Prevalence of the Trp64Arg missense mutation of the beta3-adrenergic receptor gene in Japanese subjects.

Prompted by the recent findings that a tryptophan to arginine (Trp64Arg) mutation in the beta3-adrenergic receptor gene was associated with an earlier onset of non-insulin-dependent diabetes mellitus (NIDDM) in Pima Indians, with abdominal obesity and insulin resistance in Finns, and with an increased capacity to gain weight in French whites, we studied the prevalence of this mutation in 231 diabetic and 95 nondiabetic Japanese subjects and assessed its contribution to the development of obesity and NIDDM. The allelic frequencies of the mutation were 0.18 in diabetic and 0.23 in nondiabetic subjects, showing no significant difference between the two groups (P = .067). In nondiabetic subjects, body mass index (BMI) did not differ between those with and without the mutation (22.2 +/- 3.5 v 21.4 +/- 3.2 kg/m2, P = .252). In NIDDM subjects, BMI at the time of study and maximal BMI before the start of treatment did not differ between those with and without the mutation (22.8 +/- 2.6 v 23.2 +/- 3.7 kg/m2, P = .678, and 24.7 +/- 2.6 v 24.9 +/- 3.1 kg/m2, P = .277). Homozygotes for the mutation did not have trends to have increased BMI in either diabetic or nondiabetic subjects. The age at diagnosis of NIDDM also did not differ between the two groups (48.8 +/- 9.9 v 47.8 +/- 12.5 years, P = .796). Fasting serum cholesterol and triglyceride levels and systolic and diastolic blood pressure before the start of treatment did not differ between NIDDM subjects with and without the mutation. In conclusion, although the Trp64Arg mutation is not uncommon in Japanese, it does not appear to be associated with obesity, NIDDM, age at diagnosis of NIDDM, or dyslipidemia. Our results suggest that the mutation has minor effects, if any, on the development of obesity and NIDDM in Japanese.

Massive actin polymerization induced by phosphatidylinositol-4-phosphate 5-kinase in vivo.

The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Recently we isolated several isoforms of type I PI4P5K. Here we report that PI4P5K Ialpha induces massive actin polymerization resembling "pine needles" in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity were lost. Although the dominant negative form of Rho, RhoN19, alone decreased actin fibers, those induced by PI4P5K were not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity.

Overexpression of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase does not correct glucose-stimulated insulin secretion from diabetic GK rat pancreatic islets.

Glucose-stimulated insulin secretion is impaired in GK (Goto-Kakizaki) rats, perhaps because of abnormalities in glucose metabolism in pancreatic islet beta cells. The glycerol phosphate shuttle plays a major role in glucose metabolism by reoxidizing cytosolic NADH generated by glycolysis. In the pancreatic islets of GK rats, the activity of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate shuttle, is decreased and this abnormality may be responsible, at least in part, for impaired glucose-stimulated insulin secretion. To investigate this possibility, we overexpressed mGPDH in islets isolated from GK rats via recombinant adenovirus-mediated gene transduction, and examined glucose-stimulated insulin secretion. In islets isolated from diabetic GK rats at 8 to 10 weeks of age, glucose-stimulated insulin secretion was severely impaired, and mGPDH activity was decreased to 79 % of that in non-diabetic Wistar rats. When mGPDH was overexpressed in islets from GK rats, enzyme activity and protein content increased 2- and 6-fold, respectively. Basal (3 mmol/l glucose) and glucose-stimulated (20 mmol/l) insulin secretion from the Adex1CAlacZ-infected GK rat islets were, respectively, 4.4 +/- 0.7 and 8.1 +/- 0.7 ng. x islet(-1) x 30 min(-1), and those from mGPDH-overexpressed GK rat islets 4.7 +/- 0.3 and 9.1 +/- 0.8 ng x islet(-1) x 30 min(-1), in contrast to those from the AdexlCAlacZ-infected non-diabetic Wistar rat islets (4.7 +/- 1.6 and 47.6 +/- 11.9 ng x islet(-1) x 30 min(-1)). Thus, glucose-stimulated insulin secretion is severely impaired in GK rats even in the stage when mGPDH activity is modestly decreased, and at this stage, overexpression of mGPDH cannot restore glucose-stimulated insulin secretion. We conclude that decreased mGPDH activity in GK rat islets is not the defect primarily responsible for impaired glucose-stimulated insulin secretion.

Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family.

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.

Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase.

Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic beta-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic beta-cell line MIN6. Two novel cDNAs, designated PI4P5K-Ialpha and PI4P5K-Ibeta, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.

Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C beta1 or delta1 inhibits stimulus-induced insulin release in insulinoma MIN6 cells.

To study the effects of enhanced phosphoinositide hydrolysis on insulin secretion, phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) or PLCdelta1 was overexpressed in insulinoma MIN6 cells via adenoviral vectors. Inositol phosphate production stimulated by NaF (with AlCl3) in PLCbeta1-overexpressing cells and that stimulated by KCl or glucose in both PLCbeta1- and PLCdelta1-overexpressing cells were greater than that in control cells. In addition, reduced phosphatidylinositol-4,5-bisphosphate levels were observed in these cells stimulated by NaF or KCl. The greater phosphoinositide hydrolysis was accompanied by 25-45% inhibition of insulin secretion. These data suggest that excessive phosphoinositide hydrolysis inhibits secretagogue-induced insulin release in MIN6 cells.