Roles of Acyl-CoA:Diacylglycerol Acyltransferases 1 and 2 in Triacylglycerol Synthesis and Secretion in Primary Hepatocytes

Abstract

Objective-Very low-density lipoprotein assembly and secretion are regulated by the availability of triacylglycerol.Although compelling evidence indicates that the majority of triacylglycerol in very low-density lipoprotein is derived from re-esterification of lipolytic products released by endoplasmic reticulum-associated lipases, little is known about roles of acyl-CoA:diacylglycerol acyltransferases (DGATs) in this process. We aimed to investigate the contribution of DGAT1 and DGAT2 in lipid metabolism and lipoprotein secretion in primary mouse and human hepatocytes. Approach and Results-We used highly selective small-molecule inhibitors of DGAT1 and DGAT2, and we tracked storage and secretion of lipids synthesized de novo from [ 3 H]acetic acid and from exogenously supplied [ 3 H]oleic acid. Inactivation of individual DGAT activity did not affect incorporation of either radiolabeled precursor into intracellular triacylglycerol, whereas combined inactivation of both DGATs severely attenuated triacylglycerol synthesis. However, inhibition of DGAT2 augmented fatty acid oxidation, whereas inhibition of DGAT1 increased triacylglycerol secretion, suggesting preferential channeling of separate DGAT-derived triacylglycerol pools to distinct metabolic pathways. Inactivation of DGAT2 impaired cytosolic lipid droplet expansion, whereas DGAT1 inactivation promoted large lipid droplet formation. Moreover, inactivation of DGAT2 attenuated expression of lipogenic genes. Finally, triacylglycerol secretion was significantly reduced on DGAT2 inhibition without altering extracellular apolipoprotein B levels. Conclusions-Our data suggest that DGAT1 and DGAT2 can compensate for each other to synthesize triacylglycerol, but triacylglycerol synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes triacylglycerol destined for very low-density lipoprotein assembly. an indirect transfer of triacylglycerol for VLDL maturation. 11 It has been established that the majority (60%-80%) of triacylglycerol in VLDL is derived from re-esterification of lipolytic products in hepatocytes. [12][13][14] This sinuous supply of triacylglycerol for VLDL maturation overcomes the inability of triacylglycerol stored in cytosolic lipid droplets (LDs) to cross the lipid bilayer of the endoplasmic reticulum (ER) and provides a mechanism for the regulation of VLDL secretion independently of plasma FA and intracellular triacylglycerol concentration. 15 We have previously shown that lipases involved in the hydrolysis of preformed triacylglycerol include ER-localized carboxylesterase 1d/triacylglycerol hydrolase and arylacetamide deacetylase [16][17][18] ; however, it has not been determined which DGAT catalyzes the re-esterification of diacylglycerol to support VLDL maturation.Topological studies separated DGAT activities in hepatic microsomes into overt, cytosolic side-localized, and latent, lumenal side-localized. [19][20][21] This led to a hypothesis that the overt DGAT activity might be responsible for the synthesis of tria...

Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat

“…The CLD morphology observed in enterocytes of Dgat1 Int and Dgat1 −/− mice (Figure 4 and 7) support the hypothesis that Dgat1 restricts and Dgat2 promotes CLD expansion reported in other models [12, 50]. In primary hepatocytes, which express both Dgat1 and Dgat2, inhibition of Dgat2 reduced CLD size whereas inhibition of Dgat1 increased CLD size [12]. In addition, the overexpression of Dgat2 in HEK293T cells resulted in increased CLD size [50].…”

“…Topological studies demonstrate that the active site of DGAT1 may be present on either side of the ER membrane as the protein has dual topology [10], while the active site of DGAT2 is oriented toward the cytoplasm [11]. However, additional studies investigating the roles of DGAT1 and DGAT2 in hepatocytes suggest that this partitioning may be more complex [12–15]. Furthermore, the distinct physiological roles of Dgat1 and Dgat2 have been demonstrated in knockout mouse models.…”

A unifying mathematical model of lipid droplet metabolism reveals key molecular players in the development of hepatic steatosis

“…TIP-47 dependent synthesis of nascent LDs as function of the TAG content of the ER and amount of TIP-47) are technically challenging and not available yet. However, also an increase in cellular TAG content with decreased activity of DGAT1 has been observed in primary hepatocytes [21]. Therefore, we varied the numerical values for the reaction rates and binding constants such that the final model parametrization enables simulations that were in good qualitative concordance with data from a larger number of independent knockdown and overexpression experiments carried out with liver tissue or hepatocytes.…”

Gene-based therapies in lipidology

“…ANGPTL3-ASO is thus developed for the treatment of HoFH, severe heterozygous familial hypercholesterolemia, and severe combined dyslipidemia. DGAT isoforms (DGAT1 and DGAT2) both catalyze the final step in triglyceride synthesis [27]. DGAT2 ASO treatment selectively reduces DGAT2 mRNA levels in liver and significantly reduces hepatic lipids (diacylglycerol and triglyceride but not long chain acyl CoAs) and improves hepatic insulin sensitivity in rats.…”