The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds to a specific receptor for activated C kinase (RACK) Although PKC activation enhances insulin release, the specific function of each isoenzyme is unknown. Here we show that following stimulation with glucose, ␣PKC and ⑀PKC translocate to the cell's periphery, while ␦PKC and PKC translocate to perinuclear sites. ␤C2-4, a peptide derived from the RACK1-binding site in the C2 domain of ␤PKC, inhibits translocation of ␣PKC and reduces insulin response to glucose. Likewise, ⑀V1-2, an ⑀PKC-derived peptide containing the site for its specific RACK, inhibits translocation of ⑀PKC and reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both ␣PKC and ⑀PKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of ␣PKC but not ⑀PKC and lowers insulin response by 50%. These findings illustrate the unique ability of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.(Protein kinase C is a family of 11 lipid-dependent serine/ threonine kinases involved in a wide spectrum of signal transduction (7,8). Upon activation, PKC 1 isoenzymes translocate to new cellular sites, including the plasma membrane (9, 10), cytoskeletal elements (11,12), and the nucleus (13,14), as well as other subcellular compartments (15). Many cells are known to contain several isoenzymes (16,17), each localizing to a different cellular site upon stimulation (18). The multiplicity of isoforms of a single enzyme renders the analysis of enzymefunction relationship difficult. Recent work revealed that activated PKC isoenzymes bind anchoring proteins termed RACKs (1-3), believed to be positioned in close proximity to the isoenzyme's substrate. It was further shown that the functional specificity of the PKC isoenzyme is determined, in part, by the differential localization of the isoenzyme-specific RACKs (19). The RACK for ␤PKC, RACK1, has been cloned, and at least part of its binding site on ␤PKC has been mapped to a short sequence within the C2 domain (1). ␤C2-4, a nonopeptide derived from this region, inhibits phorbol ester-induced translocation of the C2-containing isoenzymes but not the translocation of C2-less isoenzymes such as ␦-and ⑀PKC when tested in intact cells (1). A short peptide derived from the V1 region of ⑀PKC, ⑀V1-2, was similarly shown to inhibit the translocation of ⑀PKC, but not ␣-, ␤-, and ␦PKC (20). Furthermore, these isozyme-specific inhibitors blocked the specific function of individual isoenzymes; for example, ⑀V1-2, but not ␤C2-4, inhibited phorbol 12-myristate 13-acetate-induced regulation of the contraction rate in intact cardiomyocytes. Here we use these novel PKC isozyme-specific inhibitors to determine that PKC activation is part of the signals involved in the regulation of glucose-induced insulin secretion and to identify the specif...

cAMP activates multiple signal pathways, crucial for the pancreatic beta-cells function and survival and is a major potentiator of insulin release. A family of phosphodiesterases (PDEs) terminate the cAMP signals. We examined the expression of PDEs in rat beta-cells and their role in the regulation of insulin response. Using RT-PCR and Western blot analyses, we identified PDE3A, PDE3B, PDE4B, PDE4D, and PDE8B in rat islets and in INS-1E cells and several possible splice variants of these PDEs. Specific depletion of PDE3A with small interfering (si) RNA (siPDE3A) led to a small (67%) increase in the insulin response to glucose in INS-1E cells but not rat islets. siPDE3A had no effect on the glucagon-like peptide-1 (10 nmol/liter) potentiated insulin response in rat islets. Depletion in PDE8B levels in rat islets using similar technology (siPDE8B) increased insulin response to glucose by 70%, the potentiation being of similar magnitude during the first and second phase insulin release. The siPDE8B-potentiated insulin response was further increased by 23% when glucagon-like peptide-1 was included during the glucose stimulus. In conclusion, PDE8B is expressed in a small number of tissues unrelated to glucose or fat metabolism. We propose that PDE8B, an 3-isobutyl-1-methylxanthine-insensitive cAMP-specific phosphodiesterase, could prove a novel target for enhanced insulin response, affecting a specific pool of cAMP involved in the control of insulin granule trafficking and exocytosis. Finally, we discuss evidence for functional compartmentation of cAMP in pancreatic beta-cells.

Summary.Insulin release kinetics were studied in perifused islets of Langerhans, isolated from mildly hyperglycaemic and from normoglycaemic spiny mice (Acomys cahirinus), a rodent predisposed to develop spontaneously non-ketotic diabetes. In both groups, insulin response to glucose (16.7 mmol/1) was delayed in comparison with that of rat islets, the release kinetics being analogous to that of human Type 2 (non-insulin-dependent) diabetes. Thirty min priming of the isolated Acomys islets with glucose (16.7 retool/l) resulted in potentiation of the insulin release to a second stimulation. The degree of potentiation decreased exponentially with the time interval between stimulations, showing a tl/2 of 18 min. Induction of potentiation by glucose was time-dependent, giving a maximal effect after 20 min of priming. In addition to overall amplification of the insulin response, printing with glucose accelerated markedly the initial release rates, correcting the dynamics of the response. We conclude that: (1) decreased and delayed insulin secretion is found in Acomys cahirinus before the development of hyperglycaemia; (2) induction of time-dependent potentiation in the islet by priming with glucose corrects the diabetic-type dynamics of insulin release; (3) therefore the deficient insulin release of Acomys is of a functional nature, the mechanism of potentiation bypassing the defect; (4) since insulin release in Acomys resembles that in prediabetic and diabetic man, similar conclusions might apply to the islet dysfunction in Type 2 diabetes.Key words: Insulin secretion, time-dependent potentiation, non-insulin dependent-diabetes, Acomys cahirinus.Glucose intolerance and hyperglycaemia in Type 2 diabetes are the result of an inappropriate pancreatic response to changes in the blood glucose level, accompanied by some decrease in peripheral response to insulin [1]. The inappropriate pancreatic response is characterized by reduction in the sensitivity of fl cells to glucose and delay or absence of the acute insulin response [2][3][4][5][6][7]. To elucidate the metabolic defect(s) of the/3 cells, suitable animal models for Type 2 diabetes are needed. The spiny mouse (Acomys cahirinus) is a semi-desert rodent which has been studied extensively as a model for Type 2 diabetes in man [8][9][10][11][12][13][14][15][16]. Bred in captivity, these animals demonstrate various degrees of glucose intolerance, become obese with age and develop hyperglycaemia. At all stages of glucose intolerance, whether obese or lean, bred in captivity or newly captured, the spiny mouse exhibits decreased insulin response to glucose stimulation, both in vivo and in vitro [10][11][12][13][14]16].Studies in recent years demonstrated that apart from initiating immediate insulin release, glucose generates a time-dependent state of potentiation in the islets of Langerhans, which leads to the amplification of insulin secretion upon subsequent stimulation [17][18][19][20][21][22]. The present study examines the effect of such glucose priming on insulin relea...

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCepsilon in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCepsilon) or expression of inactive PKCepsilon (PKCepsilon-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCepsilon in the perinuclear region, an area known to be rich in ER-Golgi systems, associated with insulin-containing structures. ss'COP1 (RACK2) is the anchoring protein for PKCepsilon. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCepsilon-KD, and 60% in islets expressing RACK2 binding protein (epsilonV1-2); total protein biosynthesis was not affected. In islets expressing epsilonV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCepsilon plays a specific role in mediating the glucose-signal into insulin production: binding to ss'COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.

Summary. The spiny mouse (Acomys cahirinus) exhibits low insulin responsiveness to glucose with a nearly absent early phase release. The alternative fuel-secretagogue glyceraldehyde (10 retool/l) produced a maximal early insulin response in rat islets but failed to affect early response in Acomys; however, it potentiated the late insulin response in both species alike. Glucagon (1.5 ~mol/1) potentiated the early insulin response to intermediate (8.3 retool/l) glucose in rat and Aeomys islets by two-and four-fold, respectively. Glucose doubled cyclic AMP levels in rat islets but no significant response was noted in Aeomys islets. Isobutylmethylxanthine (0.1 retool/l) and forskolin (25 ~mol/1) caused a significant rise in islet cyclic AMP levels in both types of islets; however, neither agent restored the glucose stimulation of cyclic AMP in spiny mouse islets. Forskolin and isobutylmethylxanthine potentiated early and late phase insulin release in both species; however, neither augmented the early response in the Acomys to the degree observed in rat islets. Thus: (1) The early stages of glucose intolerance in man are characterized by severe reduction in early phase insulin response to glucose, and a delayed and often reduced late phase response. The nature of the metabolic defect(s) responsible for the poor responsiveness of the B cell is unknown. The spiny mouse, Aeomys cahirinus, exhibits such diabetic-like kinetics of insulin response to glucose in vivo and in vitro [11][12][13][14][15], and often develops glucose intolerance when raised in captivity. The spiny mouse is, therefore, a convenient animal model for mild Type 2 (non-insulin-dependent) diabetes. Using islets from this animal, it could thus be shown that the early phase of insulin release is more reduced than the late response [12,14,15], and that a first phase insulin response can be partially restored by priming the islets with glucose for 20-60 min, a finding that is pertinent also for glucose intolerant man [16,17].Cumulative evidence suggests that a normal first phase insulin response necessitates activation of B cell adenylate cyclase by glucose stimulation, resulting in a normal cyclic AMP response [1,4,18,19]. The cyclic AMP response of Acomys islets to glucose was found deficient [20]. In the present study we examined further the responsiveness of Acomys islets to a variety of secretagogues in an attempt to define the nature of the deficient signal that causes a low insulin response to glucose, with emphasis on agents that elevate the islet cyclic AMP concentration. The spiny mouse belongs to rodent species unrelated to any common laboratory animal. In previous studies we examined the overall rates of insulin response as well as its dynamics in this species and compared it to those seen in the rat and the mouse [11][12][13][14][15]17]. In the present study emphasis is given to the kinetics of insulin release rather than to its absolute magnitude when comparing results obtained in Acomys and in the control rat islets.