Role of the adenylate cyclase system in altered insulin release from islets of Langerhans of aging rats

In attempting to understand the causes of the hyperglycaemia observed in aging populations and to determine the mechanism(s) for the diminished in vitro insulin release from islets of Langerhans of older rats, the adenylate cyclase-cyclic AMP system was studied in isolated islets from 12 month old and 2 1/2 month old (control) male rats to determine its role in this altered insulin secretion. Islets of Langerhans were isolated by collagenase digestion and then either incubated in the presence of low or high concentrations of glucose for studies of insulin release or were sonicated and assayed for determinations of activities of adenylate cyclase and phosphodiesterase. Insulin release was identical from islets of 12 month old and 2 1/2 month old rats to 2.8 mM D-glucose, while in the presence of 16.7 mM D-glucose, insulin release was decreased by 33% (P less than 0.02) from islets of the older animals. Adenylate cyclase activity was diminished by 60% (P less than 0.005) from the 12 month old rats as compared with islets from the 2 1/2 month old controls, while low Km phosphodiesterase activity was similar in islets from both groups of animals. From these studies it appears that the adenylate cyclase-cyclic AMP system may play a role in the altered insulin release from islets of aging rats.     

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.     

Increased insulin binding to pancreatic islets of aged rats

In rat pancreatic islets, the effect of old age (24-month-old) on [125I]insulin binding, glucose-induced insulin release and inhibition of insulin secretion by exogenous insulin were studied. The results were compared with corresponding data obtained from young (3-month-old) rats. Specifically bound [125I]insulin in islets of old rats was increased by 40% (P less than 0.02) compared to that in young rats. Scatchard plots of displacement studies indicated an increase in receptor number rather than receptor affinity. The insulin-releasing capacity of 16.7 mM glucose did not differ between islets of old and young rats when medium insulin was bound to added antiinsulin serum. In the presence of 16.7 mM glucose (without the addition of antiinsulin serum), insulin secretion was less in islets of old rats compared to that in young rats (283 +/- 38 vs. 528 +/- 29 microU/ml; P less than 0.001). Exogenous insulin inhibited glucose (16.7 mM)-induced insulin release more in islets of old rats than in those of young rats. In conclusion, the present in vitro results may be interpreted to reflect increased insulin binding to islets of aged rats and, consequently, increased inhibition of glucose-mediated insulin secretion due to increased feedback of insulin.     

Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period.

At 3-4 degrees C, the transport of 3-O-methyl-D-glucose (30 mM) was severely impaired in islets prepared from adult rats injected with streptozotocin during the neonatal period. However, at 37 degrees C, the first and second phase of glucose-stimulated insulin release were decreased to the same relative extent in perifused islets of diabetic, as compared to control, animals. Moreover, the time-related increase in the oxidative response of the islets to 16.7 mM D-glucose was less pronounced in diabetic than control rats. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in islet homogenates of diabetic rats only represented one-fifth of that found in control rats, whereas the activity of the cytosolic NAD-glycerophosphate dehydrogenase was comparable in both types of rats. This coincided with the fact that a rise in D-glucose concentration from 2.8 to 16.7 mM failed to increase significantly L-[2-3H]glycerol conversion to 3HOH in islets from diabetic rats, in contrast to the situation found in control animals. The activity of 2-ketoglutarate dehydrogenase in islet homogenates when expressed per microgram protein was not different in control and diabetic rats. Likewise, the ratio between D-[6-14C]glucose oxidation and D-[3,4-14C]glucose oxidation and the capacity of either a non-metabolized analog of L-leucine or 3-phenylpyruvate to preferentially stimulated D-[6-14C]glucose oxidation relative to D-[5-3H]glucose utilization were both unaffected in islets from diabetic rats. These findings argue against the existence of a primary defect in the Krebs cycle of diabetic rats. It is proposed that, despite an obvious alteration of the hexose transport system in the islet cells of diabetic rats, the preferential impairment of the B-cell secretory response to D-glucose, as distinct from other secretagogues, in this model of non-insulin-dependent diabetes is mainly attributable to the low activity of FAD-linked glycerophosphate dehydrogenase, resulting in a decreased metabolic flow through the glycerol phosphate shuttle and a reduced rate of aerobic glycolysis.     

Acute effects of fatty acids on insulin secretion from rat and human islets of Langerhans

Fatty acids have both stimulatory and inhibitory effects on insulin secretion. Long-term exposure to fatty acids results in impaired insulin secretion whilst acute exposure has generally been found to enhance insulin release. However, there are conflicting data in the literature as to the relative efficacy of various fatty acids and on the glucose dependency of the stimulatory effect. Moreover, there is little information on the responses of human islets in vitro to fatty acids. We have therefore studied the acute effects of a range of fatty acids on insulin secretion from rat and human islets of Langerhans at different glucose concentrations. Fatty acids (0.5 mM) acutely stimulated insulin release from rat islets of Langerhans in static incubations in a glucose-dependent manner. The greatest effect was seen at high glucose concentration (16.7 mM) and little or no response was elicited at 3.3 or 8.7 mM glucose. Long-chain fatty acids (palmitate and stearate) were more effective than medium-chain (octanoate). Saturated fatty acids (palmitate, stearate) were more effective than unsaturated (palmitoleate, linoleate, elaidate). Stimulation of insulin secretion by fatty acids was also studied in perifused rat islets. No effects were observed at 3.3 mM glucose but fatty acids markedly potentiated the effect of 16.7 mM glucose. The combination of fatty acid plus glucose was less effective when islets had been first challenged with glucose alone. The insulin secretory responses to fatty acids of human islets in static incubations were similar to those of rat islets. In order to examine whether the responses to glucose and to fatty acids could be varied independently we used an animal model in which lactating rats are fed a low-protein diet during early lactation. Islets from rats whose mothers had been malnourished during lactation were still able to respond effectively to fatty acids despite a lowered secretory response to glucose. These data emphasise the complex interrelationships between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis during undernutrition.     

Secretory, enzymatic, and morphological characterization of rat pancreatic endocrine tumours induced by streptozotocin and nicotinamide

Rat pancreatic endocrine tumours were induced by administration of streptozotocin plus nicotinamide. Fifteen to eighteen months later tumours with wet weights of 0.1 to 224 mg were isolated. These tumours were compared with normal rat pancreatic islets. Insulin release from perifused tumours was stimulated by D-glucose, L-leucine, 2-ketoisocaproate, and D-glyceraldehyde, potentiated by theophylline and inhibited by norepinephrine. Compared with isolated rat pancreatic islets, however, insulin secretory responsiveness to glucose stimulation and insulin content were reduced in tumour tissue. Hypoglycaemia in tumour bearing rats and impaired diffusion of insulin out of the tumours may explain this difference. The pattern of enzyme activities observed in tumour tissue was typical for pancreatic endocrine tissue. The activities of succinate dehydrogenase, the two types of the monoamine oxidase, and alpha-glucosidase were in the normal range in tumour tissue. Only the activities of 5'nucleotidase and glutamate dehydrogenase were decreased. Immunocytochemical analysis of the tumours revealed that they contained an average of 91% B-cells. In addition 8% of D-cells were encountered. Proportions of A-cells and PP-cells ranged below 1%. Thus this endocrine tumour of the pancreas with a high proportion of functionally intact B-cells is an interesting model for studying regulation of secretion and endocrine tumour development.     

Perturbation of pancreatic islet function in glucose-infused rats

The secretory behavior of insulin- and glucagon-producing cells was found to be perturbed in isolated perfused pancreases removed from rats infused with hypertonic solutions of glucose for 48 hours. The anomalies included a high basal release of insulin and a paradoxical increase in insulin output and decrease in glucagon release in response to a fall in D-glucose concentration. Likewise, in isolated islets prepared from the glucose-infused rats, L-arginine or theophylline stimulated insulin release at a low ambient concentration of D-glucose, at variance with the situation found in islets removed from normal rats. These secretory perturbations could not be attributed to any obvious defect in either the transport of D-glucose into islet cells or its further utilization and oxidation, but coincided with the abnormal accumulation of glycogen in the B-cell. It is proposed that the latter anomaly may play a role in the altered dynamics of insulin release found in animals or patients with long-term hyperglycemia.     

Can desensitization of the B-cell to D-glucose be simulated in cultured pancreatic islets?

Summary In order to investigate the phenomenon of B-cell desensitization to D-glucose, rat pancreatic islets were cultured for 20–44h in the presence of increasing concentrations of D-glucose in the 5.6 to 27.8 mM range, and then incubated for 30 to 120 min for measurement of secretory, metabolic and ionic variables. After culture in the presence of 5.6 mM D-glucose, the release of insulin evoked by D-glucose (16.7 mM) was less marked than that seen in islets cultured in the presence of 11.1 mM D-glucose. In the latter islets, the secretory response to D-glucose (8.3 mM or more) was still modest, especially over short periods of incubation, but was markedly enhanced by either theophylline or forskolin. The release of insulin evoked by D-glucose in the presence of theophylline was little affected by either Ca²⁺ concentration of the culture medium or length of culture period (20hvs 44h). The culture-induced alteration in the responsiveness to D-glucose coincided with a smaller relative increase of D-[5³H]glucose utilization, D-[U-¹⁴C]glucose oxidation or net⁴⁵Ca uptake at increasing concentrations of the hexose. It contrasted with a well-preserved secretory response to nonnutrient secretagogues. Although these findings could be interpreted as evidence of B-cell desensitization to D-glucose, the fact that the secretory behavior of the islets was not vastly different whether they were first cultured at physiological (8.3 mM) or higher (11.1 to 27.8 mM) concentrations of D-glucose suggests that this experimental design may not be an optimal model for the functional alteration of the B-cell in hyperglycemic non-insulin-dependent diabetic subjects.     

Regulation of insulin secretion by glucose during aging

The purpose of this study was to investigate the sensitivity of insulin secretion to glucose and its possible relationship to changes in islet glucose utilization during aging. Pancreatic islets of Langerhans were isolated from male Sprague-Dawley rats aged 2- to 24-months and perifused in vitro with glucose. Insulin concentration was determined by radioimmunoassay. Glucose utilization was assessed by measuring the rate of conversion of 3H-5-D-glucose to 3H-H20. The sensitivity of isolated, perifused islets to glucose was reduced during aging. Less glucose metabolism, however, may be required to elicit comparable release of insulin by islets from old than from young rats.     

The desensitization of normal B-cells to glucose in vitro is transient and not related to high glucose levels

To examine the postulated phenomenon of glucotoxicity toward the B-cell, islets isolated from normal adult rats were cultured for 1-6 days in RPMI medium at various glucose concentrations. Insulin release and (pro)insulin biosynthesis by these islets were then measured in short term incubations. The 1-day cultured islets (at 9.7 mM glucose) displayed a deficient glucose-stimulated insulin release which was partially restored in the presence of forskolin (5 microM). By contrast they exhibited a consistent insulin release in response to ketoisocaproate (10 mM), 12-O-tetradecanoylphorbol-13-acetate (2 microM), or the combination of Ba2+ (2 mM) and theophylline (1.4 mM) in the absence of extracellular Ca2+. Desensitization of their B-cells was not specific for glucose, since glyceraldehyde (10 mM) or leucine (10 mM) also failed to stimulate insulin release. Desensitization was not related to glucose concentration (5.6, 9.7, or 16.7 mM) in the medium during the 1-day culture period and was restricted to the secretory function, with no impairment of the biosynthesis process. The desensitization to glucose was transient, and high glucose levels (9.7 and 16.7 mM) in the culture medium favored restoration of the subsequent secretory response to the hexose. Under conditions of recovery of B-cell sensitivity to glucose in vitro (5 days at 9.7 mM glucose), the secretory response to acute glucose was in fact significantly enhanced after an additional exposure (1 day) to very high glucose levels (22 or 55 mM). The present results argue against 1) the possibility that islets suffer from some unspecific decreased viability after a 1-day culture period; and 2) the hypothesis that glucose insensitivity in the 1-day cultured islets is primarily caused by a direct deleterious effect of high glucose concentrations on the B-cells. They, rather, reinforce the view that high glucose levels are actually crucial in the preservation of the insulin secretory response to glucose of islets maintained in tissue culture.     

Glyburide and tolbutamide induce desensitization of insulin release in rat pancreatic islets by different mechanisms.

Insulin secretion was studied in rat pancreatic islets after 24-h exposure to various glyburide or tolbutamide concentrations. Glucose-induced insulin release was significantly (P < 0.05) reduced in islets cultured with 0.1 microM glyburide or 100 microM tolbutamide (2098 +/- 187, 832 +/- 93, and 989 +/- 88 pg/islet.h in control, glyburide-exposed, and tolbutamide-exposed islets, respectively). When glyburide-treated islets were stimulated with glyburide or tolbutamide, insulin release was also impaired compared to that in control islets (P < 0.05). In contrast, tolbutamide-exposed islets showed an impaired response to tolbutamide, but a normal response to glyburide. To investigate the mechanism of the sulfonylurea-induced impairment of insulin secretion, we measured insulin release and Rb+ efflux (a marker of the K+ channel activity) in a perifusion system and islet Ca2+ uptake under static conditions. Insulin release in response to 16.7 mM glucose increased in control islets from 9.4 +/- 1.1 to 131 +/- 19 pg/islet.min (first phase secretion peak). Simultaneously, the fractional 86Rb+ efflux declined from 0.015 +/- 0.002% to 0.006 +/- 0.001% (change in decrement, -63.5%). Glucose-induced insulin release in glyburide- and tolbutamide-treated islets was significantly reduced (first phase peak, 22.1 +/- 5 and 39.7 +/- 8 pg/islet.min, respectively; P < 0.05), and the fractional 86Rb+ efflux decrement was -21 +/- 6% for glyburide (P < 0.005 vs. control islets) and -65 +/- 4% (not different from control) for tolbutamide. When glyburide- or tolbutamide-exposed islets were stimulated with the corresponding sulfonylurea, insulin release was impaired compared to that in control islets (P < 0.05), but, again, 86Rb+ efflux was impaired (P < 0.05) only in glyburide-exposed islets. When 45Ca2+ uptake was studied, the increase in glucose concentration from 2.8 to 16.7 mM increased calcium uptake in control islets from 1.76 +/- 0.58 to 7.27 +/- 1.36 pmol/islet.2 min (n = 4). Preexposure to 0.1 microM glyburide did not change calcium uptake at a glucose concentration of 2.8 mM (1.44 +/- 0.45 pmol/islet.2 min) but significantly reduced calcium uptake stimulated by 16.7 mM glucose (3.21 +/- 0.35 pmol/islet.2 min; n = 4; P < 0.005 compared to control islets). In contrast, preexposure to 100 microM tolbutamide did not change either basal or glucose-stimulated calcium uptake (1.44 +/- 0.45 and 6.90 +/- 0.81 pmol/islet.2 min, respectively; n = 4). These data show that in vitro chronic exposure of pancreatic islets to the sulfonylureas glyburide and tolbutamide impairs their ability to respond to a subsequent glucose or sulfonylurea stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gestational hyperglycaemia and insulin release by the fetal rat pancreas in vitro: effect of amino acids and glyceraldehyde

Summary Unrestrained pregnant rats were infused with glucose during the last week of pregnancy to produce slight or high gestational hyperglycaemia. Control rats were infused with distilled water. Insulin secretion of the fetuses at term was studied in vitro using a perifusion system. Compared with controls, perifused pancreases of slightly hyperglycaemic fetuses showed a similar pattern of insulin secretion in response to 10 mmol/l leucine. Arginine-induced insulin secretion at 20 mmol/1 was higher than in controls. In both groups, 10 mmol/l a-ketoisocaproate had a poor stimulatory effect on insulin release, and 5 mmol/1 D-glyceraldehyde was ineffective in eliciting insulin secretion. In highly hyperglycaemic fetuses all the secretagogues, with the exception of arginine, which induced a sustained monophasic insulin secretory response, had no effect on insulin release. These data show that long-term exposure of fetal B cells to high plasma glucose levels in utero suppresses or alters further insulin secretory response not only to glucose but also to other nutrient secretagogues. The partially spared insulin secretory response to arginine suggests that the defect may concern stimulus-secretion coupling rather than insulin releasing machinery.     

Structure-function relationships in pancreatic islets: support for intraislet modulation of insulin secretion

Pancreatic islet B cell function was studied in vitro using three structurally different preparations of islet tissues: isolated, intact islets, dispersed islet cells attached singly to microcarrier beads, and reaggregated islet cells. Mechanisms of intercellular communication are eliminated with single cell preparations, whereas in aggregates cell to cell communications are reestablished and a defined microenvironment restored. Perifusion studies measured nonstimulated and glucose- and arginine-stimulated insulin release from the three islet tissues. Insulin secretion rates were expressed as a function of cellular DNA content, permitting direct comparison between tissues. During perifusion with low (2.8 or 5.5 mM) glucose concentrations, secretion rates of single islet cells were up to 6-fold greater (P less than 0.001) than those of intact islets. Perifusion of islet cells with 2.8 mM glucose and 100 or 500 pg glucagon/ml had no effect whereas GH-release-inhibiting factor (330 and 1000 pg/ml) decreased nonstimulated insulin secretion rates by 15% (P less than 0.05). After reaggregation, basal insulin secretion rates were restored toward those of intact islets. Glucose (5.5-30 mM) and L-arginine (5-20 mM) elicited first phase insulin responses from single islet cells that were not significantly different from those observed with intact islets; in contrast, second phase responses of single islets to glucose were approximately 50% those seen with intact islets, and their second phase responses to arginine were absent. Single islet cell first and second phase insulin responses to 5.5 mM glucose were enhanced 2.2-fold (P less than 0.01) and 2.8-fold (P less than 0.05), respectively, in the presence of exogenous glucagon, resulting in secretory profiles characteristic of intact islets. Reaggregation of single islet cells was associated with markedly increased first and second phase insulin responses to both glucose and arginine stimulation. These data show that disruption of the islet microanatomy results in alteration of insulin secretory responses and that these effects can be reversed, in part by exogenous glucagon and GH-release-inhibiting factor, and by reaggregation. Although different mechanisms appear important for nonstimulated, first and second phase insulin release, the findings support a role for both direct intercellular communication and hormonal secretion by islet A and D cells in the modulation of B cell function.     

Influence of the oral hypoglycemic agent linogliride (McN-3935) on insulin secretion from isolated rat islets of Langerhans

The influence of a new orally effective hypoglycemic compound, linogliride (McN-3935), on insulin release from isolated perifused rat islets was investigated. At a concentration of 100 microM, linogliride was without effect on insulin secretion in the absence of glucose. While 5.5 mM glucose alone produced a weak secretagogue effect, the secretory response was dramatically (5- to 6-fold) increased by the addition of 100 microM linogliride. This concentration of linogliride did not affect the conversion of [5-3H]glucose to 3H2O, a measure of the rate of glycolysis by the islet. Insulin secretion in response to the combination of 100 microM linogliride and 5.5 mM glucose was abolished by the omission of extracellular calcium. Mannoheptulose (10 mM), an inhibitor of glucose phosphorylation and glucose-stimulated insulin secretion, markedly attenuated the insulinotropic effect of linogliride in parallel with reduced glucose usage. Paradoxically, in the presence of another metabolic inhibitor, 2-deoxyglucose (10 mM), the insulinotropic effect of linogliride (100 microM) in the presence of 5.5 mM glucose was not diminished despite the reduced glucose usage. Linogliride significantly increased the secretory response to other secretagogues, including 2.5 mM D-glyceraldehyde, 15 mM N-acetylglucosamine, 10 mM leucine, and 100 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. These data suggest that linogliride amplifies a cellular signal generated during beta-cell activation by various stimulants.     

Abnormal calcium handling by perifused pancreatic islets from neonatal streptozotocin diabetic model rats

To elucidate the mechanism of impaired insulin release in case of non-insulin-dependent diabetes (NIDDM), we investigated insulin release and 45Ca++ efflux from perifused islets obtained from neonatal streptozotocin diabetic model rats. The model rats were prepared by the intraperitoneal administration of 65 mg/kg streptozotocin (STZ) to neonatal males. Rats treated with STZ did not differ from controls in body weight from 1 week to 16 weeks. The model rats had significant hyperglycemia both in the fasting state and after intraperitoneal administration of 2 g/kg glucose. Although the diameter of the islets from the model rats was not significantly different from that of controls, immunoreactivity to anti-insulin was slightly diminished, and degranulation was slightly observed in B-cells. Insulin content was reduced to 45.6% of the control. Insulin release from the perifused islets of STZ-treated rats responded little to 16.7 mmol/L glucose, but normally to 20 mmol/L arginine in the presence of 5.5 mmol/L glucose. In experiments to test the 45Ca++ efflux from the perifused islets prelabeled with 45Ca++, a rise of 45Ca++ efflux concomitant with the second phase of insulin release from the islets of the model rats was inhibited although a sharp increase of 45Ca++ efflux concomitant with the first phase of insulin release was maintained. 45Ca++ uptake for 30 minutes was reduced in the islets from the model rats in the basal and stimulated state of insulin secretion although the incremental 45Ca++ uptake was similar. It is possible that the abnormal calcium handling in pancreatic B-cells may be one of the causes of defect in insulin release in our model rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Nuclear factor-kappaB and advanced glycation end-products expression in lacrimal glands of aging rats

Advanced glycation end products (AGEs) increase with aging and induce signaling alterations that lead to inflammation and dysfunction in several tissues. Aging reduces function and insulin signaling in lacrimal glands (LGs). To evaluate whether AGE signaling and insulin secretion in LGs are altered in aging, 24- and 2-month-old male Wistar rats were compared. Immunohistochemistry with confocal microscopy was used to evaluate AGE, AGE receptor (RAGE) and nuclear factor-kappaB (NF-kappaB) expression in LGs. Basal tear secretion volume, insulin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) levels in tears and LGs and peroxidase activity in LG tissue were measured. Insulin secretion from isolated LGs and pancreatic beta-cells was compared in the supernatant of aging and control rats in vitro by RIA after stimulation with 2.8-16.7 mM glucose, carbachol and KCl. AGE, RAGE and NF-kappaB expression was higher in LGs of aging compared with young rats. Basal tear secretion and peroxidase activity were significantly lower in the aging group (P=0.016 for both assays). IL-1beta and TNF-alpha levels were higher in tears of aging rats compared with young rats (P=0.007 and 0.05 respectively); however, even though aging rats were insulin-resistant (as confirmed by the insulin-tolerance test), the insulin levels in the tear film of aging and control rats were similar in vivo and in vitro. The higher expression of AGEs, RAGE and NF-kappaB in LGs of aging rats is accompanied by systemic insulin resistance and may be involved in LG and tear film alterations but does not affect insulin secretion in the tear film. These observations indicate that metabolic events may be related to LG and tear film dysfunctions in aging.     

Phosphoenolpyruvate in rat pancreatic islets: A possible intracellular trigger of insulin release?

Summary The content of phosphoenolpyruvate (PEP) has been measured in isolated rat islets of Langerhans incubated in vitro. Islet PEP was higher in islets incubated with 16.7 mmol/l glucose than in islets incubated with zero or 2.8 mmol/l glucose. Islet PEP content was also increased in islets incubated with 5 mmol/l D-glyceraldehyde. Mannoheptulose abolished the glucose-induced rise in PEP content but not that elicited by D-glyceraldehyde. These results are consistent with a role for PEP as an intracellular mediator of glucose- and glyceraldehyde-induced insulin release. The kinetics of pyruvate kinase in extracts of rat islets were studied. The maximal extractable activity was considerably higher than known rates of glycolytic flux. The Km values were found to be 0.16 mmol/l for PEP and 0.5 mmol/l for ADP. The control of islet PEP content and the possible role of PEP in insulin release are discussed.