Androgen receptor over expression drives lipid

accumulation in human hepatocytes.

N Nikolaou1, M Nasiri1, LL Gathercole1, S Parajes1, N Krone1, G Valsamakis2,

G Mastorakos2, JW Tomlinson1

1University of Birmingham, Centre for Endocrinology, Diabetes and Metabolism, United Kingdom

2Endocrine Unit, Aretaieion Hospital, University of Athens, Medical School, Athens, Greece

Non alcoholic fatty liver disease (NAFLD) is rapidly becoming the most common cause of hepatic dysfunction in the western world. It encompasses a

spectrum of disease ranging from simple lipid accumulation within hepatocytes to steatohepatitis (NASH) with fibrosis and leading finally to cirrhosis and

liver failure. Recent studies have shown an association between NAFLD and androgen deficiency, yet in the majority of patients with NASH, androgen

levels are normal. In contrast, in patients with polycystic ovarian syndrome (PCOS) which is characterised by androgen excess, hepatic steatosis is

prevalent. Our hypothesis is that androgen exposure may be a critical regulator of lipid flux within human hepatocytes.

Background

Methods

Results

Conclusion

Increased mRNA expression of FASN, ACC1 and ACC2 as well decreased CPT1 mRNA expression contribute to the increase in de novo lipogenesis that

is observed with testosterone and DHT treatment. Surprisingly, we also observed that AR over-expression alone, in the absence of ligand, also regulates

hepatic lipid metabolism by increasing both the expression of key components of the lipogenic pathway (FASN, ACC1, ACC2) and functional lipid

accumulation. In conclusion, these data demonstrate that enhanced androgen action is able to stimulate lipid accumulation in human hepatocytes and

this may be crucial in understanding the association between PCOS and NAFLD.

C3A human hepatoma cells were cultured and treated with Testosterone [T] (5nM, 50nM) or the more potent androgen, Dihydrotestosterone [DHT] (1nM,

10nM) for 24h. Lipid accumulation was measured by C14 acetate incorporation into triglyceride and gene expression by real-time PCR. As an additional

model of androgen excess, cells were transfected with an androgen receptor (AR) construct (pcDNA3.1+AR) or vector alone as a control. Between-group

comparisons were made with T-Test and ANOVA.

Despite androgen receptor (AR) expression being undetectable in C3A cells, FASN, ACC1, ACC2 and CPT1 mRNA expression was significantly increased

after treatment with testosterone and DHT in a dose-dependent manner (figure 1) suggesting a non-genomic action. Endorsing these data, both

testosterone and DHT increased de novo lipogenesis as measured by C14-acetate incorporation into triglyceride (ctrl 7002±259 vs. T 8748± 433, DHT

8970±330, p<0.05). Following AR transfection (figure 3, 4), even in the absence of ligand, lipogenic gene expression increased (FASN: ctrl 13.9±2.0 vs.

AR 66.8±6.2, ACC1: ctrl 1.0±0.3 vs. AR 3.5±0.3, ACC2: ctrl 0.5±0.1 vs. AR 1.0±0.1, CPT1: ctrl 1.8±0.3 vs. AR 4.3±0.2, p<0.05) (figure 5) as did de

novo lipogenesis (figure 6) suggesting a ligand-independent action (ctrl 7002±259 vs. AR 14193±755, p<0.05). Lipogenesis was further increased

following incubation of AR over-expressing C3A cells with testosterone and DHT.

Impact of androgens on lipid metabolism

in C3A human hepatoma cells.

AR over expression as a model

of androgen excess.

Impact of AR over expression

on lipid metabolism.

Gene expression.

Lipogenesis.

Gene expression. Lipogenesis.

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Figure 2: De novo lipogenesis

in C3A cells increased across

treatment with testosterone and

DHT measured by scintillation

counting. Data shown as the

mean of disintegrations per

minute (dpm).

Figure 3: C3A hepatoma cells after

transfection with green fluorescence

protein.

Figure 4: AR mRNA expression increased across

increasing concentrations measured by real time

PCR. Data shown as the mean of arbitrary units (AU).

Figure 5: mRNA expression in C3A human

hepatoma cells increased across

transfection with AR measured by real time

PCR. Data shown as the mean of arbitrary units

(AU).

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Figure 6: De novo lipogenesis increased

across transfection in C3A cells. Data

shown as disintegrations per minute (dpm).

Figure 1: mRNA expression of FASN, ACC1, ACC2 and CPT1 in C3A human hepatoma cells

increased across dose dependent treatment with testosterone and DHT. Data shown as the mean

of arbitrary units (AU).

Fatty Acid Synthase (FASN) Acetyl-CoA Carboxylase 1

(ACC1) Acetyl-CoA Carboxylase 2

(ACC2)

Carnitine

palmitoyltransferase (CPT1)

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