Multi-technique comparison of atherogenic and MCD NASH models highlights changes in sphingolipid metabolism - PubMed (original) (raw)
Multi-technique comparison of atherogenic and MCD NASH models highlights changes in sphingolipid metabolism
Sophie A Montandon et al. Sci Rep. 2019.
Abstract
Lipotoxicity is a key player in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive subtype of nonalcoholic fatty liver disease (NAFLD). In the present study, we combine histological, transcriptional and lipidomic approaches to dissociate common and specific alterations induced by two classical dietary NASH models (atherogenic (ATH) and methionine/choline deficient (MCD) diet) in C57BL/6J male mice. Despite a similar degree of steatosis, MCD-fed mice showed more pronounced liver damage and a worsened pro-inflammatory and pro-fibrogenic environment than ATH-fed mice. Regarding lipid metabolism, the ATH diet triggered hepatic counter regulatory mechanisms, while the MCD diet worsened liver lipid accumulation by a concomitant increase in lipid import and reduction in lipid export. Liver lipidomics revealed sphingolipid enrichment in both NASH models that was accompanied by an upregulation of the ceramide biosynthesis pathway and a significant rise in dihydroceramide levels. In contrast, the phospholipid composition was not substantially altered by the ATH diet, whereas the livers of MCD-fed mice presented a reduced phosphatidylcholine to phosphatidylethanolamine (PC/PE) ratio and a strong depletion in phospholipids containing the sum of 34-36 carbons in their fatty acid chains. Therefore, the assessment of liver damage at the histological and transcriptional level combined with a lipidomic analysis reveals sphingolipids as shared mediators in liver lipotoxicity and pathogenesis of NASH.
Conflict of interest statement
The authors declare no competing interests.
Figures
Figure 1
ATH and MCD diets induce similar degree of steatosis, but different NASH severity. Liver weight expressed in grams (A) and as percentage of body weight. (B) Plasma levels of liver enzymes: ALT (C) and AST. (D) Liver histology: hematoxylin and eosin (H&E), Oil Red O, Sirius Red and periodic acid Schiff (PAS). (E) SAF score with Steatosis, Activity (lobular inflammation + ballooning) and Fibrosis. (F) Evaluation of the percent of hepatocyte containing micro- and macrosteatosis. (G) Quantification of lipids (H), collagen (I) and glycogen (J) expressed as percent of area stained respectively with Oil red O, Sirius Red and PAS. Scale bars 100 µm, black arrows: immune cell infiltration, white arrows: macrosteatosis, grey arrow: microsteatosis, yellow dashed circle: lipogranuloma. One-way ANOVA: *p value ATH/MCD vs CTRL < 0.05, #p value MCD vs ATH < 0.05.
Figure 2
Real-time quantitative PCR of hepatic lipid genes of ATH- and MCD-fed mice. Hepatic relative gene expression (mean and SEM) ordered by metabolic function. Normalized to RPS29. Mean values in bold: p value vs CTRL group < 0.05. Color code according to the LOG2 of the mean from dark green (highly downregulated vs CTRL group), white (no change) to dark red (highly upregulated).
Figure 3
Lipidomic analysis of 944 lipid species of CTRL, ATH and MCD mouse livers. LOG2 of mean concentration of each lipid metabolite sorted by lipid class (PC, PE, PI, PS, CL, Cer, GlcCer, SM) and represented as heat maps. CTRL group = left column, ATH group = central column, MCD group = right column. (A) Hierarchical clustering of 801 lipid species presented as LOG2 of fold change vs Ctrl group and most represented lipid classes per cluster. (B) The predominant species per lipid group is shown in bold. Absolute concentration of lipid classes normalized to phosphate content. (C) Principal component analysis of the 801 lipid species with N = 6 animals per group. (D) PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PS: phosphatidylserine, CL: cardiolipin, Cer: ceramide, GlcCer: glycosylceramide, SM: sphingomyelin, DHCer: dihydroceramide, GlcDHCer: dihydroglycosylceramide, S1P: sphingosine-1-phosphate, SL: sphingolipids. One-way ANOVA: *p value ATH/MCD vs CTRL < 0.05, #p value MCD vs ATH < 0.05.
Figure 4
Sphingolipid metabolism is dysregulated in livers of ATH- and MCD-fed mice. Concentration of sphingolipid classes normalized to the phosphate content. (A) Gene expression of main enzymes involved in the ceramide biosynthesis pathways. (B) Concentrations of lipid species: ceramides (C), glycosylceramides (D) and sphingomyelins. (E) Cer: ceramide, DHCer: dihydroceramide, GlcCer: glycosylceramide, GlcDHCer: dihydroglycosylceramide, C1P: ceramide-1-phosphate, S1P: sphingosine-1-phosphate, SM: sphingomyelin. One-way ANOVA: *p value ATH/MCD vs CTRL < 0.05, #p value MCD vs ATH < 0.05.
Figure 5
Liver lipidomics highlights effects of ATH and MCD diets on phospholipid homeostasis including phospholipid composition, fatty acid chain length and level of desaturation. Relative concentration of lipid classes. (A) Phosphatidylcholine to phosphatidylethanolamine ratio. (B) Total amount of PC, PE, PI and PS represented according to the nature of the fatty acid linkage (diacyl vs alkyl-acyl (ether) or monoacyl (lyso) (C), degree of saturation (D) or chain length (E). Same as in ‘C-E’ but separately for phosphatidylcholine (F), phosphatidylethanolamine (G), phosphatidylinositol (H) or phosphatidylserine. (I) PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PS: phosphatidylserin1e, SL: sphingolipids, CL: cardiolipin, diacyl: diacylphospholipids, ether: alkyl-acyl-containing phospholipids, lyso: lysophospholipids, SFA: saturated fatty acids, MUFA: monounsaturated fatty acids (total sum of 1 double bond in both fatty acyl chains), PUFA: polyunsaturated fatty acids (total sum of 2–6 double bonds in both fatty acyl chains), LC: long chains (total sum of 28–34 carbons in both fatty acyl chains), VLC: very long chains (total sum of 36–44 carbons in both fatty acyl chains). One-way ANOVA: *p value ATH/MCD vs CTRL < 0.05, #p value MCD vs ATH < 0.05.
Figure 6
Transcriptional and lipidomic approaches highlights common and specific alterations in ATH- and MCD-fed mice. Illustration of the main alterations of gene expression (liver and ileum), liver lipid content and blood parameters induced by the ATH diet (left panel) and the MCD diet (right panel). In red: alterations that are divergent in the two dietary-induced NASH models. Unmodified drawings were downloaded from the free medical image database Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.
References
- Chalasani N, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology. 2012;55:2005–2023. doi: 10.1002/hep.25762. - DOI - PubMed
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