Pro- and antiapoptotic proteins regulate apoptosis but do not protect against cytokine-mediated cytotoxicity in rat islets and beta-cell lines.
Type 1 diabetes results from islet beta-cell death and dysfunction induced by an autoimmune mechanism. Proinflammatory cytokines such as interleukin-1beta and gamma-interferon are mediators of this beta-cell cytotoxicity, but the mechanism by which damage occurs is not well understood. In the current study, we present multiple lines of evidence supporting the conclusion that cytokine-induced killing of rat beta-cells occurs predominantly by a nonapoptotic mechanism, including the following: 1) A rat beta-cell line selected for resistance to cytokine-induced cytotoxicity (833/15) is equally sensitive to killing by the apoptosis-inducing agents camptothecin and etoposide as a cytokine-sensitive cell line (832/13). 2) Overexpression of a constitutively active form of the antiapoptotic protein kinase Akt1 in 832/13 cells provides significant protection against cell killing induced by camptothecin and etoposide but no protection against cytokine-mediated damage. 3) Small interfering RNA-mediated suppression of the proapoptotic protein Bax enhances viability of 832/13 cells upon exposure to the known apoptosis-inducing drugs but not the inflammatory cytokines. 4) Exposure of primary rat islets or 832/13 cells to the inflammatory cytokines causes cell death as evidenced by the release of adenylate kinase activity into the cell medium, with no attendant increase in caspase 3 activation or annexin V staining. In contrast, camptothecin- and etoposide-induced killing is associated with robust increases in caspase 3 activation and annexin V staining. 5) Camptothecin increases cellular ATP levels, whereas inflammatory cytokines lower ATP levels in both beta-cell lines and primary islets. We conclude that proinflammatory cytokines cause beta-cell cytotoxicity primarily through a nonapoptotic mechanism linked to a decline in ATP levels.
Collier
J Jason
JJ
Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Duke Independence Park Facility, 4321 Medical Park Drive, Suite 200, Durham, NC 27704, USA.
Fueger
Patrick T
PT
Hohmeier
Hans E
HE
Newgard
Christopher B
CB
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014162:Transfection; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000235:genetics
C494774:Bak1 protein, rat; D016207:Cytokines; D007375:Interleukin-1; D034741:RNA, Small Interfering; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D007371:Interferon-gamma; D051057:Proto-Oncogene Proteins c-akt