Adipocyte-Mineralocorticoid Receptor Alters Mitochondrial Quality Control Leading to Mitochondrial Dysfunction and Senescence of Visceral Adipose Tissue

Abstract

Mineralocorticoid receptor (MR) expression is increased in the adipose tissue (AT) of obese patients and animals. We previously demonstrated that adipocyte-MR overexpression in mice (Adipo-MROE mice) is associated with metabolic alterations. Moreover, we showed that MR regulates mitochondrial dysfunction and cellular senescence in the visceral AT of obese db/db mice. Our hypothesis is that adipocyte-MR overactivation triggers mitochondrial dysfunction and cellular senescence, through increased mitochondrial oxidative stress (OS). Using the Adipo-MROE mice with conditional adipocyte-MR expression, we evaluated the specific effects of adipocyte-MR on global and mitochondrial OS, as well as on OS-induced damage. Mitochondrial function was assessed by high throughput respirometry. Molecular mechanisms were probed in AT focusing on mitochondrial quality control and senescence markers. Adipo-MROE mice exhibited increased mitochondrial OS and altered mitochondrial respiration, associated with reduced biogenesis and increased fission. This was associated with OS-induced DNA-damage and AT premature senescence. In conclusion, targeted adipocyte-MR overexpression leads to an imbalance in mitochondrial dynamics and regeneration, to mitochondrial dysfunction and to ageing in visceral AT. These data bring new insights into the MR-dependent AT dysfunction in obesity.

Keywords: adipose tissue; metabolic syndrome; mineralocorticoid receptor; mitochondrial dysfunction; oxidative stress; senescence.

Figure 1

Mineralocorticoid receptor overexpression induces target genes overexpression, showing the existence of mineralocorticoid receptor (MR) overactivation in adipose tissue. Ipo8: importin 8; Ptgds: Prostaglandin D2 Synthase; Lcn2: Lipocalin 2. * p < 0.05, *** p < 0.001, Adipo-MROE vs. Ctrl. Adipo-MROE: adipocyte-MR overexpression in mice.

Figure 2

Adipocyte-MR overexpression induces EVAT and plasmatic oxidative stress (OS), with consequent DNA damage in adipocyte-MR overexpressing mice. (a,b) H₂O₂ concentrations, as measured with Amplex Red assay, are increased in EVAT and plasma of Adipo-MROE mice, showing organ and systemic OS. The order of magnitude is the nmol/mg (tissue) or nmol/mL (plasma). (c,d) EVAT phosphorylation of histone H2A.X—as measured by Western blot—and plasma 8-OHdG levels—as measured by ELISA—are increased in Adipo-MROE mice, showing the existence of oxidative DNA damage. Data are expressed as violin plots with quartiles and individual dots. n = 6–8 mice per group; * p < 0.05; ** p < 0.01; *** p < 0.001, Adipo-MROE vs. control. 8-OHdG: 8-Oxo-2’-deoxyguanosine; EVAT: epididymal visceral adipose tissue (AT); MR: mineralocorticoid receptor.

Figure 3

MR-dependent AT OS is linked to mitochondrial OS and altered mitochondrial respiration in the EVAT of adipocyte-MR overexpressing mice. (a) EVAT mitochondrial superoxide-as measured by electron paramagnetic resonance (EPR)-and (b) the mitochondrial hydrogen peroxide concentration-as measured with Amplex Red assay-are increased in Adipo-MROE mice, showing mitochondrial OS; (c) measurement of mitochondrial respiration with the Oroboros O2k-Oxygraph shows decreased state 4 leak respiration and unchanged state 3 maximal coupled respiration in Adipo-MROE mice; leak respiration is not linked to uncoupling proteins (UCP) activation, as shown by the absence of UCP-dependent uncoupling (no effect of the UCP-specific inhibitor GDP) in both groups. Maximal electron transfer chain (ETC) capacity, reflecting ETC function, and reserve capacity-an indicator of energy furniture flexibility-are decreased in Adipo-MROE mice. Data are expressed as violin plots with quartiles and individual dots. n = 6 mice per group; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant, Adipo-MROE vs control. Contr.: control; ETC: electron transport chain; EVAT: epididymal visceral adipose tissue; GDP: guanosine diphosphate; MR: mineralocorticoid receptor; OS: oxidative stress; Resp.: respiration; UCP: uncoupling protein.

Figure 4

Mitochondrial biogenesis is impaired in the EVAT of adipocyte-MR overexpressing mice. (a) mRNA Pgc1α levels, as measured by qRT-PCR, are unchanged in the EVAT of Adipo-MROE mice, whereas Tfam levels are decreased; (b) Consequently, average mtDNA copy number per cell normalised over nuclear DNA copy number is decreased in the EVAT of Adipo-MROE mice. (c) Protein levels of SIRT1 and SIRT3 are decreased in the EVAT of Adipo-MROE mice; data are expressed as violin plots with quartiles and individual dots. n = 8–10 mice per group; ** p < 0.01; **** p < 0.0001; ns = not significant, Adipo-MROE vs. control. CytB: cytochrome B; EVAT: epididymal visceral adipose tissue; Ipo8: importin 8; MR: mineralocorticoid receptor; mtDNA: mitochondrial DNA; Pgc1a: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; SIRT: sirtuin; Tfam: mitochondrial transcription factor A.

Figure 5

Mitochondrial dynamics are shifted towards fission and mitochondrial degradation, through mitophagy, is impaired in the EVAT of adipocyte-MR overexpressing mice. (a) DRP1 phosphorylation and (b) MFN2 protein levels, as measured by Western blot, are decreased in the EVAT of Adipo-MROE mice, indicating a shift of dynamics towards fission. (c) Protein levels of TOM20 are decreased in the EVAT of Adipo-MROE mice, indicating increased degradation through mitophagy. TOM20 protein levels are normalised by COX4 (mitochondrial protein) to exclude the variation linked to mitochondrial mass. Data are expressed as violin plots with quartiles and individual dots. n = 8 mice per group; * p < 0.05; ** p < 0.01; *** p < 0.001, Adipo-MROE vs. control. COX4: cytochrome c oxidase subunit 4; DRP1: dynamin-related protein 1; MFN2: mitofusin-2; MR: mineralocorticoid receptor; TOM20: translocase of outer membrane subunit 20.

Figure 6

Cellular senescence and OS induced apoptotic pathways are induced in the EVAT of adipocyte-MR overexpressing mice. (a) p53 and p21 mRNA levels as assessed by qRT-PCR are increased in the EVAT of Adipo-MROE mice, while p16 mRNA levels are unchanged; (b) The phosphorylation of the P21 target p66SHC is increased in the EVAT of Adipo-MROE mice, indicating cellular response to OS. Data are expressed as violin plots with quartiles and individual dots. n = 8 mice per group; *** p < 0.001; ns = not significant, Adipo-MROE vs. control. Ipo8: importin 8.

Figure 7

AT MR upregulates mitochondrial fission and mitophagy and inhibits biogenesis and fusion. MR activation in adipocytes (i) disrupts MQC through increased fission and mitophagy and decreased fusion and biogenesis, compromising mitochondrial integrity; (ii) alters mitochondrial respiration and increases mitochondrial OS; as a result, (iii) causes oxidative damage and senescence, leading to AT dysfunction and senescence in obesity. Created with BioRender.com.