Rapid chromatographic method to decipher distinct alterations in lipid classes in NAFLD/NASH

Abstract

AIM:To establish a simple method to quantify lipid classes in liver diseases and to decipher the lipid profile in p62/IMP2-2/IGF2BP2-2 transgenic mice. METHODS:Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein p62/ IMP2-2/IGF2BP2-2 was used as a model for steatosis. Steatohepatitis was induced by feeding a methioninecholine deficient diet. Steatosis was assessed histologically. For thin layer chromatographic analysis, lipids were extracted from freeze-dried tissues by hexane/2-propanol, dried, redissolved, and chromatographically separated by a two-solvent system. Dilution series of lipid standards were chromatographed, detected, and quantified. The detection was performed by either 2',7'-dichlorofluoresceine or a sulfuric acid/ethanol mixture. RESULTS:Histological analyses confirmed steatosis and steatohepatitis development. The extraction, chromatographic, and detection method showed high inter-assay reproducibility and allowed quantification of the different lipid classes. The analyses confirmed an increase of triglycerides and phosphatidylethanolamine and a decrease in phosphatidylcholine in the methionine-choline deficient diet. The method was used for the first time to asses the lipid classes induced in the p62-overexpressing mouse model and showed a significant increase in all detected lipid species with a prominent increase of triglycerides by 2-fold. Interestingly, the ratio of phosphatidylcholine to phosphatidylethanolamine was decreased, as previously suggested as a marker in the progression from steatosis to steatohepatitis. CONCLUSION:The thin layer chromatography analysis allows a reliable quantification of lipid classes and provides detailed insight into the lipogenic effect of p62.© 2013 Baishideng. All rights reserved.Key words: Non alcoholic steatohepatitis; Non alcoholic fatty liver disease; Thin layer chromatography; IMP2/ IGF2BP2; p62; Methionine choline deficient diet; Polar lipids; neutral lipids; Phosphatidylcholine/phosphatidylethanolamine ratio; Triglycerides Core tip: We describe a new method to quantify lipid classes in steatosis/steatohepatitis having advantages over both histology and classical analytical methods. Since lipid classes exert differential pathophysiological actions our method should be of interest for all researchers dealing with mechanisms of steatosis and steatohepatitis. We employ our method to investigate the lipid profile in the steatotic p62 transgenic mouse 558October 27, 2013|Volume 5|Issue 10|

The IGF2 mRNA binding protein p62/IGF2BP2-2 induces fatty acid elongation as a critical feature of steatosis

“…The p62-induced steatosis is characterized by an increase in almost all lipid classes, with the most distinct effect on triglycerides ( 26 ). Accordingly, we here observed an elevated abundance of almost all fatty acids ( Table 1 ).…”

“…Furthermore, p62 has been shown to promote NASH development ( 25 ). Most lipid species are elevated in p62-induced steatosis, with triglycerides showing the strongest increase ( 26 ). p62 was originally identifi ed as an autoantigen overexpressed in about one-third to two-thirds of HCC patients and correlates with poor outcome (27)(28)(29)(30).…”

Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis

“…Only physiological roles have been described for IMP2, which is required for proper nerve cell migration and morphology during development by controlling cytoskeletal remodeling and dynamics (reviewed in [17] ). We recently reported that mice with liver-specific overexpression of p62 develop a fatty liver and show increased development towards NASH-induced fibrosis [4,18,19] . This amplification of an inflammatory response was observed in a feeding model that omitted dietary cholesterol.…”

“…The composition of fatty acids in the liver has emerged as a critical factor promoting the development of non-alcoholic steatohepatitis (NASH) and potentially HCC [3][4][5][6][7] , with monounsaturated fatty acids (MUFA) implicated in a pathophysiological role [6,8] . Recent observations also highlighted the accumulation of free cholesterol as an important trigger for the progression from simple steatosis to severe NASH [9][10][11] .…”

Comparative evaluation of starting traditional and fermented mixed fodders for juveniles of pacific salmon

“…Идентификацию хроматографических пиков про-водили с использованием индексов удерживания ECL (Christie, 1988). Содержание отдельных классов липидов в сравнении со стандартами анализировали методом тон-кослойной хроматографии на аналитических пластинах «Sorbfil» с использованием системы растворителей гексан : диэтиловый эфир -1 : 1 (по объему) с добавлением 0,1 % уксусной кислоты в качестве элюента (Laggai et al, 2013).…”