Endogenous asymmetric dimethylarginine accumulation contributes to the suppression of myocardial mitochondrial biogenesis in type 2 diabetic rats

Figures

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  Fig. 1. Impairment of glucose tolerance in T2DM rats. The glucose tolerance of control and type 2 diabetes mellitus (T2DM) rats was evaluated by oral glucose tolerance test (OGTT), and the area under concentration-time curve (AUC) of blood glucose concentration was calculated by the proximity ladder shaped formula: AUC (mmol/L*min) = 1/2 × (BG 0 min + BG 30 min) × 30 min + 1/2 × (BG 30 min + BG 60 min) × 30 min + 1/2 × (BG 60 min + BG 90 min) × 30 min + 1/2 × (BG 90 min + BG 120 min) × 30 min. A is the plasma glucose levels, and B is the AUC. Data are expressed as mean ± S.E.M., n = 5. **P < 0.01 vs Control

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  Fig. 2. HE staining of myocardial tissue in T2DM rats. Panel a & b show the hematoxylin-eosin (HE) staining of left ventricular longitudinal sections in control and type 2 diabetic (T2DM) rats; Panel c & d represent the HE staining of left ventricular transverse sections in control and T2DM rats. Scale bar indicate 50 μm

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  Fig. 3. Impairments of mitochondrial function and biogenesis as well as their regulation in the myocardium of T2DM rats. Myocardial ATP content was measured to reflect mitochondrial function (panel a), and mitochondrial biogenesis was evaluated by mitochondrial DNA content, which was expressed by the copy number ratio of mitochondrial genes such as cytochrome C oxidase subunit I (COX I) and nuclear genes like β-actin indirectly (panel b), the activity of mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD, panel c) and the content of lipid peroxidation product malondialdehyde (MDA, panel d) were determined to estimate oxidative stress, the transcription and protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) & uncoupling protein 2 (UCP2) genes were detected by RT-PCR (panel e) and Western blotting (panel f) in the myocardium of control and type 2 diabetic (T2DM) rats. Graphs in panel b, e & f represent the quantification of COX I vs β-actin, PGC-1α and UCP2 vs GAPDH (mRNA) or β-actin (Protein), respectively. Data are expressed as mean ± S.M.E.; n = 4 ~ 5. *P < 0.05, **P < 0.01 vs Control

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  Fig. 4. Disorder of ADMA signal pathway in the myocardium of T2DM rats. Levels of asymmetric dimethylarginine (ADMA) in serum and myocardium were measured in control and type 2 diabetic (T2DM) rats. Activities of dimethylarginine dimethylaminohydrolase (DDAH) and nitric oxide synthase (NOS) as well as content of nitric oxide (NO) reflected by its stable end products nitrite/nitrate were also detected; protein expressions of protein arginine N-methyltransferase 1 (PRMT1), DDAH 1 and DDAH2 as well as endothelial NOS (eNOS) were determined by Western blotting in the myocardium of control and T2DM rats. Panel A & B show the serum ADMA concentration and cardiac ADMA content; panel C displays the activities of DDAH and NOS, panel D presents nitrite/nitrate contents; panel E demonstrates the gel electrophoresis bands of indicated protein expression in the myocardium of rats, and panel F is the quantification of relative protein expression vs β-actin. Data are expressed as mean ± S.M.E., n = 5 ~ 6. *P < 0.05, **P < 0.01 vs Control

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  Fig. 5. Dose- and time-dependent impairments of mitochondrial biogenesis in rat cardiomyocytes induced by ADMA and OA. Rat cardiomyocytes were incubated with various dose of high glucose (15 ~ 30 mM) and asymmetric dimethylarginine (ADMA, 3 ~ 100 μM) or oleic acid (OA, 10 ~ 100 μM) and for different time (12 ~ 72 h) as indications. The content of ADMA was measured by enzyme linked immunosorbent assay (ELISA) and normalized to the protein content of the supernatants of cell lysis. The ratio of the copy number of mitochondrial gene COX Ι to nuclear gene β-actin was used to evaluate the mitochondrial biogenesis. Panel a & b show the dose-response and time course of high glucose and OA on ADMA elaboration; Panel c & d represent the dose-response and time course of ADMA on mitochondrial biogenesis; Panel e displays the dose-response of OA, and panel f displays effects of ADMA combination with high glucose or OA or even both on mitochondrial biogenesis in cardiomyocytes. Graphs in panel c, d, e & f represent the quantifications of COX Ι vs β-actin from 3 independent experiments, respectively. Date are expressed as mean ± S.E.M., n = 3, *P < 0.05, **P < 0.01 vs control or zero group

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  Fig. 6. Exogenous NOS inhibitor L-NNA suppressed mitochondrial DNA and ATP contents, PGC-1α, NRF and UCP2 transcriptions, and PGC-1α expression in the myocardium of rats. Panel a shows ATP content, panel b represents mitochondrial DNA content, panel c exhibits the relative mRNA levels of PGC-1α, NRF and UCP2, panel d displays the relative protein level of PGC-1α in the myocardium of rats treated with L-Nitro-arginine (L-NNA, 50 mg/Kg/d, i.g.) for 8w, respectively. Graphs in panel b ~ d represent the quantifications of COX I vs β-actin (DNA), PGC-1α or NRF or UCP2 vs GAPDH (mRNA) and PGC-1α vs GAPDH (Protein), respectively. Data are expressed as mean ± S.E.M., n = 4 ~ 5, **P < 0.01 vs Control

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  Fig. 7. ADMA suppressed the expression and transcription of PGC-1α by inhibiting its promoter activity but boosting its protein acetylation and phosphorylation. Western blotting was applied to measure expressions of peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), Sirt1, total and phosphorylated Akt and AMPK proteins. Reversed transcription PCR was used to detect the transcription of PGC-1α, luciferase assay was performed to analyze the promoter activity of PGC-1α, and immunoprecipitation was employed to determine the post-translational modification including acetylation and phosphorylation of PGC-1α in cardiomyocytes incubated with ADMA (30 μM) and glucose (30 mM), or oleic acid (OA, 100 μM) for 48 h. Panel a & b demonstrate the dose-response (10 ~ 100 μM) and time course (24 ~ 72 h) of asymmetrical dimethylarginine (ADMA) on PGC-1α expression, respectively; Panel c shows the PGC-1α transcription, and panel d displays the promoter activity of PGC-1α. Panel e & f diagram the acetylation (resveratrol as the negative control) and phosphorylation (AICAR as the positive control) of PGC-1α protein, respectively. Panel g & f are the dose-response and time course of ADMA on…

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  Fig. 8. Rosiglitazone reversed inhibitions of mitochondrial function and biogenesis as well as their regulation disorders in cardiomyocytes incubated with ADMA. ATP content was measured to reflect mitochondrial function (Panel a), and the ratio of the DNA copy number of COX Ι and β-actin genes was used to evaluate mitochondrial biogenesis (mtDNA, Panel b); Transcriptions (mRNA, Panel c) and expression (Protein, Panel d) of PGC-1α and UCP2 genes were detected to assess regulations of mitochondrial biogenesis and function; manganese superoxide dismutase (Mn-SOD) activity and malonyldialdehyde (MDA) content were measured to estimate oxidative stress (Pane e & f) in cardiomyocytes incubated without and with asymmetric dimethylarginine (ADMA, 30 μM) or high glucose (30 mM) or oleic acid (OA, 100 μM) for 48 h, and plus rosiglitazone (RSG, 1 μM) pretreatment for 2 h and then co-incubated with ADMA or high glucose or OA for 48 h, respectively. Graphs in panel b, c & d represent the quantifications of COX Ι vs β-actin (mtDNA), PGC-1α and UCP2 vs GAPDH (mRNA) or vs β-actin (Protein) from 3 independent experiments, respectively. Date are expressed as mean ± S.E.M., n = 3, **P < 0.01 vs control, #P…