Impact of MR on mature adipocytes in high-fat/high-sucrose diet-induced obesity

in Journal of Endocrinology
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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.

Downloadable materials

  • Supplemental methods
  • 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 &#x00B1; 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 &#x00B1; 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 &#x00B1; 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 &#x00B1; 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%, PPAR&#x03B3;: 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 &#x00B1; SEM. **p < 0.01

 

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    Generation of adipocyte-specific MR KO mice. (A) Mr mRNA expression level relative to cyclophilin A was assessed by qRT-PCR in the liver, kidney, interscapular brown adipose tissue (BAT), subcutaneous white adipose tissue (Sub fat) and epididymal fat (Epi fat) of MR flox/flox and Adipocyte-specific MR-knockout mice (AdipoMR-KO). (n = 3, for each). (B) Mr mRNA expression relative to cyclophilin A was also assessed in the mature adipocyte fraction (MAF) and stromal vascular fraction (SVF) of epididymal white adipose tissue (n = 3, for each). Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± s.e.m. *P < 0.05, ***P < 0.001.

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    Effect of adipocyte MR deletion on body and organ weight. (A) Male MR flox/flox mice and AdipoMR-KO mice were fed HFHSD for 18 weeks from 5 weeks of age (n = 11 for mice aged 5–13 weeks, n = 10 for mice aged 14–16 weeks, n = 9 for mice aged 17 weeks, n = 7 for mice aged 18–19 weeks, n = 6 for mice aged 20–21 weeks and n = 5 for mice aged >22 weeks in the MR flox/flox group; n = 12 for mice aged 5–13 weeks, n = 11 for mice aged 14–16 weeks, n = 10 for mice aged 17 weeks, n = 8 for mice aged 18–19 weeks, n = 7 for mice aged 20–21 weeks and n = 6 for mice aged >22 weeks in the AdipoMR-KO group). (B) Organ weights of MR flox/flox and AdipoMR-KO mice. Mice fed HFHSD from 5 weeks of age were killed at 23 weeks of age and the weights of different tissues were measured. The liver, kidney, mesenteric fat (Mes fat), epididymal fat (Epi fat), right soleus and gastrocnemius muscles (muscle), subcutaneous fat (Sub fat) and brown adipose tissue (BAT) were weighed (n = 5 in the MR flox/flox group; n = 6 in the AdipoMR-KO group). Data are presented as mean ± s.e.m. *P < 0.05.

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    Effect of adipocyte MR deletion on glucose or lipid metabolism and plasma adipocytokine or steroid levels. HFHSD-fed male mice were fasted from 09:00 and blood samples were collected after 4 h. (A) Fasting glucose levels 0, 4, 8, 12, 14, 16 weeks after HFHSD feeding (n = 11 for 0, 4 and 8 weeks, n = 9 for 12 weeks, n = 7 for 14 weeks, and n = 6 for 16 weeks in the MR flox/flox group; n = 12 for 0, 4 and 8 weeks, n = 10 for 12 weeks, n = 8 for 14 weeks, and n = 7 for 16 weeks in the AdipoMR-KO group). (B) Blood insulin levels 12 weeks after HFHSD feeding (n = 7 in the MR flox/flox group; n = 8 in the AdipoMR-KO group). (C) Plasma NEFA, (D) TG, (E) T-Chol (F) HDL-C, (G) aldosterone, (H) corticosterone, (I) adiponectin, (J) leptin, (K) resistin and (L) MCP-1 levels in blood samples collected close to sacrifice (n = 5 in the MR flox/flox group; n = 6 in the AdipoMR-KO group). Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are mean ± s.e.m.

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    Effect of adipocyte MR deletion on insulin sensitivity and glucose handling. Mice fed HFHSD from 5 weeks of age underwent insulin tolerance test (ITT) 14 weeks after HFHSD feeding (A, n = 7 in the MR flox/flox group; n = 8 in the AdipoMR-KO group) and intra-peritoneal glucose tolerance test (GTT) 16 weeks after HFHSD feeding (B, n = 6 in the MR flox/flox group; n = 7 in the AdipoMR-KO group). Data are presented as mean ± s.e.m.

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    Epididymal fat mRNA analysis and 8-isoprostane levels, and BAT Ucp1 levels in HFHSD model. (A) Epididymal fat mRNA levels of steroid receptor and steroid metabolic enzyme genes. (B) Lipogenic genes. (C) Lipolytic genes. (D) Adipocytokine genes and (E) other secretary cytokine genes. (F) 8-Isoprostane, a marker of oxidative stress in epididymal fat. (G) Epididymal fat mRNA levels of oxidative stress-associated genes. (H) Thermogenesis genes. (I) BAT Ucp1 mRNA levels. (n = 5 in the MR flox/flox group; n = 6 in the AdipoMR-KO group.) Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± s.e.m.

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    mRNA levels and appearance of mature primary adipocytes from MR flox/flox and AdipoMR-KO mice. (A) Magnification image (200×) of mature primary adipocytes on day 7 from MR flox/flox and AdipoMR-KO mice. The levels of (B) steroid-related genes, (C) lipogenic genes, (D) lipolytic genes, (E) adipocytokine genes and (F) inflammation genes. Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± s.e.m. **P < 0.01. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0026.

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    Liver mRNA levels and TG content in MR flox/flox and AdipoMR-KO mice of the HFHSD model. (A) mRNA expression levels of lipogenic genes. (B) Liver TG content, as measured after HFHSD feeding. (C) Gluconeogenesis-related genes (G6Pase and PEPCK). (n = 5 in the MR flox/flox group; n = 6 in the AdipoMR-KO group.) Open bars, MR flox/flox mice; solid bars, AdipoMR-KO mice. Data are presented as mean ± s.e.m.

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