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The activation of glucose-6-phosphatase (G6Pase), a key enzyme of endogenous glucose production, is correlated with type 2 diabetes. Type 2 diabetes is characterized by sustained hyperglycemia leading to glucotoxicity. We investigated whether glucotoxicity mechanisms control the expression of the G6Pase catalytic unit (G6pc). We deciphered the transcriptional regulatory mechanisms of the G6pc promoter by glucotoxicity in a hepatoma cell line then in primary hepatocytes and in the liver of diabetic mice. High glucose exposure induced the production of reactive oxygen species (ROS) and, in parallel, induced G6pc promoter activity. In hepatocytes, glucose induced G6pc gene expression and glucose release. The decrease of ROS concentrations by antioxidants eliminated all the glucose-inductive effects. The induction of G6pc promoter activity by glucose was eliminated in the presence of small interfering RNA, targeting either the hypoxia-inducible factor (HIF)-1α or the CREB-binding protein (CBP). Glucose increased the interaction of HIF-1α with CBP and the recruitment of HIF-1 on the G6pc promoter. The same mechanism might occur in hyperglycemic mice. We deciphered a new regulatory mechanism induced by glucotoxicity. This mechanism leading to the induction of HIF-1 transcriptional activity may contribute to the increase of hepatic glucose production during type 2 diabetes.

Objective: Since the pioneering work of Claude Bernard, the scientific community has considered the liver to be the major source of endogenous glucose production in all postabsorptive situations. Nevertheless, the kidneys and intestine can also produce glucose in blood, particularly during fasting and under protein feeding. The aim of this study was to better define the importance of the three gluconeogenic organs in glucose homeostasis., Research Design and Methods: We investigated blood glucose regulation during fasting in a mouse model of inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc(-/-) mice), encoding a mandatory enzyme for glucose production. Furthermore, we characterized molecular mechanisms underlying expression changes of gluconeogenic genes (G6pc, Pck1, and glutaminase) in both the kidneys and intestine., Results: We show that the absence of hepatic glucose release had no major effect on the control of fasting plasma glucose concentration. Instead, compensatory induction of gluconeogenesis occurred in the kidneys and intestine, driven by glucagon, glucocorticoids, and acidosis. Moreover, the extrahepatic action of glucagon took place in wild-type mice., Conclusions: Our study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting endogenous glucose production under the control of glucagon, regardless of the contribution of the liver. Thus, the current dogma relating to the respective role of the liver and of extrahepatic gluconeogenic organs in glucose homeostasis requires re-examination.

The aim of this study was to understand by which intrahepatic mechanism metformin (Met) may inhibit basal hepatic glucose production (HGP) in type 2 diabetes. We studied rats that were fed for 6 weeks a high-fat (HF) diet, supplemented (HF-Met) or not (HF) with Met (50 mg x kg(-1) x day(-1)). Basal HGP, assessed by 3-[(3)H]glucose tracer dilution, was lower by 20% in HF-Met rats compared with HF-rats: 41.6 +/- 0.7 vs. 52 +/- 1.5 micromol x kg(-1) x min(-1) (means +/- SE, n = 5; P < 0.01). Glucose-6 phosphatase (Glc6Pase) activity, assayed in a liver lobe freeze-clamped in situ, was lower by 25% in HF-Met rats compared with HF-rats (7.9 +/- 0.4 vs. 10.3 +/- 0.9 micromol x min(-1) x g(-1) wet liver; P < 0.05). Glucose-6 phosphate and glycogen contents, e.g., 42 +/- 5 nmol/g and 3.9 +/- 2.4 mg/g, respectively, in HF-rats were dramatically increased by three to five times in HF-Met rats, e.g., 118 +/- 12 nmol/g and 19.6 +/- 4.6 mg/g (P < 0.05 and P < 0.01, respectively). Glucose-6 phosphate dehydrogenase activity was increased in HF-Met compared with HF rats (1.51 +/- 0.1 vs. 1.06 +/- 0.08 micromol x min(-1) x g(-1); P < 0.01). Intrahepatic lactate concentration tended to be lower in the Met-group (-30%; NS), whereas plasma lactate concentration was higher in HF-Met rats (1.59 +/- 0.15 mmol/l) than in HF rats (1.06 +/- 0.06 mmol/l; P < 0.05). We concluded that Met decreases HGP in insulin-resistant HF-fed rats mainly by an inhibition of hepatic Glc6Pase activity, promoting glycogen sparing. Additional mechanisms might involve the diversion of glucose-6 phosphate into the pentose phosphate pathway and an inhibition of hepatic lactate uptake.

At variance with the current view that only liver and kidney are gluconeogenic organs, because both are the only tissues to express glucose-6-phosphatase (Glc6Pase), we have recently demonstrated that the Glc6Pase gene is expressed in the small intestine in rats and humans and that it is induced in insulinopenic states such as fasting and diabetes. We used a combination of arteriovenous balance and isotopic techniques, reverse transcription-polymerase chain reaction, Northern blot analysis, and enzymatic activity assays. We report that rat small intestine can release neosynthesized glucose in mesenteric blood in insulinopenia, contributing 20-25% of total endogenous glucose production. Like liver glucose production, small intestine glucose production is acutely suppressed by insulin infusion. In the small intestine, glutamine and, to a much lesser extent, glycerol are the precursors of glucose, whereas alanine and lactate are the main precursors in liver. Accounting for these metabolic fluxes: 1) the phosphoenolpyruvate carboxykinase gene (required for the utilization of glutamine) is strongly induced at the mRNA and enzyme levels in insulinopenia; 2) the glycerokinase gene is expressed, but not induced; 3) the pyruvate carboxylase gene (required for the utilization of alanine and lactate) is repressed by 80% at the enzyme level in insulinopenia. These studies identify small intestine as a new insulin-sensitive tissue and a third gluconeogenic organ, possibly involved in the pathophysiology of diabetes.

The GK rat model of type 2 diabetes is especially convenient to dissect the pathogenic mechanism necessary for the emergence of overt diabetes because all adult rats obtained in our department (GK/Par colony) to date have stable basal mild hyperglycemia and because overt diabetes is preceded by a period of normoglycemia, ranging from birth to weaning. The purpose of this article is to sum up the information so far available related to the biology of the beta-cell in the GK/Par rat. In terms of beta-cell function, there is no major intrinsic secretory defect in the prediabetic GK/Par beta-cell, and the lack of beta-cell reactivity to glucose (which reflects multiple intracellular abnormalities), as seen during the adult period when the GK/Par rats are overtly diabetic, represents an acquired defect (perhaps glucotoxicity). In terms of beta-cell population, the earliest alteration so far detected in the GK/Par rat targets the size of the beta-cell population. Several convergent data suggest that the permanently reduced beta-cell mass in the GK/Par rat reflects a limitation of beta-cell neogenesis during early fetal life, and it is conceivable that some genes among the set involved in GK diabetes belong to the subset of genes controlling early beta-cell development.

PEPCK is a key enzyme of gluconeogenesis in liver and kidney. Recently, we have shown that small intestine also contributes to the endogenous glucose production in insulinopenia in rats and that glutamine is the main precursor of glucose synthesized in this tissue. The expression of the PEPCK gene in rat and human small intestine and the effect of streptozotocin-induced diabetes and fasting have been studied using reverse transcriptase-polymerase chain reaction, Northern blot analysis, and determination of enzyme activity. The PEPCK gene is expressed along the whole small intestine in adult rat and human. The abundance of PEPCK mRNA was increased approximately 30 times in the duodenum, 15 times in the jejunum, and only 3 times in the ileum from diabetic rats. PEPCK mRNA was downregulated in all parts of the tissue upon insulin treatment for 10 h. In 48-h fasted rats, the PEPCK mRNA abundance was increased 17 times in the duodenum and the jejunum and 3 times in the ileum, and it was normalized upon refeeding for 7 h. PEPCK activity was also increased 2-5 times in diabetic and fasted rats in the duodenum and jejunum but not in the ileum. We conclude that PEPCK is a crucial enzyme contributing to the induction of gluconeogenesis in small intestine, just as it is well known to be in the liver and kidney.

The mRNA and the activity of glucose-6-phosphatase (Glc-6-Pase) were present in the liver, kidney, and small intestine of 15-day-old suckling rats, but were absent from the stomach, colon, lung, white and brown adipose tissues, muscle, heart, brain, and spleen. The mRNA encoding Glc-6-Pase was present in the liver of 21-day-old fetal rats and increased markedly immediately after birth. From 5 days after birth to the end of the suckling period, it returned to 50% of the level found in the liver of 48-h starved adult rats. When rats were weaned at 21 days onto a high-carbohydrate, low-fat (HCLF) diet, the concentration of liver Glc-6-Pase mRNA was markedly increased. In the fetal rat jejunum, the activity and mRNA of Glc-6-Pase were very low. It increased during the 5 days after birth and then declined to reach very low levels. Neither mRNA nor activity of Glc-6-Pase was present in the fetal kidney. They appeared and increased slowly during the suckling period to reach maximal levels 15 days after birth and then remained constant. Weaning onto the HCLF diet did not change the Glc-6-Pase gene expression, neither in the jejunum nor in the kidney. The regulation of Glc-6-Pase gene expression by hormones and nutrients was studied in cultured hepatocytes from 20-day-old rat fetuses. Bt2cAMP stimulated the Glc-6-Pase gene expression in a dose-dependent manner. This probably resulted from an increased gene transcription since the half-life of the transcript was not affected by dibutyryl cAMP (Bt2cAMP). The Bt2cAMP-induced Glc-6-Pase mRNA accumulation was antagonized by insulin in a dose-dependent manner. Long-chain fatty acids (LCFAs), but not medium-chain fatty acids, induced the accumulation of Glc-6-Pase mRNA and the stabilization of the transcript. The peroxisome proliferator, clofibrate, induced a threefold increase in Glc-6-Pase mRNA concentration. Both stimulation of Glc-6-Pase mRNA by LCFAs and clofibrate were inhibited by insulin. Increasing concentrations of glucose (from 0 to 20 mmol/l) did not affect the Bt2cAMP-induced Glc-6-Pase gene expression. By contrast, high glucose concentration (25 mmol/l) markedly induced the Glc-6-Pase gene expression in fed adult rat hepatocytes. The difference in the response to glucose between fetal and adult rat hepatocytes is discussed. We conclude that the rapid increase in hepatic Glc-6-Pase mRNA levels that accompanies the fetal-to-neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled to the rise in liver cAMP concentration.

Using Northern blot with a specific glucose-6-phosphatase (Glc6Pase) cDNA probe and enzymatic activity determination, we studied the effect of streptozotocin-induced diabetes on Glc6Pase in rat gluconeogenic tissues. The Glc6Pase mRNA abundance was increased four to five times in both the liver and kidney of diabetic rats. This was correlated with a concomitant 130% increase in Glc6Pase catalytic subunit in both tissues. The elevated level of Glc6Pase mRNA was significantly corrected in both the liver and kidney of diabetic rats after a 12-h insulin treatment. We also studied Glc6Pase mRNA and activity in gluconeogenic tissues during the fed-fasted and fasted-refed transitions in normal rats. In the liver, the abundance of Glc6Pase mRNA was sharply increased about four times after 24 or 48 h of fasting. In the kidney, the Glc6Pase mRNA level was gradually increased some three and five times after 24 and 48 h of fasting, respectively. The increase of Glc6Pase mRNA in both organs was matched with a doubling of the activity of Glc6Pase catalytic subunit: rapid in the liver and gradual in the kidney. The liver Glc6Pase mRNA abundance in 48-h fasted rats was acutely and importantly decreased upon refeeding. The kidney Glc6Pase mRNA level was also significantly lowered under these conditions, albeit less rapidly. These data demonstrate that efficient control of Glc6Pase takes place in both gluconeogenic organs at the pretranslational level and suggest that insulin might play an important role in this control. In addition, using reverse transcription-polymerase chain reaction and Northern blot, we report that Glc6Pase mRNA is not detectable in several other tissues previously assumed to express the enzyme.