Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes.

Effects of a glucose meal on energy metabolism in patients with cirrhosis before and after liver transplantation.

HYPOTHESIS: Liver transplantation results in hepatic denervation. This may produce alterations of liver energy and substrate metabolism, which may contribute to weight gain after liver transplantation. DESIGN: Prospective clinical study. SETTING: Liver transplantation clinics in a university hospital. PATIENTS: Seven nondiabetic patients with cirrhosis were recruited while on a waiting list for liver transplantation. Seven healthy subjects were recruited as controls. INTERVENTION: Orthotopic liver transplantation. MAIN OUTCOME MEASURES: Evaluation of energy and substrate metabolism after ingestion of a glucose load with indirect calorimetry was performed before, 2 to 6 weeks after, and 5 to 19 months after transplantation. Whole-body glucose oxidation and storage and glucose-induced thermogenesis were calculated. RESULTS: Patients with cirrhosis had modestly elevated resting energy expenditure and normal glucose-induced thermogenesis and postprandial glucose oxidation and storage. These measures remained unchanged after liver transplantation despite a significant increase in postprandial glycemia. Patients, however, gained an average of 3 kg of body weight after 5 to 19 months compared with their weight before transplantation. CONCLUSION: Liver denervation secondary to transplantation does not lead to alterations of energy metabolism after ingestion of a glucose load.

Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium.

The role of cell type-specific Na+,K+-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 micromol/L), as well as by glutamate (100 micromol/L) and the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) (10 micromol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10(-6) mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type-specific expression of Na+,K+-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na+,K+-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.

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.

Systemic effects of epidural Methylprednisolone injection on glucose tolerance in diabetic patients.

ABSTRACT: BACKGROUND: Several studies have shown that in diabetic patients, the glycemic profile was disturbed after intra-articular injection of corticosteroids. Little is known about the impact of epidural injection in such patients. The goal of this study was double, at first comparing the glycaemic profile in diabetic patients after a unique injection of 80 mg of acetate methylprednisolone either intra-articular or epidural and secondly to compare the amount of systemic diffusion of the drug after both procedures. METHODS: Seventeen patients were included. Glycemic changes were compared in 9 diabetic patients following intra-articular (4 patients) and epidural injections (5 patients). Epidural injections were performed using the sacral route under fluoroscopic control in patients with lumbar spinal stenosis. Diabetes control had to stable for more than 10 days and the renal function to be preserved. Blood glucose was monitored using a validated continuous measuring device (GMS, Medtronic) the day before and for two days following the injection. Results were expressed in the form of daily glycemic profiles and as by mean, peak and minimal values +/ SD. The urinary excretion of methylprednisolone after the 2 routes of injection was analyzed in 8 patients (4 in each group). Urine samples were cropped one hour before the injections, then 4 times during the first day and 3 times a week for 2 weeks. The measurements included the free and conjugated fraction RESULTS: The glycaemic profile remains unchanged with no significant changes in the group of the 5 diabetic patients receiving epidural injections. On the other end, the average peak and mean values were enhanced up to 3 mmol/l above baseline two days after the infiltration in the groups of the 4 diabetic patients infiltrated intra-articular. The mean urinary excretion of the steroid was about ten times higher in the intra-articular versus epidural group: 7000 ng/ml versus 700 ng/ml. Looking at each individual there were marked differences especially after intra-articular injections. CONCLUSION: This is the first study to show that a single epidural steroid injection of 80 mg depot methylprednisolone had no effect on the glycemic control in diabetic patients. The absence of glycemic control changes correlated well with the very low urinary excretion of the drug after epidural injection. Trial registration NCT01420497.

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.

A prospective randomised multi-centre controlled trial on tight glucose control by intensive insulin therapy in adult intensive care units: the Glucontrol study.

PURPOSE: An optimal target for glucose control in ICU patients remains unclear. This prospective randomized controlled trial compared the effects on ICU mortality of intensive insulin therapy (IIT) with an intermediate glucose control. METHODS: Adult patients admitted to the 21 participating medico-surgical ICUs were randomized to group 1 (target BG 7.8-10.0 mmol/L) or to group 2 (target BG 4.4-6.1 mmol/L). RESULTS: While the required sample size was 1,750 per group, the trial was stopped early due to a high rate of unintended protocol violations. From 1,101 admissions, the outcomes of 542 patients assigned to group 1 and 536 of group 2 were analysed. The groups were well balanced. BG levels averaged in group 1 8.0 mmol/L (IQR 7.1-9.0) (median of all values) and 7.7 mmol/L (IQR 6.7-8.8) (median of morning BG) versus 6.5 mmol/L (IQR 6.0-7.2) and 6.1 mmol/L (IQR 5.5-6.8) for group 2 (p < 0.0001 for both comparisons). The percentage of patients treated with insulin averaged 66.2 and 96.3%, respectively. Proportion of time spent in target BG was similar, averaging 39.5% and 45.1% (median (IQR) 34.3 (18.5-50.0) and 39.3 (26.2-53.6)%) in the groups 1 and 2, respectively. The rate of hypoglycaemia was higher in the group 2 (8.7%) than in group 1 (2.7%, p < 0.0001). ICU mortality was similar in the two groups (15.3 vs. 17.2%). CONCLUSIONS: In this prematurely stopped and therefore underpowered study, there was a lack of clinical benefit of intensive insulin therapy (target 4.4-6.1 mmol/L), associated with an increased incidence of hypoglycaemia, as compared to a 7.8-10.0 mmol/L target. (ClinicalTrials.gov # NCT00107601, EUDRA-CT Number: 200400391440).

Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor.

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg. min. In P-mice, glucose clearance increased to 67.5 +/- 3.7 mg/kg. min as compared with 24.1 +/- 1.5 mg/kg. min in S-mice, and glycemia decreased from 5.0 +/- 0.1 to 3.3 +/- 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg. min (P-GLP-1 mice) did not increase the glucose clearance rate (57.4 +/- 5.0 ml/kg. min) and hypoglycemia (3.8 +/- 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg. min (P-Ex mice) reduced glucose clearance to 36.1 +/- 2.6 ml/kg. min and transiently increased glycemia to 9.2 +/- 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 +/- 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 +/- 4.6 ml/kg. min and glycemia decreased to 3.1 +/- 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor(-/-) mice failed to increase the glucose clearance rate (26.7 +/- 2.9 ml/kg. min). Glycemia increased to 8.5 +/- 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 +/- 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.

Abrupt decrease in serum testosterone levels after an oral glucose load in men: implications for screening for hypogonadism.

OBJECTIVE: This study examines the physiological impact of a glucose load on serum testosterone (T) levels in men with varying glucose tolerance (GT). DESIGN: Cross-sectional study. PATIENTS AND METHODS: 74 men (19-74 years, mean 51·4 ± 1·4 years) underwent a standard 75-g oral glucose tolerance test with blood sampling at 0, 30, 60, 90 and 120 min. Fasting serum glucose, insulin, total T (and calculated free T), LH, SHBG, leptin and cortisol were measured. RESULTS: 57% of the men had normal GT, 30% had impaired GT and 13% had newly diagnosed type 2 diabetes. Glucose ingestion was associated with a 25% decrease in mean T levels (delta = -4·2 ± 0·3 nm, P < 0·0001). T levels remained suppressed at 120 min compared with baseline (13·7 ± 0·6 vs 16·5 ± 0·7 nm, P < 0·0001) and did not differ across GT or BMI. Of the 66 men with normal T levels at baseline, 10 (15%) had levels that decreased to the hypogonadal range (<9·7 nm) at one or more time points. SHBG, LH and cortisol levels were unchanged. Leptin levels decreased from baseline at all time points (P < 0·0001). CONCLUSIONS: Glucose ingestion induces a significant reduction in total and free T levels in men, which is similar across the spectrum of glucose tolerance. This decrease in T appears to be because of a direct testicular defect, but the absence of compensatory changes in LH suggests an additional central component. Men found to have low nonfasting T levels should be re-evaluated in the fasting state.

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.

Effects of gastric bypass and gastric banding on glucose kinetics and gut hormone release.

BACKGROUND: Bariatric surgery markedly improves glucose homeostasis in patients with type 2 diabetes even before any significant weight loss is achieved. Procedures that involve bypassing the proximal small bowel, such as Roux-en-Y gastric bypass (RYGBP), are more efficient than gastric restriction procedures such as gastric banding (GB). OBJECTIVE: To evaluate the effects of RYGBP and GB on postprandial glucose kinetics and gastro-intestinal hormone secretion after an oral glucose load. METHODS AND PROCEDURES: This study was a cross-sectional comparison among non-diabetic, weight-stable women who had undergone RYGBP (n = 8) between 9 and 48 months earlier or GB (n = 6) from 25 to 85 months earlier, and weight- and age-matched control subjects (n = 8). The women were studied over 4 h following ingestion of an oral glucose load. Total glucose and meal glucose kinetics were assessed using glucose tracers and plasma insulin, and gut hormone concentrations were simultaneously monitored. RESULTS: Patients who had undergone RYGBP showed a a more rapid appearance of exogenous glucose in the systemic circulation and a shorter duration of postprandial hyperglycemia than patients who had undergone GB and C. The response in RYGBP patients was characterized by early and accentuated insulin response, enhanced postprandial levels of glucagon-like peptide-1 (GLP-1) and polypeptide YY (PYY), and greater postprandial suppression of ghrelin. DISCUSSION: These findings indicate that RYGBP is associated with alterations in glucose kinetics and glucoregulatory hormone secretion. These alterations are probably secondary to the anatomic rearrangement of the foregut, given the fact that they are not observed after GB. Increased PYY and GLP-1 concentrations and enhanced ghrelin suppression are compatible with reduced food intake after RYGBP.

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants.

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.

Lactate and glucose metabolism in severe sepsis and cardiogenic shock.

OBJECTIVE: To evaluate the relative importance of increased lactate production as opposed to decreased utilization in hyperlactatemic patients, as well as their relation to glucose metabolism. DESIGN: Prospective observational study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: Seven patients with severe sepsis or septic shock, seven patients with cardiogenic shock, and seven healthy volunteers. INTERVENTIONS: C-labeled sodium lactate was infused at 10 micromol/kg/min and then at 20 micromol/kg/min over 120 mins each. H-labeled glucose was infused throughout. MEASUREMENTS AND MAIN RESULTS: Baseline arterial lactate was higher in septic (3.2 +/- 2.6) and cardiogenic shock patients (2.8 +/- 0.4) than in healthy volunteers (0.9 +/- 0.20 mmol/L, p < .05). Lactate clearance, computed using pharmacokinetic calculations, was similar in septic, cardiogenic shock, and controls, respectively: 10.8 +/- 5.4, 9.6 +/- 2.1, and 12.0 +/- 2.6 mL/kg/min. Endogenous lactate production was determined as the initial lactate concentration multiplied by lactate clearance. It was markedly enhanced in the patients (septic 26.2 +/- 10.5; cardiogenic shock 26.6 +/- 5.1) compared with controls (11.2 +/- 2.7 micromol/kg/min, p < .01). C-lactate oxidation (septic 54 +/- 25; cardiogenic shock 43 +/- 16; controls 65 +/- 15% of a lactate load of 10 micromol/kg/min) and transformation of C-lactate into C-glucose were not different (respectively, 15 +/- 15, 9 +/- 18, and 10 +/- 7%). Endogenous glucose production was markedly increased in the patients (septic 14.8 +/- 1.8; cardiogenic shock 15.0 +/- 1.5) compared with controls (7.2 +/- 1.1 micromol/kg/min, p < .01) and was not influenced by lactate infusion. CONCLUSIONS: In patients suffering from septic or cardiogenic shock, hyperlactatemia was mainly related to increased production, whereas lactate clearance was similar to healthy subjects. Increased lactate production was concomitant to hyperglycemia and increased glucose turnover, suggesting that the latter substantially influences lactate metabolism during critical illness.

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.