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. Parallel studies demonstrated that phospholipase C (PLC) was markedly activated in rat, but not mouse, islets by both high glucose concentrations and the amino acid combination. Additional studies documented that glucose and amino acid responses of both rat and mouse islets were amplified by carbachol or forskolin. However, wortmannin, a phosphatidylinositol 3-kinase inhibitor, amplified only the responses to glucose leaving the responses to the amino acid mixture unaltered. These observations support the concept that mitochondrial metabolism alone is minimally effective in stimulating insulin secretion from islets. The activation of the supplementary second messenger systems (PLC and/or cAMP) appears essential for the emergence of their full secretory potential. The mechanism regulating the potency and specificity of wortmannin's impact on glucose-induced secretion remains to be identified; however a unique mechanism is supported by these findings.

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. The inclusion of 50 nM wortmannin or 10 microM LY294002 significantly amplified 10 mM glucose-induced release from mouse islets. The effect of wortmannin was abolished by the calcium channel antagonist nitrendipine or by lowering the glucose level to 3 mM. Wortmannin had no effect on 10 mM alpha-ketoisocaproate-induced secretion. In contrast to its potentiating effect on islets from CD-1 mice, wortmannin had no effect on 10 mM glucose-induced release from ob/ob mouse islets. Western blot analyses revealed the presence of the insulin receptor, insulin receptor substrate proteins 1 and 2 and PI3K in CD-1 islets. These results support the concept that a PI3K-dependent signaling pathway exists in beta-cells and that it may function to restrain glucose-induced insulin secretion from beta-cells. They also suggest that, as insulin resistance develops in peripheral tissues, a potential result of impaired PI3K activation, the same biochemical anomaly in beta-cells promotes a linked increase in insulin secretion to maintain glucose homeostasis.

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. When added together with stimulatory glucose, 5-hydroxytryptamine (0.5 mm) significantly reduced both phases of 8 mm glucose-induced secretion and reduced the first phase of 15 mm glucose-induced release by 60% without any effect on sustained insulin release rates. Preloading of beta-cells with 0.5 mm 5-hydroxytryptamine for 3 h resulted in a more severe impairment of 15 mm glucose-induced secretion. First and second phase release rates were reduced by 70 and 55%, respectively. In addition, this pretreatment protocol also abolished 200 microm tolbutamide-induced insulin secretion from perifused islets. These findings confirm that 5-hydroxytryptamine is a powerful inhibitor of stimulated insulin secretion. The responses of 5-hydroxytryptamine-preloaded beta-cells may not accurately reflect the biochemical events occurring during the physiologic regulation of insulin secretion. The suggestion that insulin stimulates its own secretion based exclusively on amperometric measurements should be reconsidered.

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. The conventional protein-kinase-C isoform inhibitor Gö 6976 (1 microM) completely abolished PMA-induced secretion. However, the combination of forskolin plus PMA significantly enhanced secretion from Gö 6976-treated islets. Similar to previous findings made with staurosporine, Gö 6976 (1 microM) enhanced the first phase and reduced the second phase of 20 mM glucose-induced secretion from rat islets. Additional studies were conducted comparing the secretory responses of perifused rat or mouse islets to glucose. Dramatic species differences to the hexose were observed. For example, 35-40 min after the onset of stimulation with 8, 10 or 20 mM glucose insulin release rates from mouse islets averaged 32+/-6, 84+/-27 or 131+/-17 pg/islet per minute, respectively. The responses from rat islets averaged 115+/-28, 561+/-112 or 800+/-46 pg/islet per minute at this time point. Islet insulin stores were comparable in both species. The addition of 5 microM carbachol, 500 nM forskolin or 20 mM KCl to mouse islets together with 20 mM glucose resulted in a dramatic augmentation of insulin output. The responses to carbachol or forskolin, but not KCl, were inhibited by 50 nM staurosporine. However, staurosporine (50 nM) reduced insulin secretion from rat islets stimulated with KCl plus 20 mM glucose. Gö 6976 potentiated 20 mM glucose-induced secretion from mouse islets. These studies demonstrate that 1 microM Gö 6976 completely abolishes PMA-induced release from rat islets and has a modest inhibitory effect on 20 mM glucose-induced secretion. Gö 6976 (1 microM) had no inhibitory effect on 20 mM glucose-induced release from mouse islets. These studies also confirm that staurosporine inhibits both PKC- and PKA-mediated events in islets and this lack of specificity may account for its more pronounced inhibition of release when compared to Gö 6976. Finally, significant species differences to PKC inhibitors exist between mouse and rat islets.

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. M. caroli responses were intermediate between these two extremes; a modest rising second-phase response to 20 mM glucose was observed. Prior, brief stimulation of rat islets with 20 mM glucose results in an amplified insulin secretory response to a subsequent 20 mM glucose challenge. No such potentiation or priming was observed from M. musculus islets. In contrast, M. caroli islets displayed a modest twofold potentiated first-phase response upon subsequent restimulation with 20 mM glucose. Inositol phosphate (IP) accumulation in response to 20 mM glucose stimulation in [(3)H]inositol-prelabeled rat or mouse islets paralleled the insulin secretory responses. The divergence in 20 mM glucose-induced insulin release between these species may be attributable to differences in phospholipase C-mediated IP accumulation in 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. When mouse islets were perifused under similar conditions, KIC stimulated the largest first-phase insulin response, while comparable acute insulin secretion rates were obtained with glucose- or MP-stimulated islets. In contrast to rat islets, the sustained second phase of insulin secretion from mouse islets was minimal regardless of the nutrient secretagogue used. This anomalous response of mouse islets as compared with rat islets could not be ascribed to any obvious difference in the glucose usage rate or insulin content between these 2 species. Glucose, KIC, or MP stimulated significant increases in 3H-inositol phosphates in rat islets. Significantly smaller increases were measured in mouse islets. Comparative Western blot analyses showed pronounced species differences in the expression of phospholipase Cbeta1 (PLCbeta1), PLCbeta2, PLCbeta3, and PLCdelta1 but not PLCgamma1 or protein kinase Calpha (PKCalpha) between rat and mouse islets. PLCbeta4 or PLCdelta2 could not be identified in either species. These findings are consistent with the concept that the underexpression of the nutrient-activated PLC isozyme may account for the minimal inositol phosphate (IP) and second-phase insulin secretory response from mouse islets.

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. The potentiating action of wortmannin on 8 mM glucose-induced release was duplicated by LY294002. Wortmannin had no effect on glucose usage rates or inositol phosphate accumulation in [3H]inositol-prelabeled islets. Of particular significance, although 50 nM wortmannin potentiated 8 mM glucose-induced secretion from islets of lean Zucker control rats, the fungal metabolite had little effect on 8 mM glucose-induced release from islets of insulin-resistant Zucker fatty rats. These findings support the concept that the same biochemical process, inhibition ofphosphatidyinositol 3-kinase, that causes peripheral tissue insulin resistance enhances beta-cell sensitivity to glucose and produces a compensatory increase in insulin secretion from these cells. The efficacy of wortmannin depends on the in vivo status of the donor's insulin signaling pathways. This elegant biochemical control mechanism in beta-cells ensures the maintenance of glucose homeostasis despite a reduction in insulin action on peripheral tissues.

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 27.5 mM resulted in enhanced insulin secretion from islets of fasted animals. (3) Fasting did not reduce islet insulin content. (4) Responses to 8 or 27.5 mM glucose were not affected if fatty acid-free albumin was used during the perifusion. (5) Inclusion of alpha-ketoisocaproate (5 mM), monomethyl succinate (10 mM) or carbachol (10 microM) significantly amplified insulin release from fasted islets in the simultaneous presence of 8 mM glucose. (6) Phospholipase C activation by glucose, carbachol or their combination was not adversely affected by fasting. (7) The response to the protein kinase C activator, phorbol 12-myristate 13-acetate (500 nM), was reduced by about 60% after fasting. (8) Extending the fast to 48 h resulted in a severe decline in response to 11 mM glucose; however, the further addition of 10 microM carbachol still enhanced release from these islets. The results confirm that caloric restriction impairs islet sensitivity to glucose stimulation and that protein kinase C may be involved in the reduction of glucose-induced insulin release from these islets. The activation of phospholipase C by cholinergic stimulation may contribute to the maintenance of insulin secretion from calorically restricted animals. These results also demonstrate that free fatty acids are not essential for glucose to evoke secretion from isolated islets of fasted donors.

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%. Restimulation of glucose-desensitized islets with 20 mM glucose plus 500 nM forskolin resulted in a dramatic enhancement of both phases of secretion. In contrast to the desensitization of rat islets induced by prior 20 mM glucose exposure, mouse islets were immune to this adverse effect of the hexose. Prior exposure to 20 mM glucose had no adverse effect on glucose usage rates. The activation of phospholipase C in glucose-desensitized rat islets was compromised when compared with control islets. The impairment could not be accounted for by a decrease in immunoreactive content of several major phospholipase C isozymes (beta1 or delta1) or their partitioning between the membrane and cytosolic compartments. In contrast to rat islets, prior exposure of mouse islets to 20 mM glucose for 180 min had no effect on inositol phosphate accumulation. These observations document an additional difference between rat and mouse islets and suggest that the evolution of desensitization is a consequence of the impaired activation of phospholipase C in rat islets.

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. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKC alpha by > 95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPA slightly improved the insulin secretory response to 15 mM glucose compared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly less than the responses of freshly isolated islets. Islets cultured in TPA responded to 30 mM potassium with a markedly amplified insulin secretory response that was abolished by nitrendipine. Enhanced phosphorylation of several islet proteins was also observed in TPA-cultured islets compared with control-cultured islets. These findings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins in TPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.

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. After the labeling of islet phosphoinositide pools with 2-[3H]inositol, the accumulation of labeled inositol phosphates (IP) in response to 30 mM K+ addition in the simultaneous presence of 20 mM glucose plus diazoxide was assessed. The addition of 30 mM K+ significantly increased IP accumulation approximately 300% in rat islets, whereas only an insignificant 25-30% increase was observed in mouse islets. The protein kinase C inhibitor staurosporine (50 nM) dramatically reduced the sustained secretory response from rat islets in the presence of 30 mM K+, 20 mM glucose, and diazoxide. Its effect was minimal on mouse islets and a significant inhibitory effect on insulin secretion was observed only during the final 5 min of the perifusion. The further addition of carbachol, an agonist that activates an isozyme of phospholipase C distinct from that activated by glucose, together with K+, 20 mM glucose, plus diazoxide resulted in a sustained amplification of insulin secretion from mouse but not rat islets. K+ (30 mM)-induced insulin secretion in the presence of 3 mM glucose was similar from perifused rat or mouse islets, a finding that would seem to preclude the activation of voltage-regulated Ca2+ channels as the pertinent difference. These results confirm previous observations with these species and document another anomaly that exists between the responses of rat islets compared with mouse islets. The inability to activate a nutrient- and calcium-regulated phospholipase C isozyme in mouse islets to the same extent as in rat islets appears to account, at least in part, for these different insulin secretory responses under these unique conditions.

Signal transduction in pancreatic beta-cells: regulation of insulin secretion by information flow in the phospholipase C/protein kinase C pathway.

The physiologic regulation of glucose-induced insulin secretion is dependent upon the activation of information flow in the phospholipase C (PLC)/protein kinase C (PKC) signal transduction system. In both rat and human pancreatic beta-cells, glucose has several time-dependent effects on secretory responsiveness including the regulation of biphasic insulin secretion, time-dependent potentiation and time-dependent suppression. PLC/PKC activation has been implicated in all three response patterns. Islets of Langerhans contain the three major PLC isozyme classes (beta1, gamma1 and delta1) and the available evidence suggests that one class is activated by fuel secretagogues and another by neurohumoral agonists. The expression and activation of PLC is labile. When rat islet are cultured for short periods, the content and activation of PLC in response to glucose decreases and this biochemical defect in signal transduction is paralleled by significant reductions in glucose-induced insulin secretion. Similar defects are observed when human islets are cultured as well. Mouse islet responses to glucose stimulation differ in several major and dramatic ways from rat and human islet responses. When stimulated by 15mM glucose, mouse islets fail to develop a rising second phase secretory response, they fail to exhibit either time-dependent potentiation or time-dependent suppression to the hexose. Biphasic insulin secretion can be evoked and time-dependent potentiation can be induced in mouse islets by carbachol, an agonist that activates an isozyme of PLC distinct from that activated by glucose. These divergent response patterns are attributable to the underexpression in mouse islets, when compared to rat islets, of a fuel-sensitive PLC isozyme. When taken in their entirety, the experimental evidence suggests that the activation of PLC is an essential component in the physiologic regulation of insulin secretion and that disordered activation of the enzyme culminates in disordered insulin release.

Influence of pyruvic acid methyl ester on rat pancreatic islets. Effects on insulin secretion, phosphoinositide hydrolysis, and sensitization of the beta cell.

The methyl ester of pyruvic acid (methyl pyruvate) stimulated a dose-dependent increase in insulin secretion from isolated perifused rat islets. The threshold level for release was about 10 mM, and at 20 mM the addition of MP to perifused islets resulted in a large first phase of secretion followed by an insulin-secretory response that was sustained for at least 40 min. When compared to the effects of 20 mM glucose, peak first-phase release rates in response to 20 mM methyl pyruvate were comparable, but the second phase of release was only about 10-15% of that observed with an equimolar level of the hexose. The stimulatory effects of 20 mM methyl pyruvate on secretion were abolished by the K1+-ATP channel blocker diazoxide (200 microM) and by the calcium channel antagonist nitrendipine (500 nM). The glucokinase inhibitor mannoheptulose (20 mM) had no adverse effect on the secretory response to 20 mM methyl pyruvate, whereas 10 microM forskolin amplified the insulinotropic action of MP. Sodium pyruvate alone or in combination with 10 microM forskolin had no insulinotropic effect. In additional experiments islet phosphoinositide pools were labeled with myo-2-[3H]inositol, and the subsequent accumulation of labeled inositol phosphates was used to monitor the activation of phospholipase C. Methyl pyruvate stimulated a dose-dependent increase in inositol phosphate levels when measured after a 30-min incubation period with a maximal increase of about 300% at 20 mM methyl pyruvate. The increase in phosphoinositide hydrolysis caused by methyl pyruvate (20 mM) was, like insulin secretion, reduced by both diazoxide and nitrendipine but was immune to inhibition by mannoheptulose. Pyruvate (20 mM) had no effect on inositol phosphate accumulation. Prior short-term exposure to methyl pyruvate sensitized islets to subsequent stimulation with 15 mM glucose. Sodium pyruvate did not sensitize islets. These findings support the concept that the mitochondrial metabolism of nutrient molecules is an event sufficient to acutely augment insulin release from the beta cell, to increase phospholipase C-mediated phosphoinositide hydrolysis, and to induce time-dependent potentiation of insulin secretion.

Regulation of insulin secretion by phospholipase C.

Biphasic insulin secretion in response to a sustained glucose stimulus occurs when rat or human islets are exposed to high levels of the hexose. A transient burst of hormone secretion is followed by a rising and sustained secretory response that, in the perfused rat pancreas, is 25- to 75-fold greater than prestimulatory insulin release rates. This insulin secretory response is paralleled by a significant five- to sixfold increase in the phospholipase C (PLC)-mediated hydrolysis of islet phosphoinositide (PI) pools by high glucose. In contrast, mouse islets, when stimulated under comparable conditions with high glucose, display a second-phase response that is flat and only slightly (two- to threefold) greater than prestimulatory release rates. The minimal second-phase insulin secretory response to high glucose is accompanied by the minimal activation of PLC in mouse islets as well. However, stimulation of mouse islets with the protein kinase C (PKC) activator tetradecanoyl phorbol acetate (TPA) or the muscarinic agonist carbachol, which significantly activates an isozyme of PLC distinct from that activated by high glucose, induces a rising and sustained second-phase insulin secretory response. When previously exposed to high glucose, both rat and human islets respond to subsequent restimulation with an amplified insulin secretory response. They display priming, sensitization, or time-dependent potentiation. In contrast, mouse islets primed under similar conditions with high glucose fail to display this amplified insulin secretory response on restimulation. Mouse islets can, however, be primed by brief exposure to either TPA or carbachol. Finally, whereas rat islets are desensitized by chronic exposure to high glucose, mouse islet insulin secretory responses are relatively immune to this adverse effect of the hexose. These and other findings are discussed in relationship to the role being played by agonist-induced increases in the PLC-mediated hydrolysis of islet phosphoinositide pools and the activation of PKC in these species-specific insulin secretory response patterns.

Time-dependent effects of cholinergic stimulation on beta cell responsiveness.

The effects of cholinergic stimulation on beta cell insulin secretory and phosphoinositide (PI) responses were determined in freshly isolated rat islets. Increasing the glucose level perifusing the islet from 5.6 to 8mM was accompanied by a modest insulin secretory response. The further addition of 10 microM carbachol increased peak first- and second-phase responses by 2.6- and 6. 8-fold, respectively. In the presence of 5.6 mM glucose, this low level (10 microM) of carbachol increased insulin release two- to three-fold, a response that was maintained for at least 60 min. In contrast to these acute stimulatory actions in the presence of glucose, chronic 3.5-h exposure of islets to 10 microM carbachol abolished beta cell insulin secretory responses to stimulation, with the combination of 8 mM glucose plus 10 microM carbachol. However, the further addition of 200 microM tolbutamide to these islets increased insulin secretory rates significantly. To establish the role of islet cell PI hydrolysis in these secretory responses, additional studies were conducted with islets whose PI pools were labeled with [3H]inositol. Acute exposure to 10 microM carbachol alone significantly increased inositol phosphate accumulation and the efflux of [3H]inositol, even in the absence of glucose. Including 10 microM carbachol during the labeling period with [3H]inositol resulted in significant impairments in subsequently measured inositol phosphate accumulation and [3H]inositol efflux responses to 8 mM glucose plus carbachol stimulation. Prior long-term exposure to 10 microM carbachol also induced heterologous desensitization: 20 mM glucose-stimulated insulin release and inositol phosphate accumulation were impaired in a parallel fashion. Chronic carbachol exposure had no deleterious effect on the usage of 8 or 20 mM glucose or on the insulin content of the islet. The acute stimulatory effects of carbachol on inositol phosphate accumulation as well as its inhibitory effect on 20 mM glucose-stimulated insulin release after prolonged exposure to the muscarinic agonist were significantly reduced by atropine. These findings demonstrate that changes in PI hydrolysis parallel those observed with insulin secretion and suggest that alterations in phospholipase C activation may account, at least in part, for the insulin secretory responses observed.

Signal transduction in isolated islets from the ob/ob mouse: enhanced sensitivity of protein kinase C to stimulation.

The insulin secretory responses of islets isolated from ob/ob mice or their lean litter mates to glucose or the phorbol ester tetradecanoyl phorbol acetate were determined. Glucose-induced phospholipase C activation was also monitored. Even though lean mouse islets contained more insulin than ob/ob mouse islets, the first and second phases of 15mM glucose-induced secretion were significantly greater from ob/ob mouse islets. The kinetics of this amplified response were similar to those seen from lean islets as was the ability of 15mM glucose to activate phospholipase C. A striking dichotomy in responsiveness to the protein kinase C activator tetradecanoyl phorbol acetate was observed between lean and ob/ob mouse islets: while islets from lean animals were unresponsive to tetradecanoyl phorbol acetate (500nM), a rising and sustained insulin secretory response was evoked from ob/ob mouse islets. The combination of 7.5mM glucose plus tetradecanoyl phorbol acetate resulted in dramatic and sustained insulin secretory responses from ob/ob mouse islets, responses that could be duplicated by stimulation with the combination of 3mM glucose, 500nM tetradecanoyl phorbol acetate and 30mM potassium. Significantly smaller responses to these agonist combinations were observed from lean mouse islets. These findings demonstrate that the sensitivity of ob/ob mouse islet protein kinase C to stimulation is markedly enhanced when compared to islets from lean mice and that the activation of protein kinase C or processes distal to and dependent on the enzyme may account, at least in part, for the amplified insulin secretory responses of these islets.

Species differences in the induction of time-dependent potentiation of insulin secretion.

The secretory responsiveness of the pancreatic beta-cell can be markedly improved by prior short term exposure to a stimulatory glucose level. Termed time-dependent potentiation (TDP), priming, or sensitization, this phenomenon has been documented to occur in both human and rat islets and my involve, at least in part, information flow in the phospholipase C and protein kinase C (PKC) signal transduction pathway. In contrast to human and rat islets, however, mouse islets fail to exhibit TDP in response to priming with high glucose. In the present series of studies, we explored in more detail the conditions and stimulants necessary for the induction of TDP in mouse islets and compared these responses with those obtained in rat islets. In agreement with previous reports, high (15 mM) glucose alone primed the rat beta-cell, but not the mouse beta-cell, to subsequent restimulation with 15 mM glucose. However, muscarinic stimulation of mouse islets with carbachol (100 microM) in the presence of 15 mM glucose primed the beta-cell to a subsequent 15-mM glucose stimulus. In addition, prior exposure to 50 nM of the PKC activator tetradecanoyl phorbol acetate dramatically amplified the subsequent insulin secretory responses of mouse islets to 15 mM glucose. In contrast to its significant inhibitory effect on glucose-induced insulin release from rat islets, the PKC inhibitor staurosporine (50 nM) had not effect on 15 mM glucose-induced release from control or prior glucose-exposed mouse islets. However, staurosporine significantly reduced the priming effect of tetradecanoyl phorbol acetate or carbachol on 15 mM glucose-induced insulin secretion from mouse islets. These findings emphasize the dramatic species differences that exist in the capacity of prior high glucose stimulation to induce TDP in rat and, presumably, human islets, on the one hand, and mouse islets, on the other. They also serve to emphasize the role of phosphoinositide hydrolysis and PKC activation in the induction of TDP.

Glucagon-like peptide-1 stimulates insulin secretion but not phosphoinositide hydrolysis from islets desensitized by prior exposure to high glucose or the muscarinic agonist carbachol.

In the present series of experiments, the ability of the postulated incretin factor, glucagon-like peptide-1 (GLP-1), to stimulate insulin release from desensitized islets was determined. Compared with responses observed from control islets incubated for 3.5 hours with 5.6 mmol/L glucose alone, prior exposure to 10 mmol/L glucose, 20 mmol/L glucose, or 10 micromol/L carbachol reduced peak second-phase insulin release rates to a subsequent 20-mmol/L glucose stimulus by 63%, 81%, or 70%, respectively. Efflux of 3H-inositol from prior high-glucose- or carbachol-exposed islets was abolished and accumulation of inositol phosphates (IPs) in response to 20 mmol/L glucose was reduced. Further addition of 10 nmol/L GLP-1 together with 20 mmol/L glucose significantly increased insulin output from desensitized islets. Carbachol (10 micromol/L) preexposure also abolished the subsequent insulin secretory and 3H-inositol efflux responses to 8 mmol/L glucose plus 10 micromol/L carbachol. Inclusion of 10 nmol/L GLP-1 together with 8 mmol/L glucose plus 10 micromol/L carbachol improved but did not normalize secretion from these islets. These improvements in secretory responsiveness from high-glucose- or carbachol- desensitized islets occurred despite the lack of any apparent restorative effect of GLP-1 on agonist-induced increases in phosphoinositide (PI) hydrolysis. Finally, unlike the situation observed with carbachol or high-glucose preexposure, chronic exposure of islets to GLP-1 (100 nmol/L) did not desensitized islets to a subsequent 20 mmol/L glucose stimulus. We conclude from these studies that the incretin factor GLP-1 may play an important role in maintaining insulin output from islets in which phospholipase C (PLC)-mediated hydrolysis of islet PI pools in impaired. GLP-1 may prevent a further decline in beta-cell function and the associated deterioration in glucose tolerance that accompanies chronic exposure of islets to one of several agonists, including high glucose.

Regulation of insulin release by phospholipase C activation in mouse islets: differential effects of glucose and neurohumoral stimulation.

Rat islets respond to glucose stimulation with a marked first and second phase increase in insulin secretion. In contrast, mouse islets have a similar first phase response but little second phase secretion. In these studies, we determined if activation of phospholipase C (PLC) accounts for these differences in second phase insulin secretion in these two species. Stimulation of freshly isolated mouse and rat islets with 15 mM glucose resulted in comparable first phase insulin secretion; however, the second phase response from mouse islets was only doubled from 28 +/- 6 to 60 +/- 7 pg/islet.min compared with an increase from 24 +/- 4 to 1064 +/- 93 pg/islet.min from rat islets. The addition of the muscarinic agonist carbachol (100 microM) in the presence of 15 mM glucose, however, markedly increased second phase insulin release from mouse islets to 801 +/- 80 pg/islet.min. Similar increases in second phase insulin release from mouse islets were obtained with the addition of 500 nM of the protein kinase C activator tetradecanoyl phorbol acetate in the presence of 15 mM glucose. However, the incretin factor glucagon-like peptide-1, which elevates islet cAMP levels, had little effect on second phase insulin release in the mouse. An analysis of PLC-mediated phosphoinositide (PI) hydrolysis revealed that 15 mM glucose increased inositol phosphate (IP) accumulation 0.5-fold above baseline in mouse islets compared with 3.7-fold in rat islets. In contrast, carbachol stimulated IP accumulation 3.5-fold in both mouse and rat islets. Analysis of PLC isozymes with isozyme specific monoclonal antibodies, demonstrated that mouse islets express 14 +/- 4% of PLC-delta 1 and 18 +/- 6% of PLC-beta 1 compared with rat islets but similar amounts of the PLC-gamma 1 (117 +/- 16%). These findings suggest that the decreased second phase insulin secretory response in mouse compared with rat islets results, at least in part, from an inability of high glucose to stimulate comparable increments in PI hydrolysis. This lack of glucose responsiveness may be due to the pronounced underexpression of specific PLC isozymes in the mouse.

Effects of short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol.

The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20-24 h cultured rat islets was assessed. In freshly isolated, 3H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in 3H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18 +/- 4 pg/islet/min, peak first and second phase responses now averaged 422 +/- 61 and 1016 +/- 156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20-24 h in CMRL-1066 medium attenuated all measured responses. The increases in 3H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone 68 +/- 9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358 +/- 85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.