Identification and functional characterization of proteins involved in hepatic triglyceride metabolism

Triglycerides are the main form of energy in the tissues and liver, along with the adipose tissue, is the main organ of triglyceride metabolism and storage in the lipid-droplet organelles. A number of proteins are involved in the regulation of the triglyceride metabolism in human liver, however their specific role is still not thoroughly known. The aim of this thesis is to evaluate the functional role of three proteins in triglyceride regulation in an experimental model of human liver.

In Paper I we identified the gene Transmembrane 6 superfamily member 2 (TM6SF2) as the putative cause for the association between the 19p12 locus with plasma triglyceride levels and non-alcoholic fatty liver disease, by employing expression studies and expression quantitative trait locus analysis in 206 human liver samples. TM6SF2 encodes a protein of 351 amino acids localized in the Endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment, as investigated in human hepatoma cells. Functional studies showed that TM6SF2 siRNA inhibition led to reduced secretion of triglyceride-rich lipoproteins (TRLs) and increased cellular triglyceride concentration and number of lipid-droplets, however the putative pathophysiological mechanism of these observations is still unclear.

In Paper II we investigated the physiological functions of Patatin-like phospholipase domain containing proteins 2, 3 and 4 (PNPLA2, PNPLA3 and PNPLA4), as potential triglyceride hydrolases in Huh7 and HepG2 human hepatomas. We found that siRNA inhibition of PNPLA3 or PNPLA4 is not associated with changes in triglyceride hydrolysis, TRL secretion or cellular triglyceride accumulation. However, PNPLA2 siRNA inhibition reduced intracellular triglyceride hydrolysis and decreased TRL secretion, both in the absence or presence of oleate-containing medium or of the PNPLA2 inhibitor Atglistatin. In contrast, we found no effects of PNPLA2 inhibition on lipid-droplet homeostasis. Visualization analysis with confocal microscopy found significant co-localization of PNPLA2 with the ER, but no clear evidence for PNPLA2 localization around the lipid-droplets. This data indicates that PNPLA2 hydrolyses a triglyceride compartment comprising of very small lipid-droplets that are involved in the regulation of TRL secretion, but are not detectable by confocal microscopy.

In Paper III we studied the likely role of Abhydrolase domain-containing 5 (ABHD5) as the co-activator of PNPLA2 in the regulation of hepatic triglyceride metabolism. We employed siRNA inhibition techniques in Huh7 hepatoma cells and showed that ABHD5 siRNA inhibition reduced triglyceride hydrolysis and decreased TRL secretion while there was no effect on cellular triglyceride content. These results are similar to the effects of PNPLA2 siRNA inhibition on triglyceride metabolism as examined in Paper II. We also found no additive effects of combined ABHD5-PNPLA2 siRNA inhibition in hepatic triglyceride metabolism. We employed confocal microscopy analysis and observed localization of ABHD5 in the ER, but not in Golgi or around the lipid-droplets, while a significant colocalization of ABHD5 and PNPLA2 was observed. These observations suggest that ABHD5 is a co-activator of PNPLA2 with no separate triglyceride hydrolysis activity in human hepatocytes.

Overall, this Thesis identifies TM6SF2 as a membrane protein regulating the TRL secretion in Huh7 and HepG2 hepatoma cells. It also demonstrates the role of triglyceride hydrolysis in the regulation of TRL secretion where PNPLA2 is the main triglyceride hydrolase activated by ABHD5. Finally, it suggests the existence of very small lipid-droplets containing the substrate compartment of the PNPLA2- and ABHD5-mediated triglyceride hydrolysis.

List of scientific papers

I. Mahdessian H, Taxiarchis A, Popov S, Silveira A, Franco-Cereceda A, Hamsten A, Eriksson P, Hooft FV. TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content. Proceedings of the National Academy of Sciences. 2014;111(24):8913-8918.
https://doi.org/10.1073/pnas.1323785111

II. Taxiarchis A, Mahdessian H, Silveira A, Fisher RM, Hooft FMVT. PNPLA2 influences secretion of triglyceride-rich lipoproteins by human hepatoma cells. Journal of Lipid Research. 2019;60(6):1069-1077.
https://doi.org/10.1194/jlr.M090928

III. Taxiarchis A, Silveira A, Fisher RM, Hooft FMVT. The PNPLA2 coactivator ABHD5/CGI-58 influences the secretion of triglyceride-rich lipoproteins by human hepatoma cells. [Manuscript]