Deletion of Macrophage Mineralocorticoid Receptor Protects Hepatic Steatosis and Insulin Resistance Through ERα/HGF/Met Pathway - PubMed (original) (raw)

. 2017 Jun;66(6):1535-1547.

doi: 10.2337/db16-1354. Epub 2017 Mar 21.

Yu-Yao Zhang 1 2 3, Chao Li 1 2 3, Lin-Juan Du 1 2 3, Yuan Liu 1 2 3, Xiao-Jun Zheng 1 2 3, Shuai Yan 3, Jian-Yong Sun 3, Yan Liu 1 2, Ming-Zhu Liu 3, Xiaoran Zhang 4, Gang Wei 4, Wenxin Tong 5, Xiaobei Chen 5, Yong Wu 6 7, Shuyang Sun 2 8, Suling Liu 9, Qiurong Ding 3, Ying Yu 10, Huiyong Yin 3, Sheng-Zhong Duan 11 2

Affiliations

Deletion of Macrophage Mineralocorticoid Receptor Protects Hepatic Steatosis and Insulin Resistance Through ERα/HGF/Met Pathway

Yu-Yao Zhang et al. Diabetes. 2017 Jun.

Abstract

Although the importance of macrophages in nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) has been recognized, how macrophages affect hepatocytes remains elusive. Mineralocorticoid receptor (MR) has been implicated to play important roles in NAFLD and T2DM. However, cellular and molecular mechanisms are largely unknown. We report that myeloid MR knockout (MRKO) improves glucose intolerance, insulin resistance, and hepatic steatosis in obese mice. Estrogen signaling is sufficient and necessary for such improvements. Hepatic gene and protein expression suggests that MRKO reduces hepatic lipogenesis and lipid storage. In the presence of estrogen, MRKO in macrophages decreases lipid accumulation and increases insulin sensitivity of hepatocytes through hepatocyte growth factor (HGF)/Met signaling. MR directly regulates estrogen receptor 1 (Esr1 [encoding ERα]) in macrophages. Knockdown of hepatic Met eliminates the beneficial effects of MRKO in female obese mice. These findings identify a novel MR/ERα/HGF/Met pathway that conveys metabolic signaling from macrophages to hepatocytes in hepatic steatosis and insulin resistance and provide potential new therapeutic strategies for NAFLD and T2DM.

© 2017 by the American Diabetes Association.

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Figures

Figure 1

Figure 1

Improved glucose homeostasis and insulin sensitivity in female MRKO-ob mice. A_–_E: Weekly random-fed blood glucose levels, GTT, area under the curve (AUC) of GTT, ITT, and AUC of ITT of female mice. F: Plasma insulin levels of fed or fasted female mice. G: Plasma TGs of female mice. H: Immunoblotting analysis of p-IR, total IR (T-IR), p-Akt (Ser473), and total Akt (T-Akt) in livers of female mice. I: Quantification of immunoblotting results as p-IR/T-IR and p-Akt/T-Akt. n = 6–8. *P < 0.05, **P < 0.01, ***P < 0.001. −, before insulin injection; +, after insulin injection; n.s., not significant.

Figure 2

Figure 2

Improved glucose homeostasis and insulin sensitivity in male MRKO-ob mice treated with estrogen. A_–_E: Weekly random-fed blood glucose levels, GTT, area under the curve (AUC) of GTT, ITT, and AUC of ITT of male mice. F: Plasma insulin levels of fed or fasted male mice. G: Plasma TGs of male mice. H: Immunoblotting analysis of p-IR, total IR (T-IR), p-Akt (Ser473), and total Akt (T-Akt) in livers of male mice. I: Quantification of immunoblotting results as p-IR/T-IR and p-Akt/T-Akt. n = 5–7. *P < 0.05, **P < 0.01, ***P < 0.001. −, before insulin injection; +, after insulin injection; n.s., not significant; Pl, placebo.

Figure 3

Figure 3

Attenuated hepatic steatosis in female and E2-implanted male MRKO-ob mice. A and B: LW/BW and hepatic H&E staining of female mice (n = 6–8). Scale bar = 200 μm. C: Hepatic TGs of female mice (n = 6–8). D and E: LW/BW and hepatic H&E staining of male obese mice implanted with placebo (Pl) or E2 (n = 5–7). Scale bar = 500 μm. F: Hepatic TGs of male obese mice implanted with Pl or E2 (n = 5–7). G: Quantification of TG species based on lipidomic analysis of hepatic lipids extracted from female mice (n = 4). Data are ratios of the MRKO-ob value of each species relative to the mean value of FC-ob group after normalization with hepatic protein concentration. Red represents higher and green lower concentration in MRKO-ob mice compared with FC-ob mice. H: Quantification of PC species based on lipidomic analysis of hepatic lipids from female mice (n = 4). Data are presented the same as G. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

Figure 4

Figure 4

Myeloid MRKO downregulates molecules important in hepatic lipogenesis and lipid storage in female ob/ob mice. A: qRT-PCR analysis of FAS, ACC, SCD-1, Ly6d, and Cidea gene expression in liver samples from female mice (n = 8). B: Immunoblotting analysis of FAS, ACC, SCD-1, Ly6d, and Cidea in liver samples from female mice. C: Quantification of immunoblotting results (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

Figure 5

Figure 5

MR deficiency in KCs reduces lipid accumulation and increases insulin sensitivity in hepatocytes through the ERα/HGF/Met pathway. A: Oil Red O staining of hepatocytes cocultured with FCKC or KOKC in the presence of FFAs and E2. Scale bar = 200 μm. B: Quantification of Oil Red O staining. C: Immunoblotting analysis and quantification of p-IR in hepatocytes from the above-described coculture system. D: Immunoblotting analysis and quantification of p-Akt (Ser473) in hepatocytes from the above-described coculture system. E: ELISA analysis of HGF in supernatants of FCKCs and KOKCs in the presence of FFAs and E2. F: Immunoblotting analysis and quantification of phosphorylated Met (p-Met) in hepatocytes cocultured with FCKCs or KOKCs in the presence of FFAs and E2. G: Immunoblotting analysis of p-Met and total Met (T-Met) in hepatocytes after transfection with scrambled siRNA or Met siRNA. Cells were stimulated with 40 ng/mL HGF for 15 min before collection. H: Quantification of Oil Red O staining of hepatocytes transfected with scrambled siRNA (−) or Met siRNA (+) followed by coculture with FCKCs or KOKCs in the presence of E2 and FFAs. I: Immunoblotting analysis and quantification of p-IR in hepatocytes from the coculture system in H. J: Immunoblotting analysis and quantification of p-Akt (Ser473) in hepatocytes from the coculture system in H. K: qRT-PCR analysis of Cidea gene expression in hepatocytes from the coculture system in H. Data are from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Hep, hepatocytes; −, before insulin injection; +, after insulin injection; n.s., not significant; Sc, scrambled.

Figure 6

Figure 6

MR directly regulates Esr1 in macrophages. A: qRT-PCR analysis of Esr1 gene expression in FCKCs and KOKCs in the presence of E2. B: qRT-PCR analysis of Esr1 gene expression in stable MROV RAW264.7 cells. C: Schematic illustration of the five putative MREs on mouse Esr1 promoter. MREs are labeled by region 1∼5. Arrows flanking each region represent locations of specific primers. D and F: Schematic illustrations of luciferase reporter constructs containing truncated mouse Esr1 promoters with various numbers of putative MREs (R1∼ R5). E and G: Luciferase reporter assays using HEK293FT cells cotransfected with MR-flag plasmid or empty vector (Ctrl) and truncated Esr1 promoter luciferase reporter plasmids (_Esr1_-luc) or control plasmid pGL3-basic. H: qRT-PCR analysis of ChIP products. ChIP was performed by using stable MROV RAW264.7 cells. Antibody against FLAG or IgG was used for immunoprecipitation. I: Gel electrophoresis image of regular PCR products with use of the ChIP products. R1∼ R5, amplification of ChIP products pulled down with anti-FLAG antibody (primers were specific to R1∼ R5, respectively). Data are from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. bp, base pair; M, 100-base pair DNA ladder; n.s., not significant; PC, positive control with primers specific to mouse Sgk1 (a known target gene of MR) promoter; RLU, relative luciferase unit; TSS, transcription start site.

Figure 7

Figure 7

Hepatic Met mediates the protective effects of myeloid MR deficiency in vivo. A and B: Random-fed blood glucose and fasting plasma insulin levels of female FC-ob and MRKO-ob mice injected with AdshScr or AdshMet. C_–_G: GTT, ITT, LW/BW, hepatic TGs, and hepatic H&E staining of female FC-ob and MRKO-ob mice injected with AdshScr or AdshMet. Scale bar = 400 μm. H: Immunoblotting analysis of p-IR, total IR (T-IR), p-Akt (Ser473), and total Akt (T-Akt) in livers of female FC-ob and MRKO-ob mice injected with AdshScr or AdshMet before and after insulin injection. I: Quantifications of immunoblotting results as p-IR/T-IR and p-Akt/T-Akt. J: Working model of the MR/ERα/HGF/Met axis that conveys metabolic signaling from macrophages to hepatocytes. In the liver where macrophages and hepatocytes are situated in proximity, MR deficiency leads to elevated ERα expression followed by enhanced HGF secretion from macrophages in the presence of estrogen. HGF subsequently phosphorylates and activates hepatocyte Met, which mediates the decrease of lipid accumulation and the increase of insulin signaling in hepatocytes and improves hepatic steatosis and insulin resistance in female and E2-implanted male ob/ob mice. n = 6–10. *P < 0.05, **P < 0.01, ***P < 0.001. −, before insulin injection; +, after insulin injection; n.s., not significant.

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