as increased reactive oxygen species (ROS), macrophage infiltration and endoplasmic reticulum (ER) stress, are also present in both young POKO and young ob/ob mice, indicating the lack of predictive power with regards to the severity of β-cell failure. Of interest, the relatively preserved lipidomic profile in islets from young POKO mice contrasted with the large changes in lipid composition and the differences in the chain length of triacylglycerols in the serum, liver, muscle and adipose tissue in adult POKO mice. Later lipotoxic insults in adult β cells contribute to the failure of the POKO β cell. Our results indicate that the rapid development of insulin resistance and β-cell failure in POKO mice makes this model a useful tool to study early molecular events leading to insulin resistance and β-cell failure. Furthermore, comparisons with ob/ob mice might reveal important adaptive mechanisms in β cells with either therapeutic or diagnostic potential.

Changes in the neuroendocrine control of energy homeostasis by adiposity signals during aging

Energy balance in mammals is modulated by peripheral signals that inform the brain about the magnitude of fat stores, the amount of food in the gastrointestinal tract, and the level of nutrients such as glucose in the circulation. Among these, insulin and leptin are considered adiposity signals involved in the long-term maintenance of fat stores. Here we review the mechanisms of action of leptin and insulin in the hypothalamus and how these mechanisms are altered during aging in rat models. Aged rats are characterized by increased fat mass, central leptin and insulin resistance, and hyperleptinemia. Leptin resistance is manifested by its failure to inhibit food intake, deplete fat stores, down regulate its own expression in adipose tissue, and increase energy expenditure. Moreover, leptin and insulin signaling are decreased in hypothalamus from aged rats. Calorie restriction and fasting studies provide controversial data on the cause-effect interrelationship between increased adiposity and development of central leptin resistance. Although in the absence of obesity leptin resistance seems to be a characteristic of aged animals, adiposity could either reinforce it or cause an early onset of this resistance. More studies are necessary to clarify the role of the hypothalamus in the development of age-associated obesity and insulin resistance.

Age-associated development of inflammation in Wistar rats: Effects of caloric restriction

Context: Insulin resistance and type 2 Diabetes have been associated to a low grade of inflammation and their prevalence increase with ageing. Objective: To analyse the development of inflammation in adipose tissue, liver, muscle and hypothalamus during ageing and the effects of caloric restriction. Materials and methods: We have analysed the expression of inflammatory cytokines (TNFα, IL1-β, IL-12B and IL-6), proteins involved in macrophage recruitment (MCP-1, CCR2), TLR4 and macrophage markers (CD11c, CD11b and arginase1). Immunohistochemistry of macrophages has also been performed. Results: All studied tissues present signs of inflammation during ageing, but with different pattern and intensity. Caloric restriction decreases the expression of most of inflammatory markers. Discussion and conclusions: These data indicate a role of adiposity in the development of inflammation and insulin resistance during ageing. Dietetic intervention could be a useful tool to ameliorate the development of inflammation and insulin resistance associated with ageing.

Isolation and biological characterization of a 6-kDa protein from hepatopancreas of lobster Panulirus argus with insulin-like effects

A protein with insulin-like effects was isolated from the hepatopancreas of the lobster Panulirus argus following a classic method for mammalian insulin purification from the pancreas. After acid-alcoholic extraction and ethanol-ether precipitation followed by molecular filtration chromatography, a protein with an apparent molecular weight of 6 kDa was isolated. This protein is characterized by its ability to interact with anti-insulin antibodies and by mimicking insulin actions as the stimulation of glucose oxidation to CO(2) and lipogenesis in isolated rat adipocytes. In addition, this insulin immunoreactive protein (IIP) was able to stimulate the autophosphorylation of the insulin receptor present in rat adipocyte plasma membranes, in a dose-dependent manner. The immunological and biochemical results obtained are consistent with the hypothesis that protein(s) with insulin-like effects occur in the digestive gland of the lobster P. argus and may be of significance to control metabolic and growth related processes in crustaceans.