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

OBJECTIVE—Apoptotic destruction of insulin-producing pancreatic β-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 β-cell apoptosis and in islet cell death posttransplantation. Here, we examine the importance of β-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8+ T-cells. RESEARCH DESIGN AND METHODS— Clonal MIN6 β-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-α, interleukin-1β, and interferon-γ. 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 β-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 β-cell destruction and may increase the efficacy of islet allografts in type 1 diabetes

Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells

Aims/hypothesis: This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out. Methods: Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells. Results: TCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death. Conclusions/interpretation: Regulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis. © 2010 Springer-Verlag

Human Mutation within Per-Arnt-Sim (PAS) Domain-containing Protein Kinase (PASK) Causes Basal Insulin Hypersecretion*

Background: The glucose sensor PAS kinase (PASK) plays a fundamental role in pancreatic islet physiology

MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway

Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA) - anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion. © 2012 Brozzi et al