Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress

Abstract

Liver, the central hub for bodily lipid metabolism, accumulates surplus lipids under several pathological conditions, such as obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD) ( 1 ). NAFLD covers the development of steatosis, progression to nonalcoholic steatohepatitis (NASH), and on to chronic liver diseases like cirrhosis, hepatocellular carcinoma, and fi nally liver failure ( 2 ). Hepatic steatosis starts initially with accumulation of triacy lglycerols (TGs) in hepatocytes. This abnormal TG accumu lation is due either to fasting or increased fat absorption ( 3 ), or to decreased hydrolysis ( 4, 5 ). Some reports have indicated a signifi cant increase in the TG-to-diacylglycerol (DG) ratio and in the free cholesterol-to-phosphatidylcholines (PCs) ratio when normal human livers are compared to livers with NAFLD ( 6 ). Moreover, increased levels of DG may contribute to altered TG levels and of DG-derived phospholipids (PLs), which further leads to progression of NAFLD ( 7 ). It is well known that methionine-and choline-defi cient diets lead to NASH in rodents ( 8 ); such livers have a reduced rate of PC and choline biosynthesis. The decreased ratio of PC to phosphatidylethanolamine (PC/PE) can also affect membrane integrity and leads to progression from steatosis into NASH ( 9 ). It is not clear, however, whether a change in the PC/PE ratio initiates steatosis or is the end point of NASH.Abstract Liver steatosis can be induced by fasting or highfat diet. We investigated by lipidomic analysis whether such metabolic states are refl ected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profi les and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species.Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fi tted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such pheno typing, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.

The impact of genetic stress by ATGL deficiency on the lipidome of lipid droplets from murine hepatocytes

“…C 50 to C 58 species predominated the TG species profi le for the WT-FED group as expected from our previous study ( 1 ). Yet, individual features for each sample group over the whole carbon number range were found upon construction of respective heat maps.…”

“…ATGL was discovered in 2004 to be the rate-limiting enzyme for degradation of TGs to diacylglycerols (DGs) species ( 1 ). Here, we extend these studies to genetic stressinduced lipidomes in hepatocyte LDs that emerge from aberrant lipid metabolism in ATGL-defi cient mice.…”

“…Interestingly, in-depth analysis using PCA did single out main TG species (see supplementary Table II) responsible for the separation of classes found in hepatocyte LDs. In addition, structures of lipid molecular species derived from respective lipid species by MS/MS analysis allowed recognition of metabolic relationships between molecular species of these lipid classes ( 1 ). Similarly, the effects of genetic stress studied here led to lipid species and lipid molecular species, particularly for DG and PC classes (data in supplementary Tables IV and V, respectively), whose structures provided insights on metabolic relationships as presented in Table 2 .…”

“…The mice were housed and handled in accordance with good animal practice as defi ned by FELASA ( 1 ). The animal welfare committees of the University of Graz and the national authorities approved all animal experiments.…”

“…Lipidomic analysis by LC-MS enabled profi ling of all species from glycerolipid and glycerophospholipid classes and revealed particularly the triacylglycerol (TG) lipidome to be best suited for phenotyping nutritional stresses applied to the animals. In addition, structural analysis by MS/MS at lipid molecular species level provided metabolic relationships characteristic for hepatocyte lipid metabolism ( 1 ). To further probe the phenotypic information potential inherent in the lipidome of hepatocyte LDs, we have now extended the question to genetic stress; we have characterized a mouse model lacking adipocyte triglyceride lipase (ATGL), which shows a marked defect in lipid metabolism in many tissues including the liver .…”