Treatment of isolated, perifused rat islets with exogenous PLA2 in amounts ranging from 1 to 1000 mU/ml caused a dose-dependent increase in the rate of insulin secretion. This effect of PLA2 was rapid and seen in the absence of added exogenous fuel. It differed from glucose-induced insulin release in temporal pattern: high concentrations of PLA2 caused a single phase of secretion, and high levels of glucose caused a biphasic pattern of secretion. Like glucose-induced release, PLA2-induced release was partially dependent on extracellular calcium because D600 caused a significant inhibition of release induced by PLA2 at 5 mU/ml. Concentrations of BW755c and NDGA, inhibitors of both the cyclooxygenase and lipoxygenase or only the lipoxygenase pathways of arachidonic acid metabolism, which completely blocked the insulin secretory response to 10 mM glucose, had no effect on the secretory response to 5 mU/ml of PLA2. These inhibitors also inhibited glucose usage by the islets. Finally, although repeated brief exposure of islets to stimulatory concentrations of glucose lead to a progressive increase in the magnitude of both the first and second phases of insulin secretion, repeated brief exposures to PLA2 lead to a progressive decrease in response to each new exposure. Nonetheless, those islets that had been exposed several times to exogenous PLA2, and no longer displayed a response to a further PLA2 exposure, responded normally to the addition of 10 mM glucose. These results indicate that PLA2 is a potent insulin secretagogue, that it shares some of the characteristics of glucose as a secretagogue, but that in many significant ways differs markedly from glucose in its effects on insulin release from isolated islets.
Isolated rat islets of Langerhans were incubated for 2 h in a [3H]inositol-containing medium supplemented with 7 mM glucose and the sulfonylurea tolbutamide (50–200 μM). After labeling, the ability of these islets to respond during a subsequent perifusion to 20 mM glucose or 15 mM α-ketoisocaproate (KIC) was assessed. The following major observations were made. Prior exposure to tolbutamide inhibited [3H]inositol efflux, inositol phosphate accumulation, and the insulin secretory responses of subsequently perifused islets to 20 mM glucose stimulation. When present during the 2-h labeling period, the calcium channel blocker nitrendipine (500 nM), a compound that abolishes tolbutamide-induced increases in PI hydrolysis, blocked these inhibitory effects of tolbutamide. In addition, the diacylglycerol kinase inhibitor monooleoylglycerol (50 μM) restored the impaired second phase insulin secretory response noted after a 2-h tolbutamide exposure. Prior exposure to tolbutamide (200 μM) also desensitized the islet, in terms of [3H] inositol phosphate accumulation, [3H]inositol efflux, and insulin secretory responses, to 15 mM KIC. The inclusion of monooleoylglycerol during the stimulatory period with KIC restored second phase insulin secretion. The results support the conclusion that chronic tolbutamide-induced increases in PI hydrolysis render the β-cell insensitive to a subsequent 20-mM glucose or 15-mM KIC stimulus. Blocking tolbutamide-induced increases in PI hydrolysis during the labeling period eliminates the adverse effects of the sulfonylurea. The ineffectiveness of glucose and KIC to maintain insulin secretory responses from prior tolbutamide-exposed islets appears to be the result of the inability of these agonists to appropriately activate PI hydrolysis.
Prior, short-term exposure of isolated perifused islets to cholecystokinin (CCK8S) sensitizes them to subsequent glucose stimulation. This sensitization effect develops quickly and persists long after the removal of CCK8S from the perifusion medium. Continued binding of CCK8S to its receptor on the β-cell and the increase in glucose metabolism noted with glucose stimulation are essential for the full expression of this response. This sensitization process may play an integral role in the postulated incretin effect of the peptide.
The C-terminal eight-amino acid derivative of CCK, sulfated on the tyrosine residue (CCK8S), stimulated a dose-dependent biphasic pattern of insulin secretion from isolated perifused islets in the presence of 7 mm glucose. It was without any effect if glucose were absent from the medium or maintained at 4 mm. The response to CCK8S was readily reversible and dependent on the presence of extracellular calcium. While CCK8S did not increase glucose usage rates above those noted with 7 mm glucose alone, inclusion of the metabolic inhibitor 2-deoxyglucose lowered glucose usage rates to values obtained with 3–5 mm glucose and abolished the influence of CCK8S on insulin output. Removal of the metabolic inhibitor restored the secretory response. N-Acetylglucosamine (15 mm) or glyceraldehyde (2.5 mm) substituted for glucose and permitted CCK8S to evoke secretion. The nonsulfated eight-amino acid derivative of CCK, CCK8, provoked insulin secretion in the presence of 7 mm glucose, but only at 10–100 times greater levels than CCK8S. CCK4 (1 μm) did not influence insulin output in the presence of 7 mm glucose. On an equimolar basis, CCK8S was significantly more effective than gastric inhibiting polypeptide in augmenting insulin output. The results support a role for CCK8S in the regulation of insulin levels in vivo. (Endocrinology119: 616–621, 1986)
The impact of modest but prolonged (3 h) exposure to high physiological glucose concentrations and hyperkalemia on the insulin secretory and phospholipase C (PLC) responses of rat pancreatic islets was determined. In acute studies, glucose (5-20 mM) caused a dose-dependent increase in secretion with maximal release rates 25-fold above basal secretion. When measured after 3 h of exposure to 5-10 mM glucose, subsequent stimulation of islets with 10-20 mM glucose during a dynamic perifusion resulted in dose-dependent decrements in secretion and PLC activation. Acute hyperkalemia (15-30 mM) stimulated calcium-dependent increases in both insulin secretion and PLC activation; however, prolonged hyperkalemia resulted in a biochemical and secretory lesion similar to that induced by sustained modest hyperglycemia. Glucose- (8 mM) desensitized islets retained significant sensitivity to stimulation by either carbachol or glucagon-like peptide-1. These findings emphasize the vulnerability of the beta-cell to even moderate sustained hyperglycemia and provide a biochemical rationale for achieving tight glucose control in diabetic patients. They also suggest that PLC activation plays a critically important role in the physiological regulation of glucose-induced secretion and in the desensitization of release that follows chronic hyperglycemia or hyperkalemia.
Isolated rat islets were incubated with myo-[2-3H]inositol to label their phosphoinositides (PI). Labeling was carried out in the presence of various glucose levels (2.75-10 mM) with or without human recombinant interleukin-1α (IL-1). After the labeling period, insulin release, [3H]inositol efflux, and the accumulation of labeled inositol phosphates in perfused islets were assessed under various conditions. The following major observations were made. 1) In islets labeled for 2 h with [3H] inositol in the presence of 2.75 mM glucose, subsequent perifusion with 5.0 nM IL-1 increased insulin output, [3H] inositol efflux, and [3H] inositol phosphate accumulation in the simultaneous presence of 7 mM, but not 2.75 mM, glucose. 2) Mannoheptulose, a competitive inhibitor of islet glucokinase, blocked the stimulatory effects of IL-1 noted in the presence of 7 mM glucose. In other experiments, the conditions used during the 2-h labeling period with myo-[2-3H]inositol were varied. The following major observations were made in islets subsequently stimulated during the perifusion with 20 mM glucose. 3) Islets labeled with [3H]inositol in the presence of 2.75 mM glucose with or without 5.0 nM IL-1 responded with similar increases in PI hydrolysis and insulin output. 4) Compared to that with 2.75 mM glucose alone, labeling in the presence of 7 mM glucose alone was without any adverse effect on the subsequent PI and insulin responses of perifused islets to 20 mM glucose. 5) Labeling in the presence of 7 mM glucose plus 5.0 nM IL-1 resulted in a significant reduction in the subsequent PI and insulin responses. 6) These inhibitory effects of IL-1 were abolished if mannoheptulose was included during the 2-h incubation with 7 mM glucose plus 5.0 nM IL-1. 7) The diacylglycerol kinase inhibitor 1-monooleoylglycerol (100 μM) significantly restored insulin output after IL-1 exposure (with 7 mM glucose). 8) Similar to the results obtained with 7 mM glucose plus IL-1, incubation of islets with 8-10 mM glucose alone produced dose-dependent impairments of [3H] inositol efflux patterns and inositol phosphate accumulation. Insulin secretion was also impaired. These results demonstrate that IL-1 has glucose-dependent stimulatory and inhibitory effects on β-cell function. Both effects appear to involve alterations in islet PI hydrolysis.(Endocrinology124: 2350-2357, 1989)
Isolated perifused rat islets were stimulated with glucose, exogenous insulin, or carbachol. C-peptide and, where possible, insulin secretory rates were measured. Glucose (8–10 mm) induced dose-dependent and kinetically similar patterns of C-peptide and insulin secretion. The addition of 100 nm bovine insulin had no effect on C-peptide release in response to 8–10 mm glucose stimulation. The addition of 100 nm bovine insulin or 500 nm human insulin together with 3 mm glucose had no stimulatory effect on C-peptide secretion rates from perifused rat islets. Stimulation with carbachol plus 7 mm glucose enhanced both C-peptide and insulin secretion, and the further addition of 100 nm bovine insulin had no inhibitory effect on C-peptide secretory rates under this condition. Perifusion studies using pharmacologic inhibitors (genistein and wortmannin) of the kinases thought to be involved in insulin signaling potentiated 10 mm glucose-induced secretion. The results support the following conclusions. 1) C-peptide release rates accurately reflect insulin secretion rates from collagenase-isolated, perifused rat islets. 2) Exogenously added bovine insulin exerts no inhibitory effect on release to several agonists including glucose. 3) In the presence of 3 mm glucose, exogenously added bovine or human insulin do not stimulate endogenous insulin secretion. Isolated perifused rat islets were stimulated with glucose, exogenous insulin, or carbachol. C-peptide and, where possible, insulin secretory rates were measured. Glucose (8–10 mm) induced dose-dependent and kinetically similar patterns of C-peptide and insulin secretion. The addition of 100 nm bovine insulin had no effect on C-peptide release in response to 8–10 mm glucose stimulation. The addition of 100 nm bovine insulin or 500 nm human insulin together with 3 mm glucose had no stimulatory effect on C-peptide secretion rates from perifused rat islets. Stimulation with carbachol plus 7 mm glucose enhanced both C-peptide and insulin secretion, and the further addition of 100 nm bovine insulin had no inhibitory effect on C-peptide secretory rates under this condition. Perifusion studies using pharmacologic inhibitors (genistein and wortmannin) of the kinases thought to be involved in insulin signaling potentiated 10 mm glucose-induced secretion. The results support the following conclusions. 1) C-peptide release rates accurately reflect insulin secretion rates from collagenase-isolated, perifused rat islets. 2) Exogenously added bovine insulin exerts no inhibitory effect on release to several agonists including glucose. 3) In the presence of 3 mm glucose, exogenously added bovine or human insulin do not stimulate endogenous insulin secretion. 5-hydroxytryptamine connecting-peptide phorbol 12-myristate 13-acetate Insulin secretion from the pancreatic β-cell is tightly regulated by stimulatory signals generated during the intracellular metabolism of glucose and by neurohumoral agonists operative at the cell membrane (1Henquin J.C. Bozem M. Schmeer W. Nenquin M. Biochem. J. 1987; 246: 393-399Crossref PubMed Scopus (30) Google Scholar, 2Malaisse W.J. Sener A. Herchuelz A. Hutton J.C. Metabolism. 1979; 28: 373-386Abstract Full Text PDF PubMed Scopus (266) Google Scholar, 3Zawalich W.S. Zawalich K.C. Am. J. Physiol. 1996; 271: E409-E416Crossref PubMed Google Scholar, 4Ashcroft S.J. Diabetologia. 1980; 18: 5-15Crossref PubMed Scopus (215) Google Scholar, 5Best L. Malaisse W.J. Endocrinology. 1984; 115: 1820-1831Crossref Scopus (4) Google Scholar). Most recently an additional layer of complexity has been added by reports suggesting that insulin exerts an autocrine stimulatory effect on insulin secretion from the β-cell (6Aspinwall C.A. Lakey J.R.T. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Aspinwall C.A. Qian W.-J. Roper M.G. Kulkarni R.N. Kahn C.R. Kennedy R.T. J. Biol. Chem. 2000; 275: 22331-22338Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). This concept was based primarily on amperometric measurements using β-cells preincubated in 5-hydroxytryptamine (5HT).1 Because 5HT exposure exerts inhibitory effects on insulin release (8Lernmark A. Horm. Metab. Res. 1971; 3: 305-309Crossref PubMed Scopus (50) Google Scholar, 9Gagliardino J.J. Nierle C. Pfeiffer E.F. Diabetologia. 1974; 10: 411-414Crossref PubMed Scopus (42) Google Scholar, 10Zawalich W.S. Tesz G.J. Zawalich K.C. J. Biol. Chem. 2001; 276: 37120-37123Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), the precise physiologic significance of findings made with 5HT-preloaded β-cells is unclear. Moreover, previous studies exploring the potential role of insulin on stimulated secretion showed no effect (11Schatz H. Pfeiffer E.F. J. Endocrinol. 1977; 74: 243-249Crossref PubMed Scopus (19) Google Scholar) or supported the concept that insulin exerts a negative, not positive, feedback on its own secretion (12Iversen J. Miles D.W. Diabetes. 1971; 20: 1-9Crossref PubMed Scopus (155) Google Scholar, 13Ammon H.P. Reiber C. Verspohl E.J. J. Endocrinol. 1991; 128: 27-34Crossref PubMed Scopus (34) Google Scholar, 14Loreti L. Dunbar J.C. Chen S. Foà P.P. Diabetologia. 1974; 10: 309-315PubMed Google Scholar, 15Sodoyez J.-C. Sodoyez-Goffaux F. Foà P.P. Proc. Soc. Exp. Biol. Med. 1969; 130: 568-571Crossref PubMed Scopus (55) Google Scholar). There are at least three major issues that must be addressed in attempting to establish the impact of exogenously added insulin on endogenous insulin secretory rates. The first is technical; it is difficult to accurately measure endogenous insulin release rates in the presence of exogenous insulin. The second relates to concentration of insulin necessary to establish an effect of exogenously added hormone on the β-cell. Considering that the β-cell continually releases insulin into a small volume of interstitial fluid, the level of insulin bathing the β-cell may be quite high. For example, calculations based on islet cell volume, the insulin diffusion constant and insulin secretory rates, suggest that these levels may exceed 100 nm during glucose stimulation (16Zawalich W.S. Karl R.C. Ferrendelli J.A. Matschinsky F.M. Diabetologia. 1975; 11: 231-235Crossref PubMed Scopus (59) Google Scholar). Even at rest, levels of insulin far in excess of circulating plasma levels must exist at the interface of the β-cell membrane and the interstitium. Third, the contribution of constitutive insulin release to the secretory responses observed has to be considered. The necessary insulin signaling components identified in insulin-sensitive tissues including insulin receptors, insulin receptor substrate proteins, and phosphatidylinositol 3-kinase have been found in β-cells (17Verspohl E.J. Ammon H.P. J. Clin. Invest. 1980; 65: 1230-1237Crossref PubMed Scopus (78) Google Scholar, 18Xu G.G. Rothenberg P.L. Diabetes. 1998; 47: 1243-1252PubMed Google Scholar, 19Harbeck M.C. Louie D.C. Howland J. Wolf B.A. Rothenberg P.L. Diabetes. 1996; 45: 711-717Crossref PubMed Google Scholar). Because there is constitutive secretion of insulin, a tonic level of insulin signaling in these cells may influence acute stimulatory responses and thus obscure any effect of exogenously added insulin. These three issues, in addition to the use of different species and disparate methodological approaches, may account in part for the discrepancies regarding the impact of insulin on its own secretion (11Schatz H. Pfeiffer E.F. J. Endocrinol. 1977; 74: 243-249Crossref PubMed Scopus (19) Google Scholar, 12Iversen J. Miles D.W. Diabetes. 1971; 20: 1-9Crossref PubMed Scopus (155) Google Scholar, 14Loreti L. Dunbar J.C. Chen S. Foà P.P. Diabetologia. 1974; 10: 309-315PubMed Google Scholar, 20Ammon H.P.T. Verspohl E. Endocrinology. 1976; 99: 1469-1476Crossref PubMed Scopus (40) Google Scholar, 21Rappaport A.M. Ohira S. Coddling J.A. Empey G. Kalnins A. Lin B.J. Haist R.E. Endocrinology. 1972; 91: 168-176Crossref PubMed Scopus (31) Google Scholar). To circumvent the first problem, and to establish the effect of exogenously added insulin on its own secretion, we have measured connecting (C)-peptide secretion rates in response to a variety of agonists from isolated perifused rat islets. Finally, the impact of insulin signaling on 10 mm glucose-induced secretion was explored using several compounds known to antagonize the kinases activated by insulin. The detailed methodologies employed to assess insulin output from collagenase-isolated rat islets have been described previously (22Zawalich W.S. Zawalich K.C. J. Endocrinol. 2000; 166: 111-120Crossref PubMed Scopus (14) Google Scholar, 23Zawalich W.S. Zawalich K.C. Endocrinology. 2000; 141: 3287-3295Crossref PubMed Scopus (60) Google Scholar). Male Sprague-Dawley rats (350–475g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All animals were treated in a manner that complied with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH publication no. 85–23, revised 1985). The animals were fed ad libitum. After intraperitoneal Nembutal (pentobarbital sodium, 50 mg/kg; Abbott Laboratories, Abbott Park, IL)-induced anesthesia, islets were isolated by collagenase digestion and handpicked, using a glass loop pipette, under a stereomicroscope. They were free of exocrine contamination. Groups of 14–18 freshly isolated islets were perifused with Krebs-Ringer bicarbonate at a flow rate of 1 ml/min for 30 or 45 min with 3 mm glucose to establish basal and stable insulin and C-peptide secretory rates. After this stabilization period they were then perifused with the appropriate agonist or agonist combinations as indicated in the figure legends and under "Results." Perifusate solutions were gassed with 95% O2/5% CO2 and maintained at 37 °C. Insulin (24Albano J.D.M. Ekins R.P. Maritz G. Turner R.C. Acta Endocrinol. 1972; 70: 487-509Crossref PubMed Scopus (544) Google Scholar) and rat C-peptide released into the medium were measured by radioimmunoassay; in the case of C-peptide measurements the protocol provided by the vendor was followed rigorously. C-peptide and, when possible, insulin release rates were measured in the same perifusate samples. Groups of 14–18 islets were cultured as described previously (25Zawalich W.S. Bonnet-Eymard M. Zawalich K.C. Yaney G.C. Am. J. Physiol. 1998; 274: C1388-C1396Crossref PubMed Google Scholar, 26Zawalich W.S. Zawalich K.C. Kelley G.G. Eur. J. Physiol. 1996; 432: 589-596Crossref PubMed Scopus (14) Google Scholar) for 18 h in CMRL-1066 containing 5.5 mm glucose and supplemented with penicillin (50 units/ml), streptomycin (50 μg/ml) and glutamine to achieve a final concentration of 2 mm. After this, the islets were perifused as described above. Hanks' solution was used for the islet isolation. The Krebs-Ringer bicarbonate perifusion medium consisted of 115 mm NaCl, 5 mm KCl, 2.2 mmCaCl2, 1 mm MgCl2, 24 mm NaHCO3, and 0.17 g/dl bovine serum albumin. The 125I-labeled insulin used for the insulin assay was purchased from PerkinElmer Life Sciences. Bovine serum albumin (RIA grade), glucose, carbachol, wortmannin, genistein, glutamine, phorbol 12-myristate 13-acetate (PMA), bovine insulin (Cat. no. I5500), human recombinant insulin, and the salts used to make the Hanks' solution and perifusion medium were purchased from Sigma. Genistein and wortmannin were dissolved in Me2SO, and equivalent amounts of diluent were used in control studies. Rat insulin standard (lot 615-ZS-157) was the generous gift of Dr. Gerald Gold, Eli Lilly (Indianapolis, IN). CMRL-1066 and the antibiotics employed for the culture studies were purchased from Invitrogen. Collagenase (Type P) was obtained from Roche Molecular Biochemicals. Rat C-peptide was measured using kits purchased from Linco Research, St. Charles, MO. Statistical significance was determined using the Student's t test for unpaired data or analysis of variance in conjunction with the Newman-Keuls test for unpaired data. Ap value ≤0.05 was taken as significant. Values presented in the figure legends and under "Results" represent means ± S.E. of at least three observations. In the initial series of experiments isolated perifused rat islets were stimulated with 10 mm glucose. Insulin and C-peptide secretory rates were measured in the same perifusate samples. As shown in Fig. 1, islet responses to 10 mm glucose (in terms of peptide secretory rates) were kinetically and quantitatively very similar. Both the C-peptide and insulin responses were biphasic in nature and, when compared with prestimulatory release rates measured in the presence of 3 mm glucose, the addition of 10 mm glucose resulted in ∼15-fold increments in the output of both peptides. Similar, although amplified, C-peptide and insulin responses were obtained when the perifusate glucose level was increased to 15 mm (results not shown). In the next experiment, islets were stimulated with 8 mm glucose, a level of the hexose that resulted in a modest 4–5-fold increase in insulin secretory rates (Fig.2). For example, in the presence of 3 mm glucose islets released 30–35 pg of insulin/islet/min. After 40 min of stimulation with 8 mm glucose, the secretory rate increased to 143 ± 17 (n = 5) pg/islet/min. A similar response in terms of C-peptide secretion was also observed. Prestimulatory secretion rates (5–6 pg/islet/min) increased to 31 ± 5 pg/islet/min. To assess the potential impact of exogenous insulin on glucose-induced secretion, additional groups of islets were stimulated with 8 mm glucose plus 100 nm bovine insulin. The addition of exogenous insulin precluded the measurement of endogenous insulin secretion. However, using C-peptide secretion rates as an index of the endogenous insulin secretory response, no effect of added bovine insulin on C-peptide secretory rates was seen. 40 min after the onset of 8 mmglucose stimulation, C-peptide secretory rates averaged 32 ± 3 pg/islet/min in the presence of 100 nm bovine insulin. Although not shown, we could detect no inhibitory effect of 100 nm bovine insulin on C-peptide responses to 7 or 10 mm glucose, levels of the hexose that increase endogenous insulin secretion about 2- or 10–15-fold, respectively, above those observed with 3 mm glucose. It has been reported that 100 nm exogenous bovine insulin in the presence of 3 mm glucose increases insulin secretion from β-cells, a response monitored not by the release of insulin but by 5HT release from 5HT-prelabeled β-cells (6Aspinwall C.A. Lakey J.R.T. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). We directly tested this concept in perifused islets, which retain a level of physiologic sensitivity to glucose stimulation comparable with that observed with the perfused rat pancreas preparation (27Gerich J.E. Charles M.A. Grodsky G.M. J. Clin. Invest. 1974; 54: 833-841Crossref PubMed Scopus (190) Google Scholar, 28Curry D.L. Endocrinology. 1986; 118: 170-175Crossref PubMed Scopus (59) Google Scholar, 29Grill V. Adamson U. Cerasi E. J. Clin. Invest. 1978; 61: 1034-1043Crossref PubMed Scopus (123) Google Scholar). After a 45-min stabilization period in the presence of 3 mm glucose, islets were perifused with 100 nm bovine insulin or 500 nm human insulin in the continued presence of 3 mm glucose. There was no stimulatory effect of either insulin preparation on C-peptide secretion rates (Fig.3). Throughout the perifusion, basal and stable rates of C-peptide secretion were noted. We considered that the process of collagenase isolating islets may disrupt β-cell membrane integrity and that the lack of any effect of insulin on C-peptide secretion may be a consequence of this potential adverse effect of the isolation procedure. To address this concern, two sets of additional experiments were conducted. In the first set of experiments (Fig. 4) islets were stimulated with 7 mm glucose plus 5 μm carbachol, a cholinergic agonist that activates phospholipase C via a membrane muscarinic receptor (30Loubatieres-Mariani M.M. Chapal J. Alric R. Loubatieres A. Diabetologia. 1973; 9: 439-476Crossref PubMed Scopus (33) Google Scholar, 31Zawalich W.S. Zawalich K.C. Rasmussen H. Endocrinology. 1989; 125: 2400-2406Crossref PubMed Scopus (94) Google Scholar, 32Kelley G.G. Zawalich K.C. Zawalich W.S. Am. J. Physiol. 1995; 269: E575-E582PubMed Google Scholar). Islets stimulated with 7 mm glucose plus 5 μm carbachol responded with an approximate 4-fold increase in both insulin and C-peptide secretion rates. The response to this agonist combination was ∼2.5-fold greater than the response to 7 mm glucose alone (results not shown). For example, 20, 30 or 40 min after the onset of stimulation with 7 mm glucose alone insulin release rates averaged 53 ± 9, 57 ± 10, or 58 ± 12 pg/islet/min (n = 8), respectively. The addition of carbachol increased the values at these times to 109 ± 29, 125 ± 18, or 126 ± 19 pg/islet/min (n = 5), respectively. The further addition of 100 nm insulin had no effect on C-peptide secretion to glucose plus carbachol stimulation (Fig. 4). In the next set of experiments, islets were first cultured for 18 h to allow more complete recovery of any potential adverse impact of the collagenase isolation procedure. Islets were then perifused with 3 mm glucose to establish basal C-peptide secretion rates prior to stimulation with 500 nm human insulin. Similar to the observations made with freshly isolated islets, the addition of human insulin was without any stimulatory effect on C-peptide secretion rates. Because culturing impairs islet sensitivity to glucose stimulation alone (25Zawalich W.S. Bonnet-Eymard M. Zawalich K.C. Yaney G.C. Am. J. Physiol. 1998; 274: C1388-C1396Crossref PubMed Google Scholar, 33Malaisse-Lagae F. Sener A. Malaisse W.J. Acta Diabetol. Lat. 1987; 24: 17-25Crossref PubMed Scopus (15) Google Scholar, 34Metz S.A. Diabetes. 1988; 37: 3-7Crossref PubMed Google Scholar), these islets were then stimulated with the combination of 20 mm glucose plus 500 nmPMA (Fig. 5). This agonist combination resulted in an ∼25-fold increase in C-peptide secretion rates from both control and prior insulin-stimulated islets. In an attempt to disrupt the contribution of constitutive insulin signaling on glucose-stimulated β-cell responses of our perifused islet preparation, additional studies were conducted with the tyrosine kinase inhibitor genistein (10 μm) and the phosphatidylinositol 3-kinase inhibitor wortmannin (50 nm). Both types of kinases are established participants in insulin signaling (35Virkamäki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (726) Google Scholar, 36Pessin E.J. Saltiel R.A. J. Clin. Invest. 2000; 106: 165-169Crossref PubMed Scopus (685) Google Scholar). As shown in Fig. 6, both inhibitors significantly potentiated 10 mm glucose-induced secretion. Several important considerations have emerged in our attempt to establish the precise impact of exogenously added insulin on endogenous insulin secretory rates. First is the realization that the levels of insulin bathing the β-cell, even under basal nonstimulatory conditions, must far exceed those normally bathing other tissues. This has to do with islet cell volume, limited interstitial space distribution, and constitutive rates of hormone output (16Zawalich W.S. Karl R.C. Ferrendelli J.A. Matschinsky F.M. Diabetologia. 1975; 11: 231-235Crossref PubMed Scopus (59) Google Scholar). Any attempt to establish either an inhibitory or excitatory effect of exogenous insulin on endogenous secretion must contend with this. For example, if insulin does indeed inhibit its own release as suggested in other reports (12Iversen J. Miles D.W. Diabetes. 1971; 20: 1-9Crossref PubMed Scopus (155) Google Scholar, 14Loreti L. Dunbar J.C. Chen S. Foà P.P. Diabetologia. 1974; 10: 309-315PubMed Google Scholar, 20Ammon H.P.T. Verspohl E. Endocrinology. 1976; 99: 1469-1476Crossref PubMed Scopus (40) Google Scholar), an inhibitory effect of added exogenous insulin on the secretory response to glucose might not be observed if saturating (with regards to insulin signaling) endogenous hormone concentrations already exist in the interstitial space and tonically influence secretion. In addition, it is technically difficult to measure endogenous insulin release rates in the presence of high levels of exogenously added hormone. In an attempt to circumvent these issues two approaches were utilized. First, we used C-peptide secretion rates as a surrogate marker for endogenous insulin secretion (37Steiner D.F. Diabetes. 1977; 26: 322-340Crossref PubMed Scopus (121) Google Scholar). Second, constitutive insulin signaling in the β-cell was disrupted using inhibitors known to interfere with the kinases involved in the insulin signaling cascade. Several salient points emerge from the present studies. First, C-peptide secretion rates accurately reflect both quantitatively and qualitatively the kinetics and amplitude of insulin secretion. Like insulin output in response to 10 mm glucose, it is biphasic in nature. Most importantly in terms of sensitivity, small increments in glucose-induced insulin release evoked by 7–8 mmglucose evoke small, easily measurable increments in C-peptide release. Third, in terms of inhibition of insulin secretion, we could not detect any inhibitory effect of exogenously added bovine insulin on glucose-induced insulin secretion. Fourth, neither bovine nor human insulin at levels of 100–500 nm had any discernible stimulatory effect on C-peptide secretion rates and, based on the tight coupling between C-peptide and insulin secretion demonstrated in these studies, exerted no stimulatory effect on insulin release as well. Fifth, addition of a membrane-active agonist, carbachol, evoked substantial insulin and C-peptide responses indicating that at least for this agonist the functional integrity of its membrane receptor has been maintained during the isolation procedures. However, because the insulin receptor may be more vulnerable to collagenase than the muscarinic cholinergic receptor, islets were allowed to recover from the isolation procedure during an 18-h culturing period. These islets still failed to respond to exogenously added insulin by increasing C-peptide secretion rates. We were unable to document any inhibitory effect of exogenously added insulin on its own secretion using C-peptide as the surrogate marker for insulin release. Does this failure exclude an inhibitory effect of endogenously released insulin on the insulin release process? Does constitutive insulin secretion tonically influence stimulated secretion? We attempted to address this issue by using two different inhibitors known to disrupt the kinases involved in insulin signaling. Our findings with isolated perifused rat islets confirm the observations made using mouse islets (38Jonas J.C. Plant T.D. Gilon P. Detimary P. Nenquin M. Henquin J.C. Br. J. Pharmacol. 1995; 114: 872-880Crossref PubMed Scopus (78) Google Scholar) or neonatal cultured rat islets (39Sorenson R.L. Brelje T.C. Roth C. Endocrinology. 1994; 134: 1975-1978Crossref PubMed Scopus (75) Google Scholar): genistein is a potentiator of glucose-induced secretion. Furthermore, our findings also confirm previous studies in both rat (23Zawalich W.S. Zawalich K.C. Endocrinology. 2000; 141: 3287-3295Crossref PubMed Scopus (60) Google Scholar, 40Nunoi K. Yasuda K. Tanaka H. Kubota A. Okamoto Y. Adachi T. Shihara N. Uno M., Xu, L.M. Kagimoto S. Seino Y. Yamada Y. Tsuda K. Biochem. Biophys. Res. Commun. 2000; 270: 798-805Crossref PubMed Scopus (29) Google Scholar) and mouse (41Eto K. Yamashita T. Tsubamoto Y. Terauchi Y. Hirose K. Kubota N. Yamahita S. Taka J. Satoh S. Sekihara H. Tobe K. Iino M. Noda M. Kimura S. Kadowaki T. Diabetes. 2002; 51: 87-97Crossref PubMed Scopus (62) Google Scholar) islets as well as in MIN cells (42Hagiwara S. Sakurai T. Tashiro F. Hashimoto Y. Matsuda Y. Nonomura Y. Miyazaki J. Biochem. Biophys. Res. Commun. 1995; 214: 51-59Crossref PubMed Scopus (42) Google Scholar); wortmannin is a potentiator of glucose-induced secretion. Although it is known that these compounds may interfere with kinases not involved with insulin signaling and that these effects may complicate the interpretation of the data, the main point to be made is that these compounds significantly potentiated glucose-induced release. Whether or not these inhibitors act as we assume or influence other pathways (43Leahy J.L. Vandekerkhove K.M. Endocrinology. 1990; 126: 1593-1598Crossref PubMed Scopus (118) Google Scholar) still makes these observations noteworthy and the pursuit of the precise underlying mechanisms involved an important goal for future studies. The findings also suggest that constitutive secretion may negatively affect stimulated secretion and that disruption of insulin signaling in the β-cell improves glucose-stimulated insulin secretion. Our working hypothesis is that constitutive insulin release from islets, even under nonstimulatory conditions, exerts a tonic inhibitory effect on stimulated secretion. The addition of exogenous insulin to a system already tonically inhibited has little further inhibitory effect as demonstrated here, but its disruption using several inhibitors markedly improves the secretory performance of the β-cell. This hypothesis is consistent with findings made using knockout mice where insulin signaling has been disrupted and by pharmacologic inhibition of kinases thought to be involved in insulin signaling. For example, islets deficient in the insulin receptor substrate-2 protein or the p85α regulatory subunit of phosphatidylinositol 3-kinase hyper-respond to glucose stimulation (41Eto K. Yamashita T. Tsubamoto Y. Terauchi Y. Hirose K. Kubota N. Yamahita S. Taka J. Satoh S. Sekihara H. Tobe K. Iino M. Noda M. Kimura S. Kadowaki T. Diabetes. 2002; 51: 87-97Crossref PubMed Scopus (62) Google Scholar, 44Kubota N. Tobe K. Terauchi Y. Eto K. Yamauchi T. Suzuki R. Tsubamoto Y. Komeda K. Nakano R. Miki H. Satoh S. Sekihara H. Sciacchitano S. Lesniak M. Aizawa S. Nagai R. Kimura S. Akanuma Y. Taylor S.I. Kadowaki T. Diabetes. 2000; 49: 1880-1889Crossref PubMed Scopus (432) Google Scholar). Of particular physiologic significance, perhaps, is the observation that basal secretion is not augmented from these β-cells. The impact of these genetic manipulations becomes manifest only in studies in which glucose stimulates release. This finding suggests that only under conditions in which insulin release is stimulated by glucose does insulin signaling negatively impact release. This situation is analogous to the α2 adrenergic effects in the central nervous system where presynaptically released catecholamines feed back in a negative fashion on the cell that released it in order to restrain the secretion of additional neurotransmitter (45Langer S.Z. Pharmacol. Rev. 1981; 32: 337-362Google Scholar). Our studies, as well as those of others, using pharmacologic inhibition of the kinase involved with insulin signaling are also consistent with the hypothesis that constitutive insulin signaling acts to restrain stimulated insulin secretion. For example, neither wortmannin nor genistein potentiates release from islets in the presence of low nonstimulatory glucose (23Zawalich W.S. Zawalich K.C. Endocrinology. 2000; 141: 3287-3295Crossref PubMed Scopus (60) Google Scholar, 38Jonas J.C. Plant T.D. Gilon P. Detimary P. Nenquin M. Henquin J.C. Br. J. Pharmacol. 1995; 114: 872-880Crossref PubMed Scopus (78) Google Scholar). Their positive effect on secretion only becomes manifest when stimulatory glucose is employed. Although the specificity of these inhibitors on islet kinases remains to be determined, it has to be emphasized that these inhibitors reversibly potentiate glucose-induced insulin secretion, thus ruling out any untoward nonspecific toxic action. For wortmannin at least, and from a quantitative perspective, its potentiating effect is comparable with clinically utilized insulin secretagogues. Recent interest in the potential role of insulin signaling on β-cell response patterns has been generated largely as a result of studies in insulin signaling knockout animals (46Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 47Kulkarni R.N. Winnay J.N. Daniels M. Brüning J.C. Flier S.N. Hanahan D. Kahn C.R. J. Clin. Invest. 1999; 104: R69-R75Crossref PubMed Scopus (239) Google Scholar, 48Eto, K., Tsubamoto, Y., Terauchi, Y., Waki, K., Kubota, N., Taka, J., Tamemoto, H., Tobe, K., Noda, M., and Kadowaki, T. (2000)Diabetes 49, (A45)Google Scholar). Amperometric measurements of 5HT release from normal or abnormal β-cells have been used to support the concept that insulin stimulates its own secretion (6Aspinwall C.A. Lakey J.R.T. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Aspinwall C.A. Qian W.-J. Roper M.G. Kulkarni R.N. Kahn C.R. Kennedy R.T. J. Biol. Chem. 2000; 275: 22331-22338Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Our C-peptide measurements failed to reveal any stimulatory effect of exogenous insulin on C-peptide release, a surrogate marker of insulin secretion that accurately reflects small changes in the kinetics and amplitude of insulin release. The assay used to measure C-peptide secretion appears sensitive enough to measure small increments in insulin secretion. For example, the modest 4–5-fold increase in 8 mm glucose-induced release was paralleled by a modest 4–5-fold increase in C-peptide secretion. Even with 7 mmglucose alone, small but parallel 2-fold increments in both insulin and C-peptide release were recorded. In conclusion, C-peptide release rates from perifused rat islets reflect accurately the kinetics and magnitude of glucose- and carbachol-induced insulin release. In perifused rat islets, exogenously added insulin has no inhibitory effect on endogenous insulin secretion monitored by C-peptide secretion rates. Exogenously added bovine or human insulin do not stimulate C-peptide release from islets and, by inference, also fail to exert any autocrine insulin stimulatory effect. Human insulin failed to affect C-peptide secretion from cultured islets as well. Known inhibitors of the kinases that participate in insulin signaling in other tissues significantly amplify glucose-induced secretion. Although the tonic impact of endogenously released insulin makes it technically difficult to establish an inhibitory effect of exogenously added hormone on the release process, any small autocrine stimulatory effect of added insulin, if it occurred, should have been readily detected considering the secretory capacity of the β-cell and the sensitivity of the C-peptide assay. This was, however, not the case. We thank John Cassidy for helpful comments and suggestions.
It has been suggested that the gut hormone cholecystokinin (CCK), by modulating insulin output from pancreatic β-cells, plays an important role in the enteroinsular axis. To investigate this hypothesis, eight rats were studied on two different occasions: after injection of L 364718, a specific antagonist of CCK binding to its membrane receptor, and after vehicle injection. In both studies a mixture of casein (11%) and glucose (9%) was infused through a chronic indwelling intraduodenal catheter to evoke CCK secretion. Plasma was analyzed for insulin, glucose, glucagon, and tyrosine many times during the procedure. Prior administration of the CCK antagonist significantly attenuated the increase in plasma insulin and glucagon after casein infusion. These results support the concept that cholecystokinin plays an important physiologic role in the in vivo regulation of postprandial plasma insulin and glucagon concentrations after protein ingestion.
Isolated rat islets of Langerhans were incubated for 2 h in a myo-[2-3H]inositol-containing solution to label their phosphoinositides. Also included during this labeling period was forskolin (0.1-5 μM), a compound established to elevate islet cAMP levels. These islets were subsequently perifused, and their insulin secretory responses to 20 mM glucose or 1 μM of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were assessed. Determined in parallel with secretion were [3H] inositol efflux patterns and, at the termination of the perifusion, labeled inositol phosphate accumulation. The following major observations were made. 1) Forskolin had no deleterious effect on the total amount of [3H]inositol incorporated by the islets during the labeling period. 2) However, labeling in forskolin resulted in subsequent dose-dependent decreases in 20 mM glucose- induced insulin secretion, [3H]inositol efflux and inositol phosphate accumulation. 3) Inclusion of the diacylglycerol (DAG) kinase inhibitor monooleoylglycerol (50 μM) restored to a significant degree glucose-induced release from forskolin-desensitized islets. 4) Pretreatment with 5 μU forskolin had no deleterious effect on TPA-induced insulin release. 5) Prior exposure to forskolin also impaired phosphoinositide hydrolysis in response to cholecystokinin stimulation. 6) Similar to forskolin, labeling in isobutylmethylxanthine (1 mM) reduced in a parallel fashion islet [3H]inositol efflux and insulin secretion in response to 20 mM glucose stimulation. These findings demonstrate that prior chronic elevation of islet cAMP levels suppresses the activation of phospholipase-C in response to subsequent stimulation. Defective insulin secretory responsiveness of these islets appears to be the result of impaired generation of phosphoinositide- derived second messenger molecules, particularly DAG. By substituting for DAG, however, TPA circumvents this biochemical lesion and evokes a normal insulin secretory response from forskolin-pretreated islets. (Endocrinology126: 2307–2312,1990)
Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the post-prandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates.PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion. Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the post-prandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.
