Bile acids regulate lipid metabolism through selective actions on fatty acid absorption

Abstract

Intestinal lipid absorption, the entry point for fats into the body, requires the coordinated actions of bile acids and lipases. Here, we uncover distinct yet cooperative roles of bile acids in driving the differential uptake of dietary fatty acids. We first decreased the bile acid pool size by disrupting the rate-limiting enzyme in bile acid synthesis, Cyp7a1, using liver-directed gene editing in mice. Compared with lipase inhibition, reduced bile acids prevented diet-induced obesity, increased anorectic hormones, suppressed excessive eating, and improved systemic lipid metabolism. Remarkably, decreasing bile acids selectively reduced the absorption of saturated fatty acids but preserved polyunsaturated fatty acids. By targeting additional bile acid enzymes, we identified specific functions of individual bile acid species. Mechanistically, we show that cholic acid preferentially solubilizes polyunsaturated fatty acids into mixed micelles for intestinal uptake. Our studies demonstrate that bile acids can selectively control fatty acid uptake, revealing insights for future interventions in metabolic diseases.

Keywords: CYP2A12; CYP2C70; CYP7A1; CYP8B1; GLP-1; bile acids; fatty acids; lipid absorption; lipogenesis.

Figure 1.. Manipulating absorption by distinct mechanisms leads to differences in energy balance.

(A) Overview of dietary fat digestion and absorption. (B) Diagram (left) of AAV-CRISPR tool. U6, U6 promoter; gRNA, guide RNA; HLP, hybrid liver promoter; SaCas9, Staphylococcus aureus Cas9. Western blot analysis (right) of hepatic CYP7A1 expression in representative mice. (C) Total bile acid pool from gallbladder, liver, and small intestine in Cyp7a1 CRISPR mice. n = 9–10 mice per group. Lagodeoxycholic acid and 7-ketolithocholic acid not shown in the legend but included in the total bile acid pool analysis. (D) Thin layer chromatography of fecal lipids from representative orlistat-treated mice. STD, standard; TG, triglyceride; FFA, free fatty acid; DG, diacylglyceride; MG, monoglyceride. (E) Experimental schematics to assess energy balance in Cyp7a1 CRISPR (top) and orlistat-treated mice (bottom). (F) Body mass for Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. n = 9–10 mice per group. (G) Fecal energy loss for Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. n = 7–10 per group. (H) Continuous (left) and average (right) energy expenditure during dark (grayed background) and light (white background) cycles over final one week of the experiment for Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. n = 7–8 mice per group. For the average energy expenditure graph, each data point represents one half-day timepoint (seven dark cycles, six light cycles). (I) Food intake for Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. n = 7–8 mice per group. MCA, muricholic acid; LCA, lithocholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; CA, cholic acid; UDCA, ursodeoxycholic acid; HDCA, hyodeoxycholic acid; T, taurine-conjugated. Data are represented as mean ± standard error of mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, by two-sided t-tests (C, G), multiple t-tests (C), two-way ANOVA with Šídák’s multiple comparisons test (F), ANCOVA using body mass as a covariate (H, I). See also Figure S1.

Figure 2.. Unabsorbed lipids promote enteroendocrine incretin hormone secretion.

(A) Experimental schematics to measure plasma hormone levels in Cyp7a1 CRISPR (top) and orlistat-treated mice (bottom) following fast and refeed. (B-E) Plasma concentrations of (B) ghrelin, (C) leptin, (D) GLP-1, and (E) PYY during fasted and refed states for Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. n = 6–8 mice per group. (F) Visualization of neutral lipid accumulation in cross sections of small intestinal Swiss rolls from representative mice. Schematic (left) and confocal immunofluorescence imaging of intestinal Swiss roll (right). LipidTOX (red); DAPI (blue). Scale bar is 1000 μm. (G) Experimental schematic to measure plasma GLP-1 in Gpr120^−/− mice treated with Cyp7a1 CRISPR. (H) Western blot analysis of hepatic CYP7A1 expression in representative Gpr120^−/− mice treated with Cyp7a1 CRISPR. (I) Plasma concentration of GLP-1 in Gpr120^−/− mice treated with Cyp7a1 CRISPR following fast and refeed. n = 10 mice per group. (J) Experimental schematic to profile the microbiome in Cyp7a1 CRISPR mice by metagenomic shotgun sequencing. (K) Relative abundance of taxa at the phylum level in microbiomes of Cyp7a1 CRISPR mice. n = 9–10 mice per group. (L) Alpha diversity at the species level was assessed by Shannon index (left) and number of observed species (right) in microbiomes of Cyp7a1 CRISPR mice. n = 9–10 mice per group. H, hour; Sac, sacrifice; F, fasted; R, refed; GLP-1, glucagon-like peptide-1; PYY, peptide YY. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-sided paired t-tests between fasted and refed states within groups (B-E, I), multiple t-tests (K), two-sided unpaired t-tests (L). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, two-sided unpaired t-tests between control and treatment groups for fasted and refed states (B-E, I). Color of the significance annotation indicates the group with the greater mean value. See also Figure S2.

Figure 3.. Targeting dietary fat absorption modulates systemic lipid metabolism.

(A and E) Histological images of representative liver sections stained with hematoxylin and eosin from (A) Cyp7a1 CRISPR and (E) orlistat-treated mice. Scale bar is 100 μm. (B and F) Lipidomic analyses of hepatic lipid classes from (B) Cyp7a1 CRISPR and (F) orlistat-treated mice. n = 8–10 mice per group. Directionality of triangle indicates significantly increased or decreased compared to respective controls. (C and G) Lipidomic analyses of hepatic TG species from (C) Cyp7a1 CRISPR and (G) orlistat-treated mice. Dashed line on volcano plot is the threshold at which differences reach statistical significance. TG species that are significantly different are colored yellow, TG species not significant are colored grey. PUFA-containing TG species that are significantly increased in Cyp7a1 CRISPR mice are colored blue. n = 8–10 mice per group. (D and H) Bubble plot of fold change in hepatic fatty acid abundance from (D) Cyp7a1 CRISPR or (H) orlistat-treated mice. n = 8–10 mice per group. Bubble color and size indicate significance. (I and L) Hepatic mRNA expression of Acaca, Fasn, and Scd1 in (I) Cyp7a1 CRISPR and (L) orlistat-treated mice. n = 7–10 mice per group. (J and M) Western blot analyses of hepatic ACC, FASN, and SCD1 expression in representative (J) Cyp7a1 CRISPR and (M) orlistat-treated mice. (K and N) Percentage of total newly synthesized deuterium-labeled fatty acids in livers of (K) Cyp7a1 CRISPR and (N) orlistat-treated mice. n = 10 mice per group. (O-Q, S) Lipidomic analyses of plasma and perigonadal white adipose tissue for (O, Q) total TG concentration and (P, S) TG species abundance by fold change in Cyp7a1 CRISPR mice. n = 7–9 mice per group. (R) Histological images of representative perigonadal white adipose tissue sections stained with hematoxylin and eosin from Cyp7a1 CRISPR mice. Scale bar is 100 μm. TG, triglyceride; SM, sphingomyelin; PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; LacCER, lactosylceramide; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; HexCER, hexosylceramide; FFA, free fatty acid; DG, diacylglyceride; Cer d18:1, ceramide 18:1; Cer d18:0, ceramide 18:0; CE, cholesterol ester; PUFA, polyunsaturated fatty acid; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid. Data are represented as mean ± SEM (B, F, I, K, L, N, O, Q), fold change (C, D, G, H, P, S). *p < 0.05, **p < 0.01, ***p < 0.001, by two-sided t-tests (B, F, I, K, L, N, O, Q), multiple t-tests (B, D, F, H). See also Figure S3, Table S1, Table S2.

Figure 4.. Selective fatty acid absorption following the reduction of bile acids.

(A) Experimental schematics to measure fatty acid absorption in Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. Sucrose polybehenate was added to the diet at timepoints indicated by chevrons. (B) Cumulative fatty acid excretion for Cyp7a1 CRISPR (top) and orlistat-treated (bottom) mice. n = 6–10 mice per group. (C and D) Absorption of (C) total fatty acids (FA) and (D) saturated, monounsaturated, and polyunsaturated fatty acids in Cyp7a1 CRISPR (top) and orlistat-treated mice (bottom). n = 7–10 mice per group. Data are represented as mean ± SEM. **p < 0.01, ***p < 0.001, by two-way ANOVA with Šídák’s multiple comparisons test (B), two-sided t-tests (C, D). Color of significance annotation indicates the group with the greater mean value. See also Figure S4.

Figure 5.. Fatty acids are differentially solubilized in bile.

(A) Experimental schematic to determine the volume of mouse or human gallbladder bile required to solubilize equal amounts of fatty acids varying in acyl chain length and degree of unsaturation. Above a critical volume of bile, the bile, fatty acid, and fluorophore aggregate and increase fluorescence emission upon micelle formation. (B and E) Pie charts of bile acid composition from (B) gallbladder bile pooled from wild-type mice (n = 37) and (E) human gallbladder bile (n = 1). Lagodeoxycholic acid and 7-ketolithocholic acid are not shown in the legend but included in the mouse bile acid pool analysis. (C and F) Scatter plots of (C) mouse and (F) human bile volume against fluorescence intensity. Inflection point (red) represents the minimum volume of bile required to micellize in an aqueous solution. (D and G) Table (top) of critical volumes of (D) mouse and (G) human bile mixed with free fatty acids C18:0, C18:1, C18:2, and resulting scatter plots (bottom). MCA, muricholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; HDCA, hyodeoxycholic acid; UDCA, ursodeoxycholic acid; T, taurine-conjugated; G, glycine-conjugated. See also Figure S5.

Figure 6.. Fatty acid absorption changes with alterations in bile acid pool composition.

(A) Overview of hepatic bile acid synthesis pathway. (B) Experimental schematic (top) to measure fatty acid absorption in Cyp8b1 CRISPR, Cyp2c70 CRISPR, and Cyp2a12 CRISPR mice. Donut chart of total bile acid pool from the gallbladder, liver, and small intestine for each respective group (bottom). n = 9–10 mice per group. (C) Absorption of total fatty acids in Cyp8b1 CRISPR, Cyp2c70 CRISPR, and Cyp2a12 CRISPR mice. n = 7–10 mice per group. (D) Experimental schematic (top) to measure fatty acid absorption in Cyp7a1 CRISPR and Cyp7a1 + Cyp2c70 CRISPR mice supplemented with CA or CDCA. Donut chart of total bile acid pool from the gallbladder, liver, and small intestine for each respective group (bottom). Size of donut charts are proportional to total bile acid pool size. n = 4 mice per group. (E) Absorption of total fatty acids in Cyp7a1 CRISPR and Cyp7a1 + Cyp2c70 CRISPR mice supplemented with CA or CDCA. Text inside each bar denotes what dietary bile acid was supplemented. For Cyp7a1 CRISPR mice supplemented with CDCA, the CDCA is converted to MCA by endogenous CYP2C70, which is reflected in the total bile acid pool (see Figure 6D). n = 3–4 mice per group. MCA, muricholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; HDCA, hyodeoxycholic acid; UDCA, ursodeoxycholic acid; T, taurine-conjugated. Lagodeoxycholic acid and 7-ketolithocholic acid are not shown in the legends but included in the total bile acid pool analyses (B, D). Data are represented as a percentage (B, D), mean ± SEM (C, E). ***p < 0.001, by one-way ANOVA. ^#p < 0.05, ^###p < 0.001, by one-way ANOVA between control CRISPR without dietary bile acids and all treatment conditions. Color of the significance annotation indicates the group with the greater mean value. See also Figure S6.

Figure 7.. Fatty acids are differentially solubilized by bile acids into mixed micelles.

(A) Experimental schematic to determine the influence of fatty acids on the CMC of specific bile acids. Above a CMC, the bile acid, fatty acid, and fluorophore aggregate and increase fluorescence emission upon micelle formation. (B) CMC plots for T-CA mixed with the fatty acids C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3. CMC (red) was determined by the intersection of the two tangents created in the plot. (C) Table of CMC values of various bile acids mixed with specific fatty acids. Bile acids highlighted in blue differentially solubilize fatty acids. (D) Experimental schematic to determine the effect of T-CA on the uptake efficiency of saturated, monounsaturated, and polyunsaturated fatty acids in human enteroids. (E) Rate of deuterium-labeled fatty acids C18:0, C18:1, and C18:2 uptake in enteroids over 15 minutes. n = 3 technical replicates. CMC, critical micelle concentration; CA, cholic acid; MCA, muricholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; T, taurine-conjugated; G, glycine-conjugated. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. Color of the significance annotation indicates the group with the greater mean value. See also Figure S7.