Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (VCS) and pyruvate carboxylase flux (VPC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of VCS and VPC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-13C]lactate and glucose tracer. PINTA measures VCS and VPC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic VCS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the VPC/VCS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism.

Hepatic mitochondrial function plays a critical role in the regulation of liver and whole-body glucose and fat metabolism and there is great interest in understanding the potential role for alterations in hepatic mitochondrial activity in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and type 2 diabetes (T2D) as well for the evaluation of potential novel therapies targeting hepatic mitochondrial fat oxidation to treat these diseases. Although recent studies by our group have demonstrated the utility of in vivo 13C magnetic resonance spectroscopy (MRS) to directly assess rates of hepatic mitochondrial oxidation flux (VCS) and pyruvate carboxylase flux (VPC) in humans1, 2, application of this method is expensive, time- and labor-intensive, and requires an in vivo wide-bore (>0.8 m), high-field (≥4 Tesla) magnetic resonance imaging system modified to do 13C MRS, which is available at only a few academic medical centers worldwide. To date the only non-invasive tracer method that has been described to assess hepatic mitochondrial fluxes in vivo utilizes [13C3]propionate3,4,5,6. A disadvantage of the propionate method is that it can alter hepatic mitochondrial metabolism in part through generation of high concentrations of propionyl-CoA7, 8. A key motivation for this study is to develop an alternative non-invasive tracer method to model hepatic metabolism in vivo with minimal perturbation of hepatic mitochondrial metabolism.

To address this unmet need, we have developed a simple positional isotopomer Nuclear Magnetic Resonance (NMR) tracer analysis (PINTA) method by which hepatic glucose production, anaplerosis and citrate synthase flux can be non-invasively assessed based on NMR and gas chromatography/mass spectrometry (GC/MS) analysis of plasma following an infusion of [3-13C]lactate (Supplementary Fig. 1a, b). To validate this method, we performed independent cross-validation studies and observed an excellent correlation (P < 10−15, R2 = 0.99) between VPC/VCS calculated using PINTA and our previous ex vivo NMR tracer technique in rats9, 10, and similar VPC/VCS ratios measured in healthy human subjects using two independent tracer methods (infusion of [3-13C]lactate or [1-13C]acetate1, 2). Validation studies demonstrate the ability of the PINTA method to measure an increase in VCS flux following treatment with a controlled-release mitochondrial protonophore…