Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels.

Keywords: Genistein; NAFLD; NASH; PPARα; SREBP-1c; liver; primary human hepatocytes; steatosis.

Figure 1

Amount of neutral lipids in primary human hepatocytes (PHHs) after treatment with free fatty acids (FFAs). PHHs were treated with 1 mM FFAs for 24 h and lipid levels were quantified using the Oil Red O assay normalized to the protein amount measured by sulforhodamine B (SRB) assay. (A) Evaluation of steatosis in FFA-treated PHHs in comparison to control. Data are shown as the mean + SD, n = 5, paired t-test, p < 0.01 (**). (B) Microscopic evaluation of the lipid accumulation in representative PHH cultures (magnification 200×), the scale bar is 10 µm.

Figure 2

Evaluation of lipotoxicity on FFA-treated PHHs. PHHs were treated with 1 mM FFAs for 24 h and steatotic and control PHHs were investigated for (A) cell activity measured by the conversion of XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid); (B) enzyme activities of AST (aspartate transaminase) and (C) LDH (lactate dehydrogenase) determined in cell culture supernatants to evaluate disruptions of cell membrane integrity and (D) metabolic capacity examined by quantification of their urea production. Data are shown as the mean + SD, n = 5, paired t-test, p < 0.05 (*).

Figure 3

Adaption of gene expression of key players of lipid homeostasis in steatotic PHHs. PHHs were treated with 1 mM FFAs for 24 h and relative mRNA expression levels of gene targets were determined using RT-qPCR. Investigation of genes centrally involved in lipid catabolism (PPARα, ACSL1 and CPT1L) and anabolism (SREBP-1c and FASN) in PHHs compared to control. Data are shown as the mean + SD, n = 5, paired t-test, statistical analyses were conducted on ΔC_T values, p ≤ 0.05 (*), p < 0.01 (**).

Figure 4

Activation of key signaling pathways in lipid homeostasis in steatotic hepatocytes. PHHs were treated with 1 mM FFAs for 24 h and protein levels of PPARα and SREBP-1c were investigated. Western blot analyses were performed after protein extraction from different subcellular fractions of FFA-treated PHHs and control. Densitometric measurements of (A) cytosolic PPARα normalized to the expression levels of α-tubulin, nucleic PPARα normalized to p84, (B) ER membrane-bound SREBP-1c normalized to Na⁺/K⁺-ATPase and nucleic SREBP-1c normalized to p84 expression. Data are shown as the mean + SD, n = 5, paired t-test, p ≤ 0.05 (*). (C) Representative Western blot images show the specific bands of PPARα, α-tubulin, p84, SREBP-1c, and Na⁺/K⁺-ATPase in control and steatotic PHHs.

Figure 5

Evaluation of the cytotoxic effect of different concentrations of the soy isoflavone genistein on FFA-treated and control PHHs. PHHs were treated with 1 mM FFAs for 24 h, followed by 24 h of treatment with genistein (0, 1, 5, 10, 50, and 100 µM). Toxicity was measured by evaluation of (A) cell activity determined with the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid) assay and (B) LDH (lactate dehydrogenase) release as a marker for impaired cell integrity. Data are shown as the mean + SD, n = 3, two-way ANOVA and post hoc Tukey or Sidak test, p ≤ 0.05 (*), p < 0.01 (**). Selected comparisons are shown; for details on the statistical evaluation, see Table S1A, Supplementary Materials.

Figure 6

Transcriptional and translational response of genistein-treated steatotic PHHs on (A) PPARα and (B) SREBP-1c, two central regulators of hepatic lipid homeostasis. PHHs were treated with 1 mM FFAs for 24 h, followed by 24 h of additive treatment with genistein (0, 1, 5, 10, 50, and 100 µM). (Ai) PPARα mRNA levels were determined by RT-qPCR; (Aii) cytosolic and; (Aiii) nucleic PPARα protein levels were assessed by Western blot (α-tubulin and p84 served as the respective reference proteins). (Bi) Relative mRNA expression levels of SREBP-1c as measured by RT-qPCR; (Bii) densitometric measurements of ER membrane-bound SREBP-1c normalized to Na⁺/K⁺-ATPase and (Biii) nucleic SREBP-1c normalized to p84 expression. Data are shown as the mean + SD, n = 5 for RT-qPCR data, n = 4 for Western blot measurements, two-way ANOVA with Tukey or Sidak post hoc test. p ≤ 0.05 (*), p < 0.01 (**). Selected comparisons are shown; for details on the statistical evaluation, see Table S1B, Supplementary Materials. (C) Representative Western blot images show the specific bands of PPARα, α-tubulin, p84, SREBP-1c, and Na⁺/K⁺-ATPase in control and steatotic PHHs after genistein treatment.