Signaling involving FAK, PI3K-Akt and ERK and the pancreatic beta cell

Although FAK plays a central role in several crucial biological functions (such as cell migration and survival, in particular), no study assessing its expression and/or its function in the pancreatic beta cell was reported previous to, or during this thesis project. By contrast, both PI3K-Akt and the MAP kinase ERK pathways had been reported to be involved in the regulation of the function and/or the survival of the pancreatic beta cell, as described below.

1.5.1. PI3K-Akt and the pancreatic beta cell

The expression of the three Akt isoforms Akt1, Akt2 and Akt3 has been reported for the pancreatic islets and for the transformed beta cell lines HIT15, INS-1 and RINm5F (186). Akt expression in islets is weak as compared to adipocytes, and it is prominently expressed in beta cells as compared to non-beta cells (186). Several stimuli have been shown to promote beta cell survival through the PI3K-Akt pathway. These include glucose (at the 10mM-12mM range of concentration) (187), insulin-like growth factor-I (IGF-I) (188; 189), and glucagon-like peptide-1 (GLP-1) (190; 191). Furthermore, constitutive active Akt1 has been shown to protect INS-1 cells against free fatty-acid induced apoptosis (192). However, whether ECM and/or integrins may affect the activity of Akt in the beta cell has not yet been reported.

Several studies describing transgenic mice expressing constitutively active Akt mutants or dominant-negative (kinase-dead) Akt mutants specifically in pancreatic beta cells have been reported. Overexpression of constitutive active Akt1 (i.e. Akt1 lacking the PH domain, and containing an N-terminal Src-myristoylation domain) specifically in the pancreatic beta cells (using the rat insulin II promoter) has been reported by two groups, and both made similar observations (193; 194). It was observed that this mutation leads to an increase of islet beta cell mass that is due both to an increase in the number of beta cells and an increase in beta cell size (193; 194). In addition, Bernal-Mizrachi et al. reported an increase in the proliferation of islet cells (194). An increase in the number of beta cells scattered throughout the exocrine pancreas was also observed in the transgenic mice, leading to the suggestion that active Akt1 might induce neogenesis of beta cells (194). Both groups reported an improvement of glucose

might differ from the physiological effects of activated Akt1 in vivo. Second, concerns have been raised about the use of the rat insulin promoter (RIP), because it has been reported that this promoter might affect expression of genes in the hypothalamus (195). Bernal-Mizrachi and colleagues have recently reported the generation of transgenic mice overexpressing a kinase-dead mutant of Akt1 (kdAkt1, which serves as a dominant-negative inhibitor of Akt activity), specifically in the pancreatic beta cells (196). The possibly “leaky” RIP promoter was used, however it was verified that there is no aberrant expression of the RIP-kdAkt1 transgene in the hypothalamus. These mice exhibit impaired glucose tolerance and defective insulin secretion, suggesting that Akt1 is essential for normal beta cell function. Intriguingly, islet morphology and susceptibility to apoptosis were reported to not be altered in these mice compared to wild-type mice.

In summary, these different models provide contrasting results. The in vitro studies performed on a wide range of cell types, including beta cells, using different tools, including constitutive active and/or dominant-negative Akt mutants, indicate that Akt plays a general pro-survival role in the pancreatic beta cell. However, in vivo overexpression of the constitutive active Akt mutant specifically in beta cells was reported to induce an increase in basal beta cell apoptosis, while in vivo overexpression of the dominant-negative Akt1 mutant specifically in beta cells was reported to not affect islet morphology or beta cell apoptosis. However, quantification of apoptosis in vivo can be problematic, due to rapid clearance of apoptotic cells from tissues by macrophages (26), and this may explain the discrepancies observed between the in vitro and in vivo models. Furthermore, in vivo overexpression of dominant-negative and/or of dominant-positive Akt mutants might induce non-specific side effects. Therefore, further in vivo studies are mandatory to better define the role of Akt1 in beta cell survival. One potentially interesting model could be beta-cell specific, conditional Akt knock-out mice.

1.5.2. MAP kinase ERK pathway and the pancreatic beta cell

Several stimuli have been shown to activate ERK in the pancreatic beta cell, including cytokines (IL-1β, (197-199)), glucose (200-202), phorbol esters (202), GLP-1 (203; 204) and KCl-induced depolarization (202). However, whether ECM is able to activate the MAP kinase ERK pathway in the pancreatic beta cell has not yet been reported, either before or during this thesis work.

ERK has been involved in the regulation of function (glucose-stimulated insulin secretion) and survival of beta cells, but both of these proposed functions of ERK are controversial. It has been reported by some groups that glucose-induced ERK activity is not involved in glucose-stimulated insulin secretion (200; 205), but that glucose-induced insulin gene transcription is regulated by ERK (201; 206). However, a recent paper has reported that ERK is involved in glucose-stimulated insulin secretion in MIN6 cells by controlling the

phosphorylation of the cytosolic protein synapsin I (207), and it was proposed that different experimental procedures might explain these contrasting results.

The role of the MAP kinase ERK pathway in beta cell survival is also controversial.

Indeed, in the context of cytokine-induced ERK activity, ERK has been reported to be involved in IL-1β-induced nitric oxide (NO) synthesis and NO synthase (NOS) mRNA expression (197), as well as in IL-1β-induced apoptosis (199). Furthermore, the ERK inhibitor PD98059 has been reported to prevent glucose-induced IL-1β release and beta cell dysfunction and death (208).

On the other hand, it has been reported that inhibition of ERK aggravates IL-1β-induced death of the unusual cell lines AN-ins and AN-glu (198). It has also been reported that inhibition of ERK activity does not alter glucose-induced beta cell survival (187), nor serum or IGF-I-induced cell survival (189). Finally, it has been proposed by a rather superficial study that there could be a correlation between suppressed ERK phosphorylation and diminished islet survival consecutive to islet isolation and purification (209).

In summary, although both the PI3K-Akt and MAP kinase ERK pathways have been involved in signaling leading to increased beta cell survival, their anti-apoptotic role remains to be confirmed (for the PI3K-Akt pathway), or is still controversial (for the ERK pathway).

Furthermore, some reports examining their role in pancreatic beta cells remain to be confirmed, due to lack of conclusive results. Finally, the possible effect of ECM on the activity of these pathways in the pancreatic beta cell has not yet been examined.