Active glucocorticoid levels are elevated in the adipose tissue of obesity due to the enzyme 11 beta-hydroxysteroid dehydrogenase type 1. Glucocorticoids can bind and activate both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and pharmacological blockades of MR prevent high-fat diet-induced obesity and glucose intolerance. To determine the significance of MR in adipocytes, we generated adipocyte-specific MR-knockout mice (AdipoMR-KO) and fed them high-fat/high-sucrose diet. We found that adipocyte-specific deletion of MR did not affect the body weight, fat weight, glucose tolerance or insulin sensitivity. While liver weight was slightly reduced in AdipoMR-KO, there were no significant differences in the mRNA expression levels of genes associated with lipogenesis, lipolysis, adipocytokines and oxidative stress in adipose tissues between the control and AdipoMR-KO mice. The results indicated that MR in mature adipocytes plays a minor role in the regulation of insulin resistance and inflammation in high-fat/high-sucrose diet-induced obese mice.
Supplemental Table 1. Primers used in RT-PCR.
Supplemental Figure 1. Cre-mediated excision of floxed alleles in the adipose tissue. Null alleles are seen only in adipose tissues of the AdipoMR-KO mouse. MR f/f, MR flox/flox (control); neg.con., negative control; lad, ladder. More details are described in Supplemental method.
Supplemental Figure 2. Fractionation of adipose tissue. The reliability of the fractionation process was checked by quantification of the mRNA expression levels of (A) adiponectin and (B) Dpp4, relative to those of cyclophilin A by qRT-PCR. Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± SEM. ***p < 0.001(MR flox/flox MAF vs MR flox/flox SVF, n = 3 for each), ###p < 0.001 (AdipoMR-KO MAF vs AdipoMR-KO SVF, n = 3 for each).
Supplemental Figure 3. The size of adipocytes of MR flox/flox and AdipoMR-KO mice. (A) Distribution of adipocyte size. (B) Average adipocyte size (n = 3 for each). Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± SEM.
Supplemental Figure 4. Body weight ,organ weight, plasma glucose and insulin levels of normal-chow-fed mice. (A) Body weight of 28-week-old mice (n = 3 each for MR flox/flox and AdipoMR-KO). (B) Organ weight of 28-week-old mice. (C) 4-h fasting blood glucose and (D) insulin levels of male MR flox/flox and AdipoMR-KO mice (n = 3, for each) at 13 and 17 weeks of age. Mes fat, mesenteric fat; Epi fat, epididymal fat; muscle, right soleus and gastrocnemius muscles; Sub fat, subcutaneous fat; BAT, brown adipose tissue; Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± SEM.
Supplemental Figure 5. mRNA expression levels assumed by Ct value of real-time PCR for reference. Original values were calculated from 2-Ct value. Epi fat, epididymal fat; Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± SEM. The amplification efficiencies were as follows. Mr: 100.9%, Gr: 91.6%, Er alpha: 118.9%, 11bHSD1: 99.7%, Acaca: 102.2%, Fasn: 104.4%, Scd1: 100.9%, Acly: 96.8%, HSL: 99.3%, Atgl: 100.9%, Angptl4: 98.4%, Adiponectin: 101.8%, Leptin: 104.4%, PPARγ: 104%, IL-6: 168.9%, Lipocalin 2: 101.8%, PAI1: 104.4%, Cyba: 93.4%, Sod1: 104%, Catalase: 104.4%, Ucp1: 97.6%, Ppargc1a: 112.7%, G6Pase: 109.2%, PEPCK: 107.2%
Supplemental Figure 6. The mRNA level of Mr in adipocytes. (A) Mr expression in the kidney and epididymal fat of control mice (n = 3, for each). (B) Differences in mRNA expression levels between mature adipocyte fraction and stromal vascular fraction. Epididymal fat of control mice were fractioned and quantified by qRT-PCR (n = 3, for each). (C) Mr expression levels in 3T3-L1 preadipocytes and mature 3T3-L1 adipocytes. mRNA was extracted from 3T3-L1 cells (10-15 passages) and quantified using qRT-PCR (n = 3, for each). Data are presented as mean ± SEM. **p < 0.01