High-fat diet impact on intestinal cholesterol conversion by the microbiota and serum cholesterol levels

Abstract

Cholesterol-to-coprostanol conversion by the intestinal microbiota has been suggested to reduce intestinal and serum cholesterol availability, but the relationship between intestinal cholesterol conversion and the gut microbiota, dietary habits, and serum lipids has not been characterized in detail. We measured conserved proportions of cholesterol high and low-converter types in individuals with and without obesity from two distinct, independent low-carbohydrate high-fat (LCHF) dietary intervention studies. Across both cohorts, cholesterol conversion increased in previous low-converters after LCHF diet and was positively correlated with the fecal relative abundance of Eubacterium coprostanoligenes. Lean cholesterol high-converters had increased serum triacylglycerides and decreased HDL-C levels before LCHF diet and responded to the intervention with increased LDL-C, independently of fat, cholesterol, and saturated fatty acid intake. Our findings identify the cholesterol high-converter type as a microbiome marker, which in metabolically healthy lean individuals is associated with increased LDL-C in response to LCHF.

Keywords: Genomics; Health sciences; Human metabolism; Microbiology.

Graphical abstract

Figure 1

Equal distributions of cholesterol high and low-converter types among humans with and without obesity (A) Sterol and stanol concentrations as determined by LC-MS/HRMS in 28 fecal samples from the KETO study participants before dietary intervention. (B) Negative correlation (Spearman’s rank) between fecal coprostanol and cholesterol concentrations and bimodal distribution of cholesterol high (N = 17), intermediate (N = 4) and low-converter (N = 7) types, as classified based on the fecal coprostanol/cholesterol ratio. (C and D) Comparable negative correlations (Spearman’s rank) between the fecal concentrations of the phytosterols sitosterol and campesterol and the corresponding stanol conversion products stigmastanol (n = 26), and 5β-campestanol (n = 23). (E) Similar fecal sterol and stanol concentration profiles in individuals with obesity from the CARBFUNC study before dietary intervention (n = 145 samples), compared to lean KETO study participants (N = 89/26/30 for cholesterol high/intermediate/low-converters). (F) Negative correlation (Spearman’s rank) of fecal coprostanol and cholesterol concentrations in CARBFUNC study participants and bimodal distribution into high and low-converter types. Spearman’s rank correlation, Benjamini-Hochberg (BH) corrected: q > 0.05 ns, q < 0.01 ∗∗, q < 0.001 ∗∗∗. Pooled data are represented as mean ± SD.

Figure 2

Distinct microbiota associations with fecal cholesterol and coprostanol (A and B) Reduced diversity (Wilcoxon rank-sum) and altered composition (ANOSIM) of taxonomic microbiota profiles of cholesterol high (N = 89) compared to low-converters (N = 30) with obesity from the CARBFUNC study, but no difference between lean cholesterol high (N = 7) and low-converters (N = 12) from the KETO study. p > 0.05 ns, p < 0.001 ∗∗∗. (C) Positive and negative associations of bacterial taxa with fecal coprostanol and cholesterol concentrations, as identified by a generalized linear mixed model (GLMM) for the combined dataset of KETO (N = 23) and CARBFUNC (N = 145) study participants. For the GLMM input, zero values were replaced with a pseudocount and cohort and gender added as random and fixed effects (see STAR Methods for details). (D and E) Across both cohorts combined, Eubacterium coprostanoligenes.group (N = 158) was positively correlated with fecal coprostanol and negatively correlated with fecal cholesterol concentrations, whereas Lachnoclostridium (N = 125), was positively correlated with fecal cholesterol concentrations. (F) E. coprostanoligenes.group relative abundance was the most informative microbiota feature for predicting the cholesterol converter type with a random forest model, based on leave-one-out cross-validation (LOOCV). (G) Increased relative abundance of E. coprostanoligenes.group and Christensenellaceae.R.7.group in cholesterol high-converters. Dashed lines indicate pseudocount values (0.0001% relative abundance) of samples with zero taxon counts. Benjamini-Hochberg (BH) corrected: q > 0.1 ns, q < 0.1 ∗, q < 0.05 ∗∗, q < 0.01 ∗∗∗, q < 0.001 ∗∗∗∗. Pooled data are represented as mean ± SD.

Figure 3

Distinct associations of fecal cholesterol and coprostanol concentrations with short and branched-chain fatty acids (A and B) Positive correlation of fecal cholesterol with the concentrations of the SCFAs acetate, propionate and butyrate (A) and of fecal coprostanol with the BCFA isobutyrate (B) in fecal samples from lean KETO study participants (N = 28) and in individuals with obesity from the CARBFUNC study (N = 145) before the dietary intervention. Spearman’s rank correlation, BH-corrected: q > 0.05 ns, q < 0.05 ∗, q < 0.01 ∗∗, q < 0.001 ∗∗∗.

Figure 4

Circulating blood lipids in cholesterol high and low-converters (A) Comparable total cholesterol, but increased serum TAG and decreased HDL-C levels in lean cholesterol low (N = 7) compared to high-converters (N = 17) from the KETO study. (B) No significant difference in blood lipid levels between cholesterol high (N = 89) and low-converters (N = 30) with obesity from the CARBFUNC study. Wilcoxon rank-sum, p > 0.05 ns, p < 0.05 ∗. Pooled data are represented as mean ± SD.

Figure 5

Diet impact on cholesterol-to-coprostanol conversion (A) Cholesterol high-converters with obesity (CARBFUNC study, N = 89) but not without obesity (KETO study, N = 17) exhibited an increased fecal coprostanol/stigmastanol ratio compared to low-converters (N_CARBFUNC = 26, N_KETO = 5), suggesting a higher proportion of animal vs. plant-derived dietary fat intake (Wilcoxon rank-sum, p < 0.001 ∗∗∗). Zero values were replaced with a pseudocount (1 nmol/mg dry weight [DW]). (B) LCHF diets induced consistent taxonomic microbiota alterations in the KETO and CARBFUNC cohorts, based on a combined GLMM analysis (N_PRE = 173, N_LCHF = 62). Bacterial taxa with significant changes in relative abundance (q < 0.1, BH-corrected Tukey’s test, see horizontal blue line) and a positive or negative fold-change of > 0.25 in estimated marginal means (EMM) are marked with red dots and labels, unless they were detected by the GLMM as cohort and/or sex-associated (black dots). (C) Distribution of cholesterol high and low-converters among all KETO and CARBFUNC study participants before (N = 143) and after (N = 54) LCHF dietary intervention (gray lines connecting pre and post-intervention samples). (D) Increased cholesterol-to-coprostanol conversion in low-converters from both cohorts on the LCHF diets (N_PRE = 37, N_LCHF = 11), as evidenced by reduced fecal cholesterol and increased fecal coprostanol levels and increased coprostanol/cholesterol ratios. (E) The increased cholesterol-to-coprostanol conversion in low-converters on LCHF diets was accompanied by an increased fecal relative abundance of E. coprostanoligenes.group (N_{PRE, LOW} = 37, N_{LCHF, LOW} = 11), resulting in similar relative abundances in high and low-converters on the LCHF diets (N_PRE, HIGH = 106, N_{LCHF, HIGH} = 43). (F) Decreased cholesterol-to-coprostanol conversion in high-converters from both cohorts after LCHF diet intervention (N_PRE = 106, N_LCHF = 43), at least based on increased fecal cholesterol levels and an increased coprostanol/cholesterol ratio. Individuals were classified as cholesterol high/low-converters based on pre-intervention time points, with symbol colors indicating the classification during the LCHF diet. Significance determined by GLMM and post-hoc Tukey’s test (Benjamini-Hochberg-corrected): q > 0.1 ns, q < 0.1 ∗, q < 0.05 ∗∗, q < 0.01 ∗∗∗, q < 0.001 ∗∗∗∗. Pooled data are represented as mean ± SD.

Figure 6

Cholesterol converter type-specific dietary impact on serum lipids (A and B) Decreased serum TAG levels in both cholesterol low-converters (A, N[PRE] = 37, N[LCHF] = 11) and high-converters (B, N[PRE] = 106, N[LCHF] = 43), based on estimated marginal means (EMMs), as determined by the GLMM for the combined KETO and CARBFUNC cohorts. (C) Increased serum LDL-C levels in lean cholesterol high-converters from the KETO study on the LCHF diet (N[PRE] = 17, N[LCHF] = 18). Individuals were classified as cholesterol high/low-converters based on pre-intervention time points, with symbol colors indicating the classification during the LCHF diet. Significance determined by GLMM and post-hoc Tukey’s test (Benjamini-Hochberg-corrected): q > 0.1 ns, q < 0.1 ∗, q < 0.05 ∗∗, q < 0.01 ∗∗∗, q < 0.001 ∗∗∗∗. Pooled data are represented as mean ± SD.