Enhanced activation of phospholipase C and insulin secretion from islets incubated in fatty acid-free bovine serum albumin.

Incubation in 100 micromol/L fatty acid-free bovine serum albumin (FAF-BSA) significantly amplifies insulin secretion from isolated, perifused rat islets. When compared with the responses of control islets incubated in 100 micromol/L radioimmunoassay-grade BSA, insulin secretion rates were increased 2- to 3-fold when these islets were stimulated with 10 mmol/L glucose alone or with the combination of 10 mmol/L glucose, 15 mmol/L KCl, and 100 micromol/L diazoxide. These amplified secretory responses were paralleled by significant increases in the phospholipase C (PLC) activation monitored by fractional increases in (3)H-inositol efflux from these same islets. Amplified PLC responses were also observed with the cholinergic agonist carbachol (50 micromol/L). No differences in the secretory responses to the protein kinase C activator phorbol 12-myristate 13-acetate (200 nmol/L) could be detected between control and FAF-BSA-pretreated rat islets. Mouse islets were also immune to the amplifying impact of this treatment protocol. These findings demonstrate that short-term incubation in FAF-BSA significantly augments the activation of PLC in rat islets by a number of agonists. This proximal event provides the impetus for the distal activation of protein kinase C. If applicable to human islets, this manipulation may provide a mechanism to enhance the secretory responses from islets destined for transplantation, thus improving their in vivo secretory capacity.

Biphasic insulin secretion from freshly isolated or cultured, perifused rodent islets: comparative studies with rats and mice.

In the present report, we compared the insulin secretory responses of freshly isolated, perifused rat and mouse islets to glucose. Prestimulatory glucose levels were changed to assess their influence on the subsequent secretory responses. Additional studies included experiments with the incretin factor glucagon-like peptide-1 (GLP-1), the cholinergic agonist carbachol, and the alpha2 agonist epinephrine. Our findings demonstrate that under conditions where glucose (8.5-11.1 mmol/L) evokes a dramatic biphasic insulin secretory response from perifused rat islets, mouse islets exhibit little response. Increasing the prestimulatory glucose level to 8.5 mmol/L dramatically distorts subsequently measured glucose-induced insulin secretion from rat islets but allows the evocation of a modest but clear biphasic response from mouse islets in response to 30 mmol/L, but not 11.1 or 16.7 mmol/L, glucose. In the presence of a minimally effective glucose level (10 mmol/L), mouse islets remain exquisitely sensitive to the combined stimulatory effects of GLP-1 (2.5 nmol/L) plus carbachol (0.5 micromol/L) and to the inhibitory influence of epinephrine (10 nmol/L). Short-term culture of rat islets in CMRL 1066 containing 5.6 mmol/L glucose resulted in a significant decrease in the secretory response to 11.1 mmol/L glucose, whereas the same manipulation improved mouse islet responses. It is concluded that the process of collagenase isolating islets does not alter mouse islet sensitivity in any adverse way and that increasing the prestimulatory glucose level can indeed alter the pattern of insulin secretion in either a positive or negative manner depending upon the species being investigated. Prior short-term culture of rodent islets differentially affects secretion from these 2 species.

Divergent effects of epinephrine and prostaglandin E2 on glucose-induced insulin secretion from perifused rat islets.

The impact of the catecholamine epinephrine and the postulated inhibitory second messenger prostaglandin E(2) (PGE(2)) on the kinetics and magnitude of glucose-induced insulin secretion were compared and contrasted. In agreement with a number of studies, epinephrine was a most effective antagonist of glucose-induced insulin secretion. Dose-response studies using 8 to 10 mmol/L glucose as stimulant established that levels as low as 1 to 10 nmol/L of the catecholamine were effective at inhibiting release. Glucose (20 mmol/L) caused an approximately 25-fold increase in insulin secretion, an effect that was completely abolished by 1 micromol/L epinephrine. Under conditions where it completely abolished 20 mmol/L glucose-induced insulin release, epinephrine (1 micromol/L) reduced, but did not abolish, the stimulatory effect of glucose on phospholipase C activation. Chronic 3-hour exposure to 10 mmol/L glucose alone desensitized the islet to subsequent stimulation by glucose. Despite its ability to completely suppress secretion to 10 mmol/L glucose, epinephrine failed to protect the islet from hyperglycemia-induced desensitization. In sharp contrast to epinephrine, PGE(2) at levels ranging from 1 to 10 micromol/L had no discernible adverse effect on 10 mmol/L glucose-induced secretion. These findings suggest that multiple mechanisms contribute to the inhibitory impact of epinephrine on release and, in conjunction with other studies, cast serious doubt on the concept that PGE(2) plays any significant inhibitory role in the regulation of glucose-induced secretion.

Inhibitory effects of antipsychotics on carbachol-enhanced insulin secretion from perifused rat islets: role of muscarinic antagonism in antipsychotic-induced diabetes and hyperglycemia.

Treatment with the atypical antipsychotics olanzapine and clozapine has been associated with an increased risk for deterioration of glucose homeostasis, leading to hyperglycemia, ketoacidosis, and diabetes, in some cases independent of weight gain. Because these events may be a consequence of their ability to directly alter insulin secretion from pancreatic beta-cells, we determined the effects of several antipsychotics on cholinergic- and glucose-stimulated insulin secretion from isolated rat islets. At concentrations encompassing therapeutically relevant levels, olanzapine and clozapine reduced insulin secretion stimulated by 10 micromol/l carbachol plus 7 mmol/l glucose. This inhibition of insulin secretion was paralleled by significant reductions in carbachol-potentiated inositol phosphate accumulation. In contrast, risperidone or ziprasidone had no adverse effect on cholinergic-induced insulin secretion or inositol phosphate accumulation. None of the compounds tested impaired the islet secretory responses to 8 mmol/l glucose alone. Finally, in vitro binding and functional data show that olanzapine and clozapine (unlike risperidone, ziprasidone, and haloperidol) are potent muscarinic M3 antagonists. These findings demonstrate that low concentrations of olanzapine and clozapine can markedly and selectively impair cholinergic-stimulated insulin secretion by blocking muscarinic M3 receptors, which could be one of the contributing factors to their higher risk for producing hyperglycemia and diabetes in humans.

Dexamethasone suppresses phospholipase C activation and insulin secretion from isolated rat islets.

Dexamethasone inhibits insulin secretion from isolated islets. In the present experiments, possible underlying biochemical mechanisms responsible for defective secretion were explored. Dexamethasone (1 micromol/L) had no immediate deleterious effect on 15 mmol/L glucose-induced insulin release from perifused rat islets. However, a 3-hour preincubation period with 1 micromol/L dexamethasone resulted in parallel reductions in both the first (64%) and second phases (74%) of 15 mmol/L glucose-induced insulin secretion monitored during a dynamic perifusion. When measured after the perifusion, there were no differences in insulin content or in the capacity of control or dexamethasone-treated islets to use glucose. Dexamethasone (1 micromol/L) preexposure also reduced phorbol ester- and potassium-induced secretion. In additional experiments, islets were labeled for 3 hours with 3H-inositol in the presence or absence of 1 micromol/L dexamethasone. The steroid did not affect total 3H-inositol incorporation during the labeling period. However, the capacity of 15 mmol/L glucose, 30 mmol/L KCl, and 100 micromol/L carbachol to activate phospholipase C (PLC), monitored by the accumulation of labeled inositol phosphates, was significantly reduced in dexamethasone-pretreated islets. Inclusion of the nuclear glucocorticoid receptor antagonist RU486 (mifepristone, 10 micromol/L) abolished the adverse effects of dexamethasone on both glucose-induced inositol phosphate accumulation and insulin secretion. Quantitative Western blot analyses revealed that the islet contents of PLCdelta1, PLCbeta1, beta2, beta3, and protein kinase C alpha were unaffected by dexamethasone pretreatment. These findings demonstrate that dexamethasone pretreatment impairs insulin secretion via a genomic action and that impaired activation of the PLC/protein kinase C signaling system is involved in the evolution of its inhibitory effect on secretion.

Estrogen can prevent or reverse obesity and diabetes in mice expressing human islet amyloid polypeptide.

Type 2 diabetes is characterized by loss of beta-cell mass and concomitant deposition of amyloid derived from islet amyloid polypeptide (IAPP). Previously we have shown that expression of human IAPP (huIAPP) in islets of transgenic mice results in either a rapid onset of hyperglycemia in mice homozygous for the huIAPP transgene on a lean background (FVB/N) or a gradual hyperglycemia in mice hemizygous for the huIAPP transgene on an obese background (A(vy)/A). In both strains, only the males routinely develop diabetes. To investigate this sexual dimorphism, we treated young prediabetic A(vy)/A mice transgenic for huIAPP (huIAPP-A(vy)) with 17beta-estradiol (E2). The treatment completely blocked the progression to hyperglycemia but also prevented the associated weight gain in these mice. Immunohistochemistry of pancreatic sections demonstrated normal islet morphology with no apparent deposition of islet amyloid. E2 treatment of 1-year-old huIAPP-A(vy) diabetic males rapidly reverses obesity and hyperglycemia. To determine the effects of E2 in a nonobese model, we also treated prediabetic, ad libitum-fed and pair-fed Lean-huIAPP transgenic males. E2 completely blocked the progression to hyperglycemia with no significant effect on body weight. Pancreatic insulin content and plasma insulin concentration of Lean-huIAPP transgenic mice increased in a dose-dependent manner. We demonstrated the presence of estrogen receptor (ER)-alpha mRNA in mouse and human islets. By also confirming the presence of ER-alpha protein in islets, we discovered a novel 58-kDa ER-alpha isoform in mice and a 52-kDa isoform in humans, in the absence of the classic 67-kDa protein found in most tissues of both species. The demonstrated presence of ER-alpha in mouse and human islets is consistent with a direct effect on islet function. We conclude that exogenous E2 administered to male mice may block human IAPP-mediated beta-cell loss both by direct action on beta-cells and by decreasing insulin demand through inhibition of weight gain or increasing insulin action.

Aggregation and lack of secretion of most newly synthesized proinsulin in non-beta-cell lines.

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function.

Islet isolation is a time-consuming process. Islet yields vary, and previous in vitro studies suggest that Ficoll may be an islet toxin. Here, we describe an alternative, Ficoll-free method to purify murine islets by filtration through a cell strainer. Collagenase digestion of pancreata was carried out using standard procedures. The pancreatic digest was divided into aliquots and purified either by Ficoll or by filtration. Following filtration, islets were intact and separated from nondigested tissue. Purity was similar to that achieved using Ficoll. However, purification by filtration was faster, increased islet yield, and resulted in higher insulin secretion in vitro. Moreover, when syngeneic diabetic hosts were transplanted with a marginal islet mass, islets purified by filtration restored normoglycemia significantly faster than those isolated by Ficoll. This suggests that Ficoll exposure negatively impacts islet function. In conclusion, islet filtration is a simple and rapid procedure for purification of islets that demonstrate improved functional mass.

Contrasting effects of nateglinide and rosiglitazone on insulin secretion and phospholipase C activation.

When stimulated with 6 mmol/L glucose, a minimal, transient insulin secretory response was observed from perifused rat islets. The inclusion of 5 micromol/L nateglinide significantly amplified release. Elevating glucose to 8 or 10 mmol/L resulted in an increasing insulin secretory response that was again markedly potentiated by the further inclusion of nateglinide. The calcium channel antagonist, nitrendipine, abolished secretion to 8 mmol/L glucose plus nateglinide. Unlike nateglinide, rosiglitazone (5 micromol/L), troglitazone (1 to 10 micromol/L), or darglitazone (10 micromol/L), 3 peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, were without any acute stimulatory effect on insulin release in the simultaneous presence of 6 to 10 mmol/L glucose. Glucose (8 to 10 mmol/L) significantly increased inositol phosphate accumulation. Nateglinide amplified this response. Nitrendipine reduced inositol phosphate (IP) accumulation in response to the combination of 8 mmol/L glucose plus 5 micromol/L nateglinide. Rosiglitazone had no effect on IP accumulation. These results confirm the efficacy of nateglinide as a potent glucose-dependent insulin secretagogue that exerts its stimulatory effect, at least in part, through the activation of phospholipase C (PLC). No acute potentiating effect of rosiglitazone on either insulin secretion or IP accumulation could be detected in isolated rat islets.

Physiologic implications of phosphoinositides and phospholipase C in the regulation of insulin secretion.

The secretion of insulin from the pancreatic beta-cell must be commensurate to satisfy the insulin requirements of the organism. This cell has a great flexibility to meet these requirements which are increased not only by the ingestion of nutrients (increase of plasma glucose) but also by the sensitivity of target tissues to insulin as well. The insulin secretion is a complex biochemical event regulated by a host of potential second messenger molecules acting alone or in concert. These events include the cation calcium, which gains access to the beta-cell via the opening of voltage-regulated channels, cAMP and phosphoinositide-derived second messenger molecules, generated as a consequence of phospholipase C (PLC) activation. In this review, we focused on phosphoinositides, PLC/Phosphokinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) cascade in the regulation of insulin secretion. We also described our studies on the mechanism of the beta-cell desensitization using perifused islets. It is suggested that a failure of the signaling events contribute to the pathogenesis of diabetes in which the beta-cell can no longer secrete the required amounts of insulin. It has been observed that chronic exposure to high glucose desensitizes the beta-cells to subsequent stimulation. We suggested that the failure of PLC activation can be attributed in the impairment of insulin secretion by chronic sustained glucose exposure. It may contribute to the vicious circle of impaired insulin secretion leading up to diabetes.

Beneficial metabolic effects caused by persistent activation of beta-cell M3 muscarinic acetylcholine receptors in transgenic mice.

Previous studies have shown that ß-cell M(3) muscarinic acetylcholine receptors (M3Rs) play a key role in maintaining blood glucose homeostasis by enhancing glucose-dependent insulin release. In this study, we tested the hypothesis that long-term, persistent activation of ß-cell M3Rs can improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet. To achieve the selective and persistent activation of ß-cell M3Rs in vivo, we generated transgenic mice that expressed the Q490L mutant M3R in their pancreatic ß-cells (ß-M3-Q490L Tg mice). The Q490L point mutation is known to render the M3R constitutively active. The metabolic phenotypes of the transgenic mice were examined in several in vitro and in vivo metabolic tests. In the presence of 15 mm glucose and the absence of M3R ligands, isolated perifused islets prepared from ß-M3-Q490L Tg mice released considerably more insulin than wild-type control islets. This effect could be completely blocked by incubation of the transgenic islets with atropine (10 µm), an inverse muscarinic agonist, indicating that the Q490L mutant M3R exhibited ligand-independent signaling (constitutive activity) in mouse ß-cells. In vivo studies showed that ß-M3-Q490L Tg mice displayed greatly improved glucose tolerance and increased serum insulin levels as well as resistance to diet-induced glucose intolerance and hyperglycemia. These results suggest that chronic activation of ß-cell M3Rs may represent a useful approach to boost insulin output in the long-term treatment of type 2 diabetes.

Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galphai in MMECs but coupled to Galphas in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.

Acute and chronic effects of glucose and carbachol on insulin secretion and phospholipase C activation: studies with diazoxide and atropine.

The acute and chronic effects of 20 mM glucose and 10 microM carbachol on beta-cell responses were investigated. Acute exposure of rat islets to 20 mM glucose increased glucose usage rates and resulted in a large insulin-secretory response during a dynamic perifusion. The secretory, but not the metabolic, effect of 20 mM glucose was abolished by simultaneous exposure to 100 microM diazoxide. Glucose (20 mM) significantly increased inositol phosphate (IP) accumulation, an index of phospholipase C (PLC) activation, from [(3)H]inositol-prelabeled islets. Diazoxide, but not atropine, abolished this effect as well. Unlike 20 mM glucose, 10 microM carbachol (in the presence of 5 mM glucose) increased IP accumulation but had no effect on insulin secretion or glucose (5 mM) metabolism. The IP effect was abolished by 50 microM atropine but not by diazoxide. Chronic 3-h exposure of islets to 20 mM glucose or 10 microM carbachol profoundly reduced both the insulin-secretory and PLC responses to a subsequent 20 mM glucose stimulus. The adverse effects of chronic glucose exposure were abolished by diazoxide but not by atropine. In contrast, the adverse effects of carbachol were abolished by atropine but not by diazoxide. Prior 3 h of exposure to 20 mM glucose or carbachol had no inhibitory effect on glucose metabolism. Significant secretory responses could be evoked from 20 mM glucose- or carbachol-pretreated islets by the inclusion of forskolin. These findings support the concept that an early event in the evolution of beta-cell desensitization is the impaired activation of islet PLC.

Desensitization of the pancreatic beta-cell: effects of sustained physiological hyperglycemia and potassium.

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.

Effects of glucose, exogenous insulin, and carbachol on C-peptide and insulin secretion from isolated perifused rat islets.

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.

Effects of prior 5-hydroxytryptamine exposure on rat islet insulin secretory and phospholipase C responses.

Glucose-induced insulin secretion is inhibited by 5-hydroxytryptamine (5HT). In the present studies the specificity of 5HT inhibition of release and the potential biochemical mechanisms involved were investigated. Dose-dependent inhibition of 15 mM glucose-induced secretion was induced by a prior 3 h incubation with 5HT. At the highest 5HT concentration (500 microM) employed, both first and second phase responses to 15 mM glucose were reduced 50-60%. In addition, this level (500 microM) of 5HT virtually abolished 10 mM glucose-induced secretion. In contrast, secretion in response to the protein kinase C activator phorbol 12-myristate 13-acetate (500 nM) was immune to 500 microM 5HT pre-treatment. Glucose usage rates were comparable in both control and 500 microM 5HT-pretreated islets. However, the generation of inositol phosphates and the efflux of 3H-inositol from 3H-inositol-prelabeled islets in response to stimulatory glucose were impaired in parallel with insulin secretion. Based on these observations the following conclusions were reached: (1) 5HT impairs glucose-induced insulin release by altering glucose-induced activation of phospholipase C. (2) Biochemical events distal to phospholipase C remain intact despite this proximal biochemical lesion. (3) Amperometric analysis of 5HT release from 5HT-pretreated islets must take into consideration its profound adverse impact on glucose-induced insulin secretion.

Effects of muscarinic receptor type 3 knockout on mouse islet secretory responses.

The impact of muscarinic type 3 receptor knockout (M3KO) on the cholinergic regulation of insulin secretion and phospholipase C (PLC) activation was determined. Islets isolated from control, wild-type mice or heterozygotes responded with comparable insulin secretory responses to 15 mM glucose. This response was markedly amplified by the inclusion of 10 microM carbachol. While 15 mM glucose-induced release remained similar to wild-type and heterozygote responses in M3KO mice, the stimulatory impact of carbachol was abolished. Stimulation with 15 mM glucose plus 50 microM carbachol increased fractional efflux rates of myo-[2-3H]inositol from control wild-type and heterozygote islets but not from M3KO islets. Fed plasma insulin levels of M3KO mice were reduced 68% when compared to values obtained from combined wild-type and heterozygote animals. These studies support the conclusion that the M3 receptor in islets is coupled to PLC activation and insulin secretion and that cholinergic stimulation of the islets may play an important role in the regulation of plasma insulin levels.