Modulation of insulin secretion from beta-cells by phosphoinositide-derived second-messenger molecules.

In isolated islets, the hydrolysis of membrane phosphoinositides (PI) participates in the transduction of both extracellular and intracellular signals into an effective insulin secretory response. A wide variety of potential second-messenger molecules are generated during the phospholipase C-mediated cleavage of these strategically situated membrane phospholipids. Several distinct but interrelated issues are addressed in this perspective. These include 1) methodological approaches utilized to assess PI turnover, 2) the synergistic relationship between PI-derived second messengers and cAMP, 3) the contribution of changing PI turnover rates to the biphasic pattern of insulin output induced by 20 mM glucose, and 4) the role played by PI turnover in the phenomenon of "memory" displayed by islets after prior stimulation with various agonists. The concept that events unique to PI turnover contribute to beta-cell activation is well founded. Because of uncertainty regarding the exact nature of all PI-derived messengers, however, it is not yet possible to mold the available information into a comprehensive theory of beta-cell activation. Future studies will have to address various important unresolved issues.

Induction of memory in rat pancreatic islets by tolbutamide. Dependence on ambient glucose level, calcium, and phosphoinositide hydrolysis.

The ability of the sulfonylurea tolbutamide to induce insulin output, increase phosphoinositide (PI) hydrolysis, and modulate the insulin response to other agonists was assessed. At 200 microM, tolbutamide increased both insulin release and the efflux of 3H from [3H]inositol-prelabeled islets only in the presence of 5.5 or 7 mM glucose. When the glucose level was maintained at 2.75 mM, tolbutamide (200 microM) had no positive impact on either parameter. The calcium-influx inhibitor nitrendipine (200 nM) blocked the effects of 200 microM tolbutamide (with 7 mM glucose) on 3H efflux and insulin output. Prior exposure of islets to tolbutamide (200 microM) in the presence of 7 mM glucose amplified their subsequent insulin response to 10 mM glucose and 5 mM glyceraldehyde. The effect of 200 microM tolbutamide (with 7 mM glucose) was blocked by nitrendipine. Furthermore, the effect of 200 microM tolbutamide was not observed with 2.75 mM glucose; however, if the level of tolbutamide was increased to 1 mM, both PI hydrolysis and potentiated release to subsequent stimulation with 10 mM glucose were observed. Tolbutamide (200 microM with 7 mM glucose) stimulation for 20 min resulted in an increase in 3H efflux from [3H]inositol-prelabeled islets. Despite the rapid fall in insulin secretion, elevated rates of 3H efflux persisted long after the removal of the sulfonylurea from the medium. The duration of the 3H-efflux response paralleled the duration of potentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose.

The ability of glucose to promote the hydrolysis of prelabeled [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP1), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Prior cholecystokinin exposure sensitizes islets of Langerhans to glucose stimulation.

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 beta-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.

Interleukin 1 inhibits insulin secretion from isolated perifused rat islets.

Preincubation of collagenase-isolated rat islets for 150 min with 100 U/ml purified human interleukin 1 (IL-1) altered their ability to secrete insulin. Whereas basal release rates with 4 mM glucose were comparable in control and IL-1-treated islets, both the first and second phases of release in response to 20 mM glucose were significantly reduced from IL-1-treated tissue. IL-1 pretreatment also impaired the secretory response to the combination of 100 nM cholecystokinin plus 7 mM glucose. However, the secretory response to 10 mM alpha-ketoisocaproate was comparable in control and IL-1-pretreated islets. Reducing the IL-1 exposure time to 60 min was accompanied by an augmented first phase of release to 20 mM glucose. Second phase secretion was diminished. The use of glucose measured after the perifusion was similar in control and IL-1-treated islets. Similar to other compounds that adversely impact on beta-cell viability, the inhibitory effect of IL-1 on release may presage a cytotoxic action of monokine.

Physiological role of cholecystokinin in meal-induced insulin secretion in conscious rats. Studies with L 364718, a specific inhibitor of CCK-receptor binding.

It has been suggested that the gut hormone cholecystokinin (CCK), by modulating insulin output from pancreatic beta-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.

Influence of cAMP and calcium on [3H]inositol efflux, inositol phosphate accumulation, and insulin release from isolated rat islets.

The influence of cyclic AMP (cAMP) and extracellular calcium on phosphoinositide (PI) hydrolysis in isolated islets was assessed and related to insulin output. Three stimulants were chosen to activate the beta-cell: sulfated cholecystokinin (CCK-8S, 200 nM), high-level glucose (20 mM), and the sulfonylurea tolbutamide (200 microM). The insulin secretory response to all three agonists was amplified by forskolin (which increases cAMP levels) and reduced by nitrendipine (which decreases calcium influx). All three stimulants increased the hydrolysis of inositol-containing phospholipids, an event monitored by an increase in [3H]inositol efflux from [3H]inositol-prelabeled islets and by the accumulation of labeled inositol phosphates. Forskolin, despite its positive impact on insulin secretion, reduced [3H]inositol efflux and inositol phosphate accumulation in response to all agonists. A similar inhibitory effect on these parameters was noted with nitrendipine; however, nitrendipine abolished secretion in response to all agonists. These findings support the following conclusions: 1) an increase in cellular cAMP levels reduces the quantitative impact of various agonists on these indices of PI hydrolysis; 2) despite this inhibitory effect, cAMP amplifies the insulin secretory response to these agonists; and 3) extracellular calcium is a crucial determinant of both PI hydrolysis and the ensuing insulin secretory response.

Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected].

The influence of L 364718 on islet responsiveness to sulfated cholecystokinin (CCK-8S) was investigated. In islets whose inositol-containing phospholipids were prelabeled during a 2-h incubation period, subsequent exposure to L 364718 (1 nM) significantly impaired the secretion of insulin usually noted in response to 200 nM CCK-8S in the simultaneous presence of 7 mM glucose. A higher level of the antagonist (10 nM) completely abolished insulin secretion. L 364718 (1-10 nM) reduced the efflux of 3H from myo-[2-3H]-inositol prelabeled islets in parallel with the reduction in secretion. L 364718 (10 nM) significantly reduced the accumulation of 3H-containing inositol phosphates usually noted with CCK-8S addition. L 364718, at levels 10- to 100-fold greater than those necessary to attenuate CCK-8S-induced insulin secretion, had no adverse effect on the insulin secretory response of freshly isolated islets to 10 mM glucose alone, 5 mM D-glyceraldehyde, 15 mM alpha-ketoisocaproate, or 50 ng/ml gastric inhibitory polypeptide. L 364718 (1000 nM) had no adverse influence on carbamylcholine (1 mM)-induced phosphoinositide hydrolysis. These results establish L 364718 as a potent and highly selective antagonist of cholecystokinin's stimulatory actions on beta-cells. Because of its potency, selectivity, and oral effectiveness, in vivo studies with L 364718, aimed at unraveling the pleiotropic effects of CCK-8S on glucose and insulin homeostasis, seem feasible.

Comparative effects of monomethylsuccinate and glucose on insulin secretion from perifused rat islets.

In the absence of any other exogenously added fuel, monomethylsuccinate, the methyl ester of succinic acid, at 10-20 mM stimulates insulin release in a biphasic pattern. In quantitative terms, first-phase release evoked by 20 mM MMSucc was comparable to that observed with 20 mM glucose but second-phase release was only 20% of the glucose-induced response. Secretion to both MMSucc and glucose was virtually abolished by the calcium channel antagonist nitrendipine (0.5 microM). In islets that had phosphoinositide pools labeled with [3H]inositol for 2 h, subsequent stimulation with 20 mM MMSucc results in dramatic and sustained increases in [3H]inositol efflux rates. Inositol phosphate levels are also increased. In contrast to secretion, the increase in phosphoinositide hydrolysis caused by MMSucc was largely resistant to nitrendipine, whereas significant reductions in glucose-induced phosphoinositide hydrolysis were observed in the presence of the calcium channel antagonist. MMSucc (2.75-10 mM) substitutes for glucose in that MMSucc supported the insulinotropic effects of the sulfonylurea tolbutamide (200 microM) and the gut hormone cholecystokinin (200 nM). A prior 15-min exposure to 20 mM MMSucc also sensitized islets to the stimulatory effects of 7.5 mM glucose. Finally, a 2-h exposure to 20 mM MMSucc desensitized the islet, in terms of both phosphoinositide hydrolysis and insulin secretion, to a subsequent exposure to 10 mM glucose. Thus, appropriate concentrations of MMSucc can cause qualitatively many of the effects induced by glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Pirogliride: an oral hypoglycemic drug which accelerates glucose usage and insulin secretion by islets of Langerhans.

Pirogliride potentiates glucose-induced insulin secretion from isolated islets. This effect is accompanied by a facilitated glucose metabolism. Pirogliride partially prevents the known inhibitory effects of mannoheptulose on glucose-induced secretion and utilization. Pirogliride might be useful not only to probe the underlying mechanism of stimulated insulin secretion but also in the therapy of those diabetics who have an impaired beta-cell responsiveness to glucose.

Cholecystokinin-induced alterations in beta-cell sensitivity. Duration, specificity, and involvement of phosphoinositide metabolism.

Prior exposure of isolated perifused rat islets to the sulfated gut hormone cholecystokinin-8 (CCK-8S) dramatically increased their insulin secretory response to 7.5 mM glucose, 10 mM arginine, and 10 mM alpha-ketoisocaproate. In the case of glucose, the heightened secretory response was still apparent 60-80 min after CCK-8S removal from the perifusion medium. Prior exposure of perifused islets to arginine (10 mM), tolbutamide (25 microM), or forskolin (1.0 microM) did not sensitize them to 7.5 mM glucose. CCK-8S exposure increased 3H efflux from islets prelabeled with [3H]inositol, and the increase in 3H efflux was sustained after CCK-8S removal from the perifusion medium. The duration of this increase in 3H efflux paralleled the temporal characteristics of this sensitization process and was significantly attenuated by 25 microM asperlicin, a competitive antagonist of CCK binding to its membrane receptor. Arginine, tolbutamide, or forskolin treatment of islets did not increase 3H efflux from [3H]inositol-prelabeled islets. The results suggest that the turnover of membrane phosphoinositides induced by CCK-8S is largely responsible for this heightened state of secretory responsiveness to various stimulants. Second-messenger molecules generated during phosphoinositide turnover may be responsible for the phenomenon of sensitization displayed by islet tissue to CCK-8S addition.

Glucose and 3-O-methylglucose protection against alloxan poisoning of pancreatic alpha and beta cells.

Alloxan infused into the isolated perfused rat pancreas caused transient insulin secretion release. Alloxan poisoning also prevented subsequent induction of glucose-mediated unsulin release and also prevented the inhibition of glucagon release by glucose. Glucose or 3-O-methylglucose infused simultaneously with alloxan protected the alpha- and beta-cell, allowing subsequent glucose inhibition of glucagon secretion and stimulation of insulin release. The above alloxan effects were dose-related, the alpha-cell being one fourth as sensitive to alloxan as the beta-cell. The data indicate that (1) alloxan and glucose suppression of amino-acid-stimulated glucagon secretion is independent of concomitant insulin secretion; (2) alloxan, like glucose, affects alpha-and beta-cells directly, stimulating the beta-cell and inhibiting the alpha-cell; and (3) alloxan acts on a glucoreceptor system with comparable physicochemical characteristics common to both cell types.

Regulation of glucose metabolism in pancreatic islets.

We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about 1/4 of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; 3/4 of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 x g. The supernatant fraction was enriched for glucokinase. About 1/2 of the glucose phosphorylating activity in this fraction was due to glucokinase and 1/2 was due to hexokinase. The glucokinase activity in islet homogenates was !23 of the activity of hexokinase, 1/40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 microM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed, emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of beta-cells [J. Biol. Chem. 243:2730 (1968)] is greatly strengthened by the present studies.

Physiology and pathophysiology of insulin secretion.

Mechanisms by which various classes of extracellular signals regulate insulin secretion are discussed regarding their cellular and molecular actions. Under physiological circumstances, the small postprandial changes in plasma glucose concentrations (approximately 4.4-6.6 mM) primarily serve as a conditioned modifier of insulin secretion and dramatically alter the responsiveness of islets to a combination of neurohormonal agonists. These agonists have two functions. Cholecystokinin (CCK) and acetylcholine activate the hydrolysis of polyphosphoinositides, and gastric inhibitory polypeptide (GIP) and glucagonlike peptide 1 activate adenylate cyclase. These two functional classes of neurohumoral agonists act synergistically to enhance insulin secretion when plasma glucose is greater than 6.0 mM but not when it is less than or equal to 4 mM. On the other hand, an increase in plasma glucose concentration to 8-10 mM induces an increase in insulin secretory rate in the absence of any of the neurohormonal agonists. Remarkably, high glucose leads to an increase in the same intracellular signals, as does a combination of acetylcholine and GIP. On the basis of these data, a model of how insulin secretion is regulated under physiological circumstances is proposed. This model emphasizes that the regulation of insulin secretion occurs in three stages: cephalic, early enteric, and later enteric. In this view, the crucial event occurring during the first two phases is the agonist-induced, translocation of protein kinase C (PKC) to the plasma membrane under conditions in which an increase in Ca2+ influx does not occur. PKC is now in a cellular location and a Ca2(+)-sensitive conformation such that an increase in Ca2+ influx rate occurring during the third phase leads to its immediate activation and an enhanced rate of insulin secretion. Furthermore, under physiological circumstances, an optimal insulin secretory response is dependent on a correct temporal pattern of signals arising from neural and enteric sources. If this pattern is deranged, an abnormal pattern of insulin secretion is observed. An important new insight is provided by the observation that agonists (e.g., CCK or acetylcholine) that act to stimulate the hydrolysis of phosphatidylinositides, when acting for a short period (10-20 min), induce an enhanced responsiveness of islets to glucose, i.e., proemial sensitization. However, when acting unopposed for several hours, these agonists will induce a time-dependent suppression of responsiveness to glucose and other agonists. The latter observation implies that optimal insulin secretion is dependent on periodic rather than a continuous exposure to the correct pattern of extracellular signals.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphoinositide hydrolysis and insulin secretion in response to glucose stimulation are impaired in isolated rat islets by prolonged exposure to the sulfonylurea tolbutamide.

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 microM). After labeling, the ability of these islets to respond during a subsequent perifusion to 20 mM glucose or 15 mM alpha-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 microM) restored the impaired second phase insulin secretory response noted after a 2-h tolbutamide exposure. Prior exposure to tolbutamide (200 microM) 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 beta-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.

Forskolin-induced desensitization of pancreatic beta-cell insulin secretory responsiveness: possible involvement of impaired information flow in the inositol-lipid cycle.

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 microM), a compound established to elevate islet cAMP levels. These islets were subsequently perifused, and their insulin secretory responses to 20 mM glucose or 1 microM 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 microM) restored to a significant degree glucose-induced release from forskolin-desensitized islets. 4) Pretreatment with 5 microM 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.

Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion.

Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.

Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle.

The influence of glucosamine on beta-cell response characteristics of collagenase-isolated rat islets was determined. Groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide (PI) pools. Also included in some experiments was glucosamine (0.1-10 mM). Subsequently, these islets were perifused, and their responses to 10 mM glucose, 10 mM alpha-ketoisocaproate (KIC), and 1 microM of the phorbol ester phorbol 12-myristate 13-acetate were assessed. Increases in PI hydrolysis were monitored during the perfusion by measuring fractional efflux rates of [3H]inositol. The accumulation of inositol phosphates after the perifusion was also determined. In other experiments, the use of 10 mM glucose was measured after a 2-h exposure to 5 or 10 mM glucosamine. Finally, the ability of glucosamine itself to augment release and activate PI hydrolysis was assessed. The following observations were made. 1) A prior 2-h exposure to 5-10 mM glucosamine resulted in parallel dose-dependent impairments in 10 mM glucose-induced insulin release and PI hydrolysis. 2) Glucosamine (5-10 mM) also impaired the subsequent response to alpha-ketoisocaproate (KIC). Parallel deficits in KIC-induced PI hydrolysis were noted under conditions where insulin secretion was impaired. 3) Under several conditions where glucosamine impaired glucose-induced secretion, it had no adverse effect on phorbol 12-myristate 13-acetate-induced release. 4) The desensitizing effect of 10 mM glucosamine on 10 mM glucose-induced release and PI hydrolysis developed within 30 min of exposure to it. 5) Glucosamine (5-10 mM) preexposure had no adverse effect on the use of 10 mM glucose by desensitized islets. 6) Short term (5-min) exposure to glucosamine (10 mM) alone stimulated PI hydrolysis, while a 30-min exposure to the same level of the hexosamine depressed it. 7) In the presence of 0.25 microM forskolin, 10 mM glucosamine also had a transient stimulatory effect on insulin release. These findings support the concept that the acute and chronic effects of glucosamine on the beta-cell result at least in part from its ability to influence PI hydrolysis in 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.

Sequential analysis of the releasing and fuel function of glucose in isolated perifused pancreatic islets.

Isolated islets were continuously perifused with glucose to test their secretory capacity in a dynamic fashion, and were subsequently transferred to an incubation vial to measure their capacity for metabolizing glucose. Insulin release was measured by radioimmunoassay and metabolism of glucose by determining the rate of 3H2O formation from glucose tritiated on carbon atom 2 or 5 and by lactate accumulation. Insulin release was induced by glucose at a threshold of 5 mM, was half maximal at 8 mM, maximal at 15 mM and showed biphasic kinetics, which is consistent with published data. However, in contrast to most previous reports, utilization of glucose and lactate formation showed hyperbolic concentration dependency curves. Maximal and half maximal rates of glucose use were obtained at 30 and 8 mM, respectively, and lactate formation reached highest rates at 8 mM glucose. Physiological changes of glucose levels (from 5 to 10 mM) increased hormone release 4-fold (from 0.49 to 1.94 muU/islet/min) whereas glucose use was changed only slightly (from 52 to 75 pmol/islet/h), and lactate formation not at all. These data show that there is only limited association between metabolic and insulin releasing efficiency of glucose in pancreatic islets in vitro and also implicate and threshold phenomenon triggered by either a glucose metabolite or glucose itself.