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 [3-13C]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.

Assessment of Hepatic Mitochondrial Oxidation and Pyruvate Cycling in NAFLD by 13C Magnetic Resonance Spectroscopy

“…Such high rates of hepatic pyruvate cycling would use a significant fraction of the ATP available, and place the hepatocyte in a metabolically precarious position. In a recent study we found that an intra-arterial infusion of propionate, that was lower than (Hasenour et al, 2015; Jin et al, 2005; Jin et al, 2004; Satapati et al, 2015) or similar to (Sunny et al, 2011) the total amount of sodium propionate administered in previous studies, dose-dependently increased: hepatic propionyl CoA concentrations up to 18 fold, hepatic pyruvate cycling up to 20–30 fold, hepatic TCA intermediates (malate and succinate) and aspartate by 2–3 fold, and rates of hepatic glucose production by up to 2 fold in awake rats (Perry et al, 2016). It is well established that propionyl CoA potently stimulates pyruvate carboxylase activity and flux (Perry et al, 2016; Scrutton, 1974) and this may explain at least in part the large increases in hepatic pyruvate carboxylase flux and pyruvate cycling that Sunny et al observed in their [1,2,3- 13 C 3 ]propionate labeling studies of hepatic mitochondrial metabolism in humans (Sunny et al, 2011).…”

“…We also directly assessed rates of hepatic pyruvate cycling defined by: (V PK +V ME )/(V PC +V PDH ), where V PK is pyruvate kinase flux, V ME is malic enzyme flux, V PC is pyruvate carboxylase flux and V PDH is pyruvate dehydrogenase flux, by monitoring the relative 13 C labeling in hepatic [2- 13 C]alanine relative to hepatic [2- 13 C]glutamate during an intravenous infusion of [3- 13 C]lactate (Befroy et al, 2015; Perry et al, 2016). Following the start of the [3- 13 C]lactate infusion plasma 13 C lactate enrichments (Supplemental Fig.…”

“…1B) and concentrations (~1.7 mM) reached steady state by 90 minutes in both groups. We have previously observed that under fasting conditions rates of hepatic V ME flux and V PDH flux are relatively low compared to V PK flux and V PC flux, respectively, and therefore rates of pyruvate cycling can be approximated to the rate of pyruvate kinase flux/rate of pyruvate carboxylase flux (V PK /V PC ) (Perry et al, 2016). Using this approach we found that hepatic pyruvate cycling was similar in the Control and NAFLD subjects and that rates of hepatic pyruvate kinase flux were less than 30% that of hepatic pyruvate carboxylase flux (Table 2).…”

“…However, direct 13 C NMR measurements of the ratio of [2- 13 C]glutamate to [3- 13 C]glutamate enrichment during an infusion of [3- 13 C]lactate were found to be ~1.0 demonstrating essentially complete equilibration of 13 C label between [2- 13 C]glutamate and [3- 13 C]glutamate across fumarase (Perry et al, 2016). In contrast to propionate, lactate and acetate do not perturb hepatic mitochondrial metabolite concentrations or hepatic metabolic fluxes at the infusion rates used in our studies (Perry et al, 2014, Perry et al, 2016). …”

“…In addition given the very high rates of hepatic pyruvate cycling previously reported by Sunny et al we also directly assessed rates of hepatic pyruvate cycling relative to anaplerosis in subgroups of Control and NAFLD subjects. This was accomplished by monitoring the 13 C labeling in hepatic [2- 13 C]alanine relative to hepatic [2- 13 C]glutamate during an intravenous infusion of [3- 13 C]lactate (Befroy et al, 2015, Perry et al, 2016). …”