Yoshiji Ogawa

Loss of glucose-induced insulin secretion and GLUT2 expression in transplanted β-cells

Either 200 or 400 syngeneic islets were transplanted under the kidney capsule of normal or streptozocin-induced diabetic B6/AF1 mice. The diabetic mice with 400 islets became normoglycemic, but those with 200 islets, an insufficient number, were still diabetic after the transplantation (Tx). Two weeks after Tx, GLUT2 expression in the islet grafts was evaluated by immunofluorescence and Western blots, and graft function was examined by perfusion of the graft-bearing kidney. Immunofluorescence for GLUT2 was dramatically reduced in the β-cells of grafts with 200 islets exposed to hyperglycemia. However, it was plentiful in grafts with 400 islets in a normoglycemic environment. Densitometric analysis of Western blots on graft homogenates demonstrated that GLUT2 protein levels in the islets, when exposed to chronic hyperglycemia for 2 weeks, were decreased to 16% of those of normal recipients. Moreover, these grafts had defective glucose-induced insulin secretion, while the effects of arginine were preserved. We conclude that GLUT2 expression in normal β-cells is promptly down-regulated during exposure to hyperglycemia and may contribute to the loss of glucose-induced secretion of diabetes.

Intracellular Ca2+ Modulation of ATP-Sensitive K+ Channel Activity in Acetylcholine-Induced Activation of Rat Pancreatic β-Cells

We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic beta-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca(2+) channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic beta-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K(+) channels (K(ATP)). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca(2+) chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K(ATP) with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPbetaS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca(2+) concentration inside the membrane from 10 nM-10 microM decreased K(ATP) activity only in the presence of ATP. The affinity of ATP to K(ATP) became 4.5 times higher with the higher concentration of Ca(2+). These results suggest that Ca(2+) from ACh receptor signaling modulates the sensitivity of K(ATP) to ATP. A positive-feedback mechanism of intracellular Ca(2+)-dependent Ca(2+) influx was also demonstrated.

Protein kinase C-dependent and -independent inhibition of Ca2+ influx by phorbol ester in rat pancreatic β-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic β-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca2+ concentration ([Ca2+]i) elevated by the glucose stimulation when tested in isolated rat β-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized β-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca2+ currents through L-type Ca2+ channels. BIM prevented the PMA inhibition of the Ca2+ currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic β-cells, defeating its own inhibition of the Ca2+ influx through L-type Ca2+ channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.

Modulation of Ca2+ influx by corticotropin-releasing factor (CRF) family of peptides via CRF receptors in rat pancreatic β-cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors type 1 (CRF1 receptor) and type 2 (CRF2 receptor). In a previous study, we reported that CRF, an endogenous ligand for CRF1 receptor, modulated Ca2+ influx in rat pancreatic beta-cells. In addition to CRF, other additional members of the family, urocortins, have been identified in mammals. Urocortin 1 (UCN 1), a peptide of the CRF family, binds both CRF1 receptor and CRF2 receptor with equal affinities. Urocortin 3 (UCN 3), a highly selective ligand for CRF2 receptor with little affinity for CRF1 receptor, has been shown in rat pancreatic beta-cells. The present study focused on the effects of the CRF family peptides on intracellular Ca2+ ([Ca2+]i) concentration via CRF receptors in rat pancreatic beta-cells. Microfluorimetric experiments showed that CRF (0.2 nM) and UCN 1 (0.2 nM) elevated [Ca2+]i levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF2 receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca2+]i. The CRF effect on the elevation of [Ca2+]i was inhibited by the addition of UCN 3. Taken together, the activation of CRF2 receptor antagonizes the CRF1 receptor-stimulated Ca2+ influx.

Novel Mechanism of Chronic Exposure of Oleic Acid-Induced Insulin Release Impairment in Rat Pancreatic beta-Cells

A sustained, high circulating level of free fatty acids (FFAs) is an important risk factor for the development of insulin resistance, islet β-cell dysfunction, and pathogenesis of type 2 diabetes. Here, we report a novel mechanism of chronic exposure of oleic acid (OA)-induced rat insulin release impairment. Following a 4-day exposure to 0.1 mM OA, there was no significant difference in basal insulin release when comparing OA-treated and untreated islets in the presence of 2.8 mM glucose, whereas 16.7 mM glucose-stimulated insulin release increased 2-fold in control, but not in OA-treated, islets. Perforated patch-clamp recordings showed that untreated β-cells exhibited a resting potential of -62.1 ± 0.9 mV and were electrically silent, whereas OA-treated β-cells showed more positive resting potentials and spontaneous action potential firing. Cell-attached single-channel recordings revealed spontaneous opening of ATP-sensitive potassium (KATP) channels in control, but not in OA-treated, β-cells. Inside-out excised patch recordings showed similar activity in both OA-treated and untreated β-cells in the absence of ATP on the inside of the cellular membrane, whereas in the presence of ATP, KATP channel activity was significantly reduced in OA-treated β-cells. Electron microscopy demonstrated that chronic exposure to OA resulted in the accumulation of triglycerides in β-cell cytoplasm and reduced both the number of insulin-containing granules and insulin content. Collectively, chronic exposure to OA closed KATP channels by increasing the sensitivity of KATP channels to ATP, which in turn led to the continuous excitation of β-cells, depletion of insulin storage, and impairment of glucose-stimulated insulin release.

Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of KATP channels

BackgroundPhorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic β-cells by using patch-clamp techniques.ResultsIn current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 μM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized β-cells in a concentration-dependent manner (0.8–240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K+ channel (KATP) activity. Based on inside-out patch-clamp recordings, PMA had little effect on KATP activity if no ATP was in the bath, while PMA restored KATP activity that was suppressed by 10 μM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 μM), PMA did not increase, but rather reduced insulin secretion.ConclusionIn rat pancreatic β-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing β-cell excitability in a PKC-independent manner. The enhancement of KATP activity by reducing sensitivity of KATP to ATP seems to underlie the PMA-induced impairment of β-cells electrical excitation in response to glucose stimulation.

Effect of doxazosin on the size of LDL particle in the type 2 diabetic patients with hypertension

Hypertension is common in patients with type 2 diabetes mellitus (DM), and contributes to the progression of its complications in patients with diabetes. Doxazosin is a selective alpha1-adrenoceptor-blocking anti-hypertensive agent and has a favorable impact upon lipid metabolism. We investigated the effect of doxazosin on the lipid metabolism in hypertensive patients with type 2 diabetes, especially low-density lipoprotein (LDL) particle size that is associated with many elements of the insulin resistance syndrome. Cross-sectional study (n=19) was designed to determine whether doxazosin, administered with an angiotensin II-converting enzyme inhibitor (ACEI) and a Ca antagonist, affects LDL particle size. As a follow-up study (n=6), lipid and glucose metabolism and LDL particle size were followed for 12 weeks before and after the initiation of doxazosin administration (1-4 mg/day). The average size of LDL particle was significantly larger in the patients treated with doxazosin (LDL-migration index (LDL-MI): 0.348+/-0.027) than those in the patient treated without doxazosin (0.378+/-0.035), although LDL cholesterol levels did not differ between the two groups. The plasma glucose and HbA1c levels remained unchanged. Lipid profile showed normolipemia throughout the period of the study. However, LDL particle size was demonstrated to become larger during the following period. Small LDL fraction (LDL3-7) diminished remarkably and large LDL (LDL1-2) increased on the polyacrylamide gel electrophoresis (PAGE) LDL system (LipoPrint). From this pilot study, it was concluded that doxazosin is a useful anti-hypertensive agent for hypertensive type 2 diabetic patients in improving the size of LDL particle.

Study on Pancreatic Insufficiency (Chronic Pancreatitis) and Steatorrhea in Japanese Patients with Low Fat Intake

The incidence of steatorrhea is said to be lower and its grade milder in Japanese because their fat intake is lower than that of Europeans and Americans. Failure to take this into account creates difficulties when attempting to compare data on pancreatic exocrine insufficiency in different countries. The authors examined the incidence and grade of steatorrhea in Japanese chronic pancreatitis (CP) patients whose daily fat intake was <40 g (25 patients) or ≥40 g (35 patients). In addition, 23 CP patients with steatorrhea and daily fecal fat excretion ≥5 g were given a pancreatic enzyme preparation at a dose 3–8 times higher than the usual dose to investigate its effect on fecal fat excretion. Among CP patients whose fat intake was <40 g, the incidence of fecal fat excretion <5 g was 56% and that of fecal fat excretion ≥10 g (severe steatorrhea) was 8%. In CP patients whose fat intake was ≥40 g, the incidences were 27.9 and 34.9%, respecetively; a significant increase in the number of affected patients was noted when fat intake was ≥40 g. The fat absorption rate was 76.2% among patients whose fat intake was <40 g and 77.8% among patients whose fat intake was ≥40 g, revealing no significant difference between the two groups. The proportion of CP patients whose fat absorption rate ≦80% was 32% at a fat intake <40 g and 39% at a fat intake ≥40 g, revealing no significant difference between the two groups.

A CASE OF CYSTIC PHEOCHROMOCYTOMA WITH HYPERTENSION AND HEADACHES MIMICKING A LARGE PANCREATIC CYSTIC TUMOR

ABSTRACT Objective: Cystic pheochromocytomas can become enlarged without abdominal symptoms and can potentially be confused with other abdominal cystic tumors. Methods: We report here a case of a 45-mm cystic pheochromocytoma that was initially considered a pancreatic cystic tumor. The patient had a 4-year history of treatment for hypertension and occasional headaches. Left cystic pheochromocytoma was diagnosed based on excessive catecholamine levels, 18F-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (18F-FDG PET/CT), and 123I-metaiodobenzylguanidine (123I-MIBG) scintigraphy. Results: Left adrenalectomy ameliorated the patients hypertension and headache. Pathologic findings showed an enlarged cyst with an irregular, thick wall adherent to the normal adrenal tissues. Conclusion: A large cystic pheochromocytoma can be misdiagnosed as a large pancreatic cystic tumor. CT and magnetic resonance imaging (MRI) are less important in excluding the possibility of pheochromocytoma than b...

A Case of Nonalcoholic Steatohepatitis in a Cured Acromegalic Patient With Severe Growth Hormone Deficiency

ABSTRACT Objective: Acromegaly is associated with metabolic and neoplastic complications, but successful treatment of acromegaly can sometimes result in growth hormone deficiency (GHD). Nonalcoholic fatty liver disease may develop in GHD, although there are few reports of such cases after cured acromegaly. Methods: We report the case of a 41-year-old woman with nonalcoholic steatohepatitis associated with GHD after surgical cure of acromegaly. Results: Growth hormone (GH) replacement therapy improved serum triglyceride, hyaluronic acid, and type IV collagen levels as well as liver function and quality of life. Computed tomography (CT) showed reduced volumes of both visceral and subcutaneous fat and an increased liver/spleen CT attenuation value. Conclusion: Cured acromegalic patients should be carefully examined if GHD and associated metabolic disorders arise. Appropriate diagnosis of GHD and prompt treatment with GH replacement should ameliorate liver dysfunction and the metabolic changes associated with...