Serum bile acid and unsaturated fatty acid profiles of non-alcoholic fatty liver disease in type 2 diabetic patients

Figure 1 The flow diagram depicts the incorporation and distribution of the study cohort. T2DM: Type 2 diabetes mellitus; HC: Healthy control; NAFLD: Non-alcoholic fatty liver disease; F_0-1: No or mild clinically fibrosis with fibrosis 4 score (FIB-4) < 1.3; F_2-4: Clinically significant fibrosis with FIB-4 ≥ 1.3; BAs: Bile acids; UFAs: Unsaturated fatty acids.

Figure 2 The distribution of bile acids and unsaturated fatty acids distribution among healthy control, type 2 diabetes mellitus without non-alcoholic fatty liver disease, and type 2 diabetes mellitus with non-alcoholic fatty liver disease is examined. A: Clustering analysis is conducted to analyze the levels of serum bile acids (BAs) and unsaturated fatty acid (UFA). Serum levels of BAs and UFAs in three groups. Wilcoxon test (^aP < 0.05); B: Spearman’s correlation analysis between UFAs and clinical indicators in type 2 diabetes mellitus with or without non-alcoholic fatty liver disease (^aP < 0.05, ^bP < 0.01). T2DM: Type 2 diabetes mellitus; HC: Healthy control; NAFLD: Non-alcoholic fatty liver disease; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; C-P: C-peptide; TBIL: Total bilirubin; DBIL: Direct bilirubin; IB: Indirect bilirubin; BMI: Body mass index; TyG: Triglyceride glucose index; TG: Triglyceride; DBP: Diastolic blood pressure; BUN: Blood urea nitrogen; FPG: Fasting plasma glucose; TC: Total cholesterol; LDL-C: Low-density lipoprotein cholesterol; UA: Uric acid; SCR: Serum creatinine; HDL-C: High-density lipoprotein cholesterol; SBP: Systolic blood pressure; HbA1c: Glycosylated hemoglobin; CDCA: Chenodeoxycholic acid; TLCA: Taurolithocholicacid; GDCA: Glycodeoxycholic acid; UDCA: Ursodeoxycholic acid; TDCA: Taurodeoxycholic acid.

Figure 3 Multivariate analysis of bile acids and unsaturated fatty acids in orthogonal partial least squares-discriminant analysis model. A: Orthogonal partial least squares-discriminant analysis (OPLS-DA) 3D model between type 2 diabetes (T2DM) with or without non-alcoholic fatty liver disease (NAFLD) groups. R²X = 0.227, R²Y = 0.054, and Q²Y = 0.026; B: The 200−permutation test demonstrated no overfitting in the OPLS-DA model [Q² = (0, -0.025), R² = (0, 0.028)]; C: The contribution of the metabolite to distinguish T2DM with or without NAFLD is indicated by variable influence on projection values. NAFLD: Non-alcoholic fatty liver disease; CDCA: Chenodeoxycholic acid; TLCA: Taurolithocholicacid; GDCA: Glycodeoxycholic acid; UDCA: Ursodeoxycholic acid.

Figure 4 Logistic regression risk prediction model about non-alcoholic fatty liver disease in type 2 diabetes mellitus. A: Odds ratio (OR) was represented in restricted cubic splines (RCS) nested showing association of palmitoleic acid (C16:1) level on a continuous scale and non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes mellitus (T2DM); B: OR was represented in RCS nested showing association of alpha-octadecatrienoic acid (α-C18:3) level on a continuous scale and NAFLD in T2DM. Shaded areas represent 95% confidence interval (95%CI). 12.9 and 9.21 μmol/L were set as the reference values for C16:1 and α-C18:3 respectively; C: C16:1 was analyzed for their relationship with Triglyceride glucose index (TyG) by threshold; D: α-C18:3 was analyzed for their relationship with TyG by threshold. The statistical comparison was performed by the Wilcoxon test (statistically significant: P < 0.05); E: Comparison of three NAFLD predictive model assessments of the 95%CI of their area under the curve values; F: Comparison of model performance from Decision Curve analysis. Model 1 consists of C16:1, α-C18:3, eicosapentaenoic acid, ω-3 docosapentaenoic acid, γ-linolenic acid. Model 2 consists of sex, age, body mass index, systolic blood pressure, duration of diabetes, TyG, high-density lipoprotein cholesterol, glycosylated hemoglobin, fasting c-peptide, and c-peptide 2 h postprandial. Model 3 was constructed by the factors of model 2 and model 1 together. C16:1: Association of palmitoleic acid; α-C18:3: Alpha-octadecatrienoic acid; 95%CI: 95% confidence interval; AUC: Area under the curve.

Figure 5 Performances of two unsaturated fatty acids with the ability to identify clinically significant fibrosis in nonalcoholic fatty liver disease. A: Serum levels of alpha-octadecatrienoic acid (α-C18:3) in healthy control (HC), type 2 diabetes mellitus (T2DM), no or mild clinically fibrosis with fibrosis 4 score (FIB-4) < 1.3 (F_0-1) and clinically significant fibrosis with FIB-4 ≥ 1.3 (F_2-4) groups; B: Serum levels of γ-linolenic acid (γ-C18:3) in HC, T2DM, F_0-1 and F_2-4 groups; C: Comparison of three clinically significant fibrosis predictive model assessments of the 95%CI on their area under the curve values. Model 1 consists of α-C18:3 and γ-C18:3. Model 2 consists of sex, age, body mass index, systolic blood pressure, duration of diabetes, Triglyceride glucose index, high-density lipoprotein cholesterol, glycosylated hemoglobin, fasting c-peptide, and c-peptide 2 h postprandial. Model 3 was constructed by the factors of model 2 and model 1 together. Wilcoxon test (^aP < 0.05). α-C18:3: Alpha-octadecatrienoic acid; HC: Healthy control; γ-C18:3: γ-linolenic acid; T2DM: Type 2 diabetes mellitus; 95%CI: 95% confidence interval; HC: Healthy control; AUC: Area under the curve; F_0-1: No or mild clinically fibrosis with fibrosis 4 score (FIB-4) < 1.3; F_2-4: Clinically significant fibrosis with FIB-4 ≥ 1.3.