A novel, cupin-type phosphoglucose isomerase in Escherichia coli.

BACKGROUND: Escherichia coli cells contain a homolog of presumed 5-keto-4-deoxyuronate isomerase (KduI) from pectin-degrading soil bacteria, but the catalytic activity of the E. coli protein (o-KduI) was never demonstrated. METHODS: The known three-dimensional structure of E. coli o-KduI was compared with the available structures of sugar-converting enzymes. Based on the results of this analysis, sugar isomerization activity of recombinant o-KduI was tested against a panel of D-sugars and their derivatives. RESULTS: The three-dimensional structure of o-KduI exhibits a close similarity with Pyrococcus furiosus cupin-type phosphoglucose isomerase. In accordance with this similarity, o-KduI was found to catalyze interconversion of glucose-6-phosphate and fructose-6-phosphate and, less efficiently, conversion of glucuronate to fructuronate. o-KduI was hexameric in crystals but represented a mixture of inactive hexamers and active dimers in solution and contained a tightly bound Zn2+ ion. Dilution, substrate binding and Zn2+ removal shifted the hexamer ⇆ dimer equilibrium to the dimers. CONCLUSIONS: Our findings identify o-KduI as a novel phosphosugar isomerase in E. coli, whose activity may be regulated by changes in oligomeric structure. GENERAL SIGNIFICANCE: More than 5700 protein sequences are annotated as KduI, but their enzymatic activity has not been directly demonstrated. E. coli o-KduI is the first characterized member of this group, and its enzymatic activity was found to be different from the predicted activity.

Inhibition of Glycogen Synthase II with RNAi Prevents Liver Injury in Mouse Models of Glycogen Storage Diseases.

Glycogen storage diseases (GSDs) of the liver are devastating disorders presenting with fasting hypoglycemia as well as hepatic glycogen and lipid accumulation, which could lead to long-term liver damage. Diet control is frequently utilized to manage the potentially dangerous hypoglycemia, but there is currently no effective pharmacological treatment for preventing hepatomegaly and concurrent liver metabolic abnormalities, which could lead to fibrosis, cirrhosis, and hepatocellular adenoma or carcinoma. In this study, we demonstrate that inhibition of glycogen synthesis using an RNAi approach to silence hepatic Gys2 expression effectively prevents glycogen synthesis, glycogen accumulation, hepatomegaly, fibrosis, and nodule development in a mouse model of GSD III. Mechanistically, reduction of accumulated abnormally structured glycogen prevents proliferation of hepatocytes and activation of myofibroblasts as well as infiltration of mononuclear cells. Additionally, we show that silencing Gys2 expression reduces hepatic steatosis in a mouse model of GSD type Ia, where we hypothesize that the reduction of glycogen also reduces the production of excess glucose-6-phosphate and its subsequent diversion to lipid synthesis. Our results support therapeutic silencing of GYS2 expression to prevent glycogen and lipid accumulation, which mediate initial signals that subsequently trigger cascades of long-term liver injury in GSDs.

Liver-directed gene therapy for murine glycogen storage disease type Ib.

Glycogen storage disease type-Ib (GSD-Ib), deficient in the glucose-6-phosphate transporter (G6PT), is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenoma (HCA). We examined the efficacy of G6PT gene therapy in G6pt-/- mice using recombinant adeno-associated virus (rAAV) vectors, directed by either the G6PC or the G6PT promoter/enhancer. Both vectors corrected hepatic G6PT deficiency in murine GSD-Ib but the G6PC promoter/enhancer was more efficacious. Over a 78-week study, using dose titration of the rAAV vectors, we showed that G6pt-/- mice expressing 3-62% of normal hepatic G6PT activity exhibited a normalized liver phenotype. Two of the 12 mice expressing < 6% of normal hepatic G6PT activity developed HCA. All treated mice were leaner and more sensitive to insulin than wild-type mice. Mice expressing 3-22% of normal hepatic G6PT activity exhibited higher insulin sensitivity than mice expressing 44-62%. The levels of insulin sensitivity correlated with the magnitudes of hepatic carbohydrate response element binding protein signaling activation. In summary, we established the threshold of hepatic G6PT activity required to prevent tumor formation and showed that mice expressing 3-62% of normal hepatic G6PT activity maintained glucose homeostasis and were protected against age-related obesity and insulin resistance.

S-adenosylmethionine-dependent protein methylation is required for expression of selenoprotein P and gluconeogenic enzymes in HepG2 human hepatocytes.

Cellular methylation processes enable expression of gluconeogenic enzymes and metabolism of the nutrient selenium. Selenium status has been proposed to relate to type II diabetes risk, and plasma levels of selenoprotein P (SEPP1) have been positively correlated with insulin resistance. Increased expression of gluconeogenic enzymes glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) has negative consequences for blood glucose management in type II diabetics. Transcriptional regulation of SEPP1 is directed by the same transcription factors that control the expression of G6PC and PCK1, and these factors are activated by methylation of arginine residues. We sought to determine whether expression of SEPP1 and the aforementioned glucoconeogenic enzymes are regulated by protein methylation, the levels of which are reliant upon adequate S-adenosylmethionine (SAM) and inhibited by S-adenosylhomocysteine (SAH). We treated a human hepatocyte cell line, HepG2, with inhibitors of adenosylhomocysteine hydrolase (AHCY) known to increase concentration of SAH before analysis of G6PC, PCK1, and SEPP1 expression. Increasing SAH decreased 1) the SAM/SAH ratio, 2) protein-arginine methylation, and 3) expression of SEPP1, G6PC, and PCK1 transcripts. Furthermore, hormone-dependent induction of gluconeogenic enzymes was reduced by inhibition of protein methylation. When protein-arginine methyltransferase 1 expression was reduced by siRNA treatment, G6PC expression was inhibited. These findings demonstrate that hepatocellular SAM-dependent protein methylation is required for both SEPP1 and gluconeogenic enzyme expression and that inhibition of protein arginine methylation might provide a route to therapeutic interventions in type II diabetes.

Potential role of branched-chain amino acids in glucose metabolism through the accelerated induction of the glucose-sensing apparatus in the liver.

Branched-chain amino acids (BCAAs) have a potential to improve glucose metabolism in cirrhotic patients; however, the contribution of liver in this process has not been clarified. To estimate the effect of BCAA on glucose metabolism in liver, we evaluated the mRNA expression levels of glucose-sensing apparatus genes in HepG2 cells and in rat liver after oral administration of BCAA. HepG2 cells were cultured in low glucose (100 mg/dl) or high glucose (400 mg/dl) in the absence or presence of BCAA. The mRNA expression levels and protein levels of GLUT2 and liver-type glucokinase (L-GK) were estimated using RT-PCR and immunoblotting. The expression levels of transcriptional factors, including SREBP-1c, ChREBP, PPAR-γm and LXRα, were estimated. The mRNA expression levels of transcriptional factors, glycogen synthase, and genes involved in gluconeogenesis were evaluated in rat liver at 3 h after the administration of BCAA. BCAA accelerated the expression of GLUT2 and L-GK in HepG2 cells in high glucose. Expression levels of ChREBP, SREBP-1c, and LXRα were also increased in this condition. BCAA administration enhanced the mRNA expression levels of L-GK, SREBP-1c, and LXRα and suppressed the expression levels of G-6-Pase in rat liver, without affecting the expression levels of glycogen synthase or serum glucose concentrations. BCAA administration enhanced the bioactivity of the glucose-sensing apparatus, probably via the activation of a transcriptional mechanism, suggesting that these amino acids may improve glucose metabolism through the accelerated utility of glucose and glucose-6-phosphate in the liver.

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.

Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro.

AIM: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. METHODS: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentiation of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. RESULTS: Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3beta (HNF-3beta, albumin (ALB), cytokeratin 18 (CK18), transthyretin (TTR), glucose-6-phosphate (G-6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3? ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. CONCLUSION: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats.

The terminal step in hepatic gluconeogenesis is catalyzed by glucose-6-phosphatase, an enzyme activity residing in the endoplasmic reticulum and consisting of a catalytic subunit (glucose-6-phosphatase (G6Pase)) and putative accessory transport proteins. We show that Zucker diabetic fatty rats (fa/fa), which are known to exhibit impaired suppression of hepatic glucose output, have 2.4-fold more glucose-6-phosphatase activity in liver than lean controls. To define the potential contribution of increased hepatic G6Pase to development of diabetes, we infused recombinant adenoviruses containing the G6Pase cDNA (AdCMV-G6Pase) or the beta-galactosidase gene into normal rats. Animals were studied by one of three protocols as follows: protocol 1, fed ad libitum for 7 days; protocol 2, fed ad libitum for 5 days, fasted overnight, and subjected to an oral glucose tolerance test; protocol 3, fed ad libitum for 4 days, fasted for 48 h, subjected to oral glucose tolerance test, and then allowed to refeed overnight. Hepatic glucose-6-phosphatase enzymatic activity was increased by 1.6-3-fold in microsomes isolated from AdCMV-G6Pase-treated animals in all three protocols, and the resultant metabolic profile was similar in each case. AdCMV-G6Pase-treated animals exhibited several of the abnormalities associated with early stage non-insulin-dependent diabetes mellitus, including glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral (muscle) triglyceride stores. These animals also exhibited significant decreases in circulating free fatty acids and triglycerides, changes not normally associated with the disease. Our studies show that overexpression of G6Pase in liver is sufficient to perturb whole animal glucose and lipid homeostasis, possibly contributing to the development of metabolic abnormalities associated with diabetes.

Catabolite repression by glucose 6-phosphate, gluconate and lactose in Escherichia coli.

While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA(Glc) of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of beta-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non-PTS carbon sources.