Tumor necrosis factor-alpha-induced changes in insulin-producing beta-cells.
The migration of macrophages and lymphocytes that produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) causes beta-cell death, leading to type 1 diabetes. Similarly, in type 2 diabetes, the adipocyte-derived cytokines including TNF-alpha are elevated in the circulation, causing inflammation and insulin resistance. Thus, the studies described in this article using TNF-alpha are relevant to furthering our understanding of the pathogenesis of diabetes mellitus. We used RINr1046-38 (RIN) insulin-producing beta-cells, which constitutively express calbindin-D(28k), to characterize the effect of TNF-alpha on apoptosis, replication, insulin release, and gene and protein expression. Western blots of TNF-alpha-treated RIN cells revealed a decrease in calbindin-D(28k). By ELISA, TNF-alpha-treated beta-cells had 47% less calbindin-D(28k) than controls. In association with the decline in calbindin-D(28k), TNF-alpha treatment of RIN cells led to a 73% greater increase in changes in intracellular calcium concentration (Delta[Ca(2+)](i)) in TNF-alpha-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 microM 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-alpha treatment such that the two were colocalized with mitochondria in the perinuclear region. We conclude that the proapoptotic action of TNF-alpha on beta-cells is manifested via decreased expression of calbindin-D(28k) and is mediated at least in part by [Ca(2+)](i).
Parkash
Jai
J
Joan C. Edwards School of Medicine, Department of Anatomy, Cell and Neurobiology, Marshall University, Huntington, West Virginia 25704, USA. parkash@marshall.edu
Chaudhry
Muhammad A
MA
Rhoten
William B
WB
D017209:Apoptosis / Q000187:drug effects; D020013:Calcium Signaling; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D020869:Gene Expression Profiling; D020287:In Situ Nick-End Labeling; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007328:Insulin; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha