Identification and functional characterization of novel proteins involved in
hepatic triglyceride metabolism in man
Supervisors: Professors Rachel Fisher and Per Eriksson
Department: Dept. of Medicine, Solna
Background: Disturbances in hepatic triglyceride (TG) metabolism are central in the
dysregulation of plasma TG concentrations and the development of hepatic steatosis, conditions
that increase risk for cardiovascular disease, severe liver disease, insulin resistance and Diabetes
Mellitus (reviewed in 1 and 2). Therapeutic options to treat these conditions are extremely
limited, largely as a consequence of an incomplete understanding of the physiology of TGmetabolism in human liver. The aim of this project is to uncover and functionally characterize
novel proteins involved in hepatic TG-metabolism, with a focus on the identification of the as
yet elusive lipid-droplet associated TG-hydrolase enzyme with a critical role in hepatic TGmetabolism in man.
Our laboratory has developed protocols to study key aspects of TG-metabolism in two wellcharacterized human liver cell-lines, the Huh7 and HepG2 hepatoma cell-lines. We have
previously used this system to evaluate the role of candidate proteins in hepatic TG-metabolism
and identified - amongst others - TM6SF2 as a regulator of liver fat metabolism influencing the
secretion of TG-rich lipoproteins (TRLs) and hepatic lipid-droplet metabolism (3). Subsequent
candidate-studies have uncovered unique functions of the TG-hydrolase PNPLA2 and its
activator ABHD5 in selectively influencing the hepatic secretion of TRLs (in the absence of
effects on lipid-droplet metabolism), while no evidence was found that CES1, a prominent
hepatic TG-hydrolase in rodents, is involved in human TG-metabolism (studies in progress).
However, our studies have thus far failed to identify the elusive lipid-droplet-related TGhydrolase(s) in human liver, the proposed ‘missing link’ that plays a critical role in hepatic TGmetabolism in man.
PNPLA2, ABHD5 and CES1 are all members of the metabolic serine-hydrolase protein-family,
which includes several enzymes involved in lipid metabolism (reviewed in 4). However, the
majority of the ~115 members of this family are still not annotated with respect to their
physiological substrates and functions. Our hypothesis is that one or more of these un-annotated
members of the metabolic serine-hydrolase family play major roles in lipid-droplet-related TGmetabolism in human liver. The post-doctoral fellow will therefore: 1) develop an efficient
selection procedure to identify putative candidate proteins; and 2) perform a detailed functional
analysis of these candidate proteins for their involvement in hepatic TG-metabolism.
Objective: To identify and functionally characterize novel TG-hydrolase enzymes with a
critical role in hepatic TG-metabolism in man.
Methodology and work plan:
1. Bioinformatics and gene-expression screening of the metabolic serine-hydrolase family.
The extensive bioinformatics and gene-expression resources available in our laboratory (5) will
be utilized to screen all members of the metabolic serine-hydrolase family to identify suitable
candidate enzymes for experimental validation. Analysis will make use of - amongst other
resources - a unique gene-expression database from 260 human liver samples (6). It is projected
that 10-20 enzymes will be prioritized for detailed analysis in step 2.
2. Screening of putative enzymes for involvement in hepatic TG-metabolism.
The selected candidates will be investigated for their involvement in TRL secretion and/or
cellular lipid-droplet metabolism in both Huh7 and HepG2 cell-lines, using gene-specific
inhibition methods described in (4). Effects on TRL secretion will be monitored by quantifying
both the protein and the lipid moieties of the secreted TRLs, while measurements of cellular
TG-concentration will be used to detect effects on lipid-droplet-metabolism. It is expected that
2-5 candidate genes/proteins will be identified for more detailed analysis in step 3.
3. Functional evaluation of the candidate enzymes’ roles in hepatic TG-metabolism.
The physiological function of the candidate enzymes will be studied using assays recently
developed in our laboratory for the analysis of TG-metabolism in Huh7 and HepG2 cell-lines,
including whole-cell assays for fatty acid uptake, TG-synthesis and TG-hydrolysis. In addition,
confocal microscopy will be used to identify the subcellular localization of the candidate
enzymes following expression of GFP-tagged full-length proteins and comparing colocalization with maker-proteins. Confocal microscopy will also be used to quantify changes in
the number/size of lipid-droplets following siRNA inhibition of the candidate gene.
Significance: This project will identify and functionally characterize protein(s) involved in
hepatic TG-metabolism in man to reveal the elusive lipid-droplet-associated TG-hydrolase. Our
competitive edge is the availability of extensive expertise in bioinformatics and functional
analysis of TG-metabolism in human hepatoma cell-lines, all in the setting of a center for
translational cardio-metabolic research. The post-doc will profit from our participation in the
TG-metabolism Consortium in collaboration with Robert Farese and Tobias Walther (Harvard,
Boston), Sekar Kathiresan (Broad, Boston) USA and Vivek Malhotra (Barcelona).
References
1. Rosenson RS, Davidson MH, Hirsh BJ, Kathiresan S, Gaudet D. Genetics and causality of
triglyceride-rich lipoproteins in atherosclerotic cardiovascular disease. J Am Coll Cardiol.
2014;64:2525-40.
2. National Guideline Centre (UK). Non-Alcoholic Fatty Liver Disease: Assessment and
Management. London: National Institute for Health and Care Excellence (UK). 2016.
3. Mahdessian H, Taxiarchis A, Popov S, Silveira A, Franco-Cereceda A, Hamsten A,
Eriksson P, van 't Hooft FM. TM6SF2 is a regulator of liver fat metabolism influencing
triglyceride secretion and hepatic lipid droplet content. Proc Natl Acad Sci U S A.
2014;111:8913-8.
4. Bachovchin DA, Cravatt BF. The pharmacological landscape and therapeutic potential of
serine hydrolases. Nat Rev Drug Discov. 2012;11:52-68.
5. Folkersen L, Wågsäter D, Paloschi V, Jackson V, Petrini J, Kurtovic S, Maleki S, Eriksson
MJ, Caidahl K, Hamsten A, Michel JB, Liska J, Gabrielsen A, Franco-Cereceda A, Eriksson
P. Unraveling divergent gene expression profiles in bicuspid and tricuspid aortic valve
patients with thoracic aortic dilatation: the ASAP study. Mol Med. 2011;17:1365-73.
6.
Folkersen L, van 't Hooft FM, Chernogubova E, Agardh HE, Hansson GK, Hedin U, Liska
J, Syvänen AC, Paulsson-Berne G, Franco-Cereceda A, Hamsten A, Gabrielsen A, Eriksson
P, BiKE and ASAP study groups. Association of genetic risk variants with expression of
proximal genes identifies novel susceptibility genes for cardiovascular disease. Circ
Cardiovasc Genet. 2010;3:365-73.
Contact details:
Rachel Fisher, Professor
[email protected]
+46 8 517 732 45
Per Eriksson, Professor
[email protected]
+46 8 517 732 02
Cardiovascular Medicine Unit
Department of Medicine, Solna
Karolinska Institutet
http://ki.se/en/meds/research-group-per-eriksson