Overexpression Of GLUT4 Research Articles

Abstract 19265: Adipose Tissue Overexpression of a Constitutively Active Isoform of Chrebp Protects Against Obesity and Insulin Resistance in Mice

Obesity is associated with increased risk for T2DM and CVD in humans, and nutrients typically consumed in excess in the Western diet, such as various saturated fats, cholesterol, and simple sugars, are thought to regulate the activity of metabolic genes that modulate risk for obesity and insulin resistance. The glucose-sensing transcription factor carbohydrate response element binding protein (ChREBP) has been shown to regulate hepatic intracellular glucose and lipid homeostasis independent of the effects of insulin. Recent publications have reported that white adipose tissue (WAT) and brown adipose tissue (BAT) depots also highly express ChREBP, that ChREBP expression is upregulated during adipocyte differentiation, and that increased ChREBP activation in adipocytes is associated with obesity, but not insulin resistance, in the context of adipose tissue-specific GLUT4 overexpression in mice. However, independent modulation of ChREBP expression in adipose tissue has not yet been achieved. To better understand the impact of adipocyte-specific ChREBP activity on intracellular and whole-body metabolism, we have generated a novel transgenic mouse model that overexpresses a constitutively active ChREBP (caChREBP) isoform in adipose tissue (ap2-Cre;eGFPflox-caChREBP mice). When fed either standard chow diet or Western-type diet (WD) for 10 weeks, total fat mass, but not lean mass, was significantly decreased in male ap2-Cre;eGFPflox-caChREBP mice versus ap2-Cre littermate controls. Both glucose tolerance testing and insulin tolerance testing revealed no differences between groups of chow-fed mice, but significantly improved insulin sensitivity in WD-fed male ap2-Cre;eGFPflox-caChREBP mice. RER during the daily active cycle was increased in WD-fed ap2-Cre;eGFPflox-caChREBP mice, implying greater utilization of carbohydrate for energy expenditure. Transgenic overexpression of caChREBP resulted in increased expression of lipogenic genes, but not genes involved in adipocyte differentiation, in gonadal fat. Taken together, these data indicate that upregulation of ChREBP activity in adipose tissue may defend against WD-induced obesity and insulin resistance, despite increased adipocyte expression of lipogenic genes.

Novel mechanisms by which fat cells regulate systemic insulin sensitivity and diabetes risk

The adipose cell functions as an endocrine organ in addition to its role in energy storage. Adipocytes secrete hormones, cytokines and other factors that influence energy balance, glucose homeostasis, insulin sensitivity and vascular biology through effects in peripheral tissues and the central nervous system. In humans with obesity and type 2 diabetes, expression of the Glut4 glucose transporter is down-regulated selectively in adipocytes and this has systemic metabolic effects. Knocking out Glut4 selectively in adipocytes in mice causes insulin-resistant and increases the risk of diabetes while adipose-specific Glut4 overexpression confers enhanced glucose tolerance and insulin sensitivity in spite of increased adiposity. To discover novel pathways that regulate glucose homeostasis and insulin sensitivity, we used DNA microarray analysis of adipose tissue from mice with adipose-specific alterations in Glut4 expression. We found novel adipocyte-secreted proteins that alter systemic insulin sensitivity. In addition, gene set enrichment analysis showed coordinate regulation of lipogenic genes in adipose tissue. This led to the discovery that the glucose-responsive transcription factor, Carbohydrate responsive-element binding protein (ChREBP), is a dominant regulator of lipogenesis in adipose tissue and that its expression in adipose tissue is highly associated with insulin sensitivity in humans even in the presence of obesity. Furthermore, we identified a novel, potent ChREBP isoform and defined a new “feed forward” mode of regulation of ChREBP by which adipose-lipogenesis regulates systemic glucose homeostasis. This could provide new strategies for prevention and treatment of insulin resistance and type 2 diabetes.

Expression and prognostic value of glucose transporters and hexokinases in tonsil and mobile tongue squamous cell carcinoma.

The aim of this study was to assess the expression pattern and prognostic value of the high affinity glucose transporters GLUT-1, 3, 4, 8 and 9, SGLT-1 and of hexokinases (HK) I, II and III in squamous cell carcinoma of the tonsil and mobile tongue (TTSCC) by means of immunohistochemistry. Seventy-one consecutive patients suffering from TTSCC were included. The intensity and amount of positive tumour cells in the immunoreaction (histology score (H-score)) for GLUT-1, 3, 4, 8 and 9 as well as for HK-I, II and III were assessed independently by two experienced observers, blinded to the clinical results. H-scores as well as clinical variables were related to patient outcome. Median follow-up time was 49 months (range 1-123 months). Mean H-scores for GLUT expression in decreasing order of magnitude were respectively 10.99 for GLUT-1 (sd 3.9), 5.7 for GLUT-8 (sd 4.0), 5.4 for GLUT-3 (sd 3.7), 1.0 for GLUT-4 (sd 2.0), 1.1 (sd 1.3) for SGLT-1, and 0.4 for GLUT-9 (sd 0.6); GLUT-1 > GLUT-8 = GLUT-3 > GLUT-4 = GLUT-9 = SGLT-1 (with > meaning significantly (p<0.05 on ANOVA + posthoc Bonferroni correction) higher than and =, meaning not significantly different from). Mean H-scores for hexokinase expression were respectively 5.8 for HK-I (sd 3.5), 4.6 for HK-II (sd 3.0) and 2.0 for HK-III (sd 2.0); HK-I > HK-II > HK-III. Finally high H-scores for GLUT-4 were favourably related to disease-free and overall survival on multivariate analysis. To conclude, TTSCC expresses a wide variety of glucose transporter systems and hexokinase enzymes with the "housekeeping" GLUT-1 and HK-I being the most intensely expressed. GLUT-4 over-expression appears to confer a favourable prognosis in squamous cell carcinoma of the tonsil and mobile tongue. Additional studies confirming this finding in larger cohorts of patients are mandatory.

Islet apoptosis inhibition secondary to glut4 overexpression in skeletal muscles of GK rats after Roux-en-Y gastric bypass surgery

Journal of the American College of Surgeons: September 2011 - Volume 213 - Issue 3 - p S14 doi: 10.1016/j.jamcollsurg.2011.06.011

Abstract 1268: Aberrant plasma membrane localization of GLUT4 sustains the glycolytic phenotype in multiple myeloma

Abstract The incurable plasma cell malignancy multiple myeloma (MM) is characterized by the progressive development of chemoresistance, thus warranting the design of new therapeutic strategies tailored to unique molecular mechanisms driving tumor propagation. One aspect of myeloma pathogenesis which has not been exploited therapeutically is an abnormal avidity for glucose. Inhibition of glucose metabolism has broad applicability in cancer and has demonstrated in vitro potency; unfortunately, previous attempts focusing on hexokinase inhibition have been largely unsuccessful. Targeting the upstream process of glucose transport has not been a major area of investigation due to the common association between GLUT1 and cancer and the importance of GLUT1 in many normal tissues. However, the specific glucose transporters responsible for maintaining the glycolytic phenotype in myeloma have not been identified, leaving open the possibility that family members other than GLUT1 play important roles in this cellular context. Therefore, we undertook an unbiased real time PCR-based screen of GLUT gene expression profiles in myeloma cell lines and control B lymphocytes. GLUTs 4, 8, and 11 exhibit widespread overexpression in MM cells with no significant difference noted in GLUT1 levels. Examination of microarray studies of MM patient samples and western blot analyses of MM cell lines and normal B cells confirm the upregulation of GLUT8 and GLUT11 in clinical specimens and at the protein level. However, we did not find further evidence for GLUT4 overexpression. After evaluating the subcellular localization of GLUT4 through microscopy, we determined that a substantial fraction of GLUT4 protein is constitutively mislocalized to the plasma membrane in myeloma cells, an event normally regulated by insulin in non-malignant tissues. Indeed, cell fractionation studies demonstrate a dramatic increase in plasma membrane GLUT4 content in MM cell lines relative to PBMC. Lentiviral transduction of GLUT4-targeted shRNA verifies the functional relevance of GLUT4 activity, as MM cells with reduced GLUT4 expression exhibit dramatic deficiencies in glucose transport and lactate production rates, which leads to growth inhibition and significant cell death. Similar studies with a GLUT1-targeted shRNA reveal comparable trends but less potent effects. Next, we investigated a class of HIV protease inhibitors which elicit off-target inhibitory effects on GLUT4 in vivo. Treatment with these compounds recapitulates the effects of GLUT4 knockdown, thus supporting the relevance of GLUT4 in myeloma. These studies represent the first time a prominent, functional role for GLUT4 has been demonstrated in any cancer and highlight a therapeutic strategy involving the repositioning of an FDA-approved class of drugs to target myeloma cell metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1268. doi:10.1158/1538-7445.AM2011-1268

AMP-Activated Protein Kinase Regulates GLUT4 Transcription by Phosphorylating Histone Deacetylase 5

Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms. Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription. These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Farnesoid X Receptor Induces GLUT4 Expression Through FXR Response Element in the GLUT4 Promoter

GLUT4, the main insulin-responsive glucose transporter, plays a critical role in maintaining systemic glucose homeostasis and is subject to complicated metabolic regulation. GLUT4 expression disorder might cause insulin resistance, and over-expression of GLUT4 has been confirmed to ameliorate diabetes. Here, we reported that farnesoid X receptor (FXR) and its agonist chenodeoxycholic acid (CDCA) could induce GLUT4 transcription in 3T3-L1 and HepG2 cells. Furthermore, CDCA could increase the GLUT4 protein amount in C57BL/6J mice sex-dependently. The following progressive 5''-deletion analysis and site-mutation investigation further suggested that FXR could induce GLUT4 expression through FXR response element (FXRE) in the GLUT4 promoter. EMSA and knock-down of retinoid X receptor (RXR) indicated that FXR binds to the GLUT4-FXRE as a monomer and RXR does not participate in the FXR stimulation of GLUT4 expression. In addition, we demonstrated that FXR does not interfere with insulin-induced GLUT4 translocation to plasma membrane. All these data thereby implied that FXR is a new transcription factor of GLUT4, further elucidating the potential role for FXR in glucose metabolism.

Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

Exercise increases MEF2‐ and GEF DNA‐binding activities in human skeletal muscle

Overexpression of GLUT4 exclusively in skeletal muscle enhances insulin action and improves glucose homeostasis. Transgenic studies have discovered two regions on the GLUT4 promoter conserved across several species that are required for normal GLUT4 expression in skeletal muscle. These regions contain binding motifs for the myocyte enhancer factor 2 (MEF2) family and GLUT4 enhancer factor (GEF). A single bout of exercise increases both GLUT4 transcription and mRNA abundance; however, the molecular mechanisms mediating this response remain largely unexplored. Thus, the aim of this study was to determine whether a single, acute bout of exercise increased the DNA-binding activities of MEF2 and GEF in human skeletal muscle. Seven subjects performed 60 min of cycling at approximately 70% of VO2peak. After exercise, the DNA-binding activities of both the MEF2A/D heterodimer and GEF were increased (P<0.05). There was no change in nuclear MEF2D or GEF abundance after exercise, but nuclear MEF2A abundance was increased (P<0.05). These data demonstrate that exercise increases MEF2 and GEF DNA binding and imply that these transcription factors could be potential targets for modulating GLUT4 expression in human skeletal muscle.

Overexpression of GLUT4 in Muscle and Heart Leads to Increased Cardiac Myosin Glycation and Early Death: Suppression by Caloric Restriction

Overexpression of GLUT4 in Muscle and Heart Leads to Increased Cardiac Myosin Glycation and Early Death: Suppression by Caloric Restriction

Control of muscle glucose uptake: test of the rate‐limiting step paradigm in conscious, unrestrained mice

The aim of this study was to test whether in fact glucose transport is rate-limiting in control of muscle glucose uptake (MGU) under physiological hyperinsulinaemic conditions in the conscious, unrestrained mouse. C57Bl/6J mice overexpressing GLUT4 (GLUT4(Tg)), hexokinase II (HK(Tg)), or both (GLUT4(Tg) + HK(Tg)), were compared to wild-type (WT) littermates. Catheters were implanted into a carotid artery and jugular vein for sampling and infusions at 4 month of age. After a 5-day recovery period, conscious mice underwent one of two protocols (n = 8-14/group) after a 5-h fast. Saline or insulin (4 mU kg(-1) min(-1)) was infused for 120 min. All mice received a bolus of 2-deoxy[(3)H]glucose (2-(3)HDG) at 95 min. Glucose was clamped at approximately 165 mg dl(-1) during insulin infusion and insulin levels reached approximately 80 microU ml(-1). The rate of disappearance of 2-(3)HDG from the blood provided an index of whole body glucose clearance. Gastrocnemius, superficial vastus lateralis and soleus muscles were excised at 120 min to determine 2-(3)HDG-6-phosphate levels and calculate an index of MGU (R(g)). Results show that whole body and tissue-specific indices of glucose utilization were: (1) augmented by GLUT4 overexpression, but not HKII overexpression, in the basal state; (2) enhanced by HKII overexpression in the presence of physiological hyperinsulinaemia; and (3) largely unaffected by GLUT4 overexpression during insulin clamps whether alone or combined with HKII overexpression. Therefore, while glucose transport is the primary barrier to MGU under basal conditions, glucose phosphorylation becomes a more important barrier during physiological hyperinsulinaemia in all muscles. The control of MGU is distributed rather than confined to a single rate-limiting step such as glucose transport as glucose transport and phosphorylation can both become barriers to skeletal muscle glucose influx.

Mechanisms regulating GLUT4 glucose transporter expression and glucose transport in skeletal muscle

Skeletal muscle is a major glucose-utilizing tissue in the absorptive state and the major glucose transporter expressed in muscle in adulthood is GLUT4. GLUT4 expression is exquisitely regulated in muscle and this seems important in the regulation of insulin-stimulated glucose uptake by this tissues. Thus, muscle GLUT4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabetes. Recent information indicates that glut4 gene transcription is regulated by a number of factors in skeletal muscle that include MEF2, MyoD myogenic proteins, thyroid hormone receptors, Kruppel-like factor KLF15, NF1, Olf-1/Early B cell factor and GEF/HDBP1. In addition, studies in vivo indicate that under normal conditions the activity of the muscle-specific GLUT4 enhancer is low in adult skeletal muscle compared with the maximal potential activity that it can attain at high levels of the MRF transcription factors, MEF2, and TRalpha1. This finding indicates that glut4 transcription may be greatly up-regulated via activation of this enhancer through an increase in the levels of expression or activity of these transcription factors. Understanding the molecular basis of the expression of glut4 will be useful for the appropriate therapeutic design of treatments for insulin-resistant states. The nature of the intracellular signals that mediate the stimulation of glucose transport in response to insulin or exercise is also reviewed.

Control of Exercise-stimulated Muscle Glucose Uptake by GLUT4 Is Dependent on Glucose Phosphorylation Capacity in the Conscious Mouse

Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during exercise because GLUT4 overexpression does not increase exercise-stimulated MGU. Instead of glucose transport, glucose phosphorylation is a primary limitation of exercise-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair exercise-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity was increased. Thus, C57BL/6J mice with hexokinase II (HKII) overexpression (HK(Tg)), a GLUT4 partial knock-out (G4(+/-)), or both (HK(Tg) + G4(+/-)) and wild-type (WT) littermates were implanted with carotid artery and jugular vein catheters for sampling and infusions at 4 months of age. After a 7-day recovery, 5-h fasted mice remained sedentary or ran on a treadmill at 0.6 mph for 30 min (n = 9-12 per group) and received a bolus of 2-deoxy[3H]glucose to provide an index of MGU (Rg). Arterial blood glucose and plasma insulin concentrations were similar in WT, G4(+/-), HKTg, and HKTg + G4(+/-) mice. Sedentary Rg values were the same in all genotypes in all muscles studied, confirming that glucose transport is a significant barrier to basal glucose uptake. Gastrocnemius and soleus Rg were greater in exercising compared with sedentary mice in all genotypes. During exercise, G4(+/-) mice had a marked increase in blood glucose that was corrected by the addition of HK II overexpression. Exercise Rg (micromol/100g/min) was not different between WT and G4(+/-) mice in the gastrocnemius (24 +/- 5 versus 21 +/- 2) or the soleus (54 +/- 6 versus 70 +/- 7). In contrast, the enhanced exercise Rg observed in HKTg mice compared with that in WT mice was absent in HKTg + G4(+/-) mice in both the gastrocnemius (39 +/- 7 versus 22 +/- 6) and the soleus (98 +/- 13 versus 65 +/- 13). Thus, glucose transport is not a significant barrier to exercise-stimulated MGU despite a 50% reduction in GLUT4 content when glucose phosphorylation capacity is normal. However, when glucose phosphorylation capacity is increased by HK II overexpression, GLUT4 availability becomes a marked limitation to exercise-stimulated MGU.

GLUT4 Overexpression or Deficiency in Adipocytes of Transgenic Mice Alters the Composition of GLUT4 Vesicles and the Subcellular Localization of GLUT4 and Insulin-responsive Aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased approximately 10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

Exercise and myocyte enhancer factor 2 regulation in human skeletal muscle.

Overexpression of GLUT4 in skeletal muscle enhances whole-body insulin action. Exercise increases GLUT4 gene and protein expression, and a binding site for the myocyte enhancer factor 2 (MEF-2) is required on the GLUT4 promoter for this response. However, the molecular mechanisms involved remain elusive. In various cell systems, MEF-2 regulation is a balance between transcriptional repression by histone deacetylases (HDACs) and transcriptional activation by the nuclear factor of activated T-cells (NFAT), peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1), and the p38 mitogen-activated protein kinase. The purpose of this study was to determine if these same mechanisms regulate MEF-2 in contracting human skeletal muscle. Seven subjects performed 60 min of cycling at approximately 70% of VO2(peak). After exercise, HDAC5 was dissociated from MEF-2 and exported from the nucleus, whereas nuclear PGC-1 was associated with MEF-2. Exercise increased total and nuclear p38 phosphorylation and association with MEF-2, without changes in total or nuclear p38 protein abundance. This result was associated with p38 sequence-specific phosphorylation of MEF-2 and an increase in GLUT4 mRNA. Finally, we found no role for NFAT in MEF-2 regulation. From these data, it appears that HDAC5, PGC-1, and p38 regulate MEF-2 and could be potential targets for modulating GLUT4 expression.

Metabolic adaptations in skeletal muscle overexpressing GLUT4: effects on muscle and physical activity.

To understand the long-term metabolic and functional consequences of increased GLUT4 content, intracellular substrate utilization was investigated in isolated muscles of transgenic mice overexpressing GLUT4 selectively in fast-twitch skeletal muscles. Rates of glycolysis, glycogen synthesis, glucose oxidation, and free fatty acid (FFA) oxidation as well as glycogen content were assessed in isolated EDL (fast-twitch) and soleus (slow-twitch) muscles from female and male MLC-GLUT4 transgenic and control mice. In male MLC-GLUT4 EDL, increased glucose influx predominantly led to increased glycolysis. In contrast, in female MLC-GLUT4 EDL increased glycogen synthesis was observed. In both sexes, GLUT4 overexpression resulted in decreased exogenous FFA oxidation rates. The decreased rate of FFA oxidation in male MLC-GLUT4 EDL was associated with increased lipid content in liver, but not in muscle or at the whole body level. To determine how changes in substrate metabolism and insulin action may influence energy balance in an environment that encouraged physical activity, we measured voluntary training activity, body weight, and food consumption of MLC-GLUT4 and control mice in cages equipped with training wheels. We observed a small decrease in body weight of MLC-GLUT4 mice that was paradoxically accompanied by a 45% increase in food consumption. The results were explained by a marked fourfold increase in voluntary wheel exercise. The changes in substrate metabolism and physical activity in MLC-GLUT4 mice were not associated with dramatic changes in skeletal muscle morphology. Collectively, results of this study demonstrate the feasibility of altering muscle substrate utilization by overexpression of GLUT4. The results also suggest that as a potential treatment for type II diabetes mellitus, increased skeletal muscle GLUT4 expression may provide benefits in addition to improvement of insulin action.

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter.

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.

GLUT4: a key player regulating glucose homeostasis? Insights from transgenic and knockout mice (review).

Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in skeletal muscle is important to develop effective strategies to prevent or reduce the incidence of Type II diabetes mellitus. In patients with Type II diabetes mellitus, reduced glucose transport in skeletal muscle is a major factor responsible for reduced whole body glucose uptake. Overexpression of GLUT4 in skeletal muscle improves glucose homeostasis in animal models of diabetes mellitus and protects against the development of diabetes mellitus. Thus, GLUT4 is an attractive target for pharmacological intervention strategies to control glucose homeostasis. This review will focus on the current understanding of the role of GLUT4 in regulating cellular glucose uptake and whole body glucose homeostasis.

Amelioration of insulin resistance but not hyperinsulinemia in obese mice overexpressing GLUT4 selectively in skeletal muscle

The effects of gold-thioglucose (GTG) treatment were examined in mice overexpressing GLUT4 selectively in skeletal muscle (MLC-GLUT4 mice) and in age-matched controls. Groups of MLC-GLUT4 and control mice were injected with GTG or saline at 5 weeks of age. At 12 weeks following the injections, GTG-treated control mice exhibited a 35% increase in body weight versus saline-treated controls. Similarly, a 30% increase in body weight was observed in GTG-treated MLC-GLUT4 mice compared with saline-treated MLC-GLUT4 mice 12 weeks after the injections. In saline-treated lean MLC-GLUT4 and control mice, intraperitoneal injection of insulin decreased blood glucose in 1 hour by 63% and 38%, respectively. Insulin also decreased blood glucose by 40% in GTG-treated obese MLC-GLUT4 mice after 1 hour. However, insulin did not reduce blood glucose levels in GTG-treated obese control mice. The ability of insulin to clear blood glucose in GTG-treated obese MLC-GLUT4 mice is associated with increased skeletal muscle GLUT4 content and white adipose tissue (WAT) GLUT4 content as compared with GTG-treated obese controls. However, fasting blood glucose levels in GTG-treated obese MLC-GLUT4 and control mice were elevated by approximately 30% compared with saline-treated groups. Lastly, although GTG-treated obese MLC-GLUT4 mice exhibited improved glucose clearance in response to insulin, they nevertheless remained as hyperinsulinemic as GTG-treated obese control mice. These results suggest that genetic overexpression of GLUT4 in skeletal muscle may ameliorate the development of insulin resistance associated with obesity but cannot restore normal glucose and insulin levels.