Tonic Sympathetic Nervous System Inhibition of Insulin Secretion Is Diminished in Obese Zucker Rats

It has long been known that the central nervous system (CNS) directly affects pancreatic insulin release. This study was undertaken to determine the effect of the CNS on pancreatic insulin release in three-month-old female lean (Fa/Fa) and hyperinsulinemic obese (fa/fa) Zucker rats. Chloral hydrate (400 mg/kg) was used as the anesthetic agent. The in situ brain-pancreas perfusion model with intact pancreatic innervation was used in this investigation. The study measured insulin secretion in response to a 60-minute glucose stimulus (200 mg/dl). CNS-intact and CNS-functionally ablated obese and lean rats were used. During the 60-minute perfusion period significantly more insulin was released by pancreata from obese rats compared to those from lean rats. In lean rats, about twice as much insulin was released by pancreata from CNS-ablated rats than from CNS-intact rats (P < 0.05), demonstrating a CNS tonic inhibition of insulin secretion. In obese rats, there was no significant difference in insulin released by the pancreata of the CNS-intact and CNS-ablated rats. To determine if there was a masking effect of predominant PNS activity over the SNS in the CNS-intact obese rats, bilateral vagotomy was performed in a group of otherwise CNS-intact obese rats prior to the onset of perfusion. Tonic inhibition was still not observed in the CNS-vagotomized obese rats. In conclusion, hypersecretion of insulin in obese rats is partially due to diminished tonic sympathetic nervous system inhibition of insulin release. These results provide additional evidence regarding abnormal CNS control of insulin secretion in obese Zucker rats.     

Impaired glucose priming of insulin secretion from perfused pancreas in aged female rats.

Effects of age and glucose levels on insulin secretion and synthesis were studied in the perfused pancreas of young (2-month-old) and older (10-month-old) female Wistar rats. Insulin secretion induced by 16.7 mM glucose showed a triphasic pattern: an early spike and fall (first phase, 0-6 min), followed by a sustained gradual increase (second phase, 7-120 min) and a gradual decreased release thereafter (third phase, 121-360 min) during the perfusion period of 360 min. First and second phase insulin secretion, but not third phase, were lower in older rats than in young rats. Insulin synthesis in old rat pancreas perfused with 16.7 nM glucose for 360 min was much greater than that of young rats. Second phase insulin secretion was restored to comparable levels by 28 mM glucose in older rats. Repeated pulses of 28 mM glucose potentiated subsequent insulin secretion in young rats, but not in older rats. These findings provide further evidence that sensitivity to glucose in pancreatic B cells is altered by aging.     

Comparison of insulin and glucagon pulsatile secretion between the rat and dog pancreas in-vitro

Sustained pulses of insulin and glucagon were obtained from the isolated perfused in vitro rat pancreas. The respective periodicity of hormone release (peak to peak interval) was calculated by the Pulsar computer algorithm as insulin 5.8 +/- 0.3 min and glucagon 6.5 +/- 0.25 min. Because pulsatile insulin secretion is absent in type II diabetics, pulsatile islet hormone secretion could theoretically be regulated directly by intra-islet hormone interactions or indirectly by hormone sensitive nerve feedback, possibly from a venous hormone sensitive receptor system within the pancreas. To test the possible contributions of these systems in pulse regulation, the direction of perfusion was reversed in both rat and dog pancreata to prevent hormone contact with putative venous hormone receptors. The periodicity of hormone secretion was unchanged by reversed perfusion in both species. As vascular perfusion of islet cells is normally B to A to D, these results suggest that neither intra-islet hormone interactions nor intra-pancreatic insulin or glucagon sensitive nerve feedback systems are responsible, on an acute basis, for the regulation of pulsatile insular secretion from the normal pancreas. Insulin regulates net glucagon secretion but does not acutely influence glucagon pulses. The presence of pulses during retrograde perfusion may be the result of the entrainment of the pacemaker-islet system. These observations are consistent with the presence of an independent pacemaker and neural coordinating system within the dog and rat pancreas which may influence both the A- and B-cell.     

Effect of aging on the sensitivity of pancreatic beta-cells to insulin secretagogues in rats.

Glucose-stimulated insulin release from rat pancreas is known to be blunted by aging. In the present study, we examined the effect of aging on insulin release induced by various secretagogues using the isolated perfused pancreas of female rats. Insulin release from the perfused pancreas in response to 16.7 mM glucose in 8-month-old rats (older rats) was much less than that in 2-month-old rats (young rats). The first phase of insulin release after glucose stimulation was attenuated in older rats. The addition of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) potentiated glucose-induced insulin secretion in both groups of rats. However, the second phase of insulin secretion in older rats was lower than that in younger rats. The phorbol ester 12-O-tetradecanoyl phorbol ester (TPA, 200 nM) enhanced both the first and the second phases of insulin release induced by glucose in both groups of rats. The amount of first phase insulin release induced by TPA with glucose in young rats was greater than that in older rats, whereas the second phase of insulin release was similar in both groups of rats. On the other hand, tolbutamide (200 uM) similarly stimulated the first phase of insulin release in both age groups of rat. In addition, the amount of cumulative insulin secretion induced by tolbutamide during the second phase was slightly but significantly greater in older rats than in young controls. Insulin content in the pancreas was significantly greater in older rats than in young rats and increased after the stimulation with TPA and tolbutamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.     

Amylin secretion from the perfused pancreas: dissociation from insulin and abnormal elevation in insulin-resistant diabetic rats

Amylin has been co-secreted from pancreatic islet beta-cells in constant proportion with insulin in some studies. We measured basal and glucose-stimulated amylin and insulin secretion from isolated perfused pancreases of normal and diabetic fatty Zucker rats. Glucose concentrations in the perfusion buffer were increased then decreased in small steps to mimic physiologic changes occurring after a meal. The absolute rate of amylin secretion and the molar ratio of amylin to insulin secreted from diabetic pancreases increased dramatically when infused glucose concentrations fell. Similar changes also occurred in normal pancreases, although the absolute change in amylin secretion was smaller. These studies provide the first evidence that (i) there is a mechanism within the pancreas whereby independent secretion of amylin and insulin can occur; (ii) the molar ratio of amylin to insulin secreted from both normal and diabetic pancreases can vary over a wide range; and (iii) there are important differences in the kinetics of amylin and insulin secretion or their coupling to stimulation by glucose between the isolated pancreases of normal rats and those with genetically transmitted insulin resistance and diabetes mellitus.     

Biotin enhances glucose-stimulated insulin secretion in the isolated perfused pancreas of the rat

The effects of biotin on insulin secretion in pair-fed control rats and biotin-deficient rats were investigated using the method of isolated pancreas perfusion. Isolated pancreas perfusion was performed using 20 mM glucose, 10 mM arginine, and 20 mM glucose plus various concentrations of biotin (20 mM glucose + biotin solution) as stimulants of insulin secretion. The insulin response to 20 mM glucose in biotin-deficient rats was approximately 22% of that seen in control rats. The level of the insulin response to 10 mM arginine was also significantly lower in biotin-deficient rats than in control rats. These results indicate that insulin release from the pancreas was disturbed in biotin-deficient rats. The insulin responses to 20 mM glucose + 1 mM biotin in biotin-deficient and control rats increased to 165% and 185%, respectively, of that to 20 mM glucose. These biotin-induced increases in glucose-stimulated insulin release were evident within the first few minutes of the infusion. An enhancement of the arginine-induced insulin response in control rats was not found when arginine and biotin was administered. These results suggest that biotin may play an important role in the mechanism by which glucose stimulates insulin secretion from the beta cells of the pancreatic islets.     

Dual effects of calcitonin gene-related peptide on insulin secretion in the perfused dog pancreas

Calcitonin gene-related peptide (CGRP) is an intrapancreatic neuropeptide with potential effects on islet hormone secretion. To investigate its pancreatic actions, we examined the effects of a 10 min perfusion of synthetic human CGRP on islet hormone release from the isolated dog pancreas (n = 6) at 5.5 mM glucose. At 0.1 nM, CGRP inhibited insulin secretion (P less than 0.01), which was already observed at 2 min after its introduction. After CGRP perfusion was stopped, a stimulatory off-response occurred. In contrast, at higher dose levels, CGRP stimulated insulin secretion. At 1.0 nM, the stimulation was weak and transient (P less than 0.01), occurring only during the first 3 min of CGRP perfusion. At 10 nM, the stimulation continued for 6 min (P less than 0.05), and at 50 nM, the stimulation was marked and sustained throughout the 10 min perfusion period (P less than 0.01). After the CGRP perfusion at 1.0 and 10 nM, but not at 50 nM, a marked stimulatory off-response in insulin secretion was seen. Glucagon and somatostatin secretion were not significantly affected by CGRP at any of the examined concentrations. We conclude that CGRP exerts dual effects on insulin secretion from the perfused dog pancreas: inhibition at low concentrations and stimulation at high concentrations. This pattern of effect might represent a new regulatory concept for neural influences on islet function: the qualitative response being determined by the amount of neurotransmitter released.     

Characteristics of insulin and glucagon release from the perfused pancreas, intact isolated islets, and dispersed islet cells

There are a variety of different tissue preparations which have been used to study secretion from the endocrine pancreas and there are considerable differences in the results obtained from these. The purpose of this study was to compare several preparations in one laboratory using the same rats, buffers, and radioimmunoassays. The preparations included the isolated perfused rat pancreas, fresh isolated intact islets and dispersed cells, and cultured islets and cells. Insulin release from the perfused rat pancreas at 2.8 mM glucose was so low that it could not be measured, such that over a 90-min time period the amount of insulin released was less than 0.004% of pancreatic insulin content. In contrast, islets in static incubation appear to release 2.0% of their stored content and dispersed cells appear to release 2.6% of their content. Samples were taken at early time points during incubations of fresh islets and dispersed cells, and it was found that almost all of the insulin found at the end of a 90-min incubation period was present during the first 5 min. It is therefore suspected that the true secretory rate of insulin at a low glucose concentration is far lower than had been generally appreciated. Glucagon release patterns showed similarities in that with isolated islets and dispersed cells a disproportionate amount of glucagon release was found during a 0- to 30-min incubation period when compared with the 30- to 90-min period. In summary, artifacts have been identified in some of the in vitro systems used for the study of endocrine pancreatic secretion and these deserve greater recognition.     

Direct neural effect of lateral hypothalamic stimulation on insulin secretion by pancreases of normal and obese rats

Perfusion of CNS intact pancreases with 200 mg/dl glucose with concomitant lateral hypothalamic area (LHA) stimulation significantly inhibited insulin secretion both in normal and obese rats. Sprague-Dawley, Zucker lean (FaFa) and Zucker obese (fafa) rats all responded in a similar manner, suggesting a general effect unrelated to metabolic state. Insulin secretion during mins 25-40 of perfusion was inhibited in Sprague Dawley, lean Zucker and obese Zucker rats by 31%, 42% and 33%, even though LHA stimulation took place from mins 20-25. Thus, the duration of inhibition was greater than the period of LHA stimulation, indicating that this pathway can induce prolonged changes in the responsiveness of the pancreas. The data presented in this study demonstrate that LHA stimulation, in the absence of humoral factors, results in a direct CNS-mediated suppression of insulin secretion which is relatively long lasting. This effect may illustrate a basic control mechanism by the CNS to regulate the endocrine pancreas.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional L-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of L-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (less than 30 s) paired-tracer dilution technique. In the non-diabetic pancreas L-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 microU/ml). Transport of L-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of L-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

Sensory nerve inactivation by resiniferatoxin improves insulin sensitivity in male obese Zucker rats

Recent studies have suggested that sensory nerves may influence insulin secretion and action. The present study investigated the effects of resiniferatoxin (RTX) inactivation of sensory nerves (desensitization) on oral glucose tolerance, insulin secretion and whole body insulin sensitivity in the glucose intolerant, hyperinsulinemic, and insulin-resistant obese Zucker rat. After RTX treatment (0.05 mg/kg RTX sc given at ages 8, 10, and 12 wk), fasting plasma insulin was reduced (P < 0.0005), and oral glucose tolerance was improved (P < 0.005). Pancreas perfusion showed that baseline insulin secretion (7 mM glucose) was lower in RTX-treated rats (P = 0.01). Insulin secretory responsiveness to 20 mM glucose was enhanced in the perfused pancreas of RTX-treated rats (P < 0.005) but unaffected in stimulated, isolated pancreatic islets. At the peak of spontaneous insulin resistance in the obese Zucker rat, insulin sensitivity was substantially improved after RTX treatment, as evidenced by higher glucose infusion rates (GIR) required to maintain euglycemia during a hyperinsulinemic euglycemic (5 mU.kg(-1).min(-1)) clamp (GIR(60-120min): 5.97 +/- 0.62 vs. 11.65 +/- 0.83 mg.kg(-1).min(-1) in RTX-treated rats, P = 0.003). In conclusion, RTX treatment and, hence, sensory nerve desensitization of adult male obese Zucker rats improved oral glucose tolerance by enhancing insulin secretion, and, in particular, by improving insulin sensitivity.     

Islet amyloid polypeptide (IAPP) does not inhibit glucose-stimulated insulin secretion from isolated perfused rat pancreas

Islet amyloid polypeptide (IAPP) is a recently discovered pancreatic islet hormone which is stored with insulin in the secretory vesicles of beta cells. Several lines of evidence suggested that IAPP might affect glucose-stimulated insulin secretion and, therefore, might play a role in the development of impaired insulin secretion which is typical of type 2 diabetes. In this study, the effects of human IAPP (amide) on glucose-stimulated insulin secretion was evaluated in the isolated perfused rat pancreas. IAPP in concentrations from 5 x 10(-12) to 10(-7) M had no significant effects on insulin secretion. IAPP, therefore, does not appear to be a significant modulator of glucose-stimulated insulin secretion at concentrations that are physiologically relevant.     

Combined administration of sodium chloro-2-propionate and insulin on the secretion of glucagon by the pancreas in the diabetic rat

This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional l-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of l-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (< 30 s) paired-tracer dilution technique. In the non-diabetic pancreas l-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 μU/ml). Transport of l-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of l-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

The role of arginine vasopressin in diabetes-associated increase in glucagon secretion

The purpose of this study was to investigate the role of arginine vasopressin (AVP) on glucagon secretion in both normal and diabetic rats. Diabetes was induced by intravenous administration of 50 mg/kg streptozotocin, 14 days before pancreatic perfusion. Diabetic rats were maintained on insulin replacement therapy until approximately 48 h before the perfusion experiments. Both glucagon and AVP were determined in the effluent of the perfused pancreas using RIA. Both normal and diabetic rats had similar basal glucagon secretion. AVP (3-30 pM) increased glucagon secretion from both normal and diabetic rats in a concentration-dependent manner. However, diabetic subjects were more sensitive to AVP administration than normal subjects with regard to glucagon secretion. By comparison of the areas under the curves, AVP-induced glucagon secretion in diabetic rats was approximately 2-fold that of the normal rats. In addition, immunoreactive AVP was detected in the effluent of the perfused pancreas, and diabetic rats had 70% higher AVP concentrations in the pancreatic effluent than normal rats. We conclude that AVP is secreted from the pancreas and diabetic rats can secrete more AVP from the pancreas than normal rats. Consequently, AVP may have a greater impact on glucagon secretion in diabetic subjects than normal ones. AVP might play an important role in the hypersecretion of glucagon in diabetic subjects.     

Leptin perfusion affects insulin secretion but not insulin receptors in rats

The aim of the study was to investigate acute leptin effects on insulin secretion and liver insulin binding in rats in vitro. In the in situ experiments leptin changed the pattern of insulin secretion from the pancreas but did not influence insulin binding in the liver. Perfusion of the pancreas with leptin (1, 10, and 100 nmol/l, respectively) at physiological and supraphysiological levels of glucose (6.66 and 25.0 mmol/l, respectively) did not evoke the inhibition of insulin output observed by the authors previously in the in vivo manners. On the contrary, leptin perfusion resulted in stimulation of insulin secretion. Simultaneously, liver perfusion with leptin for 30 min did not influence specific insulin binding. Analysis of Scatchard's plots indicated no changes in the number of high- and low-affinity insulin receptors and in their affinity to the hormone. Additionally, leptin did not influence general carbohydrate and lipid metabolism of the perfused liver. After the treatment with leptin, the output of glucose, free fatty acids and triglycerides to perfusate and the final contents of glycogen and triglycerides in liver were comparable to values obtained in control animals. The results indicate that some in vitro effects exerted by leptin differ from those observed in vivo.     

Effect of age on the insulin secretory response of perfused rat pancreas to arginine and tolbutamide

In this study we compared the ability of perfused pancreases from 2 1/2 month-old and 12 month-old rats to secrete insulin in response to arginine or tolbutamide. The results indicate that the insulin secretory response to either secretagogue was between 25-85% greater (two-way analysis of variance, P less than .01) by perfused pancreases of older rats. On the other hand, islet cell mass was approximately three-fold greater in the pancreases of the older rats. When this difference in mass of insulin secretory tissue was taken into consideration, it became apparent that insulin secretion per beta cell by perfused pancreases of the older rats was only half that of the younger rats in response to either arginine or tolbutamide (two-way analysis of variance, P less than 0.001). Thus, the decline with age in the ability of the beta cell to secrete insulin, previously noted in response to glucose, involves other insulin secretagogues as well.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Effect of beta-hydroxybutyrate and acetoacetate on insulin and glucagon secretion from perfused rat pancreas

To elucidate the physiological significance of ketone bodies on insulin and glucagon secretion, the direct effects of beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc) infusion on insulin and glucagon release from perfused rat pancreas were investigated. The BOHB or AcAc was administered at concentrations of 10, 1, or 0.1 mM for 30 min at 4.0 ml/min. High-concentration infusions of BOHB and AcAc (10 mM) produced significant increases in insulin release in the presence of 4.4 mM glucose, but low-concentration infusions of BOHB and AcAc (1 and 0.1 mM) caused no significant changes in insulin secretion from perfused rat pancreas. BOHB (10, 1, and 0.1 mM) and AcAc (10 and 1 mM) infusion significantly inhibited glucagon secretion from perfused rat pancreas. These results suggest that physiological concentrations of ketone bodies have no direct effect on insulin release but have a direct inhibitory effect on glucagon secretion from perfused rat pancreas.