Kohei Kaku

Circadian cycles in VIP content and VIP stimulation of cyclic AMP accumulation in the rat pineal gland

VIP content in the rat pineal gland and cyclic AMP accumulation in response to VIP in the daily light and dark cycle were examined. VIP content in the pineal varied significantly during the day and night; the content decreased during exposure to light and was lowest at the onset of darkness, 6 p.m. (mean +/- SE, 23 +/- 5 pg/pineal), and increased during the night and was highest at the onset of light, 6 a.m. (72 +/- 12 pg/pineal). Response of cyclic AMP accumulation to VIP varied with a periodicity inversely related to the daily light and dark cycle of VIP content; cyclic AMP accumulation in response to 10(-7) M VIP increased in proportion to periods of exposure to light and peaked at 6 p.m., and decreased with the onset of darkness.

HepG2/erythrocyte glucose transporter (GLUT1) gene in NIDDM : a population association study and molecular scanning in Japanese subjects

SummaryTo evaluate the role of mutations in the glucose transporter (GLUT1) gene in Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM), we first conducted a population association study using the XbaI polymorphism of the gene. A polymerase chain reaction (PCR)-based assay was developed and used for the analysis. When analysed in 91 diabetic patients and 87 non-diabetic control subjects, the distribution of the genotype frequency was significantly different between the two groups (p=0.0025). The (−) allele was significantly associated with NIDDM (odds ratio 2.317, 95% confidence interval 1.425−3.768). To identify possible mutation(s) in the GLUT1 gene, which was in linkage disequilibrium with the (−) allele, all ten exons of the gene were analysed by PCR single-strand conformation polymorphism (SSCP) analysis in 53 diabetic patients with at least one (−) allele. Variant SSCP patterns were detected in exons 2, 4, 5, 7, 9 and 10. Sequence analysis revealed that all the variants represented silent mutations. One of the variants in exon 2, GCT (Ala15)→GCC(Ala), created a HaeIII restriction site. This polymorphism was common in Japanese subjects with heterozygosity of 0.36 and polymorphism information content 0.29. We conclude that the structural mutation of GLUT1 is rare and not likely to be a major genetic determinant of NIDDM in Japanese subjects. The Xbal (−) allele of the GLUT1 gene appeared to be a genetic marker of NIDDM in Japanese subjects. The possibility of the presence of mutation(s) in the regulatory region of the gene or in another locus nearby could not be excluded.

Myelofibrosis and Systemic Lupus erythematosus: Reversal of Fibrosis with High-Dose Corticosteroid Therapy

A case of myelofibrosis in association with systemic lupus erythematosus (SLE) is reported. Acute thrombocytopenia and a bleeding tendency developed in a 24-year-old woman with SLE. Bone marrow aspiration was unsuccessful due to myelofibrosis. Pulse therapy with methylprednisolone reversed both thrombocytopenia and myelofibrosis. A review of the literature revealed that the coexistence of SLE and myelofibrosis is a rare occurrence. Only 7 cases, to our knowledge, have ever been reported in detail. The present case is the 3rd in which myelofibrosis was reversed by corticosteroids.

Effects of Long-Term Treatment with Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor in Patients with Myelodysplastic Syndrome

We examined the effects of long-term treatment with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in 61 patients with myelodysplastic syndrome (MDS). Patients were randomly assigned to receive daily subcutaneous injection of 60 micrograms/m2, 125 micrograms/m2 or 250 micrograms/m2 for 8 weeks. A significant increase in granulocyte counts including neutrophils and eosinophils was shown from one week after the start of the treatment in all three dose groups. The increase in granulocyte counts reached a plateau at the 4th week and was sustained during the treatment period. However, no consistent change in other cell lineages including monocytes, lymphocytes, reticulocytes and platelets were observed. Nevertheless peak-levels of these cells were significantly higher than the pre-treatment levels. In higher dose groups, the number of patients developing infections was reduced. There was no significant difference in the incidence of adverse events among the 3 dose groups, and the toxicity was generally well-tolerated. These observations indicate that treatment with rhGM-CSF can be of potential therapeutic benefit to patients with MDS.

Extrapancreatic effects of sulfonylurea drugs

Extrapancreatic action of sulfonylurea (SU) drugs were extensively summarized. Hypoglycemic SU drugs stimulate glycolytic pathway and inhibit gluconeogenic pathway in the liver through regulating key enzymes such as the bifunctional enzyme PFK2/F-2,6-P2ase and PEPCK. It is possible that SUs improve the primary defects in NIDDM through both pancreatic and extrapancreatic actions.

CS-045, a new oral antidiabetic agent, stimulates fructose-2,6-bisphosphate production in rat hepatocytes.

Fructose-2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key enzyme in glycolysis. We previously revealed that sulfonylureas stimulate fructose-2,6-bisphosphate production in the rat liver by activating 6-phosphofructo-2-kinase. In the present study, we show that CS-045, a new antidiabetic agent, activated 6-phosphofructo-2-kinase and raised fructose-2,6-bisphosphate levels in dispersed rat hepatocytes. This action was time- and dose-dependent. Ten micromolar CS-045 raised the fructose-2,6-bisphosphate content linearly to the submaximal level in 20 min. Dose dependency was observed in the range of 1-30 microM. Thirty micromolar CS-045 completely reversed the inhibitory effect of 0.1 nM glucagon on fructose-2,6-bisphosphate production. CS-045 activated 6-phosphofructo-2-kinase by decreasing the Km value for the substrate (fructose-6-phosphate) without affecting the Vmax. The combination of suboptimal doses of CS-045 and tolbutamide increased fructose-2,6-bisphosphate content more than that induced by each agent alone. These results indicate that CS-045 may reduce plasma glucose by facilitating glycolysis in the liver.

Neuropeptide Y Inhibits β‐Adrenergic Agonist– and Vasoactive Intestinal Peptide–Induced Cyclic AMP Accumulation in Rat Pinealocytes Through Pertussis Toxin–Sensitive G Protein

Abstract: The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose‐dependent manner during a 10‐min incubation of pinealocytes. NPY (1 × 10−7M) also inhibited vasoactive intestinal peptide (VIP)‐ and cholera toxin–induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol‐induced cAMP accumulation was completely abolished by a 5‐h pretreatment of pinealocytes with 1 μg/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT‐sensitive G protein. Yohimbine, an α2‐adrenergic antagonist, blocked NPY inhibition of isoproterenol‐stimulated cAMP accumulation. On the other hand, the α2‐adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits β‐adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT‐sensitive G protein. Our results also suggest that NPY exerts its action to affect α2‐adrenoceptor function.

A novel mutation in the ferrochelatase gene associated with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a hereditary disorder caused by mutations of the ferrochelatase gene. We investigated a Japanese patient with a dominant form of erythropoietic protoporphyria for a ferrochelatase mutation. Sequence analysis of the probands ferrochelatase cDNA revealed a T to C point mutation at nucleotide 557. This mutation resulted in the replacement of Ile by Thr at amino acid position 186, a novel mutation in erythropoietic protoporphyria. An increase in ferrochelatase activity was not observed in the crude extract of E. coli over‐expressing the mutant protein compared with the control, whereas a marked increase in activity was observed in that over‐expressing the wild type. Prediction of the secondary structure of ferrochelatase suggested that the Ile186 → Thr mutation changed the original β‐sheet structure to an α helix in the region including amino acid residue of mutation. We conclude that, in this patient, the Ile186 → Thr mutation had abolished enzyme activity, possibly by disrupting the secondary structure, thereby causing erythropoietic protoporphyria.

Effect of tolbutamide on fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase in rat liver.

The effects of tolbutamide on the activities of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were examined using rat hepatocytes. Tolbutamide stimulated fructose-6-phosphate,2-kinase activity and inhibited fructose-2,6-bisphosphatase activity, resulting in an increase of fructose-2,6-bisphosphate level. Changes in the activities of the enzyme by tolbutamide were due to variation in the Km value, but not dependent on alteration of Vmax. Glucagon inhibition of fructose-2,6-bisphosphate formation resulting from an inactivation of fructose-6-phosphate,2-kinase and an activation of fructose-2,6-bisphosphatase was released by tolbutamide. Tolbutamide stimulation of fructose-2,6-bisphosphate formation through regulation of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase may produce enhancement of glycolysis and inhibition of gluconeogenesis in the liver.

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

Abstract In the red cell incubated with ortho-[32P] phosphate, CaCl 2 and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl 2 . In the presence of purified erythrocyte calmodulin, CaCl 2 and [γ- 32 P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl 2 . From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.