Islets Of Control Rats Research Articles

Increased Phagocyte-Like NADPH Oxidase and ROS Generation in Type 2 Diabetic ZDF Rat and Human Islets

OBJECTIVETo determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.RESEARCH DESIGN AND METHODSExpression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.RESULTSLevels of phosphorylated p47phox, active Rac1, Nox activity, ROS generation, Jun NH2-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.CONCLUSIONSWe provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.

Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure

To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin 1 mRNA was incubated with the islet cytosolic fraction, the RNA–protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins.

Abnormal expression of pancreatic islet exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptors in Goto-Kakizaki rats is partially restored by phlorizin treatment and accentuated by high glucose treatment.

The role of glucotoxicity in dysregulation of islet exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins and insulin response was explored in the hyperglycemic Goto-Kakizaki (GK) rat. Syntaxin-1A and vesicle-associated membrane protein isoform 2, which drive insulin granule exocytotic fusion, and the associated nSec1, which modulates the SNARE complex assembly, were diminished in GK pancreatic islets to approximately 40% of the levels in control Wistar rat islets. Phlorizin treatment (12 d) induced normoglycemic control in GK rats, resulting in partial restoration of the insulin response to glucose. Furthermore, islet SNARE complex and nSec1 proteins increased by about 40%. Phlorizin treatment did not affect levels of islet SNARE proteins in controls or on the same SNARE complex proteins in GK rat brain. To examine the role of hyperglycemia per se, GK and control rat islets were exposed for 5 d in culture to 5.5 and 16.7 mM glucose. High glucose treatment greatly increased the levels of synaptosomal-associated membrane protein of 25 kDa and, less markedly, the levels of syntaxin-1A and nSec1 in control islets more than in GK rat islets, whereas levels were reduced in both. This was accompanied by sustained impairment of the insulin response to glucose in GK islets and a normal response in control islets. Thus, GK islets demonstrate dysregulation of SNARE protein expression, and their compensatory increase by high glucose exposure is abrogated. Conversely, normoglycemic control results in partial replenishment of these critical components of the insulin exocytotic machinery and improvement in the insulin response. We propose that dysregulation of SNARE proteins is an important mechanism behind glucotoxicity-mediated impairment of the insulin response to glucose.

Overexpression of Protein-Tyrosine Phosphatase PTPσ Is Linked to Impaired Glucose-Induced Insulin Secretion in Hereditary Diabetic Goto-Kakizaki Rats

The impaired glucose-induced insulin release in type 2 diabetes mellitus may be accounted for by reduced B-cell ATP/ADP ratio or decreased phosphorylation of proteins regulating exocytosis of insulin. This, in turn, could be due to enhanced phosphatase activity. Using in situ hybridization techniques to assess the expression of 11 different phosphotyrosine phosphatases (PTPases), known to be present in the B-cells, overexpression by approximately 60% of PTPσ (also known as LAR-PTP2 or PTP NE-3) was demonstrated in pancreatic islets and liver of spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. In agreement with these findings Western blot of islet lysates, using a polyclonal PTPσ antiserum, showed increased amounts of the protein in GK relative to control rat islets. Exposure of isolated islets for 20 h to 5 μM antisense to PTPσ, composed of an antisense PNA sequence of 15 bases linked to the cell penetrating peptide transportan, increased glucose-induced insulin secretion from GK rat islets, but not from control islets. In parallel, the amounts of the phosphatase decreased. In conclusion, increased expression of PTPσ may be of pathogenetic significance for the defective insulin secretion in GK rat islets.

Decreased expression of t-SNARE, syntaxin 1, and SNAP-25 in pancreatic beta-cells is involved in impaired insulin secretion from diabetic GK rat islets: restoration of decreased t-SNARE proteins improves impaired insulin secretion.

The physiological role of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in insulin exocytosis has been reported in pancreatic beta-cells. To determine whether the beta-cells of GK rats, a nonobese rodent model of type 2 diabetes, exhibit abnormalities in their SNARE proteins, we studied the expression and function of target (t)-SNAREs, syntaxin 1A, and synaptosomal-associated protein of 25 kDa (SNAP-25) in GK rat islets. Although insulin release and insulin content of islets isolated from 12-week-old GK rats were reduced, the proinsulin biosynthetic rate was about twofold higher than that in control rat islets, and no change in the preproinsulin mRNA level was observed. Pulse-chase experiments suggested the increased degradation of insulin in GK rat islets. Immunoblot analysis revealed that protein levels of syntaxin 1A and SNAP-25 in GK rat islets decreased to approximately 60% of the levels in control rat islets. We then examined whether the restoration of the decreased expression of t-SNAREs to the normal level in GK rat islets affected insulin secretion. Restoration was achieved by the overexpression of syntaxin 1A and SNAP-25 via the recombinant adenovirus-mediated gene transduction system, which recovered levels of these proteins to almost control levels. Glucose-stimulated insulin release from AdexlCA syntaxin 1A and Adex1CA SNAP-25-infected GK rat islets increased up to approximately 135 and 200%, respectively, of those from uninfected GK rat islets, although no difference in basal (2.2 mmol/l glucose) insulin release was evident between them. We conclude that decreased expression of t-SNAREs in GK rat islets is in part the defect responsible for impaired insulin secretion.

Troglitazone prevents mitochondrial alterations, beta cell destruction, and diabetes in obese prediabetic rats.

To determine whether the antidiabetic action of troglitazone (TGZ), heretofore attributed to insulin sensitization, also involves protection of beta cells from lipoapoptosis, we treated prediabetic Zucker Diabetic Fatty rats with 200 mg/kg per day of TGZ. Their plasma-free fatty acids and triacylglycerol fell to 1.3 mM and 111 mg/dl, respectively, compared with 2.0 mM and 560 mg/dl in untreated controls. Their islet triacylglycerol content was 34% below controls. In islets of control rats, beta cells were reduced by 82% and the islet architecture was disrupted; beta-cell glucose transporter-2 was absent, 85% of their mitochondria were altered, and they were unresponsive to glucose. In treated rats, the loss of beta cells was prevented, as were the loss of beta cell glucose transporter-2, the mitochondrial alterations, and the impairment of glucose-stimulated insulin secretion. We conclude that the antidiabetic effect of TGZ in prediabetic Zucker Diabetic Fatty rats involves prevention of lipotoxicity and lipoapoptosis of beta cells, as well as improvement in insulin sensitivity.

Preserved initiatory and potentiatory effect of alpha-ketoisocaproate on insulin release in islets of glucose intolerant rats.

Insulin responses to glucose and non-glucose secretagogues were studied in short-term cultured pancreatic islets and perfused pancreata of the glucose intolerant F1 hybrid rats of spontaneously diabetic Goto-Kakizaki and control Wistar rats. After culture at 5.5 mmol/l glucose, hybrid islet responses to 11.1, 16.7 and 27.0 mmol/l glucose were between 60 and 40% of control islet responses. A combination of 1 mmol/l isobutylmethylxanthine and 16.7 mmol/l glucose induced a pronounced insulin release, which was of similar magnitude in hybrid and control rat islets. This response was not further augmented by addition of glibenclamide and arginine. The slope of potentiation of arginine (10 mmol/l)-stimulated insulin secretion by glucose (5.5-16.7 mmol/l) was greatly impaired in hybrid islets. In contrast to glucose, alpha-ketoisocaproate (KIC), which is metabolized in Krebs cycle, dose-dependently stimulated insulin secretion to similar levels in hybrid and control islets, cultured at 5.5 mmol/l glucose. Also in hybrid islets depolarized by potassium chloride (30 mmol/l) and with adenosine triphosphate-sensitive K+-channels kept open by diazoxide, insulin responses to glucose were greatly impaired but intact to KIC. Furthermore, KIC potentiated normally the insulin response to arginine in hybrid islets. In the isolated perfused pancreas, KIC induced similar insulin responses in hybrid rats and control rats. The potentiating effect by 5.5 mmol/l glucose on the KIC-stimulated insulin responses was, however, greatly reduced in isolated islets and absent in the perfused pancreata of hybrid rats. Taken together, these findings suggest an intact capacity for insulin release, although the initiating and potentiating effect by glucose on insulin release are defective in the Goto-Kakizaki-hybrid rats. An abnormal beta-cell glucose metabolism proximal to the Krebs cycle is likely to account for the impairment of insulin release.

Impaired Coupling of Glucose Signal to the Exocytotic Machinery in Diabetic GK Rats

The GK rat is a spontaneous model of NIDDM. The insulin response to 16.7 mmol/l glucose was markedly impaired in both isolated perfused pancreas and isolated islets from GK rats compared with control Wistar rats. Depolarization with 30 mmol/l KC1 in the presence of 3.3 mmol/l glucose and 250 μmol/l diazoxide induced similar insulin responses in perfused pancreases of GK and control rats. In contrast, the glucose-stimulated insulin release was also severely impaired in GK pancreases in the depolarized state. Forskolin (1 μmol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 ± 15 vs. 3 ± 1 pmol/l0 min, P < 0.001). Dibutyryl cAMP (1 mmol/l) exerted effects similar to forskolin on insulin release in the perfused pancreas. In depolarized pancreases of GK but not control rats, forskolin also induced a marked insulin response at 3.3 mmol/l glucose (163 ± 48 vs. 16 ± 1 pmol/20 min,< P < 0.03). Similarly, in studies on isolated islets from GK rats cultured in 5.5 or 16.7 mmol/l glucose for 48 h, forskolin (5 μmol/l) restored insulin release in response to 16.7 mmol/l glucose but had no effect on islet glucose utilization at 3.3 or 16.7 mmol/l glucose. Forskolin markedly stimulated insulin release at 3.3 mmol/l glucose in GK but not control rat islets cultured for 48 h in 5.5 mmol/l glucose, whereas 20 mmol/l arginine had an almost identical effect in both islet varieties. However, in islets cultured in 16.7 mmol/l glucose, forskolin stimulated insulin release similarly both in control and GK islets at 3.3 mmol/l glucose. In conclusion, this study suggests that the insulinotropic effects of glucose are coupled to a direct regulation of the exocytotic machinery in the pancreatic 3-cell. This pathway is markedly impaired in GK rats, contributing to defective insulin response to glucose. In this model, cAMP generation restores the insulin response to 16.7 mmol/l glucose and exerts a marked insulin release even at 3.3 mmol/l glucose.

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide.

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.

Evidence of a paracrine role of neuropeptide-Y in the regulation of insulin release from pancreatic islets of normal and dexamethasone-treated rats.

Recent evidence suggests that a number of a paracrine regulatory peptides, including neuropeptide-Y (NPY), are synthesized within pancreatic islets. We have, therefore, assessed the role of NPY on insulin release from isolated perifused rat islets. NPY release was detectable at 2.1 (95% confidence interval, 1.6-2.6) attomoles/islet.min from the perifused islets of control rats when the glucose concentration was 2.8 mM and decreased by 62% (P = 0.01) on changing to 20 mM glucose. The initial insulin release was 0.7 (0.3-1.6) fmol/islet.min, and it was increased 4-fold (P < 0.001) by high glucose. Simultaneously, pancreatic glucagon release was decreased 85% (P < 0.001), and somatostatin release was decreased 25% (P = 0.06). In contrast, in islets from rats given dexamethasone (4 mg/kg.day) for 10 days, NPY release was 6-fold increased by high glucose. NPY (100 nM) decreased insulin release by 44% (P < 0.001). To determine the influence of naturally present islet NPY on insulin release, immunoneutralization with NPY antiserum was employed. With control rat islets, insulin release was increased 3-fold with NPY antiserum (P = 0.02) in the presence of 2.8 mM glucose and elevated 2-fold at 8 mM glucose (P < 0.001). NPY immunoneutralization similarly increased insulin release from the perifused islets of rats treated with dexamethasone. Our results suggest that NPY is produced by pancreatic islets, and its expression is dependent on the prevailing endocrine environment. Islet NPY appears to constrain insulin release under a variety of conditions. NPY may be an important intraislet paracrine hormone.

Evidence of a paracrine role of neuropeptide-Y in the regulation of insulin release from pancreatic islets of normal and dexamethasone- treated rats

Recent evidence suggests that a number of a paracrine regulatory peptides, including neuropeptide-Y (NPY), are synthesized within pancreatic islets. We have, therefore, assessed the role of NPY on insulin release from isolated perifused rat islets. NPY release was detectable at 2.1 (95% confidence interval, 1.6-2.6) attomoles/islet.min from the perifused islets of control rats when the glucose concentration was 2.8 mM and decreased by 62% (P = 0.01) on changing to 20 mM glucose. The initial insulin release was 0.7 (0.3-1.6) fmol/islet.min, and it was increased 4-fold (P < 0.001) by high glucose. Simultaneously, pancreatic glucagon release was decreased 85% (P < 0.001), and somatostatin release was decreased 25% (P = 0.06). In contrast, in islets from rats given dexamethasone (4 mg/kg.day) for 10 days, NPY release was 6-fold increased by high glucose. NPY (100 nM) decreased insulin release by 44% (P < 0.001). To determine the influence of naturally present islet NPY on insulin release, immunoneutralization with NPY antiserum was employed. With control rat islets, insulin release was increased 3-fold with NPY antiserum (P = 0.02) in the presence of 2.8 mM glucose and elevated 2-fold at 8 mM glucose (P < 0.001). NPY immunoneutralization similarly increased insulin release from the perifused islets of rats treated with dexamethasone. Our results suggest that NPY is produced by pancreatic islets, and its expression is dependent on the prevailing endocrine environment. Islet NPY appears to constrain insulin release under a variety of conditions. NPY may be an important intraislet paracrine hormone.

Increased insulin secretion in response to glucose in isolated islets of Langerhans from bile duct-occluded rats.

In a recent study, it was demonstrated that following i.v. injection of glucose, plasma insulin levels increased more in cholestatic rats than in control rats. This could theoretically be due to either a potentiated insulin secretion or an inhibited liver extraction of insulin in cholestatic rats. In the present study, we report the influence of obstructive jaundice on insulin secretion from isolated islets of Langerhans 3 weeks after bile duct occlusion or sham operation in rats. We found that insulin secretion from islets of control rats (250 +/- 29 microU/ml) and of bile duct-occluded rats (260 +/- 24 microU/ml) was not significantly different in a medium with a low glucose concentration (3.3 mM). In contrast, at a high glucose level (16.7 mM), an increased insulin secretion was seen from islets of cholestatic rats (469 +/- 14 microU/ml) as compared with control rats (370 +/- 19 microU/ml) (p less than 0.001). These results indicate that the increased plasma insulin levels found in vivo during cholestasis may not merely reflect a decreased liver clearance but be attributed to a potentiation of glucose-induced insulin secretion.

Ethanol influence on insulin secretion from isolated rat islets.

This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP.

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)

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.

Effects of growth hormone on insulin release in the rat.

Growth hormone injected intravenously in the rat elicited a 6-fold spike change in immunoreactive insulin with little variation in glucose. Subcutaneous administration of growth hormone for 4 days augmented by 56% the insulin-secretory response to glucose of isolated islets from hypophysectomised rats but not the response of control rat islets. When islets were cultured in the presence of growth hormone, the glucose-induced insulin release was increased by 35% in batch incubations of islets from both normal and hypophysectomised rats and by 70--110% in perifused islets. Thus the capacity for stimulated release of insulin is limited by hypophysectomy, and growth hormone is capable of directly influencing the secretory function of the beta-cell.

Effect of tolbutamide on aminophylline-, 3,5-AMP-dibutyrate-or glucagon-induced insulin release from pancreatic islets after impairment of pyridine nucleotide metabolism caused by 6-aminonicotinamide (6-AN)

The effect of tolbutamide on pyridine nucleotides and insulin secretion stimulated by aminophylline, 3,5-AMP-dibutyrate or glucagon was studied in pancreatic islets of rats previously treated with 6-aminonicotinamide (6-AN), an inhibitor of pyridine nucleotide synthesis. After being incubated for 60 min in a Krebs-Ringer-Bicarbonate-Buffer in the absence of glucose, pancreatic islets of rats i.p. injected with 35 mg/kg of 6-AN 6 hrs before pancreas removal contained about 30% less NADP and NADPH than did islets of control rats. No changes of NDA or NADH were observed in islets of 6-AN-treated animals. Addition of 16.5 mM glucose led to an increase of NADH, NADPH and a decrease of NADP in islets of both groups of animals; NAD levels remained unchanged. In vitro addition of tolbutamide to islets of control rats did not affect the levels of NADPH or NADP in the presence of 5.5 mM glucose. When 16.5 mM glucose were present, a decrease of NADPH and an increase of NADP was obvious. No effect of tolbutamide on insular NADPH or NADP was observed in islets of rats previously treated with 6-AN be it in the presence of 5.5 or 16.5 mM glucose. In islets of 6-AN-treated rats insulin release in response to aminophylline or 3,5-AMP-dibutyrate in the presence of 5.5 mM glucose was significantly depressed, when compared to islets of untreated controls. Addition of tolbutamide increased insulin release due to aminophylline, 3,5-AMP-dibutyrate or glucagon islets of controls. Tolbutamide alone was without effect. In islets of 6-AN-treated rats aminophylline, 3,5-AMP-dibutyrate or glucagon stimulated insulin release only when tolbutamide was present. Our data suggest that there is no direct interference of tolbutamide with pyridine nucleotides of pancreatic islets, and that tolbutamide increases the secretory response of the beta-cell to aminophylline, 3,5-AMP-dibutyrate or glucagon when insulin release due to these agents is inhibited during decrease of insular NADP and NADPH, caused by 6-AN.

Glucose oxidation (14-CO2 production)and insulin secretion by pancreatic islets isolated from hyperglycemic and normoglycemic rats.

Pancreatic islets from glucose-infused rats retained a “memory” of the hyperglycemic environment, (a) They oxidized (14-CO2 production) glucose-U-14-C and glucose-6-14-C at rates three to four times higher than islets from normoglycemic rats. Inhibitors of insulin secretion and synthesis did not affect the rates of 14-C2 production. In contrast, ouabain (1 mM) decreased glucose oxidation by nearly 50 per cent, (b) They secreted insulin in vitro at rates similar to those of islets of control rats, in spite of the heavy degranulation of their beta cells.