Increase In GLUT4 Research Articles

Regulation of glucose transporters and hexose uptake in 3T3-L1 adipocytes: glucagon-like peptide-1 and insulin interactions.

Glucagon-like peptide-1 (7-36 amide) (GLP-1) is known to increase insulin release when given as a bolus in the fasted and fed state. GLP-1 also increases glucose uptake and lipid synthesis in cultured adipocytes. In this study we investigated the effects of GLP-1 on glucose uptake and on the levels of expression of the facilitative glucose transporters, GLUT1 and GLUT4, in fully differentiated 3T3-L1 adipocytes. Cells were incubated with GLP-1 (10 nM) with or without insulin (10 and 100 nM) for 24 h. Under these conditions, GLP-1 alone caused an increase in basal and acute insulin-stimulated glucose uptake along with an increase in GLUT1 and GLUT4 protein levels. However, there was no change in the expression of GLUT1 and GLUT4 mRNAs. In the absence of GLP-1, prolonged exposure to insulin caused a marked reduction in the levels of GLUT4 mRNA and protein, and an inhibition of glucose uptake after an acute exposure to insulin. This insulin-induced down-regulation of GLUT4 was prevented when GLP-1 was present during the 24-h treatment. In contrast, the acute insulin-stimulated glucose uptake could not be restored by GLP-1. GLP-1 is therefore the first gut hormone shown to be capable of modulating glucose transporter levels in cultured adipocytes.

Glucose transporter expression in L6 muscle cells: regulation through insulin- and stress-activated pathways.

We addressed the effect of long-term treatment with insulin, 2,4-dinitrophenol (DNP; an uncoupler of oxidative phosphorylation that increases energy demand) and 300 mM mannitol (hyperosmolarity) on glucose transporter (GLUT) expression in L6 muscle cells and the signaling pathways involved. We found the following. 1) The insulin-mediated increase in GLUT-1 is 70-kDa ribosomal protein S6 kinase (p70 S6 kinase) and p38 mitogen-activated protein kinase (MAPK) dependent but extracellular signal-regulated protein kinase (ERK) and MAPK/ERK kinase (MEK) independent. The hypertonicity-stimulated elevation in GLUT-1 is p70 S6 kinase, p38 MAPK, and MEK dependent yet ERK independent. DNP also increased GLUT-1 protein but did not depend on any of the above pathways, 2) Insulin increased GLUT-3 protein in a p70 S6 kinase-independent but MEK/ERK-dependent fashion. Inhibition of p38 MAPK potentiated the effect of insulin on GLUT-3. Hypertonicity increased GLUT-3 via p70 S6 kinase- and p38 MAPK-dependent pathways. In conclusion, we have dissected the molecular mechanisms used by insulin and hypertonicity that culminate in the induction of GLUT-1 and GLUT-3. The mechanism(s) used by DNP remains unknown.

Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes.

Phosphatidylinositol 3-kinase (PI 3-kinase) activation promotes glucose transporter 4 (Glut 4) translocation in adipocytes. In this study, we demonstrate that protein kinase B, a serine/threonine kinase stimulated by PI 3-kinase, is activated by both insulin and okadaic acid in isolated adipocytes, in parallel with their effects on Glut 4 translocation. In 3T3-L1 adipocytes, platelet-derived growth factor activated PI 3-kinase as efficiently as insulin but was only half as potent as insulin in promoting protein kinase B (PKB) activation. To look for a potential role of PKB in Glut 4 translocation, adipocytes were transfected with a constitutively active PKB (Gag-PKB) together with an epitope tagged transporter (Glut 4 myc). Gag-PKB was associated with all membrane fractions, whereas the endogenous PKB was mostly cytosolic. Expression of Gag-PKB led to an increase in Glut 4 myc amount at the cell surface. Our results suggest that PKB could play a role in promoting Glut 4 appearance at the cell surface following exposure of adipocytes to insulin and okadaic acid stimulation.

Phosphatidylinositol 3-kinase and dynamics of insulin resistance in denervated slow and fast muscles in vivo.

Regulation of glucose uptake by 1- and 3-day denervated soleus (slow-twitch) and plantaris (fast-twitch) muscles in vivo was investigated. One day after denervation, soleus and plantaris muscles exhibited 62 and 65% decreases in insulin-stimulated 2-deoxyglucose uptake, respectively, compared with corresponding control muscles. At this interval, denervated muscles showed no alterations in insulin receptor binding and activity, amount and activity of phosphatidylinositol 3-kinase, and amounts of GLUT-1 and GLUT-4. Three days after denervation, there was no increase in 2-deoxyglucose uptake in response to insulin in soleus muscle, whereas plantaris muscle exhibited a 158% increase in basal and an almost normal absolute increment in insulin-stimulated uptake. Despite these differences, denervated soleus and plantaris muscles exhibited comparable decreases in insulin-stimulated activities of the insulin receptor (approximately 40%) and phosphatidylinositol 3-kinase (approximately 50%) and a pronounced decrease in GLUT-4. An increase in GLUT-1 in plantaris, but not soleus, muscle 3 days after denervation is consistent with augmented basal 2-deoxyglucose uptake in plantaris muscle at this interval. These results demonstrate that, in denervated muscles, there is a clear dissociation between insulin-stimulated 2-deoxyglucose uptake and upstream events involved in insulin-stimulated glucose uptake.

The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue.

The regulation of ob gene expression in abdominal subcutaneous adipose tissue was investigated using a reverse transcription-competitive PCR method to quantify the mRNA level of leptin. Leptin mRNA level was highly correlated with the body mass index of 26 subjects (12 lean, 7 non-insulin-dependent diabetic, and 7 obese patients). The effect of fasting on ob gene expression was investigated in 10 subjects maintained on a hypocaloric diet (1045 KJ/d) for 5 d. While their metabolic parameters significantly changed (decrease in insulinemia, glycemia, and resting metabolic rate and increase in plasma ketone bodies), the caloric restriction did not modify the leptin mRNA level in the adipose tissue. To verify whether insulin regulates ob gene expression, six lean subjects underwent a 3-h euglycemic hyperinsulinemic (846 +/- 138 pmol/liter) clamp. Leptin and Glut 4 mRNA levels were quantified in adipose tissue biopsies taken before and at the end of the clamp. Insulin infusion produced a significant threefold increase in Glut 4 mRNA while leptin mRNA was not affected. It is concluded that ob gene expression is not acutely regulated by insulin or by metabolic factors related to fasting in human abdominal subcutaneous adipose tissue.

Reciprocal GLUT-1 and GLUT-4 expression and glucose transport in denervated muscles.

We investigated in 3-day-denervated muscles 1) the expression of GLUT-1 in perineurial sheaths (PNS) and muscle, 2) the muscle fiber-specific changes in GLUT-1 and GLUT-4, and 3) changes in basal and insulin-stimulated 3-O-methylglucose transport. GLUT-1 was increased in both the PNS (P < 0.05) and in the muscle membranes (P < 0.05). GLUT-1 and GLUT-4 concentrations were changed reciprocally, in a fiber-dependent fashion [GLUT-1: red gastrocnemius (RG), +31%; white gastrocnemius (WG), +10%; GLUT-4: RG, -53%; WG, -16%]. Basal glucose transport was increased (P < 0.05), and this increase was correlated with the oxidative nature of the muscles (r = 0.97). Insulin-stimulated glucose transport was decreased in denervated muscles (P < 0.05). This was also related to the oxidative nature of the muscles (r = -0.88). The increase in basal glucose transport was correlated with the loss of insulin-stimulated transport (r = 0.95). Thus the increase in GLUT-1 compensates for the loss of GLUT-4, resulting in a 56% regain of the reduced insulin-stimulated glucose transport.

PLACENTAL GLUCOSE TRANSPORTER (GLUT 1) IN FETAL SHEEP IS REGULATED BY TIME-DEPENDENT CHANGES IN GLUCOSE AND INSULIN CONCENTRATIONS. ▴ 1828

Fetal glucose supply under basal conditions is solely derived from the maternal circulation via placental transport. Placental glucose transport is mediated by facilitative glucose transporters, the predominant isoform being Glut 1. We examined the time-dependent effect of maternal glucose(HYPERglycemic) and insulin (HYPOglycemic) infusions on placental Glut 1 in late gestation chronically catheterized pregnant sheep. HYPER (+80% normal; n=14) was produced by maternal continuous infusions of 50% glucose and HYPO( 48hr-41d) and hypoglycemia (<50%) with consistent fetal hypoinsulinemia, respectively. Placental tissue was analyzed for Glut 1 levels(50-55 kD) at these different time points by quantitative Western blot analysis using a rabbit anti-rat Glut 1 COOH IgG. HYPER caused a transient but minimal increase in Glut 1 protein levels at 2-3 hr (p<0.05) followed by a normalization between the 10 and 20d duration. In contrast, HYPO led to a 50% decline starting at 24-48 hr and lasting through 25 to 41d (p<0.05). We conclude that 1] changes in placental Glut 1 are time-dependent, and 2] HYPER causes a transient but minimal increase while HYPO leads to a persistent decline in placental Glut 1 concentrations. We speculate to the extent that changes in placental glucose transporter characteristics influence the rate of maternal-fetal glucose transport & fetal glucose availability, the HYPO induced decline in placental Glut 1 may mediate the development of fetal hypoglycemia and concomitant intra-uterine growth restriction.

Expression and Regulation by Insulin of Glut 3 in UMR 106-01, a Clonal Rat Osteosarcoma Cell Line

Expression of the glucose transporter GLUT 3 is mainly restricted to neuronal tissues, with lower levels in testis and placenta. In addition, GLUT 3 has recently been reported in neonatal rat calvaria byin situhybridisation. We report the co-expression of GLUT 1 and GLUT 3 mRNA and protein in UMR 106-01, a clonal osteosarcoma cell line. By semi-quantitative analysis we show that GLUT 3 protein is expressed at levels comparable to GLUT 1. Insulin stimulates glucose uptake in UMR 106-01 cells. GLUT 3 mRNA and protein are increased by chronic (16 h) treatment with insulin, and the increase in GLUT 3 mRNA is not inhibited by cycloheximide. Regulation of GLUT 3 mRNA by insulin has not been previously shown. UMR 106-01 represents a useful model for investigating regulation of GLUT 3 expression.

Upregulation of Blood-Brain Barrier GLUT1 Glucose Transporter Protein and mRNA in Experimental Chronic Hypoglycemia

An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of < 2.3 mmol/l (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 +/- 0.1 mmol/l (36 +/- 1 mg/dl) vs. 4.9 +/- 0.1 mmol/l (88 +/- 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeability-surface area (PS) product of 40% (P < 0.0005) in the chronically hypoglycemic animals as compared with controls. Western blotting of solubilized isolated brain capillaries demonstrated a 51% increase (P < 0.05) in immunoreactive BBB GLUT1 in the chronically hypoglycemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1-to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A+) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glucose transport activity that occurs with chronically low circulating blood glucose levels.

Glucosamine induces insulin resistance in vivo by affecting GLUT 4 translocation in skeletal muscle. Implications for glucose toxicity.

Glucosamine (Glmn), a product of glucose metabolism via the hexosamine pathway, causes insulin resistance in isolated adipocytes by impairing insulin-induced GLUT 4 glucose transporter translocation to the plasma membrane. We hypothesized that Glmn causes insulin resistance in vivo by a similar mechanism in skeletal muscle. We performed euglycemic hyperinsulinemic clamps (12 mU/kg/min + 3H-3-glucose) in awake male Sprague-Dawley rats with and without Glmn infusion at rates ranging from 0.1 to 6.5 mg/kg/min. After 4h of euglycemic clamping, hindquarter muscles were quick-frozen and homogenized, and membranes were subfractionated by differential centrifugation and separated on a discontinuous sucrose gradient (25, 30, and 35% sucrose). Membrane proteins were solubilized and immunoblotted for GLUT 4. With Glmn, glucose uptake (GU) was maximally reduced by 33 +/- 1%, P < 0.001. The apparent Glmn dose to reduce maximal GU by 50% was 0.1 mg/kg/min or 1/70th the rate of GU on a molar basis. Control galactosamine and mannosamine infusions had no effect on GU. Relative to baseline, insulin caused a 2.6-fold increase in GLUT 4 in the 25% membrane fraction (f), P < 0.01, and a 40% reduction in the 35%f, P < 0.05, but had no effect on GLUT 4 in the 30% f, P= NS. Addition of Glmn to insulin caused a 41% reduction of GLUT 4 in the 25%f, P < 0.05, a 29% fall in the 30%f, and prevented the reduction of GLUT 4 in the 35% f. The 30%f membranes were subjected to a second separation with a 27 and 30% sucrose gradient. Insulin mobilized GLUT 4 away from the 30%f, P < 0.05, but not the 27% f. In contrast, Glmn reduced GLUT 4 in the 27%f, P < 0.05, but not the 30%f. Thus Glmn appears to alter translocation of an insulin-insensitive GLUT 4 pool. Coinfusion of Glmn did not alter enrichment of the sarcolemmal markers 5'-nucleotidase, Na+/K+ATPase, and phospholemman in either 25, 30, or 35% f. Thus Glmn completely blocked movement of Glut 4 induced by insulin. Glmn is a potent inducer of insulin resistance in vivo by causing (at least in part) a defect intrinsic to GLUT 4 translocation and/or trafficking. These data support a potential role for Glmn to cause glucose-induced insulin resistance (glucose toxicity).

The Insulin-dependent Biosynthesis of GLUT1 and GLUT3 Glucose Transporters in L6 Muscle Cells Is Mediated by Distinct Pathways: ROLES OF p21rasAND pp70 S6 KINASE

Insulin binding results in rapid phosphorylation of insulin receptor substrate-1 to activate p21ras and mitogen-activated protein kinase. Insulin also activates the ribosomal protein S6 kinase (pp70 S6 kinase) independently of the Ras pathway. Chronic (18 h) treatment of L6 muscle cells with insulin increases glucose transport activity severalfold due to biosynthetic elevation of the GLUT1 and GLUT3 but not the GLUT4 glucose transporters. Here we investigate the roles of p21ras and pp70 S6 kinase in the insulin-mediated increases in GLUT1 and GLUT3 expression. L6 cells were transfected with the dominant negative Ras(S17N) under the control of a dexamethasone-inducible promoter. Induction of Ras(S17N) failed to block the insulin-mediated increase in GLUT1 glucose transporter protein and mRNA; however, it abrogated the insulin-mediated increase in GLUT3 glucose transporter protein and mRNA. Inhibition of pp70 S6 kinase by rapamycin, on the other hand, eliminated the insulin-mediated increase in GLUT1 but had no effect on that of GLUT3 in both parental and Ras(S17N) transfected L6 cells. These results suggest that the biosynthetic regulation of glucose transporters is differentially determined, with pp70 S6 kinase and p21ras playing active roles in the insulin-stimulated increases in GLUT1 and GLUT3, respectively.

Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.

We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA.

Growth factor-induced stimulation of hexose transport in 3T3-L1 adipocytes: Evidence that insulin-induced translocation of glut4 is independent of activation of MAP kinase

We have examined the effect of growth factors on the rate of hexose transport in 3T3-L1 adipocytes. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were found to stimulate deoxyglucose transport by about 2-fold. The concentrations of EGF and PDGF which elicited half maximal responses were 100 and 350 pM, respectively. The increases in transport rate were acute effects; the stimulations were evident whithin minutes of exposure to growth factors. By contrast, insulin stimulated deoxyglucose transport ∼16-fold over similar time periods. We have measured the appearance of both the insulin-responsive glucose transporter (GLUT4) and the erytghrocyte-type glucose transporter (GLUT1) at the cell surface in response to insulin, EGF and PDGF. We show that both EGF and PDGF inducea 2-fold increase in GLUT1 at the cell surface, but both these growth factors were without effect on GLUT4 levels at the cell surface. In contrast, insulin induced a 13-fold increase in cell surface GLUT4. We further show that insulin, EGF and PDGF all activate MAP kinase as determine by a shift in electrophoretic mobility of this protein on SDS-PAGE. However, since the large translocation of GLUT4 to the cell surface is specific for insulin, we suggest that activation of MAP kinase is not the sole requisite for this process.

Glut 4 content in the plasma membrane of rat skeletal muscle: comparative studies of the subcellular fractionation method and the exofacial photolabelling technique using ATB-BMPA

Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2-fold increase in the Glut 4 protein content in the plasma membrane of skeletal muscle from rats. Data based upon this technique are, however, impeded by poor plasma membrane recovery and cross-contamination with intracellular membrane vesicles. The present study was undertaken to compare the subcellular fractionation technique with the technique using [ 3H]ATB-BMPA exofacial photolabelling and immunoprecipitation of Glut 4 on soleus muscles from 3-week-old Wistar rats. Maximal insulin stimulation resulted in a 6-fold increase in 3- O-methylglucose uptake, and studies based on the subcellular fractionation method showed a 2-fold increase in Glut 4 content in the plasma membrane, whereas the exofacial photolabelling demonstrated a 6- to 7-fold rise in cell surface associated Glut 4 protein. Glucose transport activity was positively correlated with cell surface Glut 4 content as estimated by exofacial labelling. In conclusion: (1) the increase in glucose uptake in muscle after insulin exposure is caused by an augmented concentration of Glut 4 protein on the cell surface membrane, (2) at maximal insulin stimulation (20 mU ml ) approximately 40% of the muscle cell content of Glut 4 is at the cell surface, and (3) the exofacial labelling technique is more sensitive than the subcellular fractionation technique in measuring the amount of glucose transporters on muscle cell surface.

GLUT 4 and insulin receptor binding and kinase activity in trained human muscle.

1. Physical training enhances sensitivity and responsiveness of insulin-mediated glucose uptake in human muscle. This study examines if this effect of physical training is due to increased insulin receptor function or increased total concentration of insulin-recruitable glucose transporter protein (GLUT 4). 2. Seven healthy young subjects carried out single leg bicycle training for 10 weeks at 70% of one leg maximal oxygen uptake (VO2,max). Subsequently biopsies were taken from the vastus lateralis muscle of both legs. 3. Single leg VO2,max increased for the trained leg (46 +/- 3 to 52 +/- 2 ml min-1 kg-1 (means +/- S.E.M., P < 0.05), and cytochrome c oxidase activity was higher in this compared to the untrained leg (2.0 +/- 0.1 vs. 1.4 +/- 0.1 nmol s-1 (mg muscle)-1, P < 0.05). Insulin binding as well as basal- and insulin-stimulated receptor kinase activity did not differ between trained and untrained muscle. The concentration of GLUT 4 protein was higher in the former (14.9 +/- 1.9 vs. 11.6 +/- 1.0 arbitrary units (micrograms protein)-1 in crude membranes, P < 0.05). The training-induced increase in GLUT 4 (26 +/- 11%) matched a previously reported increase in maximum insulin-stimulated leg glucose uptake (25 +/- 7%) in the same subjects, and individual values of the two variables correlated (correlation coefficient (r) = 0.84, P < 0.05). 4. In conclusion, in human muscle training induces a local contraction-dependent increase in GLUT 4 protein, which enhances the effect of insulin on glucose uptake. On the other hand, insulin receptor function in muscle is unlikely to be affected by training.

Molecular Mechanisms of the Increased Glucose Transport in Transformed FRTL-5 Rat Thyroid Cells

We investigated the molecular mechanisms involved in the glucose carrier (Glut 1) regulation in FRTL-5 cells and two derived transformed clones (SRC and Ki-Mol cells). When compared to the wild-type strain, SRC and Ki-Mol cells showed an increase in both glucose consumption and uptake (about 60 fold), associated with 6-8 fold higher Glut 1 mRNA levels. Transcriptional studies revealed a 2- to 3 fold increased activation of the gene in the transformed cells, suggesting that transcription alone cannot fully account for the higher Glut 1 gene expression. Western blot studies showed an increase of the Glut 1 protein in SRC and Ki-Mol cells, associated with a different gel migration pattern and a disparate distribution rate between the plasma membrane and the microsomal fraction. These data indicate that the higher rate of glucose uptake observed in SRC and Ki-Mol cells is associated to an increase in Glut 1 gene expression, and that also changes in the subcellular distribution and probably in the structure of Glut 1 protein are present.

Effect of glucose deprivation of GLUT 1 expression in 3T3-L1 adipocytes.

Elevated glucose transport rates during glucose deprivation are phenomena that have been observed in several different types of cells in culture. We show here that glucose transport rates in 3T3-L1 adipocytes increased by 10-fold within 18 h in response to glucose deprivation, confirming earlier work by Van Putten and Krans (Van Putten, J. P. M., and Krans, H. M. J. (1985) J. Biol. Chem. 260, 7996-8001). Mannose and 3-O-methylglucose (a nonmetabolizable glucose analog), but not fructose or galactose, blocked the increase in transport activity. Although the increase in transport was dependent on new protein synthesis, only a small and transient increase in GLUT 1 mRNA (less than 2-fold) was observed. In addition, the level of the normal isoform of GLUT 1 (46 kDa) did not increase. A lower molecular mass isoform (37 kDa) was observed but not until 15 h after glucose removal, the appearance of which was clearly not correlated with the increase in activity. Further, the extracellular glucose concentration required to elicit accumulation of this form (p37) was 2 orders of magnitude less than that required for transport stimulation (5 microM versus 500 microM glucose; p37 accumulation and transport activation, respectively). Interestingly, p37 was seen in the presence of galactose, but not fructose, despite elevated transport activity with either sugar. The p37 isoform was slightly larger than N-glycosidase F-treated GLUT 1 (36 kDa), implying that this form is still glycosylated, albeit incompletely. It is not known if p37 is functional, but the time- and sugar-dependent appearance of the lower isoform suggests that p37 is not responsible for starvation-induced transport but potentially represents an underglycosylated precursor of the normal, 46-kDa isoform of GLUT 1.

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.

Developmental Regulation of the Distribution of Rat Brain Insulin-Insensitive (Glut 1) Glucose Transporter*

We examined the immunohistochemical localization of Glut 1 in sections of developing rat brain [gestational (G) days 18 and 20, postnatal (P) days 1, 10, 21, and greater than 40; term = approximately G21] and characterized the abundance of Glut 1 in isolated brain microvessels by Western blot analysis. Further quantitation of total glucose transporters by [125I]3-iodo-4-azido-phenethyl-amido-7-O-succinyldeacetyl-forskoli n photoaffinity labeling was performed. Glut 1 was prominently distributed in G18 and P1 brain vascular endothelial cells, with comparatively little immunoreactivity observed in brain parenchyma. Conversely, at P10, Glut 1 was prominent in brain parenchyma (undifferentiated cells) and less evident in the vascular endothelium. However, at P21, a resurgence of Glut 1 in vascular endothelial cells was observed, as was a sustained presence in parenchymal cells. On P greater than 40, a distribution in vascular endothelial cells and, to a minor extent, in parenchymal perivascular stellate cells was noted. Microvessel preparation Glut 1 (approximately 55-60 kDa) and [125I] 3-iodo-4-azido-phenethyl-amido-7-O-succinyldeacetyl-forskolin photoaffinity-labeled glucose transporters gradually increased from G18 through P10 to P greater than 40. This developmental increase in Glut 1 was also seen in whole homogenates (approximately 45-47 kDa), but not in crude brain membranes. Thus, isolated microvascular preparations indicated that Glut 1 levels constantly increased with maturation, but direct visualization of brain sections revealed that the localization in different cellular compartments changed with development. These alterations in cellular localization of glucose transporters, appear to occur in concert with the changing glucose metabolic needs of the brain, which, during the various stages of development, exhibits constantly changing phases of cellular growth and proliferation.

Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation

Glucose transport in 3T3L1 adipocytes is mediated by two facilitated diffusion transport systems. We examined the effect of chronic glucose deprivation on transport activity and on the expression of the HepG2 (GLUT 1) and adipocyte/muscle (GLUT 4) glucose transporter gene products in this insulin-sensitive cell line. Glucose deprivation resulted in a maximal increase in 2-deoxyglucose uptake of 3.6-fold by 24 h. Transport activity declined thereafter but was still 2.4-fold greater than the control by 72 h. GLUT 1 mRNA and protein increased progressively during starvation to values respectively 2.4- and 7.0-fold greater than the control by 72 h. Much of the increase in total immunoreactive GLUT 1 protein observed later in starvation was the result of the accumulation of a non-functional or mistargeted 38 kDa polypeptide. Immunofluorescence microscopy indicated that increases in GLUT 1 protein occurred in presumptive plasma membrane (PM) and Golgi-like compartments during prolonged starvation. The steady-state level of GLUT 4 protein did not change during 72 h of glucose deprivation despite a greater than 10-fold decrease in the mRNA. Subcellular fractionation experiments indicated that the increased transport activity observed after 24 h of starvation was principally the result of an increase in the 45-50 kDa GLUT 1 transporter protein in the PM. The level of the GLUT 1 transporter in the PM and low-density microsomes (LDM) was increased by 3.9- and 1.4-fold respectively, and the GLUT 4 transporter content of the PM and LDM was 1.7- and 0.6-fold respectively greater than that of the control after 24 h of glucose deprivation. These data indicate that newly synthesized GLUT 1 transporters are selectively shuttled to the PM and that GLUT 4 transporters undergo translocation from an intracellular compartment to the PM during 24 h of glucose starvation. Thus glucose starvation results in an increase in glucose transport in 3T3L1 adipocytes via a complex series of events involving increased biosynthesis, decreased turnover and subcellular redistribution of transporter proteins.