Jeffrey W. Ryder

5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice

Abstract.Aims/hypothesis: The 5AMP-activated protein kinase is an important mediator of muscle contraction-induced glucose transport and a target for pharmacological treatment of Type II (non-insulin-dependent) diabetes mellitus. The 5AMP-activated protein kinase can be activated by 5-aminoimidazole-4-carboxamide ribonucleoside. We hypothesised that 5-aminoimidazole-4-carboxamide ribonucleoside treatment could restore glucose homeostasis in ob/ob mice. Methods: Lean and ob/ob mice were given 5-aminoimidazole-4-carboxamide ribonucleoside (1 mg · g body wt–1· day–1 s.c) or 0.9 % NaCl (vehicle) for 1–7 days. Results: Short-term 5-aminoimidazole-4-carboxamide ribonucleoside treatment normalised glucose concentrations in ob/ob mice within 1 h, with effects persisting over 4 h. After 1 week of daily injections, 5-aminoimidazole-4-carboxamide ribonucleoside treatment corrected hyperglycaemia, improved glucose tolerance, and increased GLUT4 and hexokinase II protein expression in skeletal muscle, but had deleterious effects on plasma non-esterified fatty acids and triglycerides. Treatment with 5-aminoimidazole-4-carboxamide ribonucleoside increased liver glycogen in fasted and fed ob/ob mice and muscle glycogen in fasted, but not fed ob/ob and lean mice. Defects in insulin-stimulated phosphatidylinositol 3-kinase and glucose transport in skeletal muscle from ob/ob mice were not corrected by 5-aminoimidazole-4-carboxamide ribonucleoside treatment. While ex vivo insulin-stimulated glucose transport was reduced in isolated muscle from ob/ob mice, the 5-aminoimidazole-4-carboxamide ribonucleoside stimulated response was normal. Conclusion/interpretation: The 5-aminoimidazole-4-carboxamide ribonucleoside mediated improvements in glucose homeostasis in ob/ob mice can be explained by effects in skeletal muscle and liver. Due to the apparently deleterious effects of 5-aminoimidazole-4-carboxamide ribonucleoside on the blood lipid profile, strategies to develop tissue-specific and pathway-specific activators of 5AMP-activated protein kinase should be considered in order to improve glucose homeostasis. [Diabetologia (2002) 45: 56–65]

Effect of Contraction on Mitogen-activated Protein Kinase Signal Transduction in Skeletal Muscle INVOLVEMENT OF THE MITOGEN- AND STRESS-ACTIVATED PROTEIN KINASE 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38MAPK phosphorylation. Phosphorylation of ERK1/2 or p38MAPK was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38MAPK was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38MAPKinhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90Rsk), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90Rsk and MAPKAP-K2 most closely reflects ERK and p38MAPK stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38MAPK. These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.

Muscle fiber type specificity in insulin signal transduction

We determined the muscle fiber type-specific response of intracellular signaling proteins to insulin. Epitrochlearis (Epi; 15% type I, 20% type IIa, and 65% type IIb), soleus (84, 16, and 0%), and extensor digitorum longus (EDL; 3, 57, and 40%) muscles from Wistar rats were incubated without or with 120 nM insulin (3-40 min). Peak insulin receptor (IR) tyrosine phosphorylation was reached after 6 (soleus) and 20 (Epi and EDL) min, with sustained activity throughout insulin exposure (40 min). Insulin increased insulin receptor substrate (IRS)-1 and IRS-2 tyrosine phosphorylation and phosphotyrosine-associated phosphatidylinositol (PI)-3-kinase activity to a maximal level after 3-10 min, with subsequent downregulation. Akt kinase phosphorylation peaked at 20 min, with sustained activity throughout insulin exposure. Importantly, the greatest insulin response for all signaling intermediates was observed in soleus, whereas the insulin response between EDL and Epi was similar. Protein expression of the p85α-subunit of PI 3-kinase and Akt kinase, but not IR, IRS-1, or IRS-2, was greater in oxidative versus glycolytic muscle. In conclusion, increased function and/or expression of key proteins in the insulin-signaling cascade contribute to fiber type-specific differences in insulin action in skeletal muscle.We determined the muscle fiber type-specific response of intracellular signaling proteins to insulin. Epitrochlearis (Epi; 15% type I, 20% type IIa, and 65% type IIb), soleus (84, 16, and 0%), and extensor digitorum longus (EDL; 3, 57, and 40%) muscles from Wistar rats were incubated without or with 120 nM insulin (3-40 min). Peak insulin receptor (IR) tyrosine phosphorylation was reached after 6 (soleus) and 20 (Epi and EDL) min, with sustained activity throughout insulin exposure (40 min). Insulin increased insulin receptor substrate (IRS)-1 and IRS-2 tyrosine phosphorylation and phosphotyrosine-associated phosphatidylinositol (PI)-3-kinase activity to a maximal level after 3-10 min, with subsequent downregulation. Akt kinase phosphorylation peaked at 20 min, with sustained activity throughout insulin exposure. Importantly, the greatest insulin response for all signaling intermediates was observed in soleus, whereas the insulin response between EDL and Epi was similar. Protein expression of the p85alpha-subunit of PI 3-kinase and Akt kinase, but not IR, IRS-1, or IRS-2, was greater in oxidative versus glycolytic muscle. In conclusion, increased function and/or expression of key proteins in the insulin-signaling cascade contribute to fiber type-specific differences in insulin action in skeletal muscle.

Skeletal Muscle Reprogramming by Activation of Calcineurin Improves Insulin Action on Metabolic Pathways

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow-versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-γ co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4‐deficient and wild‐type mice were studied aftera3h swim exercise. In wild‐type mice, insulin and swimming each increased 2‐deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2‐deoxyglucose glucose uptake in muscle from GLUT4‐null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4‐null mice, with no effect noted in fasted GLUT4‐null mice. This exercise‐associated 2‐deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4‐null muscle was increased 1.6‐fold over basal levels after electrical stimulation. Contraction‐induced glucose transport activity was fourfold greater in wild‐type vs. GLUT4‐null muscle. Glycogen content in gastrocnemius muscle was similar between wild‐type and GLUT4‐null mice and was reduced ~50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild‐type, with no change in GLUT4‐null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4‐null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise‐induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild‐type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4‐null mice were totally restored after 24 h carbohydrate refeeding.—Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg‐Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4‐deficient mice. FASEB J. 13, 2246–2256 (1999)

Insulin- and Glucose-Induced Phosphorylation of the Na+,K+-Adenosine Triphosphatase α-Subunits in Rat Skeletal Muscle

Phosphorylation of the α-subunits of Na+,K+-adenosine triphosphatase in response to insulin, high extracellular glucose concentration, and phorbol 12-myristate 13-acetate was investigated in isolated rat soleus muscle. All three stimuli increased α-subunit phosphorylation approximately 3-fold. Phorbol 12-myristate 13-acetate- and high glucose-induced phosphorylation of the α-subunit was completely abolished by the PKC inhibitor GF109203X, whereas insulin-stimulated phosphorylation was only partially reduced. Notably, insulin stimulation resulted in phosphorylation of the α-subunit on serine, threonine, and tyrosine residues, whereas high extracellular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosphorylation only on serine and threonine residues. Insulin stimulation resulted in translocation of Na+,K+-adenosine triphosphatase α2-subunit to the plasma membrane and increased Na+,K+-adenosine triphosphatase activity in the same membrane fraction. High glucose had no effect onα -subunits...

Neuregulins mediate calcium-induced glucose transport during muscle contraction.

Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction.

Changes in glucose transport and protein kinase Cβ2 in rat skeletal muscle induced by hyperglycaemia

Aims/hypothesis. We have previously reported that hyperglycaemia activates glucose transport in skeletal muscle by a Ca2+-dependent pathway, which is distinct from the insulin-signalling pathway. The aim of this study was to explain the signalling mechanism by which hyperglycaemia autoregulates glucose transport in skeletal muscle. Methods. Isolated rat soleus muscle was incubated in the presence of various concentrations of glucose or 3-O-methylglucose and protein kinase C and phospholipase C inhibitors. Glucose transport activity, cell surface glucose transporter 1 and glucose transporter 4 content and protein kinase C translocation was determined. Results. High concentrations of 3-O-methylglucose led to a concentration-dependent increase in [3H]-3-O-methylglucose transport in soleus muscle. Dantrolene, an inhibitor of Ca2+ released from the sarcoplasmic reticulum, decreased the Vmax and the Km of the concentration-response curve. Protein kinase C inhibitors (H-7 and GF109203X) inhibited the stimulatory effect of high glucose concentrations on hexose transport, whereas glucose transport stimulated by insulin was unchanged. Incubation of muscle with glucose (25 mmol/l) and 3-O-methylglucose (25 mmol/l) led to a three fold gain in protein kinase Cβ2 in the total membrane fraction, whereas membrane content of protein kinase Cα, β1, δ, ɛ and ϑ were unchanged. A short-term increase in the extracellular glucose concentration did not change cell surface recruitment of glucose transporter 1 or glucose transporter 4, as assessed by exofacial photolabelling with [3H]-ATB-BMPA bis-mannose. Conclusion/interpretation. Protein kinase Cβ2 is involved in a glucose-sensitive, Ca2+-dependent signalling pathway, which is possibly involved in the regulation of glucose transport in skeletal muscle. This glucose-dependent increase in 3-0-methylglucose transport is independent of glucose transporter 4 and glucose transporter 1 translocation to the plasma membrane and may involve modifications of cell surface glucose transporter activity. [Diabetologia (1999) 42: 1071–1079]

Restoration of Hypoxia-stimulated Glucose Uptake in GLUT4-deficient Muscles by Muscle-specific GLUT4 Transgenic Complementation

To investigate whether GLUT4 is required for exercise/hypoxia-induced glucose uptake, we assessed glucose uptake under hypoxia and normoxia in extensor digitorum longus (EDL) and soleus muscles from GLUT4-deficient mice. In EDL and soleus from wild type control mice, hypoxia increased 2-deoxyglucose uptake 2–3-fold. Conversely, hypoxia did not alter 2-deoxyglucose uptake in either EDL or soleus from either male or female GLUT4-null mice. Next we introduced the fast-twitch skeletal muscle-specific MLC-GLUT4 transgene into GLUT4-null mice to determine whether changes in the metabolic milieu accounted for the lack of hypoxia-mediated glucose transport. Transgenic complementation of GLUT4 in EDL was sufficient to restore hypoxia-mediated glucose uptake. Soleus muscles from MLC-GLUT4-null mice were transgene-negative, and hypoxia-stimulated 2-deoxyglucose uptake was not restored. Although ablation of GLUT4 in EDL did not affect normoxic glycogen levels, restoration of GLUT4 to EDL led to an increase in glycogen under hypoxic conditions. Male GLUT4-null soleus displayed reduced normoxic glycogen stores, but female null soleus contained significantly more glycogen under normoxia and hypoxia. Reduced normoxic levels of ATP and phosphocreatine were measured in male GLUT4-null soleus but not in EDL. However, transgenic complementation of GLUT4 prevented the decrease in hypoxic ATP and phosphocreatine levels noted in male GLUT4-null and control EDL. In conclusion, we have demonstrated that GLUT4 plays an essential role in the regulation of muscle glucose uptake in response to hypoxia. Because hypoxia is a useful model for exercise, our results suggest that stimulation of glucose transport in response to exercise in skeletal muscle is totally dependent upon GLUT4. Furthermore, the compensatory glucose transport system that exists in GLUT4-null soleus muscle is not sensitive to hypoxia/muscle contraction.

Effects of calcineurin activation on insulin‐, AICAR‐ and contraction‐induced glucose transport in skeletal muscle

Skeletal muscle is composed of fast‐ and slow‐twitch fibres with distinctive physiological and metabolic properties. The calmodulin‐activated serine/threonine protein phosphatase calcineurin activates fast‐ to slow‐twitch skeletal muscle remodelling through the induction of the slow‐twitch skeletal muscle fibre gene expression programme, thereby enhancing insulin‐stimulated glucose uptake and offering protection against dietary‐induced insulin resistance. Given the profound influence of skeletal muscle fibre type on insulin‐mediated responses, we determined whether the fast‐ to slow‐twitch fibre‐type transformation leads to alterations in insulin‐independent glucose uptake in transgenic mice expressing a constitutively active form of calcineurin (MCK‐CnA* mice). We determined whether skeletal muscle remodelling by activated calcineurin alters glucose transport in response to the AMP‐activated protein kinase (AMPK) activator 5‐aminoimidazole‐4‐carboxamide‐β‐d‐ribofuranoside (AICAR) or muscle contraction, two divergent insulin‐independent activators of glucose transport. While insulin‐stimulated glucose transport was increased 52%, the AICAR effect on glucose transport was 27% lower in MCK‐CnA* mice versus wild‐type mice (P < 0.05). In contrast, glucose transport was similar between genotypes after in vitro muscle contraction. Fibre‐type transformation was associated with increased AMPKγ1, decreased AMPKγ3 and unchanged AMPKγ2 protein expression between MCK‐CnA* and wild‐type mice (P < 0.05). The loss of AICAR‐mediated glucose uptake is coupled to changes in the AMPK isoform expression, suggesting fibre‐type dependence of the AICAR responses on glucose uptake. In conclusion, improvements in skeletal muscle glucose transport in response to calcineurin‐induced muscle remodelling are limited to insulin action.