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.

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.

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.

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.

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.

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.

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.

Comparative effects of amino acids and glucose on insulin secretion from isolated rat or mouse islets.

Glucose and the combination of leucine and glutamine were used to stimulate insulin secretion from rat islets during a dynamic perifusion and the responses obtained were compared with those elicited from mouse islets under identical conditions. In rat islets, glucose (15 mM) or the amino acid combination of 10 mM glutamine plus 20 mM leucine were most efficacious and peak second-phase insulin release responses were 20- to 30-fold above prestimulatory rates. In contrast to rat islet responses, sustained second-phase insulin secretory responses to the same agonists were minimally increased 1- to 2-fold from mouse islets.

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.

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.

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.

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.

Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice.

We examined the effects of phosphatidylinositol 3-kinase (PI3K) inhibition by wortmannin or LY294002 on glucose-induced secretion from mouse islets. Islets were collagenase isolated and perifused or subjected to Western blot analyses and probed for insulin receptor-signaling components. In agreement with previous studies, mouse islets, when compared with rat islets, were minimally responsive to 10 mM glucose stimulation.

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.

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.

Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion.

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form.

Are 5-hydroxytryptamine-preloaded beta-cells an appropriate physiologic model system for establishing that insulin stimulates insulin secretion?

The release and oxidation of 5-hydroxytryptamine from 5-hydroxytryptamine-preloaded beta-cells has been used as a surrogate marker for insulin secretion. Findings made using this methodology have been used to support the concept that insulin stimulates its own release. In the present studies, the effects of 5-hydroxytryptamine on stimulated insulin secretion from isolated perifused rat islets was determined.

Effects of protein kinase C inhibitors on insulin secretory responses from rodent pancreatic islets.

The contribution of protein kinase C (PKC) to the regulation of insulin release from perifused islets was explored using staurosporine or Gö 6976 to inhibit the enzyme. Phorbol 12-myristate 13-acetate (PMA, 500 nM) addition to rat islets resulted in a slowly rising insulin secretory response. While minimally effective alone, the addition of 500 nM forskolin together with PMA resulted in a synergistic secretory response.

Insulin secretion and IP levels in two distant lineages of the genus Mus: comparisons with rat islets.

Islet responses of two different Mus geni, the laboratory mouse (Mus musculus) and a phylogenetically more ancient species (Mus caroli), were measured and compared with the responses of islets from rats (Rattus norvegicus). A minimal and flat second-phase response to 20 mM glucose was evoked from M. musculus islets, whereas a large rising second-phase response characterized rat islets.

Insulin secretion, inositol phosphate levels, and phospholipase C isozymes in rodent pancreatic islets.

During a dynamic perifusion, 20 mmol/L glucose, 20 mmol/L alpha-ketoisocaproate (KIC) or 20 mmol/L methyl pyruvate (MP) stimulate biphasic insulin secretory responses from collagenase-isolated rat islets. Peak first-phase insulin responses were comparable for all 3 nutrient agonists. The largest second-phase insulin secretory response was evoked by 20 mmol/L glucose (30-fold above basal release rates), and this response was more sustained than that observed with either 20 mmol/L KIC or 20 mmol/L MP.

A link between insulin resistance and hyperinsulinemia: inhibitors of phosphatidylinositol 3-kinase augment glucose-induced insulin secretion from islets of lean, but not obese, rats.

Wortmannin (5-100 nM), a specific phosphatidyinositol 3-kinase inhibitor, augmented 8 mM glucose-induced insulin secretion from control Sprague Dawley rat islets in a dose-dependent manner. This effect persisted after its removal from the perifusion medium; however, this augmenting effect was reduced by the calcium channel inhibitor nitrendipine or by lowering the glucose level to 3 mM. Wortmannin amplified insulin release induced by the combination of 6-8 mM glucose plus 1 microM carbachol; however, it had no effect on phorbol ester- or alpha-ketoisocaproate-induced insulin secretion.

Glucose-induced insulin secretion from islets of fasted rats: modulation by alternate fuel and neurohumoral agonists.

Islets from fed and 24-h-fasted rats were studied immediately after collagenase isolation. (1) After a 24-h fast, the insulin secretory responses to 8 mM glucose measured during perifusion were reduced by more than 90% from islets of fasted donors. (2) Increasing glucose to 11 or 27.

Glucose-induced desensitization of the pancreatic beta-cell is species dependent.

The capacity of 20 mM glucose to desensitize insulin release was determined. A prior exposure to 20 mM glucose impaired the response of rat islets to subsequent restimulation. Compared with control islets, insulin secretory rates measured 25-30 min after the onset of 20 mM glucose stimulation were reduced by 75%.

Chronic exposure to TPA depletes PKC alpha and augments Ca-dependent insulin secretion from cultured rat islets.

The insulin secretory responses of rat islets to glucose (15 mM), 12-O-tetradecanoylphorbol 13-acetate (TPA; 500 nM), and potassium (30 mM) were determined from perifused islets cultured for 22-24 h in CMRL-1066 medium (control cultured) or islets cultured in the additional presence of 500 nM TPA. Islet content of protein kinase C alpha (PKC alpha) and serine and threonine phosphoprotein patterns were also monitored after the culture period. Compared with freshly isolated islets, culturing alone had no adverse effect on the capacity of TPA or 30 mM potassium to stimulate secretion or on the islet content of PKC alpha.

Regulation of insulin secretion via ATP-sensitive K+ channel independent mechanisms: role of phospholipase C.

Groups of rat or mouse islets were isolated and perifused with 20 mM glucose plus 200 microM diazoxide. The further addition of 30 mM K+ resulted in a rapid and sustained biphasic insulin secretory response. The onset of secretion in response to the addition of K+ was comparable in both species, but the magnitude of the response was significantly greater from rat islets.