Dietary medium-chain fatty acids reduce hepatic fat accumulation via activation of a CREBH-FGF21 axis

Abstract

Objective: Dietary medium-chain fatty acids (MCFAs), characterized by chain lengths of 8-12 carbon atoms, have been proposed to have beneficial effects on glucose and lipid metabolism, yet the underlying mechanisms remain elusive. We hypothesized that MCFA intake benefits metabolic health by inducing the release of hormone-like factors.

Methods: The effects of chow diet, high-fat diet rich in long-chain fatty acids (LCFA HFD) fed ad libitum or pair-fed to a high-fat diet rich in MCFA (MCFA HFD) on glycemia, hepatic gene expression, circulating fibroblast growth factor 21 (FGF21), and liver fat content in both wildtype and Fgf21 knockout mice were investigated. The impact of a single oral dose of an MCFA-rich oil on circulating FGF21 and hepatic Fgf21 mRNA expression was assessed. In flag-tagged Crebh knockin mice and liver-specific Crebh knockout mice, fed LCFA HFD or MCFA HFD, active hepatic CREBH and hepatic Fgf21 mRNA abundance were determined, respectively.

Results: MCFA HFD improves glucose tolerance, enhances glucose clearance into brown adipose tissue, and prevents high-fat diet-induced hepatic steatosis in wildtype mice. These benefits are associated with increased liver expression of CREBH target genes (Apoa4 and Apoc2), including Fgf21. Both acute and chronic intake of dietary MCFAs elevate circulating FGF21. Augmented hepatic Fgf21 mRNA following MCFA HFD intake is accompanied by higher levels of active hepatic CREBH; and MCFA-induced hepatic Fgf21 expression is blocked in mice lacking Crebh. Notably, while feeding male and female Fgf21 wildtype mice MCFA HFD results in reduced liver triacylglycerol (TG) levels, this liver TG-lowering effect is blunted in Fgf21 knockout mice fed MCFA HFD. The reduction in liver TG levels observed with MCFA HFD was independent of weight loss.

Conclusions: Dietary MCFAs reduce liver fat accumulation via activation of a CREBH-FGF21 signaling axis.

Keywords: FGF21; Hepatokines; Insulin resistance; Liver metabolism; Medium-chain fatty acids.

Figure 1

Dietary MCFAs improve glycemia, prevent high-fat diet-associated steatosis and regulate hepatic genes associated with systemic lipid homeostasis in male mice. Study schematic (A) related to results presented in (B–H). Daily energy intake (B), body weight (C), fat mass (D), and fat-free mass (E) determined 16 days after putting mice on the indicated diets; glucose excursion curves (F) and the incremental area under the curve (iAUC) (G) based on an i.p. glucose tolerance test (2 g of glucose per kg body weight) conducted 13 days after putting mice on the indicated diets (B–G; n = 8). Glucose clearance (H) in indicated tissues determined 16 days after putting mice on the indicated diets (n = 6–8). H&E staining (I, representative of n = 9) and associated scoring of steatosis status (J, n = 9) in livers collected after 4 h of fasting from male C57BL/6J mice fed indicated diets for five weeks. Whole-genome sequencing of the same livers (as in I) with a volcano plot (K) depicting detected liver transcripts over the threshold of one fragment per kilobase million (FPKM) comparing LCFA HFD vs. MCFA HFD. Regulated (adjP < 0.05 and log₂ FC > 1 or <−1) transcripts by MCFA HFD relative to LCFA HFD (L) of genes encoding for secreted factors (K, L; n = 10). Data (B–H) were analyzed by one-way ANOVA (B–E, G), or two-way ANOVA (H). Tukey (for B–H) post-hoc tests were conducted. Data in (j) were analyzed by a Chi-Square and Fisher's exact test. ∗∗p < 0.01, ∗∗∗p < 0.001 for differences between MCFA HFD and LCFA HFD; #p < 0.05, ##p < 0.01, ###p < 0.001 for differences between chow and LCFA HFD; §p < 0.05, §§§p < 0.001 for differences between chow and MCFA HFD.

Figure 2

Acute and chronic intake of MCFAs increase hepatic Fgf21 mRNA and circulating FGF21 in male mice. mRNA abundance of Fgf21 in liver from male mice fed indicated diets for five weeks (A; n = 10), or from mice in indicated organs that were orally gavaged at 0 min with 200 μL of C8:0 MCFA-rich oil (B; n = 6). Plasma FGF21 concentrations in mice fed indicated diets for five weeks (C; n = 10); fed indicated diets for 16 days (D; n = 7–8); in lean (E; n = 10) or in diet-induced obese mice (F; n = 10–14) orally gavaged at 0 min with 200 μL of C8:0 MCFA-rich oil or LCFA-rich oil (corn oil) as control. Relative expression of each sample was normalized to the housekeeping gene (Tbp in (A); Rpl13a in (B)), then relative units (RU) were calculated by normalizing each relative expression values to the mean of LCFA HFD (A) or 0 min (B). Data were analyzed by unpaired t test (A, C), one-way AVONA (D), or two-way ANOVA (B, E, F). Tukey (for B, D) and Bonferroni (for E–F) post-hoc tests were conducted. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to LCFA HFD (or compared to 0 min (b)); #p < 0.05 for difference between chow and LCFA HFD.

Figure 3

MCFA-induced increase in hepatic Fgf21 mRNA requires CREBH in male mice. Representative image (A) and quantification of full length (B) and active CREBH (C) in livers from Crebh knockin male mice expressing flag-tagged CREBH fed indicated diets for five weeks (B–D; n = 4). mRNA abundance of the indicated genes (E–H) from liver of Crebh wildtype (WT) or liver-specific Crebh knockout (LiKO) mice fed indicated diets for five weeks (E–H; n = 4–6). Data were analyzed by unpaired t test (C, D); and two-way ANOVA (E–H). Tukey (for F–H) post hoc tests were conducted. ∗∗p < 0.01, ∗∗∗p < 0.001 for differences between LCFA HFD and MCFA HFD within Crebh knockin and Crebh WT mice. #p < 0.05 for differences between LCFA HFD and MCFA HFD within Crebh LiKO mice.

Figure 4

FGF21 is required for the liver fat-lowering effects of dietary MCFAs in male mice. Male age-matched Fgf21 KO mice (10–20 weeks old) and WT littermates (10–20 weeks old) were fed indicated diets for five weeks and body weight (A) and food intake (B) were recorded at designated time points; glucose excursion curves (C) and the incremental area under these curves (iAUC) (D) determined during an i.p. glucose tolerance test (2 g of glucose per kg body weight) conducted four weeks after placing mice on the indicated diets (A–D; n = 6–11). Plasma insulin (E), liver triacylglycerol (F), plasma triacylglycerol (G), and plasma fatty acids (h) determined after a 4 h fast, five weeks after putting mice on indicated diets (E–H; n = 9–19). Data in (A, B) were analyzed by two-way repeated-measured ANOVA. Data in (D–H) were log 2-transformed before analysis by two-way ANOVA. Tukey (for A, B, F, H) post hoc tests were conducted. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for differences between LCFA HFD and MCFA HFD within Fgf21 wildtype mice. #p < 0.05, ##p < 0.01, ###p < 0.001 for differences between LCFA HFD and MCFA HFD within Fgf21 KO mice. §§§p < 0.001 for main effect of diet.

Figure 5

Improved glycemia and liver fat lowering by dietary MCFA are partly independent of body weight loss in male mice. Diet-induced obese C57BL/6j mice were fed indicated diets for 21 days with one group being fed pair-fed (PF) LCFA HFD (LCFA HFD PF) to the MCFA HFD group. Daily body weight (A) and food intake (B) were recorded; glucose excursion curves (C) and the incremental area under these curves (iAUC) (D) determined during an i.p. glucose tolerance test (1.5 g of glucose per kg body weight) conducted on day 16 (A–D; n = 7–8). Liver triacylglycerol (E), plasma triacylglycerol (F), liver glycerol (G) and plasma glycerol (H) determined after a 4 h fast, 21 days after putting mice on indicated diets (E–H; n = 6–8). Data in (A, B) were analyzed by two-way repeated-measured ANOVA. Data in (D–H) were analyzed by one-way ANOVA. Tukey (for A, B, D, E) post hoc tests were conducted. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 for differences between LCFA HFD and MCFA HFD. #p < 0.05, ##p < 0.01, and ###p < 0.001 for difference between LCFA HFD and LCFA HFD pair-fed. §p < 0.05 for difference between MCFA HFD and LCFA HFD pair-fed.

Figure S1

Dietary MCFAs stimulate lysine acetylation in the liver. Representative western blot (A) and quantification (B) of pan-lysine acetylation in livers from mice fed indicated diets for five weeks. Data were analyzed by unpaired t test. ∗∗∗p < 0.001 for differences between LCFA HFD and MCFA HFD.

Figure S2

Dietary MCFAs in male wildtype or Fgf21 knockout mice. See Figure 4 for more details. Body weights of mice at indicated time points (A; n = 6–11). Plasma β-Hydroxybutyrate (B) from blood collected after a 4 h fast, five weeks after putting mice on indicated diets (n = 6–10). Representative western blot (C) and quantification (D, E) of indicated phosphorylation sites on Akt in livers from mice collected after a 4 h fast, five weeks after putting mice on indicated diets (n = 6–10). Data were analyzed by repeated measures (A) or regular (B, D, E) two-way ANOVA, and a Tukey post-hoc test was performed for (A). ∗p < 0.05 and ∗∗p < 0.01 for differences between LCFA HFD and MCFA HFD within Fgf21 wildtype mice. #p < 0.05 for differences between LCFA HFD and MCFA HFD within Fgf21 KO mice. §§p < 0.01 for main effect of diet.

Figure S3

FGF21 is required for the full liver fat-lowering effects of dietary MCFAs in female mice. Female Fgf21 wildtype or Fgf21 knockout (KO) mice were fed indicated diets for five weeks. Body weight (A; n = 8–10) after five weeks is shown, as well as blood glucose (B; n = 8–9), plasma insulin (C; n = 6–10), liver triacylglycerol (D; n = 8–10), plasma triacylglycerol (E; n = 8–10), and plasma fatty acids (F; n = 7–10) determined after a 4 h fast at the end of the study five weeks after putting mice on the indicated diets. Data were log 2-transformed before analysis by two-way ANOVA. Tukey (for C, D) post hoc tests were conducted. ∗∗p < 0.01, ∗∗∗p < 0.001 for differences between LCFA HFD and MCFA HFD within Fgf21 wildtype mice. §p < 0.05, §§§p < 0.001 for main effects of diet.