Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the ␤-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (␣ and ⑀) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9712-9716). Using confocal microscopy, we have now established that in response to glucose, PKC-⑀ but not PKC-␣ associates with insulin granules and that green fluorescent protein-tagged PKC-⑀ changes its distribution within the cell periphery upon stimulation of ␤-cells with glucose. Definite evidence of PKC-⑀ requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wildtype PKC-⑀ led to a significant increase in insulin exocytosis. These results suggest that association of PKC-⑀ with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose.In pancreatic ␤-cells, glucose metabolism generates a great variety of intracellular signals that in concert promote insulin exocytosis (for review see Refs. 1 and 2). There are other insulin secretagogues; however, their mode of action occasionally involves the regulation of signaling molecules that are different from the ones regulated in response to glucose. Among those signals, particular attention has been focused on the role of protein kinase C (PKC) 1 because it is known that protein phosphorylation/dephosphorylation can rapidly affect the function of a given protein or signaling molecule in response to a given agonist (3, 4). Definite proof of the role that PKC may have in the process of insulin exocytosis has been difficult to obtain, primarily because of the great diversity of PKC isoforms, the lack of specific inhibitors, and the use of different experimental designs and cell models to study their functions (for review see Ref. 5).Previous attempts to establish the identity of the PKC isoforms that are responsive to glucose in pancreatic ␤-cells, although complex, indicated that the ␣ and ⑀ isoforms are likely candidates (6, 7). Moreover, cell fractionation assays of intact islets exposed to high glucose confirm that both PKC-␣ and PKC-⑀ isoforms indeed translocate to membranes, an event that was even fast enough to coincide with the initial phase of insulin secretion (8). Using antibodies against specific PKC isoforms and selective anchoring peptides that block their activation, it was possible to assess the role of different isoforms during glucose-stimulated insulin secretion (9). Although the results of that study (9) further supported a pivotal role of PKC-␣ and PKC-⑀ by their translocation to the plas...

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl‐CoA thioesterase (MTE‐I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid‐preferring acyl‐CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP‐stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate‐limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE‐I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate‐limiting step. Overexpression of MTE‐I resulted in an increase in cAMP‐induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE‐I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a K m of 2 lM. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC 50 for NDGA was reduced from 50 lM to 25, 7.5 and 4.5 lM in the presence of 0.1, 0.5 and 2 lM triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.