Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment.

The HepG2-type glucose transporter (HepG2-GT) is expressed in 3T3-L1 fibroblasts and adipocytes. In contrast, the acutely insulin-regulatable glucose transporter (IRGT) is expressed only in the adipocytes. In the present study, the expression of the IRGT was shown to increase in parallel with the acquisition of acutely insulin-stimulated glucose uptake during differentiation of these cells, whereas the level of the HepG2-GT decreased during the course of differentiation in parallel with a decline in basal glucose uptake. We examined the effects of chronic insulin and tolbutamide treatment on glucose transporter activity in conjunction with the expression of these two glucose transporter species in 3T3-L1 adipocytes. Treatment of adipocytes with insulin, tolbutamide, or both agents in combination increased 2-deoxyglucose uptake, HepG2-GT protein, and HepG2-GT mRNA levels in parallel. The effect of combined insulin/tolbutamide administration on these three parameters was greater than the effect of either treatment alone. In contrast, these treatments either had no significant effect or decreased levels of IRGT protein and mRNA. We conclude that chronic treatment of 3T3-L1 adipocytes with insulin or tolbutamide increases glucose uptake primarily by means of a selective increase in the expression of the HepG2-GT. We suggest that part of the in vivo hypoglycemic effect of insulin and sulfonylureas may involve an increased expression of the HepG2-GT.     

Effects of adenoviral gene transfer of wild-type, constitutively active, and kinase-defective protein kinase C-lambda on insulin-stimulated glucose transport in L6 myotubes

We used adenoviral gene transfer methods to evaluate the role of atypical protein kinase Cs (PKCs) during insulin stimulation of glucose transport in L6 myotubes. Expression of wild-type PKC-lambda potentiated maximal and half-maximal effects of insulin on 2-deoxyglucose uptake, but did not alter basal uptake. Expression of constitutively active PKC-lambda enhanced basal 2-deoxyglucose uptake to virtually the same extent as that observed during insulin treatment. In contrast, expression of kinase-defective PKC-lambda completely blocked insulin-stimulated, but not basal, 2-deoxyglucose uptake. Similar to alterations in glucose transport, constitutively active PKC-lambda mimicked, and kinase-defective PKC-lambda completely inhibited, insulin effects on GLUT4 glucose transporter translocation to the plasma membrane. Expression of kinase-defective PKC-lambda, in addition to inhibition of atypical PKC enzyme activity, was attended by paradoxical increases in GLUT4 and GLUT1 glucose transporter levels and insulin-stimulated protein kinase B enzyme activity. Our findings suggest that in L6 myotubes, 1) atypical PKCs are required and sufficient for insulin-stimulated GLUT4 translocation and glucose transport; and 2) activation of protein kinase B in the absence of activation of atypical PKCs is insufficient for insulin-induced activation of glucose transport.     

Hexose specificity for downregulation of HepG2/brain-type glucose transporter gene expression in L6 myocytes

Summary Glucose deprivation of L6 myocytes results in the upregulation of glucose transporter activity, protein and mRNA. We have investigated the downregulation of transporter gene expression by glucose and other hexoses in glucose-deprived L6 myocytes. Glucose transport activity was measured as the uptake of ³H-2-deoxyglucose. Transporter protein and mRNA were detected by immunoblot and Northern blot analysis, respectively, with probes to the rat brain glucose transporter. Glucose deprivation of myocytes, in the absence and presence of insulin, increased ³H-2-deoxyglucose uptake, transporter protein and mRNA levels. Refeeding with glucose reversed the glucose deprivation effects on transport activity and mRNA within 12 h, with half-maximal effects at 1–2 mmol/l glucose. Mannose fully substituted for glucose. Refeeding with the non-metabolisable glucose analogues 2-deoxyglucose and 3-0-methylglucose, or with glucosamine or mannitol, downregulated ³H-2-deoxyglucose uptake but had little or no effect on transporter protein and mRNA expression. In contrast, glucose-6-phosphate markedly increased ³H-2-deoxyglucose uptake but partly downregulated transporter mRNA levels, whereas galactose had a small stimulatory effect on both ³H-2-deoxyglucose uptake and transporter mRNA; neither affected transporter protein levels. The transporter mRNA level was not affected by several metabolites (pyruvate, glyceraldehyde, glycerol) and amino acids (alanine, glutamine). These findings indicate that (i) there are independent pathways for hexose regulation of transport activity, protein and mRNA and (ii) down-regulation of transporter mRNA requires metabolism beyond hexose phosphate whereas glucose uptake may be regulated by direct interaction of hexoses with the transporter.     

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin.

We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.     

Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells

In the present study we determined the uptake and disposition of glucose in serum-deprived rabbit coronary microvessel endothelial (RCME) cells. RCME cells exhibited stereospecific hexose uptake inhibited by cytochalasin B. Pretreatment of the RCME cells with potassium cyanide or 2,4-dinitrophenol inhibited 2-deoxyglucose uptake but not 3-O-methylglucose transport. A major proportion (30-60%) of the 2-deoxyglucose present in the RCME cells was not phosphorylated. These two observations suggested that the rate-limiting step in the uptake of 2-deoxyglucose was not transport but rather the phosphorylation of 2-deoxyglucose to 2-deoxyglucose 6-phosphate. When glucose-deprived cells were incubated 2 hr with [U-14C]glucose the disposition of the label was as follows: glycogen 60%, acid-soluble fraction 30%, and lipid less than 5%. In contrast glucose-fed cells exhibited lower overall glucose incorporation, and a slightly different disposition: glycogen 45%, acid-soluble fraction 50%, and lipid 5%. Glucose-deprived RCME cells also exhibited greater basal levels of 2-deoxyglucose uptake compared to glucose-fed cells. RCME cells incubated in the absence of glucose and serum for 16 hr exhibited dose-dependent insulin stimulation of hexose uptake and subsequent metabolism to macromolecules (i.e., glycogen and the acid-soluble fraction). Significant effects of insulin were observed with concentrations as low as 2 x 10(-10) M, well within the physiological range. In contrast, cells preincubated in serum-free culture medium containing 5.5 mM glucose did not exhibit insulin-enhanced hexose uptake or glucose metabolism (even at doses as high as 10(-7) M). These studies indicate that the effects of insulin on rabbit coronary microvascular endothelial cell glucose uptake and metabolism require both serum and glucose deprivation.     

High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes

OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.     

The ras signaling pathway mimics insulin action on glucose transporter translocation.

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.     

Stimulation of hexose transport by metformin in L6 muscle cells in culture.

L6 muscle cells grown in culture to the stage of fused myotubes were incubated with the oral hypoglycemic drug metformin to test the effects of this drug on glucose transport. Metformin increased the initial rate of uptake of 2-deoxyglucose and 3-O-methylglucose. The effect was time dependent, with half-maximal stimulation at 5-6 h and maximal stimulation by about 16 h. The stimulation of hexose uptake was not prevented by cycloheximide. In 15 mM glucose medium, the basal rate of transport was lower than in 5 mM glucose medium. The stimulation of hexose uptake by metformin was comparable in absolute units in both media; hence, relative to basal uptake, stimulation was greater in the high glucose medium than in the low glucose medium. In 5 mM glucose medium, half-maximal stimulation was obtained with 800 microM metformin when tested for 24 h. The stimulation of hexose transport by metformin was only detectable in fused myotubes and not in perfusion myoblasts. No significant changes were observed in glucose transporter levels in total cell membranes from L6 myotubes (measured as D-glucose-protectable binding sites for cytochalasin-B) or in the total levels of the immunoreactive glucose transporter isoforms GLUT4 or GLUT1. It is concluded that metformin stimulates hexose transport into differentiated muscle cells by acting at a posttranslational level. We speculate that this might also constitute the basis for the ability of the drug to lower glycemia in diabetic individuals.     

High concentration of glucose decreases glucose transporter-1 expression in mouse placenta in vitro and in vivo

Facilitative glucose transporter-1 (GLUT1) is expressed abundantly and has an important role in glucose transfer in placentas. However, little is known about the regulation of GLUT1 expression in placental cells. We studied the changes in placental GLUT1 levels in relation to changes in glucose concentration in vitro and in vivo. In in vitro experiments, dispersed mouse placental cells were incubated under control (5.5 mM) and moderately high (22 mM) glucose concentrations, and 2-deoxyglucose uptake into cells was studied on days 1-5 of culture. After 4 days of incubation under both conditions, GLUT1 mRNA and proten levels were examined by Northern and immunoblot analyses. Treatment of cells with 22 mM glucose resulted in a significant decrease in 2-deoxyglucose uptake compared with control, from day 2 to day 5 of culture. Moreover, GLUT1 mRNA and protein levels on day 4 of culture were significantly reduced in cells incubated with 22 mM glucose compared with control. Next, we rendered mice diabetic by administering 200 micrograms/g body weight streptozotocin (STZ) on day 8 of pregnancy. Animals were killed on day 12 of pregnancy and placental tissues were obtained. [3H]Cytochalasin B binding study was carried out to assess total GLUTs, and GLUT1 mRNA and protein were measured as above. [3H]Cytochalasin B binding sites in placentas from STZ-treated mice were significantly less than those in control mice. Northern and immunoblot analyses revealed a significant decrease in GLUT1 mRNA and protein levels in diabetic mice compared with the controls. These findings suggest that the glucose concentration may regulate the expression of placental GLUT1.     

Stimulation of glucose transport by thyroid hormone in ARL 15 cells: increased abundance of glucose transporter protein and messenger ribonucleic acid.

We have studied regulation of the glucose transporter by thyroid hormone in ARL 15 cells, a thyroid hormone-responsive cell line derived from rat liver, T3 treatment (5 x 10(-8) M for 48 h) of confluent cell monolayers grown in thyroid hormone-deficient medium increased the rate of uptake of [3H] 2-deoxyglucose by 2.3 +/- 0.2-fold; this effect was half-maximal at a T3 concentration of 5 nM. The uptake of the nonmetabolizable hexose [3H]3-O-methylglucose was comparably increased, confirming a stimulation of glucose transport by thyroid hormone in these cells. In addition to enhancing glucose transporter activity, T3 increased the utilization of medium glucose to a similar degree. To elucidate the mechanism of the stimulation of glucose transport by T3, the number of glucose transporter units in crude membrane preparations was quantitated by measuring the glucose-inhibitable binding of [3H]cytochalasin-B. The Kd for specific (glucose-inhibitable) binding of [3H]cytochalasin-B was 50-60 nM, a value typical for nonhepatic glucose transporters. T3 treatment caused an increase in the glucose-inhibitable binding of this ligand that was similar in magnitude to the stimulation of [3H]2-deoxyglucose uptake (2.5 +/- 0.6-fold). Northern blot analysis of total cellular RNA using a cDNA probe for the rat brain glucose transporter showed a strong 2.9-kilobase hybridization signal after stringent washing, indicating that ARL 15 cells express the specific mRNA for this type of glucose transporter. T3 treatment increased the abundance of this mRNA by 2.3 +/- 0.2-fold. It is concluded that thyroid hormone stimulates glucose transport in ARL 15 cells, which express the brain type of glucose transporter. This effect is attributable at least in part, if not entirely, to an increase in the level of glucose transporter mRNA and an accompanying increase in the number of glucose transporter units. These findings suggest that thyroid hormone may be an important regulator of glucose transporter gene expression.     

Enhancement of glucose uptake in 3T3-L1 adipocytes by Toona sinensis leaf extract

The effects of substances extracted from Toona sinensis leaves, using 50% alcohol/water, on cellular [3H]-2-deoxyglucose uptake in differentiated cultured 3T3-L1 adipocytes were investigated. Following treatment of cells with 0.001, 0.01, or 0.1 mg/mL extracts for 60 minutes, [3H]-2-deoxyglucose uptake increased from a basal value of 0.23 nmol/min/mg protein to 0.30, 0.33, and 0.38 nmol/min/mg protein, respectively. In insulin-stimulated cells, cellular [3H]-2-deoxyglucose uptake was enhanced by Toona sinensis leaf extract from a basal value of 0.35 nmol/min/mg protein to 0.41, 0.46, and 0.52 nmol/min/mg protein, respectively. Cellular glucose uptake was also enhanced by Toona sinensis leaf extract after incubation of cells with 20 mM glucose for 48 hours. Cellular glucose uptake with a combination of Toona sinensis leaf extract and insulin was significantly inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide and the protein kinase C inhibitor calphostin C in normal-, medium- and high-glucose media. However, the glucose uptake-enhancing effect of Toona sinensis leaf extract was not diminished by cycloheximide and calphostin C in the absence of insulin. These results indicate that enhancement of cellular glucose uptake by Toona sinensis leaf extract in basal and insulin-stimulated 3T3-L1 adipocytes may be mediated by distinct mechanisms.     

Inhibition of insulin gene expression by long-term exposure of pancreatic beta cells to palmitate is dependent on the presence of a stimulatory glucose concentration

Long-term exposure of pancreatic beta cells to elevated levels of fatty acids (FAs) impairs glucose-induced insulin secretion. However, the effects of FAs on insulin gene expression are controversial. We hypothesized that FAs adversely affect insulin gene expression only in the presence of elevated glucose concentrations. To test this hypothesis, isolated rat islets were cultured for up to 1 week in the presence of 2.8 or 16.7 mmol/L glucose with or without 0.5 mmol/L palmitate. Insulin release, insulin content, and insulin mRNA levels were determined at the end of each culture period. Palmitate increased insulin release at each time point independently of the glucose concentration. In contrast, insulin content was unchanged in the presence of palmitate at 2.8 mmol/L glucose, but was markedly decreased in the presence of 0.5 mmol/L palmitate and 16.7 mmol/L glucose after 2, 3, and 7 days of culture. In the presence of a basal concentration of glucose, insulin mRNA levels were transiently increased by palmitate at 24 hours but were unchanged thereafter. In contrast, palmitate significantly inhibited the stimulatory effects of 16.7 mmol/L glucose on insulin mRNA levels after 2, 3, and 7 days. To determine whether the inhibitory effect of palmitate on glucose-stimulated insulin mRNA levels was associated with decreased insulin promoter activity, HIT-T15 cells were cultured for 24 hours in 11.1 mmol/L glucose in the presence or absence of palmitate, and insulin gene promoter activity was measured in transient transfection experiments using the insulin promoter-reporter construct INSLUC. INSLUC activity was decreased more than 2-fold after 24 hours of exposure to 0.5 mmol/L palmitate. We conclude that long-term exposure of pancreatic beta cells to palmitate decreases insulin gene expression only in the presence of elevated glucose concentrations, in part through inhibition of insulin gene promoter activity.     

Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells.

The effects of the oral hypoglycemic drug metformin on glucose and amino acid transporter activity and subcellular localization of GLUT1 and GLUT4 glucose transporters were tested in cultured L6 myotubes. In muscle cells preexposed to maximal doses of metformin (2 mM, for 16 h), 2-deoxyglucose uptake was stimulated by over 2-fold from 5.9 +/- 0.3 to 13.3 +/- 0.5 pmol/min.mg protein. Uptake of the nonmetabolizable amino acid analog methylaminoisobutyrate was unaffected by treatment with the drug under identical conditions. Extracellular calcium was required to preserve the full response to the biguanide. Exposure of muscle cells to insulin in the presence of metformin resulted in further activation of 2-deoxyglucose transport. The latter effect was additive to the maximum effect of metformin, suggesting that the biguanide stimulates hexose uptake into muscle cells by an insulin-independent mechanism. Glucose transporter number quantified by performing studies of D-glucose-protectable binding of cytochalasin-B in plasma membranes (PM) and internal membranes (IM) prepared from L6 myotubes revealed that a 16-h treatment with 800 microM metformin significantly elevated glucose transporter number in the PM (by 47%), with an equivalent decrement in glucose transporter number (47%) in the IM. Western blot analysis using antisera reactive with the GLUT1 and GLUT4 isoforms of glucose transporters showed that metformin caused a reduction in GLUT1 content in the IM fraction and a concomitant increase in the PM. Unlike insulin, metformin treatment had no effect on the subcellular distribution of GLUT4. We propose that the molecular basis of metformin action in skeletal muscle involves the subcellular redistribution of GLUT1 proteins from an intracellular compartment to the plasma membrane. Such a recruitment process may form an integral part of the mechanism by which the drug stimulates glucose uptake (and utilization) in skeletal muscle and facilitates lowering of blood glucose in the management of type II diabetes.     

Glucose uptake and phosphorylation in fat cells of fasted and hypophysectomized rats

Fat cells of hypophysectomized and fasted rats metabolize 10 times less glucose than adipocytes of normal rats in the presence of insulin. Glucose transport (3-O-methylglucose influx), transport plus phosphorylation (2-deoxyglucose uptake), hexokinase, pyruvate dehydrogenase and glucose-6-phosphate dehydrogenase activities were determined in an attempt to localize the metabolic defects. Insulin stimulates 3-O-methylglucose influx 5-fold in normal cells and 3-fold in cells of fasted rats. The basal influx in cells of fasted rats is increased and even more so in cells of hypophysectomized rats where the rate of basal influx is the same as that in cells of normal rats under maximal insulin stimulation. It cannot be further stimulated by insulin. In contrast to 3-O-methylglucose influx, basal uptake and phosphorylation of 2-deoxyglucose in cells of fasted and hypophysectomized rats is drastically decreased and stimulation by insulin is abolished. Total hexokinase and pyruvate dehydrogenase activities are drastically reduced in the homogenate of fat cells of hypophysectomized and fasted rats. Phosphorylation by hexokinase appears to become one of the rate-limiting steps of glucose metabolism in cells of hypophysectomized rats.     

Effect of Vanadate and Insulin on Glucose Transport in Isolated Adult Rat Cardiomyocytes

It is now widely accepted that insulin stimulation of glucose uptake by muscle cells is due to the activation of protein kinase B, leading to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane. Vanadate is a protein tyrosine phosphatase (PTP) inhibitor and a known insulin mimetic agent. Vanadate causes an increase of glucose transport in various tissues, but the mechanism of stimulation is not clearly understood. Hence in the present study, we have compared the mechanism of 2-deoxy-D-glucose transport induced by vanadate and insulin in isolated rat cardiomyocytes. Vanadate stimulated deoxyglucose transport in a time- and concentration-dependent manner. Insulin (100 nM) and vanadate (5 mM) stimulated 2-deoxy-D-glucose transport on an average by 3- and 2-fold respectively over basal values. The stimulation of glucose transport was accompanied by an activation of protein kinase B (PKB). This study also revealed that the activation of PKB and stimulation of 2-deoxyglucose uptake by vanadate and insulin are inhibited by treatment with wortmannin, a specific inhibitor of phoshatidylinositol 3-kinase (PI 3-kinase). Hence, we conclude that both insulin and vanadate follow the same signalling pathway downstream of PI 3-kinase to stimulate 2-deoxy-D-glucose transport.     

Partial insulin resistance in the mouse BC3H-1 cell line: absent hexose-independent actions of insulin

We have studied the regulation of glycogen metabolism by insulin in the insulin-sensitive nonfusing muscle cell line BC3H-1. The basal percentage of glycogen synthase I activity was not altered by insulin alone at any concentration, time of exposure, or age of cells tested. The addition of glucose or 2-deoxyglucose to the glucose- and serum-free incubation medium caused a 2-fold increase in glycogen synthase I activity over basal levels, and the effect was enhanced to 3-fold if insulin was added to the medium. Glycogen phosphorylase a activity was not altered by incubation in the presence of insulin, but was lowered by the addition of 2-deoxyglucose. This effect was also enhanced in the presence of insulin. The effect of exogenously added sugar occurred only if a 6-phosphorylatable hexose was used. The effect seen with 2-deoxyglucose was stable to Sephadex G-25 desalting, suggesting that activation of glycogen synthase was the result of a stable (covalent) modification of the enzyme. We were also able to demonstrate the presence of glucose-6-phosphate-activatable glycogen synthase phosphatase activity in the myocytes. The effect of 2-deoxyglucose in the presence or absence of insulin could be completely reversed by including cytochalasin B in the medium, suggesting that both the effect of hexose and the insulin enhancement of its effect were entirely dependent on carrier-mediated hexose uptake. Four insulin-mimetic agents, H2O2 Concanavalin A, Na orthovanadate, and antiinsulin receptor B2 serum, were also tested. Despite different mechanisms of action, each agent qualitatively mimicked insulin in the myocytes. All stimulated hexose transport, glucose incorporation into glycogen, and hexose-dependent activation of glycogen synthase in a manner not additive with insulin, but none increased basal glycogen synthase I activity in the absence of hexose. These results suggest that although insulin is capable of regulating glycogen metabolism both by increasing the uptake of sugar and by altering the activation state of glycogen synthase and phosphorylase, these effects are entirely due to the stimulation of hexose uptake, and hexose-independent actions of insulin are absent in BC3H-1 cells.