CAMP Dose-dependently prevents palmitate-induced apoptosis by both protein kinase A- and cAMP-guanine nucleotide exchange factor-dependent pathways in beta-cells.

Guim Kwon,Connie A Marshall,Michael L Mcdaniel,Jean E Schaffer,Kirk L Pappan

doi:10.1074/jbc.m310330200

Guim Kwon, Connie A Marshall + Show 3 more

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https://doi.org/10.1074/jbc.m310330200

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Abstract

Lipid accumulation in pancreatic beta-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the beta-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.

Highlights

Obesity is a leading factor associated with the development of type 2 diabetes
The cyclic AMP analogs, chlorophenylthio-cAMP or dibutyryl cAMP mimicked the effects of IBMX and forskolin, suggesting that the action of IBMX and forskolin is mediated by increases in intracellular cAMP (Figure 2)
Our studies have demonstrated that palmitic acid induces apoptosis in the β-cell line, RINm5F, and intracellular concentrations of cAMP exert a key role in modulating antiapoptotic responses

Summary

Introduction

Obesity is a leading factor associated with the development of type 2 diabetes. Elevated levels of FFA in the circulation of obese subjects have been postulated to cause peripheral and hepatic insulin resistance and impairment of insulin secretion from pancreatic β-cells [1]. The mechanisms responsible for the protective action of cAMP against apoptosis include the synthesis of antiapoptotic proteins, inactivation of pro-apoptotic proteins, and activation of PI 3K-dependent Akt. Interestingly, cAMP has been shown to promote apoptosis in leukemia and glioma cells by abnormal cell cycle regulation [7, 8]. Recent studies indicate that cAMP causes an even broader range of cellular responses by activating signaling pathways that are independent of PKA. Recent evidence indicates that Ca2+induced Ca2+ release is regulated by the cAMP-GEF pathway in the β-cell line, INS-1 [19], and the ability of incretins to potentiate glucose-stimulated insulin secretion from isolated mouse islets involves both a PKAdependent pathway and a PKA-independent pathway. The PKA-independent pathway involves the interaction of GEF-2 and Rim, an insulin granule-associated protein [18]

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