Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling. Am J Physiol Endocrinol Metab 290: E154-E162, 2006. First published August 23, 2005; doi:10.1152/ajpendo. 00330.2005.-Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser645, Ser649, Ser653, Ser657) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. Insulin resistance is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane: Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma ), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.

Effect of fatty acids, glucose, and insulin on hepatic glucose uptake and glycolysis

Objective:. There is evidence from in vitro studies that fatty acids can inhibit glucose uptake in liver. However, it is uncertain whether this happens in vivo when the liver is exposed to high levels of glucose and insulin, in combination with fatty acids, after a mixed meal. This study determined the effects of a combination of fatty acids and insulin on glucokinase (GK) activity and glycolysis in primary rat hepatocytes. Methods: Hepatocytes were cultured with 15 mM glucose and 2 or 10 nM insulin in combination with the fatty acids palmitate, oleate, linoleate, eicosapentaenoic acid, or docosahexaenoic acid. Total GK activity and the proportion of GK in the,active, unbound state were measured to determine the effect of fatty acid on the activity and cellular localization of GK. Glucose phosphorylation and glycolysis were measured in intact cells. Lactate and pyruvate synthesis and the accumulation of ketone bodies were also estimated. Results: Palmitate and eicosapentaenoic acid lowered total GK activity in the presence of 2 nM insulin, but not with 10 nM insulin. In contrast, oleate, linoleate, and docosahexaenoic acid did not alter GK activity. None of the fatty acids tested inhibited glucose phosphorylation or glycolysis in intact rat hepatocytes. In addition, GK activity was unaffected by insulin concentration. Conclusion: Some fatty acids can act to inhibit GK activity in primary hepatocytes. However, there was no,evidence that this decrease in GK activity impaired glucose phosphorylation or glycolysis. Glucose and high concentrations of insulin, which promote glucose uptake, appear to counteract any inhibitory action of fatty acids. Therefore, the presence of fatty acids in a normal mixed meal is likely to have little effect on the capacity of the liver to take up, phosphorylate, and oxidize glucose. (C) 2006 Elsevier Inc. All rights reserved.

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 mum nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 mum nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 mum) significantly inhibited basal and insulin-stimulated glucose uptake in adi. pocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 muM nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 muM nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degreesC. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Skeletal muscle and nuclear hormone receptors: Implications for cardiovascular and metabolic disease

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of the total body mass and a major player in energy balance. It accounts for > 30% of energy expenditure, is the primary tissue of insulin stimulated glucose uptake, disposal, and storage. Furthermore, it influences metabolism via modulation of circulating and stored lipid (and cholesterol) flux. Lipid catabolism supplies up to 70% of the energy requirements for resting muscle. However, initial aerobic exercise utilizes stored muscle glycogen but as exercise continues, glucose and stored muscle triglycerides become important energy substrates. Endurance exercise increasingly depends on fatty acid oxidation (and lipid mobilization from other tissues). This underscores the importance of lipid and glucose utilization as an energy source in muscle. Consequently skeletal muscle has a significant role in insulin sensitivity, the blood lipid profile, and obesity. Moreover, caloric excess, obesity and physical inactivity lead to skeletal muscle insulin resistance, a risk factor for the development of type II diabetes. In this context skeletal muscle is an important therapeutic target in the battle against cardiovascular disease, the worlds most serious public health threat. Major risk factors for cardiovascular disease include dyslipidemia, hypertension, obesity, sedentary lifestyle, and diabetes. These risk factors are directly influenced by diet, metabolism and physical activity. Metabolism is largely regulated by nuclear hormone receptors which function as hormone regulated transcription factors that bind DNA and mediate the pathophysiological regulation of gene expression. Metabolism and activity, which directly influence cardiovascular disease risk factors, are primarily driven by skeletal muscle. Recently, many nuclear receptors expressed in skeletal muscle have been shown to improve glucose tolerance, insulin resistance, and dyslipidernia. Skeletal muscle and nuclear receptors are rapidly emerging as critical targets in the battle against cardiovascular disease risk factors. Understanding the function of nuclear receptors in skeletal muscle has enormous pharmacological utility for the treatment of cardiovascular disease. This review focuses on the molecular regulation of metabolism by nuclear receptors in skeletal muscle in the context of dyslipidemia and cardiovascular disease. (c) 2005 Published by Elsevier Ltd.

Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, som: of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected P-32-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

GLUT4 - At the cross roads between membrane trafficking and signal transduction

GLUT4 is a mammalian facilitative glucose transporter that is highly expressed in adipose tissue and striated muscle. In response to insulin, GLUT4 moves from intracellular storage areas to the plasma membrane, thus increasing cellular glucose uptake. While the verification of this 'translocation hypothesis' (Cushman SW. Wardzala LJ. J Biol Chem 1980;255: 4758-4762 and Suzuki K, Kono T. Proc Natl Acad Sci 1980;77: 2542-2545) has increased our understanding of insulin-regulated glucose transport, a number of fundamental questions remain unanswered. Where is GLUT4 stored within the basal cell? How does GLUT4 move to the cell surface and what mechanism does insulin employ to accelerate this process) Ultimately we require a convergence of trafficking studies with research in signal transduction. However, despite more than 30 years of intensive research we have still not reached this point. The problem is complex, involving at least two separate signal transduction pathways which feed into what appears to be a very dynamic sorting process. Below we discuss some of these complexities and highlight new data that are bringing us closer to the resolution of these questions.

Aging per se does not influence glucose homeostasis - In vivo and in vitro evidence

OBJECTIVE - To assess the effect of age on glucose metabolism by examining 1) glucose metabolism in young and middle-aged subjects when total or regional adiposity is taken into account and 2) in vitro glucose transport in adipose tissue explants from young and middle-aged women paired for total and abdominal adiposity. RESEARCH DESIGN AND METHODS - Study 1: body composition, subcutaneous abdominal and visceral adipose tissue areas, and fasting and oral glucose-stimulated glucose and insulin were measured in 84 young and 81 middle-aged men and in 110 young and 91 middle-aged women. Study 2: glucose uptake in subcutaneous abdominal and visceral adipose tissue explants were measured in eight young and eight middle-aged women. RESULTS - Study 1: young and middle-aged men showed similar subcutaneous abdominal tissue area, whereas fat mass and visceral adipose tissue were greater in middle-aged than in young men (P < 0.01). Fat mass and subcutaneous and visceral adipose tissue areas were greater in middle-aged as compared with young women (P < 0.01). Fasting plasma glucose and the glucose response to an oral glucose tolerance test were significantly higher in middle-aged than in young men and women (P < 0.001). Statistical control for visceral adipose tissue area eliminated the difference seen in glucose response in men and women. Study 2: glucose transport in subcutaneous and omental adipose tissue did not differ between young and middle-aged women. CONCLUSIONS - 1) Visceral obesity, more than age per se, correlates with glucose intolerance in middle-aged subjects; 2) aging does not influence in vitro adipose tissue glucose uptake.

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro

Reasons for performing study: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. Objectives: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. Methods: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). Results: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. Conclusions: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). Potential relevance: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.