Evidence for the expression of both the hydrolase and translocase components of hepatic glucose-6-phosphatase in murine pancreatic islets

Glucose-6-phosphatase (G6Pase) is a multicomponent enzyme system which regulates the catalysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6Pase can antagonize glucose phosphorylation, a step prerequisite in the regulation of insulin secretion from pancreatic beta cells, and G6Pase activity is increased in islets isolated from animal models of type II diabetes. Using RT-PCR with hepatic G6Pase catalytic subunit primers, we demonstrate that the sizes of amplified products from ob/ob mouse islets are identical to those from liver cDNA. This was confirmed by PCR-based cloning and sequencing of the hepatic G6Pase catalytic subunit open reading frame from islet cDNA. The expression in islets of the G6P transporter, G6PT1, was also demonstrated, suggesting that all of the identified hepatic G6Pase system genes are expressed in pancreatic islets. Finally, the expression of islet-specific G6Pase-related protein (IGRP) in pancreatic islets was confirmed and its expression in liver was also observed.     

Peroxisome proliferator-activated receptor {alpha} is responsible for the up-regulation of hepatic glucose-6-phosphatase gene expression in fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.     

Correction of glycogen storage disease type 1a in a mouse model by gene therapy

Glycogen storage disease type 1a (GSD-1a), characterized by hypoglycemia, liver and kidney enlargement, growth retardation, hyperlipidemia, and hyperuricemia, is caused by a deficiency in glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. To evaluate the feasibility of gene replacement therapy for GSD-1a, we have infused adenoviral vector containing the murine G6Pase gene (Ad-mG6Pase) into G6Pase-deficient (G6Pase(-/-)) mice that manifest symptoms characteristic of human GSD-1a. Whereas <15% of G6Pase(-/-) mice under glucose therapy survived weaning, a 100% survival rate was achieved when G6Pase(-/-) mice were infused with Ad-mG6Pase, 90% of which lived to 3 months of age. Hepatic G6Pase activity in Ad-mG6Pase-infused mice was restored to 19% of that in G6Pase(+/+) mice at 7-14 days post-infusion; the activity persisted for at least 70 days. Ad-mG6Pase infusion also greatly improved growth of G6Pase(-/-) mice and normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles. Furthermore, liver and kidney enlargement was less pronounced with near-normal levels of glycogen depositions in both organs. Our data demonstrate that a single administration of a recombinant adenoviral vector can alleviate the pathological manifestations of GSD-1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy.     

Liver glucose-6-phosphatase proteins in suckling and weaned grey seal pups: structural similarities to other mammals and relationship to nutrition, insulin signalling and metabolite levels

Phocid seals have been proposed as models for diabetes because they exhibit limited insulin response to glucose, high blood glucose and increasing insulin resistance when fasting. Liver glucose-6-phosphatase (G6Pase) catalyses the final step in glucose production and is central to glucose regulation in other animals. G6Pase comprises a translocase (SLC37A4) and a catalytic subunit (G6PC). G6PC and SLC37A4 expression and activity are normally regulated by nutritional state and glucostatic hormones, particularly insulin, and are elevated in diabetes. We tested the hypotheses that (1) grey seal G6PC and SLC37A4 cDNA and predicted protein sequences differ from other species’ at functional sites, (2) relative G6Pase protein abundances are lower during feeding than fasting and (3) relative G6Pase protein abundances are related to insulin, insulin receptor phosphorylation and key metabolite levels. We show that G6PC and partial SLC37A4 cDNA sequences encode proteins sharing 82–95 % identity with other mammals. Seal G6PC contained no differences in sites responsible for activity, stability or subcellular location. Several substitutions in seal SLC37A4 were predicted to be tolerated with low probability, which could affect glucose production. Suckling pups had higher relative abundance of both subunits than healthy, postweaned fasting pups. Furthermore, relative G6PC abundance was negatively related to glucose levels. These findings contrast markedly with the response of relative hepatic G6Pase abundance to feeding, fasting, insulin, insulin sensitivity and key metabolites in other animals, and highlight the need to understand the regulation of enzymes involved in glucose control in phocids if these animals are to be informative models of diabetes.     

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.     

New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system.

The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.     

O-GlcNAc modification of FoxO1 increases its transcriptional activity: A role in the glucotoxicity phenomenon?

O-GlcNAc glycosylations on serines or threonines are reversible post-translational modifications that control the localisation, the activity or the stability of cytosolic and nuclear proteins. These dynamic modifications are tightly dependent on the availability of glucose and on its flux through the hexosamine biosynthetic pathway. We recently showed that treatments that increase protein O-GlcNAc glycosylation (high-glucose concentrations, glucosamine) or inhibit their deglycosylation (PUGNAc), induced O-GlcNAc modification of FoxO1 in HEK293 cells. O-GlcNAc glycosylation of FoxO1 resulted in an increased of its activity towards a glucose 6-phosphatase promoter-luciferase reporter gene (G6Pase-luc). This effect appeared to be independent of FoxO1 sub-cellular re-localisation, since it was also observed with the constitutively nuclear FoxO1-AAA mutant. In liver-derived HepG2 cells, glucosamine and PUGNAc increased the expression of G6Pase mRNA, and synergistic effects were observed when both agents were present together. In addition, the expression of PGC1 alpha gene, which is known to be under the control of FoxO1, was also increased by glucosamine and PUGNAc. In HepG2 cells stably expressing the G6Pase-luc reporter gene, glucosamine and PUGNAc also increased the activity of the G6Pase promoter. The stimulation of the G6Pase reporter gene by these agents was abolished by two different FoxO1 siRNAs, thereby demonstrating the involvement of endogenous FoxO1 in the observed effects. Since G6Pase plays a key role in glucose production by the liver, increased in its expression through FoxO1 O-GlcNAc modification may be of considerable importance in the context of glucotoxicity associated with chronic hyperglycaemia. Moreover, since FoxO1 also plays important roles in several aspects of cell biology, including cell proliferation, survival and apoptosis, the regulation of FoxO1 activity by O-GlcNAc modification may have implications for other crucial biological processes.     

Inhibition of hepatic glucose 6-phosphatase system by the green tea flavanol epigallocatechin gallate

Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.     

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.     

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.     

The glucose-6-phosphatase/glucokinase ratio in the liver of obese-diabetic subjects

The study of G6Pase and GK activities in human liver (needle biopsies) in overnight fasted obese NIDDM patients has shown that, while G6Pase was unchanged, GK was higher (+ 55%, P less than 0.05) than in control subjects. Consequently, the G6Pase/GK ratio (which roughly reflects hepatic glucose production) was significantly reduced (-36%) in the obese diabetic group, due to more GK activity (glucose uptake). This contrasts with the activity in IDDM and nonobese NIDDM patients (where the G6Pase/GK ratio is elevated and normal, respectively) and would suggest that in the obese diabetic subjects, hepatic glucose production is not a major factor contributing to the maintenance of hyperglycemia in the overnight fasting state (leaving peripheral insulin resistance as the major cause of hyperglycemia).     

Hypoglycemic effect of isoleucine involves increased muscle glucose uptake and whole body glucose oxidation and decreased hepatic gluconeogenesis

Isoleucine, a branched chain amino acid, plays an important role in the improvement of glucose metabolism as evidenced by the increase of insulin-independent glucose uptake in vitro. This study evaluated the effect of isoleucine on glucose uptake and oxidation in fasted rats and on gluconeogenesis in vivo and in vitro. Oral administration of isoleucine decreased the plasma glucose level by 20% and significantly increased muscle glucose uptake by 71% without significant elevation of the plasma insulin level compared with controls at 60 min after administration. Furthermore, expiratory excretion of 14CO2 from [U-14C]glucose in isoleucine-administered rats was increased by 19% compared with controls. Meanwhile, isoleucine decreased AMP levels in the liver but did not affect hepatic glycogen synthesis. Under insulin-free conditions, isoleucine significantly inhibited glucose production when alanine was used as a glucogenic substrate in isolated hepatocytes. This inhibition by isoleucine was also associated with a decline in mRNA levels for phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) and a decreased activity of G6Pase in isolated hepatocytes. These findings suggest that a reduction of gluconeogenesis in liver, along with an increase of glucose uptake in the muscle, is also involved in the hypoglycemic effect of isoleucine. In conclusion, isoleucine administration stimulates both glucose uptake in the muscle and whole body glucose oxidation, in addition to depressing gluconeogenesis in the liver, thereby leading to the hypoglycemic effect in rats.