TCF7L2 transcriptionally regulates Fgf15 to maintain bile acid and lipid homeostasis through gut-liver crosstalk

Abstract

FGF19/FGF15 is an endocrine regulator of hepatic bile salt and lipid metabolism, which has shown promising effects in the treatment of NASH in clinical trials. FGF19/15 is transcribed and released from enterocytes of the small intestine into enterohepatic circulation in response to bile-induced FXR activation. Previously, the TSS of FGF19 was identified to bind Wnt-regulated TCF7L2/encoded transcription factor TCF4 in colorectal cancer cells. Impaired Wnt signaling and specifical loss of function of its coreceptor LRP6 have been associated with NASH. We, therefore, examined if TCF7L2/TCF4 upregulates Fgf19 in the small intestine and restrains NASH through gut-liver crosstalk. We examined the mice globally overexpressing, haploinsufficient, and conditional knockout models of TCF7L2 in the intestinal epithelium. The TCF7L2^+/- mice exhibited increased plasma bile salts and lipids and developed diet-induced fatty liver disease while mice globally overexpressing TCF7L2 were protected against these traits. Comprehensive in vivo analysis revealed that TCF7L2 transcriptionally upregulates FGF15 in the gut, leading to reduced bile synthesis and diminished intestinal lipid uptake. Accordingly, Vilin^Creert2 ; Tcf7L2^fl/fl mice showed reduced Fgf19 in the ileum, and increased plasma bile. The global overexpression of TCF7L2 in mice with metabolic syndrome-linked LRP6^R611C substitution rescued the fatty liver and fibrosis in the latter. Strikingly, the hepatic levels of TCF4 were reduced and CYP7a1 was increased in human NASH, indicating the relevance of TCF4-dependent regulation of bile synthesis to human disease. These studies identify the critical role of TCF4 as an upstream regulator of the FGF15-mediated gut-liver crosstalk that maintains bile and liver triglyceride homeostasis.

Keywords: Fgf19; NASH; lipid absorption; tcf7l2.

FIGURE 1

Tcf7L2 limits gut absorption of fatty acids. (A, B) Fasting plasma TG (n > 15 mice each genotype) and TC (n > 7 mice each genotype) in the littermate controls and Tcf7l2-bac mice. Each dot in the dot plot is a biological replicate. Unpaired t-test, two-tailed, normality-tested. (C) Hepatic TG secretion assay showing plasma TG at indicated time points after injection of mice, fasted for 6 h, with polyoxamer P-407. Mice were fed a high fat diet for 3 months. n > 6 mice each genotype. (D, E) Tcf7L2 expression in the jejunum of indicated mice by IHC (D) and RT-qPCR (E) (n > 6 mice each genotype). scale bar = 70 μm. Unpaired t-test, two-tailed. (F) Plasma TG at indicated time points after feeding mice with corn oil and co-injecting with polyoxamer P-407. The mice were fasted for 6 h prior to the start of experiment. n > 7 mice each genotype, holm-sidak posthoc test was performed. (G) BODIPY-FA uptake visualized by confocal microscopy in the jejunum of indicated mice 45 min after administration of corn oil + BODIPY-FA by oral gavage. Top panel: longitudinal section, bottom panel: cross-section. White arrows point to fluorescent BODIPY-FA in the controls and much-reduced fluorescence, indicating uptake of FA in Tcf7L2-bac. n > 5 mice each genotype, scale bar = 150 μm. (H) Quantification of percent area of BODIPY-FA in the indicated genotypes. Unpaired t-test, two-tailed, normality-tested

FIGURE 2

Tcf7L2 increases Fgf15 in the ileum to suppress triglyceride synthesis in the liver. (A) RT-qPCR showing mRNA levels of indicated genes from the isolated microvilli from distal jejunum and ileum of control and Tcf7l2-bac mice in either fast or fast/refeed conditions (n > 4 mice each condition and each genotype), unpaired t test, 2-tailed. (B, C) Total bile acid content in the plasma of fasted mice 45′ post prandially, n > 7 mice each condition (B) and fecal pellets, n > 5 mice each genotype (C) of littermate controls and Tcf7L2-bac mice, unpaired t test, 2-tailed. (D) mRNA expression of the designated genes in the liver of Tcf7L2-bac versus littermate mice after 6 h fast, n > 10 mice each genotype, unpaired t test, 2-tailed. (E) Oil Red O staining and quantification depicting neutral lipids in the liver of the designated mice, n > 5 mice each genotype, unpaired t test, 2-tailed, scale bar = 150 μm. (F–I) Measurement of indicated parameters measured when littermate control and Tcf7l2-bac mice were subjected to metabolic cages, n = 3 mice control and n = 6 mice for Tcf7l2-bac, unpaired t test, two-tailed. (J, K) Fatty acid uptake measured in epididymal WAT, BAT, and liver in the controls and Tcf7L2-bac mice. n = 3 mice each. unpaired t test, two-tailed

FIGURE 3

Tcf4 directly binds Fgf15 promoter. (A) snapshot of the UCSC genome browser showing the Fgf15 locus in the mouse genome; primers used to probe Tcf4 binding are indicated. Two primers in the promoter (P1, P2) and two in the intergenic regions of Fgf15 (S1, S2) were tested. (B) Chromatin Immunoprecipitation (ChIP) assay showing % input enrichment with IgG or TCF4 in fast (F) or Fast/Refeed (F/RF) conditions at the indicated regions at Fgf15 locus in the microvilli of distal jejunum and ileum. Unpaired t test, two-tailed, normality-tested, *p < .05 Unpaired t test, two-tailed, normality-tested, *p < .05

FIGURE 4

Tcf7L2 is necessary for the suppression of hepatic BA production by Fgf15 in the ileum. (A, B) Fasting plasma TG, and total cholesterol in TCF7L2^+/− versus wild-type mice on chow diet. n > 12 mice each genotype, each dot is data from individual mice, unpaired t test, 2-tailed. (C) Bile salt concentration in TCF7L2^+/− versus wild-type mice, unpaired t test, 2-tailed, n > 8 mice each genotype. (D) Ileal Fgf15 mRNA by RT-qPCR in the designated mice, unpaired t test, two-tailed, n = 8 mice each. (E, F) Liver Cyp27a1, and Cyp8b in fasted and fed states in TCF7L2^+/− versus wild-type mice. unpaired t-test, two-tailed, n = 8 mice each. (G) TG synthesis assayed by ¹⁴C Acetate incorporation in palmitate in TCF7L2^+/− versus wild-type mice on a high-fat diet. unpaired t test, two-tailed, n = 6 mice each genotype. (H, I) Oil red O staining (H) and TAG quantification (I) of the liver in TCF7L2^+/− versus wild-type mice on high fat diet, scale bar = 150 μm, unpaired t test, two-tailed, n = 5 mice each genotype. (J) Plasma TG secretion after P-407 injection in TCF7L2^+/− versus wild-type mice on chow diet. unpaired t test, two-tailed. (K, L) FPLC of plasma TG and cholesterol in TCF7L2^+/− versus wild-type mice on high fat diet, n = 3 mice each. unpaired t test, two-tailed

FIGURE 5

Loss of Tcf7L2 in the intestinal epithelium reduces Fgf15 expression and FA absorption. (A) Schematic displaying the strategy to knock out Tcf7L2 in the intestinal epithelium. (B) RT-qPCR showing Tcf7L2 and Fgf19 expression in the ileum of mice with the designated genotypes, n = 7 mice each genotype, unpaired t test, 2-tailed. (C) Plasma bile acids in the designated genotypes 45′ post corn oil gavage, n = 7 mice each genotype. Unpaired t test, two-tailed, normality-tested, *p < .05

FIGURE 6

Tcf7L2-bac rescues steatohepatitis in Lrp6 RC/RC mice. (A, B) Oil red O (A) staining and hepatic TG (B) in the indicated mice. Each dot in the dotplot indicates a biological replicate, n = 7 for Lrp6 RC/RC and n = 9 for Lrp6 RC/RC; Tcf7L2-bac. Mice were fed a high-calorie diet (HCD) with 40% calories from fat, and 40% from carbohydrates, scale bar = 150 μm, unpaired t test, two-tailed. (C) Plasma TG after 6 h fast in the indicated mice fed with HCD, n > 7 mice each genotype, unpaired t test, two-tailed. (D) Time course of plasma TG after intraperitoneal injection of weight-normalized P-407 in the indicated mice, fed with HCD, after 6 h-fast, holm-sidak post hoc test was performed. (E) Expression and quantification of DNL enzymes Acc1 and Fasn in the indicated mice by WB. The mice were fasted overnight, followed by 6 h refeeding before sacrifice. n = 4 mice each genotype, unpaired t test, two-tailed. (F) Expression and quantification of enzymes in the DNL pathway and Insig1 expression in Tcf7L2^+/− mice. n = 3 mice each genotype, unpaired t test, two-tailed. (G) Expression of pro-inflammatory macrophage marker F4/80 in the indicated mice by IF. The single-channel (F4/80) images are depicted in white in the last two panels, scale bar = 70 μm, unpaired t test, two-tailed, normality-tested, *p < .05

FIGURE 7

Expression of TCF4 and CYP7A1 in the non-NASH and NASH human samples. (A–C) Expression and quantification of TCF4. n = 6 controls; n = 19 samples. (D–F) Expression and quantification of CYP7A1, n = 6 controls, n = 19 NASH samples. Unpaired t test, two-tailed, *p < .05. scale bar = 70 μm

FIGURE 8

Model. Graphical representation of TCF4 mediated regulation of enterohepatic regulation of bile and lipid synthesis. TCF4 transcriptionally activates Fgf19 which then suppresses bile synthesis and lipid synthesis in the liver