OBJECTIVEThe hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α2β1 was tested.RESEARCH DESIGN AND METHODSThe association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α2β1-null (itga2−/−), integrin α1β1-null (itga1−/−), and wild-type littermate mice fed chow or HF. Integrin α2β1 and integrin α1β1 signaling pathways have opposing actions.RESULTSHF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2−/− mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1−/− mice was unchanged. Insulin sensitivity in chow-fed itga1−/− and itga2−/− mice was not different from wild-type littermates.CONCLUSIONSECM collagen expansion is tightly associated with muscle IR. Studies with itga2−/− mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α2β1 interaction.
Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate–based respiration is decreased while fatty acid–based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function.
Muscle insulin resistance is associated with a reduction in vascular endothelial growth factor (VEGF) action and muscle capillary density. We tested the hypothesis that muscle capillary rarefaction critically contributes to the etiology of muscle insulin resistance in chow-fed mice with skeletal and cardiac muscle VEGF deletion (mVEGF−/−) and wild-type littermates (mVEGF+/+) on a C57BL/6 background. The mVEGF−/− mice had an ∼60% and ∼50% decrease in capillaries in skeletal and cardiac muscle, respectively. The mVEGF−/− mice had augmented fasting glucose turnover. Insulin-stimulated whole-body glucose disappearance was blunted in mVEGF−/− mice. The reduced peripheral glucose utilization during insulin stimulation was due to diminished in vivo cardiac and skeletal muscle insulin action and signaling. The decreased insulin-stimulated muscle glucose uptake was independent of defects in insulin action at the myocyte, suggesting that the impairment in insulin-stimulated muscle glucose uptake was due to poor muscle perfusion. The deletion of VEGF in cardiac muscle did not affect cardiac output. These studies emphasize the importance for novel therapeutic approaches that target the vasculature in the treatment of insulin-resistant muscle.
Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[ 14 C]deoxyglucose to assess tissue-specific glucose uptake and [3-3 H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
Glucagon-like peptide-1 (GLP-1) diminishes postmeal glucose excursions by enhancing insulin secretion via activation of the beta-cell GLP-1 receptor (Glp1r). GLP-1 may also control glucose levels through mechanisms that are independent of this incretin effect. The hyperinsulinemic-euglycemic clamp (insulin clamp) and exercise were used to examine the incretin-independent glucoregulatory properties of the Glp1r because both perturbations stimulate glucose flux independent of insulin secretion. Chow-fed mice with a functional disruption of the Glp1r (Glp1r(-/-)) were compared with wild-type littermates (Glp1r(+/+)). Studies were performed on 5-h-fasted mice implanted with arterial and venous catheters for sampling and infusions, respectively. During insulin clamps, [3-(3)H]glucose and 2[(14)C]deoxyglucose were used to determine whole-body glucose turnover and glucose metabolic index (R(g)), an indicator of glucose uptake. R(g) in sedentary and treadmill exercised mice was determined using 2[(3)H]deoxyglucose. Glp1r(-/-) mice exhibited increased glucose disappearance, muscle R(g), and muscle glycogen levels during insulin clamps. This was not associated with enhanced muscle insulin signaling. Glp1r(-/-) mice exhibited impaired suppression of endogenous glucose production and hepatic glycogen accumulation during insulin clamps. This was associated with impaired liver insulin signaling. Glp1r(-/-) mice became significantly hyperglycemic during exercise. Muscle R(g) was normal in exercised Glp1r(-/-) mice, suggesting that hyperglycemia resulted from an added drive to stimulate glucose production. Muscle AMP-activated protein kinase phosphorylation was higher in exercised Glp1r(-/-) mice. This was associated with increased relative exercise intensity and decreased exercise endurance. In conclusion, these results show that the endogenous Glp1r regulates hepatic and muscle glucose flux independent of its ability to enhance insulin secretion.
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