Decreased cyclic AMP and Insulin responses to glucose in pancreatic islets of diabetic chinese hamsters

Summary The dose as well as the time kinetics of insulin and adenosine-3,5-monophosphate (cyclic AMP) responses to glucose were compared in pancreatic islets isolated from normal and diabetic Chinese hamsters. The insulin content in diabetic islets was about one-half that in normal islets. Insulin release in diabetic islets incubated for 10 min with glucose 60–1000 mg/l00 ml was from one-third to one-half that in normal islets. Glucose 1000 mg/l00 ml stimulated three-fold increases in insulin release without increasing the accumulation of [³H] cyclic AMP in either normal or diabetic islets prelabelled with [³H] adenine. However, in the presence of 1.0 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), glucose 150 mg/l00 ml elicited significant increases of insulin release (+ 134%) and [³H] cyclic AMP accumulation in islets (+ 44%) and incubation medium (+ 48%) of islets of normal but not diabetic hamsters. Also, in perifusion experiments with 0.1 mM IBMX, glucose 500 mg/l00 ml produced threefold greater increases in insulin release and two-fold greater increases in efflux of cyclic AMP in normal than diabetic islets. By contrast with the lesser effects of glucose in diabetic islets, 1.0 mM IBMX increased islet and medium cyclic AMP, as well as insulin release, similarly in normal and diabetic islets. It is suggested that the impairment of glucose induced insulin release in islets of the diabetic Chinese hamster may be due to a defective interaction of glucose with the adenylate cyclase-cyclic AMP system in the pancreatic B cell.This work was presented in part at a meeting of the European Association for the Study of Diabetes, Sept. 1975, Munich, Germanyon leave from the Department of Endocrinology, Karolinska Hospital, Stockholm     

Studies on the mechanisms of somatostatin action on insulin release. IV. effect of somatostatin on cyclic AMP levels and phosphodiesterase activity in isolated rat pancreatic islets.

The effects of somatostatin on insulin release and cyclic AMP metabolism were studied in collagenase-isolated islets of Langerhans from the rat. Ceoncentrations from 500 to 2000 ng/ml significantly inhibited glucose stimulated insulin release, while 100 and 200 ng/ml were ineffective. Somatostatin (2000 ng/ml) inhibited insulin release and [3H]-cyclic AMP accumulation induced by 16.7 mM glucose after 10 and 30 min of incubation. In dose-response studies, the inhibition by somatostatin of the effect of glucose on [3H]cyclic AMP and insulin release could be overcome by a high concentration of the hexose (44.9 mM), suggesting competitive inhibition. In the absence of glucose, somatostatin inhibited [3H]cyclic AMP accumulation induced by the phosphodiesterase inhibitor, IBMX, while no inhibition was seen, again in the absence of hexose, when the [3H]cyclic AMP levels had been raised by the adenyl cyclase stimulator, cholera toxin. Somatostatin did not affect phosphodiesterase activity when added to islet homogenates, but preincubation of the islets with the peptide before homogenization decreased the activity by about 30%. It is suggested that somatostatin-induced inhibition of insulin release is, at least partially, mediated by cyclic AMP, probably through an action on islet adenyl cyclase.     

Reduced early and late phase insulin response to glucose in isolated spiny mouse (Acomys cahirinus) islets: a defective link between glycolysis and adenylate cyclase

Summary The spiny mouse (Acomys cahirinus) exhibits low insulin responsiveness to glucose with a nearly absent early phase release. The alternative fuel-secretagogue glyceraldehyde (10 mmol/l) produced a maximal early insulin response in rat islets but failed to affect early response in Acomys; however, it potentiated the late insulin response in both species alike. Glucagon (1.5 mol/l) potentiated the early insulin response to intermediate (8.3 mmol/l) glucose in rat and Acomys islets by two- and four-fold, respectively. Glucose doubled cyclic AMP levels in rat islets but no significant response was noted in Acomys islets. Isobutylmethylxanthine (0.1 mmol/l) and forskolin (25 mol/l) caused a significant rise in islet cyclic AMP levels in both types of islets; however, neither agent restored the glucose stimulation of cyclic AMP in spiny mouse islets. Forskolin and isobutylmethylxanthine potentiated early and late phase insulin release in both species; however, neither augmented the early response in the Acomys to the degree observed in rat islets. Thus: (1) A deficient link exists in Acomys between glycolysis and subsequent signals. (2) These islets contain a glucose-insensitive adenylate cyclase. (3) The early insulin response may be potentiated by direct activation of adenylate cyclase. (4) The glucose effects on early and late phase insulin release are probably mediated by distinct pathways. (5) In the spiny mouse the signals mediating the early response are deranged to a greater extent than those activating the late phase insulin release.     

Effects of short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol

The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20–24 h cultured rat islets was assessed. In freshly isolated,³H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in³H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18±4 pg/islet/min, peak first and second phase responses of freshly isolated islets to 20 mM glucose averaged 126±24 and 520±82 pg/islet/min, respectively. In the presence of both glucose (20 mM) and carbachol (1 mM), peak first and second phase responses now averaged 422±61 and 1016±156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20–24 h in CMRL-1066 medium attenuated all measured responses. The increases in³H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone (68±9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358±85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.     

Correction of diabetic pattern of insulin release from islets of the spiny mouse (Acomys cahirinus) by glucose priming in vitro

Summary Insulin release kinetics were studied in perifused islets of Langerhans, isolated from mildly hyperglycaemic and from normoglycaemic spiny mice (Acomys cahirinus), a rodent predisposed to develop spontaneously non-ketotic diabetes. In both groups, insulin response to glucose (16.7 mmol/l) was delayed in comparison with that of rat islets, the release kinetics being analogous to that of human Type 2 (non-insulin-dependent) diabetes. Thirty min priming of the isolated Acomys islets with glucose (16.7 mmol/l) resulted in potentiation of the insulin release to a second stimulation. The degree of potentiation decreased exponentially with the time interval between stimulations, showing a t 1/2 of 18 min. Induction of potentiation by glucose was time-dependent, giving a maximal effect after 20 min of priming. In addition to overall amplification of the insulin response, priming with glucose accelerated markedly the initial release rates, correcting the dynamics of the response. We conclude that: (1) decreased and delayed insulin secretion is found in Acomys cahirinus before the development of hyperglycaemia; (2) induction of time-dependent potentiation in the islet by priming with glucose corrects the diabetic-type dynamics of insulin release; (3) therefore the deficient insulin release of Acomys is of a functional nature, the mechanism of potentiation bypassing the defect; (4) since insulin release in Acomys resembles that in prediabetic and diabetic man, similar conclusions might apply to the islet dysfunction in Type 2 diabetes.     

The role of reduced glucose transporter content and glucose metabolism in the immature secretory responses of fetal rat pancreatic islets

Summary Isolated fetal islets show an immature or poor secretory response to nutrient secretagogues which may result from impaired mitochondrial oxidative processes. Insulin secretion, glucose metabolism and detection of metabolic enzymes by radiolabelling and immunoprecipitation were compared in islets isolated from neonatal (aged 5 days) and fetal rats (at 20 days gestation). The insulin secretory dynamics of fetal islets were abnormal in response to stimulation by glucose (10 mmol/l); a rapid release of insulin reaching a maximum 6 min after stimulation was observed with no rising second phase release. However, when the data were expressed as percentage of islet insulin content released, fetal islets released significantly more insulin than neonatal islets in response to glucose (4.86±0.45 % vs 1.81±0.62 %, p <0.01) or 100 nmol/l glibenclamide (2.49±0.17 % vs 0.25±0.06 %, p < 0.001). Fetal islets however, failed to release insulin in response to stimulation by glyceraldehyde (10 mmol/l) unlike neonatal islets. Both glucose utilisation (as measured by the formation of [³H] H₂O from 5-[³H] glucose) and glucose oxidation (as measured by the formation of [¹⁴C] CO₂ from U-[¹⁴C] glucose) did not increase significantly in response to increasing the medium glucose concentration to 10 mmol/l whereas in neonatal islets, glucose utilisation and glucose oxidation were significantly increased 2.5- and 2.7-fold, respectively. When islets were incubated with both radiolabelled glucoses simultaneously, the rate of glucose oxidation was shown to be directly proportional to the rate of glucose utilisation. The relationship between glucose utilisation and glucose oxidation was similar in fetal and neonatal islets. Finally, in experiments to detect and semiquantify metabolic enzymes, the level of GLUT-2 transporter protein was significantly reduced by 50 % (p <0.02) whereas the levels of pyruvate dehydrogenase peptides were similar in fetal and neonatal islets. In conclusion, these data do not support the hypothesis that abnormal mitochondrial oxidation is responsible for the immature secretory responses to nutrient secretagogues found in fetal islets but rather that step(s) earlier in the glycolytic pathway are important for development of normal secretory function. [Diabetologia (1994) 37: 134–140] Received: 1 June 1993 and in revised form: 16 August 1993     

Effect of GIP on the secretion of insulin and somatostatin and the accumulation of cyclic AMP in vitro in the rat

The effects of gastric inhibitory polypeptide (GIP) on insulin secretion as well as on the intra-islet accumulation of [3H]cyclic AMP were investigated in isolated pancreatic islets of the rat. In the presence of 6.7 mmol/l of glucose, 3.0 and 30 nmol/l of GIP induced both insulin and [3H]cyclic AMP responses, while lower and higher concentrations of the peptide were ineffective. A coupling of the two parameters was also found with regard to interaction between glucose and GIP. Thus while 30 nmol/l of GIP was stimulatory together with 6.7, 16.7 or 33.3 mmol/l of glucose, the peptide stimulated neither insulin release, nor the accumulation of [3H]cyclic AMP in the presence of a low concentration of glucose (3.3 mmol/l). The concomittant release of insulin and somatostatin was studied in the perfused pancreas in order to assess a possible influence by somatostatin on the dose-response pattern for GIP-induced insulin release. In this preparation 1.0 to 10 nmol/l of GIP stimulated insulin and somatostatin secretion; however while these concentrations were equipotent on insulin release, 10 nmol/l of GIP stimulated somatostatin release more than 1 nmol/l, indicating differences in dose-response curves for the GIP-induced stimulation of the two hormones. It is concluded that 1) modulation of GIP-induced insulin release is coupled to changes in cyclc AMP response in the islet, 2) GIP-induced somatostatin secretion may influence the concomittant insulin response.     

The role of islet secretory function in the development of diabetes in the GK Wistar rat

Summary Insulin secretion and glucose metabolism were compared in islets isolated from GK Wistar rats (a non-obese, spontaneous model of non-insulin-dependent diabetes mellitus) and control Wistars aged 8 and 14 weeks. By 8 weeks of age, GK Wistar rats were clearly diabetic as indicated by non-fasting plasma glucose concentrations and impaired glucose tolerance. Islet insulin content was not significantly different to controls at either age. In islets from 14-week-old GK Wistar rats glucose-stimulated insulin release (6–16 mmol/l glucose) was significantly reduced to 25–50% of controls in static incubations (p<0.001). In perifusion, glucose-stimulated insulin release was reduced by 90% for first phase (p<0.01) and by 75% for second phase (p<0.05). The responses to arginine and 2 Ketoisocaproate in islets were similar to those in controls. In contrast, islets isolated from 8-week-old GK Wistar rats exhibited no significant reduction in glucose-stimulated insulin secretion in static incubations. In perifusion, although both first and second phases of glucose-stimulated insulin release were slightly reduced, these were not significantly different to controls. Islets from 8-week-old GK Wistar rats failed however to respond to stimulation by glyceraldehyde. Raising the medium glucose concentration to 16 mmol/l significantly increased rates of glucose utilisation ([³H] H₂O production from 5-[³H] glucose) and oxidation ([¹⁴C] CO₂ production from U-[¹⁴C] glucose) in islets isolated from 8-week-old control and GK Wistar rats, respectively. The rates of oxidation were not significantly different at stimulatory glucose concentrations whereas the rates of utilisation were significantly higher in islets from the diabetic animals (p<0.05). Production of [³H] H₂O from 2-[³H] glycerol metabolism was increased (p<0.05) at 2 mmol/l glucose but was not significantly different to controls at 16 mmol/l glucose in islets from 8-week-old GK Wistar rats. This data would suggest that abnormalities in islet function are present in 8-week-old diabetic animals although these do not seriously impair glucose-stimulated insulin release from isolated islets. This in turn would indicate that a defect in the glucose signalling pathway in beta cells is not a primary cause of the diabetes of GK Wistar rats and that deterioration of the secretory response is the consequence of some factor associated with the diabetic condition.Abbreviations KIC 2 Ketoisocaproate - BSA bovine serum albumin - GLUT glucose transporter     

Insulin secretory responses of a clonal cell line of simian virus 40-transformed B cells

Summary We have evaluated the potential of the clonal insulin-secretory cell line HIT-T15 as a model system for investigating stimulus-secretion coupling in pancreatic B cells. In contrast to other cell lines, HIT cell insulin secretion was consistently stimulated 2- to 3-fold by D-glucose. The maximally effective concentration of glucose was 10 mmol/1; between 2 and 10 mmol/l glucose the increase in insulin release was paralleled by an increased rate of glucose oxidation. The main characteristics of glucose-stimulated insulin release by HIT cells were essentially similar to those of normal islets. Thus, the response was (1) specific for metabolizable sugars (D-mannose and D-glyceraldehyde stimulated insulin release but L-glucose and D-galactose were ineffective); (2) markedly dependent on extracellular Ca²⁺ concentration; (3) potentiated by forskolin, glucagon, acetylcholine and 12-0-tetradecanoyl phorbol 13-acetate; (4) inhibited by adrenaline or somatostatin; (5) showed a biphasic pattern of release in perifusion experiments, with both phases being potentiated by forskolin. The secretory response of the HIT cells to amino acids was also similar to that of normal islets. Thus, L-leucine and its deamination product 2-ketoisocaproate were effective stimuli, whereas L-isoleucine and L-glutamine were ineffective. Insulin release from HIT cells could also be evoked by the sulphonylureas glibenclamide and tolbutamide and by an increase in concentration of extracellular K⁺ to 40 mmol/1. The content of cyclic AMP in HIT cells was increased modestly by glucose but not by an increase in extracellular K⁺. Forskolin elicited a 4-fold increase in cyclic AMP content. We conclude that HIT cells retain the essential features of the insulin secretory response of normal B cells and represent an important tool for further biochemical characterisation of the secretory system.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

The role of cyclic AMP in the pathogenesis of glucose desensitization in rat pancreatic islets

Adenosine-3',5'-cyclic monophosphate (cyclic AMP) promotes exocytosis of insulin in pancreatic beta cells. This study was performed to investigate the role of cyclic AMP in the pathogenesis of glucose desensitization in rat pancreatic islets. In islets cultured with high glucose for 48 hours, 27 mmol/L glucose-induced insulin release was markedly impaired, while 3.3 mmol/L glucose-or arginine-induced insulin release was enhanced, indicating glucose desensitization. Islet cyclic AMP content was 190% enhanced in high glucose-culture islets for 48 hours. In islets cultured with dibutyryl-cyclic AMP (dbcAMP) or 3-isobutyl methy-xanthine (IBMX), islet insulin content or 27 mmol/L glucose-induced insulin release was deteriorated. In contrast, 3.3 mmol/L glucose- or arginine-induced insulin release was increased, which was similar to glucose-desensitized islets. Wash-out of dbc AMP for the last 24 hours of the 48-hour culture period restored impaired high glucose-induced insulin release in the same manner as wash-out of high glucose. Diazoxide, the KATP channel opener, also restored impaired high glucose-induced insulin release from dbcAMP-cultured islets. The data suggest that enhancement of cyclic AMP in high glucose-culture islets may be one of the pathogenesis of glucose desensitization.     

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.     

86Rb+ fluxes and K+-stimulated nitrophenyl phosphatase acitvity in the pancreatic islets of genetically diabetic mice (C57BL/KsJ-db/db)

Summary Fluxes of ⁸⁶Rb⁺ and hydrolysis of p-nitrophenyl phosphate were measured in collagenase-isolated islets of diabetic C57BL/KsJ-db/db-mice and normal controls (C57BL/KsJ-+/+). Both types of islets accumulated Rb⁺ avidly, as originally reported for hand-dissected islets of non-inbred ob/ ob-mice. KsJ-db/db-mouse islets showed enhanced accumulation of Rb⁺ and normal acitivity of K⁺-activated nitrophenyl phosphatase. D-glucose, 20 mmol/l, inhibited Rb⁺ efflux in normal islets but not in those from KsJ-db/db-mice. The glucose insensitivity of Rb⁺ efflux was observed in young animals, which exhibit glucose-induced insulin release, as well as in old animals, which do not secrete insulin in response to glucose. The anomalous regulation of Rb⁺ efflux already present in young animals may bear on the liability of KsJ-db/db-mouse B-cells to develop defective control of membrane potential, an abnormal metabolism of cyclic AMP, and a marked failure of insulin secretory capacity.     

Mechanisms underlying the insulinostatic effect of peptide YY in mouse pancreatic islets

Summary Peptide YY is an insulinostatic peptide which is released into the circulation from the intestinal mucosa upon food intake. Peptide YY is also co-stored with glucagon in the secretory granules of the pancreatic alpha cells. We examined the mechanisms underlying the insulinostatic effect of peptide YY in isolated mouse pancreatic islets. We found that peptide YY (0.1 nmol/l-1 mol/l) inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from incubated isolated islets, with a maximal inhibition of approximately 70% observed at a dose of 1 nmol/ 1 (p<0.001). Also in perifused islets the peptide (1 nmol/l) inhibited insulin secretion in response to 11.1 mmol/l glucose (p<0.001). Furthermore, peptide YY inhibited glucose-stimulated cyclic AMP formation (by 67%, p<0.05), and insulin secretion stimulated by dibutyryl cyclic AMP (p<0.01). In contrast, the peptide was without effect both on the cytoplasmic Ca²⁺ concentration in dispersed mouse islet-cell suspensions as measured by the FURA 2-AM technique, and on insulin release in isolated islets, when stimulated by the protein kinase C-activator 12-O-tetradecanoyl phorbol 13-acetate. Finally, in pre-labelled perifused islets, peptide YY caused a small and transient increase in the ⁸⁶Rb⁺ efflux (p<0.001), but only in the absence of extracellular Ca²⁺. We conclude that peptide YY inhibits glucose-stimulated insulin secretion from isolated mouse islets by inhibiting two different steps in the cyclic AMP cascade, that is, both the accumulation and the action of the cyclic nucleotide. In contrast, the data suggest that protein kinase C, K⁺ channels, the cytoplasmic Ca²⁺ concentration or other processes directly regulating the exocytosis are not involved in the signal transduction underlying peptide YY-induced inhibition of insulin secretion.Abbreviations PYY Peptide YY - TPA 12-O-tetradecanoylphorbol 13-acetate     

Islet cell function in gold thioglucose-induced obesity in mice

Summary Blood insulin, blood glucose and the biosynthesis and release of insulin have been studied in mice made obese with a single injection of gold thioglucose. In such mice, blood glucose levels were normal, though serum insulin rose in parallel with the development of obesity. When compared with controls, insulin secretion and synthesis were increased in isolated islets of Langerhans from obese mice, over a wide range of glucose concentrations. However, in obese animals, insulin biosynthesis was augmented above control levels at 2 mmol/l glucose, whilst the increase in insulin secretion accompanying obesity only became evident at glucose concentrations > 5 mmol/l. After 2 min incubation, cyclic AMP rose more in islets from obese mice than in controls, though cyclic AMP levels did not significantly differ in either group after 10 min incubation with glucose. Glucose oxidation was also increased in islets of Langerhans from obese mice. It seems possible that changes in glucose oxidation, as well as in cyclic AMP levels, contribute to the alteration in the B cell response in this type of obesity.     

Effect of low concentrations of glucagon on insulin release and cyclic AMP content in isolated rat islets

Studies on hormone action in isolated islets have generally been carried out using concentrations far above physiologic levels. This study investigates whether glucagon at concentrations close to the physiologic level is insulinotropic in isolated islets and how this relates to islet cyclic AMP levels. Collagenase isolated rat islets were tested directly after isolation or after a 24-hour tissue culture. Insulin release and islet cyclic AMP content were determined during a 30-minute incubation by radioimmunoassay. After maintenance in tissue culture glucose-induced (16.7 mmol/L) insulin release was clearly enhanced by glucagon concentrations between 2 and 1,000 ng/mL in a dose-related manner. Islet cyclic AMP was increased only by glucagon 1 mumol/L (3.8 micrograms/mL). When phosphodiesterases were inhibited (0.1 mmol/L 3-isobutyl-1-methylxanthine) insulin release was stimulated by 1 ng/mL and cyclic AMP by 100 ng/mL. By contrast, in freshly isolated islets, glucagon concentrations in the range of 1 to 100 micrograms/mL were needed to augment glucose-induced insulin release. These findings demonstrate that the hormone sensitivity of collagenase isolated islets is markedly improved by short-term maintenance in tissue culture. The threshold level for a detectable effect on islet cyclic AMP is considerably higher than for glucose-induced insulin release. Since in hepatocytes two signal transduction systems for glucagon have recently been demonstrated, the results could mean that at low concentrations glucagon effects may not be mediated via cyclic AMP.     

Effect of 2-deoxyglucose on [32P] phosphate and insulin release from perifused rat pancreatic islets

Summary The effect of 2-deoxyglucose on glucose mediated insulin and [³²P]phosphate release was studied by perifusion of isolated rat pancreatic islets. When islets were perifused with media containing 2.8 mmol/l glucose and 20 mmol/l 2-deoxyglucose for 60 minutes and then exposed to media containing 8.3 or 16.7 mmol/l glucose and 20 mmol/l 2-deoxyglucose for the next 15 minutes, insulin release at either glucose concentration was prompt but blunted. Similarly, islets preincubated (90 min) with [³²P] orthophosphate, then perifused with 20 mmol/l 2 deoxyglucose for 75 min and stimulated by either 8.3 or 16.7 mmol/l glucose for the final 15 minutes of 2 deoxyglucose exposure demonstrated obtundation of [³²P]phosphate release. Perifusion of islets with 20 mmol/l 2-deoxyglucose alone induced no heightened³²P efflux. These studies suggest that 2-deoxyglucose affects initial events in stimulus-secretion coupling of glucose mediated insulin release.     

Direct effects of progesterone on rat islets of Langerhans in vivo and in tissue culture

Summary Progesterone and oestradiol did not alter rates of insulin secretion from isolated rat islets of Langerhans during a 60 min period of incubation in vitro. However, islets isolated from rats which had been injected daily for 15 days with progesterone (5 mg) and oestradiol (5 g) showed enhanced rates of insulin secretion in response to stimulation by 20 mmol/l glucose or 6 and 20 mmol/l glucose plus 5 mmol/l theophylline. Islets from rats which had been injected with the slow-releasing depot progesterone derivative, hydroxyprogesterone hexanoate, 3 times in 15 days, also showed enhanced rates of insulin release in the absence of any alteration in adenylate cyclase activity. In neither experiment could increased food intake, blood glucose levels or islet insulin content account for the observed changes. The possibility of a direct effect of progesterone on the secretory process was investigated in islets which had been cultured for 20 h with progesterone and oestradiol; these islets were then subjected to a variety of stimuli for secretion. They responded significantly more to glucose (6 or 20 mmol/l) in the presence of theophylline (5 mmol/l), while their insulin content was not significantly different from control islets cultured for a similar period. Islets cultured for 20 h in the presence of progesterone and oestradiol did not show any change in their adenylate cyclase activities. Similarly, direct addition of progesterone and oestradiol to islet homogenates did not alter the adenylate cyclase activity during a 30 minute incubation. These results suggest that progesterone and oestradiol affect insulin secretion directly, by a mechanism which does not involve activation of adenylate cyclase.     

Long-term effects of a high glucose concentration in vitro on the oxidative metabolism and insulin production of isolated rat pancreatic islets

The present study was performed to clarify whether exposure in tissue culture of pancreatic islet B cells to high glucose concentrations will lead to glucose insensitivity and/or toxicity. For this purpose, isolated rat islets were maintained in tissue culture for up to 7 days in the presence of either 5.6, 11, or 56 mmol/L glucose and subsequently analyzed with regard to oxidative metabolism, insulin release, islet content of insulin, and insulin mRNA. Islets maintained at 56 mmol/L glucose showed a decreased insulin content, but no changes in insulin mRNA content when compared with control islets (cultured at 11 mmol/L glucose). In short-term incubations of the high-glucose cultured islets, the rate of insulin release at 1.67 mmol/L glucose was enhanced and could not be further stimulated by a 16.7-mmol/L glucose challenge. However, the insulin release at 16.7 mmol/L was decreased when compared with islets cultured at 11 mmol/L glucose. Islets cultured at 56 mmol/L glucose showed an increased oxygen uptake when incubated at 1.67 mmol/L glucose with no further stimulation at 16.7 mmol/L glucose. These islets also showed increased rates of glucose oxidation at incubation with 1.67 mmol/L glucose, but similar rates of oxidation at 16.7 mmol/L glucose as compared with islets cultured in 11 mmol/L glucose. Islets cultured at 5.6 mmol/L glucose showed decreased insulin release when incubated at either 1.67 mmol/L or 16.7 mmol/L glucose. The rates of glucose oxidation of these islets were also decreased at 16.7 mmol/L glucose when compared with the controls, whereas the oxygen uptake was decreased only during incubation at 1.67 mmol/L glucose. There was also a decreased content of insulin mRNA in these islets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The priming effect of glucose on insulin secretion from isolated islets of Langerhans

Summary Biphasic insulin secretion from perifused rat islets of Langerhans was enhanced if islets had previously been stimulated with glucose 16.6 mmol/l. The priming effect of glucose was reduced if mannoheptulose (16.6 mmol/l), deuterium oxide (D₂O; 98% v/v) or adrenaline (10mol/l) was included in the medium during the initial stimulation period, or if Calcium was omitted. Glyceraldehyde (16.6 mmol/l) but not theophylline (5 mmol/l) could substitute for glucose during the initial stimulation and make islets more responsive to subsequent stimulation. The results suggest that the priming effect of glucose on insulin secretion may be related to 1) glucose metabolism and 2) Ca fluxes in the B cell and the consequent activation of the microtubular system. Neither the generation of intracellular cyclic AMP nor the release of insulin per se appears to be involved in the priming process.