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

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.

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.

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).

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.

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.