Effect of glucose on somatostatin secretion from isolated pancreatic islets of normal and streptozotocin-diabetic rats

Pancreatic somatostatin (SRIF) secretion was examined using the RIA described in earlier paper. Ten isolated rat pancreatic islets were incubated for 30 min in 1 ml Krebs-Ringer bicarbonate buffer. Glucose (5.6 mM) caused a small but significant increase of SRIF secretion. The maximal secretion rate was observed at 16.7 mM glucose, and the half-maximal rate was seen at about 9.7 mM. Islets preincubated with 16.7 mM glucose released higher levels of SRIF and insulin during the subsequent incubation with 16.7 mM glucose than did islets preincubated with 2.8 mM glucose. Glucose-induced SRIF secretion was suppressed by epinephrine, but beta-adrenergic stimulation (epinephrine and phentolamine) produced an increase in SRIF secretion. Islets taken from rats 2 days after streptozotocin administration released minimal amounts of insulin. Basal and glucose-induced SRIF secretion from these islets, which had relatively unchanged SRIF contents and D cell numbers, equaled SRIF secretion from control rat islets. Islets taken from rats 6 weeks after streptozotocin administration, however, had increased SRIF content and D cell numbers, and they oversecreted SRIF. We conclude that pancreatic SRIF secretion can be induced by glucose and modulated by catecholamines and preexposure to high glucose, and the duration and severity of diabetes may be an important determinant of the changes in pancreatic D cell structure and function.     

Defective early phase insulin release in perifused isolated pancreatic islets of spiny mice (acomys cahirinus)

In order to characterize pancreatic beta cell function in Geneva bred spiny mice (acomys cahirinus), the dynamics of immunoreactive insulin release were examined during perifusion of pancreatic islets isolated from normoglycemic acomys. The initial insulin response of acomys was slow: no clear-cut early (1 to 10 min) peak of insulin release was observed when glucose in the perifusion medium was abruptly raised from 2.8 mM to concentrations as high as 56 mM. This was true for islets of either young, or older more obese acomys. However, after 20 to 30 min of perifusion at the high glucose concentrations, the rate of insulin release from acomysislets became similar to that from islets of rats or mice. By contrast, glucose-induced insulin release responses observed with islets of Wistar-derived rats, Swiss albino mice, and inbred C57BL/6J lean or obese (ob/ob) mice, were clearly biphasic. Tolbutamide 1.5 mM, arginine 16 mM, and theophylline 10 mM were ineffective in stimulating insulin release from acomys islets in the presence of a substimulatory glucose concentration (2.8 mM), whereas these agents were effective in rat islets at the same substimulatory concentration of glucose. On the other hand, when these agents, as well as cyclic AMP 10 mM or cytochalasin B 10 mug/ml were applied in the presence of a stimulating concentration of glucose (16.8 mM), the glucose-stimulated insulin release from acomys islets was increased to the same or to a greater extent than from rat islets. It is suggested that the failure of all the agents tested to stimulate an early rapid phase of insulin release from acomys islets may be secondary to the observed initial insensitivity to glucose, which insensitivity may in turn reflect a selective impairment in the recognition of glucose as an insulinogenic signal in this species.     

Forskolin-induced desensitization of pancreatic beta-cell insulin secretory responsiveness: possible involvement of impaired information flow in the inositol-lipid cycle.

Isolated rat islets of Langerhans were incubated for 2 h in a myo-[2-3H]inositol-containing solution to label their phosphoinositides. Also included during this labeling period was forskolin (0.1-5 microM), a compound established to elevate islet cAMP levels. These islets were subsequently perifused, and their insulin secretory responses to 20 mM glucose or 1 microM of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were assessed. Determined in parallel with secretion were [3H] inositol efflux patterns and, at the termination of the perifusion, labeled inositol phosphate accumulation. The following major observations were made. 1) Forskolin had no deleterious effect on the total amount of [3H]inositol incorporated by the islets during the labeling period. 2) However, labeling in forskolin resulted in subsequent dose-dependent decreases in 20 mM glucose-induced insulin secretion, [3H]inositol efflux and inositol phosphate accumulation. 3) Inclusion of the diacylglycerol (DAG) kinase inhibitor monooleoylglycerol (50 microM) restored to a significant degree glucose-induced release from forskolin-desensitized islets. 4) Pretreatment with 5 microM forskolin had no deleterious effect on TPA-induced insulin release. 5) Prior exposure to forskolin also impaired phosphoinositide hydrolysis in response to cholecystokinin stimulation. 6) Similar to forskolin, labeling in isobutylmethylxanthine (1 mM) reduced in a parallel fashion islet [3H]inositol efflux and insulin secretion in response to 20 mM glucose stimulation. These findings demonstrate that prior chronic elevation of islet cAMP levels suppresses the activation of phospholipase-C in response to subsequent stimulation. Defective insulin secretory responsiveness of these islets appears to be the result of impaired generation of phosphoinositide-derived second messenger molecules, particularly DAG. By substituting for DAG, however, TPA circumvents this biochemical lesion and evokes a normal insulin secretory response from forskolin-pretreated islets.     

Evidence for metabolic regulation of pancreatic glucagon secretion by L-glutamine

To investigate effects by L-glutamine on pancreatic A-cell secretion and intermediary metabolism, isolated pancreatic islets from normal and streptozotocin treated guinea pigs (A-cell rich islets) were incubated in the presence of glucose (5.5 mM) +/- L-glutamine (10 mM). Glutamine significantly enhanced glucagon release from 297 +/- 54 to 528 +/- 53 pg/micrograms DNA/h in normal islets and from 553 +/- 31 to 806 +/- 50 pg/micrograms DNA/h in A-cell rich islets. All results were expressed on the basis of islet DNA concentration, being 66 +/- 4 ng DNA per normal islet and 32 +/- 2 ng DNA per A-cell rich islet. Simultaneously, glutamine suppressed glucose oxidation to 64 per cent in normal islets and to 47 per cent of basal oxidation in A-cell rich islets. Islet content of ATP was also reduced by glutamine to about 60 per cent in A-cell rich islets, but not significantly changed in normal islets. Glutamine oxidation, at 5.5 mM-glucose, was considerably higher in A-cell rich islets (911 +/- 65 pmol/micrograms DNA/h) than in normal islets (313 +/- 52 pmol/micrograms DNA/h). Addition of porcine insulin (25 mU/ml) counteracted these effects by glutamine, i.e. suppressed glucagon release but increased glucose oxidation and ATP content of the A-cell rich islets. The present findings demonstrate that glutamine stimulates glucagon release and is readily metabolized by the A-cells. Furthermore, the regulation of glucagon secretion by glutamine appears to be reciprocally related to factors affecting glucose metabolism and ATP-levels in the A-cell.     

Effects of porcine pancreastatin on secretion and biosynthesis of insulin and glucose oxidation of isolated rat pancreatic islets

The effects of porcine pancreastatin were studied on insulin secretion induced by glucose and nonnutrient stimuli, insulin biosynthesis, and glucose oxidation of cultured rat islets. Pancreastatin (100 nM) significantly suppressed, by 32-52%, the insulin response to 27, 16.7, 11, and 5.5 mM but not to 50 mM glucose, whereas 10 nM pancreastatin inhibited insulin release significantly only at 11 and 5.5 mM glucose. Pancreastatin (10 and 100 nM) also suppressed release induced by 20 mM arginine (by 26 and 30%) as well as by 1 microgram/ml of glibenclamide (by 56 and 72%, respectively). Pancreastatin (10 and 100 nM) furthermore inhibited insulin release induced by 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) (by 40 and 61%, respectively) and 1.0 mM IBMX (by 44 and 76%, respectively). Neither glucose oxidation nor overall insulin biosynthesis in islets was significantly affected by pancreastatin, although a slight but significant enhancement of biosynthesis was noted at 1.7 mM glucose in the presence of 100 nM pancreastatin. In conclusion, these data demonstrate that porcine pancreastatin suppresses glucose-induced insulin response from isolated rat islets in a competitive manner. This effect seems not to be exerted through a suppression of (pro)insulin biosynthesis or glucose metabolism in the islets, and thus the effect mediated by pancreastatin must be on a step distal to the coupling between islet glucose metabolism and insulin secretion. The relatively strong inhibition by the peptide of IBMX-induced insulin release suggests that it acts on the cAMP system of islet B cells.     

Biphasic insulin response to high glucose and a role of protons and calcium

In order to determine the role of protons and Ca++ in the biphasic insulin response to glucose, we studied the effect of monensin, a carboxylic ionophore, on the first phase and second phase of glucose-induced insulin release and Ca++ efflux from perifused rat pancreatic islets. The agent, 1-100 nM, dose dependently inhibited the insulin release from the islets incubated for 60 min in Krebs-Ringer bicarbonate buffer containing 16.7 mM glucose. Islet 14CO2 production rates from D-(U-14C)glucose were not affected by 10 or 100 nM monensin. Perifusion of the islets prelabeled with 45Ca++ demonstrated that 100 nM monensin had only a slight inhibitory effect on the first phase insulin response to 16.7 mM glucose and no effect on 45Ca++ efflux. This agent inhibited the second phase insulin release and depressed 45Ca++ efflux. When monensin was added at the start of the second phase release, the release was inhibited. When exposed to the agent before the stimulation by glucose, the first phase insulin release was observed, albeit significantly decreased, and the start of the insulin release and 45Ca++ efflux was delayed. The agent, added 30 min after the change to high glucose, immediately inhibited the insulin release. Thus, the first phase insulin release is mediated mainly through a mechanism which is not related to protons generated from glucose metabolism. Protons may be a crucial coupling factor in the second phase insulin release.     

Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle.

The influence of glucosamine on beta-cell response characteristics of collagenase-isolated rat islets was determined. Groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide (PI) pools. Also included in some experiments was glucosamine (0.1-10 mM). Subsequently, these islets were perifused, and their responses to 10 mM glucose, 10 mM alpha-ketoisocaproate (KIC), and 1 microM of the phorbol ester phorbol 12-myristate 13-acetate were assessed. Increases in PI hydrolysis were monitored during the perfusion by measuring fractional efflux rates of [3H]inositol. The accumulation of inositol phosphates after the perifusion was also determined. In other experiments, the use of 10 mM glucose was measured after a 2-h exposure to 5 or 10 mM glucosamine. Finally, the ability of glucosamine itself to augment release and activate PI hydrolysis was assessed. The following observations were made. 1) A prior 2-h exposure to 5-10 mM glucosamine resulted in parallel dose-dependent impairments in 10 mM glucose-induced insulin release and PI hydrolysis. 2) Glucosamine (5-10 mM) also impaired the subsequent response to alpha-ketoisocaproate (KIC). Parallel deficits in KIC-induced PI hydrolysis were noted under conditions where insulin secretion was impaired. 3) Under several conditions where glucosamine impaired glucose-induced secretion, it had no adverse effect on phorbol 12-myristate 13-acetate-induced release. 4) The desensitizing effect of 10 mM glucosamine on 10 mM glucose-induced release and PI hydrolysis developed within 30 min of exposure to it. 5) Glucosamine (5-10 mM) preexposure had no adverse effect on the use of 10 mM glucose by desensitized islets. 6) Short term (5-min) exposure to glucosamine (10 mM) alone stimulated PI hydrolysis, while a 30-min exposure to the same level of the hexosamine depressed it. 7) In the presence of 0.25 microM forskolin, 10 mM glucosamine also had a transient stimulatory effect on insulin release. These findings support the concept that the acute and chronic effects of glucosamine on the beta-cell result at least in part from its ability to influence PI hydrolysis in islets.     

Prolactin regulates adenylyl cyclase and insulin secretion in rat pancreatic islets

A role for prolactin (PRL) in the regulation of adenylyl cyclase (AC), cyclic AMP (cAMP) formation and insulin secretion was studied in isolated rat pancreatic islets cultured for 4 days at 5.5 mM glucose in the absence (control) or presence of PRL (500 ng/ml). In PRL-treated islets, stimulation by glucose (8 mM), carbamylcholine chloride (CCh) and phorbol dibutyrate increased cAMP levels 40, 89, and 151%, respectively, above similarly stimulated control islets without PRL. Moreover, insulin secretion in PRL-treated islets was more than doubled in response to 8 mM glucose plus glucagon-like peptide 1 compared with control islets. PRL also increased protein kinase C (PKC) activity in cultured islets. When islets were cultured at an insulin secretion desensitizing concentration of glucose (11 mM) for 4 days, there was a decrease in forskolin-stimulated cAMP production. However, the presence of PRL with 11 mM glucose prevented the glucose-induced decrease in cAMP production. Insulin secretion in response to 17 mM glucose was also higher (P<0.02) in islets cultured with 11 mM glucose plus PRL compared with islets cultured with 11 mM glucose alone. Islet AC types -III, -V, and -VI mRNA levels increased relative to 18s rRNA following PRL treatment. In contrast, culture at 11 mM glucose decreased relative AC-III, -V and -VI mRNA levels by as much as 50%. Culture with PRL prevented the decrease in AC expression during islet culture with 11 mM glucose, and the mRNA levels remained similar to control islets cultured at 5.5 mM glucose. Thus, PRL not only increased islet AC expression and activity and insulin secretory responsiveness, but also protected islets from chronic glucose-induced inhibition of these beta-cell activation parameters.     

Differential effects of beta-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man

The selective beta 2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to beta-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mM glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mM glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of beta-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different beta 2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to beta-agonists, whereas those of human islets are equipped with functional beta-receptors which can directly influence the rate of insulin secretion.     

Effects of protein kinase C inhibitors on insulin secretory responses from rodent pancreatic islets

The contribution of protein kinase C (PKC) to the regulation of insulin release from perifused islets was explored using staurosporine or G? 6976 to inhibit the enzyme. Phorbol 12-myristate 13-acetate (PMA, 500 nM) addition to rat islets resulted in a slowly rising insulin secretory response. While minimally effective alone, the addition of 500 nM forskolin together with PMA resulted in a synergistic secretory response. The conventional protein-kinase-C isoform inhibitor G? 6976 (1 microM) completely abolished PMA-induced secretion. However, the combination of forskolin plus PMA significantly enhanced secretion from G? 6976-treated islets. Similar to previous findings made with staurosporine, G? 6976 (1 microM) enhanced the first phase and reduced the second phase of 20 mM glucose-induced secretion from rat islets. Additional studies were conducted comparing the secretory responses of perifused rat or mouse islets to glucose. Dramatic species differences to the hexose were observed. For example, 35-40 min after the onset of stimulation with 8, 10 or 20 mM glucose insulin release rates from mouse islets averaged 32+/-6, 84+/-27 or 131+/-17 pg/islet per minute, respectively. The responses from rat islets averaged 115+/-28, 561+/-112 or 800+/-46 pg/islet per minute at this time point. Islet insulin stores were comparable in both species. The addition of 5 microM carbachol, 500 nM forskolin or 20 mM KCl to mouse islets together with 20 mM glucose resulted in a dramatic augmentation of insulin output. The responses to carbachol or forskolin, but not KCl, were inhibited by 50 nM staurosporine. However, staurosporine (50 nM) reduced insulin secretion from rat islets stimulated with KCl plus 20 mM glucose. G? 6976 potentiated 20 mM glucose-induced secretion from mouse islets. These studies demonstrate that 1 microM G? 6976 completely abolishes PMA-induced release from rat islets and has a modest inhibitory effect on 20 mM glucose-induced secretion. G? 6976 (1 microM) had no inhibitory effect on 20 mM glucose-induced release from mouse islets. These studies also confirm that staurosporine inhibits both PKC- and PKA-mediated events in islets and this lack of specificity may account for its more pronounced inhibition of release when compared to G? 6976. Finally, significant species differences to PKC inhibitors exist between mouse and rat islets.     

High glucose-induced impairment in insulin secretion is associated with reduction in islet glucokinase in a mouse model of susceptibility to islet dysfunction

Type 2 diabetes is characterized by islet dysfunction resulting in hyperglycemia, which can then lead to further deterioration in islet function. A possible mechanism for hyperglycemia-induced islet dysfunction is the accumulation of advanced glycation end products (AGE). The DBA/2 mouse develops pancreatic islet dysfunction when exposed to a high glucose environment and/or obesity-induced insulin resistance. To determine the biochemical cause of dysfunction, DBA/2 and C57BL/6 control islets were incubated in 11.1 mM or 40 mM glucose in the absence or presence of the AGE inhibitor aminoguanidine (AG) for 10 days. Basal (2.8 mM glucose) insulin release was increased in both DBA/2 and C57BL/6 islets incubated with 40 mM vs 11.1 mM glucose for 10 days. Chronic exposure to hyperglycemia decreased glucose (20 mM)-stimulated insulin secretion in DBA/2 but not in C57BL/6 islets. AG significantly increased fold-induced insulin release in high glucose cultured DBA/2 mouse islets, but did not affect C57BL/6 islet function. DBA/2 islet glucokinase was significantly reduced following 40 mM glucose culture, compared with 11.1 mM glucose cultured DBA/2 islets and 40 mM glucose cultured C57BL/6 islets. Incubation of islets with AG resulted in a normalization of DBA/2 islet glucokinase levels. In conclusion, chronic high glucose-induced increases in AGE can result in islet dysfunction and this is associated with reduced glucokinase levels in a mouse model with susceptibility to islet failure.     

Effects of prior 5-hydroxytryptamine exposure on rat islet insulin secretory and phospholipase C responses

Glucose-induced insulin secretion is inhibited by 5-hydroxytryptamine (5HT). In the present studies the specificity of 5HT inhibition of release and the potential biochemical mechanisms involved were investigated. Dose-dependent inhibition of 15 mM glucose-induced secretion was induced by a prior 3 h incubation with 5HT. At the highest 5HT concentration (500 microM) employed, both first and second phase responses to 15 mM glucose were reduced 50-60%. In addition, this level (500 microM) of 5HT virtually abolished 10 mM glucose-induced secretion. In contrast, secretion in response to the protein kinase C activator phorbol 12-myristate 13-acetate (500 nM) was immune to 500 microM 5HT pre-treatment. Glucose usage rates were comparable in both control and 500 microM 5HT-pretreated islets. However, the generation of inositol phosphates and the efflux of 3H-inositol from 3H-inositol-prelabeled islets in response to stimulatory glucose were impaired in parallel with insulin secretion. Based on these observations the following conclusions were reached: (1) 5HT impairs glucose-induced insulin release by altering glucose-induced activation of phospholipase C. (2) Biochemical events distal to phospholipase C remain intact despite this proximal biochemical lesion. (3) Amperometric analysis of 5HT release from 5HT-pretreated islets must take into consideration its profound adverse impact on glucose-induced insulin secretion.     

Beta cell desensitization to glucose induced by hyperglycemia is augmented by increased calcium

Chronic hyperglycemia has been shown to induce a decrease in beta cell sensitivity to a subsequent glucose challenge. Calcium is a necessary cofactor in the insulin secretory process and glucose elevates cytoplasmic levels. This study was designed to study whether chronic exposure to different extracellular calcium and glucose concentrations would affect the islets' subsequent response to regulatory stimuli. Islets were isolated and cultured in TC 199 plus 10% beta calf serum, glucose (5.5 or 27.5 mM) and calcium (0.5, 2.5 or 4.0 mM) for 48 h. Following culture, the islets were harvested and incubated a second time in the presence of glucose and/or arginine, theophylline, and trifluoperazine (TFP). Some islets were used for insulin content, protein synthesis studies and/or CO2 production from labelled glucose. Islets cultured in a normal glucose environment with low or normal calcium concentration maintained the capacity to respond to a subsequent glucose or arginine challenge. However, islets cultured in a high glucose or high calcium medium failed to respond to a second glucose or arginine stimulus. Theophylline stimulated insulin secretion from both glucose-sensitive and non-sensitive islets, while trifluoperazine inhibited glucose-stimulated insulin secretion in previously sensitive islets and increased insulin secretion in previously non-sensitive islets. The different culture conditions did not alter insulin content, protein synthesis or glucose conversion to labelled CO2. We conclude that chronic exposure to high glucose decreases beta cell responsiveness to glucose and amino acids. Increased extracellular calcium augmented this response. However, the beta cell remained sensitive to theophylline-induced insulin secretion, while TFP paradoxically increased insulin secretion in the glucose-insensitive beta cells. Protein synthesis and glucose oxidation were not affected by culture conditions. Thus we suggest that the glucose-induced desensitization of the beta cells may be due to alterations in the calcium-dependent release mechanism.     

Possible relationship between the B-cell threshold for glucose-induced insulin secretion and blood glucose concentrations in the normal toad

The aim of this work was to gain information on the possible relationship between basal glycemia in the toad and the B-cell threshold for glucose-induced insulin release. Hence, pieces of pancreas from Bufo arenarum were incubated with 2 to 20 mM glucose or preincubated with 2 mM glucose plus the hexokinase and glucokinase inhibitors (50 mM of 2-deoxyglucose and mannoheptulose, respectively) followed by an incubation with different glucose concentrations. The maximal rate of insulin release occurred at 8 mM glucose, while 50% of the release (K(s50)) was observed at 7 mM glucose. Regardless of the glucose concentration employed, pancreas pieces preincubated with 2-deoxyglucose released less insulin than the corresponding controls. On the other hand, mannoheptulose significantly inhibited the release of insulin at high glucose concentrations, having no effect at low glucose concentrations. The blocking effect of these two inhibitors is the first indirect evidence of the existence of the hexokinase/glucokinase enzymic system in the toad pancreas. Since the activity ratio of this system determines the glucose sensitivity of the insulin secretory mechanism, it is concluded that the possible existence of a higher ratio of these enzymes in toad B cells could explain the particular characteristics of glucose sensitivity in this animal, which in turn may explain its low blood glucose concentration.     

A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation.

Short- and long-term effects of hyperlipidemia with elevated FFA on insulin secretion were investigated. Male Sprague-Dawley rats were fed ad libitum and additionally infused with Intralipid 10%, 1.0 ml/h. After 3 h of Intralipid the response to 27 mM glucose in isolated perfused pancreas was enhanced by 86%, P less than 0.02. After 6 h of Intralipid enhancement had subsided. After 48 h of Intralipid glucose-induced insulin release was inhibited by 49%, from 1950 +/- 177 microU/min after saline to 1003 +/- 232 microU/min after Intralipid, P less than 0.02. Inhibition was glucose-selective since responses to other secretagogues (1 mM 3-isobutyl-1 methylxanthine, 10 mM octanoate, or 5 mM alpha-ketoisocaproic acid) were unaffected as were pancreatic contents of insulin (2284 +/- 111 mU/pancreas after saline, 2566 +/- 131 mU/pancreas after Intralipid). In isolated islets from 48 h lipid infused rats production of [14-C]CO2 from D[U-14-C]glucose was decreased (P less than 0.02) in parallel with the insulin response to 27 mM glucose. Glucose-induced secretion was partially normalized by in vitro exposure to a carnitine palmitoyl-transferase I inhibitor (Etomoxir). Effects of a 48 h lipid infusion were also tested during hyperglycemia. Rats were infused with glucose, and hyperglycemia was enhanced by dexamethasone (25 micrograms/24 h). Hyperglycemia depressed glucose-induced secretion from perfused pancreas from 2072 +/- 22 microU/min after saline + dexamethasone to 1185 +/- 155 microU/min after glucose + dexamethasone, P less than 0.01). Intralipid, added to the latter protocol, further inhibited glucose-induced secretion to 437 +/- 87 microU/min, P less than 0.005. Hyperlipidemia is concluded to be associated with short term stimulation but long term inhibition of glucose-induced insulin secretion. Evidence indicates that inhibition depends on fatty acid oxidation, is coupled to decreased glucose oxidation and operates both during normo- and hyperglycemia.     

Suppressive effect of intragastric nutrients infusion on insulin release from perfused rat pancreas

Using the isolated and perfused rat pancreas preparations, we examined whether or not the direct transmission of signals from the gastro-duodenum to the pancreatic islets is existent. Pancreases were isolated en bloc with stomach and the proximal portion of duodenum and were perfused without recirculation. After perfusion with 5.5mM glucose in the perfusate for 40 minutes, 1 ml solution of the following nutrients (0.9% and 9% NaCl, 5% and 50% glucose, 5% and 50% fructose) was respectively infused into the stomach. And ten minutes after the infusion, the pancreas was perfused with 16.7 mM glucose for 20 minutes. Although the glucose-induced insulin release was similar to controls in the pancreas with intragastric infusion of 0.9% NaCl, 5% glucose, and 5% fructose, it was significantly lowered in the pancreas with intragastric infusion of 9% NaCl, 50% glucose, and 50% fructose. These results indicate that there is a direct transmission of some signals from the gastro-duodenum to the pancreatic islets through intragastric high osmolarity.     

Insulin secretion is stimulated by ethanol extract of Anemarrhena asphodeloides in isolated islet of healthy Wistar and diabetic Goto-Kakizaki Rats.

BACKGROUND: The hypoglycemic effect of extract of Anemarrhena asphodeloides has been accounted for by the substance mangiferin which increases insulin sensitivity. The present study aimed to investigate whether an ethanol extract of Anemarrhena asphodeloides would stimulate insulin secretion and if so, further elucidate the mechanism behind this effect. METHODS: Isolated pancreatic islets of normal Wistar rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused to study effect of Anemarrhena asphodeloides extract (TH2) on insulin release. RESULTS: At 3.3 mM glucose, 2, 4, and 8 mg/ml TH2 increased the insulin release of Wistar rat islets 2.5-, 4.1-, and 5.7-fold, respectively (p < 0.05) and of GK rat islets 1.7-, 3.0-, and 6.3-fold, respectively (p < 0.01). Similarly at 16.7 mM glucose, 2, 4 and 8 mg/ml TH2 increased insulin release of Wistar rat islets 1.5-, 2.2-, and 3.8-fold, respectively (p < 0.05) and of GK rat 2.5-, 4.2-, and 11.9-fold, respectively (p < 0.01). In perifusions of islets, TH2 also increased insulin secretion that returned to basal levels when TH2 was omitted from the perifusate. Mangiferin had no effect on insulin secretion of islets. In islets depolarized by 30 mM KCl and B-cell K-ATP channels kept open by 0.25 mM diazoxide, TH2 (8 mg/ml) further enhanced insulin secretion at 3.3 but not at 16.7 mM glucose. Pertussis toxin suppressed the insulin stimulating effect of 2 and 8 mg/ml TH2 by 35 % and 47 % (p < 0.05 and p < 0.001, respectively). CONCLUSIONS: Ethanol extract of the roots of Anemarrhena asphodeloides contains a substance, TH2, that stimulates insulin secretion both at 3.3 and 16.7 mM glucose in islets of normal Wistar and diabetic GK rats. The mechanism behind TH2-stimulated insulin secretion involves an effect on the exocytotic machinery of the B-cell, mediated via pertussis toxin-sensitive Gi- (or Ge-) proteins.     

Effect of chloroquine on biosynthesis, release and degradation of insulin in isolated islets of rat pancreas

Insulin has been reported to degrade inside the islets and islet lysosomal proteases have been thought to take part. As chloroquine is regarded as a potent lysosomotropic agent, an attempt has been made to see whether chloroquine has an influence on intrainsular degradation of insulin. After preculture of collagenase-isolated rat islets at 11 mM glucose together with [3H]leucine for 3 days for labelling newly synthesized insulin, islets were cultured for 1 day at 2.2 or 22 mM glucose with or without 0.02 mM chloroquine. Afterwards, radioactivity was measured in the proinsulin/insulin fraction. For control, the influence of chloroquine during 3-h incubation of both freshly isolated and precultured islets was also studied. During the 1-day culture at 2.2 mM glucose, prelabelled insulin was degraded significantly and addition of chloroquine did not alter the amount of insulin degraded. At 22 mM glucose, no significant amount of insulin had been degraded. During the 3-h incubation of freshly isolated as well as precultured islets, chloroquine was found to inhibit significantly glucose-induced biosynthesis of insulin. Glucose-induced release of insulin, however, was not influenced by chloroquine. It is concluded that chloroquine does not influence glucose-induced release or intra-insular degradation of insulin, but it interferes with the biosynthesis of insulin.     

Rapid oscillation of insulin release by the rat pancreatic islets under stringent Ca2+-free conditions

Oscillation of insulin release by the pancreatic islets was evaluated under stringent Ca(2+)-free conditions for the first time. Isolated single rat islets were exposed to 16.7 mM glucose in the presence of 1.9 mM Ca(2+), or under the stringent Ca(2+)-free conditions (Ca(2+) omission with 1 mM EGTA, 6 microM forskolin and 100 nM phorbol 12-myristate 13-acetate). Fifteen minutes after the initiation of glucose stimulation, effluent was collected at a 6-s interval, insulin was determined in duplicate by a highly sensitive insulin radioimmunoassay, and oscillation and pulsatility of release statistically analyzed. Significant oscillation of insulin release was observed in all islets irrespective of presence and absence of Ca(2+). Significant pulsatility of release was detected in 7 of 11 islets in the presence of Ca(2+) and three of six isl! ets in the absence of Ca(2+). In conclusion, high glucose elicits oscillatory insulin release both in the presence and absence of extracellular Ca(2+).     

Preserved pulsatile insulin release from prediabetic mouse islets.

During the development of type I diabetes, the plasma insulin pattern changes. Because the islet secretory pattern has been implicated in this phenomenon, insulin release was measured from female nonobese diabetic (NOD) mouse islets isolated at different ages. Islets from 5-week-old mice were used as controls because they had no infiltrating mononuclear cells and insulin release rose almost 9-fold with maintained oscillatory frequency when the glucose concentration was raised from 3 to 11 mM. Islets isolated from 13- and 25-week-old mice were infiltrated with mononuclear cells. In these islets, increase in the glucose concentration from 3 to 11 mM only doubled insulin release. However, despite the cellular infiltration, insulin release was pulsatile. Islets from 13-week-old mice had reduced glucose oxidation rate. Culture of such islets for 7 days at 11.1 mM glucose causes a decrease in the number of mononuclear cells infiltrating the islets, which in the present study was accompanied by a normalization of both glucose oxidation and glucose-induced insulin release. In the presence of the mitochondrial substrate alpha-keto-isocaproate (5 mM) both control and infiltrated islets responded with pronounced insulin pulses with similar amplitudes. The results suggest that the deranged plasma insulin pattern observed during the development of type I diabetes may be related to decrease in the insulin pulse amplitude rather than loss of the pulsatile release from the islets.