Adenovirus-mediated preproinsulin gene transfer into adipose tissues ameliorates hyperglycemia in obese diabetic KKA(y) mice.

We investigated whether adenovirus-mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKA(y) mice, a genetically obese type 2 diabetic animal model, were treated with a single subcutaneous injection of recombinant adenovirus, Adex1CA-human preproinsulin (Adex1CA-pchi), into the epididymal fat pads. pchi mRNA was expressed only in adipose tissue in which mature insulin was produced. Three days after virus injection these mice showed a marked decrease of blood glucose levels (from about 400 to 200 mg/dl), and an intraperitoneal glucose tolerance test revealed the markedly improved glucose tolerance. There was no significant difference in serum insulin levels between control and recombinant adenovirus-treated KKA(y) mice. The normalized glucose levels in diabetic mice were maintained for at least 2 weeks after the virus injection. This strategy could provide a novel and, most importantly, a simple and convenient gene therapy for obese type 2 diabetes patients.

Increases in plasma ACTH and cortisol after hypertonic saline infusion in patients with central diabetes insipidus.

To clarify the mechanism for the potentiation of CRH-induced ACTH response by the infusion of hypertonic saline, we investigated changes in plasma ACTH concentration after infusion of 5% hypertonic saline in five patients with untreated central diabetes insipidus (DI). Basal levels of plasma ACTH and cortisol in the DI group were not significantly different from those in normal control subjects. The infusion of hypertonic saline produced an increase in plasma arginine vasopressin (AVP) in controls, but did not elevate ACTH. However, in patients with DI, the plasma AVP concentration did not change, but circulating ACTH increased 3.6-fold (7.7 +/- 1.5 to 23.0 +/- 2.7 pmol/liter; P < 0.01), and plasma cortisol also increased significantly (298 +/- 99 to 538 +/- 124 nmol/liter; P < 0.05). Moreover, a positive correlation was observed between plasma ACTH and osmolality (r = 0.72; P < 0.005). These results indicate that ACTH secretion in DI patients is regulated by a mechanism distinct from that in healthy subjects. It seems possible that the increase in plasma osmolality promotes ACTH secretion in DI patients through AVP and/or urocortin via the hypophyseal portal system, independent of the AVP secretion from magnocellular neurons.

Acute inhibition of proinsulin biosynthesis at the translational level by palmitic acid.

The effects of fatty acids on pancreatic beta cell are still controversial. Here, in order to determine whether free fatty acids acutely affect beta cell functions, we studied the effect of palmitic acid (PA) on proinsulin biosynthesis and insulin secretion using rat islets in vitro. Exposure of islets to PA for 1 h reduced glucose-stimulated proinsulin biosynthesis in a dose-dependent manner; in contrast, no change in insulin secretion was observed after 1 h incubation with PA. Furthermore, PA treatment did not cause any change of preproinsulin mRNA level during 1-h incubation period. Thus, our data indicate that PA primarily suppresses glucose-induced proinsulin biosynthesis within 1 h at the translational level.

Syntaxin, but not soluble NSF attachment protein (SNAP), biosynthesis by rat pancreatic islets is regulated by glucose in parallel with proinsulin biosynthesis.

Recent studies have revealed that soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE)-related proteins, originally identified in neural tissues, are also expressed in pancreatic beta cells. In this study, we investigated the effect of glucose on syntaxin 1 and alpha/beta SNAP biosynthesis in pancreatic beta cells and we demonstrated that syntaxin 1, but not alpha/beta SNAP biosynthesis by rat isolated pancreatic islets was stimulated specifically by glucose nearly in parallel with proinsulin biosynthesis. Stimulation of syntaxin 1 and proinsulin biosynthesis by glucose was dose-dependent (Km = approximately 8 mmol/l) and reached the maximum (about 8-12 fold) at concentrations over 11 mmol/l. In contrast, 22 mmol/l glucose increased alpha/beta SNAP biosynthesis about 2-fold only, similar to the increase in total protein synthesis. Stimulation of syntaxin 1 biosynthesis by glucose was also time-dependent, taking around 3 h to reach the maximum, and was not affected by actinomycin-D, suggesting regulation at the translational level. On the other hand, glucose had a similar stimulating effect on both syntaxin 1 and alpha/beta SNAP biosynthesis by mouse insulinoma betaTC3 cells as it did on proinsulin biosynthesis. The evidence showing coordinated stimulation of syntaxin 1 and proinsulin biosynthesis by glucose in rat islets suggested the critical functional role of syntaxin 1 in the insulin exocytotic mechanism.

Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway.

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic beta cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of approximately 35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic beta-cells, rat syntaxin 1A or 1B was overexpressed in mouse beta TC3 cells using the transient transfection procedure. Transfection of beta TC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable beta TC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-beta-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected beta TC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing beta TC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.

[A HBV carrier with fulminant hepatitis complicated by ATL, multiple myeloma and thyroid cancer].

A 75-year-old female, born in Tochigi Prefecture, was admitted because of lumbago in August of 1991. The leukocyte count was 11,800/microliters with 22.5% atypical lymphocytes. We demonstrated a lymphocyte surface marker, ATL-associated antigen, and proviral DNA. We also identified 2.60 g/dl of serum monoclonal protein, found to be IgG, lambda type, and punched out lesions in the skull. We made a diagnosis of ATL. She was also a HBV carrier. The patient was treated with a modification of CHOP therapy, because of increasing atypical lymphocytes in the peripheral blood in November of 1992. She died of acute hepatitis, suddenly, in March of 1993. Autopsy revealed multiple myeloma, fulminant hepatitis and occult thyroid cancer in addition to ATL.

Developmental expression of GLUT3 glucose transporter in the rat brain.

The ontogeny of the GLUT3 glucose transporter gene and protein expression was studied in rat brain. Northern blot analysis using total RNA from rat brains at different developmental stages revealed that the levels of GLUT3 mRNA were very low during the embryonic stage and increased towards the postnatal stage. Immunohistochemistry using a specific antibody showed that the expression of GLUT3 protein was barely detectable in the embryonic stage, but was clearly detected on the plasma membrane of neuronal cells from 10 days after birth to the adult. Expression of GLUT3 mRNA and protein in the cerebral neuronal cell cultures was also examined during the maturation of neurons. GLUT3 glucose transporter of primary neuronal cultured cerebral cortical neurons was only detected in mature neurons after they were cultured for 14 days. These results indicate that GLUT3 plays an important role in glucose homeostasis postnatally in neurons of the rat brain.

Agranulocytosis during treatment with fluconazole.

Fluconazole, a new effective triazole antifungal agent, has been reported to cause fewer adverse reactions than amphotericin B. A patient who was diagnosed as having agranulocytosis and thrombocytopenia after treatment with fluconazole was investigated and recovered after withdrawal of the antifungal therapy. This case suggests the need for careful haematological observation during the treatment with fluconazole.

[Urinary methylmalonic acid excretion and clinical features in megaloblastic anemia due to vitamin B12 deficiency].

Urinary methylmalonic acid (MMA) excretion in megaloblastic anemia due to vitamin B12 (B12) deficiency was studied using a colorimetric method. Average MMA excretion in 20 patients with untreated B12 deficiency was 164 mg/day, whereas it increased to 518 mg/day following oral administration of 10 g L-valine. Urinary MMA correlated significantly with platelet number, erythroblast percentage and deoxyuridine suppression test, while no correlation was found with hemoglobin, leukocyte number, reticulocyte, serum LDH, serum B12 and folate concentration. Patients with neurological disturbances excreted significantly larger amounts of MMA than those without neurological disorders. The results also indicated that MMA could be a useful adjunct for differentiation of megaloblastic anemia from myelodysplastic syndromes showing marked megaloblastic changes.

[Pernicious anemia with indicanuria].

A 67-year-old female was diagnosed as having classical pernicious anemia. Laboratory data included low serum vitamin B12 concentrations, abnormal deoxyuridine suppression test, methylmalonic aciduria, atrophic gastritis, positive anti-intrinsic factor antibody and Schilling test results typical of pernicious anemia. During hospitalization it was incidentally noted that her urine was green colored. Jaffe' reaction and Obermayer reaction for indicanuria were both positive. Dark purple crystalline material was obtained by centrifugation of her urine. The crystalline substance was soluble in methanol and its absorbance curve was identical to that of authentic indoxylsulphate potassium salt. Daily output of this substance was nearly 50 times normal. There was no increase in urinary excretion of monoamino-monocarboxyl amino acides. The exact reason for her indicanuria was not clear, although abnormal bacterial growth in the intestine remained as a possibility.