Donna Wilks

Overexpression of manganese superoxide dismutase ameliorates high-fat diet-induced insulin resistance in rat skeletal muscle

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.

Sialic Acid Modification of Adiponectin Is Not Required for Multimerization or Secretion but Determines Half-Life in Circulation

Adiponectin is an adipocyte-secreted, insulin-sensitizing hormone the circulating levels of which are reduced in conditions of insulin resistance and diabetes. Previous work has demonstrated the importance of posttranslational modifications, such as proline hydroxylation and lysine hydroxylation/glycosylation, in adiponectin oligomerization, secretion, and function. Here we describe the first functional characterization of adiponectin sialylation. Using a variety of biochemical approaches we demonstrated that sialylation occurs on previously unidentified O-linked glycans on Thr residues of the variable domain in human adiponectin. Enzymatic removal of sialic acid or its underlying O-linked sugars did not affect adiponectin multimer composition. Expression of mutant forms of adiponectin (lacking the modified Thr residues) or of wild-type adiponectin in cells defective in sialylation did not compromise multimer formation or secretion, arguing against a structural role for this modification. Activity of desialylated adiponectin was comparable to control adiponectin in L6 myotubes and acute assays in adiponectin(-/-) mice. In contrast, plasma clearance of desialylated adiponectin was accelerated compared with that of control adiponectin, implicating a role for this modification in determining the half-life of circulating adiponectin. Uptake of desialylated adiponectin by isolated primary rat hepatocytes was also accelerated, suggesting a role for the hepatic asialoglycoprotein receptor. Finally, after chronic administration in adiponectin(-/-) mice steady-state levels of desialylated adiponectin were lower than control adiponectin and failed to recapitulate the improvements in glucose and insulin tolerance tests observed with control adiponectin. These data suggest an important role for sialic acid content in the regulation of circulating adiponectin levels and highlight the importance of understanding mechanisms regulating adiponectin sialylation/desialylation.

Altered feeding differentially regulates circadian rhythms and energy metabolism in liver and muscle of rats.

Energy metabolism follows a diurnal pattern responding to the light/dark cycle and food availability. This study investigated the impact of restricting feeding to the daylight hours and feeding a high fat diet on circadian clock (bmal1, dbp, tef and e4bp4) and metabolic (pepck, fas, ucp3, pdk4) gene expression and markers of energy metabolism in muscle and liver of rats. The results show that in chow-fed rats switched to daylight feeding, the peak diurnal expression of genes in liver was shifted by 6-12h while expression of these genes in muscle remained in a similar phase to rats feeding ad libitum. High fat feeding during the daylight hours had limited effect on clock gene expression in liver or muscle but shifted the peak expression of metabolic genes (pepck, fas) in liver by 6-12h. The differential effects of daylight feeding on gene and protein expression in muscle and liver were accompanied by an 8% reduction in whole body energy expenditure, a 20-30% increased glycogen content during the light phase in muscle of day-fed rats and increased adipose tissue deposition per gram food consumed. These data demonstrate that a mismatch of feeding and light/dark cycle disrupts tissue metabolism in muscle with significant consequences for whole body energy homeostasis.

Regulation of the nuclear hormone receptor nur77 in muscle: influence of exercise-activated pathways in vitro and obesity in vivo.

Regular physical exercise is well known to improve glucose and lipid metabolism in skeletal muscle. However, the transcription factors regulating these adaptive changes are not well-characterised. Recently the nuclear orphan receptor nur77 was shown to be induced by exercise and linked to regulation of metabolic gene expression in skeletal muscle. In this study we investigated the regulation of nur77 in muscle by different exercise-activated pathways. Nur77 expression was found to be responsive to adrenergic stimulation and calcium influx, but not to activation of the AMP dependent kinase. These results identify the adrenergic-cyclic AMP-PKA pathway to be the most potent activator of nur77 expression in muscle and therefore the likely cause of increased expression after exercise. We also identified nur77 expression to be reduced in the muscle of obese/insulin resistant rats after high fat feeding. Furthermore exposure to fatty acids, insulin or inflammation was not the cause of decreased nur77 expression in insulin resistant muscle. This suggests a reduced responsiveness to adrenergic stimulation as the likely cause of diminished nur77 expression in muscle of high fat fed rats, which has been observed in obese/insulin resistant individuals. Our results suggest adrenergic stimulation as the most important stimulus for nur77 expression and point to a significant role for this transcription factor in adaptive changes in muscle after exercise and in insulin resistant states.

Overexpression of the orphan receptor Nur77 alters glucose metabolism in rat muscle cells and rat muscle in vivo

Aims/hypothesisA hallmark feature of the metabolic syndrome is abnormal glucose metabolism which can be improved by exercise. Recently the orphan nuclear receptor subfamily 4, group A, member 1 (NUR77) was found to be induced by exercise in muscle and was linked to transcriptional control of genes involved in lipid and glucose metabolism. Here we investigated if overexpression of Nur77 (also known as Nr4a1) in skeletal muscle has functional consequences for lipid and/or glucose metabolism.MethodsL6 rat skeletal muscle myotubes were infected with a Nur77-coding adenovirus and lipid and glucose oxidation was measured. Nur77 was also overexpressed in skeletal muscle of chow- and fat-fed rats and the effects on glucose and lipid metabolism evaluated.ResultsNur77 overexpression had no effect on lipid oxidation in L6 cells or rat muscle, but did increase glucose oxidation and glycogen synthesis in L6 cells. In chow- and high-fat-fed rats, Nur77 overexpression by electrotransfer significantly increased basal glucose uptake and glycogen synthesis, but no increase in insulin-stimulated glucose metabolism was observed. Nur77 electrotransfer was associated with increased production of GLUT4 and glycogenin and increased hexokinase and phosphofructokinase activity. Interestingly, Nur77 expression in muscle biopsies from obese men was significantly lower than in those from lean men and was closely correlated with body-fat content and insulin sensitivity.Conclusions/interpretationOur data provide compelling evidence that NUR77 is a functional regulator of glucose metabolism in skeletal muscle in vivo. Importantly, the diminished content in muscle of obese insulin-resistant men suggests that it might be a potential therapeutic target for the treatment of dysregulated glucose metabolism.

Central neuropeptide Y infusion and melanocortin 4 receptor antagonism inhibit thyrotropic function by divergent pathways

Weight loss inhibits thyrotropic function and reduces metabolic rate, thereby contributing to weight regain. Under negative energy balance there is an increase in the hypothalamic expression of both neuropeptide Y (NPY) and agouti related peptide (AgRP), the endogenous antagonist of melanocortin 4 (MC4) receptors. Both NPY and MC4 receptor antagonism reduce thyrotropic function centrally, but it is not known whether these pathways operate by similar or distinct mechanisms. We compared the time-course of effects of acute or chronic intracerebroventricular (ICV) administration of NPY (1.2 nmol acute bolus, or 3.5 nmol/day for 6 days) or the MC4 receptor antagonist HS014 (1.5 nmol bolus, or 4.8 nmol/day) on plasma concentrations of thyroid stimulating hormone (TSH) or free thyroxine (T4) in male rats pair-fed with vehicle-infused controls. These doses equipotently induced hyperphagia in acute studies, reduced latency to feed, and increased white adipose tissue mass after 6 days of infusion. Acute central NPY but not HS014 administration significantly reduced plasma TSH concentrations within 30-60 min and plasma free T4 levels within 90-120 min. These inhibitory effects were sustained for up to 5-6 days of continuous NPY infusion. HS014 induced a transient decrease in plasma free T4 levels that was observed only after 1-2 days of continuous ICV infusion. While both NPY and HS014 significantly increased corticosteronemia within an hour after ICV injection, the effect of NPY was significantly more pronounced and was sustained for up to 4 days of administration. Both NPY and HS014 significantly decreased the brown adipose tissue protein levels of uncoupling protein-3. We conclude that central NPY and MC4 antagonism decrease thyrotropic function via partially distinct mechanisms with different time courses, possibly involving glucocorticoid effects of NPY. MC4 receptor antagonism increases adiposity via pathways independent of increased food intake or changes in circulating concentrations of TSH, free T4 or corticosterone.

Interaction between adrenal glucocorticoids and parasympathetic activation in mediating hyperinsulinaemia during long-term central neuropeptide Y infusion in rats

Aims/hypothesis. Hypothalamic neuropeptide Y is implicated in the aetiology of obesity and insulin resistance because of its hyperinsulinaemic, hyperphagic effects. We investigated the interaction of adrenal glucocorticoids and the parasympathetic nervous system in the hyperinsulinaemia caused by neuropeptide Y infusion in rats.¶Methods. Neuropeptide Y was intracerebroventricularly given to normal or adrenalectomised rats for 3–6 days with pair-feeding, with or without subcutaneous dexamethasone infusion. We measured basal and intravenous glucose-induced insulinaemia and the effect of prior atropine injection.¶Results. Neuropeptide Y increased basal plasma insulin and C-peptide concentrations (380 ± 90 and 1000 ± 60 pmol/l, vs 190 ± 20 and 590 ± 50 pmol/l in controls, p < 0.05). Neuropeptide Y also increased the plasma concentrations of these hormones as early as 60 s after glucose injection (1630 ± 170 and 3200 ± 170 pmol/l for insulin and C peptide, respectively, vs 1080 ± 80 and 1860 ± 130 pmol/l in controls, p < 0.05). Atropine reversed the effect of neuropeptide Y on basal plasma insulin and C-peptide concentrations but had no effect on post-glucose plasma concentrations. The hyperinsulinaemic effects of neuropeptide Y were prevented by adrenalectomy, but were restored by dexamethasone infusion. Dexamethasone in itself did not statistically significantly increase insulinaemia in adrenalectomised rats. As in intact rats, atropine attenuated the basal hyperinsulinaemia of adrenalectomised rats that had been infused with neuropeptide Y and dexamethasone but had no effect on post-glucose hyperinsulinaemia.¶Conclusion/interpretation. These data suggest firstly that neuropeptide Y infused centrally induces basal hyperinsulinaemia in rats through glucocorticoid-dependant parasympathetic activation to the pancreas. Secondly, neuropeptide Y potentiates glucose-induced insulinaemia through a pathway dependant on adrenal glucocorticoids that cannot be reversed by short-term blockade of the increased parasympathetic tonus. [Diabetologia (2000) 43: 859–865]

Ablation of Grb10 Specifically in Muscle Impacts Muscle Size and Glucose Metabolism in Mice

Grb10 is an adaptor-type signaling protein most highly expressed in tissues involved in insulin action and glucose metabolism, such as muscle, pancreas, and adipose. Germline deletion of Grb10 in mice creates a phenotype with larger muscles and improved glucose homeostasis. However, it has not been determined whether Grb10 ablation specifically in muscle is sufficient to induce hypermuscularity or affect whole body glucose metabolism. In this study we generated muscle-specific Grb10-deficient mice (Grb10-mKO) by crossing Grb10flox/flox mice with mice expressing Cre recombinase under control of the human α-skeletal actin promoter. One-year-old Grb10-mKO mice had enlarged muscles, with greater cross-sectional area of fibers compared with wild-type (WT) mice. This degree of hypermuscularity did not affect whole body glucose homeostasis under basal conditions. However, hyperinsulinemic/euglycemic clamp studies revealed that Grb10-mKO mice had greater glucose uptake into muscles compared with WT mice. Insulin signaling was increased at the level of phospho-Akt in muscle of Grb10-mKO mice compared with WT mice, consistent with a role of Grb10 as a modulator of proximal insulin receptor signaling. We conclude that ablation of Grb10 in muscle is sufficient to affect muscle size and metabolism, supporting an important role for this protein in growth and metabolic pathways.

384 INCREASED GLOBAL POSTTRANSLATIONAL O-GLCNAC MODIFICATION OF LIVER PROTEINS PRECEDES THE DEVELOPMENT OF GLUCOSE-INDUCED INSULIN RESISTANCE IN VIVO

Increased flux through the hexosamine biosynthetic pathway (HBP) has been proposed as one mechanism by which hyperglycemia induces insulin resistance and complications associated with diabetes. Excess glucose entering the HBP is converted to UDP-GlcNAc, a sugar donor for the posttranslational glycosylation of Ser/Thr residues by O-linked N-acetylglucosamine monosaccharide (GlcNAc). This dynamic and reversible modification of cytosolic and nuclear proteins provides an alternative mechanism of protein regulation to phosphorylation. The purpose of this study was to assess whether increased O-GlcNAc modification of proteins precedes the development of glucose-induced insulin resistance in vivo. Methods In male Wistar rats, blood glucose was maintained at 10-12 mM by variable glucose infusion for 3 or 5 hours, after which blood glucose was returned to basal over 45 minutes by stepwise cessation of iv glucose. A 2-hour euglycemic hyperinsulinemic clamp (0.25 U/kg/hr) was then performed in these and saline-infused control rats. Insulin resistance was present in both muscle and liver after 5 but not after 3 hours of glucose infusion. Soluble protein, isolated from liver and gastrocnemius muscle of rats infused with saline or glucose for 3 or 5 hours, was separated by SDS-PAGE and transferred to nitrocellulose. Global protein O-GlcNAc modification was quantitated using an anti-O-GlcNAc antibody and normalized for protein loading using α-tubulin. Results Preliminary data suggest that in liver, but not in muscle, there is an increase in the global level of O-GlcNAc modification following 3 hours of glucose infusion. Analysis of the four most intense bands corresponding to O-GlcNAc modified proteins in the liver at 141 kDa, 130 kDa, 90 kDa, and 60 kDa showed an increase (range 20-500%) in the level of modification with glucose infusion. We found that the mean difference in protein modification between glucose- and saline-infused rats was borderline significant for the 141 kDa protein (p = .0751) and significant for the 90 kDa and 60 kDa proteins (p = .0479 and .0022, respectively) after adjusting for hour (3 vs 5) and accounting for correlation of repeated measures. Conclusions In an in vivo model of insulin resistance, a global increase in protein O-GlcNAc modification in liver precedes the development of insulin resistance.