[Frontiers in Bioscience 2, d160-172, March 15, 1997]

	[Frontiers in Bioscience 2, d160-172, March 15, 1997] Reprints PubMed CAVEAT LECTOR
	SIGNAL TRANSDUCTION IN PANCREATIC ß-CELLS: REGULATION OF INSULIN SECRETION BY INFORMATION FLOW IN THE PHOSPHOLIPASE C/PROTEIN KINASE C PATHWAY Walter S. Zawalich, Marc Bonnet-Eymard, and Kathleen C. Zawalich Yale University School of Nursing, 100 Church Street South, New Haven, CT 06536-0740 USA Received 2/24/97; Accepted 2/27/97; On-line 3/15/97 6. Species differences in PLC activation, glucose-induced insulin secretion, time-dependent potentiation and time dependent suppression 6.1 Glucose-induced insulin release from mouse islets About 16 years ago both Lenzen (77) and Berglund (78) reported that the insulin secretory responses of the perfused mouse pancreas preparation to glucose stimulation differed significantly from those seen with the perfused rat pancreas preparation studied under comparable conditions. While acute first phase release rates were similar, the mouse ß-cell response to glucose stimulation was notable for the absence of a rising second phase secretory response. For example, when rat ß-cells were stimulated with high glucose, 30-40 fold increments in insulin release rates were observed with the perfused pancreas preparation (7, 8, 79) or from freshly-studied perifused islets (58, 65) (See Figure 1). Second phase responses from mouse islets are flat and from a quantitative perspective much smaller (2-4 fold above prestimulatory rates) than those observed from rat islets (52). This deviation in second phase release from mouse ß-cells has recently been confirmed by Grodsky and coworkers (80), by our group using perifused mouse islets (52) and by Sharp and coworkers (81) studying release from ßHC-9 mouse tumoral cells . This difference in glucose sensitivity is unrelated to islet insulin content (80). Of particular importance, the deviation in mouse islet responsiveness to glucose also stands in contrast to the large and rising second phase responses from human islets studied in vivo with the hyperglycemic clamp technique (10, 12, 82). Thus, while rat and human islets respond to sustained glucose stimulation with a large and rising second phase insulin secretory response, mouse ß-cells fail to demonstrate this response to glucose. 6.2 PLC activation in mouse islets In an attempt to explain the dichotomy in the insulin secretory responses to glucose stimulation which exist between rat and human islets on the one hand and mouse islets on the other, we first focused our attention on information flow in the PLC/PKC pathway. Detailed glucose dose-response curves looking at the accumulation of inositol phosphates, a surrogate and highly sensitive marker for PLC activation, revealed a possible biochemical explanation for the anomalous behavior of mouse islets when stimulated by glucose. In response to the hexose, minimal IP accumulation was observed (Figure 2). This suggested to us that at least part of the explanation for the smaller secretory effect of glucose was attributable to the minimal generation of PI-derived signaling molecules. Since we knew from our rat islet studies that carbachol activated an isozyme of PLC different from the one activated by glucose, we next measured the IP responses to this agonist. Comparable stimulatory effects were noted (54) and, most importantly, the addition of carbachol or the protein kinase activator TPA together with 15mM glucose resulted in the emergence of a brisk biphasic insulin secretory response from mouse islets (Figure 4). Before concluding this section it might be posited that the failure of glucose to stimulate PLC to the same quantitative extent in mouse islets as in rat, and presumably human islets, would leave the animal at risk for hyperglycemia. It must be remembered however that food consumption in these animals is accompanied by an increase in vagal tone and CCK secretion. These factors provide the necessary PLC-generated signals to support insulin secretion. What makes human and rat islets so different and unique is that they have a backup or supplemental system in that, in addition to neural or incretin factors, fuel or nutrient molecules like glucose are also capable of providing significant PLC-generated second messengers to reinforce the secretory response. 6.3 Time-dependent potentiation in mouse islets The failure of glucose to evoke a rising second phase insulin secretory response from mouse islets when compared to the responses of human or rat islets studied under comparable stimulatory conditions is not the only anomaly reported to exist between these species. In follow-up studies Berglund (83) reported that prior high glucose stimulation of mouse ß-cells failed to prime or sensitize them to restimulation. This phenomenon, also referred to as time-dependent potentiation (TDP), can be induced in both rat (7, 16) and human islets (84, 85) by prior short term glucose exposure. In what may turn out to be a particularly prescient remark, Berglund (83) concluded that the failure of glucose to induce TDP in mouse ß-cells may be related to the failure of glucose to induce a rising second phase secretory response in this species. The fact that the magnitude of the second phase response may actually regulate the priming effect of glucose was initially suggested by Grodsky (4) 25 years ago. The further characterization of the factor(s) which limit glucose-induced insulin secretion from mouse ß-cells and accounts for the anomalous behavior of mouse ß-cells to glucose stimulation when compared to human or rat ß-cells may offer a most unique opportunity to elucidate the factors which regulate glucose-induced insulin secretion. Because of our interest in this process and because of our earlier suggestion that both the rising second phase response and the induction of TDP seen when rat islets are stimulated with glucose may be dependent on events associated with PLC/PKC activation (29, 70, 86, 87) , we explored these differences in more detail and attempted to explain these species-dependent response patterns to glucose stimulation. Figure 4. Stimulation of Rising Second Phase Insulin Secretory Responses from Mouse Islets. Groups of islets were isolated from CD-1 mice and perifused with 3mM glucose (G3) for 30 min. At this time one group was stimulated with 15mM glucose (G15) alone (closed circles), a second group with 15mM glucose plus 500nM of the phorbol ester tetradecanoyl phorbol acetate (TPA, open circles), and the third group was stimulated with 15mM glucose plus 100µM carbachol (closed triangles). Note that while glucose alone is without any effect in stimulating a rising second phase, the addition of carbachol or TPA together with 15mM glucose evokes a large, rising second phase response. Confirming the findings made with the perfused pancreas preparation, mouse islets are immune to the sensitizing effect of prior short term glucose exposure. When briefly exposed to glucose, rat islets respond to subsequent restimulation with a dramatically enhanced first phase insulin secretory response (88) (See Figure 5) Mouse islets could not be sensitized under conditions where rat islets so readily exhibit this response pattern. If, as we have suggested, the failure of glucose to activate PLC in mouse islets is the immediate cause of the failure of the hexose to induce priming, then it might be suggested that carbachol or TPA should be able to induce TDP. This was verified is subsequent studies (88) (See Figure 6) and further supported the concept that the species differences in the expression and activation of PLC played a major and determining role in the species-dependent response patterns to glucose stimulation. Figure 5. Glucose Stimulation Induces Time-Dependent Potentiation in Rat but not Mouse Islets. Groups of rat (left, open circles) or mouse (right, open circles) islets were perifused for 30 min with 3mM glucose (G3) followed by 15 min with 15mM glucose (G15). After a 15 min washout with 3mM glucose they were stimulated with 15mM glucose for 30 min and this is the period shown. The responses of control islets (closed circles) perifused for 60 min with 3mM glucose prior to 15mM glucose stimulation are also shown. Note that prior glucose sensitized rat but not mouse islets. Note also the change in scale between panels. Figure 6. Prior Exposure to the Phorbol Ester TPA Induces Time-Dependent Potentiation in Mouse Islets. Two groups of mouse islets were studied. One group (open circles) was perifused for 60 min with 3mM glucose (G3) prior to stimulation with 15mM glucose (G15). The second group (closed circles) was perifused for 30 min with 3mM glucose, 15 min with 3mM glucose plus 500nM TPA and for an additional 15 min with 3mM glucose alone. They were then stimulated with 15mM glucose and this is the period shown here. 6.4 Glucose fails to induce time-dependent suppression (TDS) in mouse islets While the positive stimulatory actions of glucose on the ß-cell have been the primary foci up until now, it is also clear that sustained exposure to the hexose impairs insulin secretion. Termed TDS, it can also be induced by other agonists including but not confined to those compounds which act initially to increase information flow in the PLC/PKC signaling system. Thus, TDS can be induced by long term (2-3 hour) exposure to cholecystokinin (CCK) or to carbachol as well as by sustained exposure to forskolin, which elevates cAMP to supraphysiological levels in islets (89). In islets suppressed by any one of these compounds, the common biochemical lesion and the one which appears to account for reduced insulin secretion is the impaired capacity of subsequent restimulation with glucose to activate PLC. Our working hypothesis to account for the induction of TDS is that the sustained activation of PKC or PKA results in the phosphorylation of PLC, a type of negative feedback and an event which reduces PLC's ability to subsequently respond to stimulation. This proposed model of inhibition is consistent with what has been observed in other systems as well: PKC activation inhibits PLC activation (53, 90, 91). Moreover, PLC activation is also inhibitable by cAMP as well, providing an explanation for the inhibitory action of forskolin on ß-cell PLC and its capacity to induce TDS of insulin release. If sustained PLC/PKC activation by high glucose is responsible for the induction of TDS of secretion in rat islets, then mouse islets which respond poorly in terms of PLC-mediated PI hydrolysis should be relatively immune to the desensitizing effect of the hexose. This has been confirmed using perifused mouse islets previously exposed to 20mM glucose for 3 hr (55) (See Figure 7). For example, 3 hour exposure of rat islets to 20mM glucose impaired the capacity of these islets to respond subsequently to stimulation with 20mM glucose plus 100µM carbachol; 20mM glucose induced TDS of release. Mouse islets secretory responses studied after a 3 hour incubation period with 20mM glucose were not suppressed when stimulated with 20mM glucose plus 100µM carbachol; 20mM glucose failed to induce TDS of release in mouse islets. Studies with mouse islets have afforded us a most unique opportunity to dissect out the contribution of glucose signaling via the PLC/PKC cascade to the regulation of insulin secretion. Figure 7. Effects of Prior 3hr. Exposure to 20mM Glucose on Insulin Release Rates from Rat or Mouse Islets: High Glucose Fails to Induce Suppression in Mouse Islets. Groups of islets were isolated from Sprague-Dawley (SD) rats (left) or CD-1 mice (right) and incubated for 3 hr in 5mM glucose alone (closed circles, solid line) or 20mM glucose alone (closed triangles, dashed line). All groups of islets were then perifused for 30 min with 5mM glucose (G5) and for an additional 30 min with the combination of 20mM glucose (G20) plus 100µM carbachol (Carb). This combination of agonists was used because glucose alone is such a poor stimulant for secretion from mouse islets. Note the reduction in release from rat islets due to prior 20mM glucose exposure but the slight increase from mouse islets.

[Frontiers in Bioscience 2, d160-172, March 15, 1997]
Reprints
PubMed
CAVEAT LECTOR

SIGNAL TRANSDUCTION IN PANCREATIC ß-CELLS: REGULATION OF INSULIN SECRETION BY INFORMATION FLOW IN THE PHOSPHOLIPASE C/PROTEIN KINASE C PATHWAY

Walter S. Zawalich, Marc Bonnet-Eymard, and Kathleen C. Zawalich

Yale University School of Nursing, 100 Church Street South, New Haven, CT 06536-0740 USA

Received 2/24/97; Accepted 2/27/97; On-line 3/15/97

6. Species differences in PLC activation, glucose-induced insulin secretion, time-dependent potentiation and time dependent suppression

6.1 Glucose-induced insulin release from mouse islets

About 16 years ago both Lenzen (77) and Berglund (78) reported that the insulin secretory responses of the perfused mouse pancreas preparation to glucose stimulation differed significantly from those seen with the perfused rat pancreas preparation studied under comparable conditions. While acute first phase release rates were similar, the mouse ß-cell response to glucose stimulation was notable for the absence of a rising second phase secretory response. For example, when rat ß-cells were stimulated with high glucose, 30-40 fold increments in insulin release rates were observed with the perfused pancreas preparation (7, 8, 79) or from freshly-studied perifused islets (58, 65) (See Figure 1). Second phase responses from mouse islets are flat and from a quantitative perspective much smaller (2-4 fold above prestimulatory rates) than those observed from rat islets (52). This deviation in second phase release from mouse ß-cells has recently been confirmed by Grodsky and coworkers (80), by our group using perifused mouse islets (52) and by Sharp and coworkers (81) studying release from ßHC-9 mouse tumoral cells . This difference in glucose sensitivity is unrelated to islet insulin content (80). Of particular importance, the deviation in mouse islet responsiveness to glucose also stands in contrast to the large and rising second phase responses from human islets studied in vivo with the hyperglycemic clamp technique (10, 12, 82). Thus, while rat and human islets respond to sustained glucose stimulation with a large and rising second phase insulin secretory response, mouse ß-cells fail to demonstrate this response to glucose.

6.2 PLC activation in mouse islets

In an attempt to explain the dichotomy in the insulin secretory responses to glucose stimulation which exist between rat and human islets on the one hand and mouse islets on the other, we first focused our attention on information flow in the PLC/PKC pathway. Detailed glucose dose-response curves looking at the accumulation of inositol phosphates, a surrogate and highly sensitive marker for PLC activation, revealed a possible biochemical explanation for the anomalous behavior of mouse islets when stimulated by glucose. In response to the hexose, minimal IP accumulation was observed (Figure 2). This suggested to us that at least part of the explanation for the smaller secretory effect of glucose was attributable to the minimal generation of PI-derived signaling molecules. Since we knew from our rat islet studies that carbachol activated an isozyme of PLC different from the one activated by glucose, we next measured the IP responses to this agonist. Comparable stimulatory effects were noted (54) and, most importantly, the addition of carbachol or the protein kinase activator TPA together with 15mM glucose resulted in the emergence of a brisk biphasic insulin secretory response from mouse islets (Figure 4). Before concluding this section it might be posited that the failure of glucose to stimulate PLC to the same quantitative extent in mouse islets as in rat, and presumably human islets, would leave the animal at risk for hyperglycemia. It must be remembered however that food consumption in these animals is accompanied by an increase in vagal tone and CCK secretion. These factors provide the necessary PLC-generated signals to support insulin secretion. What makes human and rat islets so different and unique is that they have a backup or supplemental system in that, in addition to neural or incretin factors, fuel or nutrient molecules like glucose are also capable of providing significant PLC-generated second messengers to reinforce the secretory response.

6.3 Time-dependent potentiation in mouse islets

The failure of glucose to evoke a rising second phase insulin secretory response from mouse islets when compared to the responses of human or rat islets studied under comparable stimulatory conditions is not the only anomaly reported to exist between these species. In follow-up studies Berglund (83) reported that prior high glucose stimulation of mouse ß-cells failed to prime or sensitize them to restimulation. This phenomenon, also referred to as time-dependent potentiation (TDP), can be induced in both rat (7, 16) and human islets (84, 85) by prior short term glucose exposure. In what may turn out to be a particularly prescient remark, Berglund (83) concluded that the failure of glucose to induce TDP in mouse ß-cells may be related to the failure of glucose to induce a rising second phase secretory response in this species. The fact that the magnitude of the second phase response may actually regulate the priming effect of glucose was initially suggested by Grodsky (4) 25 years ago. The further characterization of the factor(s) which limit glucose-induced insulin secretion from mouse ß-cells and accounts for the anomalous behavior of mouse ß-cells to glucose stimulation when compared to human or rat ß-cells may offer a most unique opportunity to elucidate the factors which regulate glucose-induced insulin secretion. Because of our interest in this process and because of our earlier suggestion that both the rising second phase response and the induction of TDP seen when rat islets are stimulated with glucose may be dependent on events associated with PLC/PKC activation (29, 70, 86, 87) , we explored these differences in more detail and attempted to explain these species-dependent response patterns to glucose stimulation.

Figure 4. Stimulation of Rising Second Phase Insulin Secretory Responses from Mouse Islets. Groups of islets were isolated from CD-1 mice and perifused with 3mM glucose (G3) for 30 min. At this time one group was stimulated with 15mM glucose (G15) alone (closed circles), a second group with 15mM glucose plus 500nM of the phorbol ester tetradecanoyl phorbol acetate (TPA, open circles), and the third group was stimulated with 15mM glucose plus 100µM carbachol (closed triangles). Note that while glucose alone is without any effect in stimulating a rising second phase, the addition of carbachol or TPA together with 15mM glucose evokes a large, rising second phase response.

Confirming the findings made with the perfused pancreas preparation, mouse islets are immune to the sensitizing effect of prior short term glucose exposure. When briefly exposed to glucose, rat islets respond to subsequent restimulation with a dramatically enhanced first phase insulin secretory response (88) (See Figure 5) Mouse islets could not be sensitized under conditions where rat islets so readily exhibit this response pattern. If, as we have suggested, the failure of glucose to activate PLC in mouse islets is the immediate cause of the failure of the hexose to induce priming, then it might be suggested that carbachol or TPA should be able to induce TDP. This was verified is subsequent studies (88) (See Figure 6) and further supported the concept that the species differences in the expression and activation of PLC played a major and determining role in the species-dependent response patterns to glucose stimulation.

Figure 5. Glucose Stimulation Induces Time-Dependent Potentiation in Rat but not Mouse Islets. Groups of rat (left, open circles) or mouse (right, open circles) islets were perifused for 30 min with 3mM glucose (G3) followed by 15 min with 15mM glucose (G15). After a 15 min washout with 3mM glucose they were stimulated with 15mM glucose for 30 min and this is the period shown. The responses of control islets (closed circles) perifused for 60 min with 3mM glucose prior to 15mM glucose stimulation are also shown. Note that prior glucose sensitized rat but not mouse islets. Note also the change in scale between panels.

Figure 6. Prior Exposure to the Phorbol Ester TPA Induces Time-Dependent Potentiation in Mouse Islets. Two groups of mouse islets were studied. One group (open circles) was perifused for 60 min with 3mM glucose (G3) prior to stimulation with 15mM glucose (G15). The second group (closed circles) was perifused for 30 min with 3mM glucose, 15 min with 3mM glucose plus 500nM TPA and for an additional 15 min with 3mM glucose alone. They were then stimulated with 15mM glucose and this is the period shown here.

6.4 Glucose fails to induce time-dependent suppression (TDS) in mouse islets

While the positive stimulatory actions of glucose on the ß-cell have been the primary foci up until now, it is also clear that sustained exposure to the hexose impairs insulin secretion. Termed TDS, it can also be induced by other agonists including but not confined to those compounds which act initially to increase information flow in the PLC/PKC signaling system. Thus, TDS can be induced by long term (2-3 hour) exposure to cholecystokinin (CCK) or to carbachol as well as by sustained exposure to forskolin, which elevates cAMP to supraphysiological levels in islets (89). In islets suppressed by any one of these compounds, the common biochemical lesion and the one which appears to account for reduced insulin secretion is the impaired capacity of subsequent restimulation with glucose to activate PLC. Our working hypothesis to account for the induction of TDS is that the sustained activation of PKC or PKA results in the phosphorylation of PLC, a type of negative feedback and an event which reduces PLC's ability to subsequently respond to stimulation. This proposed model of inhibition is consistent with what has been observed in other systems as well: PKC activation inhibits PLC activation (53, 90, 91). Moreover, PLC activation is also inhibitable by cAMP as well, providing an explanation for the inhibitory action of forskolin on ß-cell PLC and its capacity to induce TDS of insulin release. If sustained PLC/PKC activation by high glucose is responsible for the induction of TDS of secretion in rat islets, then mouse islets which respond poorly in terms of PLC-mediated PI hydrolysis should be relatively immune to the desensitizing effect of the hexose. This has been confirmed using perifused mouse islets previously exposed to 20mM glucose for 3 hr (55) (See Figure 7). For example, 3 hour exposure of rat islets to 20mM glucose impaired the capacity of these islets to respond subsequently to stimulation with 20mM glucose plus 100µM carbachol; 20mM glucose induced TDS of release. Mouse islets secretory responses studied after a 3 hour incubation period with 20mM glucose were not suppressed when stimulated with 20mM glucose plus 100µM carbachol; 20mM glucose failed to induce TDS of release in mouse islets. Studies with mouse islets have afforded us a most unique opportunity to dissect out the contribution of glucose signaling via the PLC/PKC cascade to the regulation of insulin secretion.

Figure 7. Effects of Prior 3hr. Exposure to 20mM Glucose on Insulin Release Rates from Rat or Mouse Islets: High Glucose Fails to Induce Suppression in Mouse Islets. Groups of islets were isolated from Sprague-Dawley (SD) rats (left) or CD-1 mice (right) and incubated for 3 hr in 5mM glucose alone (closed circles, solid line) or 20mM glucose alone (closed triangles, dashed line). All groups of islets were then perifused for 30 min with 5mM glucose (G5) and for an additional 30 min with the combination of 20mM glucose (G20) plus 100µM carbachol (Carb). This combination of agonists was used because glucose alone is such a poor stimulant for secretion from mouse islets. Note the reduction in release from rat islets due to prior 20mM glucose exposure but the slight increase from mouse islets.