Biomedical subjects

M T Guillam

Publications and source records attributed to M T Guillam.

9 recordsLinked to original sources

PubMed2000

Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion.

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse alpha to beta cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic beta cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of alpha to beta cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in beta cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

Glucose uptake, utilization, and signaling in GLUT2-null islets.

We previously reported that pancreatic islet beta-cells from GLUT2-null mice lost the first phase but preserved the second phase of glucose-stimulated insulin secretion (GSIS). Furthermore, we showed that the remaining secretory activity required glucose uptake and metabolism because it can be blocked by inhibition of oxidative phosphorylation. Here, we extend these previous studies by analyzing, in GLUT2-null islets, glucose transporter isoforms and glucokinase expression and by measuring glucose usage, GSIS, and glucose-stimulated insulin mRNA biosynthesis. We show that in the absence of GLUT2, no compensatory expression of either GLUT1 or GLUT3 is observed and that glucokinase is expressed at normal levels. Glucose usage by isolated islets was increased between 1 and 6 mmol/l glucose but was not further increased between 6 and 20 mmol/l glucose. Parallel GSIS measurements showed that insulin secretion was not stimulated between 2.8 and 6 mmol/l glucose but was increased by >4-fold between 6 and 20 mmol/l glucose. Stimulation by glucose of total protein and insulin biosynthesis was also markedly impaired in the absence of GLUT2. Finally, we re-expressed GLUT2 in GLUT2-null beta-cells using recombinant lentiviruses and demonstrated a restoration of normal GSIS. Together, these data show that in the absence of GLUT2, glucose can still be taken up by beta-cells, albeit at a low rate, and that this transport activity is unlikely to be attributed to GLUT1 or GLUT3. This uptake activity, however, is limiting for normal glucose utilization and signaling to secretion and translation. These data further demonstrate the key role of GLUT2 in murine beta-cells for glucose signaling to insulin secretion and biosynthesis.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

Aquaporin-related proteins in the filter chamber of homopteran insects.

In the Homopteran order of insects, the plant xylem feeders exhibit a highly differentiated part of their digestive tract known as the filter chamber. In this tissue, water crosses plasma membranes through a transepithelial osmotic gradient. In previous studies on the filter chamber of Cicadella viridis, we purified and characterized from the plasma membranes a 25 kDa protein that we demonstrated to be an aquaporin (or water channel, member of the major intrinsic protein family, a group of membrane channels for small solutes). We called this protein AQPcic for Cicadella aquaporin. In the present study, we used polyclonal antibody anti-AQPcic in Western blotting and immunocytochemical analysis of the intestinal tract of Cercopis sanguinolenta, Philaenus spumarius, Aphrophora alni (Cercopidae), Euscelidius variegatus, and Scaphoideus titanus (Jassidae). Western blotting experiments revealed that immunologically related AQPcic proteins are found in those species. The molecular weight of these proteins is 15-26 kDa. Immunocytochemical studies on ultrathin filter-chamber sections revealed that the anti-AQPcic antibody systematically labelled the membrane microvilli of epithelial cells. A good correlation thus exists between the physiology of these cells and the presence of aquaporin-related proteins in their membranes.

Animal Structures↗

PubMed1997

Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2.

Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic beta-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice. In beta-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low or almost undetectable levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and beta-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, beta-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the alpha- to beta-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.

Animals↗

PubMed1996

Molecular cloning and characterization of an insect aquaporin functional comparison with aquaporin 1.

We previously described the structural organization of P25, a member of the major-intrinsic-protein family found in the digestive tract of homopteran sap-sucking insects [Beuron, F., Le Cahérec, F., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland, J. P., Hubert, J.F., Gouranton, J. & Thomas, D. (1995) J. Biol. Chem. 270, 17414-17422]. We demonstrated, by means of introducing P25 tetramers into the membranes of Xenopus oocytes, that this protein exhibits functional properties similar to those of aquaporin 1, the archetypal water channel [Le Cahérec, F., Bron, P., Verbavatz, J. M., Garret, A., Morel, G., Cavalier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert, J.F. (1996) J. Cell Sci. 109, 1285-1295]. In the present work, we cloned a full-length cDNA from a Cicadella viridis library with an open reading frame of 765 bp that encoded a 26-kDa protein whose sequence was 43, 40, 36 and 36% identical to aquaporins 1, 2, z and tonoplast intrinsic protein gamma, respectively. Translation of the corresponding RNA in Xenopus oocytes generated a polypeptide that was specifically recognized by polyclonal antibodies raised against native P25. Expression of the protein in Xenopus oocyte membranes was assessed by immunocytochemistry and led to a 15-fold increase of osmotic membrane water permeability. This increase was inhibited by HgCl2. The permeability had an Arrhenius activation energy of 11.7 kJ/mol. We called this protein Cicadella aquaporin (AQPcic). The oocytes expressing Cicadella aquaporin were less sensitive to HgCl2 than oocytes expressing aquaporin 1. In the Xenopus oocyte system, Cicadella aquaporin failed to transport glycerol, urea and ions. It exhibited permeabilities to ethylene glycol and formamide similar to those measured for aquaporin 1 under the same conditions.

Amino Acid Sequence↗

PubMed1995

Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.

Homopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes. Using polymerase chain reaction, a 360-base pair cDNA, named cic, was isolated from RNA of the FC. cic encodes a 119-amino acid polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and gamma-TIP are 38, 38, 34, and 20%, respectively. Using a specific antibody raised against a 15-amino acid peptide from the CIC sequence, we concluded that CIC and P25 are identical entities, and hence that P25 belongs to the MIP family. We investigated the quaternary structure of P25 in the membranes of the FC using biophysical analysis of P25 nondenaturing detergent micelles, scanning transmission electron microscopy, and image processing of conventional transmission electron microscopic images. All those different approaches converged to the conclusion that P25 exists as an homotetramer forming a regular two-dimensional array in the membranes.

Amino Acid Sequence↗

PubMed1992

Expression of RNA isolated from the water-shunting complex of a sap-sucking insect increases the membrane permeability for water in Xenopus oocytes.

The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.

Animals↗