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. 2016 Mar;65(3):561-73.
doi: 10.2337/db15-0823. Epub 2015 Dec 30.

Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

Piotr Zabielski  1 , Ian R Lanza  1 , Srinivas Gopala  1 , Carrie J Holtz Heppelmann  2 , H Robert Bergen 3rd  2 , Surendra Dasari  3 , K Sreekumaran Nair  4
Affiliations

Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

Piotr Zabielski et al. Diabetes. 2016 Mar.

Abstract

Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.

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Figures

Figure 1
Figure 1
Insulin deprivation adversely affects skeletal muscle mitochondrial physiology. A: Oxygen consumption (JO2) was not different across groups. Expression of mitochondrial respiratory CI and V proteins (B) and mitochondrial coupling efficiency, measured by respiratory control ratios (RCR) (C) and ADP:O ratio (D), was reduced by insulin deprivation and corrected by insulin treatment. E: Mitochondrial ATP production rate (MAPR) was significantly lower with insulin deprivation and restored with insulin therapy. F: Mitochondrial H2O2 emissions were elevated with insulin deprivation and partially reduced by insulin replacement across all respiratory states. G: O2 consumption as a percent of ROS was higher in STZ–I. H: Increase in mitochondrial ROS production was associated with downregulation of mitochondrial MnSOD. Increased expression of AMP-sensitive adenylate kinase 1 (AK1) confirms the low-energy state of skeletal muscle in insulin-deprived animals. Substrate combinations description: GM, glutamate and malate; SR, succinate and rotenone; PCM, palmitoylcarnitine and malate; PPKM, palmitoylcarnitine, pyruvate, α-ketoglutarate, and malate. Data are means ± SEM (n = 13 per group for mitochondrial functional measurements, n = 6 for Western blot measurements). AA, amino acids; AU, arbitrary unit. *P < 0.05 vs. ND; #P < 0.05 vs. STZ–I group.
Figure 2
Figure 2
The effect of insulin deprivation and treatment on skeletal muscle proteome. A: Compared with ND, STZ–I exhibited reduced expression of mitochondrial proteins but increased proteins linked with FA uptake/transport and glycolysis/glycogen breakdown. These directional trends were reversed in STZ+I (B) but were still visible compared with ND (C). D: Protein markers of oxidative stress and ubiquitination were higher in STZ–I mice compared with ND, and proteins linked with protein synthesis were lower. These directional trends were reversed in STZ+I (E) to the levels comparable with ND mice (F). Only significantly affected proteins are shown on volcano plots (–log10 P > 1.301). Individual protein names and expression values are given in Supplementary Table 2A–F for panels A to F, respectively.
Figure 3
Figure 3
Canonical pathways affected by insulin deprivation and treatment. A: Pathways affected in STZ–I compared with ND animals. B: Pathways affected by insulin treatment (STZ+I) compared with diabetic insulin-deprived (STZ–I) mice. C: Pathways that distinguish between STZ+I and ND mice. The middle column of each graph lists the pathway name and the overall pathway score presented as –log10 P value. The □ bars display the pathway –log10 P value score calculated using only downregulated proteins, whereas ■ bars depict the same variable using only upregulated proteins. Significantly affected pathways display the –log10 P value score >1.3 (P < 0.05). All significantly affected pathways from STZ–I vs. ND, STZ+I vs. STZ–I, and STZ+I vs. ND comparisons are listed in Supplementary Table 3A, B, and C, respectively.
Figure 4
Figure 4
Both insulin deprivation and treatment leads to accumulation of acyl-carnitines and suggests incomplete fatty acid β-oxidation in skeletal muscle of T1D mice. Expression of FA intracellular transport and β-oxidation proteins is affected in insulin-deprived mice and not fully corrected by insulin replacement, as revealed by high-throughput proteomic analysis (A) and Western blot (E). Insulin deprivation increases skeletal muscle short-chain acyl-carnitine content (B and C) and elevates CPT1 and SCAD inhibition indexes (D). Insulin treatment corrects expression of proteins of intracellular FA uptake and short-chain FA β-oxidation (A) and reverses short-chain acyl-carnitine (SC-AC) accumulation and CPT1 and SCAD inhibition indexes (B and C) but leads to accumulation of medium-chain (MC-AC), long-chain (LC-AC), and 3-hydroxy (total OH) acyl-carnitine species (B and C) and increases the LCAD inhibition index (D). SC-AC, C2–C5 acyl-carnitines; MC-AC, C6–C12 acyl-carnitines; LC-AC, C14–C20 acyl-carnitines. Data are means ± SEM (n = 6 per group). AU, arbitrary unit; M/SCHAD, medium-/short-chain 3-hydroxyacyl-coenzyme A dehydrogenase. *P < 0.05 vs. ND; #P < 0.05 vs. STZ–I group.
Figure 5
Figure 5
Inhibition of β-oxidation in palmitate-treated C2C12 cells mimics mitochondrial disturbances observed in skeletal muscle of diabetic animals. Inhibition of β-oxidation by MCPA (SCAD and MCAD inhibitor) significantly decreases mitochondrial O2 consumption (JO2) (A) and mitochondrial coupling efficiency (respiratory control ratio [RCR]) (B) and elevates cellular ROS content (C) in C2C12 cells treated with palmitate. The adverse effects of β-oxidation inhibition were observed only under both palmitate and MCPA treatment (PA+MCPA) and were not observed in control cells treated with MCPA. PA, palmitate-treated cells; PA+MCPA, palmitate- and MCPA-treated cells; MCPA, control cells treated with MCPA. Data are means ± SEM (n = 3 per experiment). AA, amino acids; AU, arbitrary unit. *P < 0.05 vs. control; #P < 0.05 vs. MCPA group; $P < 0.05 vs. PA group.
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