Tissue-specific remodeling of the mitochondrial proteome in type 1 diabetic akita mice

Abstract

Objective: To elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications.

Research design and methods: Mitochondrial matrix and membrane fractions were generated from liver, brain, heart, and kidney of wild-type and type 1 diabetic Akita mice. Comparative proteomics was performed using label-free proteome expression analysis. Mitochondrial state 3 respirations and ATP synthesis were measured, and mitochondrial morphology was evaluated by electron microscopy. Expression of genes that regulate mitochondrial biogenesis, substrate utilization, and oxidative phosphorylation (OXPHOS) were determined.

Results: In diabetic mice, fatty acid oxidation (FAO) proteins were less abundant in liver mitochondria, whereas FAO protein content was induced in mitochondria from all other tissues. Kidney mitochondria showed coordinate induction of tricarboxylic acid (TCA) cycle enzymes, whereas TCA cycle proteins were repressed in cardiac mitochondria. Levels of OXPHOS subunits were coordinately increased in liver mitochondria, whereas mitochondria of other tissues were unaffected. Mitochondrial respiration, ATP synthesis, and morphology were unaffected in liver and kidney mitochondria. In contrast, state 3 respirations, ATP synthesis, and mitochondrial cristae density were decreased in cardiac mitochondria and were accompanied by coordinate repression of OXPHOS and peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1alpha transcripts.

Conclusions: Type 1 diabetes causes tissue-specific remodeling of the mitochondrial proteome. Preservation of mitochondrial function in kidney, brain, and liver, versus mitochondrial dysfunction in the heart, supports a central role for mitochondrial dysfunction in diabetic cardiomyopathy.

FIG. 1.

GO enrichment analysis. Simplified hierarchical trees of selected GO terms (boxes) of the “biological process” category that are enriched to the greatest extent in matrix (A) or membrane (B) fractions of Akita mitochondria using pooled proteomic data from all tissues (complete GO enrichment analyses are presented in online appendix Tables S4 and S5). Significantly enriched GO terms (P < 0.05) are highlighted, and the degree of color saturation of each node positively correlates with the enrichment significance of the corresponding GO term (red = most significant enrichment).

FIG. 2.

Gene expression. Gene expression in liver (A), heart (B), brain (C), and kidney (D) tissue of 12-week-old wild-type and Akita mice normalized to 16S RNA transcript levels (n = 6–8). Values represent fold change in mRNA transcript levels relative to wild type, which was assigned as one (dashed line).

FIG. 3.

Mitochondrial function in the liver. Respiration rates (A, B, D, E, and G) and ATP synthesis rates (C, F, and H) of mitochondria isolated from livers of 12-week-old wild-type (WT) (■) and Akita (□) mice, measured in the presence of succinate/rotenone (A–C) or glutamate/malate (D–F) or palmitoyl-carnitine/malate (G and H) as a substrate (n = 5–7). I: State 3 respiration and ATP synthesis rates were used to calculate ATP-to-O ratios for each substrate. There were no significant differences in any parameter. glu, glutamate; pc, palmitoyl-carnitine; suc, succinate.

FIG. 4.

Mitochondrial function in the kidney. Respiration rates (A, B, D, E, and G) and ATP synthesis rates (C, F, and H) of mitochondria isolated from kidneys of 12-week-old wild-type (WT) (■) and Akita (□) mice, measured in the presence of succinate/rotenone (A–C) or glutamate/malate (D–F) or palmitoyl-carnitine/malate (G and H) as a substrate (n = 5–7). I: State 3 respiration and ATP synthesis rates were used to calculate ATP-to-O ratios for each substrate. *P < 0.05 vs. wild type. glu, glutamate; pc, palmitoyl-carnitine; suc, succinate.

FIG. 5.

Mitochondrial function in the heart. Respiration rates (A, B, D, and E) and ATP synthesis rates (C and F) of mitochondria isolated from hearts of 12-week-old wild-type (WT) (■) and Akita (□) mice, measured in the presence of succinate/rotenone (A–C) or glutamate/malate (D–F) as a substrate (n = 5–7). G: State 3 respiration and ATP synthesis rates were used to calculate ATP-to-O ratios for each substrate. *P < 0.05 vs. wild type. glu, glutamate; suc, succinate.

FIG. 6.

Mitochondrial morphology. Representative longitudinal electron microscopy images of liver (A), kidney (B), brain (C), and heart (D) at a magnification of ×40,000 and quantification of mitochondrial volume density (E) and mitochondrial number (F), in liver, kidney, and heart tissue of 12-week-old wild-type (WT) (■) and Akita (□) mice (n = 4).