Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Mutual control of membrane fission and fusion proteins.

Membrane fusion and fission are antagonistic reactions controlled by different proteins. Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles. Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles. Vps1p forms polymers that couple several t-SNAREs together. At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane. This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE. Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity. We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Inhibition of bicarbonate transport protects embryonic heart against reoxygenation-induced dysfunction.

It has not been well established whether the mechanisms participating in pH regulation in the anoxic-reoxygenated developing myocardium resemble those operating in the adult. We have specially examined the importance of Na+/H+ exchange (NHE) and HCO3-dependent transports in cardiac activity after changes in extracellular pH (pHo). Spontaneously contracting hearts isolated from 4-day-old chick embryos were submitted to single or repeated anoxia (1 min) followed by reoxygenation (10 min). The chronotropic, dromotropic and inotropic responses of the hearts were determined in standard HCO3- buffer at pHo 7.4 and at pHo 6.5 (hypercapnic acidosis). In distinct experiments, acidotic anoxia preceded reoxygenation at pHo 7.4. NHE was blocked with amiloride derivative HMA (1 micro mol/l) and HCO3-dependent transports were inactivated by replacement of HCO3 or blockade with stilbene derivative DIDS (100 micro mol/l). Anoxia caused transient tachycardia, depressed mechanical function and induced contracture. Reoxygenation temporarily provoked cardiac arrest, atrio-ventricular (AV) block, arrhythmias and depression of contractility. Addition of DIDS or substitution of HCO3 at pHo 7.4 had the same effects as acidosis per se, i.e. shortened contractile activity and increased incidence of arrhythmias during anoxia, prolonged cardioplegia and provoked arrhythmias at reoxygenation. Under anoxia at pHo 6.5/reoxygenation at pHo 7.4, cardioplegia, AV block and arrhythmias were all markedly prolonged. Interestingly, in the latter protocol, DIDS suppressed AV block and arrhythmias during reoxygenation, whereas HMA had no effect. Thus, intracellular pH regulation in the anoxic-reoxygenated embryonic heart appears to depend predominantly on HCO3 availability and transport. Furthermore, pharmacological inhibition of anion transport can protect against reoxygenation-induced dysfunction.

A gene knockout approach in mice to identify glucose sensors controlling glucose homeostasis.

A key aspect of glucose homeostasis is the constant monitoring of blood glucose concentrations by specific glucose sensing units. These sensors, via stimulation of hormone secretion and activation of the autonomic nervous system (ANS), regulate tissue glucose uptake, utilization or production. The best described glucose detection system is that of the pancreatic beta-cells which controls insulin secretion. Secretion of other hormones, in particular glucagon, and activation of the ANS, are regulated by glucose through sensing mechanisms which are much less well characterized. Here I review some of the studies we have performed over the recent years on a mouse model of impaired glucose sensing generated by inactivation of the gene for the glucose transporter GLUT2. This transporter catalyzes glucose uptake by pancreatic beta-cells, the first step in the signaling cascade leading to glucose-stimulated insulin secretion. Inactivation of its gene leads to a loss of glucose sensing and impaired insulin secretion. Transgenic reexpression of the transporter in GLUT2/beta-cells restores their normal secretory function and rescues the mice from early death. As GLUT2 is also expressed in other tissues, these mice were then studied for the presence of other physiological defects due to absence of this transporter. These studies led to the identification of extra-pancreatic, GLUT2-dependent, glucose sensors controlling glucagon secretion and glucose utilization by peripheral tissues, in part through a control of the autonomic nervous system.

Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, and somatostatin levels.

IDX-1 (islet/duodenum homeobox-1) is a transcription factor expressed in the duodenum and pancreatic beta and delta cells. It is required for embryonic development of the pancreas and transactivates the Glut2, glucokinase, insulin, and somatostatin genes. Here we show that exposure of isolated rat pancreatic islets to palmitic acid induced a approximately 70% decrease in IDX-1 mRNA and protein expression as well as 40 and 65% decreases in the binding activity of IDX-1 for its cognate cis-regulatory elements of the Glut2 and insulin promoters, respectively. The inhibitory effect of palmitic acid required its mitochondrial oxidation since it was prevented by the carnitine palmitoyltransferase I inhibitor bromopalmitic acid. The palmitic acid effect on IDX-1 was correlated with decreases in GLUT2 and glucokinase expression of 40 and 25%, respectively, at both the mRNA and protein levels. Insulin and somatostatin mRNA expression was also decreased by 40 and 60%, whereas glucagon mRNA expression was not modified. After 48 h of exposure to fatty acids, total islet insulin, somatostatin, and glucagon contents were decreased by 85, 55, and 65%, respectively. At the same time, total hormone release was strongly stimulated (13-fold) for glucagon, whereas its was only marginally increased for insulin and somatostatin (1.5- and 1.7-fold, respectively). These results indicate that elevated fatty acid levels 1) negatively regulate Idx-1 expression; 2) decrease the expression of genes transactivated by IDX-1 such as those for GLUT2, glucokinase, insulin, and somatostatin; and 3) lead to an important increase in glucagon synthesis and secretion. Fatty acids thus have pleiotropic effects on pancreatic islet gene expression, and the negative control of Idx-1 expression may be an initial event in the development of these multiple defects.

GLUTX1, a novel mammalian glucose transporter expressed in the central nervous system and insulin-sensitive tissues.

Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.

Effect of somatostatin on duodenal glucose absorption in man.

OBJECTIVE: The hyperglycemic hyperinsulinemic clamp technique using intraduodenally infused glucose is an attractive tool for studying postprandial glucose metabolism under strictly controlled conditions. Because it requires the use of somatostatin (SST), we examined, in this study, the effect of SST on intestinal glucose absorption. CONTEXT: Twenty-six normal volunteers were given a constant 3-h intraduodenal infusion of glucose (6 mg.kg(-1).min(-1)) labeled with [2-(3)H]glucose for glucose absorption measurement. During glucose infusion, 19 subjects received iv SST at doses of 10-100 ng.kg(-1).min(-1) plus insulin and glucagon, and seven subjects were studied under control conditions. In the controls, glucose was absorbed at a rate that, after a 20-min lag period, equaled the infusion rate. RESULTS: With all the doses of SST tested, absorption was considerably delayed but equaled the rate of infusion after 3 h. At that time, only 5 +/- 2% of the total amount of infused glucose was unabsorbed in the control subjects vs. 36 +/- 2% (P < 0.001) in the SST-infused subjects. In the latter, the intraluminal residue was almost totally absorbed within 40 min of the cessation of SST infusion. At the lowest dose of SST tested (10 ng.kg(-1).min(-1)), suppression of insulin secretion was incomplete. CONCLUSION: These properties of SST hamper the use of intraduodenal hyperglycemic hyperinsulinemic clamps as a tool for exploring postprandial glucose metabolism.

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

Regulated expression of GLUT2 in diabetes studied in transplanted pancreatic beta cells.

GLUT2 disappearance is a marker of the beta cell glucose-unresponsiveness associated with diabetes. Understanding the factor(s) leading to this dysfunction may shed light on pathogenesis of diabetes. Since the regulation of GLUT2 expression in diabetes can so far only be studied in in vivo experiments, we developed a novel experimental approach to study the genetic regulation of GLUT2 in diabetes. By encapsulating islets or cell lines in semi-permeable membranes, these cells can be exposed to the diabetic environment of rats or mice and can be retrieved for analysis of GLUT2 expression and for the change in the secretory response to glucose. Immunocytochemical analysis of transporter expression reveals changes in protein expression while transcriptional analysis of GLUT2 gene expression could be performed in cells transfected with promoter-reporter gene constructs. Using this last approach we hope to be able to characterize the promoter regions involved in the beta cell- and diabetes-specific regulation of GLUT2 expression and possibly to determine which factors are responsible for this regulation.

Facilitated glucose transporters in epithelial cells.

The molecular cloning of facilitated sugar transporters has led to the identification of a family of transport molecules having similar functions, but possessing specific kinetic and regulatory properties. These transporter isoforms are characterized by different primary structures, specific tissue localization, and polarized expression within the same epithelial cells. The use of Xenopus oocytes for the functional expression of different members of this transporter family has been of considerable value in defining the kinetic properties and sugar specificities of the different isoforms. The expression of chimeric or variously mutated transporters should, in the near future, permit the determination of the structural basis for their kinetic properties and sugar specificities. cDNA probes and antipeptide antibodies specific for each isoform are now being used to determine their specific regulation during development and in different states of altered glucose homeostasis. The variety of molecular forms implicated in the apparently simple task of sugar uptake or transepithelial transport has been surprising. With the available molecular tools now in hand, it will be possible to study these mechanisms in much greater detail.

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes.

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12 degreesC). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism.

OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.