Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

Distribution of insulin receptors among mouse liver endomembranes.

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. 125I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10--20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1--2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0-10(8) M-1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na+ + K+ + Mg2+)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.     

Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles.

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.     

Synergistic effect of insulin and follicle-stimulating hormone on biochemical and morphological differentiation of porcine granulosa cells in vitro

Insulin and follicle-stimulating hormone (FSH) have been shown to facilitate granulosa cell differentiation in vitro. To gain insight into this process, we evaluated the effects of these hormones, alone and in combination, upon the biochemical parameters of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction and progesterone secretion concomitantly with morphometric analysis of granulosa cell ultrastructure and LH/hCG receptor distribution by quantitative autoradiography under light microscopy. Granulosa cells isolated from small antral follicles (controls) cultured in the absence of exogenous hormones exhibited few microvilli and gap junctions; the mitochondria, endoplasmic reticulum, and Golgi complex were all poorly developed. Progesterone secretion was negligible and the cells bound little [125I]iodo-hCG. Insulin treatment increased gap junction formation, and the extent of smooth and rough endoplasmic reticulum and Golgi complex development (all p less than 0.05) but did not affect mitochondrial ultrastructure or volume. Insulin treatment modestly but significantly increased [125I]iodo-hCG binding and progesterone secretion relative to controls (p less than 0.001). FSH treatment had a similar effect to insulin on cell ultrastructure and additionally enhanced development of the mitochondria and smooth endoplasmic reticulum as well as formation of the microvilli (p less than 0.05). FSH significantly increased [125I]iodo-hCG binding and progesterone secretion relative to insulin-treated samples (p less than 0.001). Combined treatment with insulin and FSH markedly increased gap junction and microvilli formation and enhanced the development of the smooth endoplasmic reticulum and the Golgi complex relative to treatment with either hormone alone (p less than 0.05). Additionally, the combined treatment produced larger mitochondria with tubular christae. Consistent with the morphological development, the combined treatment of insulin and FSH significantly increased progesterone secretion and [125I]iodo-hCG binding (p less than 0.001). Autoradiographic analysis showed that aggregated cells in general exhibited higher LH/hCG receptor density than nonaggregated cells, and a significantly higher overall receptor density compared to nonaggregated cells or to cells treated either with insulin or FSH alone. Our results indicate that insulin and FSH facilitate morphological differentiation of the granulosa cell in a synergistic manner, stimulating gap junctions and microvilli formation and enhancing development of the mitochondria, endoplasmic reticulum, and Golgi complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of insulin receptors among mouse liver endomembranes

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. ¹²⁵I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10–20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1–2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0 · 10⁸ M^?1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na⁺ + K⁺ + Mg²⁺)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.     

Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells

Glucose is known to affect mRNA levels of several genes. In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose. Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations. These data suggest that glucose affects insulin receptor gene expression.     

Biogenesis of very-low-density lipoproteins in rat liver. Intracellular distribution of apolipoprotein B

The hepatic subcellular distribution of apolipoprotein B (apo B) was studied quantitatively by using an enzyme immunoassay developed for apo B and by immunoadsorption-precipitation of [3H]leucine-labelled apo B. Over 50% (of 0.59 microgram/mg protein) of the apo B was located in the microsomal fraction. Further subfractionation of the microsomes revealed that 47% of the microsomal apo B was in the Golgi apparatus, while another 43% was associated with the rough endoplasmic reticulum. The smooth endoplasmic reticulum accounted for only 4% of the total. When rat livers were labelled with [3H]leucine for 10 min, the rough endoplasmic reticulum accounted for 80% of the total immunoadsorbed precipitable apo B radioactivity while the smooth accounted for 20%, with no contribution from the Golgi. However, only 8.7% of the total radioactive immunoadsorbed precipitable apo B was lipoprotein-associated, the remainder being membrane-bound. Lipoprotein-associated apo B radioactivity in the smooth endoplasmic reticulum accounted for 40%, with the rough contribution attributed at 50% and the Golgi at 9%. We concluded that (a) there are two major pools of apo B in rat liver microsomes; (b) although the apo B mass may be negligible in the smooth endoplasmic reticulum, the latter does play a role in lipoprotein biogenesis. The possible function of apo B associated with membranes of the microsomes is also discussed.     

Unregulated secretion of an exogenous glycotripeptide by rat islets and HIT cells

Freshly isolated rat islets and cultured hamster insulinoma cells (HIT T15) were incubated with a membrane-permeable octanoyl tripeptide (N-octanoyl-ASN-TYR-THR-NH2), which contains an acceptor sequence for ASN-linked glycosylation. Labeled octanoyltripeptide (125[I]TYR) was glycosylated by both islets and HIT cells. The carbohydrate moiety of this glycotripeptide was removed by N-glycanase indicating that glycotripeptide was formed in the lumen of endoplasmic reticulum and, subsequently was secreted via the route for secretory protein. Secretion of glycotripeptide began more rapidly than that of insulin newly synthesized from 3[H]leucine. At 30 min glycotripeptide secretion was already significant but, over a 3-h period, it never represented more than 21% of glycotripeptide produced. Glycotripeptide secretion was not affected by compounds shown to regulate insulin secretion (glucose, forskolin, EGTA and streptozotocin). Thus in beta cells, it appears that glycotripeptide secretion is unregulated and that its cellular secretory pathway is different from that for insulin.     

Functional rest through intensive treatment with insulin and potassium channel openers preserves residual beta-cell function and mass in acutely diabetic BB rats.

Diazoxide and the diazoxide-analogue, NNC 55-0118, are potassium channel openers that interfere with insulin secretion from beta-cells. In vitro, we show that these two drugs inhibit insulin release from diabetes-resistant BB rat islets cultured at either low or high glucose concentration and cause an intracellular accumulation of insulin with high glucose. Preservation of beta-cells was investigated in newly diabetic BB rats treated with insulin implants from day 0-8 under oral diazoxide, NNC 55-0118 or solvent gavage once a day from day 0-7. Three of eight rats (37.5%) treated with diazoxide and three of ten (30%) treated with NNC 55-0118 retained near normal C-peptide responses when challenged with glucose/arginine on day 9, whereas none of eight (0%) solvent-treated rats showed a C-peptide response. Immunohistochemical staining for insulin and glucagon showed that all the C-peptide responding rats had insulin-positive cells in their islets. In contrast, islets from non-responding rats displayed marked inflammation or end-stage lesions. Furthermore, rats with C-peptide response and treated with NNC 55-0118 exhibited only minimal signs of islet inflammation, whereas C-peptide responding diazoxide-treated rats had low level islet inflammation. These results imply that it is conceivable to preserve residual beta-cells at diabetes onset by induction of target cell rest with potassium channel openers and continuous insulin treatment.     

Morphological changes of pancreatic B cells in ventromedial hypothalamic (VMH)-lesioned obese rats

Morphological changes of pancreatic B cells were investigated in ventromedial hypothalamic (VMH)-lesioned obese rats. An increase in body weight, a decrease in body length, and a marked increase in fat-pad were observed in VMH-lesioned obese rats. The volume density of pancreatic islets and that of B cells in VMH-lesioned obese rats were more increased than those in sham-operated control rats. The B cells of VMH-lesioned obese rats often showed a slight reaction for aldehyde fuchsin or anti-pig insulin serum. By the electron microscopy, the degranulated B cells were found to contain well developed Golgi apparatus and rough endoplasmic reticulum. These observations indicate increased activities of synthesis and release of insulin. We concluded that VMH-lesioned obesity was caused by accelerated lipogenesis with hypersecretion of insulin.     

Long-term effects of glucose on insulin release and glucose oxidation by mouse pancreatic islets maintained in tissue culture

Rates of glucose oxidation and insulin release in response to a wide range of glucose concentrations were studied in short-term experiments in isolated mouse pancreatic islets maintained in tissue culture for 6 days at either a physiological glucose concentration (6.7mm) or at a high glucose concentration (28mm). The curves relating glucose oxidation or insulin release to the extracellular glucose concentration obtained with islets cultured in 6.7mm-glucose displayed a sigmoid shape similar to that observed for freshly isolated non-cultured islets. By contrast islets that had been cultured in 28mm-glucose showed a linear relationship between the rate of glucose oxidation and the extracellular glucose concentration up to about 8mm-glucose. The maximal oxidative rate was twice that of the non-cultured islets and the glucose concentration associated with the half-maximal rate considerably decreased. In islets cultured at 28mm-glucose there was only a small increase in the insulin release in response to glucose, probably due to a depletion of stored insulin in those B cells that had been cultured in a high-glucose medium. It is concluded that exposure of B cells for 6 days to a glucose concentration comparable with that found in diabetic individuals causes adaptive metabolic alterations rather than degeneration of these cells.     

Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.     

Stimulation of proinsulin biosynthesis and insulin release by pyruvate and lactate.

Increasing concentrations of pyruvate failed to stimulate proinsulin biosynthesis and insulin release in freshly isolated islets. Glycolytic flux (3H2O from [5-3H]glucose) decreased by 80-85%, but decarboxylation of [1(-14)C]pyruvate was unaffected in islets tested immediately after alloxan exposure. This strongly suggested that in freshly isolated islets, beta-cells, in relation to other islet cells, hardly contribute to the decarboxylation of pyruvate. Non-alloxan-treated cultured islets decarboxylated 2-2.5 times as much pyruvate as did alloxan-treated islets cultured for 15-18h. Thus the contribution of beta-cells to the metabolism of pyruvate after culturing markedly increased. Concomitantly beta-cells became responsive to pyruvate. At 20mM-pyruvate, release of prelabelled proinsulin and insulin and incorporation of [3H]leucine into proinsulin reached values approximately half of those obtained with 20mM-glucose. Lactate was as effective as pyruvate in inducing responses in cultured islets. The experiments indicate that a critical degree of substrate utilization is necessary for the generation of signals for insulin release and proinsulin biosynthesis.     

Stereological-ultrastructural study of pancreatic B cells in metabolic alkalosis

The secretion of insulin in response to glucose and the changes in the B cell at the ultrastructural level were studied in rat pancreas perfused at pH 7.4 and 7.8 with different concentrations of glucose. Raising the extracellular pH from 7.4 to 7.8 significantly inhibits glucose-induced insulin secretion. Coincidentally, morphometric studies showed significant evidences of low secretory activity in B cells from pancreas submitted to high glucose stimulation under alkalosis, namely lower number of emiocytotic figures and microtubules as well as a decrease in the volume density of the granular endoplasmic reticulum and the Golgi complex. On the other hand, a significant increment in the number of images of granulolysis was also demonstrated. These secretory and ultrastructural results confirm the inhibitory effect of pH 7.8 upon B cell secretory activity induced by glucose. Moreover, they lend further support to the role of intracellular hormone degradation as a regulator of B cell insulin content.     

Secretory and electrophysiological characteristics of insulin cells from gastrectomized mice: evidence for the existence of insulinotropic agents in the stomach

Mice were subjected to gastrectomy (GX) or sham operation (controls). Four to six weeks later the pancreatic islets were isolated and analysed for cAMP or alternatively incubated in a Krebs-Ringer based medium in an effort to study insulin secretion and cAMP accumulation in response to glucose or the adenylate cyclase activator forskolin. Freshly isolated islets from GX mice had higher cAMP content than islets from control mice, a difference that persisted after incubation for 1 h at a glucose concentration of 4 mmol/l. Addition of forskolin to this medium induced much greater cAMP and insulin responses in islets from GX mice than in islets from control mice. In contrast, the insulin response to high glucose (16.7 mmol/l) was much weaker in GX islets than in control islets. Glucose-induced insulin release was associated with a 2-fold rise in the cAMP content in control islets. Surprisingly no rise in cAMP was noted in GX islets incubated at high glucose. Capacitance measurements conducted on isolated insulin cells from GX mice revealed a much lower exocytotic response to a single 500 ms depolarisation (from -70 mV to zero) than in control insulin cells. Addition of cAMP to the cytosol enhanced the exocytotic response in insulin cells from control mice but not from GX mice. The depolarisation-triggered inward Ca(2+) current in insulin cells from GX mice did not differ from that in control mice, and hence the reduced exocytotic response following GX cannot be ascribed to a decreased Ca(2+) influx. Experiments involving a train of ten 500 ms depolarisations revealed that the exocytotic response was prominent in control insulin cells but modest in GX insulin cells. It seems that cAMP is capable of eliciting insulin release from insulin cells of GX mice only when cAMP is generated in a specific microdomain conceivably through the intervention of membrane-associated adenylate cyclases that can be activated by forskolin. The GX-evoked impairment of depolarisation-induced exocytosis and glucose-stimulated insulin release may reflect the lack of a gastric agent that serves to maintain an appropriate insulin response to glucose and an appropriate exocytotic response to depolarisation by raising cAMP in a special glucose-sensitive compartment possibly regulated by a soluble adenylate cyclase.     

Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death

Hepatocyte growth factor (HGF) increases beta cell proliferation and function in rat insulin promoter (RIP)-targeted transgenic mice. RIP-HGF mouse islets also function superiorly to normal islets in a transplant setting. Here, we aimed to determine whether viral gene transfer of the HGF gene into mouse islets ex vivo could enhance the performance of normal islets in a streptozotocin-diabetic severe combined immunodeficient mouse marginal islet mass model in which 300 uninfected or adenovirus (Adv) LacZ-transduced islet equivalents were insufficient to correct hyperglycemia. In dramatic contrast, 300 AdvHGF-transduced islet equivalents promptly (day 1) and significantly (p < 0.01) decreased random non-fasting blood glucose levels, from 351 +/- 20 mg/dl to an average of 191 +/- 7 mg/dl over 8 weeks. At day 1 post-transplant, beta cell death was significantly (p < 0.05) decreased, and the total insulin content was significantly (p < 0.05) increased in AdvHGF-transduced islets containing grafts. This anti-beta cell death action of HGF was independently confirmed in RIP-HGF mice and in INS-1 cells, both treated with streptozotocin. Activation of the phosphatidylinositol 3-kinase/Akt intracellular-signaling pathway appeared to be involved in this beta cell protective effect of HGF in vitro. In summary, adenoviral delivery of HGF to murine islets ex vivo improves islet transplant survival and blood glucose control in a subcapsular renal graft model in immuno-incompetent diabetic mice.     

Cell fractionation analysis of human CD8 glycoprotein transport between endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells

We have set up an analytical cell fractionation procedure to dissect, by a non-morphological method, the anterograde transport of proteins from endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells. Using this procedure after pulse-chase labelling of cells expressing human CD8 glycoprotein, we obtained results that: (1) support the view that the intermediate compartment is a distinct station in the export from the endoplasmic reticulum to the Golgi complex; and (2) strongly suggests that the O -glycosylation process starts after the intermediate compartment, presumably in the cis -Golgi complex.     

Small doses of intracarotid insulin do not lower plasma glucose in the rat

We sought to confirm the observation that 500 microU of insulin injected into the carotid artery of rats lowers plasma glucose by approximately 20 mg/dl within 2 minutes. In our hands, glucose concentrations fell gradually by approximately 20-25 mg/dl over a 45-60 minute period after insertion of a carotid artery cannula. This occurred whether 500 microU of insulin and/or anti-insulin serum or saline were injected toward the heart. There was no change in glucose concentrations following injection of 500 microU of insulin toward the head 45 minutes after insertion of the cannula. Thus, the hypoglycemic response to small amounts of insulin administered to the head via the carotid artery must be very sensitive to factors that are currently difficult to recognize.