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.

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Glucose-6-phosphatase (Glu-6-Pase) catalyzes the terminal step of gluconeogenesis, the conversion of glucose 6-phosphate (Glu-6-P) to free glucose. This enzyme activity is thought to be conferred by a complex of proteins residing in the endoplasmic reticulum (ER), including a Glu-6-P translocase that transports Glu-6-P into the lumen of the ER, a phosphohydrolase catalytic subunit residing in the lumen, and putative glucose and inorganic phosphate transporters that allow exit of the products of the reaction. In this study, we have investigated the effect of adenovirus-mediated overexpression of the Glu-6-Pase catalytic subunit on glucose metabolism and insulin secretion, using a well differentiated insulinoma cell line, INS-1. We found that the overexpressed Glu-6-Pase catalytic subunit was normally glycosylated, correctly sorted to the ER, and caused a 10-fold increase in Glu-6-Pase enzymatic activity in in vitro assays. Consistent with these findings, a 4.2-fold increase in 3 H 2 O incorporation into glucose was observed in INS-1 cells treated with the recombinant adenovirus containing the Glu-6-Pase catalytic subunit cDNA (Ad-CMV-Glu-6-Pase). 3-[ 3 H]Glucose usage was decreased by 32% in AdCMV-Glu-6-Pase-treated cells relative to controls, resulting in a proportional 30% decrease in glucose-stimulated insulin secretion. Our findings indicate that overexpression of the Glu-6-Pase catalytic subunit significantly impacts glucose metabolism and insulin secretion in islet ␤-cells. However, INS-1 cells treated with AdCMV-Glu-6-Pase do not exhibit the severe alterations of ␤-cell function and metabolism associated with islets from rodent models of obesity and non-insulin-dependent diabetes mellitus, suggesting the involvement of genes in addition to the catalytic subunit of Glu-6-Pase in the etiology of such ␤-cell dysfunction.Glucose-6-phosphatase (Glu-6-Pase) 1 catalyzes the terminal step in gluconeogenesis, the hydrolysis of glucose 6-phosphate (Glu-6-P) to free glucose. In liver, Glu-6-Pase plays an important role in regulating glucose disposal and storage in concert with the opposing actions of the glucose-phosphorylating enzyme glucokinase. Glucokinase is also an important regulator of glucose metabolism and glucose-stimulated insulin secretion in ␤-cells of the islets of Langerhans (1, 2). The stimulation of insulin secretion by glucose is thought to be mediated by increases in the ATP:ADP ratio and closure of ATP-sensitive K ϩ channels (1, 3). Glu-6-Pase has also been reported to be expressed in the islets of Langerhans (4), but measurements of enzyme activity have yielded conflicting results (4, 5), and the extent to which the enzyme participates in regulation of glucose flux in normal islets remains unresolved. Measurement of glucose formation and utilization in islets from an obese and insulin-resistant strain of rodents (the ob/ob mouse) has revealed a dramatically increased rate of incorporation of 3 H 2 O into glucose in such islets relative to those from normal lean animals (6, 7). Glucose-stimulated ...

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.

The effect of arachidonic acid (A,Ach) on liver glucose-6-phosphatase (Glc6Pase) has been studied in v i m using untreated and detergent-treated microsomes prepared from fed and 48-h-fasted normal rats and from streptozotocin-induced diabetic rats. Glc6Pase of both untreated and detergenttreated microsomes (60 pg proteidml) is inhibited by d,Ach in a dose-dependent manner between 10-100 pM. The inhibition is very rapid and does not depend on preincubation of microsomes in the presence of A,Ach. It does depend on the concentration of microsomal membranes and on the concentration of glucose 6-phosphate: it is more pronounced at low Glc6P concentrations than at high. As a consequence, the enzyme displays sigmoidal kinetics in the presence of A,Ach. Hill coefficients (equal to 1 in the control experiments) of about 1.4 were determined in the presence of 50 pM d,Ach, indicating a clear positive cooperative dependency of the Glc6Pase upon its substrate in the presence of A,Ach. The A,Ach inhibition is fully reversible in the presence of bovine serum albumin. The inhibition does not depend on the metabolism of d,Ach through the prostaglandin synthase (cyclooxygenase) or arachidonate 12-lipoxygenase pathways since it is not affected by indomethacin and nordihydroguaiaretic acid. Several other unsaturated fatty acids are able to inhibit the enzyme within the same concentration range. In contrast, saturated fatty acids, the arachidonic acid methyl ester and numerous other lipid compounds containing esterified unsaturated fatty acids do not inhibit Glc6Pase within the same concentration range. The enzyme of fed rats was inhibited in the same manner as the enzyme of 48-h-fasted rats. However, Glc6Pase of untreated microsomes from diabetic rats was less inhibitable by d,Ach than the Glc6Pase of normal rats. This difference does not persist after solubilization of the membrane lipids by detergent treatment.