Dulaglutide Ameliorates Palmitic Acid-Induced Hepatic Steatosis by Activating FAM3A Signaling Pathway

Abstract

Background: Dulaglutide, a long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA), has been shown to reduce body weight and liver fat content in patients with type 2 diabetes. Family with sequence similarity 3 member A (FAM3A) plays a vital role in regulating glucose and lipid metabolism. The aim of this study was to determine the mechanisms by which dulaglutide protects against hepatic steatosis in HepG2 cells treated with palmitic acid (PA).

Methods: HepG2 cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide for 24 hours. Hepatic lipid accumulation was determined using Oil red O staining and triglyceride (TG) assay, and the expression of lipid metabolism-associated factor was analyzed using quantitative real time polymerase chain reaction and Western blotting.

Results: Dulaglutide significantly decreased hepatic lipid accumulation and reduced the expression of genes associated with lipid droplet binding proteins, de novo lipogenesis, and TG synthesis in PA-treated HepG2 cells. Dulaglutide also increased the expression of proteins associated with lipolysis and fatty acid oxidation and FAM3A in PA-treated cells. However, exendin-(9-39), a GLP-1R antagonist, reversed the expression of FAM3A, and fatty acid oxidation-associated factors increased due to dulaglutide. In addition, inhibition of FAM3A by siRNA attenuated the reducing effect of dulaglutide on TG content and its increasing effect on regulation of fatty acid oxidation.

Conclusion: These results suggest that dulaglutide could be used therapeutically for improving nonalcoholic fatty liver disease, and its effect could be mediated in part via upregulation of FAM3A expression through a GLP-1R-dependent pathway.

Keywords: Dulaglutide; FAM3A; Fatty acid oxidation; Glucagon-like peptide-1 receptor; HepG2 cells; Non-alcoholic fatty liver disease.

Fig. 1

Dulaglutide (Dula) inhibits palmitic acid (PA)-induced lipid accumulation in HepG2 cells. Cells were pre-treated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM Dula for 24 hours. Lipid accumulation were evaluated using Oil red O staining (400× magnification) (A, B) and triglyceride (TG) content assay (C). mRNA and protein expression levels of perilipin 1 (PLIN1) and PLIN2 were analyzed using quantitative real time polymerase chain reaction and Western blotting, and were normalized to that of the 18S rRNA gene and β-actin, respectively (D, E). ^aP<0.05 and ^bP<0.01 compared with control (Con); ^cP<0.05 and ^dP<0.01 compared with PA-treated Con group.

Fig. 2

Dulaglutide (Dula) decreases lipogenesis and triglyceride (TG) synthesis and increases TG secretion in HepG2 cells treated with palmitic acid (PA). HepG2 cells were pre-treated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM Dula for 24 hours. mRNA expression levels of the genes encoding sterol regulatory-binding protein 1 (SREBP-1), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) (A), diacylglycerol acyltransferase 1 (DGAT1), DGAT2 (B), microsomal triglyceride transfer protein (MTP), and apolipoprotein B (ApoB) (C) were analyzed using quantitative real time polymerase chain reaction and normalized to that of the 18S rRNA gene. ^aP<0.05 and ^bP<0.01 compared with control (Con); ^cP<0.05 and ^dP<0.01 compared with PA-treated Con group.

Fig. 3

Dulaglutide (Dula) increases lipolysis and fatty acid oxidation in HepG2 cells treated with palmitic acid (PA). HepG2 cells were pre-treated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM Dula for 24 hours. (A, B, C, D, E, F) adipose triglyceride lipase (ATGL), phospho-hormone-sensitive lipase (p-HSL), sirtuin 1 (SIRT1), phospho-adenosine monophosphate-activated protein kinase (p-AMPK), phospho-acetyl-CoA carboxylase (p-ACC), and peroxisome proliferator-activated receptor alpha (PPARα) levels were analyzed using Western blotting. ^aP<0.05 and ^bP<0.01 compared with control (Con); ^cP<0.05 and ^dP<0.01 compared with PA-treated Con group.

Fig. 4

Dulaglutide (Dula) increases the expression of family with sequence similarity 3 member A (FAM3A) via glucagon-like peptide-1 receptor (GLP-1R) in HepG2 cells treated with palmitic acid (PA). (A) HepG2 cells were pre-treated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM Dula for 24 hours. (B) Cells were pre-treated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide and 100 nM exendin (9–39), a GLP-1R antagonist, for 24 hours. FAM3A, GLP-1R, sirtuin 1 (SIRT1), phospho-adenosine monophosphate-activated protein kinase (p-AMPK), phospho-acetyl-CoA carboxylase (p-ACC), and peroxisome proliferator-activated receptor alpha (PPARα) levels were analyzed using Western blotting. ^aP<0.01 compared with control (Con); ^bP<0.05 compared with PA-treated Con group.

Fig. 5

Dulaglutide (Dula) inhibits lipid deposition and increases fatty acid oxidation via family with sequence similarity 3 member A (FAM3A). HepG2 cells transfected with 10 nM FAM3A small interfering RNA (siRNA) or control (Con) siRNA for 24 hours were pretreated with 400 μM palmitic acid (PA) for 24 hours, followed by treatment with or without 100 nM Dula for 24 hours. (A) Lipid deposition was evaluated using triglyceride (TG) content assay. (B) FAM3A, sirtuin 1 (SIRT1), phospho-adenosine monophosphate-activated protein kinase (p-AMPK), phospho-acetyl-CoA carboxylase (p-ACC), and peroxisome proliferator-activated receptor alpha (PPARα) levels were analyzed using Western blotting. scr, scrambled siRNA. ^aP<0.05 and ^bP<0.01 compared with Con; ^cP<0.01 compared with PA-treated Con group; ^dP<0.01 compared with each non-transfected groups.