Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice

Abstract

Objective: Fibroblast growth factor 21 (FGF21) has emerged as an important metabolic regulator of glucose and lipid metabolism. The aims of the current study are to evaluate the role of FGF21 in energy metabolism and to provide mechanistic insights into its glucose and lipid-lowering effects in a high-fat diet-induced obesity (DIO) model.

Research design and methods: DIO or normal lean mice were treated with vehicle or recombinant murine FGF21. Metabolic parameters including body weight, glucose, and lipid levels were monitored, and hepatic gene expression was analyzed. Energy metabolism and insulin sensitivity were assessed using indirect calorimetry and hyperinsulinemic-euglycemic clamp techniques.

Results: FGF21 dose dependently reduced body weight and whole-body fat mass in DIO mice due to marked increases in total energy expenditure and physical activity levels. FGF21 also reduced blood glucose, insulin, and lipid levels and reversed hepatic steatosis. The profound reduction of hepatic triglyceride levels was associated with FGF21 inhibition of nuclear sterol regulatory element binding protein-1 and the expression of a wide array of genes involved in fatty acid and triglyceride synthesis. FGF21 also dramatically improved hepatic and peripheral insulin sensitivity in both lean and DIO mice independently of reduction in body weight and adiposity.

Conclusions: FGF21 corrects multiple metabolic disorders in DIO mice and has the potential to become a powerful therapeutic to treat hepatic steatosis, obesity, and type 2 diabetes.

Figures

FIG. 1.

Reversal of high-fat diet–induced obesity by recombinant FGF21 in DIO mice. DIO mice were treated with recombinant murine FGF21 intraperitonally at doses of 0 (vehicle), 0.1, 1, or 10 mg · kg−1 · day−1 divided into two daily injections. An additional group of DIO mice was treated with rosiglitazone formulated in the high-fat diet to provide a dose of ∼4 mg · kg−1 · day−1. Mice on standard diet (SD) were included as controls and injected intraperitonally with vehicle. A: Photograph of representative mice either fed standard diet and administered vehicle or fed high-fat diet and administered vehicle, 10 mg · kg−1 · day−1 FGF21, or rosiglitazone for 6 weeks. B: Body weight monitored throughout the treatment. C: Body composition analyzed after 27 days of treatment. D: Average daily food intake during 6 weeks of treatment. Vehicle (open circles or open bars); FGF21 (open triangles or striped bars; 0.1, 1, and 10 denote FGF21 doses in mg · kg−1 · day−1); rosiglitazone (black squares or black bars). All data are means ± SE, n = 10 per group, ∧P < 0.05; #P < 0.01; *P < 0.001 vs. vehicle-treated high-fat diet mice. (Please see http://dx.doi.org/10.2337/db08-0392 for a high-quality digital representation of this figure.)

FIG. 2.

Recombinant FGF21 reduces plasma glucose, insulin, and lipid levels and improves glucose tolerance in DIO mice. Mice were treated with vehicle or recombinant murine FGF21 intraperitonally at doses of 0 (vehicle), 0.1, 1, or 10 mg · kg−1 · day−1 divided into two daily injections (8:00 a.m. and 4:00 p.m.). Mice were fed ad libitum, and blood was collected 1 h after the morning injection on study day 24. A: Blood glucose. B: Plasma insulin. C: Plasma cholesterol and triglyceride levels. D: Plasma nonesterified free fatty acid (NFFA) levels. E: Plasma leptin. F: GTT was initiated 1 h after the morning injection on study day 30. Fasted mice were intraperitonally injected with 2 mg/kg glucose solution, and blood glucose was measured at 0 min (before glucose injection) and at 30 and 90 min (after glucose injection). Vehicle (open circles or open bars); FGF21 (open triangles or striped bars; 0.1, 1, and 10 denote FGF21 doses in mg · kg−1 · day−1); rosiglitazone (black squares or black bars). All data are means ± SE, n = 10 per group, ∧P < 0.05; #P < 0.01; *P < 0.001 vs. vehicle-treated high-fat diet mice. NEFA, nonesterified fatty acid.

FIG. 3.

Tissue histological analysis. Tissue sections were prepared at the end of the 6-week treatment period from mice fed standard diet and treated with vehicle (A, E, I, and M) or fed high-fat diet and treated with vehicle (Vehi.; B, F, J, and N), FGF21 (10 mg · kg−1 · day−1; C, G, K, and O), or rosiglitazone (∼4 mg · kg−1 · day−1; D, H, L, and P). H-E (A–D) and oil-red-O (E–H) staining of liver sections shows reversal of vacuolation and lipid accumulation in FGF21-treated DIO mice. I–L: H-E staining of brown adipose tissue reveals the reduction of large lipid vacuoles in FGF21-treated DIO mice. M–P: H-E staining of pancreatic sections shows the neutral effects of FGF21 on islet morphology (indicated by arrows). (Please see http://dx.doi.org/10.2337/db08-0392 for a high-quality digital representation of this figure.)

FIG. 4.

Hepatic protein and gene expression analysis. Total protein and RNA were extracted from frozen liver samples of mice fed standard diet and administered vehicle (open bars, SD), fed high-fat diet and administered vehicle (open bars, HFD), FGF21 (striped bars; 0.1, 1, and 10 mg · kg−1 · day−1 doses), or rosiglitazone (black bars; ∼4 mg · kg−1 · day−1) for 6 weeks. Western analyses and RT-PCRs were conducted on pooled protein and RNA extracts (n = 5 per group). A: FGF21 reduced the amount of mature SREBP-1 without changing its precursor content in liver. Vehi, vehicle; Rosi, rosiglitazone. B: FGF21 reduced protein expression levels of total ACC, FAS, and PPARγ in liver. C: FGF21 reduced mRNAs encoding enzymes involved in hepatic lipogenesis without changing SREBP-1 mRNA expression. GK, glucokinase; PK, pyruvate kinase; SCD1, stearoyl-CoA desaturase 1; Elovl6, long-chain fatty acid elongase 6; DGAT1 and MOGAT2, diacylgycerol and monoacyglycerol acyltransferases. D: FGF21 reduced mRNA expression of PPARγ and its target genes, aP2 and CD36, in liver. E: FGF21 reduced glucose-6-phosphatase (G6Pase) mRNA expression.

FIG. 5.

Recombinant FGF21 increases energy expenditure and physical activity in DIO mice. Indirect calorimetry was conducted continuously for 19 days. DIO mice were injected intraperitonally with FGF21 at doses of 10 mg · kg−1 · day−1 (red open squares, n = 5) and 1 mg · kg−1 · day−1 (green filled circles, n = 6) or vehicle (blue open circles, n = 5). A: FGF21 treatment elevated O2 consumption. Measurements were collected every 20 min, and each data point represents a rolling average of six time points. Dark periods (6:30 p.m. to 6:30 a.m.) are shown by a shaded gray box. Arrow marks first statistically significant difference between FGF21 10 mg · kg−1 · day−1 and vehicle. All data are means of multiple mice ± SE. Data shown are the first 7 days of the treatment. O2 consumption (B), CO2 production (C), physical activity (D), energy expenditure (E), and respiratory quotient (F) in the dark periods were monitored for the entire 19 days. Data in B–F represent the average from the whole dark period. All means are shown with SE.

FIG. 6.

Recombinant FGF21 improves whole-body glucose metabolism in both lean and DIO mice. Hyperinsulinemic-euglycemic clamps were conducted on standard diet–or high-fat diet–fed mice treated with vehicle (open bars), 0.1 (striped bars), or 10 (black bars) mg · kg−1 · day−1 FGF21 for 21 days. A: Steady-state glucose infusion rate, obtained from averaged rates of 90–120 min of hyperinsulinemic-euglycemic clamps. B: Basal rates of HGP. C: Insulin-stimulated rates of HGP during clamps. D: Insulin-stimulated whole-body glucose turnover. E: Whole-body glycolysis. F: whole-body glycogen plus lipid synthesis. All data are means ± SE; standard diet–vehicle (n = 10); standard diet–FGF21 treated (n = 8); high-fat diet–all groups (n = 12). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated (diet-matched) mice, †P< 0.01, †††P < 0.0001 vs. standard diet–vehicle–treated mice.

FIG. 7.

Recombinant FGF21 improves tissue-specific glucose uptake in lean and DIO mice. Insulin-stimulated glucose uptake in individual organs during clamps was assessed using 2-[14C]deoxyglucose uptake assay on standard diet or high-fat diet mice treated with vehicle (open bars), 0.1 (striped bars), or 10 (black bars) mg · kg−1 · day−1 FGF21 for 3 weeks. Insulin-stimulated rates of glucose uptake are shown for skeletal muscle (A), white adipose tissue (WAT) (B), brown adipose tissue (BAT) (C), and heart (D). All data are means ± SE; standard diet–vehicle (n = 10); standard diet–FGF21 treated (n = 8); high-fat diet–all groups (n = 12). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated (diet-matched) mice, ††P < 0.05, †††P < 0.0001 vs. standard diet–vehicle–treated mice.