Inhibition of AMP-activated protein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis and CD8+ T-cell-induced cytotoxicity.

OBJECTIVE: Apoptotic destruction of insulin-producing pancreatic beta-cells is involved in the etiology of both type 1 and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge whose sustained activation has recently been implicated in pancreatic beta-cell apoptosis and in islet cell death posttransplantation. Here, we examine the importance of beta-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8(+) T-cells. RESEARCH DESIGN AND METHODS: Clonal MIN6 beta-cells or CD1 mouse pancreatic islets were infected with recombinant adenoviruses encoding enhanced green fluorescent protein (eGFP/null), constitutively active AMPK (AMPK-CA), or dominant-negative AMPK (AMPK-DN) and exposed or not to tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Apoptosis was detected by monitoring the cleavage of caspase-3 and DNA fragmentation. The cytotoxic effect of CD8(+) purified T-cells was examined against pancreatic islets from NOD mice infected with either null or the AMPK-DN-expressing adenoviruses. RESULTS: Exposure to cytokines, or expression of AMPK-CA, induced apoptosis in clonal MIN6 beta-cells and CD1 mouse pancreatic islets. By contrast, overexpression of AMPK-DN protected against the proapoptotic effect of these agents, in part by preventing decreases in cellular ATP, and lowered the cytotoxic effect of CD8(+) T-cells toward NOD mouse islets. CONCLUSIONS: Inhibition of AMPK activity enhances islet survival in the face of assault by either cytokines or T-cells. AMPK may therefore represent an interesting therapeutic target to suppress immune-mediated beta-cell destruction and may increase the efficacy of islet allografts in type 1 diabetes.

Methylglyoxal impairs the insulin signaling pathways independently of the formation of intracellular reactive oxygen species.

Nonenzymatic glycation is increased in diabetes and leads to elevated levels of advanced glycation end products (AGEs), which link hyperglycemia to the induction of insulin resistance. In hyperglycemic conditions, intracellularly formed alpha-ketoaldehydes, such as methylglyoxal, are an essential source of intracellular AGEs, and the abnormal accumulation of methylglyoxal is related to the development of diabetes complications in various tissues and organs. We have previously shown in skeletal muscle that AGEs induce insulin resistance at the level of metabolic responses. Therefore, it was important to extend our work to intermediates of the biosynthetic pathway leading to AGEs. Hence, we asked the question whether the reactive alpha-ketoaldehyde methylglyoxal has deleterious effects on insulin action similar to AGEs. We analyzed the impact of methylglyoxal on insulin-induced signaling in L6 muscle cells. We demonstrate that a short exposure to methylglyoxal induces an inhibition of insulin-stimulated phosphorylation of protein kinase B and extracellular-regulated kinase 1/2, without affecting insulin receptor tyrosine phosphorylation. Importantly, these deleterious effects of methylglyoxal are independent of reactive oxygen species produced by methylglyoxal but appear to be the direct consequence of an impairment of insulin-induced insulin receptor substrate-1 tyrosine phosphorylation subsequent to the binding of methylglyoxal to these proteins. Our data suggest that an increase in intracellular methylglyoxal content hampers a key molecule, thereby leading to inhibition of insulin-induced signaling. By such a mechanism, methylglyoxal may not only induce the debilitating complications of diabetes but may also contribute to the pathophysiology of diabetes in general.