Deletion of fatty acid transport protein 4 in HepG2 cells increases lipolysis lipids and lipoprotein secretion

Expression of fatty acid transport protein 4 (FATP4) is increased in adipose tissues of individuals with central and acquired obesity, and polymorphisms of FATP4 are associated with blood lipids and insulin resistance. We have reported an increase of blood triglycerides (TAG), glycerol, and non-esterified free fatty acids (NEFA) in liver-specific Fatp4-deficient mice only when fed with high-fat diet. Here we investigated whether FATP4 plays a role in hepatic TAG metabolism in HepG2 cells. Methods:

Genetic deletion of FATP4 was performed by using CRISPR/Cas9 technology. To study FATP4 effects on hyperlipidemia, control (HepCon) and FATP4-knockout (HepFATP4KO) cells were treated with 300 or 600µM oleate for 2 or 4h. We studied TAG synthesis by using isotope labelled glycerol (¹³C₃, 99%; D₈, 98%) and 13 species of ¹³C₃D₅TAG containing various fatty acids were probed in cells treated with 20µM ¹³C₃D₈-glycerol for 2h. Results:

Compared to HepCon, HepFATP4KO cells showed an increase of cellular ¹³C₃D₅TAG containing saturated and monounsaturated fatty acids (MUFA) under basal conditions. Interestingly, oleate-treated KO cells compared to treated HepCon showed an increase of ¹³C₃D₅TAG containing a combination of 18:2, 18:3 or 20:4, indicating a diversion of polyunsaturated fatty acids (PUFA) towards TAG. HepFATP4KO cells under basal conditions also showed a marked increase of released VLDL and HDL as well as lipoprotein transport MTTP mRNA expression, all of which were not further altered by oleate treatment. Gene analyses revealed that HepFATP4KO cells showed downregulation of β-oxidation CPT1B and fatty-acid uptake CD36, but upregulation of lipolysis hormone-sensitive-lipase, all of which were not affected by oleate treatment. In support of lipolysis activity, HepFATP4KO cells at basal conditions showed an increase of glycerol and NEFA levels in both cells and supernatants, and the levels of these lipids together with TAG were further increased upon oleate treatment. Taken together, FATP4 deletion in HepG2 cells under basal conditions elicited fatty-acid trafficking towards synthesis of saturated and MUFA TAG leading to an increase of released lipoproteins and lipolysis lipids. Upon oleate treatment, FATP4 deletion preferentially caused an increase of PUFA TAG and lipolysis lipids. Our results implicate the role of FATP4 on hyperlipidemia by modulating fatty-acyl chain saturation in hepatocyte TAG.