Hepatic FGF21 expression is induced at birth via PPARalpha in response to milk intake and contributes to thermogenic activation of neonatal brown fat

Abstract

Plasma FGF21 levels and hepatic FGF21 gene expression increase dramatically after birth in mice. This induction is initiated by suckling, requires lipid intake, is impaired in PPARalpha null neonates, and is mimicked by treatment with the PPARalpha activator, Wy14,643. Neonates exhibit reduced FGF21 expression in response to fasting, in contrast to the upregulation occurring in adults. Changes in FGF21 expression due to suckling or nutritional manipulations were associated with circulating free fatty acid and ketone body levels. We mimicked the FGF21 postnatal rise by injecting FGF21 into fasting neonates, and found that this enhanced the expression of genes involved in thermogenesis within brown fat, and increased body temperature. Brown adipocytes treated with FGF21 exhibited increased expression of thermogenic genes, higher total and uncoupled respiration, and enhanced glucose oxidation. We propose that the induction of FGF21 production by the liver mediates direct activation of brown fat thermogenesis during the fetal-to-neonatal transition.

Figure 1. Developmental Regulation of FGF21 Gene Expression: FGF21 Gene Expression in the Liver Is Induced and Plasma FGF21 Levels Are Increased at Birth

Plasma FGF21 levels (A) and FGF21 mRNA levels in liver (B) at the indicated times during neonatal development. HC and HF denote mice spontaneously weaned onto a high-carbohydrate (HC) or high-fat (HF) diet between days 15 and 21. Bars are means ± SEM of three to six individual samples from separate litters. Statistically significant differences (p < 0.05) with respect to fetuses are denoted by *, and those with respect to adults by #.

Figure 2. Effects of Postnatal Starvation, Lipid or Glucose Intake, and PPARα Abrogation on the Neonatal Induction of FGF21 Gene Expression in the Liver

(A) FGF21 mRNA levels in the livers of mice at birth (prior to suckling), of pups at 8 or 16 hr of life that were allowed to suckle (fed) or not allowed to suckle (fasted), and of fasted pups who received a lipid emulsion or glucose after birth. (B) FGF21 levels in plasma and FGF21 mRNA levels in the livers of wild-type and PPARα null neonates. Pups were analyzed at birth, 16 hr after birth (fed and fasted), and after injection with Wy14,643 after birth. (C) Effects of a 24 hr fast on FGF21 mRNA expression and plasma FGF21 levels in mice at distinct stages of development. UD, undernourished 21-day-old pups belonging to oversized litters (18 pups/litter). Bars are the means ± SEM of FGF21 mRNA levels, which are expressed as relative to the values in pups at birth (A and B, left) or relative to fasted 2-day-old pups (C, left). The bars in (B) (right) and (C) (right) are the means ± SEM of plasma FGF21 levels. Data are the means of four to six individual littermates obtained from four to six litters. Statistically significant differences (p < 0.05), with respect to values at birth, are denoted by * (A and B). Differences between fed and fasted mice are denoted by # (A and C), and those between PPARα null and wild-type littermates are denoted by # (B).

Figure 3. Gene Expression in Neonatal Mouse BAT, Effects of FGF21 Injection on Neonatal BAT, and Effects of FGF21 on Brown Adipocytes in Primary Culture

(A) Transcript levels of thermogenic genes in BAT from pups at birth (0 hr) and at the indicated time after birth. (B–D) Pups were injected 4 hr after birth with FGF21, and BAT was studied at the indicated times after injection. Representative immunoblots for PGC-1α and UCP1 are shown in (B), left. Levels of PGC-1α, UCP1, and Cyt c proteins are shown in (B), right. (D) Neonatal body temperature 12 hr after FGF21 injection. (E) Transcript levels of PGC-1α and UCP1 (left) and body temperature (right) in PPARα null pups and wild-type littermates. (F) Transcript levels of the indicated genes in differentiated brown adipocytes in culture treated with FGF21. (G and H) Differentiated brown adipocytes in culture were treated with 5 nM FGF21 or saline for 24 hr; (G), total respiration and uncoupled respiration; (H), ¹⁴C-CO2 dpm levels after 3 hr incubation with ¹⁴C-glucose of control and FGF21-treated brown adipocytes. Bars are means ± SEM of five to seven pups or independent cell cultures. Statistically significant differences (p < 0.05) between controls and FGF21-treated conditions are denoted by *. In (E), statistically significant differences (p < 0.05) between PPARα null pups and wild-type littermates for the same experimental condition are shown as #, and those between 0 and 16 hr are shown as +.

Figure 4. Schematic Representation of the Regulation of FGF21 Gene Expression and FGF21 Effects during the Transition from Fetal to Neonatal Metabolism

Transcription of the FGF21 gene in the liver and release of FGF21 into the circulation are induced immediately after birth in response to the high fatty acid availability associated with the initiation of suckling. PPARα mediates the milk ingestion-dependent induction of FGF21 expression at birth. The burst in FGF21 levels after birth exerts endocrine actions that are relevant to metabolic adaptations in the neonatal period, mainly the induction of thermogenesis in BAT.