Testosterone Deficiency Promotes Hypercholesteremia and Attenuates Cholesterol Liver Uptake via AR/PCSK9/LDLR Pathways

Abstract

Background: Testosterone deficiency is reportedly correlated with an elevation of cholesterol in plasma, but the mechanism remains unclear. Our objective was to investigate the effects of testosterone deficiency on cholesterol metabolism and the corresponding molecular changes in vivo and in vitro.

Methods: SD rats were randomized into three groups: sham-operated (SHAM), subtotal orchiectomized (SO), and orchiectomized (ORX) and fed for 8 weeks. HepG2 cells were cultured with medium containing testosterone with the final concentrations of 0, 10, 30, and 300 nM. Method of isotope tracing and fluorescence labelling was adopted to investigate cholesterol metabolism. Several key molecules of cholesterol metabolism were also analyzed.

Results: SO and ORX rats displayed dysfunctional liver uptake of cholesterol. HepG2 cells incubated with testosterone of lower and excessive level exhibited reduced capacity of cholesterol uptake. Further investigation revealed that lack of testosterone induced increased proprotein convertase subtilisin/kexin type 9 (PCSK9) and decreased low-density lipoprotein receptor (LDLR) both in vivo and in vitro. Moreover, the androgen receptor (AR) antagonist flutamide mimicked the effects of testosterone deficiency on PCSK9 and LDLR indicating the role of AR as a mediator in triggering attenuating liver cholesterol uptake in which testosterone instead of dihydrotestosterone (DHT) is the major functional form of androgen.

Conclusion: Testosterone deficiency attenuated cholesterol liver uptake mediated by the PCSK9-LDLR pathway, in which AR and testosterone without transforming to DHT play important roles.

Figure 1

Metabolic characteristics of sham-operated (SHAM), subtotal orchiectomized (SO) and orchiectomized (ORX) rats. (a) Serum testosterone level six weeks after operations. Rats in SO and ORX groups displayed markedly reduced testosterone levels as expected. Testosterone level of rats in ORX group were all under the lower limit of the measurement (b) Food intake of the rats during the experimentation. (c) The body weight alteration of rats within 20 weeks. (d) Representative graphs of computed tomography (CT) scanning and three-dimensional reconstructed models (yellow: subcutaneous fat; red: visceral fat) of SHAM, SO and ORX group. Volume of subcutaneous and visceral fat are analyzed from the images. Black and white arrows indicates visceral and subcutaneous fat of SHAM, SO and ORX rats respectively. Data are presented as mean ± SEM. Statistical analyses are unpaired t-test or one-way ANOVA. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001 ns: non-significant.

Figure 2

Testosterone deficiency induced disorder of cholesterol metabolism. (a) Plasma total cholesterol (TC), (b) low-density lipoprotein cholesterol (LDLc), (c) high density lipoprotein cholesterol (HDLc), (d) triglyceride (TG) of the rats. Data are presented as mean ± SEM. Statistical analyses are unpaired t-test or one-way ANOVA. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns: non-significant.

Figure 3

Testosterone deficiency attenuates cholesterol liver uptake. (a) Amount of 3H-cholesterol in the liver of rats 2 hours after the gavage with 5 μCi 3H-cholesterol and 0.1 mg unlabeled cholesterol demonstrating the liver cholesterol uptake (n = 6). (b) Cholesterol uptake of HepG2 cells in different levels of testosterone. Cells were incubated with 0, 10, 30, 300 nM testosterone for 72 h and then BODIPY-labeled low-density lipoprotein (LDL) (red) for 2 h. After wash of PBS for twice, cells were stained with DAPI (blue) and visualized using confocal microscopy. Data are presented as mean ± SEM. Statistical analyses are unpaired t-test or one-way ANOVA. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns: non-significant.

Figure 4

Testosterone deficiency reduces low-density lipoprotein receptor (LDLR) in liver by upregulating proprotein convertase subtilisin/kexin type 9 (PCSK9). Key mediators of liver cholesterol uptake LDLR and PCSK9 were analyzed in vivo and in vitro in different conditions of testosterone by western blotting and real-time quantitative PCR. ((a), (b)) LDLR and ((c), (d)) PCSK9 level in the liver of SHAM, SO, ORX rats. ((e), (f)) LDLR and ((g), (h)) PCSK9 level in HepG2 cells incubated with 0 (control), 10, 30, 300 nM testosterone for 72 h. Data are presented as mean ± SEM. Statistical analyses are one-way ANOVA. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns: non-significant.

Figure 5

Androgen receptor (AR) antagonist flutamide abolishes the testosterone-induced PCSK9 downregulation and LDLR upregulation while 5α reductase inhibitor dutasteride shows no effect. HepG2 cells were incubated with 0, 30 nM testosterone for 72 h or 30 nM testosterone plus 50 μM AR antagonist flutamide or 1.5 μM 5α reductase inhibitor dutasteride for 72 h. Then cells were harvested. ((a), (b)) PCSK9 and ((c), (d)) LDLR were analyzed by western blotting or real-time quantitative PCR. Data are presented as mean ± SEM. Statistical analyses are one-way ANOVA. ^∗P < 0.05; ^∗∗P < 0.001; ^∗∗∗P < 0.01; ns: non-significant.