Freyja D. James

The Glucagon-Like Peptide-1 Receptor Regulates Endogenous Glucose Production and Muscle Glucose Uptake Independent of Its Incretin Action

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.

Diet-Induced Muscle Insulin Resistance Is Associated With Extracellular Matrix Remodeling and Interaction With Integrin α2β1 in Mice

OBJECTIVE The 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 METHODS The 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. RESULTS HF-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. CONCLUSIONS ECM 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.

Endothelial nitric oxide synthase is central to skeletal muscle metabolic regulation and enzymatic signaling during exercise in vivo.

Endothelial nitric oxide synthase (eNOS) is associated with a number of physiological functions involved in the regulation of metabolism; however, the functional role of eNOS is poorly understood. We tested the hypothesis that eNOS is critical to muscle cell signaling and fuel usage during exercise in vivo, using 16-wk-old catheterized (carotid artery and jugular vein) C57BL/6J mice with wild-type (WT), partial (+/-), or no expression (-/-) of eNOS. Quantitative reductions in eNOS expression ( approximately 40%) elicited many of the phenotypic effects observed in enos(-/-) mice under fasted, sedentary conditions, with expression of oxidative phosphorylation complexes I to V and ATP levels being decreased, and total NOS activity and Ca(2+)/CaM kinase II Thr(286) phosphorylation being increased in skeletal muscle. Despite these alterations, exercise tolerance was markedly impaired in enos(-/-) mice during an acute 30-min bout of exercise. An eNOS-dependent effect was observed with regard to AMP-activated protein kinase signaling and muscle perfusion. Muscle glucose and long-chain fatty acid uptake, and hepatic and skeletal muscle glycogenolysis during the exercise bout was markedly accelerated in enos(-/-) mice compared with enos(+/-) and WT mice. Correspondingly, enos(-/-) mice exhibited hypoglycemia during exercise. Thus, the ablation of eNOS alters a number of physiological processes that result in impaired exercise capacity in vivo. The finding that a partial reduction in eNOS expression is sufficient to induce many of the changes associated with ablation of eNOS has implications for chronic metabolic diseases, such as obesity and insulin resistance, which are associated with reduced eNOS expression.

Muscle-Specific Vascular Endothelial Growth Factor Deletion Induces Muscle Capillary Rarefaction Creating Muscle Insulin Resistance

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.

Glucagon-Like Peptide-1 Receptor Knockout Mice Are Protected from High-Fat Diet-Induced Insulin Resistance

Glucagon-like peptide-1 augments nutrient-stimulated insulin secretion. Chow-fed mice lacking the glucagon-like peptide-1 receptor (Glp1r) exhibit enhanced insulin-stimulated muscle glucose uptake but impaired suppression of endogenous glucose appearance (endoRa). This proposes a novel role for the Glp1r to regulate the balance of glucose disposal in muscle and liver by modulating insulin action. Whether this is maintained in an insulin-resistant state is unknown. The present studies tested the hypothesis that disruption of Glp1r expression overcomes high-fat (HF) diet-induced muscle insulin resistance and exacerbates HF diet-induced hepatic insulin resistance. Mice with a functional disruption of the Glp1r (Glp1r-/-) were compared with wild-type littermates (Glp1r+/+) after 12 wk on a regular chow diet or a HF diet. Arterial and venous catheters were implanted for sampling and infusions. Hyperinsulinemic-euglycemic clamps were performed on weight-matched male mice. [3-(3)H]glucose was used to determine glucose turnover, and 2[14C]deoxyglucose was used to measure the glucose metabolic index, an indicator of glucose uptake. Glp1r-/- mice exhibited increased glucose disappearance and muscle glucose metabolic index on either diet. This was associated with enhanced activation of muscle Akt and AMP-activated protein kinase and reduced muscle triglycerides in HF-fed Glp1r-/- mice. Chow-fed Glp1r-/- mice exhibited impaired suppression of endoRa and hepatic insulin signaling. In contrast, HF-fed Glp1r-/- mice exhibited improved suppression of endoRa and hepatic Akt activation. This was associated with decreased hepatic triglycerides and impaired activation of sterol regulatory element-binding protein-1. These results show that mice lacking the Glp1r are protected from HF diet-induced muscle and hepatic insulin resistance independent of effects on total fat mass.

SIRT3 is crucial for maintaining skeletal muscle insulin action and protects against severe insulin resistance in high fat fed mice

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.

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) Effect on Glucose Production, but Not Energy Metabolism, Is Independent of Hepatic AMPK in Vivo

Background: AMPK is implicated as the mediator of AICAR action on liver metabolism. Results: AICAR suppresses glucose production independent of AMPK. Regulation of mitochondrial function is AMPK-dependent. Conclusion: Nucleotide monophosphates rely on AMPK to regulate energy metabolism but not to suppress glucose production. Significance: Targeted AMPK activation will not lower glucose production in metabolic diseases but could improve hepatic energetics. Metabolic stress, as well as several antidiabetic agents, increases hepatic nucleotide monophosphate (NMP) levels, activates AMP-activated protein kinase (AMPK), and suppresses glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 8 mg·kg−1·min−1)-euglycemic clamps were performed to elicit an increase in NMP in wild type (α1α2lox/lox) and liver-specific AMPK knock-out mice (α1α2lox/lox + Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5-h fasted α1α2lox/lox and α1α2lox/lox + Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose disappearance. These results demonstrate that AMPK is unnecessary for normal 5-h fasting glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK energy sensor paradigm. AICAR reduced hepatic energy charge by ∼20% in α1α2lox/lox, which was exacerbated by ∼2-fold in α1α2lox/lox + Albcre. This corresponded to a ∼6-fold rise in AMP/ATP in α1α2lox/lox + Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ∼30% lower in α1α2lox/lox + Albcre than α1α2lox/lox livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration.

Relaxin Treatment Reverses Insulin Resistance in Mice Fed a High-Fat Diet

The endogenous hormone relaxin increases vascular reactivity and angiogenesis. We demonstrate that acute relaxin infusion in lean C57BL/6J mice enhances skeletal muscle perfusion and augments muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. However, an acute effect was absent in mice fed a high-fat (HF) diet for 13 weeks. In contrast, mice fed an HF diet for 13 weeks and continuously treated with relaxin for the final 3 weeks of the diet exhibited decreased fasting blood glucose. Insulin-stimulated whole-body glucose disappearance and percent suppression of hepatic glucose production are corrected by chronic relaxin. The increase in peripheral glucose utilization is a result of augmented in vivo skeletal muscle glucose uptake. Relaxin intervention improves endothelial-dependent vascular reactivity and induces a two-fold proliferation in skeletal muscle capillarity. The metabolic effects of the treatment are not attributed to changes in myocellular insulin signaling. Relaxin intervention reverses the accumulation of collagen III in the liver and collagen III and collagen IV in the heart; this is induced by HF feeding. These studies show the potential of relaxin in the treatment of diet-induced insulin resistance and vascular dysfunction. Relaxin provides a novel therapeutic approach targeting the extramyocellular barriers to insulin action, which are critical to the pathogenesis of insulin resistance.

Heterozygous SOD2 Deletion Impairs Glucose-Stimulated Insulin Secretion, but Not Insulin Action, in High-Fat–Fed Mice

Elevated reactive oxygen species (ROS) are linked to insulin resistance and islet dysfunction. Manganese superoxide dismutase (SOD2) is a primary defense against mitochondrial oxidative stress. To test the hypothesis that heterozygous SOD2 deletion impairs glucose-stimulated insulin secretion (GSIS) and insulin action, wild-type (sod2+/+) and heterozygous knockout mice (sod2+/−) were fed a chow or high-fat (HF) diet, which accelerates ROS production. Hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI) clamps were performed to assess GSIS and insulin action in vivo. GSIS during HG clamps was equal in chow-fed sod2+/− and sod2+/+ but was markedly decreased in HF-fed sod2+/−. Remarkably, this impairment was not paralleled by reduced HG glucose infusion rate (GIR). Decreased GSIS in HF-fed sod2+/− was associated with increased ROS, such as superoxide ion. Surprisingly, insulin action determined by HI clamps did not differ between sod2+/− and sod2+/+ of either diet. Since insulin action was unaffected, we hypothesized that the unchanged HG GIR in HF-fed sod2+/− was due to increased glucose effectiveness. Increased GLUT-1, hexokinase II, and phospho-AMPK protein in muscle of HF-fed sod2+/− support this hypothesis. We conclude that heterozygous SOD2 deletion in mice, a model that mimics SOD2 changes observed in diabetic humans, impairs GSIS in HF-fed mice without affecting insulin action.

Mass spectrometry-based microassay of 2H and 13C plasma glucose labeling to quantify liver metabolic fluxes in vivo

Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.