Dihydroceramides in Triglyceride-Enriched VLDL Are Associated with Nonalcoholic Fatty Liver Disease Severity in Type 2 Diabetes

Abstract

Plasma dihydroceramides are predictors of type 2 diabetes and related to metabolic dysfunctions, but the underlying mechanisms are not characterized. We compare the relationships between plasma dihydroceramides and biochemical and hepatic parameters in two cohorts of diabetic patients. Hepatic steatosis, steatohepatitis, and fibrosis are assessed by their plasma biomarkers. Plasma lipoprotein sphingolipids are studied in a sub-group of diabetic patients. Liver biopsies from subjects with suspected non-alcoholic fatty liver disease are analyzed for sphingolipid synthesis enzyme expression. Dihydroceramides, contained in triglyceride-rich very-low-density lipoprotein (VLDL), are associated with steatosis and steatohepatitis. Expression of sphingolipid synthesis enzymes is correlated with histological steatosis and inflammation grades. In conclusion, association of plasma dihydroceramides with nonalcoholic fatty liver might explain their predictive character for type 2 diabetes. Our results suggest a relationship between hepatic sphingolipid metabolism and steatohepatitis and an involvement of dihydroceramides in the synthesis/secretion of triglyceride-rich VLDL, a hallmark of NAFLD and type 2 diabetes dyslipidemia.

Trial registration: ClinicalTrials.gov NCT02431234.

Keywords: NAFLD; VLDL; biomarker; diabetes mellitus; dihydroceramide; lipoproteins; liver; sphingolipids.

Conflict of interest statement

The authors declare no competing interests.

© 2020 The Author(s).

Figures

Graphical abstract

Figure 1

Correlations between Plasma Sphingolipids and Clinical Characteristics in the CERADIAB and DIACART Cohorts Shown as a Heatmap (A) Correlation coefficient based on univariate regression was calculated between the sum of each class of sphingolipid species or individual sphingolipid species and biological characteristics for the CERADIAB cohort (n = 90). (C) Similar calculations were made for the DIACART cohort (n = 149), but, for ceramide and sphingomyelin, only the sum of the different species is presented. (B and D) Positive correlations are depicted in red and negative correlations in blue. Statistical significance of the correlations is indicated in (B) for the CERADIAB cohort and in (D) for the DIACART cohort. A Benjamini-Hochberg correction procedure for multiple comparisons was used. BMI, body mass index; Cer, ceramide; Chol, cholesterol; DhCer, dihydroceramide; F, fibrotest; FLI, fatty liver index; hsCRP, high sensitivity C-reactive protein; NT, NASH test; sphingomyelin, sphingomyelin; ST, steatotest; TG, triglyceride.

Figure 2

Plasma DhCer and Total Cer Concentrations According to SteatoTest and NASH Test in the CERADIAB Cohort (A) Plasma DhCer concentrations according to SteatoTest (ST) values, S0: 0  0.70. (B) Total plasma ceramide concentrations according to SteatoTest scores. (C) Plasma DhCer concentrations according to NASH Test scores. (D) Total plasma ceramide concentrations according to NASH test scores. ∗p 

Figure 3

Principal-Component Analysis of DhCer Species in the CERADIAB Cohort A principal-component analysis was applied to the DhCer data of the CERADIAB cohort (n = 90). (A) Variable correlation plot. (B) Statistical significance of the correlations between PCs and clinical variables shown as a heatmap. Statistical significances were corrected by the Benjamini-Hochberg procedure for multiple comparisons.

Figure 4

Correlations between Plasma DhCer Concentrations, Fatty Liver Index, and NASH Test in the DIACART Cohort (A) Correlation of total plasma DhCer concentration with Fatty Liver Index. The coefficient r is shown as well as the p value; n = 149. (B) Correlation of the Fatty Liver Index with SteatoTest. The coefficient r is shown as well as the p value; n = 149. (C) Total plasma DhCer concentration (mean ± SEM, n = 149) according to the fatty liver index scores. (D) Total plasma DhCer concentration (mean ± SEM, n = 149) according to the NASH Test scores. ∗p 60: n = 86. N0: n = 48, N1: n = 77, N2: n = 24.

Figure 5

Analysis of VLDL Sphingolipids and Their Relation with SteatoTest and NASH Test Analysis were performed on 32 type 2 diabetic patients. For (A) and (B), S0: n = 7, S1-S2: n = 13, S3: n = 12. (A) Apo B concentration in the VLDL fraction according to the degree of estimated steatosis. S0: 0  0.70. (B) Ratio triglyceride (TG) concentration/Apo B concentration in the VLDL fraction according to the degree of estimated steatosis. (C) Scatterplot showing the correlation between VLDL total dihydroceramide (DhCer) concentration and SteatoTest. (D) Scatterplot showing the correlation between the ratio VLDL total DhCer/VLDL total ceramide concentration and SteatoTest. (E) Scatterplot showing the correlation between VLDL total DhCer concentration and VLDL triglycerides. (F) Scatterplot showing the correlation between VLDL total ceramide concentration and SteatoTest. (G) Scatterplot showing the correlation between LDL total DhCer concentration and SteatoTest. In the scatterplots, concentration units are given as μmol/L plasma. ∗∗∗p 

Figure 6

Analysis of the Expression (mRNA) of Enzymes of Sphingolipid Metabolism in Liver According to the NAFLD State Box-and-whisker plot showing SPTLC1 (A), DEGS1 (B), and SGMS1 (C) gene expressions according to steatosis (S0–S3), activity (A0–A4), and fibrosis (F0–F4) grades determined histologically. ∗p < 0.05 and ∗∗p < 0.01 difference significant when comparing a given grade to grade 0. mRNA expression is given as arbitrary units. S0: n = 9, S1: n = 20, S2: n = 26, S3, n = 18. A0: n = 10, A1: n = 13, A2: n = 30, A3: n:15, A4: n = 5. F0: n = 30, F1: n = 19, F2: n = 15, F3: n = 8, F4, n = 1.