Abstract
It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D). To dissect the underlying mechanisms, we performed a multiplexed proteomics study on liver mitochondria isolated from a spontaneous diabetic rat model before/after they were rendered diabetic. Altogether, we identified 1091 mitochondrial proteins, 228 phosphoproteins, and 355 hydroxyproteins. Mitochondrial proteins were found to undergo expression changes in a highly correlated fashion during T2D development. For example, proteins involved in beta-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation, and other bioenergetic processes were coordinately up-regulated, indicating that liver cells confronted T2D by increasing energy expenditure and activating pathways that rid themselves of the constitutively increased flux of glucose and lipid. Notably, activation of oxidative phosphorylation was immediately related to the overproduction of reactive oxygen species, which caused oxidative stress within the cells. Increased oxidative stress was also evidenced by our post-translational modification profiles such that mitochondrial proteins were more heavily hydroxylated during T2D development. Moreover, we observed a distinct depression of antiapoptosis and antioxidative stress proteins that might reflect a higher apoptotic index under the diabetic stage. We suggest that such changes in systematic metabolism were causally linked to the development of T2D. Comparing proteomics data against microarray data, we demonstrated that many T2D-related alterations were unidentifiable by either proteomics or genomics approaches alone, underscoring the importance of integrating different approaches. Our compendium could help to unveil pathogenic events in mitochondria leading to T2D and be useful for the discovery of diagnosis biomarker and therapeutic targets of T2D.
Highlights
It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D)
Despite numerous efforts made in investigating the etiology of T2D, we are still grappling with the enormous complexity of the disease process in which almost every aspect of the body’s metabolism goes awry
We comprehensively identified mitochondrial proteins and their post-translational modifications (PTMs) profiles in rat liver; we closely investigated T2D-derived changes in their protein expression
Summary
Chemicals and Reagents—Goto-Kakizaki rats were purchased from Shanghai Laboratory Animal Center (Jiu-Ting, Shanghai, China). The pellets was washed three times in ice-cold washing buffer containing 200 mM mannitol, 50 mM sucrose, 1 mM EDTA, 0.5 mM EGTA, 0.2 mM Na3VO4, 1 mM NaF, and 10 mM Tris-HCl at pH 7.4 and applied to Nycodenz density gradient centrifugation. For SCX-RP-MS/MS analysis, 300 g of mitochondrial protein digests were loaded onto the SCX column (320 m ϫ 100 mm; Column Technology) in pH 2.0 buffer and eluted by a continuous pH gradient (from pH 2.0 to pH 8.5). To evaluate the rate of incorrect identifications (false discovery rate (FDR)), all the filtered spectra were subjected to database searching against a composite database containing rat protein sequences in both the forward (correct) and reverse (incorrect) orientations. The parent general terms of these specific terms are obtained as well
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