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.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.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).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.
Adenosine monophosphate-activated protein kinase (AMPK), silent mating type information regulation 2 homologue 1 (SIRT 1), and peroxisome proliferator-activated receptor γ co-activator α (PGC1α) constitute an energy sensing cellular network that controls mitochondrial biogenesis. Caloric restriction activates both AMPK and SIRT-1 to increase ATP production from fat oxidation. We characterized AMPK and SIRT 1 expression and activity in human skeletal muscle in response to dietary fat or carbohydrate intake on the background of either overfeeding or caloric restriction. AMPK phosphorylation and acetylation of PGC1α (as a measure of SIRT activity) were determined. Euglycemic-hyperinsulinemic clamp and muscle biopsies were performed in human subjects participating in 2 separate studies. In study 1, 21 lean healthy individuals were overfed for 5 days, while in study 2, 18 obese otherwise healthy individuals consumed a calorie-restricted diet for 5 days. Under both conditions - overfeeding and caloric restriction - high fat/low carbohydrate (HF/LC) diet significantly increased phosphorylation of AMPK and deacetylation of PGC1α in skeletal muscle without affecting total amounts of AMPK, PGC1α, or SIRT 1. In contrast, low fat/high carbohydrate (LF/HC) hypocaloric diet reduced phosphorylation of AMPK and deacetylation of PGC1α. Our data indicate that a relative deficiency in carbohydrate intake or, albeit less likely, a relative excess of fat intake even in the absence of caloric deprivation is sufficient to activate the AMPK-SIRT 1-PGC1α energy-sensing cellular network in human skeletal muscle.
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, p85α, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85α expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85α 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 p85α or S6K1 could represent therapeutic targets to mitigate insulin resistance.
