Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.     

Effect of Diabetes on Glycogen Metabolism in Rat Retina

Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.     

Chronic Treatment with the AMP-Kinase Activator AICAR Increases Glycogen Storage and Fatty Acid Oxidation in Skeletal Muscles but Does Not Reduce Hyperglucagonemia and Hyperglycemia in Insulin Deficient Rats

This study tested whether the glycogen-accumulating effect of chronic in vivo pharmacological 5′AMP-activated protein kinase (AMPK) activation could improve glycemic control under conditions of insulin deficiency. Male Wistar rats were rendered diabetic through the administration of streptozotocin (STZ) and then treated for 7 consecutive days with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Subsequently, glycogen content and synthesis, glucose oxidation, and fatty acid oxidation (FAO) were determined in oxidative and glycolytic skeletal muscles. Glycemia, insulinemia, glucagonemia, and circulating triglycerides (TG) and non-esterified fatty acids (NEFAs) were measured after AICAR treatment. Insulin was almost undetectable in STZ rats and these animals were severely hyperglycemic. Glycogen content was markedly low mainly in glycolytic muscles of STZ rats and AICAR treatment restored it to control values. No differences were found among all muscles studied with regards to the content and phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3. Even though glycogen synthase content was reduced in all muscles from STZ rats, insulin-induced dephosphorylation/activation of this enzyme was preserved and unaffected by AICAR treatment. Glucagon and NEFAS were 2- and 7.4-fold fold higher in STZ rats than controls, respectively. AICAR did not affect hyperglycemia and hyperglucagonemia in STZ rats; however, it normalized circulating NEFAs and significantly increased FAO in glycolytic muscles. In conclusion, even though AICAR-induced AMPK activation enhanced glycogen accumulation in glycolytic muscles and normalized circulating NEFAs and TG levels, the hyperglycemic effects of glucagon likely offset the potentially glucose-lowering effects of AICAR, resulting in no improvement of glycemic control in insulin-deficient rats.     

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rate is blocked by protein synthesis inhibitor

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3′,5′-monosphosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity.In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.     

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rats is blocked by protein synthesis inhibitor.

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen, increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3',5'-monophosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity. In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.     

Oral treatment with vanadium of Zucker fatty rats activates muscle glycogen synthesis and insulin-stimulated protein phosphatase-1 activity

Since the glucose-lowering effects of vanadium could be related to increased muscle glycogen synthesis, we examined the in vivo effects of vanadium and insulin treatment on glycogen synthase (GS) activation in Zucker fatty rats. The GS fractional activity (GSFA), protein phosphatase-1 (PP1), and glycogen synthase kinase-3 (GSK-3) activity were determined in fatty and lean rats following treatment with bis(maltolato)oxovanadium(IV) (BMOV) for 3 weeks (0.2 mmol/kg/day) administered in drinking water. Skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). In both lean and fatty rats, muscle GSFA was significantly increased at 15 min following insulin stimulation. Vanadium treatment resulted in decreased insulin levels and improved insulin sensitivity in the fatty rats. Interestingly, this treatment stimulated muscle GSFA by 2-fold (p < 0.05) and increased insulin-stimulated PP1 activity by 77% (p < 0.05) in the fatty rats as compared to untreated rats. Insulin resistance, vanadium and insulin in vivo treatment did not affect muscle GSK-3 activity in either fatty or lean rats. Therefore, an impaired insulin sensitivity in the Zucker fatty rats was improved following vanadium treatment, resulting in an enhanced muscle glucose metabolism through increased GS and insulin-stimulated PP1 activity.     

The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase.

Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.     

Insulin-regulated protein kinases during postnatal development of rat heart

The control of glucose uptake and glycogen metabolism by insulin in target organs is in part mediated through the regulation of protein-serine/ threonine kinases. In this study, the expression and phosphotransferase activity levels of some of these kinases in rat heart ventricle were measured to investigate whether they might mediate the shift in the energy dependency of the developing heart from glycogen to fatty acids. Following tail-vein injection of overnight fasted adult rats with 2 U of insulin per kg body weight, protein kinase B (PKB), the 70-kDa ribosomal S6 kinase (S6K), and casein kinase 2 (CK2) were activated (30–600%), whereas the MAP/ extracellular regulated kinases (ERK)1 and ERK2 were not stimulated under these conditions. When the expression levels of the insulin-activated kinases were probed with specific antibodies in ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats, phosphatidylinositol 3-kinase (PI3K), PKB, S6K, and CK2 were downregulated (40–60%) with age. By contrast, ventricular glycogen synthase kinase-3β (GSK3β) protein levels were maintained during postnatal development. Similar findings were obtained when the expression of these kinases was investigated in freshly isolated ventricular myocytes, where they were detected predominantly in the cytosolic fraction of the myocytes. Compared to other adult rat tissues such as brain and liver, the levels of PI3K, PKB, S6K, and GSK3β were relatively low in the heart. Even though CK2 protein and activity levels were reduced by ∼60% in 365 day as compared to 1-day-old rats, expression of CK2 in the adult heart was as high as detected in any of the other rat tissues. The high basal activities of CK2 in early neonatal heart may be associated with the proliferating state of myocytes. J. Cell. Biochem. 71:328–339, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats

A role for elevated glycogen synthase kinase-3 (GSK-3) activity in the multifactorial etiology of insulin resistance is now emerging. However, the utility of specific GSK-3 inhibition in modulating insulin resistance of skeletal muscle glucose transport is not yet fully understood. Therefore, we assessed the effects of novel, selective organic inhibitors of GSK-3 (CT-98014 and CT-98023) on glucose transport in insulin-resistant muscles of Zucker diabetic fatty (ZDF) rats. Incubation of type IIb epitrochlearis and type I soleus muscles from ZDF rats with CT-98014 increased glycogen synthase activity (49 and 50%, respectively, P < 0.05) but did not alter basal glucose transport (2-deoxyglucose uptake). In contrast, CT-98014 significantly increased the stimulatory effects of both submaximal and maximal insulin concentrations in epitrochlearis (37 and 24%) and soleus (43 and 26%), and these effects were associated with increased cell-surface GLUT4 protein. Lithium enhanced glycogen synthase activity and both basal and insulin-stimulated glucose transport in muscles from ZDF rats. Acute oral administration (2 x 30 mg/kg) of CT-98023 to ZDF rats caused elevations in GSK-3 inhibitor concentrations in plasma and muscle. The glucose and insulin responses during a subsequent oral glucose tolerance test were reduced by 26 and 34%, respectively, in the GSK-3 inhibitor-treated animals. Thirty minutes after the final GSK-3 inhibitor treatment, insulin-stimulated glucose transport was significantly enhanced in epitrochlearis (57%) and soleus (43%). Two hours after the final treatment, insulin-mediated glucose transport was still significantly elevated (26%) only in the soleus. These results indicate that specific inhibition of GSK-3 enhances insulin action on glucose transport in skeletal muscle of the insulin-resistant ZDF rat. This unique approach may hold promise as a pharmacological treatment against insulin resistance of skeletal muscle glucose disposal.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Role of the PDK1-PKB-GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart

In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.     

Changes of glycogen content in liver,skeletal muscle,and heart from fasted rats

Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non‐esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h‐fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase‐3 (GSK‐3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK‐3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin‐stimulated phosphorylation of Akt and GSK‐3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK‐3 phosphorylation and glycogen content are decreased in liver and skeletal muscles, but in the heart it remain unchanged (Akt and GSK‐3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo effects of vanadate on hepatic glycogen metabolizing and lipogenic enzymes in insulin-dependent and insulin-resistant diabetic animals

The insulin-mimetic action of vanadate is well established but the exact mechanism by which it exerts this effect is still not clearly understood. The role of insulin in the regulation of hepatic glycogen metabolizing and lipogenic enzymes is well known. In our study, we have, therefore, examined the effects of vanadate on these hepatic enzymes using four different models of diabetic and insulin-resistant animals. Vanadate normalized the blood glucose levels in all animal models. In streptozotocin-induced diabetic rats, the amount of liver glycogen and the activities of the active-form of glycogen synthase, both active and inactive-forms of phosphorylase, and lipogenic enzymes like glucose 6-phosphate dehydrogenase and malic enzyme were decreased and vanadate treatment normalized all of these to near normal levels. The other three animal models (db/db mouse, sucrose-fed rats and fa/fa obese Zucker rats) were characterized by hyperinsulinemia, hypertriglyceridemia, increases in activities of lipogenic enzymes, and marginal changes in glycogen metabolizing enzymes. Vanadate treatment brought all of these values towards normal levels. It should be noted that vanadate shows differential effects in the modulation of lipogenic enzymes activities in type I and type II diabetic animals. It increases the activities of lipogenic enzymes in streptozotocin-induced diabetic animals and prevents the elevation of activities of these enzymes in hyperinsulinemic animals. The insulin-stimulated phosphorylation of insulin receptor subunit and its tyrosine kinase activity was increased in streptozotocin-induced diabetic rats after treatment with vanadate. Our results support the view that insulin receptor is one of the sites involved in the insulin-mimetic actions of vanadate.     

Activation and inactivation of glycogen phosphorylase isoenzymes purified from diabetic rat heart

1. Hearts of diabetic rats gradually accumulate glycogen, although the activities of glycogen synthase and glycogen phosphorylase are altered in favor of a depletion of glycogen. 2. Phosphorylase in diabetic hearts has been reported to be even more activated in response to adrenaline than controls. 3. The situation is further complicated by the fact that in rat heart two isoenzymes of phosphorylase are present. Therefore we have studied the properties of phosphorylases purified from diabetic rat heart in more detail. 4. This investigation revealed that compared to controls: (A) the amount of enzyme protein which could be isolated from diabetic animals is drastically lower; (B) the affinities towards glycogen and inorganic phosphate are decreased; (C) the activation by phosphorylase kinase is delayed; and (D) the inactivation by protein phosphatase-1 is accelerated. 5. We conclude that all of the reported changes in diabetes might contribute to a phosphorylase system less able to catalyze glycogen breakdown effectively.     

Chronic selective glycogen synthase kinase-3 inhibition enhances glucose disposal and muscle insulin action in prediabetic obese Zucker rats

Increasing evidence supports a negative role of glycogen synthase kinase-3 (GSK-3) in regulation of skeletal muscle glucose transport. We assessed the effects of chronic treatment of insulin-resistant, prediabetic obese Zucker (fa/fa) rats with a highly selective GSK-3 inhibitor (CT118637) on glucose tolerance, whole body insulin sensitivity, plasma lipids, skeletal muscle insulin signaling, and in vitro skeletal muscle glucose transport activity. Obese Zucker rats were treated with either vehicle or CT118637 (30 mg/kg body wt) twice per day for 10 days. Fasting plasma insulin and free fatty acid levels were reduced by 14 and 23% (P < 0.05), respectively, in GSK-3 inhibitor-treated animals compared with vehicle-treated controls. The glucose response during an oral glucose tolerance test was reduced by 18% (P < 0.05), and whole body insulin sensitivity was increased by 28% (P < 0.05). In vivo insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (50%) and IRS-1-associated phosphatidylinositol-3' kinase (79%) relative to fasting plasma insulin levels were significantly elevated (P < 0.05) in plantaris muscles of GSK-3 inhibitor-treated animals. Whereas basal glucose transport in isolated soleus and epitrochlearis muscles was unaffected by chronic GSK-3 treatments, insulin stimulation of glucose transport above basal was significantly enhanced (32-60%, P < 0.05). In summary, chronic treatment of insulin-resistant, prediabetic obese Zucker rats with a specific GSK-3 inhibitor enhances oral glucose tolerance and whole body insulin sensitivity and is associated with an amelioration of dyslipidemia and an improvement in IRS-1-dependent insulin signaling in skeletal muscle. These results provide further evidence that selective targeting of GSK-3 in muscle may be an effective intervention for the treatment of obesity-associated insulin resistance.     

Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle

Eukaryotic initiation factor eIF2B is a guanine nucleotide exchange protein involved in regulation of translation initiation. Phosphorylation of the epsilon-subunit is thought to be important in insulin-mediated changes in eIF2B activity. However, elucidation of insulin's action has proven elusive, primarily because eIF2B epsilon is a substrate in vitro for at least three different protein kinases. In the present study, we observed changes in eIF2B epsilon kinase activity only in those muscles previously shown to exhibit alterations in protein synthesis in response to insulin. Specifically, eIF2B epsilon kinase activity was increased in psoas muscle from diabetic rats compared to controls. Treating diabetic rats with insulin rapidly reduced eIF2B epsilon kinase activity below control values. Changes were not observed in heart. To identify the kinase(s) in psoas responsible for phosphorylating eIF2B epsilon, the wildtype and two variant forms of the epsilon-subunit were expressed in and purified from Sf9 insect cells, and were used as substrates in protein kinase assays. The first variant contained a point mutation in the eIF2B epsilon cDNA that converted the glycogen synthase kinase-3 (GSK-3) phosphorylation site, Ser535, to a nonphosphorylatable Ala residue. In the second variant, the putative GSK-3 'priming' site, Ser539, was converted to Asp. Based on the pattern of phosphorylation of the wildtype and two variant forms of eIF2B epsilon using casein kinase (CK)-I, CK-II, or GSK-3 as well as that observed with skeletal muscle extracts, we conclude that the predominant eIF2B epsilon kinase in psoas muscle is GSK-3. Thus, insulin-mediated changes in eIF2B activity are likely to involve GSK-3.     

Cardiac glycogen accumulation after dexamethasone is regulated by AMPK

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5'-AMP-activated protein kinase (AMPK) on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. After Dex, cardiac tissue had a limited contribution toward the development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with increased GLUT4 mRNA. Both total and phosphorylated AMPK increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa (AS160) followed by Western blot analysis demonstrated no change in Akt phosphorylation at Ser(473) and Thr(308) in Dex-treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr(172), which correlated well with AS160 phosphorylation. In Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase-3-beta phosphorylation was augmented. Our data suggest that AMPK-mediated glucose entry combined with the activation of glycogen synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53: 1790-1797, 2004) act together to promote glycogen storage. Should these effects persist chronically in the heart, they may explain the increased morbidity and mortality observed with long-term excesses in endogenous or exogenous glucocorticoids.     

Hyperglycemia impairs the insulin signaling step between PI 3-kinase and Akt/PKB activations in ZDF rat liver

Akt/PKB activation is reportedly essential for insulin-induced glucose metabolism in the liver. During the hypoinsulinemic and hyperglycemic phase in the Zucker diabetic fatty (ZDF) rat liver, insulin-induced phosphorylations of the insulin receptor (IR) and insulin receptor substrate (IRS)-1/2 were significantly enhanced. Similarly, phosphatidylinositol (PI) 3-kinase activities associated with IRS-1/2 were markedly increased in ZDF rat liver compared with those in the control lean rat liver. However, interestingly, insulin-induced phosphorylation and kinase activation of Akt/PKB were severely suppressed. The restoration of normoglycemia by sodium-dependent glucose transporter (SGLT) inhibitor to ZDF rats normalized elevated PI 3-kinase activation and phosphorylation of IR and IRS-1/2 to lean control rat levels. In addition, impaired insulin-induced Akt/PKB activation was also normalized. These results suggest that chronic hyperglycemia reduces the efficiency of the activation step from PI 3-kinase to Akt/PKB kinase and that this impairment is the molecular mechanism underlying hyperglycemia-induced insulin resistance in the liver.     

Effects of diabetes and glycogen on the distribution between soluble and particulate fractions of activities of enzymes involved in hepatic glycogen metabolism.

The large decreases in hepatic glycogen associated with alloxan diabetes in fed rats were accompanied by apparent decreases in total activities of glycogen synthase, phosphorylase, protein kinase and synthase phosphatase determined on 8000 × g supernatants of liver homogenates. Inclusion of 4% glycogen in the extraction buffer normalized total soluble activities of synthase in the diabetic. Whereas inclusion of 4% glycogen in the extraction buffer doubled total soluble phosphorylase, total activity remained lower in the diabetic than in the normal. Extraction and assay of soluble protein kinase were unaffected by added glycogen. When activities were determined on whole homogenates, total glycogen synthase activities were the same in normal and diabetic liver. Although the decreases in total activities of phosphorylase, kinase and phosphatase were less when determined on whole homogenates of livers from diabetic rats, the diabetes-related decreases in total activities remained significant. Therefore, it appears that while alloxan diabetes results in absolute decreases in total hepatic activities of phosphorylase, kinase and phosphatase, it may also result in redistribution of hepatic synthase and phosphorylase between soluble and particulate fractions, a phenomenon possibly related to tissue glycogen concentrations. Such a redistribution might be involved in the lack of control of hepatic glycogenesis observed in alloxan diabetic rats.