Acute effects of different diet compositions on skeletal muscle insulin signalling in obese individuals during caloric restriction.

OBJECTIVE: The cellular effects of restricting fat versus carbohydrate during a low-calorie diet are unclear. The aim of this study was to examine acute effects of energy and macronutrient restriction on skeletal muscle insulin signalling in obesity. MATERIALS/METHODS: Eighteen obese individuals without diabetes underwent euglycemic-hyperinsulinemic clamp and skeletal muscle biopsy after: (a) 5days of eucaloric diet (30% fat, 50% carbohydrate), and (b) 5days of a 30% calorie-restricted diet, either low fat/high carbohydrate (LF/HC: 20% fat, 60% carbohydrate) or high-fat/low carbohydrate (HF/LC: 50% fat, 30% carbohydrate). RESULTS: Weight, body composition, and insulin sensitivity were similar between groups after eucaloric diet. Weight loss was similar between groups after hypocaloric diet, 1.3±1.3kg (p<0.0001 compared with eucaloric). Whole-body insulin sensitivity was unchanged after calorie restriction and similar between groups. However, ex vivo skeletal muscle insulin signalling differed depending on macronutrient composition of calorie-restricted diet. Skeletal muscle of the LF/HC group had increased insulin-stimulated tyrosine phosphorylation of IRS-1, decreased insulin-stimulated Ser307 phosphorylation of IRS-1, and increased IRS-1-associated phosphatidylinositol (PI)3-kinase activity. Conversely, insulin stimulation of tyrosine phosphorylated IRS-1 was absent and serine 307 phosphorylation of IRS-1 was increased on HF/LC, with blunting of IRS-1-associated PI3-kinase activity. CONCLUSION: Acute caloric restriction with an LF/HC diet alters skeletal muscle insulin signalling in a way that improves insulin sensitivity, while acute caloric restriction with an HF/LC diet induces changes compatible with insulin resistance. In both cases, ex vivo changes in skeletal muscle insulin signalling appear prior to changes in whole body insulin sensitivity.

Early responses of insulin signaling to high-carbohydrate and high-fat overfeeding.

BACKGROUND: Early molecular changes of nutritionally-induced insulin resistance are still enigmatic. It is also unclear if acute overnutrition alone can alter insulin signaling in humans or if the macronutrient composition of the diet can modulate such effects. METHODS: To investigate the molecular correlates of metabolic adaptation to either high-carbohydrate (HC) or high-fat (HF) overfeeding, we conducted overfeeding studies in 21 healthy lean (BMI < 25) individuals (10 women, 11 men), age 20-45, with normal glucose metabolism and no family history of diabetes. Subjects were studied first following a 5-day eucaloric (EC) diet (30% fat, 50% CHO, 20% protein) and then in a counter balanced manner after 5 days of 40% overfeeding of both a HC (20% fat, 60% CHO) diet and a HF (50% fat, 30% CHO) diet. At the end of each diet phase, in vivo insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp technique. Ex vivo insulin action was measured from skeletal muscle tissue samples obtained 15 minutes after insulin infusion was initiated. RESULTS: Overall there was no change in whole-body insulin sensitivity as measured by glucose disposal rate (GDR, EC: 12.1 ± 4.7; HC: 10.9 ± 2.7; HF: 10.8 ± 3.4). Assessment of skeletal muscle insulin signaling demonstrated increased tyrosine phosphorylation of IRS-1 (p < 0.001) and increased IRS-1-associated phosphatidylinositol 3 (PI 3)-kinase activity (p < 0.001) following HC overfeeding. In contrast, HF overfeeding increased skeletal muscle serine phosophorylation of IRS-1 (p < 0.001) and increased total expression of p85α (P < 0.001). CONCLUSION: We conclude that acute bouts of overnutrition lead to changes at the cellular level before whole-body insulin sensitivity is altered. On a signaling level, HC overfeeding resulted in changes compatible with increased insulin sensitivity. In contrast, molecular changes in HF overfeeding were compatible with a reduced insulin sensitivity.

Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase.

Phosphorylation of insulin receptor substrate-1 (IRS-1) on serine residues has been recognized as a mechanism responsible for a diminution of insulin action and insulin resistance. Potential approaches to improve insulin sensitivity may include interference with and/or reduction in expression of certain signaling intermediates that participate in the pathogenesis of insulin resistance. In this study, we transduced fully differentiated 3T3-L1 adipocytes with a constitutively active myristoylated Akt that led to hyperactivation of mammalian target of rapamycin and p70 S6 kinase (S6K1), increased serine phosphorylation of IRS-1, and reduction in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and glucose transport. We then reduced expression of the PI 3-kinase regulatory subunit, p85alpha, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85alpha expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85alpha or S6K1 achieved with small interfering RNA in the presence of myristoylated Akt rescued 3T3-L1 adipocytes from the insulin resistance induced by serine phosphorylation of IRS-1 and completely restored insulin-stimulated activation of PI 3-kinase and glucose uptake. We conclude that reduction in expression of p85alpha or S6K1 could represent therapeutic targets to mitigate insulin resistance.

Knockdown of JNK rescues 3T3-L1 adipocytes from insulin resistance induced by mitochondrial dysfunction.

Mitochondrial dysfunction has been linked to etiology of insulin resistance, however the mechanism remains unknown. In this study we investigated whether mitochondrial dysfunction induced by cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) alters insulin sensitivity in 3T3-L1 adipocytes and which cellular signaling molecules might be involved. Fully differentiated 3T3-L1 adipocytes were treated with 10 microM FCCP for 1h, resulting in increased serine-307 phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation, association of p85alpha subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with IRS-1, decreased insulin-stimulated PI 3-kinase activity and H(3)-2-deoxyglucose (2DOG) uptake. A partial (46%) knockdown of JNK1 blocked FCCP-induced serine phosphorylation of IRS-1 and restored insulin-stimulated tyrosine phosphorylation of IRS-1, association of p85alpha subunit of PI 3-kinase with IRS-1, activation of PI 3-kinase, and stimulation of 2DOG uptake. Thus, FCCP-induced mitochondrial dysfunction may cause insulin resistance that is ameliorated by reduction of JNK1 expression.