Impact of peripheral ketolytic deficiency on hepatic ketogenesis and gluconeogenesis during the transition to birth

Abstract

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 h of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother's milk is ingested. Livers of SCOT-knock-out (SCOT-KO) neonates induce the expression of the genes encoding peroxisome proliferator-activated receptor γ co-activator-1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, and glucose-6-phosphatase, and the neonate's pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of (13)C-labeled substrates revealed that livers of SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the tricarboxylic acid cycle as acetyl-CoA is increased in SCOT-KO livers and is associated with diminished terminal oxidation of fatty acids. After mother's milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic β-hydroxybutyrate synthesis by 90%. Disruption of β-hydroxybutyrate production increases hepatic NAD(+)/NADH ratios 3-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth.

Keywords: Coenzyme A Transferase; Gluconeogenesis; Glucose Homeostasis; Ketone Body Metabolism; Liver Metabolism; NMR Substrate Fate Mapping; Neonatal Metabolism; Redox; Tricarboxylic Acid (TCA) Cycle.

FIGURE 1.

Absence of extrahepatic ketone body oxidation engages an hepatic gluconeogenic program in neonatal mice. A, relative mRNA abundance of encoded mediators of pyruvate metabolism and gluconeogenesis in livers of P1 mice. n = 5/group. B, liver glycogen content (μg of glycogen/mg of tissue) in P1 neonates. n = 8/group. p = 0.06 by Student's t test. C, blood alanine, serine, and glutamate concentrations (micromolar) in P1 mice. n = 5–7/group. D, circulating amino acid concentrations (micromolar) in blood of P1 mice. n = 5–10/group. E, plasma pyruvate pool (pyruvate + lactate) in P1 mice. n = 8–11/group. F, endogenous hepatic glucose concentration (left) and accumulated [¹³C]glucose in livers (right) of P1 mice that had been injected with [3-¹³C]pyruvate (10 μmol/g of body weight) 30 min prior to collection of tissues and generation of extracts for NMR. n = 4/group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test. Error bars, S.E.

FIGURE 2.

Alterations of terminal fatty acid oxidation and pyruvate handling in livers of SCOT-KO mice. A, hepatic fractional ¹³C enrichments of glutamate (left) and total hepatic glutamate pools (right) 20 min after intraperitoneal injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g of body weight) in P1 mice. n = 6–8/group. B, fractional ¹³C enrichments of glutamate (left) and total hepatic glutamate pools (right) 20 min after intraperitoneal injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g of body weight) + unlabeled pyruvate (20 μmol/g) in livers P1 mice. n = 6–7/group. C, fractional ¹³C enrichments of glutamate 30 min after intraperitoneal injection of sodium [3-¹³C]pyruvate (10 μmol/g of body weight) in livers of P1 mice. n = 4/group. D, short chain acylcarnitine concentrations in blood of untreated P1 mice. n = 5–10/group. E, hepatic triacylglycerol (TAG) content in livers of untreated P1 mice. n = 5–6/group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test. Error bars, S.E.

FIGURE 3.

Mother's milk-induced impairment of de novo βOHB production in neonatal SCOT-KO liver. A, hepatic fractional ¹³C enrichments of βOHB (left), total βOHB pools (middle), and ¹³C-βOHB concentration (right) 20 min after intraperitoneal injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g of body weight) in P1 mice. n = 6–8/group. B, fractional ¹³C enrichments of βOHB (left), total βOHB pools (middle), and ¹³C-βOHB concentration (right) 20 min after intraperitoneal injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g of body weight) in livers of unfed P0 mice. n = 6/group. C, plasma total ketone body (TKB) concentration (millimolar), measured in P0 wild-type and SCOT-KO mice prior to the onset of suckling. n = 4/group. D, plasma total ketone body concentration (millimolar), measured in P0 wild-type and SCOT-KO mice within 2 h after the onset of suckling. The distributions of d-βOHB and AcAc are shown. n = 8–10/group. †, p < 0.05 for AcAc; *, p < 0.05 for βOHB. E, fractional ¹³C enrichments of βOHB (left), total βOHB pools (middle), and ¹³C-βOHB concentration (right) 20 min after intraperitoneal injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g of body weight) in milk-fed P0 mice. n = 6–7/group. ***, p < 0.001 by Student's t test. Error bars, S.E.

FIGURE 4.

Normal in vitro hepatic ketogenesis of livers from SCOT-KO mice. Determination of ketone body production (pmol of ketone/mg of liver), 0.25, 1, and 8 h after stimulation with BSA-conjugated oleic acid (150 μm) was used to derive ketogenic rate (pmol/mg of liver per h) in liver explants derived from unfed (A) and fed (B) P0 mice. n = 4/group for unfed pups, and n = 8–10/group for fed pups. **, p < 0.01; ***, p < 0.001 by one-way ANOVA. Error bars, S.E.

FIGURE 5.

d-βOHB inhibits neonatal hepatic ketogenesis in vivo. Total βOHB pools (A), fractional ¹³C enrichments of βOHB (B), and ¹³C-βOHB concentrations (C) 20 min after intraperitoneal injection of sodium [1,2,3,4-¹³C₄]octanoate (10 μmol/g of body weight) alone or co-injected with [¹³C]octanoate plus 20 μmol/g of body weight of unlabeled AcAc, l-βOHB, or d-βOHB, in livers of milk-fed P0 mice. The [¹³C]octanoate alone datasets (the white bars in these panels) are reproduced from Fig. 3E for comparison. n = 5–7/group for each panel. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus wild-type neonates injected with [¹³C]octanoate alone, or as indicated by 1-way ANOVA. ††, p < 0.01; †††, p < 0.001 versus AcAc co-injected neonates. Error bars, S.E.

FIGURE 6.

Oxidized hepatic redox potential in P1 SCOT-KO mice. A, plasma AcAc/d-βOHB molar ratios in milk-fed P0 mice. n = 8–9/group. B and C, NAD⁺/NADH ratios, NAD⁺, NADH, and total NAD (NAD⁺ + NADH; NADt (nmol/g of tissue)) in livers (n = 5/group) (B) and skeletal muscles (C) of fed P0 neonates (n = 6/group). D, plasma AcAc/d-βOHB molar ratios in P1 mice. n = 11–14/group. E and F, NAD⁺/NADH ratios, [NAD⁺], [NADH], and [NADt] (nmol/g of tissue) in livers (E) and skeletal muscles (F) of P1 wild-type and SCOT-KO mice. n = 13–14/group. **, p < 0.01; ***, p < 0.001 by Student's t test. Error bars, S.E.