Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

Upregulation of hepatic glucose 6-phosphatase gene expression in rats treated with an inhibitor of glucose-6-phosphate translocase

The multicomponent hepatic glucose 6-phosphatase (Glc-6-Pase) system catalyzes the terminal step of hepatic glucose production and plays a key role in the regulation of blood glucose. We used the chlorogenic acid derivative S 3483, a reversible inhibitor of the glucose-6-phosphate (Glc-6-P) translocase component, to demonstrate for the first time upregulation of Glc-6-Pase expression in rat liver in vivo after inhibition of Glc-6-P translocase. In accordance with its mode of action, S 3483-treatment of overnight-fasted rats induced hypoglycemia and increased blood lactate, hepatic Glc-6-P, and glycogen. The metabolic changes were accompanied by rapid and marked increases in Glc-6-Pase mRNA (above 35-fold), protein (about 2-fold), and enzymatic activity (about 2-fold). Maximal mRNA levels were reached after 4 h of treatment. Glycemia, blood lactate, and Glc-6-Pase mRNA levels returned to control values, whereas Glc-6-P and glycogen levels decreased but were still elevated 2 h after S 3483 withdrawal. The capacity for Glc-6-P influx was only marginally increased after 8.5 h of treatment. Prevention of hypoglycemia by euglycemic clamp did not abolish the increase in Glc-6-Pase mRNA induced by S 3483 treatment. A similar pattern of hypoglycemia and possibly of associated counterregulatory responses elicited by treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinic acid could account for only a 2-fold induction of Glc-6-Pase mRNA. These findings suggest that the significant upregulation of Glc-6-Pase gene expression observed after treatment of rats in vivo with an inhibitor of Glc-6-P translocase is caused predominantly either by S 3483 per se or by the compound-induced changes of intracellular carbohydrate metabolism.     

O-glycosylation of FoxO1 increases its transcriptional activity towards the glucose 6-phosphatase gene

Mono-O-glycosylations post-translationally regulate the activity of nucleocytoplasmic proteins. We showed that glucosamine and an inhibitor of deglycosylation (PUGNAc) induced O-glycosylation of FoxO1, resulting in increased expression of a glucose-6-phosphatase reporter gene. This effect was independent of FoxO1 re-localisation, since it was also observed with constitutively nuclear FoxO1-AAA mutant. Moreover, in HepG2 cells, glucosamine and PUGNAc have a synergistic effect on the glucose-6-phosphatase reporter gene, and this effect was inhibited by FoxO1 siRNAs. Since glucose-6-phosphatase plays a key role in hepatic glucose production, our observation may be of importance with regard to glucotoxicity associated with chronic hyperglycaemia in diabetes.     

Resistin overexpression impaired glucose tolerance in hepatocytes

Resistin is a 12.5-kDa cysteine-rich protein secreted from adipose tissue and is an important factor linking obesity with insulin resistance. Here, we investigated the effect of resistin on glucose tolerance in adult human hepatocytes (L-02 cells). In this study, resistin cDNA was transfected into L-02 cells, and glucose concentration and glucokinase activity were determined subsequently. The data indicated resistin impaired, insulin-stimulated glucose utilization, which implied liver was a target tissue of resistin. To understand its molecular mechanism, mRNA levels of key genes in glucose metabolism and insulin signaling pathway were analyzed. The results demonstrated resistin-stimulated expression of glucose-6-phosphatase (G6Pase), sterol regulatory element-binding protein 1c (SREBP1c) and suppressor of cytokine signaling 3 (SOCS-3), repressed expression of peroxisome proliferator-activated receptor gamma (PPARgamma) as well as insulin receptor substrate 2 (IRS-2). Given that glucokinase (GK) activity and glucose transporter 2 (GLUT2) expression were not altered, we presumed that resistin did not effect them. Moreover, resistin lowered mRNA levels of IRS-2 while stimulating SOCS-3 expression, which suggests it impairs glucose tolerance by blocking the insulin signal transduction pathway.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone

The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase.     

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.     

Signal-dependent control of gluconeogenic key enzyme genes through coactivator-associated arginine methyltransferase 1

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

Glucokinase is highly induced and glucose-6-phosphatase poorly repressed in liver of rainbow trout (Oncorhynchus mykiss) by a single meal with glucose

The low dietary starch utilisation by rainbow trout (Oncorhynchus mykiss) may be attributed to a dysfunction of the nutritional regulation of the hepatic glucose/glucose-6-phosphate cycle. The present study was initiated to analyse the regulation of activity and gene expression of hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) by dietary carbohydrates in this species. We found that even a single meal containing 24% of glucose is sufficient to induce the GK expression (mRNA and activity) as in mammals. In contrast, although the inhibitory effect of dietary glucose on G6Pase expression is observed at the molecular level, the G6Pase activity is not significantly inhibited by dietary glucose. Thus, in contrast to the gluconeogenic G6Pase enzyme, a rapid adaptation of the hepatic glycolytic GK enzyme to dietary glucose seems effective in rainbow trout. These results suggest that in carnivorous rainbow trout, the liver is capable to strongly regulate the utilisation of glucose but not the synthesis of glucose.     

Regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones

The regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones has been studied following the administration of 3,3',5-triiodo-L-thyronine and/or triamcinolone to hypophysectomized rats. The apparent Km for glucose-6-P in isolated ("intact") microsomes increased following administration of either hormone; there was little or no difference in the apparent Km when microsomes were treated with sodium deoxycholate ("disrupted"). In intact microsomes, triiodothyronine caused a 2.3-fold increase in the Vmax of glucose 6-phosphatase; triamcinolone, a 4-fold increase; and both hormones together, a 4.4-fold increase. Corresponding values for disrupted microsomes were: triiodothyronine, 3.7-fold; triamcinolone, 1.8-fold; both hormones, 3.3-fold. After triiodothyronine treatment, disruption of microsomes caused an over 5-fold increase in Vmax; after triamcinolone treatment, the increase was only 1.5-fold. This difference could not be explained by a change in the energy of activation of glucose 6-phosphatase in either intact or disrupted microsomes following hormone treatment. Glucose 6-phosphatase was localized by a cytochemical procedure; the reaction product was associated with 90% of the profiles in all microsomal preparations, except for those from triiodothyronine-treated rats, where less than 50% contained lead precipitate. Vesicles free of lead phosphate were isolated from sucrose gradients and accounted for less than 10% of the protein and glucose 6-phosphatase in all preparations, again except for those from triiodothyronine-treated rats, where they represented 40% of both the protein and glucose 6-phosphatase. The results are consistent with a model for glucose 6-phosphatase in which the substrate is transported across the microsomal membrane by a specific carrier before hydrolysis within the cisternae by a phosphohydrolase. It is suggested that the effect of triiodothyronine is mainly on the activity of the phosphohydrolase, and triamcinolone, on that of the carrier.     

Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.     

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.     

Bile acids regulate hepatic gluconeogenic genes and farnesoid X receptor via G��i-protein-coupled receptors and the AKT pathway

Bile acids are important regulatory molecules that can activate specific nuclear receptors and cell signaling pathways in the liver and gastrointestinal tract. In the current study, the chronic bile fistula (CBF) rat model and primary rat hepatocytes (PRH) were used to study the regulation of gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) and the gene encoding short heterodimeric partner (SHP) by taurocholate (TCA). The intestinal infusion of TCA into the CBF rat rapidly (1 h) activated the AKT (∼9-fold) and ERK1/2 (3- to 5-fold) signaling pathways, downregulated (∼50%, 30 min) the mRNA levels of PEPCK and G-6-Pase, and induced (14-fold in 3 h) SHP mRNA. TCA rapidly (∼50%, 1–2 h) downregulated PEPCK and G-6-Pase mRNA levels in PRH. The downregulation of these genes by TCA was blocked by pretreatment of PRH with pertussis toxin (PTX). In PRH, TCA plus insulin showed a significantly stronger inhibition of glucose secretion/synthesis from lactate and pyruvate than either alone. The induction of SHP mRNA in PRH was strongly blocked by inhibition of PI3 kinase or PKCζ by specific chemical inhibitors or knockdown of PKCζ by siRNA encoded by a recombinant lentivirus. Activation of the insulin signaling pathway appears to be linked to the upregulation of farnesoid X receptor functional activity and SHP induction.     

Deletion of CaMKK2 from the liver lowers blood glucose and improves whole-body glucose tolerance in the mouse

Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca(2+)/CaM-dependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system -signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism. CaMKK2 is shown to be expressed in liver and acute, specific reduction of the kinase in the liver of high-fat diet-fed CaMKK2(floxed) mice results in lowered blood glucose and improved glucose tolerance. Primary hepatocytes isolated from CaMKK2 KO mice produce less glucose and have decreased mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α and the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and these mRNA fail to respond specifically to the stimulatory effect of catecholamine in a cell-autonomous manner. The mechanism responsible for suppressed gene induction in CaMKK2 KO hepatocytes may involve diminished phosphorylation of histone deacetylase 5, an event necessary in some contexts for derepression of the peroxisome proliferator-activated receptor γ coactivator 1-α promoter. Hepatocytes from CaMKK2 KO mice also show increased rates of de novo lipogenesis and fat oxidation. The changes in fat metabolism observed correlate with steatotic liver and altered acyl carnitine metabolomic profiles in CaMKK2 KO mice. Collectively, these results are consistent with suppressed catecholamine-induced induction of gluconeogenic gene expression in CaMKK2 KO mice that leads to improved whole-body glucose homeostasis despite the presence of increased hepatic fat content.     

Distinct hormone stimulation and counteraction by insulin of the expression of the two components of glucose 6-phosphatase in HepG2 cells

We found recently (J. Biol. Chem. 274, 33866-33869, 1999) that the expression of the catalytic subunit (p36) and putative glucose 6-phosphate translocase (p46) of the liver glucose 6-phosphatase system was stimulated by cyclic AMP and glucose and repressed by insulin. We now further show in HepG2 cells that whereas insulin (0.01-10 nM) suppressed p36 mRNA, it only reduced p46 mRNA by half at 1 microM. Cyclic AMP (0.01-100 microM) caused a 2.7-fold increase in p36 mRNA but barely increased p46 mRNA. In contrast, dexamethasone (0.1-100 nM) increased both p36 and p46 mRNA by more than 3-fold. The effects of cyclic AMP and dexamethasone were counteracted by 1 microM insulin. The endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1-100 nM) increased p36 mRNA by 2-fold but not p46 mRNA. It thus appears that the hormonal changes which affect p36 alone concur with known modifications in glucose production; those that affect both p36 and p46 are rather consistent with glucose storage.     

Diabetes affects similarly the catalytic subunit and putative glucose-6-phosphate translocase of glucose-6-phosphatase

The effect of streptozocin diabetes on the expression of the catalytic subunit (p36) and the putative glucose-6-phosphate translocase (p46) of the glucose-6-phosphatase system (G6Pase) was investigated in rats. In addition to the documented effect of diabetes to increase p36 mRNA and protein in the liver and kidney, a approximately 2-fold increase in the mRNA abundance of p46 was found in liver, kidney, and intestine, and a similar increase was found in the p46 protein level in liver. In HepG2 cells, glucose caused a dose-dependent (1-25 mM) increase (up to 5-fold) in p36 and p46 mRNA and a lesser increase in p46 protein, whereas insulin (1 microM) suppressed p36 mRNA, reduced p46 mRNA level by half, and decreased p46 protein by about 33%. Cyclic AMP (100 microM) increased p36 and p46 mRNA by >2- and 1.5-fold, respectively, but not p46 protein. These data suggest that insulin deficiency and hyperglycemia might each be responsible for up-regulation of G6Pase in diabetes. It is concluded that enhanced hepatic glucose output in insulin-dependent diabetes probably involves dysregulation of both the catalytic subunit and the putative glucose-6-phosphate translocase of the liver G6Pase system.