Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

Evidence from glut2-null mice that glucose is a critical physiological regulator of feeding.

A role for glucose in the control of feeding has been proposed, but its precise physiological importance is unknown. Here, we evaluated feeding behavior in glut2-null mice, which express a transgenic glucose transporter in their beta-cells to rescue insulin secretion (ripglut1;glut2-/- mice). We showed that in the absence of GLUT2, daily food intake was increased and feeding initiation and termination following a fasting period were abnormal. This was accompanied by suppressed regulation of hypothalamic orexigenic and anorexigenic neuropeptides expression during the fast-to-refed transition. In these conditions, however, there was normal regulation of the circulating levels of insulin, leptin, or glucose but a loss of regulation of plasma ghrelin concentrations. To evaluate whether the abnormal feeding behavior was due to suppressed glucose sensing, we evaluated feeding in response to intraperitoneal or intracerebroventricular glucose or 2-deoxy-D-glucose injections. We showed that in GLUT2-null mice, feeding was no longer inhibited by glucose or activated by 2-deoxy-D-glucose injections and the regulation of hypothalamic neuropeptide expression by intracerebroventricular glucose administration was lost. Together, these data demonstrate that absence of GLUT2 suppressed the function of central glucose sensors, which control feeding probably by regulating the hypothalamic melanocortin pathway. Furthermore, inactivation of these glucose sensors causes overeating.

Differential regulation of the glucose-6-phosphatase TATA box by intestine-specific homeodomain proteins CDX1 and CDX2.

Glucose-6-phosphatase (Glc6Pase), the last enzyme of gluconeogenesis, is only expressed in the liver, kidney and small intestine. The expression of the Glc6Pase gene exhibits marked specificities in the three tissues in various situations, but the molecular basis of the tissue specificity is not known. The presence of a consensus binding site of CDX proteins in the minimal Glc6Pase gene promoter has led us to consider the hypothesis that these intestine-specific CDX factors could be involved in the Glc6Pase-specific expression in the small intestine. We first show that the Glc6Pase promoter is active in both hepatic HepG2 and intestinal CaCo2 cells. Using gel shift mobility assay, mutagenesis and competition experiments, we show that both CDX1 and CDX2 can bind the minimal promoter, but only CDX1 can transactivate it. Consistently, intestinal IEC6 cells stably overexpressing CDX1 exhibit induced expression of the Glc6Pase protein. We demonstrate that a TATAAAA sequence, located in position -31/-25 relating to the transcription start site, exhibits separable functions in the preinitiation of transcription and the transactivation by CDX1. Disruption of this site dramatically suppresses both basal transcription and the CDX1 effect. The latter may be restored by inserting a couple of CDX- binding sites in opposite orientation similar to that found in the sucrase-isomaltase promoter. We also report that the specific stimulatory effect of CDX1 on the Glc6Pase TATA-box, compared to CDX2, is related to the fact that CDX1, but not CDX2, can interact with the TATA-binding protein. Together, these data strongly suggest that CDX proteins could play a crucial role in the specific expression of the Glc6Pase gene in the small intestine. They also suggest that CDX transactivation might be essential for intestine gene expression, irrespective of the presence of a functional TATA box.

Activation of liver G-6-Pase in response to insulin-induced hypoglycemia or epinephrine infusion in the rat.

This study was conducted to test the hypothesis of the activation of glucose-6-phosphatase (G-6-Pase) in situations where the liver is supposed to sustain high glucose supply, such as during the counterregulatory response to hypoglycemia. Hypoglycemia was induced by insulin infusion in anesthetized rats. Despite hyperinsulinemia, endogenous glucose production (EGP), assessed by [3-(3)H]glucose tracer dilution, was paradoxically not suppressed in hypoglycemic rats. G-6-Pase activity, assayed in a freeze-clamped liver lobe, was increased by 30% in hypoglycemia (P < 0.01 vs. saline-infused controls). Infusion of epinephrine (1 microg x kg(-1) x min(-1)) in normal rats induced a dramatic 80% increase in EGP and a 60% increase in G-6-Pase activity. In contrast, infusion of dexamethasone had no effect on these parameters. Similar insulin-induced hypoglycemia experiments performed in adrenalectomized rats did not induce any stimulation of G-6-Pase. Infusion of epinephrine in adrenalectomized rats restored a stimulation of G-6-Pase similar to that triggered by hypoglycemia in normal rats. These results strongly suggest that specific activatory mechanisms of G-6-Pase take place and contribute to EGP in situations where the latter is supposed to be sustained.

Polyunsaturated fatty acyl coenzyme A suppress the glucose-6-phosphatase promoter activity by modulating the DNA binding of hepatocyte nuclear factor 4 alpha.

Glucose-6-phosphatase confers on gluconeogenic tissues the capacity to release endogenous glucose in blood. The expression of its gene is modulated by nutritional mechanisms dependent on dietary fatty acids, with specific inhibitory effects of polyunsaturated fatty acids (PUFA). The presence of consensus binding sites of hepatocyte nuclear factor 4 (HNF4) in the -1640/+60 bp region of the rat glucose-6-phosphatase gene has led us to consider the hypothesis that HNF4 alpha could be involved in the regulation of glucose-6-phosphatase gene transcription by long chain fatty acid (LCFA). Our results have shown that the glucose-6-phosphatase promoter activity is specifically inhibited in the presence of PUFA in HepG2 hepatoma cells, whereas saturated LCFA have no effect. In HeLa cells, the glucose-6-phosphatase promoter activity is induced by the co-expression of HNF4 alpha or HNF1 alpha. PUFA repress the promoter activity only in HNF4 alpha-cotransfected HeLa cells, whereas they have no effects on the promoter activity in HNF1 alpha-cotransfected HeLa cells. From gel shift mobility assays, deletion, and mutagenesis experiments, two specific binding sequences have been identified that appear able to account for both transactivation by HNF4 alpha and regulation by LCFA in cells. The binding of HNF4 alpha to its cognate sites is specifically inhibited by polyunsaturated fatty acyl coenzyme A in vitro. These data strongly suggest that the mechanism by which PUFA suppress the glucose-6-phosphatase gene transcription involves an inhibition of the binding of HNF4 alpha to its cognate sites in the presence of polyunsaturated fatty acyl-CoA thioesters.

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes.

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.