Endothelial cell signalling supports pancreatic beta cell function in the rat.

AIMS/HYPOTHESIS: The proximity of endothelial cells and beta cells in islets by necessity means that they are exposed to each other's products. Whereas islet endothelial cells require signals from beta cells to function properly, endothelin-1, thrombospondin-1 and laminins, among others, have been identified as endothelial-derived molecules, although their full effects on beta cells have not been explored. We tested the hypothesis that islet endothelial-derived products affect beta cell function. METHODS: Endothelial cells from rat islets were proliferated and purified. Endothelium-conditioned culture medium (ECCM) was obtained by maintaining the endothelial cells in culture medium. Islet function was evaluated following exposure of cultured islets to standard culture medium or ECCM. Changes in mRNA levels for key beta cell metabolic enzymes were also measured in islets after ECCM exposure. RESULTS: Glucose-stimulated insulin release and islet insulin content were markedly enhanced by exposure to ECCM. This was at least partly explained by improved mitochondrial function, as assessed by glucose oxidation and an upregulation of the mitochondrial gene for glycerol-3-phosphate dehydrogenase (mGpdh [also known as Gpd2]), combined with upregulation of the rate-limiting enzyme in the glycolysis, glucokinase, in the islets. The intracellular degradation of insulin was also decreased in the islets. Islet endothelial cells produced laminins, and the positive effects of islet endothelial cells were prevented by addition of a neutralising antibody to the beta1-chain of laminin. Addition of exogenous laminin stimulated islet function. CONCLUSIONS/INTERPRETATION: This study provides proof of principle that endothelial cells can affect the function of beta cells in their vicinity and that this is at least partially mediated by laminins.

Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores.

Steroid effects on intracellular degradation of insulin and crinophagy in isolated pancreatic islets.

Under physiological conditions substantial amounts of hormone may be degraded within endocrine cells by a crinophagic process comprising fusions of secretory granules with lysosomes. Glucocorticoids may stabilise and progesterone destabilise lysosomal membranes. The effects of corticosterone and progesterone on intracellular degradation of insulin and crinophagy were determined in pancreatic beta-cells, and possible pathways mediating these effects were evaluated. Pancreatic islets were isolated from mice, intracellular degradation of insulin was measured by a pulse-chase method, and crinophagy was studied by electron microscopy. The islets were exposed to 3.3, 5.5 or 28 mM glucose with or without corticosterone, progesterone or the receptor ligands A-224817.0 and WAY-161358. Mifepristone was used to block steroid receptors and indomethacin to inhibit prostaglandin synthesis. Corticosterone caused a concentration-dependent decrease of insulin degradation at the lower glucose concentrations. Progesterone effected a concentration-dependent stimulation of insulin degradation. These results were paralleled with changes of the crinophagic activity in the beta-cells. Corticosterone decreased and progesterone increased islet production of prostaglandin E(2). Mifepristone abolished the steroid actions on insulin degradation and prostaglandin production. The effects of corticosterone were mimicked by the selective glucocorticoid receptor modulator A-224817.0, but in contrast to progesterone, the selective progesterone receptor agonist WAY-161358 had no effect on insulin degradation or prostaglandin production. Inhibition of cyclooxygenase blocked insulin degradation. The findings indicate that both corticosterone and progesterone could affect intracellular insulin degradation and crinophagy solely via the glucocorticoid receptor, and that prostaglandins may have a regulatory role in intracellular turnover of secretory material in pancreatic islet beta-cells.

Increased susceptibility to phenytoin teratogenicity: excessive generation of reactive oxygen species or impaired antioxidant defense?

Phenytoin is a human and animal teratogen. Accumulating evidence suggests that the teratogenicity is associated with a potential of phenytoin to cause embryonic cardiac arrhythmia and resultant generation of toxic reactive oxygen species via hypoxia-reoxygenation mechanisms. The A/J mouse is more susceptible to phenytoin teratogenicity than other mouse strains. The aim of this study was to investigate whether A/J mice have other antioxidant enzyme activities than C57BL/6J and CD-1 mice. Also, strain differences in phenytoin effects on embryonic heart rate and rhythm were determined. Another objective was to determine whether a spin trapping agent with capacity to capture reactive oxygen species alter the developmental toxicity of phenytoin. Treatment with this agent resulted in a marked decrease in phenytoin teratogenicity, which supports the idea that reactive oxygen species are important mediators for the teratogenic action of phenytoin. The A/J mice embryos were most susceptible to the adverse cardiac effects of phenytoin and had the highest activity of superoxide dismutase and glutathione peroxidase, while the activity of catalase was the same in embryos of the three different strains. The high activities of antioxidant enzymes in the A/J stain indicate that the sensitivity to develop malformations is caused by excessive arrhythmia-related generation of reactive oxygen species rather than impaired antioxidant defense.

Effects of alloxan on the islets of Langerhans: inhibition of leucine metabolism and insulin secretion.

The action of alloxan on the metabolism of the islets of Langerhans was studied in vitro. Isolated mouse islets were exposed to the drug at 4 degrees C to prevent its decomposition. Islet uptake of leucine was subsequently estimated at 37 degrees C, and was found not to be affected by the drug. However, islet leucine oxidation was strongly inhibited by the preceding alloxan exposure. The islets were protected against this inhibition by an incubation at a high glucose concentration prior to alloxan exposure. In contrast, a high concentration of leucine failed to provide full protection of either islet leucine oxidation or islet glucose oxidation. Furthermore, it was shown that alloxan impeded islet insulin response to both leucine and glucose. In addition, the potentiation of insulin release by theophylline was abolished after alloxan treatment of the islets. The results reinforce the hypothesis that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell, and that these sites may be protected against the action of the drug by some metabolite of glucose.

Diabetes and embryonic malformations. Role of substrate-induced free-oxygen radical production for dysmorphogenesis in cultured rat embryos.

The aim of this study was to evaluate the role of free-oxygen radicals in the embryonic maldevelopment of diabetic pregnancy. Rat embryos cultured in vitro during early organogenesis showed growth retardation and severe malformations after exposure to 50 mM glucose, 3 mM PYR, 10 mM HBT, or 3 mM KIC. Combinations of 25 mM glucose, 2.5 mM HBT, and 1 mM KIC also elicited embryonic growth retardation and malformations. The deleterious effects on embryonic development by all agents were alleviated by addition of SOD to the culture media, which yielded increased enzyme activity in the embryos and their membranes. The endogenous SOD activity also increased in embryos subjected to a high concentration of glucose or PYR in the culture medium. Addition of the mitochondrial PYR transport inhibitor CHC to the culture media blocked the dysmorphogenesis caused by glucose and PYR, but was without effect on the teratogenic actions of HBT and KIC. These findings implicate the embryonic mitochondria as a likely site for enhanced substrate-induced production of free-oxygen radicals mediating the teratogenic effect of a diabetic environment. In particular, the teratogenic process in diabetic pregnancy may depend on an increased production of free-oxygen radicals in immature embryonic mitochondria in response to a metabolic overload. This notion implies that every oxidative substrate entering the mitochondrial metabolism in excess may induce embryonic malformations and emphasizes the need for an extended metabolic surveillance of pregnant diabetic women. Consequently, optimal metabolic control should aim at normalizing the maternal serum concentrations of all possible oxidative substrates.

Lysosomes and pancreatic islet function. Time course of insulin biosynthesis, insulin secretion, and lysosomal transformation after rapid changes in glucose concentration.

The aim of this study was to estimate the time course of lysosomal transformations associated with crinophagy, i.e., the degradation of insulin within lysosomes, in the beta-cells of pancreatic islets. Primary and secondary lysosomes were identified in mouse islet beta-cells and subjected to ultrastructural morphometry. Islets from an in situ preparation were compared with isolated islets incubated in vitro. Under the in vitro conditions, the islets were initially exposed to 28 or 3.3 mM glucose for 24 h. Then the glucose concentration was rapidly changed to 3.3 and 28 mM glucose, respectively, and the islets were incubated for up to an additional 24 h. The beta-cell lysosomes were analyzed and related to alterations in insulin biosynthesis and secretion and islet insulin content after the rapid change in glucose concentration. In vivo, the beta-cell lysosomal population was predominantly composed of secondary lysosomes, which frequently contained secretory granule cores. After the initial 24-h period at 3.3 mM glucose, the volume density and the average volume of the secondary beta-cell lysosomes were increased, suggesting increased crinophagic activity. The mean diameter of the primary beta-cell lysosomes was decreased after 24 h at either 28 or 3.3 mM glucose. The change in glucose from 28 to 3.3 mM resulted in alterations in insulin biosynthesis and secretion, leading to an accumulation of insulin within the beta-cells. Lysosomal transformations suggestive of increased crinophagy were observed 24 h after the alteration in glucose concentration. The change from 3.3 to 28 mM glucose resulted in a parallel increase in insulin biosynthesis and secretion without a change in islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetic embryopathy. Studies with animal and in vitro models.

Diabetic pregnancy is associated with an increased risk for fetal maldevelopment for a largely unknown reason. A decade ago, Norbert Freinkel suggested that the altered fuel mixture offered to the growing conceptus may be the key to most of the changes in the embryogenesis of diabetic pregnancy. He coined the term fuel-mediated teratogenesis. During early pregnancy, periods of maternal hyper- and hypoglycemia may cause marked changes in the availability of glucose to the conceptus. Also, increased concentrations of lipids, notably ketone bodies, and branched-chain amino acids in the maternal circulation contribute to a changed fuel mixture for the embryo. In a recent experimental study of diabetic rats, it was found that the maternal metabolism of all three major classes of nutrients and maternal somatic growth during gestation covaried with the development of the embryo. Consequently, the maintenance of normal concentrations of metabolites from all nutrient classes may be important for prevention of adverse fetal outcome in diabetic pregnancy. In vitro, a high glucose concentration causes embryonic dysmorphogenesis by generation of free oxygen radicals. An enhanced production of such radicals in embryonic tissues may be directly related to an increased risk of congenital malformations in diabetic pregnancy. Thus, the notion that alterations in the net transfer of cellular fuels from the diabetic mother to her offspring may cause embryonic dysmorphogenesis, which suggests that combustion of the fuel may produce compounds that impair embryonic development, has obtained experimental support. If this is also true for human diabetic pregnancy, it has therapeutic implications.

The pseudotetrasaccharide acarbose inhibits pancreatic islet glucan-1,4-alpha-glucosidase activity in parallel with a suppressive action on glucose-induced insulin release.

The pseudotetrasaccharide acarbose, previously known as a potent inhibitor of intestinal alpha-glucoside hydrolases, was investigated with regard to its influence on islet lysosomal enzyme activities and the insulin secretory processes. We observed that acarbose was a potent inhibitor of mouse islet lysosomal acid glucan-1,4-alpha-glucosidase activity, EC50 approximately 5 mumol/l, as well as of acid alpha-glucosidase activity. In contrast, acarbose did not influence other lysosomal enzyme activities such as acid phosphatase and N-acetyl-beta-D-glucosaminidase. Neutral alpha-glucosidase (endoplasmic reticulum) was only moderately inhibited in homogenate and was unaffected in intact islets. Incubation of isolated mouse islets with acarbose revealed that the pseudotetrasaccharide was a strong inhibitor of glucose-induced insulin secretion, EC50 approximately 500 nmol/l, and a significant inhibition was already observed at a concentration of acarbose as low as 100 nmol/l. The acarbose analogue maltotetrose did not influence either glucose-induced insulin release or islet lysosomal enzyme activities. Further, acarbose as well as two other alpha-glucoside hydrolase inhibitors, the deoxynojirimycin derivatives miglitol and emiglitate, did not affect islet glucose oxidation at low or high glucose levels. Acarbose also inhibited insulin release induced by the sulfonylurea glibenclamide, whereas insulin secretion stimulated by the cholinergic muscarinic agonist carbachol or the phosphodiesterase inhibitor isobutylmethylxanthine was unaffected by the drug. Moreover, complementary in vivo experiments showed that pretreatment of mice with acarbose to allow for endocytosis of the compound markedly suppressed the insulin secretory response to an intravenous glucose load.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in net glucose uptake and in the pancreatic B-cell GLUT2 transporter induced by diazoxide and by secretory stimuli.

The pancreatic B-cell GLUT2 transporter and glucose metabolism were examined in isolated rat islets subjected to treatments affecting insulin secretion. Diazoxide was used to inhibit, while glipizide or depolarization of the plasma membrane with a high extracellular K(+) concentration were used to stimulate insulin release in short-term experiments. Islet GLUT2 and insulin were determined by quantitative immunohistochemistry and GLUT2 was also determined by Western blot analysis. Islet net glucose uptake and glucose oxidation were measured using radioactively labelled glucose. Exposure of the islets to diazoxide was associated with a marked increase in the B-cell plasma membrane staining for GLUT2 and increased net glucose uptake. Glucose oxidation was not changed, which may reflect a lowered energy requirement. Conversely, islets subjected to a stimulated insulin secretion with glipizide or a high extracellular K(+) concentration showed a reduced staining of the GLUT2 transporter. The net glucose uptake and glucose oxidation were also reduced. In islets exposed to the high K(+) concentration no change in the molecular weight or phosphorylation of GLUT2 was observed but a lesser amount of the transporter was found by Western blot analysis. Thus, GLUT2 and glucose uptake in the pancreatic B-cell are modified by the secretory process, which suggests that changes in the glucose transporter have a functional role in normal B-cell physiology.