Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G.
BACKGROUND
In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.


METHODS
Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.


RESULTS
The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial beta-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid beta-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-independent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.


CONCLUSIONS
Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.
Mishra
Rangnath
R
Division of Nephrology, Department of Medicine, School of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA. rxm24@po.cwru.edu
Simonson
Michael S
MS
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004852:Epoxy Compounds / Q000494:pharmacology; D006801:Humans; D019787:Linoleic Acid / Q000494:pharmacology; D050527:Mesangial Cells / Q000166:cytology* / Q000502:physiology; D008928:Mitochondria / Q000201:enzymology*; D019301:Oleic Acid / Q000494:pharmacology; D010168:Palmitates / Q000494:pharmacology*; D015398:Signal Transduction; D013228:Stearates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004852:Epoxy Compounds; D010168:Palmitates; D013228:Stearates; D019301:Oleic Acid; D045304:Cytochromes c; D019787:Linoleic Acid; D004706:Endodeoxyribonucleases; C052391:endonuclease G; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases; C054207:etomoxir