Stimulated 2-deoxyglucose Uptake Research Articles

OR31-4 Neonatal Vitamin D Deficiency and Insulin Resistance

Background: Vitamin D deficiency is prevalent in pregnant women and their offspring. Maternal vitamin D deficiency is associated with insulin resistance in children and can induce a proinflammatory macrophage phenotype [1, 2]. Purpose: We hypothesized that maternal vitamin D deficiency induces immune cell reprogramming that promotes adipose insulin resistance in the offspring and is detectable in the newborn period. Methods: We enrolled 32 normal pregnant women prior to delivery of AGA, term infants. Cord blood was obtained and used to determine vitamin D [25(OH)D] levels and qPCR of microRNA levels. Cord blood monocytes were co-cultured with 3T3L1 adipocytes for insulin-stimulated 2-deoxyglucose uptake studies, and the monocyte media and co-cultured adipocytes were analyzed for microRNA expression. 3T3L1 adipocytes were transfected with miR106b mimic and antagonist to confirm the effect of the 2-deoxyglucose results. Results: 83% of the newborn’s venous cord blood showed vitamin D deficiency with 25(OH)D <20 ng/mL (n=24, total n=29). Adipocytes co-cultured with cord blood monocytes showed an inverse correlation between insulin stimulated 2-deoxyglucose uptake and cord blood 25(OH)D levels (n=29, Spearman r=0.43, p=0.02). Adipocytes exposed to monocytes or to monocyte media elevated miR106b by 7 to 12-fold (n=8, p<0.01). Interestingly, cord blood plasma miR106b levels were found to inversely correlate with 25(OH)D levels (n=27, Spearman r=-0.41, p<0.04). Transfection of adipocytes with miR106b mimic reduced the 2-deoxyglucose uptake after insulin stimulation, whereas transfection with anti-miR-106b increased 2-deoxyglucose uptake after insulin stimulation, suggesting that monocyte secretion of miR106b induced adipose insulin resistance (n=8, p<0.05). Conclusions: This study provides evidence of a novel intercellular miRNA communication mechanism enabling monocytes programmed in utero by vitamin D deficiency to induce adipose insulin resistance. Identification of circulating miR106b as a mechanism of insulin resistance in offspring born with vitamin D deficiency is a potential advance toward precision medicine as it allows for optimal targeting of those at risk for diabetes later in life within a heterogeneous population. These findings serve as background for interventional studies evaluating the effect of maternal vitamin D supplementation on offspring insulin resistance later in life.

Regulation of subcellular distribution of GLUT4 in cardiomyocytes: Rab4A reduces basal glucose transport and augments insulin responsiveness.

Members of the Rab subfamily of small-GTP binding proteins have been suggested to be involved in insulin-regulated translocation of the glucose transporter GLUT4. To directly study this process in muscle tissue, we have established an insulin-sensitive cardiac cell line (H9K6) stably overexpressing GLUT4, which was derived from H9c2 cardiac myoblasts. H9K6-cells were transiently transfected with rab4A and rab3C with an efficiency of 65% and glucose uptake and the cellular distribution and expression of the transporter isoforms GLUT1 and GLUT4 was subsequently determined. Rab3C-overexpression caused no significant change in both basal and insulin-stimulated 2-deoxyglucose uptake compared to control cells transfected with the blank vector. Rab4A was barely detectable in membranes of H9K6 cells. However, after transient transfection this protein was expressed at a level comparable to adult cardiomyocytes. This resulted in a reduction of basal glucose uptake by 31% compared to control cells. Under these conditions insulin was able to stimulate 2-deoxyglucose uptake by 120%. Total expression of GLUT1 and GLUT4 was not affected by Rab4-overexpression. Cell surface biotinylation was used to quantify the abundance of GLUT1 and GLUT4 in the plasma membrane. A decrease of cell surface GLUT4 by about 40% compared to control cells was found in Rab4-overexpressing cells Insulin treatment increased cell surface-GLUT4 by 100% compared to only 26% in control cells. Distribution of GLUT1 was not affected under these conditions. Our data show that Rab4A but not Rab3C is able to reduce basal glucose uptake and cell surface content of GLUT4 in cardiac muscle cells. This results in an increased stimulation of glucose uptake by insulin which can be fully explained by enhanced translocation of GLUT4. We suggest that Rab4A participates in the redistribution of GLUT4 to intracellular pools and represents an essential determinant of the insulin responsiveness of GLUT4 translocation in cardiac muscle cells.

Peroxisome proliferator-activated receptors γ and α agonists stimulate cardiac glucose uptake via activation of AMP-activated protein kinase

Myocardial energy and glucose homeostasis are crucial for normal cardiac structure and function. Peroxisome proliferator-activated receptors (PPARs) play an important role in controlling transcriptional expression of key enzymes that are involved in glucose metabolism, and they have been demonstrated to significantly reduce tissue injury in cardiovascular diseases. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a sensor that maintains intracellular energy homeostasis and mediates a number of physiological signals. It has been reported that AMPK promotes glucose uptake. We hypothesize that PPARγ and α agonists may play a role in the regulation of glucose metabolism through AMPK. We tested this hypothesis by using isolated papillary muscles of rat hearts treated with PPARγ and α agonists, troglitazone and GW7647, respectively. Our results demonstrated that both troglitazone and GW7647 significantly stimulated 2-deoxyglucose uptake of cardiac muscles. Interestingly, both agonists stimulated phosphorylation of AMPK and its downstream protein target acetyl-CoA carboxylase. Endothelial nitric oxide synthase (eNOS) was also activated by both agonists. In addition, AMPK activator 5-amino-4-imidazole-1-β-D-carboxamide ribofuranoside increased glucose uptake, while AMPK inhibitor compound C and NOS inhibitor, N ω-nitro- l-arginine, significantly blocked troglitazone- and GW7647-stimulated glucose uptake in cardiac muscles. There was also a reduction of glucose uptake with a marked decrease in AMPK and eNOS phosphorylation. In conclusion, both PPARγ and α activation play a role in the regulation of glucose uptake in cardiac muscles and this regulation is mediated by the AMPK and eNOS signaling pathways.

Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and β2‐adrenoceptor mechanisms

Picomolar concentrations of the beta3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via beta2-adrenoceptors. Effects of BRL37344 and beta2-adrenoceptor agonists are compared. Mouse soleus muscles were incubated with 2-deoxy[1-(14)C]-glucose, [1-(14)C]-palmitate or [2-(14)C]-pyruvate, and BRL37344, beta2-adrenoceptor agonists and selective beta-adrenoceptor antagonists. Formation of 2-deoxy[1-(14)C]-glucose-6-phosphate or (14)CO2 was measured. 2-Deoxy[1-(14)C]-glucose uptake and beta-adrenoceptor mRNA were measured in C2C12 cells. 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33-54%. The effect of BRL37344 was prevented by 1 microM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 microM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 stimulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only beta2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant. Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via beta2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake.

The Cannabinoid CB1 Receptor Antagonist Rimonabant Stimulates 2-Deoxyglucose Uptake in Skeletal Muscle Cells by Regulating the Expression of Phosphatidylinositol-3-kinase

The endocannabinoid system regulates food intake, energy, and glucose metabolism at both central and peripheral levels. We have investigated the mechanism by which it may control glucose uptake in skeletal muscle cells. Detectable levels of the cannabinoid receptor type 1 (CB1) were revealed in L6 cells. Exposure of differentiated L6 myotubes to the CB1 antagonist rimonabant (SR141716) selectively increased 2-deoxyglucose uptake (2-DG) in a time- and dose-dependent manner. A similar effect was induced by genetic silencing of CB1 by small interfering RNA. Protein expression profiling revealed that both the regulatory p85 and the catalytic p110 subunits of the phosphatidylinositol-3-kinase (PI3K) were increased by SR141716. No significant change in the cellular content of other known molecules regulating PI3K was observed. However, phosphoinositide-dependent kinase-1, Akt/protein kinase B, and protein kinase Czeta activities were rapidly induced after SR141716 treatment of L6 cells in a PI3K-dependent manner. The stimulatory effect of SR141716 on PI3K expression and activity was largely prevented by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of the cAMP-dependent protein kinase. Moreover, SR141716-stimulated 2-DG uptake was blunted by the coincubation either with H-89 or with the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), both in L6 cells and in mouse primary myocytes. Thus, modulation of CB1 regulates glucose uptake at the level of the PI3K signaling system in skeletal muscle cells. Interfering with CB1 signaling may therefore ameliorate glucoregulatory functions in peripheral tissues.

Effects of olanzapine on glucose transport, proliferation and survival in C2C12 myoblasts

The aim of our study was to investigate the direct effects of atypical antipsychotics on muscle cell functions in order to ascertain the diabetic liability of these drugs. We investigated the effects of olanzapine, clozapine and alpha-methyl-5-hydroxytryptamine on basal glucose uptake and glucose uptake in response to insulin using in vitro cultures of mouse skeletal muscle satellite cells (C2C12). We extended our study to the effects of these compounds on cell proliferation, survival and differentiation into myotubes and on the growth of differentiated myotubes. Olanzapine and alpha-methyl-5-HT stimulated 2-deoxyglucose uptake in C2C12 myoblasts in a dose-dependent manner (minimal effective dose: 2 microM olanzapine and 10 microM alpha-methyl-5-HT). The treatment with clozapine had no effect on glucose transport. Insulin and olanzapine increased the plasma membrane (PM) abundance of glucose transporter GLUT4. We investigated whether protein kinase Akt (PKB) and AMP-dependent kinase may participate in mediating olanzapine effects on glucose transport. Clozapine and olanzapine did not induce DNA laddering in differentiating myoblasts and differentiated myotubes and did not affect myotube growth. Olanzapine-induced glucose disposal in vitro is consistent with the acute lowering of plasma glucose/insulin concentrations that occurs in rats before olanzapine-induced overeating [Albaugh, V.L., Henry, C.R., Bello, N.T., Hajnal, A., Lynch, S.L., Halle, B., Lynch, C.J., 2006. Hormonal and metabolic effects of olanzapine and clozapine related to body weight in rodents. Obesity 14, 36-50].

Skeletal Muscle Insulin Resistance in Rats Fed a High Fat Diet and Treated with Acipimox

The mechanism by which a high fat diet causes insulin resistance is not yet understood. It has been proposed by some investigators that elevations in the circulating levels of free fatty acids are responsible for the development of insulin resistance. PURPOSE: The purpose of this study was to determine if the skeletal muscle insulin resistance induced by a high fat diet would persist in the context of a weeklong treatment to reduce plasma FFAs with acipimox. METHODS: Rats were fed standard chow or a high fat diet in which 50% of calories come from fat (lard and corn oil) for a period of 4 weeks. During the 5th week, a subset of the rats fed a high fat diet were given 2 doses (100mg/kg) per day of Acipimox, which inhibits lypolysis and reduces plasma FFA concentrations. Glucose uptake, Glucose tolerance, fat pad weights, and plasma FFA were measured to determine the metabolic profile and insulin resistance in these animals. RESULTS: the concentration of plasma FFA's were reduced in response to Acipimox treatment in high fat fed rats (0.75±0.08 and 0.38±0.07 mM in High fat fed and High fat+acipimox, respectively). Whole animal glucose tolerance was improved with acipimox treatment, and was not significantly different from that of the chow animals. Skeletal muscle insulin resistance was evident in the animals fed a high fat diet alone as well as rats that received acipimox. The insulin stimulated 2-Deoxyglucose uptake in the epitrochlearis muscle was 1.17±0.11, 0.82±0.11, and 0.68±0.10 μmol 2DG/ml/20min in chow, HF and Acip+HF groups respectively. High fat feeding caused a significant increase in total abdominal fat pad weights and acipimox treatment caused a further increase in fat pad mass (7.01±0.32, 12.80±0.71 and 15.75±0.43 gms fat in Chow, High Fat and High Fat+ acipimox, respectively). CONCLUSION: Lowering of plasma FFA with Acipimox improved overall insulin action, yet did not improve insulin resistance measured in epitrochlearis muscle.

Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

Rad is a low molecular weight GTPase that is overexpressed in skeletal muscle of some patients with type 2 diabetes mellitus and/or obesity. Overexpression of Rad in adipocytes and muscle cells in culture results in diminished insulin-stimulated glucose uptake. To further elucidate the potential role of Rad in vivo, we have generated transgenic (tg) mice that overexpress Rad in muscle using the muscle creatine kinase (MCK) promoter-enhancer. Rad tg mice have a 6- to 12-fold increase in Rad expression in muscle as compared to wild-type littermates. Rad tg mice grow normally and have normal glucose tolerance and insulin sensitivity, but have reduced plasma triglyceride levels. On a high-fat diet, Rad tg mice develop more severe glucose intolerance than the wild-type mice; this is due to increased insulin resistance in muscle, as exemplified by a rightward shift in the dose-response curve for insulin stimulated 2-deoxyglucose uptake. There is also a unexpected further reduction of the plasma triglyceride levels that is associated with increased levels of lipoprotein lipase in the Rad tg mice. These results demonstrate a potential synergistic interaction between increased expression of Rad and high-fat diet in creation of insulin resistance and altered lipid metabolism present in type 2 diabetes.

Synthesis and in Vitro Evaluation of18F- and19F-Labeled Insulin: A New Radiotracer for PET-based Molecular Imaging Studies

A new and regioselective strategy was developed for the preparation of fluorine-18-labeled insulin as a novel positron emission tomography (PET) tracer. [18F]-4-Fluorobenzoic acid (4-18FBA), which was produced in 83 +/- 8% yield (n = 10), through the use of succinimidyl [18F]-4-fluorobenzoate (4-(18)FSB), was conjugated through a short spacer (6-aminohexanoic acid, AHx) to the PheB1 residue of a protected form of insulin. 18FB-AHx-insulin (8b) was repeatedly prepared in practical quantities (10-20 mCi, 370-740 MBq) in good radiochemical yield (9 +/- 5%, n = 9) and in a specific activity of 7.8 mCi/micromol. The final product was characterized by comparing the radioHPLC and radioTLC of 8b with that of the 19F-analogue (19FB-AHx-insulin, 8a) and by analyzing a carrier-added synthesis by mass spectrometry. Dithiothreitol and endoproteinase Glu-C digestion experiments on 8a confirmed that the prosthetic group was in fact conjugated to the PheB1 residue. An insulin receptor (IR) phosphorylation assay using CHO-hIR cells overexpressing recombinant human insulin receptors indicated no statistical difference in the extent of autophosphorylation stimulated by 8a as compared to that for human insulin (EC50 values of 0.82 nM and 1.0 nM, respectively). The stimulation of 2-deoxyglucose uptake in 3T3-L1 mouse adipocytes utilizing 8a versus unmodified human insulin gave similar EC50 values of 0.68 nM and 0.41 nM, respectively. The IC50 values for 8a versus native insulin for the displacement of 125I-insulin from HEK-293 cells were also the same within experimental error (2.6 nM for 8a versus 2.4 nM for unmodified human insulin). These results support the use of the 18F-insulin analogue as a PET tracer for imaging the distribution of insulin in vivo.

The v-SNARE Vti1a Regulates Insulin-stimulated Glucose Transport and Acrp30 Secretion in 3T3-L1 Adipocytes

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 μM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-β-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.

Discovery of a novel protein tyrosine phosphatase-1B inhibitor, KR61639: potential development as an antihyperglycemic agent

Protein tyrosine phosphatase-1B (PTP-1B), a negative regulator of insulin signaling, may be an attractive therapeutic target for type 2 diabetes mellitus. High throughput screening (HTS) for PTP-1B inhibitors using compounds from the Korea Chemical Bank identified several hits (active compounds). Among them, a hit with 1,2-naphthoquinone scaffold was chosen for lead development. KR61639, {4-[1-(1 H-indol-3-yl)-3,4-dioxo-3,4-dihydro-naphthalen-2-ylmethyl]-phenoxy}-acetic acid tert-butyl ester, inhibited human recombinant PTP-1B with an IC 50 value of 0.65 μM in a noncompetitive manner. KR61639 showed modest selectivity over several phosphatases and increased insulin-stimulated glycogen synthesis in HepG2 cells and stimulated 2-deoxyglucose uptake in 3T3/L1 adipocytes. In addition, in vivo study using ob/ob mouse demonstrated that KR61639 exerted a hypoglycemic action when given orally. Thus, KR61639 may be a good starting point for lead optimization in developing a novel antidiabetic agent.

Circadian Rhythm of Glucose Uptake in Cultures of Skeletal Muscle Cells and Adipocytes in Wistar-Kyoto, Wistar, Goto-Kakizaki, and Spontaneously Hypertensive Rats

Hypertension and noninsulin-dependent diabetes mellitus are usually associated with marked glucose intolerance. Hypertensive and even nonhypertensive diabetic individuals display disturbances of the normal circadian blood pressure rhythm. However, little is known about circadian changes of the glucose uptake in muscle and fat cells, the major glucose utilizing tissues. Therefore, we investigated circadian rhythms of glucose uptake in primary muscle and fat cell cultures of hypertensive and type II diabetic rats and their respective control strains. 2-Deoxy-d-(1-3H)glucose uptake was measured over 48 h after synchronization of cells by means of medium change with and without addition of insulin, phloretine, and/or staurosporine. The circadian changes of glucose uptake were assessed by fitting cosine curves to the uptake values. Insulin stimulation of deoxyglucose uptake was only present in control animals, not in hypertensive and diabetic rats. Deoxyglucose uptake displayed a circadian rhythm in control animals, and was markedly disturbed in hypertensive and diabetic animals. Blocking of glucose transporters by phloretine abolished the circadian pattern of deoxyglucose uptake indicating a role of glucose transporters in its generation. Inhibition of kinases by staurosporine inhibited the insulin-stimulated deoxyglucose uptake, but did not dampen the circadian rhythmicity of basal deoxyglucose uptake. The generation of the circadian rhythm of glucose uptake in muscle and fat cell cultures is therefore probably insulin independent and independent of protein kinases. In summary, our results show for the first time: (a) a circadian rhythm of deoxyglucose uptake in glucose utilizing muscle and fat cells in vitro, (b) a disruption of this rhythm in cells of hypertensive and diabetic rats.

Cdc42 Is a Rho GTPase Family Member That Can Mediate Insulin Signaling to Glucose Transport in 3T3-L1 Adipocytes

We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active G(q) (CA-G(q); Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-G(q) expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G alpha(q/11) in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-G(q), and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C lambda (PKC lambda) antibody or overexpression of a kinase-deficient PKC lambda construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active G(q) expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC lambda inhibition blocks CA-cdc42, CA-G(q), and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G alpha(q/11) and upstream of PI3-kinase and PKC lambda in this stimulatory pathway.

Stimulation of glucose uptake in muscle cells by prolonged treatment with scriptide, a histone deacetylase inhibitor.

Glucose incorporation is regulated mainly by GLUT4 in skeletal muscles. Here we report that treatment of L6 myotubes with scriptide, a hydroxamic acid-based histone deacetylase (HDAC) inhibitor, stimulated 2-deoxyglucose uptake. The effect appeared only after 24 hr, resulting in 2.4-fold glucose uptake at treatment day 6. Scriptide acted synergistically with insulin, indicating it stimulated a distinct pathway from the insulin signaling pathway. It was not observed in undifferentiated myoblasts or 3T3-L1 adipocytes, suggesting a muscle-specific effect of scriptide. A five-carbon chain and hydroxamic acid, essential for histone deacetylase inhibition, were indispensable for this effect, and trichostatin A stimulated glucose uptake as well. Scriptide increased the cellular content of GLUT4, and induced GLUT4 translocation, but GLUT4 mRNA level did not change, indicating scriptide functions posttranslationally. Our results indicated a novel function for HDAC inhibitors of increasing GLUT4 content and its translocation in muscle cells, resulting in stimulation of glucose uptake.

Molecular characterization and partial cDNA cloning of facilitative glucose transporters expressed in human articular chondrocytes; stimulation of 2-deoxyglucose uptake by IGF-I and elevated MMP-2 secretion by glucose deprivation

Objective Recent evidence suggests that human chondrocytes express several facilitative glucose transporter (GLUT) isoforms and also that 2-deoxyglucose transport is accelerated by cytokine stimulation. The aim of the present investigation was to determine if human articular chondrocytes express any of the recently identified members of the GLUT/SLC2A gene family and to examine the effects of endocrine factors, such as insulin and IGF-I on the capacity of human chondrocytes for transporting 2-deoxyglucose. Design/methods PCR, cloning and immunohistochemistry were employed to study the expression of GLUT/SLC2A transporters in normal human articular cartilage. The uptake of 2-deoxyglucose was examined in monolayer cultured immortalized human chondrocytes following stimulation with TNF-α, insulin and IGF-I. Levels of MMP-2 were assessed by gelatin zymography following glucose deprivation of alginate cultures. Results Using PCR we detected transcripts for eight glucose transporter isoforms (GLUTs 1, 3, 6, 8, 9, 10, 11 and 12) and for a fructose transporter (GLUT5) in human articular cartilage. Expression of GLUT1, GLUT3 and GLUT9 proteins in normal human articular cartilage was confirmed by immunohistochemistry. The uptake of 2-deoxyglucose was dependent on time and temperature, inhibited by cytochalasin B and phloretin, and significantly accelerated in chondrocyte cultures stimulated with IGF-I. However, 2-deoxyglucose uptake was unaffected by short and long-term insulin treatment, which ruled out a functional role for insulin-sensitive GLUT4-mediated glucose transport. Furthermore, secretion of MMP-2 was increased in alginate cultures deprived of glucose. Conclusions The data supports a critical role for glucose transport and metabolism in the synthesis and degradation of cartilage. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

Need for GLUT4 activation to reach maximum effect of insulin-mediated glucose uptake in brown adipocytes isolated from GLUT4myc-expressing mice.

There is a need to understand whether the amount of GLUT4 at the cell surface determines the extent of glucose uptake in response to insulin. Thus, we created a heterozygous mouse expressing modest levels of myc-tagged GLUT4 (GLUT4myc) in insulin-sensitive tissues under the control of the human GLUT4 promoter. Insulin stimulated 2-deoxyglucose uptake 6.5-fold in isolated brown adipocytes. GLUT1 did not contribute to the insulin response. The stimulation by insulin was completely blocked by wortmannin and partly (55 +/- 2%) by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Insulin increased surface exposure of GLUT4myc twofold (determined by fluorescent or enzyme-linked myc immunodetection in intact adipocytes). Such increase was completely blocked by wortmannin but insensitive to SB203580. Insulin increased the kinase activity of the p38 MAPK beta-isoform 1.9-fold without affecting p38-alpha. In summary, the GLUT4myc mouse is a promising model for measuring GLUT4 translocation in intact primary cells. It affords direct comparison between GLUT4 translocation and glucose uptake in similar cell preparations, allowing one to study the regulation of GLUT4 activity. Using this animal model, we found that stimulation of glucose uptake into brown adipocytes involves both GLUT4 translocation and activation.

G(alpha)11 signaling through ARF6 regulates F-actin mobilization and GLUT4 glucose transporter translocation to the plasma membrane.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

Sibutramine metabolites increase glucose transport by cultured rat muscle cells.

The anti-obesity agent sibutramine, a serotonin and noradrenaline reuptake inhibitor (SNRI), has been shown to reduce insulin resistance and improve glycaemic control in obese-diabetic ob/ob mice and overweight type 2 diabetic patients. To investigate whether sibutramine or its metabolites act directly on muscle cells to improve glucose uptake and insulin action. Uptake of the non-metabolized glucose analogue 2-deoxyglucose was measured in cultured L6 rat muscle cells after incubation with sibutramine, its two pharmacologically active metabolites and related agents. Sibutramine itself (10(-8)-10(-6) M) did not significantly affect 2-deoxyglucose uptake during incubations up to 72 h. The primary amine metabolite M2 (10(-7) and 10(-6) M) increased basal and insulin-stimulated 2-deoxyglucose uptake (by 12% and 34%) after 24 h incubation. These effects of M2 were lost by 72 h incubation. However, the secondary amine metabolite M1 (10(-6) M) increased basal and insulin-stimulated 2-deoxyglucose uptake (by 50%) after 72 h incubation, although M1 was ineffective after 24 h. M2 stimulated 2-deoxyglucose uptake in the presence of LY-294,002 (an inhibitor of phosphatidylinositol 3-kinase) but the effect of M2 was inhibited by cytochalasin B, which acutely blocks glucose transporters. Incubations with serotoninergic, noradrenergic and dopaminergic agents, or agents known to stimulate release or inhibit reuptake of these substances in nervous tissues indicated that the sibutramine metabolites were not affecting 2-deoxyglucose uptake via mechanisms associated with their SNRI properties. Sibutramine metabolites can improve insulin-sensitive 2-deoxyglucose uptake by cultured muscle cells independently of SNRI effects.

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.