The Role And Pathway Of Polyunsaturated Fatty Acids-induced Mitochondrial Dysfunction In Insulin Resistance

BACKGROUD: Defective mitochondrial function has been observed in metabolism syndrome, type 2 diabetes mellitus and cardiovascular disease, and is proposed to be a major contributing factor in the pathogenesis and progression of the disease. While the mechanism leading to mitochondrial dysfunction in diabetes remains under intensive investigation, a critical role for nonesterified fatty acid (NEFA) and/or fatty acid metabolites is emphasized by an increasing body of evidence. NEFA may influence mitochondrial function by alterations in gene expression, metabolism, and/or mitochondrial Ca²⁺ ([Ca²⁺]m) homeostasis. We have previously reported that polyunsaturated fatty acids (PUFA) induce Ca²⁺ efflux from mitochondria, an action that may deplete [Ca²⁺]m and thus contribute to NEFA-responsive mitochondrial dysfunction. Here we investigate the mechanism of PUFA-induced mitochondrial Ca²⁺ efflux (PIMCE) and the molecular identities required for PIMCE pathway.MOTHODS: Investigated PIMCE of various rat tissues using isolated mitochondria by fluorescent Ca²⁺ indicators. According to difference of PIMCE among different tissues, design experiments to find out the interested protein molecular identities in PIMCE pathway. Selecte the human teratocarcinoma cell line NT2 and treat NT2 to differentiate to target cell. Analysis of the mitochondrial protein from NT2 and differentiated cells with 2-dimensional gel electrophoresis indicates that the expression level of a number of proteins is largely change. The protein spots of interest in the gel are analyzed with capillary-HPLC-electrospray tandem mass spectrometry and the results identity these protein molecular. on the basis of the above results, further studies with eligible pharmacological inhibitors and RNA interference (RNAi) indicate the role of the protein in PIMCE pathway by fluorometer and laser scan confocal imaging analysis system. Show localization of the protein in mitochondria by western blot and immunofluorescence. Data are presented as means±SE. comparisons are performed using a two-tailed Student's t test or analysis of variance (ANOVA). A significant difference was defined at p<0.05.RESULTS: 1. Mitochondria from rat brain shows diminished PIMCE responses compared to mitochondria of other tissues(p<0.01, liver vs brain, n=6). 2. PIMCE is attenuated following differentiation of NT2 cells to neuronal cells with retinoic acid (RA). Analysis of the mitochondrial proteins from NT2 and differentiated cells with 2-dimensional gel electrophoresis indicated that the expression was largely reduced following RA-induced NT2 differentiation. Three protein spots of interest in the gels are analyzed with capillary-HPLC-electrospray tandem mass spectrometry, and the results identify these proteins as heat shock protein 90β1 (HSP90β1), a homologue of gp96, glucose-regulated protein 94 (GRP94), and tumor rejection antigen 1; the 150 kD oxygen-regulated protein variant 1; and leucine-rich PPR motif-containing protein, or nodal modulator 2 isoform 2. After a literature search for information on the function of the above identified proteins of interest, the protein HSP90β1 is focused on and is tested. 3. PIMCE in NT2 cells can be blocked by HSP90β1 inhibitor 17-(dimethylaminoethylamino)- 17-demethoxygeldanamycin (17-DMAG) and HSP90β1 RNAi. 4. The presence of HSP90β1 in mitochondria is demonstrated by western blot analysis and immunofluorescence analysis of NT2 cells double-labeled. And the level of HSP90β1 in mitochondria is significantly reduced by 17-DMAG and HSP90β1 RNAi. 5. The comparison of the acute effect of 17-DMAG and novobiocin, two inhibitors of HSP90β1 that bind to the N- and C-terminus in isolated mitochondria, is performed. Addition of 17-DMAG to mitochondria immediately prior to LA has no effect on PIMCE, but addition of novobiocin to mitochondria immediately prior to LA inhibits PIMCE (p < 0.02 ,novobipocin vs control,n=4).CONCLUSION: The present study has identified the essential role of HSP90β1 in LA-induced mitochondrial Ca²⁺ efflux. The data presented in this study demonstrate an important novel function for the well-known chaperone protein HSP90β1, i.e., its involvement in PIMCE, a process by which elevated NEFA in type 2 diabetes may lead to altered mitochondrial Ca²⁺ homeostasis and mitochondrial dysfunction. And the C-terminus of HSP90β1 plays a critical role in PIMCE. The data demonstrate that HSP90β1 plays an essential role in PIMCE and may be a potential therapeutic target for prevention of mitochondrial dysfunction in diseases linked to high NEFA.