cAMP Dose-dependently Prevents Palmitate-induced Apoptosis by Both Protein Kinase A- and cAMP-Guanine Nucleotide Exchange Factor-dependent Pathways in β-Cells

Abstract

Lipid accumulation in pancreatic ␤-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the ␤-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…Also interesting was the fact that FSKmediated activation of the insulin gene was not completely inhibited by PKA inhibitors (maximum 80% inhibition) in the studies by Lawrence and co-workers. This would suggest, as has been found for other GLP-1 actions on the β cell (Kwon et al, 2004b), that PKA is a major regulator of insulin transcription when the levels of cAMP are lower and more localized. There are three putative binding sites [(T/A)GGAAA(A/N)(A/T/C) where N=nucleotide] for NFAT on RIP1 (see Fig.…”

“…As discussed earlier in section 3.3 under conditions of low and localized subcellular increases in cAMP as in those stimulated by GLP-1R activation of AC the preferred pathway appears to be via PKA activation rather than through Epac2. Kwon and co-workers have demonstrated that this is the case for the antiapoptotic properties of GLP-1 in which they also found a PKA-dependent component of the mechanism in the prevention of FFA-induced apoptosis in RINm5F cells (Kwon et al, 2004b).…”

“…In a study on the protective effect of cAMP activators against palmitate-induced apoptosis in RINm5F cells, protection was conferred by an Epac-dependent mechanism upon stimulation with forskolin but a PKA-dependent component was found at the lower concentrations of cAMP generated by GLP-1 (Kwon et al, 2004b). Anchoring proteins have been shown in muscle cells to bind both PKA and Epac proteins and thus coordinate their regulation (Dodge-Kafka et al, 2005); whether such a scenario exists in β cells is not known.…”

Continuous stimulation of human glucagon-like peptide-1 (7–36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes

“…Importantly, the anti-apoptotic action of GLP-1 has also been demonstrated in freshly isolated human islets [13]. Furthermore, GLP-1 increased the survival of immortalised rodent beta cell lines after challenge with various apoptotic stimuli [12,[14][15][16][17]. Because of these actions, GLP-1 offers several potential therapeutic advantages for patients with type 2 diabetes.…”

Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B

“…Recent data show that the actions of GLP-1 that protect against diabetes also include the enhancement of beta cell mass in rodents, through inhibition of beta cell apoptosis, and stimulation of beta cell proliferation and islet neogenesis [16][17][18][19]. Furthermore, GLP-1 and its agonists increase the survival of immortalised rodent beta cell lines when challenged with various apoptotic stimulators, including hydrogen peroxide, fatty acids, streptozotocin and staurosporine [17,[20][21][22]. Importantly, exendin-4 treatment also reduces cytokine-induced apoptosis in purified rat beta cells [17].…”