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

5. Glucose-induced insulin secretion: role of protein kinase C activation

5.1 Protein kinase C activation in freshly studied islets

The response of the perfused rat pancreas preparation to glucose stimulation is a biphasic release of insulin. In freshly-isolated rat islets, the addition of a stimulatory glucose level (15-20mM) also results in a dramatic and sustained biphasic insulin secretory response (58, 59) (See also Figure 1). In particular, the magnitude of the second phase response is 30-40 fold greater than prestimulatory secretion rates. The calcium-dependent activation of PLC also occurs and IP levels, used as a surrogate marker for PLC activation, increase 300-400% (41, 47) (See Figure 2). Since, in addition to the inositol phosphates, PLC-mediated PI hydrolysis results in the generation of diacylglycerol (60), the endogenous activator of PKC (61), the activation of this enzyme might be anticipated as well. In fact, studies in vivo or in vitro with the perfused rat pancreas (62) or perifused rat islets (63) have demonstrated that PKC translocates from a predominantly cytoplasmic compartment to a membrane one, a response often used to indicate its activation. Furthermore, the phosphorylation state of the MARCKS protein, an established protein target for activated PKC, is increased (64). Blocking PKC activation with low levels of the inhibitor staurosporine reduces both MARCKS phosphorylation (64) and second phase secretion from rat islets (65) consistent with an important contributory role for PKC in determining the effects of glucose on the ß-cell. PKC activation by glucose or TPA also increases cAMP levels in islets, providing an additional second messenger mechanism to amplify insulin secretion (66, 67). However, based on studies with cultured islets, the conclusion has been reached that PKC activation may not be involved in glucose-induced insulin secretion (68, 69). The reasons for this continuing controversy and a possible resolution to this issue deserve further comment.

5.2 PKC Activation and Glucose-induced Insulin Secretion from Cultured Rat Islets

In a series of early studies using the phorbol ester TPA, an exogenous PKC activator, to stimulate the ß-cell we suggested that the activation of PKC may be playing an important role in the rising second phase secretory response evoked by glucose (70). We based this suggestion on the slowly rising and sustained release of insulin that followed ß-cell exposure to the phorbol ester (67, 70), a response that qualitatively was somewhat reminiscent to that seen with glucose stimulation. This concept-that PKC activation regulated at least in part glucose-induced insulin secretion-has been both challenged and supported. Most of the negative data concerning the role of PKC activation has been generated with islets cultured for various periods of time after isolation. However, the activation of PKC is not the only aberration cultured islets demonstrate. When stimulated by high glucose, the accumulation of IPs in one-day cultured islets is reduced (71), high glucose fails to translocate PKC to the membrane (68) , islet content of PLCdelta1 falls by about 50% (unpublished observation), residual insulin release is immune to inhibition by staurosporine (72) and, when primed by high glucose, time-dependent potentiation is not induced (unpublished observation). Most telling, however, is the minimal insulin secretory response observed when cultured rat islets are stimulated with glucose (68, 71). The magnitude of this response, 4-5 fold above basal stimulatory rates (See Figure 3), stands in contrast to the robust 30-40 fold increase seen from the perfused pancreas preparation or from our freshly-isolated rat islets. The decline in islet responsiveness to glucose observed after culturing is not confined to rat islets. Cultured human islets suffer the same functional decline. For example the human ß-cell response to glucose stimulation in vivo is a 10-20 fold increase in second phase insulin secretion rates (10, 12). In sharp contrast the second phase insulin secretory response to 28mM glucose from cultured human islets is flat (72), less than 2-fold greater than prestimulatory secretion rates and is paralleled by the minimal activation of PKC as well (72).

Figure 3. Glucose-induced Insulin Secretion is Impaired from Rat Islets after Short Term Culture. Two groups of rat islets were studied. One group (closed circles) was perifused with 3mM glucose (G3) for 30 min and with 15mM glucose (G15) immediately after isolation. The second group (open circles) was cultured 22-24 hours in CMRL-1066 medium supplemented with 10% fetal calf serum prior to perifusion. The glucose level of the medium was 5-5.5mM. Note the dramatic reduction in secretion from cultured islets.

5.3 Insulin secretion from PKC-depleted rat islets

In an attempt to establish the involvement of PKC activation in the sequence of biochemical events which regulate physiologic glucose-induced insulin secretion from rat ß-cells, several groups have cultured islets in the presence of TPA to deplete the islet of PKC. There are actually at least three issues here but only one is usually addressed. First is the effect of culturing which alone impairs glucose-induced insulin secretion (71, 73, 74) irrelevant of any other additions to the culture medium. The second is the added effect of sustained PKC stimulation on an islet whose secretory integrity is deteriorating as a result of culturing. Finally, the tacit assumption that only PKC content is altered and that all of the protein substrates for PKC remain in a basal or unstimulated state is unwarranted. Since PKC activation exerts long term effects on ß-cell sensitivity, effects presumably mediated by the sustained phosphorylation of PKC substrates (75), the fact that PKC is depleted does not address the possible contribution of these phosphoproteins to insulin release (76). To emphasize the problem with cultured islets one only has to study the secretory data from one report (69) often cited as evidence that PKC activation is not involved in the physiologic regulation of glucose-induced insulin secretion. In this study, the insulin secretory response after 20-24 hour of culture from control islets stimulated with 5.6mM glucose was 0.44 ng/h per islet. The insulin secretory response of cultured control islets to 20mM glucose increased to 0.75 ng/h per islet and less than a doubling of insulin output occurred. This observation stands in sharp contrast to the 30-fold increase in release rates we and others have obtained with freshly isolated islets and casts a considerable doubt on the physiologic significance of any further observations made with such profoundly impaired islets irrelevant of their PKC content.

To conclude that PLC/PKC activation is not involved in the physiologic regulation of insulin secretion from a preparation (cultured islets) which differs in several substantial ways from the responses of fresh islets is inappropriate. We have repeated many of these studies with cultured rat islets and arrived at the same conclusion (71). In islets whose insulin secretory responsiveness to glucose is dramatically impaired by culturing, the activation of PLC/PKC signaling events may not be involved in the minimal insulin secretory responses observed. We (54, 55) and others (39) have dealt with this issue in several articles. However, studies with cultured islets have reinforced our conviction that information flow in the PLC/PKC signaling system is an essential component of physiologic glucose-induced insulin secretion.