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Pathophysiology

Exercise Training Induces Mitochondrial Biogenesis and Glucose Uptake in Subcutaneous Adipose Tissue Through eNOS-Dependent Mechanisms

  1. Elisabetta Trevellin1,
  2. Michele Scorzeto2,
  3. Massimiliano Olivieri1,
  4. Marnie Granzotto1,
  5. Alessandra Valerio3,
  6. Laura Tedesco4,
  7. Roberto Fabris1,
  8. Roberto Serra1,
  9. Marco Quarta2,
  10. Carlo Reggiani2,
  11. Enzo Nisoli4 and
  12. Roberto Vettor1⇑
  1. 1Internal Medicine 3, Endocrine-Metabolic Laboratory, Department of Medicine DIMED, University of Padua, Padua, Italy
  2. 2Department of Biomedical Sciences, University of Padua, Padua, Italy
  3. 3Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
  4. 4Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy
  1. Corresponding author: Roberto Vettor, roberto.vettor{at}unipd.it.
  1. E.N. and R.V. contributed equally to this work.

Diabetes 2014 Aug; 63(8): 2800-2811. https://doi.org/10.2337/db13-1234
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    Figure 1

    Mitochondrial biogenesis in mouse subcutaneous adipose tissue. WT and eNOS−/− mice (n = 8 per group) were swim-trained for 6 weeks, killed, and subcutaneous adipose tissue was collected from the inguinal fat pad. A: Relative mRNA levels were measured by combined reverse transcription (RT) and qPCR techniques (qRT-PCR) using 18s rRNA as the internal control and expressed as fold change. B: mtDNA content was measured by means of real-time PCR and expressed as % of mtDNA copy number per nuclear DNA copy number. C: Representative Western blots show PGC-1α, COX IV, and β-actin immunodetected signals in WAT lysates of mice. SED, sedentary; D: Protein expression levels were measured by Western blot analysis using β-actin as the internal control and are expressed as fold change. All graphs depict mean ± SEM. Two-way ANOVA, *P < 0.05 and **P < 0.01 relative to sedentary mice; †P < 0.05 and ††P < 0.01 relative to WT mice.

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    Figure 2

    Norepinephrine-induced mitochondrial biogenesis and β-adrenergic signaling in mouse subcutaneous adipose tissue. WT and eNOS−/− mice (n = 10 per group) received one intraperitoneal injection of 0.9% saline solution (control [CTRL]) or norepinephrine (5 mg/kg), were killed, and subcutaneous adipose tissue was collected from inguinal fat pad. A: Relative mRNA levels were measured by qRT-PCR in WAT lysates of mice killed 24 h after injection, using 18s rRNA as the internal control and expressed as fold change. B: Representative Western blots show HSL, phospho (P)-HSL, ERK1/2, phospho (P)-ERK1/2, and β-actin immunodetected signals in WAT lysates of mice killed 30 min after the injection with saline (CTRL) or norepinephrine (TREATED) solution. C: Protein expression was measured by Western blot analysis using β-actin as the internal control. The signals obtained from phosphorylated proteins were normalized, each one to the corresponding total protein level. All graphs depict mean ± SEM. Two-way ANOVA, *P < 0.05 relative to control mice; †P < 0.05 relative to WT mice.

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    Figure 3

    Glucose uptake and insulin signaling in mouse subcutaneous adipose tissue. A: For glucose uptake measurement, mice (n = 8 per group) were fasted 8 h and injected with PBS (left) or insulin (0.5 units/kg body weight, right). B: Representative Western blots show AKT, phospho (P)-AKT, GLUT4, and β-actin immunodetected signals in protein lysates obtained from WAT of sedentary (SED) and trained WT and eNOS−/− mice. C: Protein expression levels were measured by Western blot analysis using β-actin as the internal control and expressed as fold change. All graphs depict mean ± SEM. Two-way ANOVA, *P < 0.05 relative to sedentary mice; †P < 0.05 relative to WT mice.

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    Figure 4

    Mitochondrial biogenesis in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated for 72 h with 100 μmol/L vehicle (CTRL) or 100 μmol/L DETA-NO. A: mRNA levels were analyzed by means of qRT-PCR using 18s rRNA as the internal control and expressed as fold change (n = 3 independent experiments). B: MTG dye was used as an indicator of mitochondrial mass in live cells. Mitochondrial area (C) and elongation (D) were analyzed as the percentages of larger (>1.5 μm2) and longer (>3 μm) mitochondria in DETA-NO–treated cells compared with untreated cells (n = 3 independent experiments). Data are expressed as mean ± SEM. Student t tests, *P < 0.05 and **P < 0.01 relative to untreated cells (CTRL).

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    Figure 5

    Glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated for 72 h with vehicle (CTRL) or 100 μmol/L DETA-NO. Cells were serum-starved for 8 h, treated with PBS (basal) or 2 μmol/L insulin for 30 min and subjected to a [3H]-DG uptake assay (A) or immunostained with anti-GLUT4 antibody (B). Fluorescence was detected by confocal microscopy, and ratio between the membrane and cytoplasmic signal was measured by means of image analysis. All data are expressed as mean ± SEM (n = 3 independent experiments). Two-way ANOVA, *P < 0.05 and **P < 0.01 relative to control cells; †P < 0.05 relative to cells in basal conditions.

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    Figure 6

    Mitochondrial biogenesis and membrane potential in human adipocytes. Fully differentiated human adipocytes isolated from subcutaneous adipose tissue were treated for 72 h with control (CTRL) vehicle or 100 μmol/L DETA-NO. A: mRNA levels were analyzed by qRT-PCR using 18s rRNA as the internal control and are expressed as fold change (n = 3 independent experiments). B: mtDNA content was measured by means of qPCR and expressed as % of mtDNA copy number per nuclear DNA copy number (n = 3 independent experiments). Data are expressed as mean ± SEM. Student t tests, *P < 0.05 and **P < 0.01 relative to control cells. C: JC-1 assay was used as an indicator of mitochondrial membrane polarization in live cells. A representative image from three independent experiments is shown.

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    Figure 7

    Glucose uptake and GLUT4 translocation in human subcutaneous adipocytes. Fully differentiated human adipocytes isolated from subcutaneous adipose tissue were treated for 72 h with control (CTRL) vehicle or 100 μmol/L DETA-NO. Cells were serum-starved for 8 h, treated with PBS (basal) or 2 μmol/L insulin for 30 min, and subjected to a [3H]-DG uptake assay (A) or immunostained with anti-GLUT4 antibody (B). C: Fluorescence was detected by confocal microscopy, and the ratio between the membrane and cytoplasmic signal was measured by means of image analysis. All data are expressed as mean ± SEM (n = 3 independent experiments). Two-way ANOVA, *P < 0.05 and **P < 0.01 relative to control cells; †P < 0.05 relative to cells in basal conditions.

Tables

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  • Table 1

    Effect of swim training on mice performance evaluated in vivo

    WTeNOS−/−
    Sedentary (n = 8)Trained (n = 8)Sedentary (n = 8)Trained (n = 8)
    Grip test force (mN/g)207.4 ± 6.6228.3 ± 20.9208.5 ± 5.5221.8 ± 11.3
    Treadmill exhaustion time (%)100.0 ± 6.9133.8 ± 14.7*100.3 ± 16.7100.4 ± 9.7
    • Grip test performance is expressed as generated force (mN) relative to mouse body mass (g). Endurance was evaluated by time to exhaustion in treadmill running at increasing speeds and is expressed relative to the exhaustion time of WT sedentary mice (37.6 ± 2.6 min).

    • * P < 0.05 relative to sedentary.

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Exercise Training Induces Mitochondrial Biogenesis and Glucose Uptake in Subcutaneous Adipose Tissue Through eNOS-Dependent Mechanisms
Elisabetta Trevellin, Michele Scorzeto, Massimiliano Olivieri, Marnie Granzotto, Alessandra Valerio, Laura Tedesco, Roberto Fabris, Roberto Serra, Marco Quarta, Carlo Reggiani, Enzo Nisoli, Roberto Vettor
Diabetes Aug 2014, 63 (8) 2800-2811; DOI: 10.2337/db13-1234

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Exercise Training Induces Mitochondrial Biogenesis and Glucose Uptake in Subcutaneous Adipose Tissue Through eNOS-Dependent Mechanisms
Elisabetta Trevellin, Michele Scorzeto, Massimiliano Olivieri, Marnie Granzotto, Alessandra Valerio, Laura Tedesco, Roberto Fabris, Roberto Serra, Marco Quarta, Carlo Reggiani, Enzo Nisoli, Roberto Vettor
Diabetes Aug 2014, 63 (8) 2800-2811; DOI: 10.2337/db13-1234
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