FGF21 does not require interscapular brown adipose tissue and improves liver metabolic profile in animal models of obesity and insulin-resistance

Abstract

FGF21 is a key metabolic regulator modulating physiological processes and its pharmacological administration improves metabolic profile in preclinical species and humans. We used native-FGF21 and a long-acting FGF21 (PF-05231023), to determine the contribution of liver and brown adipose tissue (BAT) towards metabolic improvements in Zucker rats and DIO mice (DIOs). FGF21 improved glucose tolerance and liver insulin sensitivity in Zuckers without affecting BW and improved liver function by decreased lipogenesis, increased fatty acid oxidation and improved insulin signaling. Through detailed lipidomic analyses of liver metabolites in DIOs, we demonstrate that FGF21 favorably alters liver metabolism. We observed a dose-dependent increase of [(18)F]-FDG-glucose uptake in interscapular BAT (iBAT) of DIOs upon FGF21 administration. Upon excision of iBAT (X-BAT) and administration of FGF21 to mice housed at 80 °F or 72 °F, the favorable effects of FGF21 on BW and glucose excursion were fully retained in both sham and X-BAT animals. Taken together, we demonstrate the liver as an organ that integrates the actions of FGF21 and provide metabolic benefits of FGF21 in Zucker rats and DIOs. Finally, our data demonstrates iBAT does not play a role in mediating favorable metabolic effects of FGF21 administration in DIOs housed at 80 °F or 72 °F.

Figure 1. FGF21 improves glucose tolerance in Zucker rats.

Eight week old Zucker rats were administered vehicle, 1.32 mg/kg/day native FGF21, or 3 mg/kg and 10 mg/kg PF-05231023 subcutaneously twice a week for two weeks. a) body weight (BW), b) food intake, c) oral glucose tolerance test (OGTT), d) area under the curve (AUC), e) glucose infusion rate (GIR), f) basal hepatic glucose production (HGP), g) clamp hepatic glucose production, h) suppression of hepatic glucose production. n = 8–10 animals in each group. All data represented as Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one way Anova and Dunnett’s posthoc test.

Figure 2. FGF21 improves hepatic metabolic profile in Zucker rats.

Livers from Zucker rats administered 1.32 mg/kg/day native FGF21 for two weeks were harvested. a) representative western blots for precursor and mature SREBP1, b) incorporation of 2-14C-acetate in ex vivo livers, n = 6/group, c) western blots for P-AMPK (Thr 172) and Tubulin, d) 14C incorporation into CO₂ from ex vivo liver slices, n = 6/group. e) Acute insulin response in Zuckers administered 1.32 mg/kg/day native FGF21 for two weeks. Western blots for P-Akt and total Akt from the indicated tissues. In Figures a and c, each lane represents a different animal. All gels reported in a), c) and e) were run under the same experimental conditions on the same gels respectively for each of these proteins. In a) each lane represents liver lysates from a separate animal, 4 administered PBS and 4 administered FGF21. All samples were run in the order shown in the figure. In c) data is shown for 3 animals administered PBS and 3 animals administered FGF21. Each protein was run on its own gel. Dotted line indicates where blots were cropped. In e) data is shown for 3 animals administered PBS and 3 administered FGF21. All samples were run in the order shown in the gels. Representative total Akt blot is from liver. Each protein was run on its own gel. Data represented as Mean ± SEM. * p < 0.05 by paired t-test.

Figure 3. [¹⁸F]-FDG uptake in FGF21-treated DIO mice.

a) Representative PET scan images in DIO mice administered native FGF21 at the indicated doses for two weeks. b) Region of interest (ROI) quantified from figure a) n = 6/group. c) [¹⁸F]-FDG counts from iBAT from DIOs administered native FGF21, n = 7–8/group. d) [¹⁸F]-FDG counts from indicated tissues from DIOs administered native FGF21 n = 7–8/group. Data represented as Mean ± SEM. * p < 0.05 by paired t-test.

Figure 4. Role of interscapular BAT in FGF21 pharmacology.

iBAT was excised (X-BAT), or sham surgery was performed in DIO mice and administered 0.85 mg/kg FGF21 or vehicle continuously via an osmotic minipump for 14 days. At 80 °F which is close to the thermoneutral zone in mice, a) BW, b) OGTT, c) oxygen consumption, d) CO₂ production. The same experiment was performed in mice housed at 72 °F. e) BW, f) OGTT. UCP1 mRNA from inguinal white adipose tissue normalized to cyclin B as housekeeping gene from mice housed at g) 80 °F and h) 72 °F. n = 7–8 animals/group. Data represented as Mean ± SEM. *p < 0.05 by one way Anova and Dunnett’s posthoc test.