Fibroblast growth factor 21-deficient mice demonstrate impaired adaptation to ketosis

Abstract

Fibroblast growth factor 21 (FGF21) is a key metabolic regulator. Expressed primarily in liver and adipose tissue, FGF21 is induced via peroxisome proliferator-activated receptor (PPAR) pathways during states requiring increased fatty acid oxidation including fasting and consumption of a ketogenic diet. To test the hypothesis that FGF21 is a physiological regulator that plays a role in lipid oxidation, we generated mice with targeted disruption of the Fgf21 locus (FGF21 knockout). Mice lacking FGF21 had mild weight gain and slightly impaired glucose homeostasis, indicating a role in long-term energy homeostasis. Furthermore, FGF21KO mice tolerated a 24-h fast, indicating that FGF21 is not essential in the early stages of starvation. In contrast to wild-type animals in which feeding KD leads to dramatic weight loss, FGF21KO mice fed KD gained weight, developed hepatosteatosis, and showed marked impairments in ketogenesis and glucose control. This confirms the physiological importance of FGF21 in the adaptation to KD feeding. At a molecular level, these effects were accompanied by lower levels of expression of PGC1alpha and PGC1beta in FGF21KO mice, strongly implicating these key transcriptional regulators in the action of FGF21. Furthermore, within the liver, the maturation of the lipogenic transcription factor sterol regulatory element-binding protein-1c was increased in FGF21KO mice, implicating posttranscriptional events in the maladaptation of FGF21KO mice to KD. These data reinforce the role of FGF21 is a critical regulator of long-term energy balance and metabolism. Mice lacking FGF21 cannot respond appropriately to a ketogenic diet, resulting in an impaired ability to mobilize and utilize lipids.

Figure 1

Targeted disruption of the Fgf21 gene produces mild obesity and glucose intolerance. A, Analysis of FGF21 expression by qPCR in liver, PGWAT, and BAT under KD-fed conditions reveals complete disruption of FGF21 expression (#, not detectable). B, Circulating serum FGF21 in KD-fed WT and FGF21KO (KO) mice shows negligible circulating FGF21immunoreactivity in mice with disrupted Fgf21 allele. C, Weight of WT and FGF21KO mice at age 14 and 24 wk. A trend to greater weight in the FGF21KO mice was noted at 14 wk that became significant at 24 wk of age. D, Body composition of WT and FGF21KO mice at 14 and 24 wk as measured by quantitative nuclear magnetic resonance. Initially, FGF21KO mice had no difference in fat mass and a significant elevation in lean mass; by 24 wk, FGF21KO mice had significant increment in adipose depots with maintained increase in lean mass. E, Intraperitoneal glucose tolerance test in age-matched mice (WT 33.3 ± 0.8 g vs. FGF21KO 37.8 ± 1.3 g) showed no change in basal glucose but an increased glucose excursion in FGF21KO mice. F, Area under the curve (AUC) of glucose tolerance test was significantly elevated in FGF21KO mice. *, P ≤ 0.05; error bars shown as sem for groups of five to nine animals.

Figure 2

Response of FGF21KO (KO) mice to 24 h of fasting. A, Total weight loss after a 24-h fast in CLAMS apparatus was significantly less in FGF21KO mice. B, Activity monitoring showed no difference in total 24-h ambulation between WT and FGF21KO mice in either fed or fasted conditions. C, Measurement in CLAMS apparatus showed significant decreases in VO₂ in FGF21KO mice in both fed and fasted conditions. D, Calculation of RER revealed significant reduction during light phase under fed conditions (*) and in both dark and light phases under fasted conditions (+). There was no difference between response of WT or FGF21KO was detected in either dark or light phases. E, Monitoring core temperature revealed no statistically significant change in core temperature in FGF21KO mice in either fed or fasted states. *, P ≤ 0.05; error bars shown as sem for groups of five to nine animals.

Figure 3

Response of FGF21KO (KO) mice to KD feeding. A, Body weight of WT and FGF21KO mice switched from chow feeding (Day 0) to KD feeding showed increased body weight in FGF21KO mice in contrast to normal weight loss seen in WT mice. B, Mean caloric intake of KD-fed mice over 14 d of KD feeding showed increased consumption of KD in FGF21KO mice. C, Activity monitoring showed significant reduction in ambulation in FGF21KO mice in both dark and light phases. D, Measurement in CLAMS apparatus showed a trend toward reduced VO₂ in KD-fed FGF21KO mice however this difference failed to reach statistical significance in either dark or light phases. E, Body composition analysis of WT and FGF21KO mice fed KD for 14 d revealed significant increases in body fat in FGF21KO mice. F, Postmortem organ weights from WT and FGF21KO mice revealed increases in liver, PGWAT and BAT weights in FGF21KO mice. G, Analysis of hypothalamic neuropeptide expression by qPCR revealed induction of pro-opiomelanocortin (POMC) and suppression of agouti-related peptide (AgRP) mRNA. There was no difference in expression of leptin receptor (LEPR). *, P ≤ 0.05; error bars shown as sem for groups of five to nine animals.

Figure 4

FGF21KO mice are susceptible to hepatosteatosis and maladaptive changes in gene expression when fed KD. A, Macroscopically, FGF21KO (KO) mice had enlarged livers (left lobe only shown) with an increased fatty appearance. Scale bar, 1 cm. B, Light microscopy demonstrated the increased fatty appearance of liver in KD-fed FGF21KO mice. Increased fatty infiltration was noted at the periphery of the lobule in KD-fed WT mice; however fatty infiltration had a more marked centripetal spread toward the central vein (white arrows) in FGF21KO mice. Scale bar, 200 μm. C, Electron microscopy of hepatic cells midway between central vein and portal triad revealed marked fatty infiltration in FGF21KO mice and also increased staining of α-glycogen granules within the cytoplasm of FGF21KO hepatocytes (arrow heads). D, Biochemical determination of total triglyceride content of WT and FGF21KO mice livers revealed significantly elevated hepatic stores. E, Biochemical assay of glycogen showed significant increases in hepatic glycogen stores in FGF21KO mice. F, qPCR analysis of hepatic metabolic genes showed significant increases in adipose differentiation-related peptide (ADRP) in FGF21KO mice. There was a trend to decreased expression of ACADM, ACOX1, OHBUTDH and HMGCS2 that did not reach significance. PEPCK was significantly reduced in FGF21KO mice in the absence of regulation of G6Pase. There was no significant change in any PPAR isoform analyzed, yet PGC1α and PGC1β were significantly down regulated in FGF21KO mice. G, qPCR analysis revealed no significant change in SREBP1 expression at mRNA level. H, Immunoblot analysis revealed a significant shift from precursor to mature form of SREBP1. *, P ≤ 0.05; error bars shown as sem for groups of five to nine animals.

Figure 5

FGF21KO (KO) mice develop white adipocyte hypertrophy when fed KD associated with decreased lipolytic and regulatory gene expression. A, Light microscopy showed increased adipocyte size in PGWAT depots of FGF21KO mice. Scale bar, 200 μm. B, Image analysis revealed a statistically significant increase in PGWAT cell volume in KD-fed FGF21KO, which was almost 3-fold that of WT mice. C, Gene expression analysis of PGWAT by qPCR showed a significant reduction in expression of lipases adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and also adiponutrin in FGF21KO mice. There was a significant reduction in all PPAR isoforms investigated and a concomitant reduction in PGWAT expression of PGC1α and PGC1β in KD-fed FGF21KO compared with WT mice. D, Light microscopy showed increased fatty appearance of BAT. Scale bar, 50 μm. E, Gene expression analysis of BAT transcripts revealed no significant change in PPARα or γ or PGC1 isoforms. *, P ≤ 0.05; error bars shown as sem for groups of five to nine animals.