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.     

Insulin, glucagon, somatostatin, and thyrotropin-releasing hormone content and secretion by perifused fetal rat islets during culture

In the neonatal period of the rat, pancreatic thyrotropin-releasing hormone content decreases and the sensitivity of insulin secretion to glucose increases. In adult rat islets, TRH inhibits glucose-induced insulin release. The aim of this study was to investigate whether a high TRH content and release can be part of the explanation for the functional immaturity of neonatal islets. For that purpose, we have measured the tissue content and the secretion of immunoreactive insulin, glucagon, somatostatin and TRH in islets from 21.5-day-old rat fetuses cultured for up to one week. Insulin, glucagon and somatostatin content increased during one week of culture in the presence of 11.1 mmol/l glucose. The TRH content decreased during culture, but did not equal adult values. Insulin, glucagon and somatostatin responses to glucose were present after one week of culture. Glucose had no effect on TRH release in cultured fetal islets, but inhibited TRH release in adult islets. We conclude that glucose can stimulate insulin secretion without inhibiting TRH release, but that a decrease in islet TRH content and a sensitization of TRH secretion to glucose may be important in the full maturation of fetal pancreatic islets.     

Increased somatostatin secretion from pancreatic islets of streptozotocin-diabetic rats in response to glucose

Glucose stimulates somatostatin release from perifused pancreatic islets of diabetic rats 42–47 days after the induction of diabetes, and 48 h after withdrawal of insulin replacement therapy.

The glucose effect is augmented by theophylline or glucagon.

Basal somatostatin release and glucose-induced secretion are significantly higher in diabetic islets than in controls.

It is suggested that glucose promotes somatostatin release by directly interacting with islet D cells but not via indirect pathways. Glucose-induced stimulation appears to be modulated by a D-cell adenylate cyclase/phosphodiesterase system. Reasons responsible for increased somatostatin secretion by diabetic islets include reduction in B-cell mass, suggesting that B cells may normally suppress the secretory activity of D cells.     

Paradoxical reduction in pancreatic glucagon with normalization of somatostatin and decrease in insulin in normoglycemic alloxan-diabetic dogs: a putative mechanism of glucagon irresponsiveness to hypoglycemia

In alloxan-diabetic (A-D) dogs, plasma glucagon does not increase when glycemia is decreased by insulin. Therefore, as in insulin-dependent diabetes mellitus (IDDM), increased glucose utilization is not matched by an increase in hepatic production. To explore further the abnormal effects of insulin on regulation of pancreatic glucagon, we studied content and morphology of pancreatic hormones in six normal (N) dogs, five hyperglycemic A-D (HD) dogs, and in four A-D dogs where normoglycemia was maintained by insulin (ND). Morphometric measurement of islets and of immunocytochemically localized A cells (glucagon) were performed by an image analysis system. In normal pancreas, islets of tail and body were bigger in size (tail = 4850 +/- 376 microns 2, body = 3256 +/- 198 microns 2), than the head (2009 +/- 207 microns 2). Glucagon content was 331 +/- 50 micrograms with a mean concentration of 8.5 +/- 0.9 micrograms/g in N dogs, and did not change in HD dogs (422 +/- 34 micrograms, 9.3 +/- 0.4 micrograms/g). With normoglycemia, glucagon content decreased by 5-fold (p less than 0.001). Morphometry indicated that, although A cell area per islet increased (2.7-fold), islet number decreased (70%), explaining the unchanged glucagon content in HD dogs. This decrease in islet number can also justify the dramatic glucagon decrease in ND dogs. Despite the 70% decrease in islet numbers in HD dogs, pancreatic somatostatin increased 3-fold (9.93 +/- 3.3 to 30.6 +/- 7.2 micrograms), indicating that its islet content was augmented 10-fold. Somatostatin content returned to normal with normoglycemia. Pancreatic insulin content in HD dogs was negligible (55 +/- 23 micrograms) when compared with that in N dogs (5500 micrograms) and it did not increase with normoglycemia. The distinct but markedly diminished insulin and proinsulin peaks in HD dogs nearly disappeared in ND dogs. Thus, in alloxan-diabetic HD dogs, 70% of islets are destroyed. A marked increase in glucagon in residual islets can explain the unchanged islet size despite the absence of B cells; however, the percent increase of somatostatin is larger than that of glucagon. Normoglycemia 1) normalizes somatostatin content, 2) further diminishes insulin and proinsulin synthesis presumably due to lack of hyperglycemic stimulus, and 3) paradoxically decreases pancreatic glucagon content 5-fold below its normal level. We hypothesize that with normalization of plasma insulin, glucagon content in each islet normalizes, but because of destruction of most islets, pancreatic glucagon content becomes extremely low.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peptidergic regulation of maturation of the stimulus-secretion coupling in fetal islet beta cells

The stimulus-secretion coupling of the insulin-producing pancreatic islet beta cell is subject to functional maturation during fetal life. We studied the maturation of a glucose-responsive insulin release from fetal rat islets and specifically investigated the impact of peptidergic regulation. To this end, islets were isolated from 21-day-old fetal rats and maintained for 7 days in tissue culture at 3.3 or 11.1 mM glucose and various supplements. In islets cultured in low glucose, acutely raising the ambient glucose concentration to 16.7 mM evoked a modest stimulation of short-term insulin release that was more pronounced in islets maintained in high glucose. Moreover, the insulin content was much higher in islets cultured in high than in low glucose. Culture with growth hormone (GH) markedly amplified both basal and stimulated short-term insulin secretion from islets maintained in either low or high glucose. Additionally, GH significantly elevated the insulin content in islets maintained in low glucose. Transforming growth factor alpha (TGF-alpha) increased basal, but not glucose-stimulated, insulin release and insulin content in islets cultured in low glucose. Gastrin, expressed in islets during fetal life, did not affect basal or glucose-stimulated insulin release, or insulin content, in islets maintained in either low or high glucose. The addition of gastrin to TGF-alpha did not affect the results obtained with the latter peptide. Gastrin-releasing peptide failed to influence basal or glucose-responsive insulin secretory rates, and insulin content, at either glucose concentration during culture. The somatostatin analog Sandostatin (octreotide acetate) neither influenced basal nor stimulated short-term insulin release at any glucose concentration present during culture, whereas the hormone significantly decreased the insulin content of islets cultured in high glucose. Pancreastatin, produced by porcine islet beta and delta cells, failed to influence basal or glucose-responsive insulin secretory rates, and islet insulin content, at either glucose concentration during culture. Culture with gastric inhibitory peptide (GIP) or glucagon-like peptide I (GLP-1), two proposed incretins, did not affect short-term insulin secretion in response to 3.3 or 16.7 mM glucose irrespective of the ambient glucose concentration during culture. To the contrary, GLP-1, but not GIP, increased the content of insulin in islets cultured in low glucose. We conclude that islet beta-cell differentiation and functional maturation of the stimulus-secretion coupling can be modulated in vitro in fetal rat pancreatic tissue by peptidergic regulation and glycemic stimulation. We suggest that GH and TGF-alpha stimulate, while somatostatin, through paracrine interaction, may inhibit, these processes. These effectors may be of regulatory significance in the in vivo development of glucose-sensitive beta cells, and defects in these mechanisms may result in glucose intolerance in adult subjects.     

Neurotensin inhibits glucose but not glucagon-induced insulin and somatostatin release in isolated islets.

The effects of neurotensin on insulin and somatostatin release were examined in isolated pancreatic islets prepared from 3-4 days rats, and maintained in culture for 48 h before use. In the presence of 12 mM glucose, glucagon (50-2,000 ng/ml, i.e. 14-560 nM) caused a 2-fold increase in insulin and somatostatin release. Neurotensin (150 ng/ml, i.e., 100 nM) did not affect the glucagon-stimulated release, nor did it alter the release of either peptide measured at 12 mM glucose in the absence of glucagon. In contrast, neurotension markedly inhibited the release of both insulin and somatostatin that was induced by 23 mM glucose. These observations suggest that neurotensin may modulate the release of insulin and somatostatin evoked by high glucose concentrations, but not that resulting from the action of glucagon on pancreatic islets.     

Islet secretion of immunoreactive thyrotropin-releasing hormone and the 'paracrine-like' effects of its exogenous administration

In order to know more about the secretory pattern of islet TRH in response to glucose and its possible physiological relevance, the release of this hormone as well as that of insulin, glucagon, and somatostatin was radioimmunologically measured. Whereas the secretion of immunoreactive insulin and somatostatin by incubated rat islets is known to be dose-dependently stimulated by glucose, that of glucagon and TRH was inhibited by glucose. Similarly, palmitate dose-dependently inhibited islet glucagon and TRH release. Exogenous TRH exerted strong and dose-dependent effects on islet secretion of the other hormones at the same concentration range at which its hypophysiotropic effects are produced (10(-10) to 10(-8) mol/l). It inhibited the insulin response to glucose and blocked that of glucagon, whereas it enhanced glucose-induced stimulation of somatostatin. These results are suggestive of a possible paracrine inhibitory role of islet TRH, either directly exerted on the secretion of insulin and glucagon or partially mediated through the stimulation of somatostatin release.     

Insulin,glucagon, and somatostatin release from the prediabetic Chinese hamster

Summary Diabetes mellitus in the adult Chinese hamster is characterized by subnormal pancreatic insulin release in vitro, decreased insulin content, and lack of obesity. The cause of the islet B-cell failure is not clear. We measured insulin, glucagon, and somatostatin release from in vitro perfused pancreases of young (mean age 10 and 20 weeks), genetically diabetic animals (subline AC, mean plasma glucose 8.0 and 16.6mmol/l, respectively). Compared to age- and sex-matched normal hamsters (subline M, mean plasma glucose 5.3 mmol/l), the younger diabetic animals had a significantly elevated mean plasma glucose level, but net in vitro pancreatic release of insulin, glucagon, and somatostatin was normal. Pancreatic content of insulin and glucagon was also not significantly different from normal. At age 20 weeks, when the plasma glucose of the diabetic animals was even more elevated, pancreatic content and release of insulin were significantly subnormal, whereas glucagon and somatostatin release were normal, and pancreatic content of glucagon was normal. In a similar group of young (mean age 10 weeks) diabetic animals, non-fasting plasma insulin levels were within the normal range, but the corresponding glucose levels were excessive in most of the animals (13 out of 19). In conclusion, 10-week-old diabetic hamsters show mild hyperglycaemia which cannot be accounted for directly by decreased pancreatic release in response to a glucose plus arginine stimulus in vitro. Decreased ability of the B cell to respond in vivo to hyperglycaemia or peripheral resistance to insulin may contribute to later B-cell failure in the older diabetic hamster.     

Monolayer culture of adult rat pancreatic islets on extracellular matrix: long term maintenance of differentiated B-cell function

Fragmented islets, obtained by mild overdigestion of the adult rat pancreas with collagenase, readily formed monolayer cultures on dishes coated with extracellular matrix derived from bovine corneal endothelial cells. Contaminating fibroblasts were removed by treatment with sodium ethylmercurithiosalicylate. The cultured islets remained functional for over 6 weeks in primary culture and up to 9 weeks in secondary culture, as indicated by their substantial insulin response to an acute glucose stimulus. Insulin secretion from islet monolayers showed biphasic kinetics. The functional competence of the monolayers was further evaluated by studying glucose-stimulated insulin release in the presence of various modulators of B-cell function. The response to physiological agents such as somatostatin, epinephrine, glucagon, and arginine was retained for at least 4 weeks in culture. The sensitivity to inhibition by somatostatin and epinephrine (ID50 = 10 ng/ml) and that to stimulation by glucagon (ED50 = 3 ng/ml) were similar to or better than those for freshly isolated islets. We have thus obtained a fibroblast-free monolayer culture of pancreatic islets from adult rats containing B-cells that retain normal function for long periods. This experimental system appears ideally suited for studying chronic modulations of islet cell function under controlled in vitro conditions, which can allow the stimulation of normal and diabetic environments.     

Long-term exposure of mouse pancreatic islets to oleate or palmitate results in reduced glucose-induced somatostatin and oversecretion of glucagon

AIMS/HYPOTHESIS: Long-term exposure to NEFAs leads to inhibition of glucose-induced insulin secretion. We tested whether the release of somatostatin and glucagon, the two other major islet hormones, is also affected. METHODS: Mouse pancreatic islets were cultured for 72 h at 4.5 or 15 mmol/l glucose with or without 0.5 mmol/l oleate or palmitate. The release of glucagon and somatostatin during subsequent 1 h incubations at 1 or 20 mmol/l glucose as well as the islet content of the two hormones were determined. Lipid-induced changes in islet cell ultrastructure were assessed by electron microscopy. RESULTS: Culture at 15 mmol/l glucose increased islet glucagon content by approximately 50% relative to that observed following culture at 4.5 mmol/l glucose. Inclusion of oleate or palmitate reduced islet glucagon content by 25% (at 4.5 mmol/l glucose) to 50% (at 15 mmol/l glucose). Long-term exposure to the NEFA increased glucagon secretion at 1 mmol/l glucose by 50% (when islets had been cultured at 15 mmol/l glucose) to 100% (with 4.5 mmol/l glucose in the culture medium) and abolished the inhibitory effect of 20 mmol/l glucose on glucagon secretion. Somatostatin content was unaffected by glucose and lipids, but glucose-induced somatostatin secretion was reduced by approximately 50% following long-term exposure to either of the NEFA, regardless of whether the culture medium contained 4.5 or 15 mmol/l glucose. Ultrastructural evidence of lipid deposition was seen in <10% of non-beta cells but in >80% of the beta cells. CONCLUSIONS/INTERPRETATION: Long-term exposure to high glucose and/or NEFA affects the release of somatostatin and glucagon. The effects on glucagon secretion are very pronounced and in type 2 diabetes in vivo may aggravate the hyperglycaemic effects due to lack of insulin.     

Dose-response effect of somatostatin-14 on human basal pancreatic hormones

The effect of increased doses of Somatostatin-14 (3, 10, 30, 100, 300 micrograms/h) on basal release of insulin, pancreatic glucagon and pancreatic polypeptide (PP) was investigated on eight normal volunteers. Levels of Somatostatin-like immunoreactivity (SLI) was determined in order to correlate the increased SLI levels with the degree of islet hormone inhibition (r = 0.9947, p less than 0.01). By increasing the basal levels of SLI by one-third, a significant inhibition (p less than 0.01) of insulin, glucagon, and PP was noted (78.5, 78.6, 75.2%, respectively, on basal levels). The maximal effect was obtained with 300 micrograms/h for insulin, with 30 micrograms/h for glucagon and 100 micrograms/h for PP. In evaluating the relative inhibitory potency of somatostatin, expressed as ED50, the theoretic potency of somatostatin on each peptide had similar values, ranging from 30 to 10 micrograms/h. The present data show that a minimal peripheric increase in SLI is able to regulate basal islet pancreatic hormones.     

Interleukin-6 affects insulin secretion and glucose metabolism of rat pancreatic islets in vitro

Recently it has been postulated that interleukin-1 (IL-1) locally released by infiltrating mononuclear cells may destroy the pancreatic B cells during the development of insulin-dependent diabetes mellitus. Since IL-1 is a potent inducer of interleukin-6 (IL-6) in various cells, it is conceivable that IL-6 is a second mediator of the IL-1 action. In the present study the effects of IL-6 alone or in combination with IL-1 were studied on pancreatic islet function in vitro after tissue culture and compared with the effects observed after exposure to IL-1 only. Rat pancreatic islets were cultured in medium RPMI 1640 + 10% calf serum with or without the addition of human recombinant IL-6 (500-5000 pg/ml) for 48 h. The medium insulin accumulation was increased by 40-50% after culture with 500-2000 pg/ml IL-6, but was similar to the controls at 5000 pg/ml. When islets were cultured for 18 h only, also 5000 pg/ml IL-6 stimulated the medium insulin accumulation. IL-6 did not affect the islet insulin content and the rates of islet (pro)insulin and total protein biosynthesis. It inconsistently decreased the islet DNA content. In short-term experiments after 48-h culture with IL-6, there was a dose-dependent inhibition of the glucose-stimulated insulin release. On the other hand, islets cultured with IL-6 (5000 pg/ml) exhibited an elevated glucose oxidation and oxygen uptake, but a lower ATP content at 16.7 mM glucose and an unaffected glucose utilization and glutamine oxidation compared to the controls. This raises the possibility that IL-6 had induced a condition with an increased energy expenditure, resulting in an enhanced mitochondrial metabolism of glucose. Islets cultured with human recombinant IL-1 beta (25 units/ml) showed a strong inhibition of the insulin accumulation in the culture medium and of glucose-stimulated insulin release and a marked decrease in the islet DNA and insulin content. A combination of IL-1 (25 U/ml) + IL-6 (1000 pg/ml) did not alter the inhibitory action of IL-1 alone. The present findings thus show that IL-6 induces a dissociation between insulin secretion and glucose oxidation in islets in vitro. This has not been observed in islets exposed to IL-1, which suggests that IL-6 does not solely mediate the inhibitory effects of IL-1 on islet function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of exogenous insulin, glucagon, and somatostatin on islet hormone secretion in the perfused chicken pancreas

The effects of exogenous insulin were examined in the isolated perfused chicken pancreas with the duodenum excluded. At low background glucose (50 mg/dl), exogenous insulin infused at a concentration of 20,000 microU/ml elicited clear stimulation of somatostatin secretion while simultaneously inhibiting glucagon release. When the background glucose concentration was elevated to 750 mg/dl, exogenous insulin, had no effect on either somatostatin or glucagon release. When graded doses of exogenous insulin were infused into the chicken pancreas at low background glucose, low concentrations (200 microU/ml) had little effect on somatostatin or glucagon release, but higher concentrations (2000 and 20,000 microU/ml) had clear effects on both somatostatin and glucagon secretion. Glucagon infused at 100 ng/ml stimulated both insulin and somatostatin release. When somatostatin was infused at 25 ng/ml, clear inhibition of glucagon was seen with insulin inhibited to a lesser extent. This study supports the notion of a negative feedback relation between B and D-cells of the pancreatic islets and suggests a paracrine mediation.     

Effects of somatostatin on pancreatic isolated islets and peripheral tissues of rats]

This study was performed in order to evaluate the effects of somatostatin on insulin releasing mechanisms and on glucose uptake in peripheral tissues using isolated pancreatic islets, isolated rat diaphragms and epididymal fat pads. Insulin release by various concentrations of glucose were examined, and it was found that 100 ng/ml of somatostatin significantly inhibited insulin release at the glucose concentration of 200 mg/dl. Somatostatin also significantly inhibted insulin release by the administration of 5microgram/ml of glucagon with 200 mg/dl of glucose concentration and 20 mM of orginine with 200mg/dl of glucose concentrations. But at the glucose concentration of 50mg/dl, no significant inhibition of somatostatin on insulin release was observed even when various concentrations of glucagon or arginine were added. The influence of somatostatin on peripheral tissues was examined in vitro, and no significant change on glucose uptake compared with the control group was shown in either tissues. The results indicated that somatostatin directly inhibited insulin release from rat pancreatic islets but had no effect on glucose uptake in peripheral tissues. The inhibitory effect of somatostatin on insulin release may act through the common mechanism of both glucose and other substances in leading to insulin release.     

In vivo in vitro effects of somatostatin and insulin on glucagon release in a human glucagonoma

Inhibition of pancreatic glucagon secretion has been reported to be mediated by glucose, insulin and somatostatin. As no human pancreatic α-cell lines are available to study in vitro the relative importance of insulin and glucose in the control of pancreatic glucagon release, we investigated a patient presenting with a malignant glucagonoma who underwent surgical resection of the tumour. Functional somatostatin receptors were present as octreotide administration decreased basal glucagon and insulin secretion by 52 and 74%, respectively. The removed tumour was immunohistochemically positive for glucagon, chromogranin A and pancreatic polypeptide but negative for insulin, gastrin and somatostatin. The glucagonoma cells were also isolated and cultured in vitro . Incubation experiments revealed that change from high (10 m m ) to low (1 m m ) glucose concentration was unable to stimulate glucagon secretion. A dose-dependent inhibition of glucagon release by insulin was however, observed at low glucose concentration. These findings demonstrate that insulin could inhibit glucagon secretion in vitro in the absence of elevated glucose concentrations. These data suggest, as observed in vivo in vitro in several animal studies, that glucopenia-induced glucagon secretion in humans is not mediated by a direct effect of low glucose on α-cells but possibly by a reduction of insulin-mediated α-cell suppression and/or an indirect neuronal stimulation of glucagon release.     

Persistent impairment of the insulin response to glucose both in vivo and in vitro after streptozotocin exposure: studies with grafted pancreatic islets and islets maintained in culture

The functional responses of the pancreatic B-cells after cytotoxic damage are still largely unknown. Using in vitro models to clarify this issue, we have recently observed a preferential reduction of glucose-stimulated insulin production and release in mouse pancreatic islets maintained in culture after in vitro exposure to streptozotocin. In order to evaluate the relevance of these findings in vivo, two sets of experiments were performed. First, mouse pancreatic islets were exposed in vitro to 2.2 mmol/l streptozotocin or vehicle alone, cultured for 6 days, and finally grafted under the kidney capsule of normoglycemic nude mice. Two weeks after transplantation there was no difference in the total DNA and insulin content between the two groups of islet grafts, but the insulin concentration, as expressed per microgram DNA, was decreased by 40% in the streptozotocin-treated islets. The insulin release of the grafts, during perfusion of the graft-bearing kidney in situ with 16.7 mmol/l glucose was diminished in the streptozotocin group, whilst perfusion with 16.7 mmol/l glucose plus 5 mmol/l theophylline was able partially to counteract the reduction in insulin release. In the second set of experiments, NMRI mice were injected iv with 160 mg/kg streptozotocin or vehicle alone, and their islets isolated 15 min after the injections. After 6 days in culture, there was no decrease in DNA, glucagon and somatostatin contents, but the insulin content was decreased by 40% in the streptozotocin exposed islets. These islets also showed a 60% decrease in the insulin response to glucose, which was partly counteracted by incubation with 16.7 mmol/l glucose plus 5 mmol/l theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of glucagon or somatostatin on desensitized insulin secretion

In this study we have examined the role of glucagon and somatostatin in regulating glucose-induced desensitization of insulin secretion from rat islets. Measured in batch incubations with medium routinely used to induce three phases of insulin secretion, secreted glucagon levels fell off over 24 h to 20% of peak secretion levels. Although more responsive to various secretagogues, somatostatin secretion also declined to the same degree. Thus, the A- and D-cells desensitize to chronic stimulation as does the B cell. In other experiments, added glucagon (10(-6) M) enhanced glucose (11 X 10(-3) M)-stimulated insulin secretion 34% in the first 3 h; however, islets became insensitive to continuous glucagon by 4 h. The exogenous glucagon did not prevent or delay glucose-induced desensitization of insulin secretion. When glucagon was administered as acute 1-h tests over continuous glucose administration, the degree of B-cell response did not differ in the 1st, 3rd, or 6th hours and appeared to increase in the 21st hour. When islets were perifused continuously with glucose (22 X 10(-3) M) plus 3 X 10(-7) M somatostatin, glucose-induced insulin secretion was suppressed 50% in the first 3 h, but this inhibitory effect disappeared after 6 h. Desensitization was slightly delayed, but not prevented. When somatostatin was administered as acute 1-h tests over continuous glucose perifusion, the B-cell response was relatively constant in the 3rd, 6th, and 21st hours. Results show that 1) islet release of glucagon and somatostatin desensitizes during constant stimulation; and 2) islet release of insulin desensitizes to chronic potentiation or inhibition, respectively, by these hormones. Furthermore, 3) changing B-cell sensitivity to either glucagon or somatostatin cannot account for observed desensitization of insulin secretion with chronic glucose exposure.     

Effect of glibenclamide on pancreatic hormone release from isolated perifused islets of normal and cysteamine-treated rats

The effect of glibenclamide, a sulfonylurea agent, on islet hormone secretion, particularly on glucagon was studied using isolated perifused pancreatic islets of normal and cysteamine-treated rats. In normal rat islets, glibenclamide enhanced both insulin and somatostatin release in normoglycemic (50 mg/dL) and glucopenic (0 mg/dL) states, as well as under the condition of arginine stimulation. In contrast, glibenclamide stimulated glucagon release only transiently, then suppressed it in a sustaining manner in each state. In the cysteamine-treated islets, as expected, somatostatin concentrations in the perifusate remained unchanged during the infusion of arginine and/or glibenclamide. Under this condition, glibenclamide enhanced insulin release to the same extent as seen in normal islets, and again markedly inhibited glucagon release. These observations indicate that in isolated perifused rat pancreatic islets, glibenclamide suppresses glucagon secretion independently of D cell stimulation. It is concluded that glibenclamide may exert its inhibitory effect directly on A cell rather than through paracrine action of concomitant somatostatin release, and that the suppression of glucagon secretion by glibenclamide may, in part, contribute to the antidiabetogenic effect of this compound.     

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.     

Paradoxical glucagon response in perifused islets of the diabetic Chinese hamster

Dynamic insulin and glucagon response to glucose was examined in the perifusion system to investigate the relationship between pancreatic hormone content and the pattern of hormone secretion in diabetic Chinese hamsters of the Asahikawa colony (CHA). Isolated islets of normals and diabetics from the CHA were perifused. When the medium was changed to high glucose (500 mg/dl), a low insulin response and paradoxical glucagon response were seen in diabetics compared with normals. Positive correlations were found between pancreatic insulin and the amount of perifusate insulin, and glucagon content and glucagon release, respectively. It is suggested, accordingly, that pancreatic hormone content is related to the amount of hormone release in CHA. A negative correlation between the amount of perifusate insulin and glucagon release was found. It is suggested, therefore, that an impaired suppression of glucagon release in the diabetic CHA animals could be attributed at least to insulin deficiency. These findings agree with the histological discovery of decreased B-cells and increased A-cells in the diabetic islets. Both decreased B-cells and islet numbers could be the cause of the low insulin response to glucose. Increased numbers of A-cells with hyperfunction resulting from local insulin deficiency could be the cause of the paradoxical glucagon response.