Reduction in blood and urine glucose levels in streptozotocin and alloxan diabetes by phenazine methosulfate

Summary The purpose of this investigation was to ascertain the ability of phenazine methosulfate (PMS) to improve the streptozotocin (STZ) and alloxan induced diabetic conditionin vivo as determined by changes in blood and urine glucose levels and by alteration in the secretion of insulin by isolated islets. STZ and alloxan diabetes was induced in male albino rats (200–250 g body weight). A single injection of PMS (6.0 mg/kg) or nicotinamide (500 mg/kg) simultaneously with diabetic doses of either STZ or alloxan caused a significant reduction in blood and urine glucose levels three days after the injection. The reduction in glycemic levels was greater with PMS than with nicotinamide. Daily PMS (0.5 mg/kg) injection, initiated 5, 10, 20 or 30 days after the development of STZ- and alloxan-diabetes, caused a significant decrease in blood and urine glucose levels and also increased body weight determined 60 days after STZ or alloxan administration. These effects were observed even if the injections were initiated 20 or 30 days after the onset of the diabetic syndrome. Glucose stimulated insulin secretion was significantly inhibited by pre-incubation of isolated islets for one hour at 37 °C with either STZ or alloxan. However, insulin secretion was induced by PMS in the STZ or alloxan pretreated islets. Nicotinamide neither protected nor induced insulin secretion under similar conditions. The level of insulin secretion induced by PMS whether in the normal islets or in islets previously exposed to the B-cytotoxic agents were comparable in quantity to glucose (17 mM)-stimulated insulin secretion. Data presented in this study are suggestive evidence for the potentiality of a reactive proton donor as an insulin secretagoguein vivo andin vitro.     

Insulin and glucagon releasing activity of coleonol (forskolin) and its effect on blood glucose level in normal and alloxan diabetic rats

Summary Colenol, a diterpenoid isolated from the roots ofColeus forskohlii stimulates the release of insulin and glucagon from the islets bothin vitro andin vivo. Coleonol-stimulated release of glucagon from isletsin vitro is much more pronounced as compared to that of insulin. Glucose concentration of 5.6 mM in the medium is required for the coleonol stimulation of insulin release. Feeding of coleonol to alloxan diabetic rats cause 36.5% increase in blood glucose level as compared to alloxan diabetic control. Oral feeding of coleonol for 7 days to normal rats causes increase in blood glucose, serum insulin, glucagon and free fatty acid levels with corresponding increase in glucose-6-phosphatase activity and depletion of liver glycogen. Predominant stimulation of A-cells by coleonol is suggested for the above effects. C.D.R.I. Communication n ^o 4646.     

In vitro conversion of proinsulin to insulin by cathepsin B in isolated islets and its inhibition by cathepsin B antibodies

Summary Purified bovine cathepsin B, when incubated with isolated rat islets of Langerhans, completely converts proinsulin to insulin as demonstrated by the incorporation of¹⁴C-leucine into islet proteins, releasing lysine as the only basic amino acid. Cathepsin B antibodies raised in rabbit inhibited the above conversion. CDRI Communication No. 2703     

Hypophysis and function of pancreatic islets. IV. Effect of treatment with growth hormone and corticotrophin on insulin secretion and biosynthesis in isolated pancreatic islets of normal rats.

The effects of the recognized diabetogenic hormones, growth hormone and ACTH, administered in vivo, on (pro-)insulin biosynthesis and secretion in isolated islets of normal rats were studied. Rats were treated with these hormones for 4 weeks. Afterwards, their collagenase-isolated islets were incubated with [3H]leucine for 3 h. (Pro-)Insulin biosynthesis was estimated for the incorporation data into islet protein fractions. Islets of treated rats released significantly more insulin and incorporated significantly less [3H]leucine into proinsulin and insulin compared with controls when tested at 100 mg glucose/100 ml. At 200 mg glucose/100 ml, no significant differences were found. The findings demonstrate an impact of these hormones on the B-cells derived from normal pancreas of intact rats. The alterations are, however, not very pronounced and can be easily compensated under a strong glucose stimulus. It appears that the mechanisms for (pro-)insulin biosynthesis and release in rats are more resistant against the diabetogenic actions of pituitary hormones than in other species.     

Alloxan-induced alteration of insulin release,rubidium efflux and glucose metabolism in rat islets stimulated by various secretagogues

Summary Insulin release and⁸⁶Rb efflux were studied in perifused rat islets exposedin vitro to alloxan (2 mmol/l) for 5 min. At a low glucose concentration, alloxan transiently increased⁸⁶Rb efflux. Alloxan immediately and completely abolished the secretory response to glucose (15 mmol/l) and markedly delayed the reduction in⁸⁶Rb efflux normally produced by the sugar. 3-O-methylglucose (20 mmol/l) provided complete protection against the alteration of⁸⁶Rb efflux and partial protection against the inhibition of insulin release. Immediately after alloxan treatment, glyceraldehyde,-ketoisocaproic acid and tolbutamide still induced a rapid release of insulin, but the late phase normally stimulated by glyceraldehyde and-ketoisocaproic acid was inhibited. If islets were exposed to glyceraldehyde or tolbutamide 15 min after alloxan treatment, the rapid insulin release was also markedly impaired. Alloxan failed, however, to affect the ability of these three stimuli to reduce⁸⁶Rb efflux from islet cells. Glucose oxidation and utilization were decreased in alloxan-treated islets and 3-O-methylglucose protected against this effect. The results show that the glucose recognition system in B-cells is the most rapidly and severely affected by alloxan. The drug also alters the response to other secretagogues, the insulin releasing properties of which can be impaired without alteration of their ability to reduce⁸⁶Rb efflux.     

Exendin‐4 ameliorates diabetic symptoms through activation of glucokinase

Background: Glucagon‐like peptide‐1 (GLP‐1) and its stable analogue exendin‐4 maintain glucose homeostasis by modulating insulin secretion from pancreatic β‐cells and controlling hepatic glucose output. Glucokinase (GK), by catalysing the first step in glycolysis, plays an important role in glucose‐stimulated insulin secretion and hepatic glucose metabolism. In the present study, we investigated the effects of exendin‐4 on GK in high fat‐fed and alloxan‐treated diabetic mice. Methods: The effects of alloxan (5, 10 and 20 μmol/L) on insulin release from isolated murine islets, as well as glycogen synthesis by isolated murine hepatocytes, were assessed. The effects of exendin‐4 (10 nmol/kg, twice daily for 4 weeks) were assessed in high fat‐fed, alloxan (50 mg/kg, i.v.)‐treated C57 mice. Glucokinase activity was assessed in the same model. Results: Pretreatment with exendin‐4 attenuated alloxan‐induced decreases in insulin release and glycogen synthesis in islets and hepatocytes. The alloxan‐induced decrease in the GK activity in islets and hepatocytes was also ameliorated by exendin‐4 treatment. Pretreatment with the GLP‐1 receptor antagonist exendin‐9 (100 nmol/L) blocked the effects of exendin‐4 on the liver and pancreas. Treatment of high‐fat fed, alloxan‐treated diabetic mice with exendin‐4 (10 nmol/L, i.p.) reduced the severity of diabetic symptoms. Specifically, exendin‐4 treatment reduced serum glucose by 50% and %HbA1c by 24% compared with control and significantly decreased HOMA‐IR by 39% and increased HOMA‐β by 150%. In addition, exendin‐4 treatment significantly reduced body weight by 6.8% and serum triglycerides by 35%. Conclusions: The results indicate that glucose‐stimulated insulin release and glycogen synthesis are decreased by alloxan due to reduced GK activity. These findings provide further insight into the mechanism by which exendin‐4 regulates glucose homeostasis.     

Effect of methylxanthines on alloxan inhibition of insulin release

Summary Isolated rat islets were maintainedin vitro in a perifusion system, exposed to alloxan (20 mg/100 ml) for 5 minutes in the presence of agents which affect cAMP metabolism and subsequently stimulated with glucose. The rate of insulin secretion was monitored throughout the period of perifusion. Exposure to alloxan alone produces complete inhibition of glucose-induced insulin release [18] whereas concomitant exposure to caffeine and theophylline for this brief interval provided almost complete protection of the islets from the inhibitory action of alloxan. Glucagon, cAMP and DBcAMP did not protect the islets from alloxan. Pre-treatment of the islets with either theophylline or glucagon and DBcAMP did not provide protection. These findings indicate that the protective action of theophylline and caffeine against alloxan is unrelated to the effect of these agents on cAMP metabolism in the beta cell.This research was supported in part by NIH grants AM03373 and AM06181     

Effects of hexoses on insulin biosynthesis, content, and release in channel catfish islets.

Catfish islets were incubated in the presence of glucose, mannose, fructose, or galactose at concentrations of 10, 40, or 200 mg/100 ml. Insulin biosynthesis was measured by the rate of incorporation of radiolabeled isoleucine into proinsulin and insulin; islet insulin content and secretion from islets were monitored by radioimmunoassay. The presence of 10 mg/100 ml of mannose, fructose, or galactose or the absence of hexose caused a significant decrease in incorporation when compared to that in control islets incubated in the presence of 40 mg/100 ml of glucose. This is the first demonstration that glucose can maintain insulin biosynthesis in piscine islets. Galactose at 40 or 200 mg/100 ml was also found to decrease incorporation. This sugar at all three concentrations evoked a significant increase of insulin release, which is the first demonstration that galactose can act as an insulin secretogogue. The only hexose to affect insulin content of islets over a 24-hr period was fructose, which caused a decrease of insulin retained in the islet tissue at all concentrations tested.     

Effects of alloxan on glucose-stimulated insulin secretion,glucose metabolism,and cyclic adenosine 3′, 5′-monophosphate levels in rat isolated islets of Langerhans

Summary Insulin secretion was stimulated and cyclic adenosine 3, 5-monophosphate (cAMP) levels were elevated in isolated rat islets by 27.5 mmol/l glucose. Alloxan caused a dose-dependent decrease in both variables with complete obliteration of insulin release at a concentration of 1.25 mmol/l. D-glucose, in the presence or absence of extracellular calcium, or 3-0-methyl-D-glucose (both at 27.5 mmol/l) protected completely against the effects of alloxan on both glucose-induced insulin release and cAMP levels. 3-0-Methylglucose did not stimulate insulin secretion or elevate cAMP and did not interfere with glucose-stimulated secretion or elevation of cAMP. When glucose-stimulated insulin release was abolished by alloxan, the metabolism of glucose, determined by the rate of³H₂O formation from [5-³H] glucose, was depressed by 20%. It is concluded that alloxan altered the adenylate cyclase system such that it could no longer be stimulated by glucose. Glucose-stimulated insulin secretion or elevation of cAMP did not appear essential for glucose to protect against alloxan. Protection by 3-0-methylglucose did not appear to be mediated through an alteration of cAMP metabolism. Alloxan did not inhibit glucose-induced insulin secretion by grossly altering glycolysis.     

Effects of insulin and glucose on subcellular fractions from rat adipose tissue

Summary Subcellular fractions of rat adipose tissue segments incubatedin vitro were obtained by differential centrifugation. In the presence of glucose, insulin produced the following effects: (a) increased incorporation of³²P-orthophosphate and¹⁴C-glycine into the mitochondrial and microsomal fractions; (b) increased incorporation of³²P-orthophosphate into nuclear RNA. In the absence of insulin, lack of glucose increased the incorporation of¹⁴C-orotic acid into both total RNA and nuclear RNA.     

Decreased glucose-induced insulin release and biosynthesis by islets of rats with non-insulin-dependent diabetes: effect of tissue culture

Non-insulin dependent diabetes (NIDD) was obtained in adult rats after a neonatal streptozotocin injection. In the fed state 3- to 5-month-old rats with NIDD exhibited modestly elevated plasma glucose levels (controls, 131 +/- 7 mg/dl; diabetics, 159 +/- 4 mg/dl; P less than 0.01), impaired glucose tolerance, and a very low insulin response after glucose injection. In the present study the secretion and biosynthesis of insulin were measured using isolated islets from these rats. The insulin and DNA content of islets freshly isolated from rats with NIDD were significantly lower (60% and 80%, respectively, P less than 0.001) than in the controls. The insulin content per islet cell from diabetic rats was also significantly reduced (75%) (P less than 0.01) as compared to controls. At a low glucose concentration (2.8 mM) the insulin release from islets of diabetic rats was 60% (P less than 0.05) of that the controls. At a glucose concentration of 16.5 mM it was stimulated 5-fold from the islets of NIDD rats and 7-fold from the islets of control rats. (Pro)insulin biosynthesis, assessed in the same islets by measuring the incorporation of [3H]phenylalanine into immunoprecipitable material, was significantly higher (50%) (P less than 0.01) in islets from NIDD rats when measured at 2.8 mM glucose. Although (pro)insulin biosynthesis in these islets was significantly stimulated by 16.5 mM glucose (5-fold), it was less (P less than 0.05) than in the control islet (9-fold). To determine whether the derangements described above in the islets of the rats with NIDD could be modified by changing the environmental conditions of the B cells, corresponding experiments were performed after a 5-day culture of the islets at 5.5 mM glucose or at 11 mM glucose. The insulin release and the (pro)insulin biosynthesis, either measured in basal or stimulated states, were then found to be similar in the islets of diabetic and control islets after the 5.5 mM glucose culture period. By contrast, after the 11 mM glucose culture period the insulin release and the proinsulin biosynthesis in the islets of diabetic rats were found significantly less stimulated by 16.5 mM glucose than in the control islets. This suggests that islets of rats with NIDD, once removed from the chronic in vivo exposure to diabetic metabolic disorders, can behave as isolated islets of normal rats, at least as far as insulin handling is concerned.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro restoration of insulin production in islets from adult rats treated neonatally with streptozotocin

The aim of this study was to evaluate if the impaired insulin production of the beta-cell deficient islet organ of neonatally streptozotocin (SZ) injected rats is caused by exposure of the beta-cells to a long-lasting functional demand in vivo or a persistent toxic effect of the drug. For this purpose islets were isolated from adult rats which had received an ip injection of SZ (100 mg/kg body weight) on postnatal day 1 or from control rats receiving the solvent only. The islets used were either fresh or after culture for 2, 7, or 14 days in RPMI 1640 supplemented with 5.6, 11.1, or 16.7 mM glucose. After the various culture periods determinations were performed of the islet contents of insulin and insulin mRNA and the rates of (pro)insulin biosynthesis and insulin release. Freshly isolated islets from SZ-treated rats exhibited lower contents of insulin and insulin mRNA, a lower rate of (pro)insulin biosynthesis, and an impaired glucose-sensitive insulin release. Similar results were obtained after 2 days of culture, in each of the glucose concentrations. After 7 days of culture, however, the content of insulin mRNA and the rate of (pro)insulin biosynthesis of the SZ islets were restored to the control levels. When such islets were cultured for 7 days in 5.6 mM glucose, they exhibited a glucose-sensitive insulin release similar to that of the control islets. A difference in the insulin release between the two groups nevertheless persisted after culture for 7 days at either 11.1 and 16.7 mM glucose. Also, after 14 days of culture at 16.7 mM glucose there was an impaired glucose-sensitive insulin release from SZ islets, while islets cultured at 11.1 mM glucose showed a glucose-stimulated insulin release similar to that of the controls. The present data indicate that, as far as storage and biosynthesis of insulin is concerned, the functional aberrations observed in the freshly isolated SZ-islets did not reflect a permanent cytotoxic damage. The persistent impairment of insulin release after culture at 16.7 mM glucose may reflect either an injurious effect of the mildly diabetic metabolism in vivo or of the neonatal streptozotocin injection.     

Interaction of cyclic AMP and alloxan on insulin secretion in isolated rat islets perifused in vitro.

The interaction of alloxan and cyclic AMP on glucose-induced insulin secretion was studied with the use of isolated rat islet perifusion. Simultaneous perifusion with cyclic AMP and alloxan did not protect the islets against the effect of alloxan. However, addition of dibutyryl cyclic AMP to the alloxan solution produced 40% protection of glucose-induced insulin secretion. Partial protection was obtained with either theophylline (41%) or caffeine alone (54%), and the addition of 1 mg/ml glucose to the theophylline or caffeine solution provided greater than 68% protection. The levels of islet tissue cyclic AMP were more than 2.1 times that of islets not protected against the alloxan effect, when partial or nearly complete reversal of the inhibitory action of alloxan on glucose-induced insulin secretion was effected by theophylline or caffeine. These results suggest that cyclic AMP affords partial protection against the effect of alloxan on glucose-induced insulin secretion.     

Insulinotropic action of L-rhamnose in isolated rat islets

Summary L-Rhamnose at different concentrations stimulated incorporation of³H-leucine both into islet (pro)insulin and that released into the medium. Maximum isotope incorporation with either glucose or rhamnose was seen at a concentration of 16.7 mM, although the glucose-induced effect was significantly greater. Like glucose, rhamnose also enhanced the activities of acid phosphatase and cathepsin B in isolated rat islets.Communication No. 2759.     

Effect of age on glucose oxidation by isolated rat islets

Summary Islets were isolated from pancreases of 2-month and 12-month-old rats, and the oxidation of¹⁴C-glucose to¹⁴CO₂ determined at various medium D-glucose concentration. Islets from 12-month-old rats oxidized significantly less glucose than those from 2-month-old rats at glucose concentrations of 150, 300, and 450 mg/dl, and this was true when islets were selected by hand or by Ficoll density gradient separation. The effect of age on glucose oxidation was seen when islets were incubated with [U-¹⁴C], [1-¹⁴C], or [6-¹⁴C] glucose. The results raise the possibility that previously reported age-related defects in glucose-stimulated insulin secretion may be secondary to the effect of age on islet glucose catabolism.     

Growth hormone regulation of DNA replication,but not insulin production,is partly mediated by somatomedin-C/insulin-like growth factor I in isolated pancreatic islets from adult rats

Summary We have investigated whether the previously demonstrated stimulatory actions of growth hormone on DNA synthesis and (pro)insulin biosynthesis and release of isolated adult rat islets of Langerhans are mediated by an autocrine release of somatomedin-C/insulin-like growth factor I (SM-C/IGF I). In medium containing 1% fetal calf serum, the presence of 16.7 mmol/l glucose, or 2.7 mmol/l glucose supplemented with a concentrate of essential amino acids, caused a significant increase in ³H-thymidine incorporation and insulin release compared to 2.7 mmol/l glucose alone but no increase in SM-C/IGFI release. Further supplementation with 1 g/ml growth hormone increased ³H-thymidine incorporation and SM-C/IGF I release within all groups, and insulin release in the 16.7 mmol/l glucose and 2.7 mmol/l plus amino acid groups. The ability of growth hormone to increase ³H-thymidine incorporation in the presence of 16.7 mmol/l glucose, but not its action on insulin release, was partly inhibited by a monoclonal antibody against SM-C/IGF I (control cultures 100%; growth hormone alone 261±27%, mean±SEM; growth hormone+anti-SM-C/IGFI 179±21%; p<0.05, n=18). Growth hormone, but not 100 ng/ml SM-C/IGF I, increased insulin biosynthesis assessed as immunoprecipitable ³H-labelled insulin by 45%, but this was accompanied by a similar increase in overall protein synthesis. Similarly growth hormone, but not SM-C/IGF I caused a 75% increase in glucose oxidation by islets. Both growth hormone and SM-C/IGF I failed to increase the cellular uptake of -aminoisobutyric acid or 3-O-methyl glucose over a 90 min period. The results suggest that while the stimulatory effect of growth hormone on islet cell insulin biosynthesis and release, glucose oxidation and general protein synthesis is probably direct, its action on B-cell replication is partly mediated by a paracrine release of SM-C/IGF I. This may provide a mechanism for increasing B-cell mass and consequently total insulin output during times of increased metabolic demands on insulin secretion.     

Hypophysis and function of pancreatic islets

Summary Insulin secretion and biosynthesis of proinsulin and insulin were determined in isolated pancreatic islets of hypophysectomized rats. Control rats were of both same age and weight. Hypophysectomy was performed either 13 or 5 weeks prior to the investigation, the weight of the animals being either 80 or 170 g. Biosynthesis of insulin was estimated from the amounts of radioactivity incorporated into proinsulin and insulin after incubation of isolated islets at 50 or 300 mg% glucose in the presence of³H-leucine for 3 h. Islet proteins were separated on Sephadex G 50 fine. — Hypophysectomy resulted in a significant decrease of both glucose stimulated secretion and biosynthesis of insulin. It was found that this reduction was 1) more significant when compared with controls of same age 2) more marked in rats which had been hypophysectomized 13 weeks before than in rats after an interval of 5 weeks and 3) less in rats which had been hypophysectomized at a weight of 170 g than in rats in whom pituitary ablation was performed at a weight of 80 g. At basal glucose concentrations, no significant changes of both secretion and biosynthesis of insulin were apparent. The relation of radioactivity incorporated into proinsulin and insulin was unchanged under all conditions. Insulin content of the isolated islets used was found within about the same range in all rats, apart from the animals which had been hypophysectomized 13 weeks before. In islets of these rats, a reduction to 84% was observed. — Our findings may be explained by reduced sensitivity of the pancreatic B-cell to glucose and a slower rate of insulin biosynthesis after hypophysectomy.Alexander von Humboldt-Fellow 1970–1972.H.S. was on leave from the 2nd Medical Clinic, University of Vienna, Austria.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, SFB Ulm 87, Proj. 11.     

Nitric oxide is involved in the insulin release in rats byl-arginine

The insulinogenic effect ofl-arginine has been demonstrated bothin vivo andin vitro, but the mechanism by which this amino acid stimulates the pancreatic B-cells to release insulin is not entirely clear. Recently it was shown thatl-arginine-derived nitric oxide may mediatel-arginine-induced insulin release, and data were also provided to suggest that nitric oxide (NO) has no part in this process. To further investigate whether NO is involved in the release of insulin induced byl-arginine, we infused different doses ofl- andd-arginine in rats. L-Arginine (25 and 100 mg/kg/minute) elicited dose-dependent increases of the plasma insulin levels by up to 18.65±2.13 and 48.6±6.6 U/L, respectively, and increased the plasma glucose levels by up to 1.18±0.13 and 1.43±0.1 mmol/L, respectively. D-Arginine (25 and 100 mg/kg/minute) also elicited dose-dependent increases of the plasma insulin levels by up to 9.08±1.23 and 23.33±2.33 U/L, and increased the plasma glucose levels by up to 0.32±0.09 and 0.46±0.08 mmol/L. Thus, the increases in plasma insulin and glucose levels were significantly smaller during infusion ofd-arginine. We conclude that the plasma insulin response to i.v. infusion ofl-arginine is at least partly mediated by augmented NO synthesis by the pancreatic islets, althoughl-arginine-derived NO is not an obligatory stimulus for insulin release.     

Pregnancy-associated changes in the endocrine pancreas of normoglycaemic streptozotocin-treated Wistar rats

Summary The effect of pregnancy on pancreatic insulin content and relative B-cell volume has been studied in normoglycaemic Wistar rats treated with streptozotocin 14 days before mating. A single intravenous injection of streptozotocin (30 mg/kg body weight) caused a significant reduction of pancreatic insulin content and B-cell volume. The islet insulin content was 60% of control values. However, pregnancy-associated adaptation was preserved in these streptozotocin-treated animals. Plasma insulin levels, pancreatic insulin and B-cell volume were significantly enhanced compared with non-pregnant rats investigated on the same date. The incorporation of [³H]-thymidine into islets from pregnant rats (day 10.5) was higher than that in islets isolated from non-pregnant animals. After delivery insulin content and B-cell volume returned to pre-pregnant values. Also during a longer period after streptozotocin treatment (156 days), no measurable enhancement of B-cell volume and pancreatic insulin content was observed indicating the unresponsiveness of residual B cells to compensate spontaneously for the loss despite persisting normoglycaemia.     

Effect of age on the binding of lectin125I-PHA-B to pancreatic islets of ratin vitro and stimulation of some cellular events

Summary Agaricus bisporus lectin (PHA-B) stimulates insulin releasein vitro. The stimulation is associated with increased conversion of proinsulin to insulin in the isolated islets of Langerhans of rats. Both these functions are directly proportional to the binding of I¹²⁵ PHA-B, which is more marked in the islets from younger rats. The lectin binding to islets is not affected by glucose concentration in the medium. C.D.R.I. Communication N ^o 4334.