The incidence of NAFLD has increased in modern society, with an increasing population of individuals with obesity [22]. This surge is more prevalent in males over 45 years of age than in females of the same age [8]. Considering the decline in the total androgen levels in males around the age [9], the relationship between androgens and NAFLD is an important topic, although it is established that NAFLD is strongly linked to obesity and type2 diabetes [23,24]. In line with this argument, the present study examined the effects of a fat-rich diet and showed that orchiectomized mice, deprived of endogenous androgens, are predisposed to liver steatosis compared to naive mice. This evidence suggests that the presence of androgens might protect against the development of NAFLD.

When mice were fed a fat-rich diet, body weight and hepatic TG accumulation concurrently increased than the mice fed with a normal diet. The increase of hepatic TG accumulation was prominent in orchiectomized mice, compared to naïve mice and the mice injected with testosterone after castration. Meanwhile, unlike the increased TG accumulation, body weight was lowest in the orchiectomized mice, among the mice fed with a fat-rich diet. It is considered that testosterone suppresses hepatic TG accumulation and increases the body weight due to an increase in fat-free mass [25]. Along with the degree of TG accumulation, orchiectomized mice were also more vulnerable to inflammation and fibrosis. This implies that androgens can prevent the development of NAFLD and progression from NAFLD to steatosis-induced NASH with fibrosis, under a fat-rich diet.

First, we observed the fluctuation of hepatic DNL, as a recent study showed that hepatic TG accumulation is induced via DNL in patients with NAFLD [26]. AMPK-ACC signaling plays a major role in hepatic DNL [27]. The phosphorylation of AMPK and ACC decreased in orchiectomized mice, leading to a reduction in hepatic TG accumulation. Likewise, we found that AMPK-ACC signaling was inactivated when testosterone was administered, particularly under the condition of fatty acid supplementation and AR overexpression in vitro assay. Concurrently, the mRNA levels of AR increased in vivo when androgens levels were sufficient. In addition, the mRNA levels of AR increased in testosterone-treated cells. The AR mRNA levels surged more sharply when cells were treated with bicalutamide, an AR antagonist. This increase is considered a compensatory response to the steep suppression of AR activation [28], as the AR antagonist and the media to eradicate androgens inside the cells were simultaneously used. Unlike mRNA expression, the antagonistic effect of bicalutamide affected protein expression. This suggests that testosterone which is one of the androgens, by cooperating with AR, modulates hepatic TG synthesis and accumulation via AMPK-ACC signaling.

To assess whether the complex bound with testosterone and AR (AR complex) is directly recruited to the promoter of the AMPK gene, we performed a ChIP assay using the SNU-423 cells treated with testosterone. However, the results of the ChIP assay did not show the recruitment of the AR complex to the promoter of the AMPK gene. This result suggests that testosterone does not directly modulate AMPK expression. Despite the evidence, we could not exclude the genomic response of testosterone since the increased AMPK phosphorylation paralleled activation of the AR.

A recent study revealed that LKB1, an upstream regulator of AMPK, interacts with sex steroids in a genomic manner in adipocytes [15]. Therefore, we investigated whether LKB1 cooperates with androgens in the same way in hepatocytes. The results showed that LKB1 mRNA expression increased when androgens and/or testosterone were present at sufficient levels both in vivo and in vitro, and in turn, the protein expression increased in vitro. Along with the increased LKB1 expression, the phosphorylation of AMPK and ACC was also raised. This implies that LKB1 mediates the signaling between androgens and AMPK phosphorylation in hepatocytes. Likewise, we speculated whether androgens drive LKB1 expression in a genomic manner and performed a ChIP assay to assess the expression of the LKB1 gene. The results showed that the AR complex was recruited to the promoter of the LKB1 gene. As in previous ChIP studies on AR, the AR element sequence motif at the AR binding site is presently presumed to be “ACATTTGT” in part of the LKB1 gene promoter [29].

In summary, our study suggests that androgens, particularly testosterone, can prevent hepatic TG accumulation by suppressing hepatic DNL. Androgens bound to AR modulate LKB1 transcription through direct recruitment to the promoter of the LKB1 gene. In turn, increased LKB1 expression modulates the phosphorylation of AMPK-ACC signaling, leading to the suppression of hepatic TG synthesis and accumulation. Therefore, these findings are meaningful because androgens deficiency might be another risk factor of NAFLD in males, and the control of hormone homeostasis prevents NAFLD from progressing to steatosis-induced NASH, cirrhosis, and hepatocellular carcinoma.

3. Conclusions