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. 2018 Jun 25:2018:3062319.
doi: 10.1155/2018/3062319. eCollection 2018.

Protective Effect of Sex Hormone-Binding Globulin against Metabolic Syndrome: In Vitro Evidence Showing Anti-Inflammatory and Lipolytic Effects on Adipocytes and Macrophages

Hiroki Yamazaki  1 , Akifumi Kushiyama  2 , Hideyuki Sakoda  3 , Midori Fujishiro  4 , Takeshi Yamamotoya  5 , Yusuke Nakatsu  5 , Takako Kikuchi  2 , Sunao Kaneko  6 , Hirotoshi Tanaka  1 , Tomoichiro Asano  5
Affiliations

Protective Effect of Sex Hormone-Binding Globulin against Metabolic Syndrome: In Vitro Evidence Showing Anti-Inflammatory and Lipolytic Effects on Adipocytes and Macrophages

Hiroki Yamazaki et al. Mediators Inflamm. .

Abstract

Sex hormone-binding globulin (SHBG) is a serum protein released mainly by the liver, and a low serum level correlates with a risk for metabolic syndrome including diabetes, obesity, and cardiovascular events. However, the underlying molecular mechanism(s) linking SHBG and metabolic syndrome remains unknown. In this study, using adipocytes and macrophages, we focused on the in vitro effects of SHBG on inflammation as well as lipid metabolism. Incubation with 20 nM SHBG markedly suppressed lipopolysaccharide- (LPS-) induced inflammatory cytokines, such as MCP-1, TNFα, and IL-6 in adipocytes and macrophages, along with phosphorylations of JNK and ERK. Anti-inflammatory effects were also observed in 3T3-L1 adipocytes cocultured with LPS-stimulated macrophages. In addition, SHBG treatment for 18 hrs or longer significantly induced the lipid degradation of differentiated 3T3-L1 cells, with alterations in its corresponding gene and protein levels. Notably, these effects of SHBG were not altered by coaddition of large amounts of testosterone or estradiol. In conclusion, SHBG suppresses inflammation and lipid accumulation in macrophages and adipocytes, which might be among the mechanisms underlying the protective effect of SHBG, that is, its actions which reduce the incidence of metabolic syndrome.

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Figures

Figure 1
Figure 1
SHBG inhibits inflammatory cytokine levels in peritoneal macrophages and differentiated 3T3-L1 cells. (a) Peritoneal macrophages from C57BL/6 mice were treated with SHBG overnight, followed by 1 ng/ml LPS stimulation for 8 hrs. mRNA levels of inflammatory cytokines were measured by RT-PCR. Student's t-test was performed. Data are the means ± S.D. (n = 4, ∗p < 0.05, ∗∗p < 0.01). (b) Peritoneal macrophages from C57BL/6 mice were treated with SHBG protein overnight, followed by 1 ng/ml LPS stimulation for the indicated times. Inflammatory signaling was evaluated by Western blotting. Each band was quantified using ImageJ. Relative intensities are shown. Data are the means ± S.D. (n = 3, ∗p < 0.05, ∗∗p < 0.01). (c) Differentiated 3T3-L1 cells were treated with SHBG proteins overnight, followed by 1 ng/ml LPS or 1 ng/ml TNFα stimulation for 24 hrs. mRNA levels of MCP-1 and IL-6 were measured by RT-PCR. Student's t-test was performed. Data are the means ± S.D. (n = 3, ∗∗p < 0.01).
Figure 2
Figure 2
SHBG inhibits inflammatory cytokine levels in 3T3-L1 cells cocultured with peritoneal macrophages. Differentiated 3T3-L1 cells and peritoneal macrophages from C57BL/6 were cocultured using a transwell system overnight, in culture media with or without SHBG protein. Thereafter, we added 100 pg/ml LPS to the culture media and cells were collected 12 hrs later. mRNA levels of inflammatory cytokines in each cell were measured by RT-PCR. Student's t-test was performed. Data are the means ± S.D. (n = 3, ∗p < 0.05, ∗∗p < 0.01).
Figure 3
Figure 3
SHBG reduces lipid contents of differentiated 3T3-L1 cells with alterations in corresponding protein levels. (a) Differentiated 3T3-L1 cells were treated with SHBG proteins at the indicated concentrations in serum-free media and incubated for 3 days. Oil Red O staining was performed. Representative fluorescent microscopy images are shown. (b) Differentiated 3T3-L1 cells were treated with 20 nM SHBG proteins for 18 or 35 hrs. Glycerol concentrations in culture media were measured by ELISA. Student's t-test was performed. Data are the means ± S.D. (n = 3, ∗p < 0.05). (c) Differentiated 3T3-L1 cells were treated with 20 nM SHBG proteins or 10 μM isoproterenol for 1 or 18 hrs. Intracellular cAMP concentrations were measured. Student's t-test was performed. Data are the means ± S.D. (SHBG 0 and 20 nM: n = 4). (d) Differentiated 3T3-L1 cells were treated with 20 nM SHBG proteins for 3 days. Protein levels of CEBPα and ATGL were evaluated by Western blotting. Each band was quantified using ImageJ. Relative intensities normalized by β-actin are shown. Data are means ± S.D. (n = 3, ∗p < 0.05).
Figure 4
Figure 4
SHBG alters some of the mRNA levels related to lipid metabolism in differentiated 3T3-L1 cells. Differentiated 3T3-L1 cells were treated with 20 nM SHBG proteins for 18 hrs. mRNA levels were measured by RT-PCR. Relative levels normalized by the 36B4 level are shown. Student's t-test was performed. Data are the means ± S.D. (n = 3, ∗p < 0.05, ∗∗p < 0.01).
Figure 5
Figure 5
Coincubation with testosterone or 17β-estradiol did not affect the function of SHBG. (a) Differentiated 3T3-L1 cells were treated with SHBG protein in the presence or absence of 1 μM testosterone (T) or 17β-estradiol (E2) overnight, followed by stimulation with 1 ng/ml LPS for 12 hrs. mRNA levels of MCP-1 and IL-6 were measured by RT-PCR. Student's t-test was performed. Mean ± S.D. (n = 3, ∗p < 0.05, ∗∗p < 0.01). (b) Differentiated 3T3-L1 cells were treated with SHBG protein at concentrations ranging from 0–20 nM in serum-free media containing 20 μM of testosterone (T) or 17β-estradiol (E2). Three days later, Nile Red staining was performed and fluorescence was quantified. The Jonckheere test was performed. Mean ± S.D. (n = 4, ∗∗p < 0.01).
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