Tumor necrosis factor‐α‐induced changes in insulin‐producing β‐cells

The migration of macrophages and lymphocytes that produce cytokines such as tumor necrosis factor‐α (TNF‐α) causes β‐cell death, leading to type 1 diabetes. Similarly, in type 2 diabetes, the adipocyte‐derived cytokines including TNF‐α are elevated in the circulation, causing inflammation and insulin resistance. Thus, the studies described in this article using TNF‐α are relevant to furthering our understanding of the pathogenesis of diabetes mellitus. We used RINr1046‐38 (RIN) insulin‐producing β‐cells, which constitutively express calbindin‐D 28k , to characterize the effect of TNF‐α on apoptosis, replication, insulin release, and gene and protein expression. Western blots of TNF‐α‐treated RIN cells revealed a decrease in calbindin‐D 28k . By ELISA, TNF‐α‐treated β‐cells had 47% less calbindin‐D 28k than controls. In association with the decline in calbindin‐D 28k , TNF‐α treatment of RIN cells led to a 73% greater increase in changes in intracellular calcium concentration (Δ[Ca 2+ ] i ) in TNF‐α‐treated cells as compared to that in control RIN cells upon treatment with 50 mM KCl; caused a greater increase in the [Ca 2+ ] i following the addition of 5.5 μM ionomycin; increased by more than threefold the apoptotic rate, expressed as the percentage of TUNEL‐positive nuclei to total nuclei; decreased the rate of cell replication by 36%; and increased and decreased selectively the expression of specific genes as determined by microarray analysis. The subcellular localizations of Bcl‐2, an antiapoptotic protein, and Bax, a proapoptotic protein, within RIN cells were altered with TNF‐α treatment such that the two were colocalized with mitochondria in the perinuclear region. We conclude that the proapoptotic action of TNF‐α on β‐cells is manifested via decreased expression of calbindin‐D 28k and is mediated at least in part by [Ca 2+ ] i . © 2005 Wiley‐Liss, Inc.