Fibroblast growth factor 21 prevents atherosclerosis by suppression of hepatic sterol regulatory element-binding protein-2 and induction of adiponectin in mice

Abstract

Background: Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF21 in the cardiovascular system remains elusive.

Methods and results: The roles of FGF21 in atherosclerosis were investigated by evaluating the impact of FGF21 deficiency and replenishment with recombinant FGF21 in apolipoprotein E(-/-) mice. FGF21 deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF21 protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription factor sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF21-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF21 are abrogated by hepatic expression of sterol regulatory element-binding protein-2.

Conclusions: FGF21 protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

Keywords: adipokines; atherosclerosis; fibroblast growth factor 21; hormones.

Figure 1.

Apolipoprotein (apo) E^−/− mice with fibroblast growth factor (FGF) 21 deficiency exhibit exacerbated atherosclerosis and premature death. Aortas were dissected from 24- and 52-week-old apoE^−/− mice and apoE^−/−FGF21^−/− (DKO) mice. n=8 in each group. A, En face staining of entire aortas of 24-week-old mice with oil red O. B and C, Cross-sections of aortic sinuses and brachiocephalic arteries of 24-week-old mice, respectively. D and E, Macrophage infiltration and smooth muscle proliferation in aortic sinus as determined by immunostaining for F4/80 and α-actin, respectively. F, Cholesterol ester levels in brachiocephalic arteries (BCA) of 24-week-old mice. G, The surviving rate of apoE^−/− mice (n=20) and DKO mice (n=20) on standard chow was monitored for 18 months. Data are presented as dot plots with the line indicating the median. The Mann-Whitney U test was used for 2-group comparisons (A–F); the survivals of mice were compared using Kaplan-Meier survival analysis with the log-rank test (G).

Figure 2.

Fibroblast growth factor (FGF) 21 deficiency worsens lipid profiles and exacerbates inflammation in apolipoprotein (apo) E^−/− mice. ApoE^−/−FGF21^−/− (DKO) and apoE^−/− mice fed with standard chow were euthanized at 24 weeks after birth. Plasma samples were collected for measurement of triglycerides (TG; A), total cholesterol (TC; B), high-density lipoprotein (HDL; C), low-density lipoprotein (LDL; D), and very LDL (VLDL; E). F, The mRNA expression of intercellular adhesion molecule-1 (ICAM-1) vascular cell adhesion protein-1 (VCAM-1), tumor necrosis factor-α (TNFα), and monocyte chemotactic protein-1 (MCP-1) in aortic tissue, as well as (G–J) plasma levels of these proinflammatory chemokines and cytokines, were measured with real-time polymerase chain reaction and ELISA, respectively. n=6 to 7. Data are presented as dot plots with the line indicating the median. The Mann-Whitney U test was used for comparison of 2 groups.

Figure 3.

Recombinant mouse fibroblast growth factor (FGF) 21 and adiponectin (ADN) attenuate the atherosclerotic plaque formation in apolipoprotein E^−/−FGF21^−/− (DKO) mice. Eight-week-old DKO mice were treated with recombinant mouse FGF21 (0.1 mg/kg per day), adiponectin (10 mg/kg per day), or vehicle by daily intraperitoneal injection for a period of 16 weeks. A and B, Plasma levels of adiponectin and its mRNA expression in epididymal adipose tissues (EPAT), subcutaneous (SAT), perivascular (PVAT), and perirenal (PRAT) adipose tissues. C, En face staining of entire aortas with oil red O. D and E, Cross-section analysis of aortic sinuses and brachiocephalic arteries with oil red O, respectively. n=6 to 7. Data are presented as dot plots with the line indicating the median. The Mann-Whitney U test was used to compare 2 groups (A and B). The global significance among 3 groups was determined by Kruskal-Wallis test, followed by pairwise comparisons with the Dunn-Sidak procedure (C–E).

Figure 4.

Differential effects of fibroblast growth factor (FGF) 21 and adiponectin on lipid profiles and atherosclerotic plaque composition in apolipoprotein E^−/−FGF21^−/− (DKO) mice. DKO mice were treated with recombinant mouse FGF21, adiponectin (ADN), or vehicle for 16 weeks as in Figure 3. A, Immunohistological analysis of atherosclerotic lesion areas in aortic sinuses with antibodies against the smooth muscle marker α-actin, the macrophage marker F4/80, or with Masson trichrome staining for the collagen composition as indicated. B–F, The mRNA expression of several proinflammatory chemokines and cytokines in the aortic sinus and their plasma levels as determined by real-time polymerase chain reaction and ELISA, respectively. G, Cholesterol ester content in the brachiocephalic arteries. H and I, Plasma cholesterol and triglyceride levels in DKO mice treated with recombinant mouse (rm) FGF21, ADN, or vehicle, respectively. n=5 to 7. Data are presented as dot plots with the line indicating the median. The global significance among 3 groups was determined by Kruskal-Wallis test, followed by pairwise comparisons with the Dunn-Sidak procedure.

Figure 5.

Effects of fibroblast growth factor (FGF) 21 and adiponectin (ADN) on cholesterol metabolism in mice. Apolipoprotein (apo) E^−/−FGF21^−/− (DKO) mice were treated with recombinant mouse FGF21, ADN, or vehicle for 4 weeks as in Figure 4. ApoE^−/− mice were used as a control. A, The absorption rate of dietary cholesterol was determined by oral gavage with [¹⁴C] cholesterol and [³H] sitostanol, followed by measurement of the ratio of the 2 isotopes in feces. B, Fecal cholesterol and (C) bile acids were measured with the corresponding commercial kits, respectively. D, The rate of de novo cholesterol synthesis as measured by determining the amount of [1-¹⁴C]-acetate incorporated into sterols per minute per gram liver tissue. E, Hepatic cholesterol contents determined by a cholesterol assay kit. n=6 to 7. Data are presented as dot plots with the line indicating the median. The global significance among 4 groups was determined by Kruskal-Wallis test, followed by pairwise comparisons with the Dunn-Sidak procedure.

Figure 6.

Fibroblast growth factor (FGF) 21, but not adiponectin (ADN), alters hepatic expression of the key genes involved in cholesterol biosynthesis and transport. Total RNA extracted from the liver of apolipoprotein (apo) E^−/− mice or apoE^−/−FGF21^−/− (DKO) mice treated with recombinant mouse FGF21, ADN, or vehicle as in Figure 4 was subjected to real-time polymerase chain reaction analysis. Figure shows the relative mRNA expression levels of genes involved in cholesterol synthesis, including 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3-hydroxy-3-methylglutaryl-CoA synthetase (HMGCS), squalene synthase (Sqle), and farnesyl diphosphate synthetase (Fdps; A and B) genes involved in bile acids metabolism including cholesterol 7-α-monooxygenase (CYP7A1), sterol 12-α-hydroxylase (CYP8B1), sterol 27-hydroxylase (CYP27A1), and small heterodimer partner (SHP; C), as well as genes involved in cholesterol transports including ABCG5 and ABCG8 (D). n=5 to 7. Data are presented as dot plots with the line indicating the median. The global significance among 4 groups was determined by Kruskal-Wallis test, followed by pairwise comparisons with the Dunn-Sidak procedure.

Figure 7.

Effects of fibroblast growth factor (FGF) 21 and adiponectin (ADN) on several key transcription factors involved in cholesterol metabolism. The liver samples from apolipoprotein (apo) E^−/− mice or apoE^−/−FGF21^−/− (DKO) mice treated with recombinant mouse FGF21, ADN, or vehicle as in Figure 4 were subjected to real-time polymerase chain reaction or Western blot analysis. A and B, The relative mRNA and protein expression levels of liver X receptor (LXR) α, farnesoid X receptor (FXR), and sterol regulatory element-binding protein (Srebp)-1. C and D, The relative mRNA and protein expression of Srebp-2. E and F, The DNA binding activities of Srebp-1 and Srebp-2 in the nuclear extracts of liver tissues. n=5 to 7. Data are presented as dot plots with the line indicating the median. The global significance among 4 groups was determined by Kruskal-Wallis test, followed by pairwise comparisons with the Dunn-Sidak procedure.

Figure 8.

Fibroblast growth factor (FGF) 21 decreases hypercholesterolemia via inhibition of hepatic sterol regulatory element-binding protein (Srebp)-2. A–C, Apolipoprotein (apo) E^−/−FGF21^−/− (DKO) mice were infected with adenovirus encoding small interfering RNA (siRNA) specific to Srebp-2 or scrambled control (5×10⁸ plaque-forming units per mouse) for various periods. Age-matched apoE^−/− mice were used as a control. A, Protein expression levels of hepatic Srebp-2 at day 6 and day 12 after adenoviral infection. B, Circulating levels of total cholesterol (TC) and (C) the expression levels of cholesterologenic genes determined by real-time polymerase chain reaction analysis at day 12 (n=6). D and E, DKO mice were infected with adenovirus encoding Srebp-2 (Ad-Srebp-2) or luciferase (Ad-Luc) for 6 days (as control), followed by treatment with daily intraperitoneal injection of recombinant mouse (rm) FGF21 (0.1 mg/kg per day) for another 6 days. D, The protein expression levels of Srebp-2 in the liver and (E) serum levels of total cholesterol. F, The mRNA expression of cholesterologenic genes at 12 days after adenoviral infection (n=6). Data are presented as dot plots with the line indicating the median. The global significance among 3 groups was determined by Kruskal-Wallis test, followed by pairwise comparisons with the Dunn-Sidak procedure.