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. 2020 Sep 4;3(1):479.
doi: 10.1038/s42003-020-01202-x.

Progesterone receptor membrane associated component 1 enhances obesity progression in mice by facilitating lipid accumulation in adipocytes

Ryogo Furuhata #  1   2 , Yasuaki Kabe #  3   4 , Ayaka Kanai  1 , Yuki Sugiura  1 , Hitoshi Tsugawa  1 , Eiji Sugiyama  1 , Miwa Hirai  1 , Takehiro Yamamoto  1 , Ikko Koike  1 , Noritada Yoshikawa  5 , Hirotoshi Tanaka  5 , Masahiro Koseki  6 , Jun Nakae  7 , Morio Matsumoto  2 , Masaya Nakamura  2 , Makoto Suematsu  8
Affiliations

Progesterone receptor membrane associated component 1 enhances obesity progression in mice by facilitating lipid accumulation in adipocytes

Ryogo Furuhata et al. Commun Biol. .

Abstract

Progesterone receptor membrane associated component 1 (PGRMC1) exhibits haem-dependent dimerization on cell membrane and binds to EGF receptor and cytochromes P450 to regulate cancer proliferation and chemoresistance. However, its physiological functions remain unknown. Herein, we demonstrate that PGRMC1 is required for adipogenesis, and its expression is significantly enhanced by insulin or thiazolidine, an agonist for PPARγ. The haem-dimerized PGRMC1 interacts with low-density lipoprotein receptors (VLDL-R and LDL-R) or GLUT4 to regulate their translocation to the plasma membrane, facilitating lipid uptake and accumulation, and de-novo fatty acid synthesis in adipocytes. These events are cancelled by CO through interfering with PGRMC1 dimerization. PGRMC1 expression in mouse adipose tissues is enhanced during obesity induced by a high fat diet. Furthermore, adipose tissue-specific PGRMC1 knockout in mice dramatically suppressed high-fat-diet induced adipocyte hypertrophy. Our results indicate a pivotal role of PGRMC1 in developing obesity through its metabolic regulation of lipids and carbohydrates in adipocytes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Progesterone receptor membrane-associated component 1 (PGRMC1) is required for lipid accumulation during 3T3L1 cells differentiation.
a Analyses of protein expressions in differentiated 3T3L1 cells (control, KD#1, or KD#2) by western blotting using antibodies against PGRMC1 or GAPDH. b Oil Red O staining of differentiated 3T3L1. Control or two types of stable PGRMC1-KD 3T3L1 cells (PGRMC1 KD#1 and KD#2) were differentiated and stained with Oil Red O. The microscope images are shown in the upper panels of (b). Graphs in the lower panel of (b) depict the absorbance at 490 nm by Oil Red O (n = 8). c Analyses of mRNA expression of PGRMC1, CEBPβ, PPARγ, or FABP4 in 3T3L1 cells at the indicated time periods during differentiation (control, PGRMC1 KD) by quantitative PCR (qPCR) (n = 10). Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. d Analyses of protein expressions in 3T3L1 control cells during differentiation by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. e Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (Control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data represent mean ± S.E. Statistical analyses were performed using ANOVA with Tukey’s T test. *P < 0.05. †P < 0.05 (vs control day 0 (c)). #P < 0.05 (PGRMC1 KD vs control on the same day (c)).
Fig. 2
Fig. 2. The progesterone receptor membrane-associated component 1 (PGRMC1) expression is enhanced during 3T3L1 cell differentiation.
a Analyses of mRNA expression in 3T3L1 cells treated with 15 μmol l−1 TZD for 2 days by quantitative PCR (qPCR) (n = 4). The graph shows relative fold change by normalizing with mRNA levels in 3T3L1 cells treated without TZD. b Analyses of protein expressions in 3T3L1 cells treated with 15 μmol l−1 TZD by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. c, d Reporter gene assay of mouse PGRMC1 promoter. The reporter constructs of the PGRMC1 promoter containing PPRE sequences (−1695/+1-PGRMC1-Luc, -345/+1-PGRMC1-Luc), or lacking PPRE site (-327/+1-PGRMC1-Luc) (c), or constructs containing a control SV40 promoter, or three repeats of the PPRE or the mutated PPRE (PPRE-mt) upstream of a control SV40 promoter (d) were transfected into 3T3L1 cells and were incubated for 2 days after adding 15 μmol l−1 TZD. The graph shows relative luciferase activity by normalizing with luciferase activity in 3T3L1 cells treated without TZD (n = 4). e, f Analyses of the PGRMC1 expression by treatment with TZD in mice. TZD (5 mg kg−1) was intraperitoneally injected in C57BL/6J mice for 3 consecutive days. The mRNA expressions of PGRMC1, PPARγ, and FABP4 in white adipose tissue (WAT) were analyzed by qPCR (n = 5). e The protein expressions in perirenal WAT were analyzed by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. f Data represent mean ± S.E. Statistical analysis was performed using Student’s T test (a, c, d, and e). *P < 0.05.
Fig. 3
Fig. 3. Progesterone receptor membrane-associated component 1 (PGRMC1) contributed to low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) uptake by regulating the translocation of LDL-R and VLDL-R.
a, b Analyses of the effect to LDL uptake by PGRMC1. a Images of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or PGRMC1-Y113F stained with Alexa Fluor 488-acetylated LDL (green) and DAPI (blue) are shown (scale bar: 10 μm). b Flow-cytometric analysis of fluorescence intensities of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or Y113F after incubation with Alexa Fluor 488-acetylated LDL for 60 min. The graph shows the mean of fluorescence intensities (per 10,000 cells) (n = 4). c, d Analyses of the effect to VLDL uptake by PGRMC1. c Images of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or PGRMC1-Y113F stained with DiI–VLDL (red) and DAPI (blue) are shown (scale bar: 10 μm). d Flow-cytometric analysis of fluorescence intensities of 3T3L1 control cells, PGRMC1 KD cells, or PGRMC1 KD cells expressing shRNA-resistant PGRMC1 WT or Y113F after incubation with DiI–VLDL for 60 min. (n = 4). e Analyses of regulation of the LDL-R and VLDL-R localization by PGRMC1. Protein expressions in whole-cell lysates were detected by western blotting using antibodies against LDL-R, VLDL-R, Tf-R, GAPDH, or PGRMC1. f After plasma membrane fractions of 3T3L1 cells (control and PGRMC1 KD) were extracted, protein expression in the plasma membrane was detected by western blotting using antibodies against LDL-R, VLDL-R, Tf-R, Na-K ATPase α1, or PGRMC1. f, g Co-immunoprecipitation assay for interaction between PGRMC1 and LDL-R, or PGRMC1 and VLDL-R. FLAG-PGRMC1 WT or Y113F was overexpressed in 3T3L1 cells, and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1, anti-LDL-R antibody, or anti-VLDL-R antibody. h Co-immunoprecipitation assay for interaction between endogenous PGRMC1 and LDL-R. 3T3L1 cell lysate was incubated with anti-PGRMC1 antibody or normal rabbit IgG, and then incubated with 10 µl protein A-sepharose beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-LDL-R antibody. Data are represented as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test (b, d). *P < 0.05.
Fig. 4
Fig. 4. Progesterone receptor membrane-associated component 1 (PGRMC1) contributes to glucose uptake by regulating the translocation to the plasma membrane of GLUT4.
a Analysis of the effect of insulin-stimulated 2-deoxyglucose (DG) uptake by PGRMC1. After treatment with or without 0.5 μmol l−1 insulin for 18 min, 3T3L1 cells (control or PGRMC1 KD) were incubated with 1 μmol l−1 2-DG for 20 min, and the 2-DG uptake was measured. The graph shows relative fold change by normalizing with 2-DG uptake of 3T3L1 control cells without treatment of insulin (n = 4). b Analysis of the effect of fatty acid synthesis by PGRMC1 using [13C6]-glucose. After differentiated 3T3L1 cells (control and KD) were incubated with 4.5 g l−1 [13C6]-glucose for 24 h, the fatty acids were extracted. [13C6]-glucose, [13C4–12]-palmitic acid, [13C4–16]-stearic acid, and [13C4–16]-oleic acid in cells were measured by LC/MS. (n = 3). See Supplementary Fig. 7 for more detail. c Analyses of regulation of the GLUT4 translocation by PGRMC1. Plasma membrane fractions were extracted from 3T3L1 cells (control and PGRMC1 KD) treated with or without insulin. The expressed proteins in the plasma membrane or whole-cell lysate were detected by western blotting using antibodies against GLUT4, GLUT1, PGRMC1, phosphorylated Akt (pAkt), Akt, Na-K ATPase α1, or GAPDH. d Co-immunoprecipitation assay for interaction between PGRMC1 and GLUT4. FLAG-PGRMC1 WT or Y113F was overexpressed in 3T3L1 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-GLUT4 antibody. e Co-immunoprecipitation assay for interaction between endogenous PGRMC1 and GLUT4. 3T3L1 cell lysate was incubated with anti-PGRMC1 antibody or normal rabbit IgG, and then incubated with 10 µl of protein A-sepharose beads. Co-immunoprecipitated proteins were detected with western blotting using anti-PGRMC1 or anti-GLUT4 antibody. All data are represented as mean ± S.E. Statistical analysis was performed using Student’s T test. *P < 0.05.
Fig. 5
Fig. 5. Carbon monoxide (CO) interferes with low-density lipoproteins (LDL) and glucose uptake.
a, b Analyses of the effect to LDL uptake by CO-RM. 3T3L1 cells were incubated with 10 μmol l−1 CO-RM or control RuCl3 for 2 h, then treated with 5 mg l−1 Alexa Fluor 488-acetylated LDL. a Images of 3T3L1 cells treated with or without CO-RM and stained with Alexa Fluor 488-acetylated LDL (green) and DAPI (blue) are shown (scale bar: 10 μm). b Flow-cytometry analysis of the fluorescence intensities of 3T3L1 cells after incubation with Alexa Fluor 488-acetylated LDL for 60 min. The graph shows the mean of fluorescence intensities (per 10,000 cells) (n = 4–5). c Analyses of regulation of the LDL-R or VLDL-R localization by CO-RM. Plasma membrane fraction or whole-cell lysate of 3T3L1 cells with or without CO-RM were analyzed by western blotting using antibodies against LDL-R, VLDL-R, or GAPDH. d Analysis of the effect of 2-DG uptake by CO-RM. After treatment of 0.5 μmol l−1 insulin for 18 min with or without 10 μmol l−1 CO-RM for 2 h, 3T3L1 cells were incubated with 1 μmol l−1 2-DG for 20 min, and the 2-DG uptake was measured. The graph shows relative fold change by normalizing with 2-DG uptake of cells without CO-RM (n = 4). e Analyses of regulation of the GLUT4 translocation by CO-RM. Plasma membrane fraction of 3T3L1 cells (with or without treatment with CO-RM) was incubated with or without 0.5 µmol l−1 insulin for 2 h. Protein expressions in the plasma membrane or whole-cell lysate were detected by western blotting using antibodies against either GLUT4, progesterone receptor membrane-associated component 1 (PGRMC1), pAkt, Akt, Na-K ATPase α1, or GAPDH. f 3T3L1 cells were transiently transfected with the shRNA-resistant expression vector of wild-type haem oxygenase 1 (HO-1) (WT) or the H25A mutant (H25A). The protein expressions were analyzed by western blotting using antibodies against HO-1 or GAPDH. g Analysis of the effect of HO-1 to 2-DG uptake. 2-DG uptake was measured in 3T3L1 cells (control or PGRMC1 KD) expressing HO-1 WT or H25A mutant. All data represent as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test. *P < 0.05.
Fig. 6
Fig. 6. Progesterone receptor membrane-associated component 1 (PGRMC1) contributes to the progression of adipocyte hypertrophy in mice.
a Tissue weights of perirenal white adipose tissue (WAT), subcutaneous WAT, brown adipose tissue (BAT), and liver in C57BL/6J mice (WT or PGRMC1 adipose tissue-specific knockout (AKO)) fed normal diet (ND) or high-fat diet (HFD) for 16 weeks. (n = 5–8). b Paraffin sections of mesenteric WAT and subcutaneous WAT from WT and PGRMC1-AKO mice fed ND or HFD stained with hematoxylin and eosin (scale bar: 500 μm). The graph depicts the average adipocyte area (at least 100 cells per mouse) (n = 5). c The paraffin sections of BAT in WT and PGRMC1-AKO mice fed ND and HFD stained with hematoxylin and eosin (scale bar: 500 μm). d Analyses of mRNA expressions in mesenteric WAT of WT and PGRMC1-AKO mice fed ND or HFD by quantitative PCR (qPCR) (n = 5–8). The graph shows relative fold change after normalizing with the mRNA level of GAPDH. e The analyses of protein expressions in mesenteric WAT of WT and PGRMC1-AKO mice fed ND or HFD by western blotting using the antibody against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data are represented as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 7
Fig. 7. Schematic model for the regulation of lipid accumulation in adipocytes by progesterone receptor membrane-associated component 1 (PGRMC1).
When insulin signaling induces adipogenesis, the PGRMC1 gene expression is transactivated by ATF/CREB and PPARγ. The heme-dimerized PGRMC1 interacts with LDL-R, VLDL-R, or GLUT4 and facilitates their translocation to the plasma membrane. Consequently, PGRMC1 contributes to lipid accumulation in adipocytes by regulating the lipid uptake via LDL-R or VLDL-R, or de novo fatty acid synthesis.
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