Impairments of hepatic gluconeogenesis and ketogenesis in PPARα-deficient neonatal mice

Abstract

Peroxisome proliferator activated receptor-α (PPARα) is a master transcriptional regulator of hepatic metabolism and mediates the adaptive response to fasting. Here, we demonstrate the roles for PPARα in hepatic metabolic adaptations to birth. Like fasting, nutrient supply is abruptly altered at birth when a transplacental source of carbohydrates is replaced by a high-fat, low-carbohydrate milk diet. PPARα-knockout (KO) neonatal mice exhibit relative hypoglycemia due to impaired conversion of glycerol to glucose. Although hepatic expression of fatty acyl-CoA dehydrogenases is imparied in PPARα neonates, these animals exhibit normal blood acylcarnitine profiles. Furthermore, quantitative metabolic fate mapping of the medium-chain fatty acid [(13)C]octanoate in neonatal mouse livers revealed normal contribution of this fatty acid to the hepatic TCA cycle. Interestingly, octanoate-derived carbon labeled glucose uniquely in livers of PPARα-KO neonates. Relative hypoketonemia in newborn PPARα-KO animals could be mechanistically linked to a 50% decrease in de novo hepatic ketogenesis from labeled octanoate. Decreased ketogenesis was associated with diminished mRNA and protein abundance of the fate-committing ketogenic enzyme mitochondrial 3-hydroxymethylglutaryl-CoA synthase (HMGCS2) and decreased protein abundance of the ketogenic enzyme β-hydroxybutyrate dehydrogenase 1 (BDH1). Finally, hepatic triglyceride and free fatty acid concentrations were increased 6.9- and 2.7-fold, respectively, in suckling PPARα-KO neonates. Together, these findings indicate a primary defect of gluconeogenesis from glycerol and an important role for PPARα-dependent ketogenesis in the disposal of hepatic fatty acids during the neonatal period.

Keywords: 3-hydroxymethylglutaryl-CoA synthase; glucose homeostasis; ketone body metabolism; metabolic adaptation to birth; nuclear magnetic resonance substrate fate mapping; peroxisome proliferator-activated receptor-α.

Fig. 1.

Neonatal peroxisome proliferator-activated receptor-α-konckout (PPARα-KO) mice exhibit relative hypoglycemia. A: blood glucose on postnatal days 0 (P0) and 1 (P1) in suckling wild-type and PPARα-KO mice. Glucose tolerance in P0 (B) and P1 (C) mice. Basal glycemia in B and C (0-min time point) is replicated from A, since glycemia can be safely measured only twice in neonatal mice; n > 6/group. ***P < 0.001 by 2-way ANOVA, as indicated.

Fig. 2.

PPARα promotes gluconeogenesis from glycerol in neonatal mice. A–E: mRNA abundances of gluconeogenic mediators in neonatal liver; n = 8/group. *P < 0.05 and **P < 0.01 by 2-way ANOVA, as indicated. F: glycerol tolerance test in fed P0 mice; n > 10/group. **P < 0.01 by Student's t-test, as indicated. G: hepatic fractional enrichment of [¹³C]glucose (left) and total hepatic glucose pools (right) 20 min after ip injection of [U-¹³C]glycerol (20 μmol/g body wt) in neonatal mice; n = 5/group. ****P < 0.0001 by Student's t-test, as indicated. Pck1, phosphoenolpyruvate carboxykinase 1; G6pc, glucose-6-phosphatase; Gyk, glycerol kinase; Gpd1, cytosolic glycerol phosphate dehydrogenase; Gpd2, mitochondrial glycerol phosphate dehydrogenase.

Fig. 3.

Normal hepatic terminal fatty acid oxidation of PPARα-KO neonates. A–H: hepatic mRNA abundances of mediators of fatty acid oxidation; n = 8/group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by 2-way ANOVA, as indicated. I: hepatic fractional ¹³C enrichments of glutamate (left) and total hepatic glutamate pools (right) 20 min after ip injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g body wt) in neonatal mice; n = 4–8/group. J: total hepatic taurine pools from the same mice as in I. Pparg1a, PPARγ coactivator-1α; Cpt1a, carnitine palmitoyl transferase 1a; Acox1, acyl-CoA oxidase; Acadvl, very long-chain acyl-CoA dehydrogenase; Acadl, long-chain acyl-CoA dehydrogenase; Acadm, medium-chain acyl-CoA dehydrogenase; Acads, short-chain acyl-CoA dehydrogenase.

Fig. 4.

Altered pyruvate metabolism is associated with labeling of glucose from fatty acids in livers of PPARα-KO neonates. A and B: hepatic fractional ¹³C enrichments of glucose (A) and total hepatic glucose pools (B) 20 min after ip injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g body wt) in neonatal mice; n = 4–8/group. C–F: hepatic mRNA abundances of mediators of pyruvate metabolism; n = 8/group. G: serum pyruvate/lactate ratios (left), [pyruvate] (in mM; middle), and [lactate] (in mM; right) in P1 mice. H: pyruvate tolerance test in P1 mice; n > 10/group. *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-way ANOVA or Student's t-test, as indicated. Me, malic enzyme; Pcx, pyruvate carboxylase.

Fig. 5.

PPARα-KO neonates exhibit decreased hepatic expression of ketogenic enzymes and hypoketonemia. A: serum total ketone bodies (TKB) in fed neonatal mice; n > 6/group. B–F: hepatic mRNA abundances of ketogenic enzymes; n = 8/group. G: immunoblots for mitochondrial 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), β-hydroxybutyrate dehydrogenase 1 (BDH1), and actin in neonatal liver (quantification normalized to actin below). *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-way ANOVA, as indicated. Hmgcl, 3-hydroxymethylglutaryl-CoA lyase; Acaa2, mitochondrial acetyl-CoA acyltransferase 2; Acat1, mitochondrial acetoacetyl-CoA thiolase; NS, not significant.

Fig. 6.

Suppression of de novo ketogenesis is associated with neonatal hepatic steatosis. A: hepatic fractional ¹³C enrichments of β-hydroxybutyrate (βOHB; left) and total βOHB pools (right) 20 min after ip injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g body wt) in neonatal mice; n = 4–8/group. B and C: hepatic triacylglycerol (TAG; B) and free fatty acid (FFA; C) concentrations in neonatal mice on P0 and P1; n = 6–7/group, *P < 0.05 and ****P < 0.0001 by 2-way ANOVA, as indicated.