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. Author manuscript; available in PMC: 2017 Jul 12.
Published in final edited form as: Cell Metab. 2016 Jul 12;24(1):167–171. doi: 10.1016/j.cmet.2016.06.005

Direct Assessment of Hepatic Mitochondrial Oxidation and Pyruvate Cycling in Non Alcoholic Fatty Liver Disease by 13C Magnetic Resonance Spectroscopy

Kitt Falk Petersen 1,6, Douglas E Befroy 2, Sylvie Dufour 5, Douglas L Rothman 2,3, Gerald I Shulman 1,4,5,6
PMCID: PMC4946568  NIHMSID: NIHMS797400  PMID: 27411016

Summary

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and there is great interest in understanding the potential role of alterations in mitochondrial metabolism in its pathogenesis. To address this question we assessed rates of hepatic mitochondrial oxidation in subjects with and without NAFLD by monitoring the rate of 13C labeling in hepatic [5-13C]glutamate and [1-13C]glutamate by 13C MRS during an infusion of [1-13C]acetate. We found that rates of hepatic mitochondrial oxidation were similar between NAFLD and Control subjects. We also assessed rates of hepatic pyruvate cycling during an infusion of [3-13C]lactate by monitoring the 13C label in hepatic [2-13C]alanine and [2-13C]glutamate and found that this flux also was similar between groups and more than 10-fold lower than previously reported. Contrary to previous studies we show that hepatic mitochondrial oxidation and pyruvate cycling are not altered in NAFLD and do not account for the hepatic fat accumulation.

Graphical abstract

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Introduction

Non-alcoholic fatty liver disease (NAFLD) currently affects 1 in 3 Americans and is a major predisposing factor in the pathogenesis of type 2 diabetes (T2D) (Samuel and Shulman, 2016). NAFLD is also a key predisposing factor for the development of non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma and it is anticipated that NAFLD-induced NASH will soon surpass hepatitis C and alcoholic cirrhosis as the most common indication for liver transplantation in the USA (Sanyal, 2002). Thus there is great interest in understanding the etiology of NAFLD and the potential role of alterations in mitochondrial metabolism in this process. For example reductions in rates of hepatic mitochondrial oxidation may play an important role in the pathogenesis of NAFLD by reducing rates of lipid oxidation thus promoting increased hepatic triglyceride accumulation. Conversely increased rates of hepatic mitochondrial oxidation could lead to increased ROS formation and promote the progression of NAFLD to nonalcoholic steatohepatitis (NASH). However rates of hepatic mitochondrial lipid oxidation have been difficult to assess in humans and studies using indirect approaches have been inconclusive (Croci et al., 2013; Kotronen et al., 2009; Sunny et al., 2011, Zhu et al. 2011). In this regard Sunny et al. reported that rates of hepatic mitochondrial oxidation were approximately twofold greater in subjects with high intrahepatic triglyceride content compared to subjects with low intrahepatic triglyceride content (Sunny et al., 2011). Furthermore these high rates of hepatic mitochondrial oxidation rates were associated with a 2–3 fold increase in rates of hepatic pyruvate cycling, which were 4–6 fold higher than rates of gluconeogenesis in both control and NAFLD subjects (Sunny et al., 2011). Due to this controversy and because the methods used in these previous studies relied on indirect and/or in vitro methodology we directly quantified these hepatic fluxes in subjects with and without hepatic steatosis using a novel in vivo 13C-MRS method (Befroy et al., 2014). Rates of hepatic mitochondrial oxidation (VCS) were assessed directly by monitoring the rate of 13C label incorporation into hepatic [5-13C]glutamate and [1-13C]glutamate by 13C MRS during an intravenous infusion of [1-13C]acetate (Befroy et al., 2014). 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 13C labeling in hepatic [2-13C]alanine relative to hepatic [2-13C]glutamate during an intravenous infusion of [3-13C]lactate (Befroy et al., 2015, Perry et al., 2016).

Results and Discussion

NAFLD subjects had liver triglyceride concentrations that were more than 4-fold higher than the Control subjects and were insulin resistant as reflected by higher fasting plasma insulin concentrations and increased HOMA-IR compared to the Control group (Table 1). Rates of hepatic mitochondrial oxidation were assessed in both groups by monitoring the rate of 13C label incorporation into hepatic [5-13C]glutamate and [1-13C]glutamate by in vivo 13C MRS during an intravenous infusion of [1-13C]acetate as previously described (Befroy et al., 2014). Our measurements of rates of hepatic mitochondrial oxidation depend primarily on the kinetics of enrichment in hepatic [5-13C]glutamate and are independent of hepatic anaplerotic flux (VANA) and hepatic pyruvate kinase flux (VPK) rates (Befroy et al., 2014; Befroy et al., 2015). Following the start of the [1-13C]acetate infusion plasma 13C acetate enrichments (Supplemental Fig. 1A) and concentrations (~1 mM) reached steady state within 5 minutes in both groups and the rate of appearance of 13C incorporation into hepatic [5-13C]glutamate and [1-13C]glutamate were similar in both groups (Fig. 1). Modeling the hepatic [5-13C]glutamate time course data yielded similar rates of hepatic mitochondrial oxidation in the Control and NAFLD subjects (Table 2). These rates for hepatic mitochondrial oxidation in both Control and NAFLD subjects are similar to those previously observed using this same 13C MRS approach in healthy young men with low hepatic triglyceride content (Befroy et al., 2014). Furthermore these data are consistent with our results in high fat fed rodent models of NAFLD, using an alternative isotopic MRS-LC/MS/MS approach, demonstrating that hepatic steatosis does not alter rates of mitochondrial oxidation flux (Perry et al., 2014).

Table 1.

Anthropometric and Metabolic Characteristics of the Control and NAFLD subjects

Control NAFLD P-value
Age (Years) 28±1 30±2 0.27
BMI (Kg/m2) 24.0±0.4 25.5±0.8 0.08
Hepatic Triglyceride Content (%) 1.89±0.26 8.80±1.04 2.8×10−9
Fasting Plasma Glucose (mg/dL) 93±2 95 ±2 0.62
Fasting Plasma Insulin (μU/mL) 11±1 18±3 0.02
Plasma AST (U/L) 23±2 23±3 0.82
Plasma ALT (U/L) 25±4 34±5 0.16
HbA1c (%) 5.1 ±0.1 5.4±0.1 0.05
Total Plasma Cholesterol (mg/dL) 181±11 173±14 0.64
HDL (mg/dL) 50±3 42±5 0.18
LDL (mg/dL) 119±9 108±9 0.47
Plasma Triglycerides (mg/dL) 97±8 129±15 0.05
Plasma Uric Acid (mg/dL) 5.45±0.28 6.28±0.41 0.10
Plasma Non Esterified Fatty Acids (mmol/L) 0.43±0.04 0.55±0.07 0.12
HOMA-IR 2.47±0.21 4.13±0.82 0.02
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Figure 1.

Figure 1

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Time courses for hepatic [5-13C]glutamate and [1-13C]glutamate 13C enrichment (normalized to [5-13C]glutamate at 90 minutes) during an infusion of [1-13C]acetate in Control and NAFLD subjects. Panel inserts show typical 13C MRS spectra obtained for hepatic [5-13C]glutamate in Control and NAFLD subjects and the absence of any detectable labeling in hepatic [5-13C]glutamine. Data are shown mean ± SEM.

Table 2.

Rates of Hepatic Mitochondrial Oxidation and Pyruvate Cycling (Pyruvate Kinase Flux/Pyruvate Carboxylase Flux) in Control and NAFLD subjects.

Parameter Control NAFLD P-Value
Rates of Hepatic Mitochondrial
Oxidation (VCS)
[μmol/(g-min)]		 0.48±0.03 0.49±0.05 0.91
Pyruvate Kinase Flux (VPK)/
Pyruvate Carboxylase Flux (VPC)		 0.29±0.04 0.26±0.04 0.62
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We also directly assessed rates of hepatic pyruvate cycling defined by: (VPK+VME)/(VPC+VPDH), where VPK is pyruvate kinase flux, VME is malic enzyme flux, VPC is pyruvate carboxylase flux and VPDH is pyruvate dehydrogenase flux, by monitoring the relative 13C labeling in hepatic [2-13C]alanine relative to hepatic [2-13C]glutamate during an intravenous infusion of [3-13C]lactate (Befroy et al., 2015; Perry et al., 2016). Following the start of the [3-13C]lactate infusion plasma 13C 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 VME flux and VPDH flux are relatively low compared to VPK flux and VPC flux, respectively, and therefore rates of pyruvate cycling can be approximated to the rate of pyruvate kinase flux/rate of pyruvate carboxylase flux (VPK/VPC) (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).

Taken together our results demonstrate that rates of hepatic mitochondrial oxidation and pyruvate cycling are not altered in NAFLD subjects and rates of hepatic pyruvate cycling are relatively low in both groups compared to hepatic pyruvate carboxylase flux. These results are in marked contrast to a recent study by Sunny et al. who found that rates of hepatic mitochondrial oxidation were increased twofold in subjects with NAFLD compared to control subjects and that rates of pyruvate cycling were more than 4–6 fold higher than their rates of gluconeogenesis (equivalent to VPC-VPK) in both Control and NAFLD subjects.

Pyruvate kinase catalyzes the irreversible final step of glycolysis from phosphoenolpyruvate (PEP) to pyruvate in mammals. PEP has the highest-energy phosphate bond found in living organisms and because of the large decrease in free energy, which occurs when PEP is converted to pyruvate, two enzymatic reactions [PC and phosphoenolpyruvate carboxykinase (PEPCK)], each consuming an ATP equivalent are required to make PEP from pyruvate in gluconeogenic tissues. Consequently the PK, PC and PEPCK reactions are all highly regulated steps in the regulation of glycolysis and gluconeogenesis. Under fasting conditions, when hepatic gluconeogenesis is the major contributor to whole body glucose production, the liver would therefore be expected to limit futile cycling through pyruvate kinase. In this regard it was surprising that several studies using [1,2,3-13C3]propionate as a metabolic tracer have reported extremely high rates of hepatic pyruvate cycling, which were 3–4 times the rate of hepatic mitochondrial oxidation flux and 4–6 times the rates of gluconeogenesis in both rodents (Jin et al., 2004; Satapati et al., 2015) and humans (Sunny et al., 2011). 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 [1,2,3-13C3]propionate labeling studies of hepatic mitochondrial metabolism in humans (Sunny et al., 2011).

Serious concerns about the use of propionate as a tracer have also been raised previously because in contrast to other metabolic tracers, such as alanine or lactate, propionate is subject to significant hepatic zonation (Jungermann, 1987; Jungermann and Kietzmann, 1996; Puchowicz et al., 1999). When propionate is administered orally, as it was in the Sunny et al. study, it enters the liver via the portal circulation and is almost completely cleared from the blood by the time it reaches the hepatic vein (Jungermann, 1987; Jungermann and Kietzmann, 1996; Puchowicz et al., 1999). As such, periportal hepatocytes take up and metabolize much more propionate than perivenous hepatocytes, which invalidates many of the assumptions required for the metabolic flux rate calculations using this method. The effects of zonation on [1,2,3-13C3]propionate metabolism alone could explain a large proportion of the wide variation in flux estimates from livers perfused with 0.1 vs. 0.5 mM of [1,2,3-13C3]propionate where the higher concentrations of propionate increased hepatic phosphoenolpyruvate carboxykinase flux and pyruvate carboxylase kinase flux 2 to 4 fold (Burgess et al., 2007; Satapati et al., 2015). Furthermore the impact of zonation on estimates of hepatic metabolic fluxes will be even greater with lower doses of [1,2,3-13C3]propionate when a greater proportion of [1,2,3-13C3]propionate will be metabolized by the periportal heptatocytes.

Concerns have also been raised regarding the use of acetate and lactate as tracers of hepatic metabolism (Burgess et al., 2015; Previs and Kelley, 2015; Satapati et al., 2015). In regards to acetate, it is possible that extra-hepatic or hepatic metabolism of 13C labeled acetate can lead to significant 13C labeling of glutamine and 13CO2, which in turn could potentially impact the 13C labeling in hepatic glutamate. While it is well established that infusions of 13C acetate over the course of several hours will lead to significant 13C labeling in plasma glutamine (Landau et al., 1993; Schumann et al., 1991) our 13C MRS studies were performed over a relatively short duration (<100 minutes), which resulted in negligible 13C labeling of hepatic [5-13C]glutamine (Fig. 1, insert) (Befroy et al., 2015). Furthermore our in vivo MRS method for estimating rates of hepatic mitochondrial oxidation depends primarily on the incorporation of 13C label from [1-13C]acetate into hepatic [5-13C]glutamate, which is independent of any 13C labeling into hepatic CO2 (Befroy et al., 2014, Befroy et al., 2015).

In regards to the use of lactate as a metabolic tracer it has been suggested that disequilibrium across fumarase may result in an underestimation in rates of pyruvate cycling. However, direct 13C NMR measurements of the ratio of [2-13C]glutamate to [3-13C]glutamate enrichment during an infusion of [3-13C]lactate were found to be ~1.0 demonstrating essentially complete equilibration of 13C label between [2-13C]glutamate and [3-13C]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 summary, we directly assessed rates of hepatic mitochondrial oxidation and hepatic pyruvate cycling in non obese subjects with low and high liver triglyceride content by in vivo 13C MRS. We found that these rates were not altered in subjects with NAFLD, contrary to a previous study by Sunny et al. (Sunny et al., 2011). Furthermore we found that rates of hepatic pyruvate cycling were more than 10-fold lower than previously reported (Sunny et al., 2011). Together, these data suggest that alterations in rates of hepatic mitochondrial oxidation and hepatic pyruvate cycling are not major factors in the pathogenesis of NAFLD in otherwise healthy non obese insulin resistant individuals. Whether these hepatic fluxes are altered by aging, obesity, T2D or during the progression of NAFLD to NASH remains to be determined.

Experimental Procedures

Subjects

Thirty healthy, non-obese, non-smoking men without history of liver disease, with normal liver function tests and consuming no more than 1 alcoholic drink per day were studied after prescreening with an oral glucose tolerance test (OGTT) to ensure normal glucose tolerance. Muscle and hepatic triglyceride content were measured with 1H-MRS (Petersen et al., 2003); subjects were categorized as low liver triglyceride (Control, N=20) when hepatic triglyceride content was <4% (corresponding to the lower 95% percentile of healthy non obese men (N>2,000) in the Yale/New Haven NAFLD cohort, and high liver triglyceride when hepatic triglyceride content was ≥4% (N=10) (Table 1). The study was approved by the Yale University Human Investigation Committee and written informed consent was obtained from each participant after explanation of the purpose, nature and potential complications of the study.

In Vivo 13C Magnetic Resonance Spectroscopy

The studies were performed on a 4.0 T Bruker Medspec system using a custom-built 13C/1H probe consisting of a 9cm diameter 13C surface coil with a pair of elliptical, 14.5×11cm, 1H coils arrayed in quadrature for imaging, shimming and decoupling. At 7:30 AM following a 12-hour overnight fast and placement of two antecubital intravenous catheters, the subjects were positioned supine on the bed of the MRS scanner, the probe was attached to a rigid support across the bed and positioned tightly over the right side of the abdomen over the liver. Scout MRI scans were obtained to confirm correct positioning of the probe and to define a volume for localized 13C-MRS. Field homogeneity was optimized using a respiratory-gated FASTMAP routine and 1H MR spectra were acquired from within the region of interest to confirm hepatic lipid content. Localized 1H-decoupled, 13C-MR spectra of the liver were acquired during a 20 min baseline period and during a 100 min infusion of [1-13C]acetate (99% atom percent enrichment (APE), 350 mmol/L sodium salt, Cambridge Isotope Laboratories Inc., Cambridge, MA) at a rate of 3.0 mg/(Kg-min). Spectra were acquired with nuclear Overhauser effect (NOE) to increase 13C sensitivity (Befroy et al., 2014). Blood samples were collected every 5–10 minutes for the measurements of plasma acetate concentrations and 13C APE by gas-chromatography–mass-spectrometry (GC/MS) (Befroy et al., 2008). The APE of hepatic [5-13C]glutamate and [1-13C]glutamate were determined from each difference spectrum relative to baseline and fitted to a metabolic model of the TCA cycle as previously described (Befroy et al., 2014).

To estimate hepatic pyruvate cycling, a sub-group of Control subjects (N=4) and NAFLD subjects (N=4) underwent a second 13C-MRS study with an infusion of [3-3C]lactate while monitoring the 13C-labeling of hepatic [2-13C]alanine, and [2-13C]glutamate in the 13C-MRS spectrum. The 13C-labeling of [2-13C]alanine from the [3-13C]lactate requires isotopic equilibration, which occurs due to the rapid inter-conversion of oxaloacetate-malate-fumarate followed by the regeneration of pyruvate via pyruvate kinase flux (VPK) (Befroy et al., 2015; Befroy et al., 2014, Perry et al., 2016).

Model of Hepatic Metabolism

The model of hepatic metabolism of [1-13C]acetate has been described in detail previously (Befroy et al., 2014; Befroy et al., 2015). Briefly, in the hepatocytes [1-13C]acetate is converted to [1-13C]acetyl CoA, which enters the TCA cycle where the 13C will be transferred to the C5 position of citrate and α-ketoglutarate (αKG) (Supplemental Figure 2). Glutamate and αKG are in rapid exchange via aspartate-aminotransferase, and equilibration results in the 13C labeling of [5-13C]glutamate, which can be monitored in the 13C-MRS Spectra (Befroy et al., 2014; Befroy et al., 2015). The metabolic model also accounts for the incorporation of 13C label into [1- 13C]glutamate after the second turn of the TCA cycle, which is dependent upon anaplerotic fluxes. The rate of label exchange between the mitochondria and the cytosolic glutamate pool was found to be non-limiting in our previous study (Befroy et al., 2014; Befroy et al., 2015) and therefore did not impact the rate of appearance of 13C label at [5-13C] or [1-13C]glutamate. We have performed simulations to show that the impact of anaplerosis and pyruvate recycling are minimal on the calculated rates of hepatic mitochondrial oxidation (VCS) (Befroy et al., 2014; Befroy et al., 2015).

To estimate hepatic pyruvate cycling a sub-group of Control and NAFLD subjects underwent a second 13C-MRS study with an infusion of [3-13C]lactate, while monitoring the 13C-labeling of hepatic [2-13C]alanine, and [2-13C]glutamate in the 13C-MRS spectrum as previously described (Befroy et al., 2015; Perry et al., 2016) (Supplemental Figure 3). The 13C-labeling of [2-13C]alanine from the [3-13C]lactate requires isotopic equilibration, which occurs due to the rapid inter-conversion of oxaloacetate-malate-fumarate followed by the regeneration of pyruvate via pyruvate kinase flux (VPK) (Befroy et al., 2015; Perry et al., 2016). Rates of hepatic pyruvate cycling were calculated by: (VPK+VME)/(VPC+VPDH), where VPK is pyruvate kinase flux, VME is malic enzyme flux, VPC is pyruvate carboxylase flux and VPDH is pyruvate dehydrogenase flux, by monitoring the relative 13C labeling in hepatic [2-13C]alanine relative to hepatic [2-13C]glutamate during an intravenous infusion of [3-13C]lactate (Befroy et al., 2014, Perry et al., 2016).

Statistical Analysis

Statistical analyses were performed with StatPlus software. Statistically significant differences between control and NAFLD subjects were detected using unpaired Student’s t-tests. The precision in the fit of the metabolic model to each individual subject’s raw data was determined by probability distribution analysis of 500 Monte-Carlo simulations of the model with random Gaussian noise added to the fitted time-course (Befroy et al., 2014). If the uncertainty in the calculated VCS rate due to noise was greater than 10% the data were not used in the analysis. All data are expressed as mean ± SEM.

Supplementary Material

supplement

Acknowledgments

The authors would like to thank Anne Impellizeri, B.S., Irina Smolgovsky, Mikhail Smolgovsky, Gina Solomon, R.N. and Catherine Parmelee, R.N. and the Yale Center for Clinical Investigation, Hospital Research Unit for assistance with the in vivo 4T MRS studies and Dr. Gary W. Cline, Dr. Xian-Man Zhang and Nimit Jain for assistance with the GC-MS, LC-MS/MS and in vitro and in vivo NMR analyses and modeling. This publication was supported by grants from the United States Public Health Service: R01 DK-49230, R01 AG-23686, P30 DK-45735, UL1 RR-024139, a Distinguished Clinical Investigator Award from the American Diabetes Association (KFP) and Gilead Sciences Inc. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.

Footnotes

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Author Contributions

K.F.P. D.E.B., D.L.R and G.I.S. designed the experimental protocols. K.F.P. D.E.B, and S.D. performed the studies. K.F.P., D.E.B, S.D. and D.L.R analyzed the data. K.F.P. and G.I.S. wrote this manuscript with contributions from all of the other authors.

Contributor Information

Douglas E. Befroy, Email: douglas.befroy@gmail.com.

Sylvie Dufour, Email: sylvie.dufour@yale.edu.

Douglas L. Rothman, Email: douglas.rothman@yale.edu.

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