Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism

Abstract

In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo.

Keywords: epinephrine; glucagon; gluconeogenesis; isotopic tracer; liver metabolism; pyruvate kinase.

FIGURE 1.

Flux modeling scheme using [3-¹³C]lactate as a tracer.

FIGURE 2.

Physiologic increases in plasma glucagon and epinephrine concentrations both promote increased rates of hepatic glucose production, but only glucagon suppresses V_{PK + ME}/V_{PC + PDH} flux. A, hepatic glucose production. B, hepatic V_{PK + ME}/V_{PC + PDH} flux. C, hepatic V_PDH/V_CS flux. In all panels, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control; §, p < 0.05 versus glucagon-treated rats. Symbols over each bar represent comparisons with the analogous group of controls (control or ME inhibitor treated). Data are means ± S.E. of n = 6 per group.

FIGURE 3.

Hyperinsulinemia cannot explain the changes to fluxes measured in glucagon- and epinephrine-treated rats. A, plasma insulin concentrations after 120 min. B, plasma glucose concentrations during the clamp. C, glucose infusion rate during the clamp. D, hepatic glucose production. E, V_{PK + ME}/V_{PC + PDH} flux, measured as [¹³C₂]alanine/[¹³C₅]glucose. Data are mean ± S.E. of n = 6 per group.

FIGURE 4.

Continuous intra-arterial propionate infusion (low dose = 333 μmol/kg = 2. 8 μmol/(kg-min) × 120 min; high dose = 667 μmol/kg = 5.6 μmol/[kg-min × 120 min) markedly increases plasma propionate concentrations, hepatic propionyl-CoA concentrations, hepatic glucose production rates, and stimulates PC activity as well as V_{PK + ME}/V_{PC + PDH} flux in a dose-dependent manner. A, plasma propionate concentrations. B, liver propionyl-CoA concentrations. C, hepatic glucose production. D, ex vivo PC activity. E, V_{PK + ME}/V_{PC + PDH} flux. Data are mean ± S.E. of n = 6 per group. N.S., not significant.

FIGURE 5.

Continuous intra-arterial propionate infusion (low dose = 333 μmol/kg = 2. 8 μmol/(kg-min) × 120 min; high dose = 667 μmol/kg = 5.6 μmol/[kg-min × 120 min) raises TCA cycle intermediate concentrations in a dose-dependent manner. A–C, liver succinate, malate, and aspartate concentrations. In all panels, data are mean ± S.E. of n = 6 per group. N.S., not significant.

FIGURE 6.

Infusion of [3-¹³C₃]lactate (40 μmol/(kg-min) × 120 min) does not change plasma lactate or TCA cycle intermediate concentrations. A, plasma lactate concentrations before (control) or after a 120-min infusion of C3 lactate. B–D, liver succinate, malate, and aspartate concentrations. In all panels, data are mean ± S.E. of n = 6 per group. N.S., not significant.

FIGURE 7.

Oral bolus of propionate (333 μmol/kg) raises portal vein propionate concentrations to 1300 μm and increases hepatic propionyl-CoA concentrations 100-fold. A, jugular vein propionate concentrations. B, portal vein propionate concentrations 30 min after an oral bolus. C, liver propionyl-CoA concentrations 30 min after an oral bolus. Data are mean ± S.E. of n = 6 per group.