Identification of a novel variant in the phosphoenolpyruvate carboxykinase gene promoter in Japanese patients with type 2 diabetes.

Phospho enolpyruvate carboxykinase (PEPCK) plays an important role in gluconeogenesis and hepatic glucose production. To test the hypothesis that mutations of the PEPCK gene promoter contribute to the increased hepatic glucose production that leads to diabetes, we screened for polymorphisms of the PEPCK promoter region in 252 Japanese type 2 diabetic patients and 188 non-diabetic control subjects. A novel variant at position - 232 (C to G) was found at a similar frequency in type 2 diabetes patients (32 %) and control subjects (35 %) (p = 0.26). However, patients with the - 232 G/G genotype had an earlier age of onset than those with the - 232 C/C or - 232 C/G genotypes (p = 0.028). As the variant might well otherwise influence hormonal action, we transfected PEPCK-luciferase fusion gene constructs with the variant into human hepatoma cells and examined the response to dexamethasone, insulin, and cAMP. The reporter assay showed no significant difference in hormonal responses with the fusion gene containing the variant. Accordingly, the single-base variant at position - 232 of the PEPCK gene promoter is most probably not a major contributor to the pathogenesis of type 2 diabetes. However, this variation may be useful as a genetic marker for other metabolic disorders, especially in Japanese.

Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients.

Glucose-6-phosphatase (G6Pase) catalyzes the rate-limiting step of gluconeogenesis, and hepatic G6Pase activity is increased in diabetes. We have cloned and analyzed the human G6Pase gene promoter region and identified putative regulatory sequences for insulin, cAMP, glucocorticoid, and hepatocyte nuclear factors. The promoter region of the G6Pase gene was analyzed in 154 noninsulin-dependent diabetes mellitus patients and 90 control subjects by PCR-single strand conformation polymorphism and direct sequencing methods. Polymorphisms were not found in any subjects. The results suggested that in noninsulin-dependent diabetic patients, the major cause of the hepatic glucose overproduction was not attributed to dysregulation of the G6Pase gene due to mutation/polymorphism of its promoter region.

Hepatocyte nuclear factor-4 alpha gene mutations in Japanese non-insulin dependent diabetes mellitus (NIDDM) patients.

A mutation in the hepatocyte nuclear factor-4 alpha (HNF-4 alpha) gene has been recently reported to cause maturity-onset diabetes of the young (MODY) (Yamagata, Furuta, et al., 1996). The mutation can also be a good candidate for the responsible gene of non-insulin dependent diabetes mellitus (NIDDM). The existence of the mutated allele of Q268X (C to T substitution within the exon 7 of HNF-4 alpha gene) was searched in 514 alleles of Japanese NIDDM patients by polymerase chain reaction-restriction fragment length polymorphism analysis. No mutation was found in these patients. The result showed that the Q268X mutation of HNF-4 alpha gene was not frequent among general NIDDM patients and that it cannot serve as the major diabetogenic gene in the Japanese ethnic group.

Expression and distribution of glucose-6-phosphatase catalytic subunit messenger RNA and its changes in the diabetic state.

Glucose-6-phosphatase (G6Pase) plays a major role in gluconeogenic pathway. To analyze its regulation, we have cloned a full-length cDNA for G6Pase catalytic subunit from the rat liver. In the cloned cDNA, a 492 base insertion, respective to the previously reported sequence, was found in the 3'-noncoding region. In both ends of this insertion, 5'- and 3' 2' splice site motifs were identified. However, spliced mRNA was hardly observed in the rat RNA. Abundant expression was observed in the liver and the kidney by Northern analysis. Expression was also observed in the spleen, adrenal gland and small intestines. Reverse-transcription (RT) polymerase chain reaction (PCR) analysis revealed that G6Pase mRNA was also expressed in a variety of tissues including pancreatic islets. Its expression was increased in the ketotic diabetic mice livers and was corrected by insulin treatment. However, no appreciable changes were observed in kidneys. Broad tissue distribution in expression and the tissue specificity in regulation were thus considered to be the important features of G6Pase gene.

Monophyletic origin and unique dispersal patterns of domestic fowls.

With the aim of elucidating in greater detail the genealogical origin of the present domestic fowls of the world, we have determined mtDNA sequences of the D-loop regions for a total of 21 birds, of which 12 samples belong to red junglefowl (Gallus gallus) comprising three subspecies (six Gallus gallus gallus, three Gallus gallus spadiceus, and three Gallus gallus bankiva) and nine represent diverse domestic breeds (Gallus gallus domesticus). We also sequenced four green junglefowl (Gallus varius), two Lafayette's junglefowl (Gallus lafayettei), and one grey junglefowl (Gallus sonneratii). We then constructed a phylogenetic tree for these birds by the use of nucleotide sequences, choosing the Japanese quail (Coturnix coturnix japonica) as an outgroup. We found that a continental population of G. g. gallus was the real matriarchic origin of all the domestic poultries examined in this study. It is also of particular interest that there were no discernible differences among G. gallus subspecies; G. g. bankiva was a notable exception. This was because G. g. spadiceus and a continental population of G. g. gallus formed a single cluster in the phylogenetic tree. G. g. bankiva, on the other hand, was a distinct entity, thus deserving its subspecies status. It implies that a continental population of G. g. gallus sufficed as the monophyletic ancestor of all domestic breeds. We also discussed a possible significance of the initial dispersal pattern of the present domestic fowls, using the phylogenetic tree.

Disordered expression of hepatic glycolytic and gluconeogenic enzymes in Otsuka Long-Evans Tokushima fatty rats with spontanteous long-term hyperglycemia.

Expression of key regulatory enzymes involved in glucose metabolism was studied in the livers of Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin dependent diabetes mellitus. The activity and mRNA levels of glucokinase and L-type pyruvate kinase was increased in the liver of OLETF rats compared with control rats. There was no such remarkable change in liver-type phosphofructokinase. The activities of glucose-6-phosphatase and fructose-1,6-biphosphatase also increase despite high plasma levels of glucose and insulin. The activity of phosphoenolpyruvate carboxykinase did not show any significant change. The mRNA levels for fructose-1,6-biphosphatase, and phosphoenolpyruvate carboxykinase exhibited no marked changes. These results suggest that the expression of glucose-6-phosphatase and fructose-1,6-biphosphatase is disordered in OLETF rats.

The role of prohormone convertases PC1 (PC3) and PC2 in the cell-specific processing of proglucagon.

To elucidate the mechanism of the differential processing of proglucagon, we analyzed the processing products of proglucagon in three types of rodent endocrine cells and their relation to prohormone convertases PC1 (PC3) and PC2. Proglucagon gene was transfected into AtT-20 cells and GH3 cells, which are derived from pituitary tumors. InR1-G9 cells, which are insulinoma-derived cells, express an endogenous proglucagon gene. Oxyntomodulin was the predominant processing product in AtT-20 cells, which contained abundant PC1 mRNA. In contrast, glucagon was the major product in GH3 cells, which expressed PC2 mRNA. Oxyntomodulin and glucagon were produced in equal amounts in InR1-G9 cells, which expressed both PC1 and PC2 mRNAs. These findings suggest that PC1 and PC2 preferentially cleave proglucagon into oxyntomodulin and glucagon, respectively, thus contributing to the cell-specific processing of proglucagon.