In the present study, we show that type 1 diabetes causes tissue-specific remodeling of the proteome involved in mitochondrial energy metabolism. The hepatic mitochondrial proteome was regulated to the greatest extent (41% of all identified proteins), and the cerebral mitochondrial proteome was regulated the least (13%). The tissue-specific remodeling is not surprising, considering that the mitochondrial proteome composition is quite different among tissues, even in normal animals (24
). The fact that proteins of substrate metabolism were regulated to the greatest extent in liver mitochondria of Akita appears plausible, since the liver plays a central role in the regulation of systemic glucose metabolism, such as maintenance of glucose concentrations by modulating gluconeogenesis under fed and fasted conditions (26
). Thus, hepatic energy metabolism may be particularly sensitive to diabetes-associated changes in systemic concentrations of glucose and insulin.
With the exception of the brain, mitochondrial FAO protein levels did not parallel FAO gene expression in Akita mice. In the liver, FAO gene expression was increased, whereas mitochondrial FAO protein content was reduced. Similarly, FAO gene expression was reduced but FAO protein levels were increased in cardiac tissue, and FAO protein content was increased but FAO gene expression was unchanged in kidney tissue, suggesting that mRNA levels do not predict FAO capacity in liver, heart, and kidney tissue of Akita. Alternative mechanisms that regulate mitochondrial FAO protein content could include 1
) increased mRNA translation, 2
) decreased protein turnover, or 3
) increased import of proteins into the mitochondrion. Modulation of protein translation has been suggested in studies showing that hyperglycemia and hyperinsulinemia increase mRNA elongation and translation via dephosphorylation of eukaryotic elongation factor 2 in proximal tubular epithelial cells and that eukaryotic elongation factor 2 phosphorylation is reduced in renal cortex of type 2 diabetic db/db
). Support also exists for the hypothesis that diabetes may regulate mitochondrial protein import in Akita. The translocase of the inner mitochondrial membrane 44 is induced in kidneys of streptozotocin-induced diabetic mice, and gene delivery of translocase of the inner mitochondrial membrane 44 increases mitochondrial import of manganese superoxide dismutase and glutathione reductase (28
). Thus, future studies will be conducted to determine whether changes in the regulation of mRNA translation or mitochondrial import might regulate protein levels of FAO enzymes independently of changes in gene expression.
The TCA cycle and electron transport chain are important determinants of mitochondrial function. Since the proteome of these pathways was significantly remodeled in hepatic, cardiac, and renal mitochondria of Akita, we measured mitochondrial respiration and ATP synthesis rates. Despite the coordinate induction of OXPHOS subunits in liver mitochondria and the coordinate induction of TCA cycle enzymes in kidney mitochondria, state 3 respiration, FCCP-stimulated respiration, and ATP synthesis did not increase. The absence of differences in liver, brain or kidney mitochondrial function between Akita and nondiabetic controls could indicate a true absence of mitochondrial dysfunction in these tissues. It has to be acknowledged, though, that we investigated at a relatively early stage. Six weeks of diabetes might not have been sufficient to cause mitochondrial damage in liver, brain, and kidney tissue, and whether a longer duration of diabetes could impair mitochondrial function in these tissues cannot be ruled out. Since insulin signaling may regulate mitochondrial function (30
), low but measurable levels of insulin in the Akita mouse may partially offset the detrimental effect of diabetes and/or insulin deficiency on mitochondrial function in this model. Alternatively, the increase in protein content in certain mitochondrial pathways may reflect compensatory changes that offset impaired function elsewhere. Thus, the fact that proteomic changes do not reflect or predict actual metabolic flux rates in these tissues emphasizes the importance of using a systems biology approach including metabolite measurements (metabolomics) in combination with comparative proteomics to better inform the complex interaction of transcriptional and protein changes in the adaptation of mitochondria to diabetes. Our findings contrast with other studies that have reported impaired mitochondrial function in livers and kidneys of streptozotocin-induced diabetic models (5
). Moreover, no impairment in mitochondrial morphology was observed in these tissues. Thus, the Akita model appears to be a unique model of type 1 diabetes that is relatively resistant to diabetes-induced mitochondrial damage in liver and kidney and may reflect the fact that these mice produce measurable amounts of insulin despite severe hyperglycemia (18
In contrast to liver and kidney, mitochondrial function was impaired in Akita hearts using glutamate and succinate as substrates. Functional impairment was associated with reduced protein content of TCA cycle enzymes and OXPHOS subunits in Akita. At the gene level, mRNA content of four of five OXPHOS genes examined was reduced in Akita hearts, and there was a coordinate repression of the transcriptional regulators of mitochondrial mass and function (i.e., PGC-1α, PGC-1β, TFAm, and ERRα). Thus, these results suggest that reduced signaling via the PGC-1 transcriptional regulatory cascade may contribute to reduced TCA cycle and OXPHOS subunit content, leading to compromised mitochondrial function in Akita diabetic hearts. Oxidative damage unlikely contributes to reduced respiration rates since mitochondrial reactive oxygen species production and oxidative damage are not increased in hearts of the Akita mouse model (3
). We cannot rule out that other mechanisms such as altered mitochondrial membrane lipid content or changes in glycosylation of mitochondrial proteins, which are proposed mechanisms for mitochondrial dysfunction in diabetes (31
), may contribute to impaired cardiac mitochondrial function in Akita hearts. Based on the impairment in mitochondrial function and morphology, cardiac mitochondria appear to be affected to the greatest extent in 12-week-old type 1 diabetic Akita mice, relative to other tissues, underscoring an important role for mitochondrial dysfunction in cardiac complications of type 1 diabetes.
Analysis of the cardiac mitochondrial proteome revealed increased abundance of three FAO enzymes: long-chain acetyl-CoA dehydrogenase, acetyl-CoA acyltransferase 2, and hydroxyacyl-CoA dehydrogenase, all of which are essential components of the β-oxidation spiral. This induction is consistent with increased cardiac FAO rates in the Akita mouse and other type 1 diabetic models (3
). It is widely accepted that increased PPARα activity increases fatty acid oxidative capacity in diabetic hearts. Indeed, gene expression of PPARα and its target genes increases in streptozotocin-induced diabetic mice, and transgenic overexpression of PPARα in cardiomyocytes results in a metabolic phenotype similar to the diabetic heart (34
). However, despite increased serum free fatty acid and triglyceride levels in the Akita mouse, expression of PPARα and its target gene medium-chain acyl CoA dehydrogenase was reduced in Akita hearts (3
), suggesting either that increased FAO protein content may not be regulated by PPARα in Akita hearts or the existence of additional regulatory mechanisms that determine fatty acid oxidative capacity in Akita hearts, as discussed above.
In conclusion, tissue-specific remodeling of the proteome of mitochondrial energy metabolism in type 1 diabetic Akita mice was demonstrated. This remodeling was only partially mediated by transcriptional mechanisms. Despite remodeling of the mitochondrial proteome in all tissues investigated, impaired mitochondrial function was only observed in cardiac mitochondria, which we believe reflects greater repression of PGC-1α signaling in the heart relative to other tissues. These results confirm an important role of mitochondrial dysfunction in the pathogenesis of cardiac complications in type 1 diabetes.