Frank Stümpel

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 [14C]glucose proceeded with the same kinetics in control and GLUT2−/− intestine, [14C]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.

Sensing by intrahepatic muscarinic nerves of a portal-arterial glucose concentration gradient as a signal for insulin-dependent glucose uptake in the perfused rat liver

In vivo, insulin increases net hepatic glucose uptake efficiently only in the presence of a portal‐arterial glucose gradient. In isolated perfused rat livers supplied with a glucose gradient (portal 10 mM/arterial 5 mM) insulin‐induced glucose uptake was blocked by atropine; in livers not supplied with the gradient (portal=arterial 5 mM) insulin‐dependent glucose uptake was elicited by acetylcholine. Apparently, the gradient was sensed and transformed into a metabolic signal by intrahepatic nerves, releasing acetylcholine to muscarinic receptors.

Mice without the Regulator Gene Rsc1A1 Exhibit Increased Na+-d-Glucose Cotransport in Small Intestine and Develop Obesity

ABSTRACT The product of the intronless single copy gene RSC1A1, named RS1, is an intracellular 617-amino-acid protein that is involved in the regulation of the Na+-d-glucose cotransporter SGLT1. We generated and characterized RS1 knockout (RS1− / −) mice. In the small intestines of RS1 − / − mice, the SGLT1 protein was up-regulated sevenfold compared to that of wild-type mice but was not changed in the kidneys. The up-regulation of SGLT1 was posttranscriptional. Small intestinal d-glucose uptake measured in jointly perfused small bowel and liver was increased twofold compared to that of the wild-type, with increased peak concentrations of d-glucose in the portal vein. At birth, the weights of RS1 − / − and wild-type mice were similar. At the age of 3 months, male RS1 − / − mice had 5% higher weights and 15% higher food intakes, whereas their energy expenditures and serum leptin concentrations were similar to those of wild-type mice. At the age of 5 months, male and female RS1 − / − mice were obese, with 30% increased body weight, 80% increased total fat, and 30% increased serum cholesterol. At this age, serum leptin was increased, whereas food intake was the same as for wild-type mice. The data suggest that the removal of RS1 leads to leptin-independent up-regulation of food intake, which causes obesity.

Mechanism of insulin resistance in CCl4-induced cirrhosis of rats

Insulin action was studied in rats with CCl4/phenobarbital-induced cirrhosis of the liver using the euglycemic hyperinsulinemic clamp technique coupled with isotopic measurement of individual tissue glucose uptake, glycogen formation, and lipogenesis. In cirrhotic rats, dose response curves showed a reduction of insulin-stimulated total body glucose disposal of about 30%. Insulin action on tissue glucose uptake and initial phosphorylation (assessed with [3H]2-deoxyglucose) were unchanged; however, incorporation of [14C]glucose into lipids and particularly into glycogen was reduced substantially (being most pronounced in skeletal muscle and diaphragm) at maximally as well as half-maximally effective serum insulin concentrations during euglycemic clamping. At identical IV insulin infusion rates, steady-state serum insulin concentrations were elevated up to fourfold in cirrhotic animals. Antilipolytic action of insulin was unaltered. These data suggest that the principal metabolic pathway affected in insulin resistance of rats with experimental cirrhosis appeared to be insulin-stimulated glycogen formation in muscle tissues.

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.

Abstract. Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.

Loss of regulation by sympathetic hepatic nerves of liver metabolism and haemodynamics in chronically streptozotocin-diabetic rats

SummaryThe consequences of autonomic diabetic neuropathy, a common complication of chronic diabetes mellitus, have been studied mainly with regard to heart and stomach function. Since the autonomic nervous system also regulates liver carbohydrate metabolism and haemodynamics via hepatic nerves, it was the purpose of this study to examine the function of hepatic nerves in chronically diabetic rats. Diabetes was induced by i.p. injection of streptozotocin. Rat livers were perfused via both portal vein and hepatic artery. Hepatic nerves were stimulated for 2 min using a platinum electrode placed around the portal vein and the hepatic artery; in an additional stimulation phase noradrenaline was infused into the portal vein. Stimulation of hepatic nerves as well as portal noradrenaline infusion increased hepatic glucose output and reduced flow in control and in acutely (48-h) diabetic animals, which still had almost normal glycogen content. In addition stimulation also caused an overflow of noradrenaline into the caval vein. However, nerve stimulation neither increased glucose output nor decreased flow in 4-month diabetic rats. In these rats noradrenaline overflow was nearly completely abolished and hepatic glycogen content was markedly depleted. Portal noradrenaline infusion in chronically diabetic rats reduced flow to a similar extent as in controls, yet the increase in glucose output was diminished. The lack of nerve stimulation-dependent glucose output, flow reduction and noradrenaline overflow is indicative of a profound loss of function of hepatic autonomic nerves in chronically diabetic rats.

In vivo metabolic action of insulin-like growth factor I in adult rats

SummaryThe acute metabolic actions of insulin-like growth factor I were studied in anaesthetized adult rats and its potency was compared to that of insulin. Following an i. v. bolus injection of insulin-like growth factor I a dose-dependent decrease of blood glucose and serum non-esterified fatty acid concentrations was noted with a potency of about 2% that of insulin. Stimulation of total body glucose disposal during euglycaemic clamping required ∼ 50times higher insulin-like growth factor I serum concentrations to achieve an identical half-maximal response. A similar difference in potency was observed for the stimulatory action on 2-de-oxyglucose uptake and on glycogen formation in skeletal muscle. Lipogenesis in epididymal fat pads was increased dose-dependently by both hormones requiring approximately 30 times higher half-maximally effective serum concentrations of insulin-like growth factor I. These data demonstrate that insulin-like growth factor I exerted acute insulin-like metabolic actions in vivo with low potency. These effects were probably mediated via insulin receptors. A preferential stimulation of glucose metabolism in skeletal muscle was not observed.

A new role for enteric glucagon-37: acute stimulation of glucose absorption in rat small intestine.

Glucagon‐37 is secreted by intestinal L‐cells following carbohydrate uptake. It is known to inhibit gastric acid secretion (hence also named oxyntomodulin) and appears to increase intracellular cyclic AMP concentrations. Since cyclic AMP could enhance intestinal glucose absorption, a possible stimulatory effect of glucagon‐37 on glucose transport was examined. Glucagon‐37 acutely increased glucose absorption in the isolated, vascularly perfused small intestine and in isolated enterocytes of the rat. In these cells the stimulation by glucagon‐37 could be completely blocked by the cAMP antagonist Rp‐cAMPS and was therefore mediated by cAMP. The stimulation of intestinal glucose absorption by glucagon‐37 appears to be a major new physiological function.

Impaired glucose sensing by intrahepatic, muscarinic nerves for an insulin-stimulated hepatic glucose uptake in streptozotocin-diabetic rats

Insulin‐induced net hepatic glucose uptake depends on the sensing by muscarinic, intrahepatic nerves of a glucose concentration gradient between portal vein and hepatic artery. The function of these intrahepatic nerves was examined in streptozotocin‐diabetic rats. In the presence of the glucose gradient insulin induced net glucose uptake in isolated perfused livers from control and acutely diabetic but not from chronically diabetic animals. The neurotransmitter acetylcholine still mimicked the existence of the gradient, excluding a metabolic impairment of livers of chronically diabetic animals. The impairment of the intrahepatic nerves due to diabetic neuropathy could contribute to postprandial hyperglycemia in diabetes mellitus.

In vivo metabolic activity of des-(B26-B30)-insulin-B25-amide and related analogues in the rat

Metabolic potency of des-(B26-B30)-insulin-B25-amide, [TyrB25]des- (B26-B30)-insulin-B25-amide and [HisB25]des-(B26-B30)-insulin-B25-amide was studied in anaesthetized rats. Compared to insulin, full potency for des-(B26-B30)-insulin-B25-amide and an enhanced potency for both substituted analogues has been described previously on rat adipocytes in vitro. Hypoglycaemic effects following i.v. injection of all of these analogues were almost identical to those of native insulin with a half-maximal effective dose of approximately 3 nmol.kg-1. Stimulation of glucose metabolism during euglycaemic hyperinsulin-/analogueaemic clamp studies was indistinguishable from that of the native hormone with a maximal stimulation of approximately 19 mg.kg-1.min-1 and half-maximal effective hormone concentrations of approximately 1 pmol.ml-1. Analogue action on individual peripheral tissues estimated by the uptake of 2-deoxyglucose as well as stimulation of lipogenesis in epididymal fat was not different to that of insulin. These data demonstrate that C-terminal amidation of des-(B26-B30)-insulin results in a shortened molecule with full in vivo metabolic potency. When substituting phenylalanine in position B25 by tyrosine or histidine, the insulin-identical potency is preserved.