Cinnabarinic Acid Provides Hepatoprotection Against Nonalcoholic Fatty Liver Disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a chronic condition in which excess lipids accumulate in the liver and can lead to a range of progressive liver disorders including non-alcoholic steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. While lifestyle and diet modifications have proven to be effective as NAFLD treatments, they are not sustainable in the long-term, and currently no pharmacological therapies are approved to treat NAFLD. Our previous studies demonstrated that cinnabarinic acid (CA), a novel endogenous Aryl hydrocarbon Receptor (AhR) agonist, activates the AhR target gene, Stanniocalcin 2, and confers cytoprotection against a plethora of ER/oxidative stressors. In this study, the hepatoprotective and anti-steatotic properties of CA were examined against free fatty-acid-induced in vitro and high-fat-diet fed in vivo NAFLD models. The results demonstrated that CA treatment significantly lowered weight gain and attenuated hepatic lipotoxicity both before and after the established fatty liver, thereby protecting against steatosis, inflammation, and liver injury. CA mitigated intracellular free fatty acid uptake concomitant with the downregulation of CD36/fatty acid translocase. Genes involved in fatty acid and triglyceride synthesis were also downregulated in response to CA treatment. Additionally, suppressing AhR and Stc2 expression using RNA interference in vitro verified that the hepatoprotective effects of CA were absolutely dependent on both AhR and its target, Stc2. Collectively, our results demonstrate that the endogenous AhR agonist, CA, confers hepatoprotection against NAFLD by regulating hepatic fatty acid uptake and lipogenesis. SIGNIFICANCE STATEMENT: In this study using in vitro and in vivo models, we demonstrate that cinnabarinic acid (CA), an endogenous AhR agonist, provides protection against non-alcoholic fatty liver disease. CA bestows cytoprotection against steatosis and liver injury by controlling expression of several key genes associated with lipid metabolism pathways, limiting the hepatic lipid uptake, and controlling liver inflammation. Moreover, CA-induced hepatoprotection is absolutely dependent on AhR and Stc2 expression.

Fig. 1.

Determination of (A) cell viability of HepG2 cells treated with different concentrations of palmitic acid (PA) and oleic acid (OA) for 24-hour cell viability was measured by a luminescent assay and expressed relative to BSA-treated control. Data are represented as mean ± SD (n = 3). *P < 0.05 compared with control group. (B) CA protects against palmitic acid (PA)/oleic acid (OA)-induced steatosis. Representative images of oil red O stained HepG2 cells treated with 500-µM BSA + DMSO (control), 30-µM CA, 500-µM PA, 500-µM OA, 30-µM CA+ 500-µM PA/OA, 30-µM CA after 500-µM PA/OA. (C) Quantification of oil red O-stained images; area of oil red O-stained lipid droplets was normalized to the total area (three images per treatment). (D) Quantification of accumulated oil red O by colorimetry; absorbance measured at 500 nm. Data are represented as mean ± SD (n = 3). *P < 0.05 compared with PA/OA-only treatment group.

Fig. 2.

Quantification of (A) triglyceride, (B) free fatty acid content, and (C) free fatty acid uptake in HepG2 cells treated with 500-µM BSA + DMSO (control), 30-µM CA, 500-µM PA, 500-µM OA, 30-µM CA+ 500-µM PA/OA, 30-µM CA after 500-µM PA/OA. Triglyceride content was measured using a luminescence assay, whereas free fatty acid content and free fatty acid uptake were determined fluorometrically. Data are represented as mean ± SD (n = 3). *P < 0.05 compared with PA/OA-only treatment group.

Fig. 3.

Expression of mRNAs encoding genes involved in (A) free fatty acid transport, (B) fatty acid synthesis, (C) triglyceride synthesis, and (D) inflammation. HepG2 cells were treated with 500-µM BSA+ DMSO (control), 30-µM CA, 500-µM PA, 500-µM OA, 30-µM CA+ 500-µM PA/OA, 30-µM CA after 500-µM PA/OA. mRNA message was analyzed by qRT-PCR and normalized to 18S rRNA. Results are expressed as fold of the value found in control treatment arbitrarily set at 1. For statistical analysis, a mixed-effects multivariate ANOVA (MANOVA) model was used. After an overall significant F test from MANOVA model, the post hoc multiple-comparison tests were performed for the pre-specified comparisons adjusted by Tukey procedure. Data are represented as mean ± SD (n = 3). *P < 0.05 compared with PA/OA-only treatment group.

Fig. 4.

CA treatment reduces body and liver weight of high-fat-diet fed mice. C57BL6 mice were fed with a control diet (CD) for 16 weeks, high-fat diet (HFD) for 16 weeks, high-fat diet and treated with CA for 16 weeks (CA + HFD), and high-fat diet for 16 weeks with CA treatment initiated after 10 weeks of exposure to HFD for a remaining 6 weeks (CA after HFD). (A) Representative image of mice after 16 weeks of diet, (B) body mass, (C) body weight at the end of the study, week 16, (D) weight of the liver at week 16, (E) percentage ratio of liver weight normalized to total body weight at the end of the study, and (F) daily food intake calculated from the average of weekly food intake. Data are represented as mean ± SD (n = 7). *P < 0.05 compared with HFD-only treatment group.

Fig. 5.

CA alleviates steatosis and hepatic injury in high-fat-diet fed mice. (A) Representative image of H&E-stained liver sections, (B) liver triglycerides, (C) liver cholesterol, and (D) serum ALT measurement of mice fed with control diet (CD) for 16 weeks, high fat diet (HFD) for 16 weeks, high-fat diet and treated with CA for 16 weeks (CA + HFD), and high-fat diet for 16 weeks with CA treatment initiated after 10 weeks of exposure to HFD for remaining 6 weeks (CA after HFD). Data are represented as mean ± SD (n = 7). *P < 0.05 compared with HFD-only treatment group.

Fig. 6.

CA lowers blood glucose levels and improves glucose tolerance in HFD-fed mice. Mice were fed with control diet (CD), high-fat diet (HFD), high-fat diet with CA treatment (CA + HFD) for 16 weeks, and with high-fat diet for 16 weeks with CA treatment initiated after 10 weeks of HFD feeding for the last 6 weeks (CA after HFD). (A) Fasting blood glucose measurement, (B) glucose tolerance test performed, and (C) the area under the curve calculated for respective groups. Data are represented as mean ± SD (n = 7). *P < 0.05 compared with HFD-only treatment group.

Fig. 7.

CA attenuates hepatic free fatty acid uptake, lipogenesis, and inflammation in vivo. C57BL6 mice were fed with a control diet (CD) for 16 weeks, high-fat diet (HFD) for 16 weeks, high-fat diet and CA treatment for 16 weeks (CA + HFD), and high-fat diet for 16 weeks with CA treatment for last 6 weeks (CA after HFD). mRNA expression of markers for (A) free fatty acid transport, (B) de novo lipogenesis, (C) triglyceride synthesis, (D) fatty acid oxidation, and (E) inflammation, were analyzed by qRT-PCR and normalized to 18S rRNA. Results are expressed as fold of the value found in control treatment arbitrarily set at 1. Data are represented as mean ± SD (n = 7). *P < 0.05 compared with HFD-only treatment group.

Fig. 8.

CA failed to protect against NAFLD in an AhR-silenced in vitro model. HepG2 cells were transiently transfected with AhR or non-targeting (scrambled) siRNA for 24 hours followed by 500-µM BSA+ DMSO (control), 30-µM CA, 500-µM PA, 500-µM OA, 30-µM CA+ 500-µM PA, 30-µM CA+ 500-µM OA and/or 30-µM CA 24 hours after 500-µM PA/OA treatments. (A) Western blotting on total lysate was performed to monitor AhR expression. Actin was probed as a control. (B) Oil red O staining to detect lipid content. Quantification of (C) triglyceride. (D) Free fatty acid content. (E) Free fatty acid uptake; mRNA expression of genes involved in (F) free fatty acid uptake. (G) Fatty acid synthesis. (H) Triglyceride synthesis. (I) Inflammation, normalized to 18S rRNA. Data are represented as mean ± SD (n = 3). *P < 0.05 compared with an untransfected control group not treated with CA.

Fig. 9.

CA-mediated protection against lipotoxicity is Stc2 dependent. HepG2 cells were transiently transfected with Stc2 or non-targeting (scrambled) siRNA for 24 hours followed by 500-µM BSA+ DMSO (control), 30-µM CA, 500-µM PA, 500-µM OA, 30-µM CA+ 500-µM PA, 30-µM CA+ 500-µM OA and/or 30-µM CA 24 hours after 500-µM PA/OA treatments. (A) Stc2 expression in total lysate was monitored by performing immunoblotting. Actin was used as a control. (B) Lipid content was detected using oil red O staining. Quantification of (C) triglyceride, (D) free fatty acid, and (E) free fatty acid uptake. Expression of genes involved in (F) free fatty acid uptake, (G) fatty acid synthesis, (H) triglyceride synthesis, and (I) inflammation, was quantitated by performing quantitative RT-PCR, normalized to 18S rRNA. Data are represented as mean ± SD (n = 3). *P < 0.05 compared with an untransfected control group untreated with CA.