Laurie J. Goodyear

Role of AMP-activated protein kinase in mechanism of metformin action

Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization. Metformins beneficial effects on circulating lipids have been linked to reduced fatty liver. AMP-activated protein kinase (AMPK) is a major cellular regulator of lipid and glucose metabolism. Here we report that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformins inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.

A Muscle-Specific Insulin Receptor Knockout Exhibits Features of the Metabolic Syndrome of NIDDM without Altering Glucose Tolerance

Skeletal muscle insulin resistance is among the earliest detectable defects in humans with type 2 diabetes mellitus. To determine the contribution of muscle insulin resistance to the metabolic phenotype of diabetes, we used the Cre-loxP system to disrupt the insulin receptor gene in mouse skeletal muscle. The muscle-specific insulin receptor knockout mice exhibit a muscle-specific > 95% reduction in receptor content and early signaling events. These mice display elevated fat mass, serum triglycerides, and free fatty acids, but blood glucose, serum insulin, and glucose tolerance are normal. Thus, insulin resistance in muscle contributes to the altered fat metabolism associated with type 2 diabetes, but tissues other than muscle appear to be more involved in insulin-regulated glucose disposal than previously recognized.

Evidence for 5′AMP-Activated Protein Kinase Mediation of the Effect of Muscle Contraction on Glucose Transport

The intracellular signaling proteins that lead to exercise-stimulated glucose transport in skeletal muscle have not been identified, although it is clear that there are separate signaling mechanisms for exercise- and insulinstimulated glucose transport. We have hypothesized that the 5′AMP-activated protein kinase (AMPK) functions as a signaling intermediary in exercise-stimulated glucose uptake. This hypothesis was based on recent studies showing the following: 1) muscle contraction increases AMPK activity and 2) perfusion of rat hindlimb skeletal muscles with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a compound that results in increased AMPK activity, increased insulin-stimulated glucose uptake. In the current study, isolated rat epitrochlearis muscles were treated to contract in vitro (via electrical stimulation for 10 min) and/or incubated in the absence or presence of AICAR (2 mmol/l), insulin (1 μmol/1), or wortmannin (100 nmolA). Both contraction and AICAR significantly increased AMPK activity, while the enzyme was not activated by insulin. AICAR, contraction, and insulin all increased 3-O-methylglucose (3MG) transport by threefold to fivefold above basal. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin completely blocked insulin-stimulated transport, but did not inhibit AICAR- or contraction-stimulated transport. The increase in glucose transport with the combination of maximal AICAR plus maximal insulin treatments was partially additive, suggesting that these stimuli increase glucose transport by different mechanisms. In contrast, there was no additive effect on glucose transport with the combination of AICAR plus contraction. These data suggest that AICAR and contraction stimulate glucose transport by a similar insulin-independent signaling mechanism and are consistent with the hypothesis that AMPK is involved in exercise-stimulated glucose uptake.

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.

Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects.

To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action.

Brown adipose tissue regulates glucose homeostasis and insulin sensitivity

Brown adipose tissue (BAT) is known to function in the dissipation of chemical energy in response to cold or excess feeding, and also has the capacity to modulate energy balance. To test the hypothesis that BAT is fundamental to the regulation of glucose homeostasis, we transplanted BAT from male donor mice into the visceral cavity of age- and sex-matched recipient mice. By 8-12 weeks following transplantation, recipient mice had improved glucose tolerance, increased insulin sensitivity, lower body weight, decreased fat mass, and a complete reversal of high-fat diet-induced insulin resistance. Increasing the quantity of BAT transplanted into recipient mice further improved the metabolic effects of transplantation. BAT transplantation increased insulin-stimulated glucose uptake in vivo into endogenous BAT, white adipose tissue (WAT), and heart muscle but, surprisingly, not skeletal muscle. The improved metabolic profile was lost when the BAT used for transplantation was obtained from Il6-knockout mice, demonstrating that BAT-derived IL-6 is required for the profound effects of BAT transplantation on glucose homeostasis and insulin sensitivity. These findings reveal a previously under-appreciated role for BAT in glucose metabolism.

Restoring Systemic GDF11 Levels Reverses Age-Related Dysfunction in Mouse Skeletal Muscle

Help the Aged Muscle function declines with age, as does neurogenesis in certain brain regions. Two teams analyzed the effects of heterochronic parabiosis in mice. Sinha et al. (p. 649) found that when an aged mouse shares a circulatory system with a youthful mouse, the aged mouse sees improved muscle function, and Katsimpardi et al. (p. 630) observed increased generation of olfactory neurons. In both cases, Growth Differentiation Factor 11 appeared to be one of the key components of the young blood. A circulating growth factor promotes youthful muscles and brains in aged mice. Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral “rejuvenating” factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.

Muscle-specific PPARγ-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by thiazolidinediones (TZDs) improves insulin resistance by increasing insulin-stimulated glucose disposal in skeletal muscle. It remains debatable whether the effect of TZDs on muscle is direct or indirect via adipose tissue. We therefore generated mice with muscle-specific PPARgamma knockout (MuPPARgammaKO) using Cre/loxP recombination. Interestingly, MuPPARgammaKO mice developed excess adiposity despite reduced dietary intake. Although insulin-stimulated glucose uptake in muscle was not impaired, MuPPARgammaKO mice had whole-body insulin resistance with a 36% reduction (P < 0.05) in the glucose infusion rate required to maintain euglycemia during hyperinsulinemic clamp, primarily due to dramatic impairment in hepatic insulin action. When placed on a high-fat diet, MuPPARgammaKO mice developed hyperinsulinemia and impaired glucose homeostasis identical to controls. Simultaneous treatment with TZD ameliorated these high fat-induced defects in MuPPARgammaKO mice to a degree identical to controls. There was also altered expression of several lipid metabolism genes in the muscle of MuPPARgammaKO mice. Thus, muscle PPARgamma is not required for the antidiabetic effects of TZDs, but has a hitherto unsuspected role for maintenance of normal adiposity, whole-body insulin sensitivity, and hepatic insulin action. The tissue crosstalk mediating these effects is perhaps due to altered lipid metabolism in muscle.

Highly Efficient, Functional Engraftment of Skeletal Muscle Stem Cells in Dystrophic Muscles

Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.

Distinct Signals Regulate AS160 Phosphorylation in Response to Insulin, AICAR, and Contraction in Mouse Skeletal Muscle

Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK α2-inactive (α2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK α2i transgenic mice. Combined AMPK α2 and Akt inhibition by wortmannin treatment of AMPK α2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK α2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.