Effect of hydroxytyrosol and olive leaf extract on 1,2-dicarbonyl compounds, hydroxymethylfurfural and advanced glycation endproducts in a biscuit model.

The antiglycative activity of hydroxytyrosol (HT) and olive leaf extract (OLE) was investigated in wheat-flour biscuits. Quercetin (QE) and gallic acid (GA) were used as reference of antiglycative activity of phenolic compounds. HT, OLE, QE and GA were added in the range of 0.25-0.75% (w/w). Samples were compared against a control recipe baked at 180°C/20min. HT biscuit was able to inhibit efficiently the formation of hydroxymethylfurfural (HMF) and 3-deoxyglucosone (3-DG), as well as reduced the formation of overall free fluorescent AGEs and pentosidine. The inhibition of the 3-DG and HMF formation was directly and significantly correlated under controlled baking conditions. However, samples formulated with OLE exerted similar antiglycative capacity against pentosidine and Nε-carboxyethyl-lysine, although the amount of HT in the biscuit was 100-fold lower than the biscuit formulated with HT. Methylglyoxal, 3-DG, and glyoxal were the predominant 1,2-dicarbonyl compounds after baking but only 3-DG was significantly reduced by HT.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

Low levels of 1,5-anhydro-D-glucitol are associated with vascular endothelial dysfunction in type 2 diabetes.

BACKGROUND: Vascular endothelial dysfunction is involved in macrovascular disease progression in type 2 diabetes mellitus (T2DM). We reported previously that blood glucose fluctuations, as evaluated by continuous glucose monitoring (CGM), correlate with vascular endothelial function, serving as a marker of vascular endothelial function. However, the use of CGM is limited, suggesting the need for another marker of vascular endothelial function. Here, we investigated the relationship between vascular endothelial dysfunction and blood levels of 1,5-anhydro-D-glucitol (1,5-AG), a marker of both postprandial hyperglycemia and fluctuations in blood glucose. METHODS: In 32 inpatients with T2DM and HbA1c less than 8.0%, the reactive hyperemia index (RHI), an index of vascular endothelial function, was determined by peripheral arterial tonometry. The relationships between RHI and 1,5-AG, blood glucose, lipid metabolism markers, and blood pressure, were examined. RESULTS: There was a strong correlation between 1,5-AG and natural logarithmic-scaled RHI (L_RHI) (r = 0.55; P = 0.001). However, there was no correlation between L_RHI and HbA1c, fasting blood glucose, IRI, LDL-C, HDL-C, TG, systolic blood pressure, or diastolic blood pressure. Multivariate analysis identified blood 1,5-AG levels to be the only significant and independent determinant of L_RHI. CONCLUSIONS: In T2DM with HbA1c <8.0%, low 1,5-AG levels were associated with vascular endothelial dysfunction, suggesting it is a potentially useful marker for vascular endothelial dysfunction. TRIAL REGISTRATION: UMIN000015317.

Pinusolide improves high glucose-induced insulin resistance via activation of AMP-activated protein kinase.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a crucial role in the maintenance of cellular energy homeostasis, and several natural compounds that activate AMPK possibly enhance glucose uptake by muscle cells. In this study, we found that pinusolide stimulated AMPK phosphorylation and glucose uptake and these effects were significantly reduced by siRNA LKB1 or compound C, suggesting that enhanced glucose uptake by pinusolide is predominantly accomplished via an LKB1-mediated AMPK activation pathway. An insulin resistance state was induced by exposing cells to 30mM glucose, as indicated by reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and glucose uptake. Under these conditions, the phosphorylation of AMPK and ACC were decreased. Surprisingly, disrupted insulin signaling and decreased AMPK activity by high glucose concentrations were prevented by pinusolide. Moreover, this treatment increased insulin-stimulated glucose uptake via AMPK activation. Taken together, our findings suggest a link between high glucose and insulin resistance in muscle cells, and provide further evidence that pinusolide attenuates blockade of insulin signaling by enhancing IRS-1 tyrosine phosphorylation by the activating the AMPK pathway. In addition, this study indicates the targeting of AMPK represents a new therapeutic strategy for hyperglycemia-induced insulin resistance and type 2 diabetes.

Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes.

Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland, therefore, plays a critical role in milk synthesis. Facilitative glucose transporters (GLUT) mediate glucose uptake in the mammary gland. Glucose transporter 1 (GLUT1) is the major facilitative glucose transporter expressed in the bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. Glucose transporter 1 is, therefore, thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 (bGLUT1) was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B, and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-D-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a Michaelis constant (K(m)) of 9.8 ± 3.0mM for 2-deoxy-d-glucose, similar to 11.7 ± 3.7 mM for human GLUT1. Transport by bGLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bGLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bGLUT1 in taking up glucose across mammary epithelial cells for milk synthesis.

Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells.

Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, exerts antitumoral and antiproliferative action. In this study, the addition of metformin to 2-deoxyglucose (2DG) inhibited mitochondrial respiration and glycolysis in prostate cancer cells leading to a severe depletion in ATP. The combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone, leading to 96% inhibition of cell viability in LNCaP prostate cancer cells. In contrast, a moderate effect on cell viability was observed in normal prostate epithelial cells. At the cellular level, the combination of metformin and 2DG induced p53-dependent apoptosis via the energy sensor pathway AMP kinase, and the reexpression of a functional p53 in p53-deficient prostate cancer cells restored caspase-3 activity. In addition to apoptosis, the combination of metformin and 2DG arrested prostate cancer cells in G(2)-M. This G(2)-M arrest was independent of p53 and correlated with a stronger decrease in cell viability than obtained with either drug. Finally, metformin inhibited 2DG-induced autophagy, decreased beclin 1 expression, and triggered a switch from a survival process to cell death. Our study reinforces the growing interest of metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anticancerous treatment.

Rip11 is a Rab11- and AS160-RabGAP-binding protein required for insulin-stimulated glucose uptake in adipocytes.

The translocation of GLUT4 to the plasma membrane underlies the ability of insulin to stimulate glucose uptake, an event that involves the activation of protein kinase B, several members of the Rab family of GTP-binding proteins and the phosphorylation of the Rab GTPase-activating protein AS160. Here, we explored the regulation by insulin of the class I Rab11-interacting proteins Rip11, RCP and FIP2. We show that Rip11, but not RCP or FIP2, translocates to the plasma membrane of 3T3-L1 adipocytes in response to insulin. This unique response of Rip11 prompted us to explore the role of this protein in more detail. We found that Rip11 partially colocalises with GLUT4 in intracellular compartments. siRNA-mediated knockdown of Rip11 inhibits insulin-stimulated uptake of 2-deoxyglucose, and overexpression of Rip11 blocks insulin-stimulated insertion of translocated GLUT4 vesicles into the plasma membrane. We additionally show that Rip11 forms a complex with AS160 in a Rab11-independent manner and that insulin induces dissociation of AS160 from Rip11. We propose that Rip11 is an AS160- and Rab-binding protein that coordinates the protein kinase signalling and trafficking machinery required to stimulate glucose uptake in response to insulin.

2-Deoxy-D-glucose combined with cisplatin enhances cytotoxicity via metabolic oxidative stress in human head and neck cancer cells.

Glucose deprivation has been hypothesized to cause cytotoxicity by inducing metabolic oxidative stress in human cancer cells. The current work tests the hypothesis that 2-deoxy-d-glucose (2DG) combined with cisplatin [cis-diamminedichloroplatinum(II)] can enhance cytotoxicity in human head and neck cancer cells (FaDu) by mechanisms involving oxidative stress. Exposure of FaDu cells to the combination of 2DG and cisplatin resulted in a significant decrease in cell survival when compared with 2DG or cisplatin alone. Treatment with 2DG and cisplatin also caused perturbations in parameters indicative of oxidative stress, including decreased intracellular total glutathione and increased percentage of glutathione disulfide. Simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC) inhibited parameters indicative of oxidative stress, as well as protected FaDu cells from the cytotoxic effects of cisplatin alone and the combination of 2DG and cisplatin. In addition, polyethylene glycol-conjugated antioxidant enzymes (PEG-superoxide dismutase and PEG-catalase) also protected FaDu cells from 2DG toxicity. An inhibitor of glutathione synthesis, l-buthionine-[S,R]-sulfoximine (BSO), sensitized FaDu cells to the cytotoxic effects of 2DG and cisplatin, and these effects were inhibited by NAC. Furthermore, the combination of 2DG, cisplatin, and BSO significantly increased the percentage of glutathione disulfide, which was also inhibited by NAC. These results support the hypothesis that exposure of human head and neck cancer cells to 2DG combined with cisplatin enhances cytotoxicity via metabolic oxidative stress. These findings provide a strong biochemical rationale for evaluating inhibitors of glucose and hydroperoxide metabolism in combination with cisplatin for the treatment of head and neck cancer.

Genistein-induced apoptosis and autophagocytosis in ovarian cancer cells.

OBJECTIVE: Genistein, a naturally occurring isoflavenoid abundant in soy products, has anti-neoplastic activity in multiple tumor types. There are several mechanisms reported for genistein's anti-neoplastic activity. In the present study, we studied the mechanism of genistein-induced cell death in ovarian cancer cells. METHODS: The effect of genistein on the induction of apoptosis, autophagy, and inhibition of glucose uptake in ovarian cancer cells was determined. The effect of genistein on the expression of phosphorylated Akt was determined by immunoblotting. RESULTS: Genistein is cytotoxic to ovarian cancer cells. The mechanism of genistein-induced cell death includes both apoptosis and autophagy. Because autophagy is typically an adaptive response to nutrient starvation, we hypothesized that genistein could induce a starvation-like signaling response. We show here that genistein treatment results in caspase-independent cell death with hallmarks of autophagy. Genistein treatment dramatically inhibits glucose uptake in ovarian cancer cells, and methyl pyruvate, a cell-permeable 3-carbon substrate for oxidative phosphorylation and fatty acid synthesis, rescues cells from genistein-induced autophagy. In addition, genistein treatment results in reduced levels of phosphorylated Akt, which may contribute towards a mechanism to limit glucose utilization. CONCLUSIONS: Most conventional chemotherapeutic agents induce apoptotic cell death. Because genistein can induce both apoptotic and autophagic cell death, it has the potential to circumvent chemoresistance due to alterations in apoptotic signaling.

The involvement of phosphatidylinositol 3-kinase /Akt signaling in high glucose-induced downregulation of GLUT-1 expression in ARPE cells.

Glucose transporters have been reported to be associated with the development of diabetic retinopathy. Retinal pigment epithelial cells (RPEs) are believed to play an important role in the pathogenesis of diabetic retinopathy. However, the effect of hyperglycemia on glucose transporters in RPEs and the related signal pathways have not yet been elucidated. Therefore, we examined the effect of high glucose on the glucose transporter 1 in ARPEs and the related signal molecules. In the present study, high glucose decreased 2-deoxyglucose uptake in a time (>2 h) and dose dependent manner. In addition, we found that high glucose downregulated the expression of glucose transporter 1 (GLUT-1). The high glucose-induced downregulation of GLUT-1 was blocked by Wortmanin, LY 294002 (PI-3 kinase inhibitors) and Akt (Akt inhibitor). The high glucose increased stimulation of Akt activation in a time dependent manner. We also investigated the upstream regulator of Akt activation. The high glucose-induced phosphorylation of Akt was blocked by bisindolymaleimide I, H-7, staurosporine (protein kinase C [PKC] inhibitors), vitamin C and catalase (antioxidants). In addition, the high glucose-induced downregulation of GLUT-1 was also blocked by PKC inhibitors and antioxidants. Moreover, high glucose increased lipid peroxide formation, which was prevented by PKC inhibitors. In conclusion, high glucose downregulated GLUT-1 by Akt pathway activation mediated by the PKC-oxidative stress signaling pathway in ARPE cells.

High glucose-induced inhibition of 2-deoxyglucose uptake is mediated by cAMP, protein kinase C, oxidative stress and mitogen-activated protein kinases in mouse embryonic stem cells.

Abnormally high glucose levels may play an important role in early embryo development and function. In the present study, we investigated the effect of high glucose on 2-deoxyglucose (2-DG) uptake and its related signalling pathway in mouse embryonic stem (ES) cells. 2. 2-Deoxyglucose uptake was maximally inhibited by 25 mmol/L glucose after 24 h treatment. However, 25 mmol/L mannitol and dextran did not affect 2-DG uptake. Indeed, 25 mmol/L glucose decreased GLUT-1 mRNA and protein levels. The glucose (25 mmol/L)-induced inhibition of 2-DG uptake was blocked by pertussis toxin (a G(i)-protein inhibitor; 2 ng/mL), SQ 22,536 (an adenylate cyclase inhibitor; 10(-6) mol/L) and the protein kinase (PK) A inhibitor myristoylated PKI amide-(14-22) (10(-6) mol/L). Indeed, 25 mmol/L glucose increased intracellular cAMP content. 3. Furthermore, 25 mmol/L glucose-induced inhibition of 2-DG uptake was prevented by 10(-4) mol/L neomycin or 10(-6) mol/L U 73,122 (phospholipase C (PLC) inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase (PK) C inhibitors). At 25 mmol/L, glucose increased translocation of PKC from the cytoplasmic fraction to the membrane fraction. The 25 mmol/L glucose-induced inhibition of 2-DG uptake and GLUT-1 protein levels was blocked by SQ 22,536, bisindolylmaleimide I or combined treatment. In addition, 25 mmol/L glucose increased cellular reactive oxygen species and the glucose-induced inhibition of 2-DG uptake were blocked by the anti-oxidants N-acetylcysteine (NAC; 10(-5) mol/L) or taurine (2 yen 10(-3) mol/L). 4. Glucose (25 mmol/L) activated p38 mitogen-activated protein kinase (MAPK) and p44/42 MAPK. Staurosporine (10(-6) mol/L), NAC (10(-5) mol/L) and PD 98059 (10(-7) mol/L) attenuated the phosphorylation of p44/42 MAPK. Both SB 203580 (a p38 MAPK inhibitor; 10(-7) mol/L) and PD 98059 (a p44/42 MAPK inhibitor; 10(-7) mol/L) blocked 25 mmol/L glucose-induced inhibition of 2-DG uptake. 5. In conclusion, high glucose inhibits 2-DG uptake through cAMP, PLC/PKC, oxidative stress or MAPK in mouse ES cells.

Nelfinavir-induced insulin resistance is associated with impaired plasma membrane recruitment of the PI 3-kinase effectors Akt/PKB and PKC-zeta.

AIMS/HYPOTHESIS: Chronic exposure of 3T3-L1 adipocytes to the HIV protease inhibitor nelfinavir induces insulin resistance, recapitulating key metabolic alterations of adipose tissue in the lipodystrophy syndrome induced by these agents. Our goal was to identify the defect in the insulin signal transduction cascade leading to nelfinavir-induced insulin resistance. METHODS: Fully differentiated 3T3-L1 adipocytes were exposed to 30 micro mol/l nelfinavir for 18 h, after which the amount, the phosphorylation and the localisation of key proteins in the insulin signalling cascade were evaluated. RESULTS: Insulin-induced interaction of phosphatidylinositol 3'-kinase (PI 3-kinase) with IRS proteins was normal in cells treated with nelfinavir, as was IRS-1-associated PI 3-kinase activity. Yet insulin-induced phosphorylation of Akt/protein kinase B (PKB), p70S6 kinase and extracellular signal-regulated kinase 1/2 was significantly impaired. This could not be attributed to increased protein phosphatase 2A activity or to increased expression of phosphoinositide phosphatases (SHIP2 or PTEN). However, insulin failed to induce translocation of the PI 3-kinase effectors Akt/PKB and protein kinase C-zeta (PKC-zeta) to plasma membrane fractions of nelfinavir-treated adipocytes. CONCLUSIONS/INTERPRETATION: We therefore conclude that nelfinavir induces a defect in the insulin signalling cascade downstream of the activation of PI 3-kinase. This defect manifests itself by impaired insulin-mediated recruitment of Akt/PKB and PKC-zeta to the plasma membrane.

Cbl PYXXM motifs activate the P85 subunit of phosphatidylinositol 3-kinase, Crk, atypical protein kinase C, and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes.

The thiazolidinedione (TZD), rosiglitazone, has previously been found to tyrosine-phosphorylate Cbl and activate Cbl-dependent phosphatidylinositol (PI) 3-kinase and atypical protein kinase Cs (aPKCs) while stimulating glucose transport in 3T3/L1 adipocytes. Presently, the role of Cbl in rosiglitazone action was further assessed in both 3T3/L1 and human adipocytes by expressing Y371F and/or Y731F mutant forms of Cbl that nullified the functionality of canonical pYXXM motifs in Cbl. These mutants diminished the interaction of Cbl with the p85 subunit of PI 3-kinase and inhibited subsequent increases in Cbl-dependent PI 3-kinase activity, aPKC activity, and glucose transport. These mutants also inhibited the interaction of Cbl with Crk, which has been implicated in the activation of other PI 3-kinase-independent signaling factors that have been found to be required during activation of glucose transport by insulin and other agonists. We conclude that pYXXM motifs in Cbl serve to activate PI 3-kinase-dependent and possibly PI 3-kinase-independent pathways that are required for TZD-dependent glucose transport in adipocytes.

Effects of peripheral administration of 5-hydroxytryptamine (5-HT ) on 2-deoxy-D-glucose-induced hyperphagia in rats.

Effects of peripheral administration of 5-HT (5-hydroxytryptamine, serotonin) on hyperphagia induced by 2-deoxy-D-glucose(2-DG) were studied in rats. It was found that 5-HT i.p. reduced 2-DG-elicited feeding in rats dose-dependently. The 5-HT-induced hypophagia was antagonized by the 5-HT2A receptor antagonist, ketanserin. It is known that 2-DG induces glucoprivation, resulting in hyperphagia and hyperglycemia. However, 5-HT did not affect hyperglycemia induced by 2-DG. These results suggest that peripheral injection of 5-HT reduces 2-DG-induced hyperphagia mediated by the peripheral 5-HT2A receptor and that its effects are not due to enhancement of hyperglycemia.

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.

Stimulatory effect of isoferulic acid on alpha1A-adrenoceptor to increase glucose uptake into cultured myoblast C2C12 cell of mice.

In an attempt to elucidate the effect of isoferulic acid on alpha1-adrenoceptor (AR), the myoblast C2C12 cells of mice were employed to investigate the change of glucose uptake in the present study. Isoferulic acid enhanced the uptake of radioactive glucose into C2C12 cells in a concentration-dependent manner, which were abolished by pretreatment with prazosin. Effect of isoferulic acid on alpha1-AR was further characterized using the displacement of [3H]YM617 binding in C2C12 cells. The radioactive glucose uptake increasing action of isoferulic acid was abolished by tamsulosin or WB 4101 at concentration sufficient to block alpha1A-adrenoceptor (alpha1A-AR) but it was not modified by chlorethylclonidine (CEC) at the concentration sufficient to abolish alpha1B-AR. An activation of alpha1A-AR by isoferulic acid in C2C12 cells can thus be considered. Pharmacological inhibition of phospholipase C (PLC) by U73312 resulted in a concentration-dependent reduction of isoferulic acid-stimulated glucose uptake in C2C12 cells. This inhibition by U73112 was specific because the inactive congener, U73343, failed to modify the action of isoferulic acid. Also, chelerythrine and GF 109203X diminished the action of isoferulic acid at concentration sufficient to inhibit the activity of protein kinase C (PKC). The obtained data suggest that an activation of alpha1A-AR by isoferulic acid may increase the glucose uptake via PLC-PKC pathway in C2C12 cells.

The antihyperglycemic drug alpha-lipoic acid stimulates glucose uptake via both GLUT4 translocation and GLUT4 activation: potential role of p38 mitogen-activated protein kinase in GLUT4 activation.

The cofactor of mitochondrial dehydrogenase complexes and potent antioxidant alpha-lipoic acid has been shown to lower blood glucose in diabetic animals. alpha-Lipoic acid enhances glucose uptake and GLUT1 and GLUT4 translocation in 3T3-L1 adipocytes and L6 myotubes, mimicking insulin action. In both cell types, insulin-stimulated glucose uptake is reduced by inhibitors of p38 mitogen-activated protein kinase (MAPK). Here we explore the effect of alpha-lipoic acid on p38 MAPK, phosphatidylinositol (PI) 3-kinase, and Akt1 in L6 myotubes. alpha-Lipoic acid (2.5 mmol/l) increased PI 3-kinase activity (31-fold) and Akt1 (4.9-fold). Both activities were inhibited by 100 nmol/l wortmannin. alpha-Lipoic acid also stimulated p38 MAPK phosphorylation by twofold within 10 min. The phosphorylation persisted for at least 30 min. Like insulin, alpha-lipoic acid increased the kinase activity of the alpha (2.8-fold) and beta (2.1-fold) isoforms of p38 MAPK, measured by an in vitro kinase assay. Treating cells with 10 micromol/l of the p38 MAPK inhibitors SB202190 or SB203580 reduced the alpha-lipoic acid-induced stimulation of glucose uptake by 66 and 55%, respectively. In contrast, SB202474, a structural analog that does not inhibit p38 MAPK, was without effect on glucose uptake. In contrast to 2-deoxyglucose uptake, translocation of GLUT4myc to the cell surface by either alpha-lipoic acid or insulin was unaffected by 20 micromol/l of SB202190 or SB203580. The results suggest that inhibition of 2-deoxyglucose uptake in response to alpha-lipoic acid by inhibitors of p38 MAPK is independent of an effect on GLUT4 translocation. Instead, it is likely that regulation of transporter activity is sensitive to these inhibitors.

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha.

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.

Ethanol inhibits G-protein-mediated glucose uptake by C6 glioma cells.

The mechanism of ethanol inhibition of glucose uptake was investigated using C6 glioma cells. Basal [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was inhibited by ethanol in a concentration-dependent manner. Fifty, 75 and 100 mM ethanol significantly inhibited basal 2DG uptake by 12, 20 and 23%, respectively (p < 0.05). Carbachol (an agonist acting via G protein-coupled receptors) stimulated the uptake by 26% (p < 0.05). In the presence of 100 mM ethanol, the ability of carbachol to stimulate 2DG uptake was abolished. In contrast, ethanol did not inhibit the ability of insulin to stimulate 2DG uptake. These results suggest that ethanol inhibits 2DG uptake by selectively interfering with G protein-mediated signal transduction pathway.

Can experimental conditions explain the discrepancy over glutamate stimulation of aerobic glycolysis?

Uncertainty reigns over whether or not glutamate uptake in astrocytes leads to strong stimulation of glucose utilization, measured as accumulation of radioactive deoxyglucose-6-phosphate. This is an important issue, not only because glutamate is the major excitatory transmitter, but also because it has been postulated that glutamate-induced stimulation of glycolysis links brain excitation with activation of energy production. The effect of glutamate on deoxyglucose utilization in cultured rat and mouse astrocytes grown in different media and incubated under various conditions during the deoxyglucose assay has, therefore, been studied. Under most conditions, no stimulation occurred but rather a decrease in deoxyglucose utilization during exposure to glutamate; under certain conditions, the contribution of non-metabolized deoxyglucose to the intracellular 14C signal was significant.