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. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Metabolism. 2008 Feb;57(2):290–298. doi: 10.1016/j.metabol.2007.09.015

Enhanced Activation of Phospholipase C and Insulin Secretion from Islets Incubated in Fatty Acid-Free BSA

Walter S Zawalich 1, Kathleen C Zawalich 1
PMCID: PMC2275802  NIHMSID: NIHMS38901  PMID: 18191063

Abstract

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

Keywords: islets, secretion, phospholipase C, 3H-inositol, fatty acids

Introduction

Glucose-induced insulin secretion is a complex biochemical event regulated by a host of potential second messenger molecules acting alone or in concert 1-3. These events include not only the cation calcium, which gains access to the β-cell via the opening of voltage-regulated channels, but also cAMP and phosphoinositide-derived second messenger molecules, generated as a consequence of phospholipase C (PLC) activation. Because of the emerging consensus 4, 5 that β-cell failure plays a major pathogenic role in the emergence of Type 2 diabetes, attention has been focused on elucidating the nature of the pathways that so elegantly control insulin secretion. Moreover, their identification may also provide insight into maneuvers that might improve their function.

Recently Straub and Sharp 6 reported that a brief 3-6 hour exposure of isolated rat islets to fatty acid-free bovine serum albumin (FAF-BSA) dramatically potentiated the subsequent insulin secretory response to 16.7 mM glucose. This amplified response was not confined to glucose; it could be observed in response to α-ketoisocaproate alone and from islets stimulated with the combination of glucose and potassium in the presence of diazoxide. However, this amplified response could not be duplicated in mouse islets or in 2 clonal cell lines, INS-1 and INS 832/13. They further demonstrated that the activation of protein kinase C (PKC) appeared to be playing a preeminent role in this amplification process since a number of PKC inhibitors proved effective in thwarting its development and evocation.

The species specificity of the amplification induced by incubating islets in FAF-BSA is reminiscent of the divergent acute stimulatory impact that glucose exerts on perfused or freshly isolated perifused rat or mouse islets 7-9. We have previously demonstrated that the smaller second phase response observed from mouse islets by a number of investigators including ourselves might be attributable to the less robust activation of PLC in this species as opposed to rat islets 1, 10, 11. Because the agonists employed in the comprehensive study by Straub and Sharp 6 activate PLC in addition to PKC, we decided to explore how incubating islets in FAF-BSA influences PLC activation in both species. In addition, we also examined how the response to phorbol 12-myristate 13-acetate (PMA) might be influenced by this manipulation, since this is a direct activator of PKC. Our findings confirm the observation that prior exposure to FAF-BSA markedly augments glucose-induced secretion from rat but not from mouse islets. Our studies also suggest that enhanced activation of PLC appears to account for this response.

Material and Methods

Islet Isolation

The detailed methodologies employed to assess insulin output from collagenase-isolated islets have been previously described 12, 13. Male Sprague-Dawley rats (weighing 325-425 g at the time of study) and male CD-1 mice (weighing 28-45 g at the time of study) were purchased from Charles River and used in all studies. All animals were treated in a manner that complied with the NIH Guidelines for the Care and Use of Laboratory Animals. The animals were fed ad lib. After Nembutal (pentobarbital sodium, 50 mg/kg; Abbott, North Chicago, IL) -induced anesthesia, 20-30 ml of cold Hanks’ solution (without any added glucose) was used to distend the pancreas via the biliary system. Islets were isolated by collagenase digestion and handpicked, using a glass loop pipette, under a stereomicroscope into Krebs-Ringer Bicarbonate (KRB) supplemented with 3mM glucose. They were free of exocrine contamination.

Islet Incubation

After isolation, a process lasting no more than 90 minutes after initial surgical removal of the pancreas, groups of 18-32 islets were loaded onto nylon filters (Sefar America Inc., Kansas City, MO). The larger groups of islets were used for the 3H-inositol efflux studies. The islets on nylon filters were then placed in Corning #3513, 12 well, cell culture cluster flat bottom containers. Two ml of CMRL culture medium1066 (Gibco #11530, without added L-glutamine) were gently added to each well. This medium contains 5.6 mM (100 mg%) glucose. Control islets were incubated in CMRL 1066 supplemented with 100 μM BSA (Sigma albumin #A7888, RIA grade, Fraction V, batch # 044KO460). Experimental islets were incubated in CMRL 1066 supplemented with 100 μM FAF-BSA (Roche heat shock, fatty acid-free, lot # 93433222). All groups of islets were then incubated for 4 hours at 37°C in an incubator with 5% CO2/95% humidified air.

In those experiments where PLC activation was monitored, 3H-inositol was included during the incubation period in CMRL 1066 to label phosphoinositide pools 14. This solution was prepared as follows. To twenty μl stock myo-[2-3H(N)]inositol (PerkinElmer, 1 mCi/ml), 2.1 ml of CMRL 1066 were added. After mixing, 2 ml of this solution was gently added to the incubation well with the islets.

Perifusion studies

After the incubation period, all islets were washed gently with 5 ml of warmed, fresh KRB perifusion medium. They were then perifused in a KRB buffer at a flow rate of 1ml (±0.1 ml)/minute for 30 minutes in the presence of 3 mM glucose unless otherwise indicated. Sigma BSA (RIA grade), 25 μM, was included during the entire perifusion period with all groups of islets irrelevant of their pretreatment. This is the standard BSA concentration we have employed in many previous reports 15, 16. After this 30 min stabilization period islets were then perifused with the appropriate agonist or agonist combinations as indicated in the figure legends and Results section. To prevent the reincorporation of 3H-inositol back into phosphoinositide pools and to facilitate the measurements of 3H-inositol efflux from these small groups of islets, 0.1 mM cold myo-inositol was included in the perifusion where efflux was monitored.

Perifusate solutions were gassed with 95% O2/5% CO2 and maintained at 37°C. Insulin released into the medium was measured by radioimmunoassay 17. From the same collection tubes, the amount of 3H-inositol effluxing from the islets was also determined 18, 19. At the termination of the efflux experiments, the number of counts remaining in the islets were assessed and efflux of 3H-inositol expressed as fractional efflux rates 19-21.

Reagents

Ice-cold Hanks’ solution without any added glucose was used for the islet isolation. The perifusion medium consisted of 115 mM NaCl, 5 mM KCl, 2.2 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3 and 0.17 g/dl (25 μM) bovine serum albumin, Sigma A7888, RIA grade. The 125I-labeled insulin used for the insulin assay and the 3H-inositol used to assess PLC activation were purchased from PerkinElmer Life Sciences (Boston, MA). Glucose, carbachol, LiCl, diazoxide, PMA and the salts used to make the Hanks’ solution and perifusion medium were purchased from Sigma (St. Louis, MO). Rat insulin standard (lot #615-ZS-157) was the generous gift of Dr. Gerald Gold, Eli Lilly Co. (Indianapolis, IN). Collagenase (Type P) was obtained from Roche Applied Science (Indianapolis, IN).

Statistics

Statistical significance was determined using the Student’s t test for unpaired data or analysis of variance. A p value ≤0.05 was taken as significant. Values presented in the figures and results represent means ± SEMs of at least three observations.

Results

In the initial series of studies we first confirmed the observations made previously by Straub and Sharp 6 that a 4 hour incubation period in culture medium containing 100 μM FAF-BSA enhances glucose-induced insulin secretion. Because it is more physiologic than the 16.7 mM glucose concentration employed in this previous report, we decided to employ a lower glucose level, 10 mM. In addition, to facilitate the assessment of PLC activation, monitored by the efflux of 3H-inositol 19, 22, 0.1 mM inositol was also included with glucose. In response to 10 mM glucose stimulation, a modest biphasic insulin secretory response was evoked from control islets (Figure 1A). Peak first phase secretory rates averaged 116±5 pg/islet/min while peak second phase release rates were 221±28 pg/islet/min (n=7). When compared to prestimulatory secretion of about 30 pg/islet/min, release rates were increased about 6-7 fold.

Figure 1. Effects of FAF-BSA on insulin secretion and 3H-inositol efflux from isolated, perifused rat islets stimulated with 10 mM glucose.

Figure 1

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Groups of islets were isolated and subjected to a 4 hour incubation in CMRL 1066 medium supplemented with 100 μM Sigma #7888 RIA grade BSA (open circles) or 100 μM Roche FAF-BSA (closed circles) and 3H-inositol. After washing to remove unincorporated label the islets were then perifused. For the initial 30 minutes of the perifusion, the glucose level was maintained at 3 mM (G3). For the next 40 minutes, onset indicated by the vertical line, both groups of islets were stimulated with 10 mM glucose (G10) plus 0.1 mM cold inositol to restrain reincorporation of labeled 3H-inositol back into phosphoinositide pools. Insulin secretion rates (A) were measured and fractional rates of 3H-inositol efflux rates (B) were calculated. This and subsequent figures have not been corrected for the dead space, about 2.5 ml or 2.5 minutes with a flow rate of 1 ml/min, in the perifusion apparatus. Mean values ±SEM of at least 7 experiments are shown. The asterisk indicates a significant difference (p≤0.05) between groups at the indicated time points. With regards to fractional efflux rates, significant differences between groups were noted during the entire perifusion.

Prestimulatory secretion rates from FAF-BSA preincubated islets were comparable to those measured from control islets. However, islets incubated for 4 hours in FAF-BSA exhibited markedly enhanced insulin release rates (Figure 1A). Both phases of secretion were amplified. Peak first and second phase responses to 10 mM glucose from FAF-BSA incubated islets averaged 202±31 pg/islet/min and 506±71 pg/islet/min respectively (n=8). When compared to prestimulatory secretion rates of about 30 pg/islet/min, release rates were increased about 16-17 fold.

A closer analysis of the data presented by Straub and Sharp 6 suggested to us that the proximal activation of PLC by glucose might result in the distal, enhanced activation of PKC as they suggested. To address this issue, the efflux of 3H-inositol from control and FAF-BSA incubated islets was next determined. As shown in Figure 1B (open circles), the efflux of 3H-inositol in response to 10 mM glucose was modestly increased from control islets in the presence of 0.1 mM inositol. Efflux rates measured in the presence of 3 mM glucose alone increased from 0.16±0.03%/min to 0.32±0.07%/min after 40 minutes of stimulation with 10 mM glucose.

A different result emerged when similar studies were conducted using islets incubated in FAF-BSA. A small increase in efflux rates was measured in the presence of 3 mM glucose from these islets (Figure 1B, closed circles). However, the addition of 10 mM glucose to these islets resulted in a significant enhancement of 3H-inositol efflux. Rates of 3H-inositol efflux increased rapidly from these islets and averaged 1.63±0.15%/min after 40 minutes of stimulation.

In their seminal report on this topic, Straub and Sharp 6 also reported that in the presence of diazoxide the combination of glucose plus potassium also evoked amplified secretion from FAF-BSA incubated islets. We confirmed this as well, as shown in Figure 2A, and also demonstrated that PLC activation is similarly enhanced from these islets (Figure 2B). In islets perifused with 100 μM diazoxide to maintain the patency of the K-ATP sensitive potassium channel, the stimulatory combination of 10 mM glucose plus 15 mM KCl evoked amplified secretory and PLC responses from FAF-BSA incubated islets.

Figure 2. Effects of FAF-BSA on insulin secretion and 3H-inositol efflux from isolated, perifused rat islets stimulated with KCl.

Figure 2

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Groups of islets were isolated and subjected to a 4 hour incubation in CMRL 1066 medium supplemented with 100 μM Sigma #7888 RIA grade BSA (open circles) or 100 μM Roche FAF-BSA (closed circles) and 3H-inositol. After washing to remove unincorporated label the islets were then perifused for 70 minutes with the combination of 10 mM glucose (G10) plus 100 μM diazoxide. After 30 minutes, 15 mM KCl was then added to the perifusion medium to stimulate the islets. Inositol (0.1 mM) was also included during this time to prevent reincorporation of labeled 3H-inositol back into phosphoinositide pools. Insulin secretion (A) and fractional rates of 3H-inositol efflux (B) were calculated. At least 3 experiments were performed under each condition. The asterisk indicates a significant difference (p≤0.05) between groups at the indicated time points.

In the next set of experiments we determined the impact of FAF-BSA incubation on PLC activation by the cholinergic agonist carbachol (50 μM). In the presence of 3 mM glucose, the addition of 50 μM carbachol to control islets modestly increased 3H-inositol efflux (Figure 3). This response was dramatically enhanced from FAF-BSA pretreated islets. In additional experiments we included 10 mM LiCl in the perifusion medium. This compound blocks the phosphatases responsible for the metabolism of inositol phosphates into membrane permeable free inositol 23. This effect facilitates cellular measurements of inositol phosphates. It reduces the efflux of free inositol from the cell and studies with lithium have been used to confirm the activation of PLC as well 19, 24, 25. Its inclusion in the perifusion medium significantly reduced the efflux of 3H-inositol from FAF-BSA incubated islets (Figure 3). Although not shown, the addition of LiCl also significantly reduced 3H-inositol efflux in response to 10 mM glucose alone or to the combination of 10 mM glucose, KCl and diazoxide from FAF-BSA incubated islets as well.

Figure 3. Effects of FAF-BSA on 3H-inositol efflux from isolated, perifused rat islets stimulated with carbachol.

Figure 3

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Groups of islets were isolated and subjected to a 4 hour incubation in CMRL 1066 medium supplemented with 100 μM Sigma #7888 RIA grade BSA (open circles) or 100 μM Roche FAF-BSA (closed circles, closed triangles) and 3H-inositol. After washing to remove unincorporated label the islets were then perifused with 3 mM glucose. For the next 40 minutes, onset indicated by the vertical line, all groups of islets were stimulated with 50 μM carbachol in the presence of 3 mM glucose plus 0.1 mM cold inositol to restrain reincorporation of labeled 3H-inositol back into phosphoinositide pools. In one group of FAF-BSA incubated islets (closed triangles), 10 mM LiCl was included during the stimulatory period with carbachol. At least 3 experiments were performed under each condition. The asterisk indicates a significant difference (p≤0.05) in efflux from islets incubated for 4 hours in CMRL 1066 (closed circles) when compared to the two other groups at the indicated time points. At all time points after minute 40 of the perifusion these differences were significant.

The amplified 3H-inositol responses observed in response to glucose, glucose plus KCl and carbachol stimulation could not be accounted for by any preferential increase in 3H-inositol incorporation during the 4 hour incubation period. Total amounts of 3H-inositol incorporation-the amount effluxing from the β-cell during the perifusion and the amount of label remaining at the end of the experiment were not significantly different from control or FAF-BSA incubated islets. They averaged 20,768±1,565 cpm/28 islets (n=17) and 22,800±1,216 cpm/28 islets (n=25) in control and FAF-BSA incubated islets respectively.

Confirming that glucose-induced insulin secretion is indeed enhanced by prior exposure to FAF-BSA, a response that was paralleled by a marked enhancement of PLC activation as well, allowed us to make several predictions that could be experimentally tested. First, Straub and Sharp 6 reported that mouse islet secretory responses to glucose were immune to the enhancing impact of incubation in FAF-BSA. If PLC activation underlies amplification, then mouse islet PLC responses should also be unaffected as well. Second, if the proximal activation of PLC results in the distal activation of PKC, then rat islet responses to PMA stimulation should not be significantly enhanced as a result of incubation in FAF-BSA. Experiments were designed to explore these two issues.

We first confirmed that mouse islet responses to 20 mM glucose stimulation are indeed immune to the enhancing impact of a prior 4 hour exposure to 100 μM FAF-BSA. This particular glucose level was chosen in this species since it is approximately equipotent, in terms of insulin secretion, to the 10 mM glucose concentration used for rat islet studies (See Figure 1). The first and second phase insulin secretory responses from control BSA and FAF-BSA pretreated groups were comparable (Figure 4A). Basal secretion rates from both groups averaged 20-25 pg/islet/min. In response to 20 mM glucose stimulation (plus 0.1 mM cold inositol) peak first phase release rates increased to 215±37 pg/islet/min (n=4) from control islets and to 238±38 pg/islet/min (n=5) from FAF-BSA incubated islets. After 40 minutes of stimulation release rates from control BSA islets now averaged 263±41 pg/islet/min (n=4) while those from FAF-BSA islets were 259±29 pg/islet/min (n=5). Consistent with the minimal impact of glucose stimulation on inositol phosphate accumulation in mouse islets 10, 11, only small, minor increments in 3H-inositol efflux rates were noted from control islets during the final minutes of the perifusion (Figure 4B). There was no enhancement of 3H-inositol efflux after incubation of mouse islets in FAF-BSA. Both groups responded to the further addition of 50 μM carbachol with comparable increments in 3H-inositol efflux and this was accompanied by enhanced insulin release as well.

Figure 4. Effects of FAF-BSA on insulin secretion and 3H-inositol efflux from isolated, perifused mouse islets.

Figure 4

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Groups of mouse islets were isolated and subjected to a 4 hour incubation in CMRL 1066 medium supplemented with 100 μM Sigma #7888 RIA grade BSA (open circles) or 100 μM Roche FAF-BSA (closed circles) and 3H-inositol. After washing to remove unincorporated label the islets were then perifused. For the initial 30 minutes of the perifusion, the glucose level was maintained at 3 mM (G3). For the next 40 minutes, onset indicated by the vertical line, both groups of islets were stimulated with 20 mM glucose (G20) plus 0.1 mM cold inositol. For the final 20 minutes, 50 μM carbachol, in the continued presence of both 20 mM glucose plus inositol was added to the medium. Insulin secretion rates (A) were measured and fractional rates of 3H-inositol efflux (B) were calculated. At least 4 experiments were performed under each condition.

The conclusion that PKC activation played a major role in the amplified secretory responses from FAF-BSA incubated islets was supported by the findings that a number of PKC antagonists inhibited this response 6. However, the findings presented above suggest that while PKC is playing an important role, its involvement may be more distal to the proximal activation of PLC. If this is indeed the case it might be predicted that rat islets should be insensitive to the secretory impact of the direct PKC activator PMA. By substituting for PLC-derived diacylglycerol (DAG), exposure of islets to PMA culminates in a PLC-independent secretory response 26. We employed a 200 nM concentration of PMA since it is approximately equipotent to the secretory impact of 10 mM glucose in rat islets and 20 mM glucose in mouse islets. In the presence of 3 mM glucose, the further addition of 200 nM PMA resulted in modest secretion of insulin from control BSA-pretreated rat islets. Peak release rates of about 200-250 pg/islet/min were noted 30-40 minutes after the onset of stimulation (Figure 5). Islets incubated for 4 hours in FAF-BSA, a pretreatment condition that significantly amplifies secretion in response to 10 mM glucose from this species, displayed similar responses to 200 nM PMA stimulation. Thus, our prediction that the proximal activation of PLC results in the distal activation of PKC was supported by the findings presented in Figure 5.

Figure 5. Effects of FAF-BSA on insulin secretion in response to phorbol ester stimulation.

Figure 5

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Groups of 18 rat islets were isolated and subjected to a 4 hour incubation in CMRL 1066 medium supplemented with 100 μM Sigma #7888 RIA grade BSA (open circles) or 100 μM Roche FAF-BSA (closed circles). They were then washed and perifused with 3 mM glucose for 30 minutes to establish stable and basal secretion rates. They were then stimulated, onset indicated by the vertical line, for the next 40 minutes with 200 nM phorbol 12-myristate 13-acetate (PMA). Four experiments were performed under each condition.

Discussion

When confronted with a hyperglycemic challenge, a carefully titrated insulin secretory response from the β-cell is necessary to maintain fuel homeostasis. Failure to activate the secretory apparatus commensurate with the elevation in glucose culminates in diabetes 27. Thus the factors that control insulin exocytosis, while of interest from a purely scientific perspective, have important clinical implications as well. A burgeoning number of studies have probed the nature of the signaling apparatus that so elegantly regulates insulin secretion. Our working hypothesis is that information flow in the PLC/PKC cascade plays a pivotal role in stimulus-response coupling. This concept is supported by a number of observations. First, a tight coupling exists between the ambient glucose level and the calcium-dependent activation of both PLC and insulin secretion 1. Second, glucose-induced insulin release from perfused or freshly studied perifused mouse islets 7-10, 28 is less robust than rat islets, a response pattern paralleled by reduced activation of PLC as well 1, 11. Third, the further addition of the PLC-activating cholinergic agonist carbachol converts the minimal mouse islet response to glucose into one that now simulates the robust response of rat islets to glucose alone 10. We have attributed this amplified response to the established ability of carbachol to activate PLC in mouse islets, a finding also supported by muscarinic receptor type 3 knock out mice 29-31. Finally in rat islets desensitized by chronic exposure to glucose, reduced activation of PLC parallels the secretory defect 1, 32-34.

The concept that PLC/PKC activation plays an important regulatory role in glucose-induced insulin secretion would, however, be substantially strengthened if the PLC response to physiologic glucose concentrations could be enhanced. In theory, at least, this should amplify secretion as well. Recently, Straub and Sharp 6 reported that a 3-6 hour incubation of rat islets in FAF-BSA culminated in a markedly enhanced insulin secretory response to a number of agonists including glucose. Of particular interest was their finding that mouse islets responses to glucose were immune to this manipulation. They focused their attention on the activation of PKC or a DAG response element as the pertinent biochemical alteration and used a variety of PKC inhibitors to substantiate the concept that PKC activation was important in this response. It is of interest to note, however, that all the stimulatory conditions employed in this detailed study also activate PLC as well. Thus not only high glucose, but also α-ketoisocaproate 35, 36, leucine 35, tolbutamide 37, 38 and high potassium in the presence of high glucose and diazoxide 15 all activate PLC. Thus the observations of Straub and Sharp are also compatible with the concept that the proximal activation of PLC results in the distal activation of PKC. We decided to utilize rat islets incubated in FAF-BSA to assess the potential role of PLC in these amplified insulin secretory responses. Comparable studies were also conducted with mouse islets. Our findings strengthen the concept that PLC activation plays a critically important role in the regulation of glucose-induced release and further comment on our findings are appropriate.

We first confirmed the findings previously reported by Straub and Sharp 6 that incubation of rat islets in FAF-BSA markedly enhances glucose-induced insulin secretion. While we employed a lower glucose level in our studies (10 mM as opposed to 16.7 mM), our results clearly support the finding that this type of manipulation markedly amplifies both phases of glucose-induced secretion.

We also demonstrated that, in parallel with augmented secretion, the activation of PLC was also dramatically enhanced in these islets. We thus confirmed the hypothesis that provided the motivating force for these experiments. It is not clear from our studies how incubation in FAF-BSA amplifies glucose-induced activation of PLC and, pari passu, PKC activation and insulin secretion. Initially proposed by Straub and Sharp was the idea that fatty acids in the β-cell exert a tonic inhibitory effect on β-cell signal transduction events. If this is an accurate portrayal of events occurring after exposure to FAF-BSA, then the action of fatty acids would appear to involve the tonic inhibition of PLC since this manipulation markedly amplifies the activation of this enzyme and subsequent secretion as well. These findings also suggest that conditions that elevate fatty acids in vivo might result in impaired PLC activation and a decompensation of insulin secretion. The lipotoxic impact of elevated fatty acids, like glucotoxicity, might thus be mediated by altered activation of PLC as well. Whatever the exact mechanism involved, it is quite clear that from a quantitative perspective this inhibition may play a crucial and previously unappreciated role in the regulation of secretion. Unraveling the biochemical identity of the perturbation that so dramatically enhances secretion promises to be a fruitful, from both a scientific and clinical perspective, avenue of investigation and warrants future investigation.

We monitored the activation of PLC by measuring the efflux of 3H-inositol from 3H-inositol-labeled islets. The strength of this methodology is that secretion along with both the time course and extent of PLC activation can be assessed in the same islets. Many groups have utilized this approach 14, 19, 39-41. There are, however, limitations with this method as well. First, the nature of the inositol phosphates that give rise to the membrane permeant free inositol cannot be determined. Second, reincorporation of free inositol occurs resulting in an underestimation of PLC activation with this approach. The inclusion of cold inositol circumvents this last problem and increases efflux rates of the label. These methodological considerations have been discussed in detail elsewhere 1, 24. Our observations that similar amounts of label are incorporated into both control and FAF-BSA incubated islets and that lithium significantly reduces efflux from stimulated islets support the concept that the activation of PLC accounts for the increase in efflux.

We also confirmed that secretion in response to the combination of glucose and potassium in the presence of diazoxide is also enhanced from FAF-BSA pretreated islets. A parallel potentiation of PLC stimulation also occurred. Augmented PLC activation also occurred in response to carbachol, an agonist thought to activate an isozyme of PLC distinct from that activated by glucose 42. Thus, whatever the nature of the effect produced by incubation in FAF-BSA, it is not confined to glucose but extends to other PLC activators as well.

Several other observations made herein also implicate PLC as the seminal alteration in the amplified insulin secretory response observed from islets incubated in FAF-BSA. While the inhibitor studies employed by Straub and Sharp clearly suggest the involvement of PKC, they do not exclude that PLC activation may also be involved as a more upstream event or as the principal pathway involved in response to FAF-BSA. If enhanced sensitivity of PKC to stimulation underlies amplified secretion from these islets, then we predicted that the response to the phorbol ester PMA should be enhanced. This did not occur and suggests to us that the provision of greater amounts of PKC activating moieties, in particular DAG derived from PLC-mediated hydrolysis of islet phosphoinositide pools, and not the enhanced sensitivity of PKC itself accounts, in large part, for amplification of exocytosis. This does not mean that PKC is not involved in the amplified response that results from this manipulation, but only that it is a downstream participant whose activation culminates in enhanced secretion from these cells.

Species differences separate mouse and rat islet insulin secretory responses to glucose stimulation 1, 7-9, 43, 44. When compared to the robust rising second phase response to glucose stimulation from freshly studied rat islets, freshly studied mouse islet responses from both perfused and perifused islet preparations are reduced and flat. This secretory dichotomy is also paralleled by reduced activation of PLC in mouse islets as well 10. Freshly studied mouse islets also fail to exhibit time-dependent potentiation to prior glucose stimulation 13, 45 and are also markedly less sensitive to the desensitizing effect of prolonged glucose exposure as well 46. We have proposed that PLC activation is involved in all of these disparate time-dependent effects of glucose on the β-cell 1. We confirmed herein that mouse islets are also immune to the amplifying effect of incubation in FAF-BSA, an anomaly previously noted by Straub and Sharp 6 as well. Our earlier studies suggested that the underexpression of a nutrient activated PLC isozyme, perhaps PLCδ, accounts for this species dichotomy 10, 11. A similar explanation may account for the failure of mouse islets to augment secretion after incubation in FAF-BSA. Thus, because they express less of the PLC isozyme that couples glucose to secretion, mouse islets are also less sensitive to the amplifying FAF-BSA results in potentiation was not determined but remains a possibility. Whatever the nature of the exact underlying mechanism involved in these differences, comparative studies using both mouse and rat islet should prove instrumental in its identification.

In the initial report by Straub and Sharp 6, they concluded that a novel PKC isoform or a DAG-binding protein was involved. With regards to PKC, a number of inhibitors were employed to establish its involvement in the amplification process. Our data supports the involvement of PKC as a distal response element in these augmented responses. However, it remains to established how the activation of PLC might account for their findings with these inhibitors. Several points pertinent to this issue deserve comment. First, multiple isozymes of PKC are present in islets and their actions may be either excitatory or inhibitory to the secretory process. For example, the inhibitor of conventional PKC isoforms Go 6976 has a small inhibitory effect on second phase glucose-induced secretion from rat islets but actually potentiates secretion from mouse islets 47. Second, the dose-response effects of calphostin and Ro31-8220 on each of these have not been established. Third, the time-dependent actions of these inhibitors on amplified responses add an additional tier of complexity as initially noted. Finally, it should also be noted that the fatty acid composition of membrane phospholipids synthesized during the 4 hour incubation period may be altered as well, and this might change the nature of the DAG generated during subsequent stimulation. It is clear is that major issues remain to be addressed and explored in more detail. However, considering the magnitude of the amplification process induced by this manipulation, their identification should proceed as expeditiously as possible.

Before concluding several other points should be made. First, from a clinical perspective at least, two future applications of the findings made using this approach should be entertained. First, considering the vulnerability of PLC to desensitization, a process that culminates in impaired secretion as well, pharmacological manipulation of PLC might be a reasonable therapeutic approach to enhance secretion from a failing β-cell, a defect that appears to result in the emergence of Type 2 diabetes. Second, prior to their transplantation, incubation of isolated human islets in FAF-BSA might be considered a useful adjunct to improve or retain their physiologic integrity. In this context it might allow the more rapid restoration of insulin secretion and even enhance their therapeutic potential, thus lowering the often prohibitively large numbers of islets necessary to restore normoglycemia 48. The implementation of these two suggestions would seem to depend, however, on the more precise identification of the exact biochemical mechanisms involved. Future studies should thus focus on this exciting biochemical area replete with potentially important clinical overtones.

Acknowledgments

These studies were supported by NIH grant # 41230

Footnotes

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References

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