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Effect of uncouplers of oxidative phosphorylation on metabolism of propionate in liver explants from dairy cows

  • Author Footnotes
    * Current address: Institute of Nutritional Physiology (“Oskar Kellner”), Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany.
    Katherine M. Kennedy
    Footnotes
    * Current address: Institute of Nutritional Physiology (“Oskar Kellner”), Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany.
    Affiliations
    Department of Animal Science, Michigan State University, East Lansing, MI 48824
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  • Shawn S. Donkin
    Affiliations
    Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
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  • Michael S. Allen
    Correspondence
    Corresponding author
    Affiliations
    Department of Animal Science, Michigan State University, East Lansing, MI 48824
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  • Author Footnotes
    * Current address: Institute of Nutritional Physiology (“Oskar Kellner”), Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany.
Open ArchivePublished:January 14, 2021DOI:https://doi.org/10.3168/jds.2020-19536

      ABSTRACT

      Our objective was to determine the effects of uncouplers of oxidative phosphorylation on the metabolism of propionate in liver tissue of dairy cows in the postpartum period. A total of 8 primiparous dairy cows were biopsied for liver tissue explants in 2 block-design experiments. Cows were 5.9 ± 2.8 (mean ± SD) days in milk, and the 2 experiments were run concurrently. Treatments for experiment 1 were 10 μM 2,4-dinitrophenol methyl ether (DNPME) or propylene carbonate (vehicle control). Treatments for experiment 2 were 5 mM sodium salicylate (SAL) or no treatment (control). Explants were incubated in 2.5 mM [13C3]propionate with treatments and terminated after 0.5, 15, and 60 min of exposure to tracer. Treatment with DNPME had no effects on measured metabolites compared with control. Treatment with SAL increased total 13C% enrichment of succinate (3.03 vs. 2.45%), but tended to decrease total 13C% enrichment of fumarate (2.86 vs. 3.10%) and decreased total 13C% enrichment of malate (3.96 vs. 4.58%) compared with the control. Treatment with DNPME appeared to have no effects on hepatic propionate metabolism, and treatment with SAL may impair the succinate dehydrogenase reaction.

      Key words

      INTRODUCTION

      The negative energy balance experienced by dairy cows during the early postpartum period can be exacerbated by the suppression of appetite (
      • Allen M.S.
      • Piantoni P.
      Metabolic control of feed intake: Implications for metabolic disease of fresh cows.
      ), which increases the risk for metabolic diseases as well as decreases milk production and impairs fertility (
      • Herdt T.H.
      Ruminant adaptation to negative energy balance: Influences on the etiology of ketosis and fatty liver.
      ;
      • Butler W.R.
      Energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows.
      ). To combat the potential for negative energy balance, energy density of diets can be increased by substituting starch-containing cereal grains for forages. Propionic acid, produced by the fermentation of starch in high-energy diets, is a primary glucose precursor for ruminants and has been shown to have hypophagic effects in dairy cows (
      • Gualdrón-Duarte L.B.
      • Allen M.S.
      Fuels derived from starch digestion have different effects on energy intake and metabolic responses of cows in the postpartum period.
      ;
      • Maldini G.
      • Allen M.S.
      Temporal effects of ruminal propionic acid infusion on feeding behavior of Holstein cows in the postpartum period.
      ). The hypophagic effects of propionate are likely from stimulating hepatic oxidation according to the hepatic oxidation theory (
      • Allen M.S.
      • Bradford B.J.
      • Oba M.
      Board-invited review: The hepatic oxidation theory of the control of feed intake and its application to ruminants.
      ).
      As a test of the hepatic oxidation theory, we previously injected dairy cows with uncouplers of oxidative phosphorylation that we expected to decrease the capacity of the liver to efficiently oxidize fuels (
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      ). Two different uncouplers of oxidation phosphorylation (OXPHOS) were used in that study: 2,4-dinitrophenol methyl ether (DNPME) and sodium salicylate (SAL).
      • Perry R.J.
      • Kim T.
      • Zhang X.M.
      • Lee H.Y.
      • Pesta D.
      • Popov V.B.
      • Zhang D.
      • Rahimi Y.
      • Jurczak M.J.
      • Cline G.W.
      • Spiegel D.A.
      • Shulman G.I.
      Reversal of hypertriglyceridemia, fatty liver disease, and insulin resistance by a liver-targeted mitochondrial uncoupler.
      showed that DNPME is preferentially extracted by the liver and converted to 2,4-dinitrophenol (DNP). Both uncouplers are protonophores and decrease the proton-motive force necessary for ATP synthesis by the electron transport chain.
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      showed that intrajugular injection of 10 mg/kg of BW0.75 of DNPME increased meal length and decreased eating rate (meal size/meal length) over the first 4 h of meals compared with the control. Intrajugular injection of 50 mg/kg of BW of SAL decreased the eating rate over the first 4 h of meals compared with control (
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      ). However, short-term, direct effects (≤1 h) of these uncouplers on hepatic metabolism in bovines are unknown.
      Our objective was to determine the effects of uncouplers of oxidative phosphorylation, DNPME, and SAL on the metabolism of propionate in liver explants from dairy cows in the immediate postpartum period (≤3 wk postpartum). We hypothesized that uncoupling the electron transport chain would increase anaplerosis by propionate as evidenced by increased 13C enrichment of tricarboxylic acid (TCA) cycle intermediates.

      MATERIALS AND METHODS

      The Institutional Animal Care and Use Committee at Michigan State University approved all animal procedures for this experiment.

      Experimental Design, Treatments, and Animal Management

      Eight primiparous Holstein Friesian dairy cows were used in 2 block-design experiments. Animals were housed in tiestalls at the Michigan State University Dairy Cattle Teaching and Research Center (East Lansing). They were fed a common ration formulated to contain 32% NDF, 23% forage NDF, 22% starch, and 18.4% CP that consisted of corn silage, ground corn, soybean meal, alfalfa silage, alfalfa hay, and a premix of minerals and vitamins. Cows were 5.9 ± 2.8 (mean ± SD) DIM at the time of biopsy and had a BCS of 3.01 ± 0.71 and BW of 585 ± 77.1 kg. Two experiments (Exp. 1 and Exp. 2) were conducted concurrently using the liver tissue biopsied from these cows, and we used the same techniques for data collection and analysis for both experiments.

      Experiment 1

      Treatments of either 20 μL of the DNPME solution [10 μM DNPME dissolved in propylene carbonate (vehicle) per vial (Alfa Aesar, Haverhill, MA, B21542)] or 20 μL of propylene carbonate (vehicle, control, CON1; Sigma Aldrich, St. Louis, MO) were added to 1.98 mL of the medium for a total volume of 2 mL per vial. Treatment dose was based on
      • Perry R.J.
      • Kim T.
      • Zhang X.M.
      • Lee H.Y.
      • Pesta D.
      • Popov V.B.
      • Zhang D.
      • Rahimi Y.
      • Jurczak M.J.
      • Cline G.W.
      • Spiegel D.A.
      • Shulman G.I.
      Reversal of hypertriglyceridemia, fatty liver disease, and insulin resistance by a liver-targeted mitochondrial uncoupler.
      , who incubated liver mitochondria from rats in 0, 3, 6, or 12.5 μM of either DNPME or DNP and measured oxygen consumption rate.

      Experiment 2

      Treatments were either 20 μL of sodium salicylate solution [5 mM SAL dissolved in medium (vehicle) per vial; Sigma Aldrich, S2679)] added to 1.98 mL of medium for a total volume of 2 mL per vial, or no treatment (control; CON2) for a total volume of 2 mL per vial. Treatment dose was based on
      • Jeffrey S.W.
      • Smith M.J.H.
      Some effects of salicylate on mitochondria from rat liver.
      who incubated isolated mitochondria from rats in 5 mM SAL and measured oxygen and inorganic phosphate uptake.

      Data Collection and Analysis

      Before the liver biopsy, animals were moved to a surgery room at the Michigan State Dairy Cattle Research and Teaching Center. Liver was biopsied before the morning feeding at approximately 0800 h. Cows were blocked from feed for approximately 30 min before biopsy, and 1 cow was biopsied per day. Liver tissue (∼300–400 mg) for the experiment was biopsied as described by
      • Kennedy K.M.
      • Donkin S.S.
      • Allen M.S.
      Effect of propionate concentration on short-term metabolism in liver explants from dairy cows in the postpartum period.
      . Tissue was rinsed in ice-cold sterile saline to remove excess blood and then placed in ice-cold supplemented Medium 199 (Sigma Aldrich, M7528) for immediate transport to the laboratory. Explants were prepared from biopsied tissue as described by
      • Zhang Q.
      • Koser S.L.
      • Bequette B.J.
      • Donkin S.S.
      Effect of propionate on mRNA expression of key genes for gluconeogenesis in liver of dairy cattle.
      and incubated as described by
      • Kennedy K.M.
      • Donkin S.S.
      • Allen M.S.
      Effect of propionate concentration on short-term metabolism in liver explants from dairy cows in the postpartum period.
      . Explants were incubated in 2 mL of Medium 199 supplemented with 1% BSA (GenDEPOT, Katy, TX, A0100), 0.6 mM oleic acid (Sigma Aldrich, O3008), 2 mM sodium l-lactate (Sigma Aldrich, L7022), 0.2 mM sodium pyruvate (Sigma Aldrich P2256), 0.5 mM l-glutamine (Sigma Aldrich, G3126), and 2.5 mM [13C3]sodium propionate (Sigma Aldrich, 99 atom % 13C, 490636) for 13C metabolite analysis in 20-mL glass scintillation vials (Research Products International, Mt. Prospect, IL, 121002) flushed with 95% O2:5% CO2 for 0.5, 15, or 60 min. At the designated end time point, samples were removed from medium with sterile tweezers, rapidly blotted (∼10 s) on Kimwipes (Kimtech Science, Kimberly-Clark Professional, Roswell, GA), placed in 2-mL screw-cap tubes with O-ring caps (Thomas Scientific; Swedesboro, NJ, 1213G01), and dropped in liquid nitrogen. Samples were stored at −80°C until analysis. Tissue not used for explants was frozen in liquid nitrogen and used as a baseline (0 min) and correction factor for isotope natural abundance.
      Explants were prepared and analyzed for DNP, salicylate, and inorganic phosphate concentration by GC-MS as described in
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      . Samples were derivatized by methoximation and silylation as described by
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      . Nonincubated tissue for each cow was analyzed and used as a baseline (0 min) concentration for treatment and control samples. Although the most reliable fragment ion of DNP was selected for monitoring (241.1 m/z), the liver is a complex matrix, and consequently, other compounds not related to DNP are likely to have a similar fragment ion. As such, baseline samples were used as a correction factor and subtracted from the measured DNP concentrations detected at each sampling for each cow.

      13C Metabolite Analysis

      Explants were prepared and analyzed for citrate, isocitrate, succinate, fumarate, malate, pyruvate, lactate, glutamate, oxaloacetate (OAA), α-ketoglutarate, and glucose abundance by GC-MS; to analyze acetyl CoA and propionyl CoA abundance, we used liquid chromatography–tandem mass spectrometry as described by
      • Kennedy K.M.
      • Donkin S.S.
      • Allen M.S.
      Effect of propionate concentration on short-term metabolism in liver explants from dairy cows in the postpartum period.
      . Area quantification of peaks were integrated using MassLynx Mass Spectrometry Software (version 4.1, Waters Corporation, Milford, MA). The abundance of isocitrate is naturally low, and as such, data were undetectable or unreliable with higher isotopologues. Therefore, we did not include the data pertaining to isocitrate in the analysis. Additionally, an unknown mass at [M+2] for OAA and α-ketoglutarate interfered with those isotopologues by masking their abundance, including unlabeled samples, and thus the data for OAA and α-ketoglutarate were also not included in the analysis. Total percentage of 13C enrichment of each metabolite was calculated as modified from
      • Wang Z.
      • Jones A.D.
      Profiling of stable isotope enrichment in specialized metabolites using liquid chromatography and multiplexed nonselective collision-induced dissociation.
      using the following equation:
      13C%enrichment=(M¯L-M¯U13.00335-12×100)ncarbonsinmetabolite,


      where M¯L is the weighted average isotopologue mass of the labeled metabolite, M¯U is the weighted average isotopologue mass of the unlabeled metabolite, and the denominator is the difference between 13C and 12C atomic masses. Additionally, this equation adjusts for the number of carbons within each metabolite, not including carbons within CoA. This equation is a simple representation of stable isotope labeling and calculates the total percent increase of 13C over its natural abundance that is present within a metabolite (i.e., it does not distinguish between the distribution of isotopologues within the metabolite). The isotope enrichment, expressed as molar percent, was calculated for the isotopologues [M+3]propionyl CoA, [M+2]pyruvate, [M+1]acetyl CoA, [M+2]acetyl CoA, [M+3]citrate, [M+4]citrate, [M+5]citrate, [M+2]glutamate, [M+3]glutamate, [M+2]glucose, and [M+3]glucose as described by
      • Kennedy K.M.
      • Donkin S.S.
      • Allen M.S.
      Effect of propionate concentration on short-term metabolism in liver explants from dairy cows in the postpartum period.
      . The enrichment of each isotopologue is relative to the mass distribution of all the isotopologues in the metabolite and is on a scale of 0 to 100%.

      Statistical Analysis

      All data were analyzed with mixed models (PROC MIXED) and repeated measures using SAS software (version 9.4; SAS Institute Inc., Cary, NC). Concentrations of hepatic metabolites, total 13C% enrichment of metabolites, and 13C% enrichment of specific isotopologues for both experiments were analyzed with the following model:
      Yijkl=μ+Ci(Rj)+Rj+Tk+Sl+TkSl+TkRj+eijkl,


      where Yijkl = the response variable; μ = overall mean; Ci(Rj) = random effect of cow i nested within run j; Rj = fixed effect of run j; Tk = fixed effect of treatment k; Sl = fixed effect of sampling time l; TkSl = interaction of treatment and sampling time; TkRj = interaction of treatment and run; and eijkl = residual. Time was used in the repeated statement to account for repeated measures, and the subject was defined as treatment by cow nested within run interaction. Run was defined as the group of samples analyzed on a mass spectrometer (GC-MS or liquid chromatography–tandem mass spectrometry) within a single session. The covariance structure was compound symmetry, and denominator degrees of freedom were estimated by using the Kenward-Roger option in the MODEL statement. Paired differences were determined using the diff option in PROC MIXED.
      Treatment effects and interactions were declared significant at P ≤ 0.05, and tendencies were declared at 0.05 < P ≤ 0.10. Response variables that violated the assumptions of normality, homogeneity of residuals, or both were transformed. Normality was determined by visual inspection of Q-Q plots, and homogeneity of residuals was determined by visual inspection of plotting the residual versus predicted values. Data transformed are noted in the tables and figures along with the transformation used for statistical analysis. All transformed data have been back-transformed for interpretation.

      RESULTS

      Experiment 1: DNPME

      The DNPME treatment increased DNP concentration compared with CON1 (Table 1; −0.12 vs. 1.66 nmol/g; P = 0.02), and DNP concentrations tended to increase with time until 15 min, but then decreased (Figure 1A; P = 0.07); no significant treatment by time interaction was detected (P = 0.61). Similarly, the DNPME treatment increased inorganic phosphate concentration compared with CON1 (9.67 vs. 8.98 μmol/g; P < 0.01), but no significant treatment by time interaction was detected (P = 0.72). Inorganic phosphate concentrations initially increased when introduced to the medium, but then decreased over time (Figure 1B; P < 0.01). The DNPME treatment had no treatment or treatment by time effects on the total 13C% enrichment of measured metabolites (Table 2). All metabolites increased total 13C% enrichment over time (Appendix, Figure A1; P < 0.05), except acetyl CoA, and significant effects of time are reported within each figure.
      Table 1Concentrations of metabolites in liver explants from dairy cows in the immediate postpartum period
      Metabolite
      Values reported as LSM (95% CI).
      ControlDNPME
      DNPME = 2,4-dinitrophenol methyl ether.
      Sodium salicylateP-value
      TreatmentTimeTreatment × time
      Experiment 1
      Explants were incubated for up to 60 min in 2.5 mM [13C3]propionate with treatments of either 10 μM DNPME or propylene carbonate (control); DNP = 2,4-dinitrophenol; phosphate = inorganic phosphate.
       DNP (nmol/g)
      Data transformed using inverse square root for statistical analysis and back-transformed for interpretation.
      −0.12(−1.96, 2.42)1.66(−0.67, 4.97)0.020.070.61
       Phosphate (μmol/g)8.98(7.88, 10.1)9.67(8.57, 10.8)<0.01<0.010.72
      Experiment 2
      Explants were incubated for up to 60 min in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control).
       Salicylate (nmol/g)
      Data transformed using natural log for statistical analysis and back-transformed for interpretation.
      7.34(5.32, 10.1)380(275, 524)<0. 01<0. 01<0. 01
       Phosphate (μmol/g)9.08(7.92, 10.2)9.06(7.91, 10.2)0.96<0. 010.14
      1 Values reported as LSM (95% CI).
      2 DNPME = 2,4-dinitrophenol methyl ether.
      3 Explants were incubated for up to 60 min in 2.5 mM [13C3]propionate with treatments of either 10 μM DNPME or propylene carbonate (control); DNP = 2,4-dinitrophenol; phosphate = inorganic phosphate.
      4 Data transformed using inverse square root for statistical analysis and back-transformed for interpretation.
      5 Explants were incubated for up to 60 min in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control).
      6 Data transformed using natural log for statistical analysis and back-transformed for interpretation.
      Figure thumbnail gr1
      Figure 1Concentrations of 2,4-dinitrophenol (DNP; A) and inorganic phosphate (phosphate; B) in liver explants from dairy cows in the postpartum period for experiment 1. Explants were incubated in [13C3]propionate with treatments of either 10 μM 2,4-dinitrophenol methyl ether (DNPME) or propylene carbonate (control; CON) and sampled at 0 (baseline), 0.5, 15, and 60 min. Graphs are presented as highest level of significant or tendency for significant interaction, and associated P-values can be found in . Time effects are presented at the bottom of each graph with a letter in order of 0, 0.5, 15, and 60 min, and different letters define significant differences from each other (P ≤ 0.05). Concentration of hepatic DNP was transformed using inverse square root for statistical analysis and back-transformed for interpretation.
      Table 2Total 13C% enrichment of metabolites from liver explants from dairy cows in the immediate postpartum period
      Metabolite
      Total 13C enrichment of metabolite above the natural abundance (%). Values reported as LSM (95%CI).
      ControlDNPME
      DNPME = 2,4-dinitrophenol methyl ether
      Sodium salicylateP-value
      TreatmentTimeTreatment × time
      Experiment 1
      Explants were incubated for up to 60 min in 2.5 mM [13C3]propionate with treatments of either 10 μM DNPME or propylene carbonate (control).
       Propionyl CoA29.9(26.1, 33.6)31.3(27.5, 35.0)0.15<0.010.91
       Succinate
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      2.12(1.93, 2.32)2.11(1.92, 2.31)0.94<0.010.29
       Fumarate9.43(8.71, 10.1)9.37(8.65, 10.1)0.78<0.010.90
       Malate9.11(8.42, 9.80)8.97(8.28, 9.66)0.50<0.010.82
       Citrate
      Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      3.01(2.67, 3.36)2.92(2.58, 3.26)0.41<0.010.55
       Glutamate
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      0.19(0.15, 0.23)0.16(0.12, 0.21)0.36<0.010.81
       Pyruvate1.23(0.68, 1.79)1.25(0.70, 1.81)0.87<0.010.99
       Lactate
      Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      0.72(0.58, 0.87)0.73(0.58, 0.87)0.84<0.011.00
       Glucose0.31(0.21, 0.40)0.34(0.25, 0.44)0.55<0.010.46
       Acetyl CoA
      Data transformed using square root (n + 10) for statistical analysis and back-transformed for interpretation.
      0.97(−0.52, 2.58)1.61(0.07, 3.26)0.540.130.98
      Experiment 2
      Explants were incubated for up to 60 min in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control).
       Propionyl CoA29.6(25.1, 34.0)30.9(26.5, 35.4)0.35<0.010.85
       Succinate
      Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      2.45(2.08, 2.84)3.03(2.63, 3.45)<0.01<0.010.09
       Fumarate
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      3.10(2.90, 3.31)2.86(2.67, 3.06)0.09<0.010.10
       Malate
      Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      4.58(4.11, 5.07)3.96(3.52, 4.43)0.01<0.010.01
       Citrate
      Data transformed using inverse square root (n+1) for statistical analysis and back-transformed for interpretation purposes.
      1.53(1.43, 1.64)1.42(1.33, 1.53)0.12<0.010.74
       Glutamate
      Data transformed using inverse square root (n+1) for statistical analysis and back-transformed for interpretation purposes.
      0.38(0.35, 0.41)0.36(0.33, 0.39)0.44<0.010.36
       Pyruvate
      Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      1.35(0.86, 1.90)1.30(0.82, 1.84)0.61<0.010.96
       Lactate
      Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      0.54(0.44, 0.64)0.58(0.48, 0.68)0.40<0.010.82
       Glucose0.42(0.28, 0.56)0.33(0.19, 0.47)0.14<0.010.48
       Acetyl CoA1.47(−0.54, 3.49)1.61(−0.40, 3.62)0.900.080.68
      1 Total 13C enrichment of metabolite above the natural abundance (%). Values reported as LSM (95%CI).
      2 DNPME = 2,4-dinitrophenol methyl ether
      3 Explants were incubated for up to 60 min in 2.5 mM [13C3]propionate with treatments of either 10 μM DNPME or propylene carbonate (control).
      4 Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      5 Data transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      6 Data transformed using square root (n + 10) for statistical analysis and back-transformed for interpretation.
      7 Explants were incubated for up to 60 min in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control).
      8 Data transformed using inverse square root (n+1) for statistical analysis and back-transformed for interpretation purposes.
      The DNPME treatment did not affect 13C% enrichment of the selected isotopologues compared with CON1 (Table 3; P > 0.10). 13C% enrichment of all selected isotopologues increased or tended to increase over time (Appendix, Figure A2; P ≤ 0.10), except for 13C% enrichment of [M+1]acetyl CoA (P = 0.56).
      Table 313C% Enrichment of select metabolite isotopologues from liver explants from dairy cows in the immediate postpartum period
      Metabolite
      Enrichment of each isotopologue is relative to the mass distribution of all the isotopologues in the metabolite (molar percent) and is on a scale of 0 to 100%. Values reported as LSM (95%CI).
      ControlDNPME
      DNPME = 2,4-dinitrophenol methyl ether
      Sodium salicylateP-value
      TreatmentTimeTreatment × time
      Experiment 1
      Explants were incubated for up to 60 min in 2.5 mM [13C3]propionate with treatments of either 10 μM DNPME or propylene carbonate (control).
       Propionyl CoA[M+3]51.5(47.4, 55.6)53.3(49.2, 57.5)0.22<0.010.98
       Pyruvate[M+2]2.13(1.65, 2.61)2.25(1.77, 2.73)0.28<0.010.51
       Acetyl CoA[M+1]0.56(−2.31, 3.42)3.19(0.32, 6.05)0.200.560.25
       Acetyl CoA[M+2]
      Data transformed using square root (n + 10) for statistical analysis and back-transformed for interpretation.
      0.94(−2.02, 4.36)−0.07(−2.88, 3.20)0.630.060.63
       Citrate[M+3]
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation.
      3.08(2.68, 3.53)2.93(2.54, 3.36)0.40<0.010.58
       Citrate[M+4]
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation.
      1.71(1.46, 1.99)1.61(1.37, 1.87)0.40<0.010.61
       Citrate[M+5]
      Data transformed using inverse square root (n + 1) for statistical analysis and back-transformed for interpretation.
      0.92(0.76, 1.10)0.96(0.80, 1.14)0.73<0.010.35
       Glutamate[M+2]
      Data transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.
      0.32(0.20, 0.46)0.31(0.20, 0.45)0.68<0.010.94
       Glutamate[M+3]
      Data transformed using inverse square root (n + 1) for statistical analysis and back-transformed for interpretation.
      0.19(0.09, 0.30)0.14(0.05, 0.25)0.41<0.010.59
       Glucose[M+2]
      Data transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.
      0.21(0.16, 0.26)0.22(0.17, 0.27)0.71<0.010.26
       Glucose[M+3]0.06(0.05, 0.07)0.07(0.05, 0.08)0.11<0.010.45
      Experiment 2
      Explants were incubated for up to 60 min in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control).
       Propionyl CoA[M+3]51.1(45.8, 56.4)53.8(48.5, 59.1)0.16<0.010.52
       Pyruvate[M+2]2.47(2.08, 2.86)2.28(1.89, 2.66)0.23<0.010.69
       Acetyl CoA[M+1]2.04(−1.82, 5.91)1.38(−2.48, 5.24)0.750.530.77
       Acetyl CoA[M+2]1.22(−1.70, 4.14)1.87(−1.05, 4.78)0.710.040.51
       Citrate[M+3]9.65(8.55, 10.7)8.12(7.02, 9.22)0.04<0.010.14
       Citrate[M+4]
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation.
      2.24(1.90, 2.62)1.99(1.68, 2.34)0.09<0.010.81
       Citrate[M+5]2.31(1.91, 2.70)2.29(1.89, 2.68)0.83<0.010.68
       Glutamate[M+2]
      Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation.
      0.82(0.74, 0.91)0.75(0.67, 0.84)0.23<0.010.27
       Glutamate[M+3]
      Data transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.
      0.40(0.35, 0.45)0.38(0.33, 0.43)0.42<0.010.30
       Glucose[M+2]0.30(0.23, 0.37)0.21(0.14, 0.28)0.01<0.010.10
       Glucose[M+3]
      Data transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.
      0.11(0.09, 0.13)0.07(0.05, 0.08)<0.01<0.010.01
      1 Enrichment of each isotopologue is relative to the mass distribution of all the isotopologues in the metabolite (molar percent) and is on a scale of 0 to 100%. Values reported as LSM (95%CI).
      2 DNPME = 2,4-dinitrophenol methyl ether
      3 Explants were incubated for up to 60 min in 2.5 mM [13C3]propionate with treatments of either 10 μM DNPME or propylene carbonate (control).
      4 Data transformed using square root (n + 10) for statistical analysis and back-transformed for interpretation.
      5 Data transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation.
      6 Data transformed using inverse square root (n + 1) for statistical analysis and back-transformed for interpretation.
      7 Data transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.
      8 Explants were incubated for up to 60 min in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control).

      Experiment 2: SAL

      The SAL treatment increased salicylate concentration compared with CON2 (Table 1; 380 vs. 7.34 nmol/g; P < 0. 01) and increased salicylate concentrations over time compared with CON2 (Figure 2A; P < 0. 01). The SAL treatment had no treatment or treatment by time effects on inorganic phosphate (P > 0.10), but inorganic phosphate concentration increased over time compared with baseline (Figure 2B; P < 0. 01). The SAL treatment increased total 13C% enrichment of succinate (Table 2; P < 0.01) and tended to increase total 13C% enrichment of succinate over time compared with CON2 (Figure 3B; interaction, P = 0.09). The SAL treatment tended to decrease total 13C% enrichment of fumarate and malate compared with CON2 (P < 0.10) and interacted or tended to interact with time to affect total 13C% enrichment of fumarate and malate (Figure 3; P ≤ 0.10). All metabolites increased in total 13C% enrichment over time (Appendix, Figure A3; P < 0. 01) except for acetyl CoA (P = 0.08), which tended to increase at 60 min compared with 15 min (3.34 vs. 0.78%; pairwise test, P = 0.06) and increased at 60 min compared with baseline (pairwise test, P = 0.02).
      Figure thumbnail gr2
      Figure 2Concentrations of salicylate (A) and inorganic phosphate (phosphate; B) in liver explants from dairy cows in the postpartum period for experiment 2. Explants were incubated in [13C3]propionate with treatments of either 5 mM sodium salicylate (SAL) or no treatment (control; CON) and sampled at 0 (baseline), 0.5, 15, and 60 min. Graphs are presented as highest level of significant or tendency for significant interaction, and associated P-values can be found in . Time effects are presented at the bottom of each graph with a letter in order of 0, 0.5, 15, and 60 min, and different letters define significant differences from each other (P ≤ 0.05). Treatment by time interactions significantly different from each other at each time point are denoted by * (P ≤ 0.05). Concentration of hepatic inorganic phosphate was transformed using the natural log for statistical analysis and back-transformed for interpretation.
      Figure thumbnail gr3
      Figure 3Total 13C% enrichment of hepatic succinate (A), fumarate (B), and malate (C) from liver explants from dairy cows in the postpartum period for experiment 2. Total 13C% enrichment is the percent increase of 13C within each metabolite above the natural abundance. Explants were incubated in [13C3]propionate with treatments of either 5 mM sodium salicylate (SAL) or no treatment (control; CON) and sampled at baseline (no enrichment, 0), 0.5, 15, and 60 min. Graphs are presented as highest level of significant or tendency for significant interaction, and associated P-values can be found in . Time effects are presented at the bottom of each graph with a letter in order of 0.5, 15, and 60 min, and different letters define significant differences from each other (P ≤ 0.05). Treatment by time interactions significantly different from each other at each time point are denoted by * (P ≤ 0.05). Total 13C% enrichment of succinate and malate were transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation. Total 13C% enrichment of fumarate was transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation.
      The SAL treatment decreased 13C% enrichment of [M+3]citrate, [M+2]glucose, and [M+3]glucose compared with CON2 (Table 3; P ≤ 0.04) and tended to decrease [M+4]citrate (P = 0.09). The SAL treatment tended to decrease 13C% enrichment of [M+2]glucose compared with CON2 over time (Figure 4A; interaction, P = 0.10) and decreased 13C% enrichment of [M+3]glucose compared with CON2 over time (Figure 4B; interaction, P = 0.01). In addition, labeling of all isotopologues increased over time (Appendix, Figure A4; P < 0.05), except for 13C% enrichment of [M+1]acetyl CoA (P = 0.53).
      Figure thumbnail gr4
      Figure 413C% enrichment of hepatic [M+2]glucose (A) and [M+3]glucose (B) from liver explants from dairy cows in the postpartum period for experiment 2. Explants were incubated in [13C3]propionate with treatments of either 5 mM sodium salicylate (SAL) or no treatment (control; CON) and sampled at 0.5, 15, and 60 min. The enrichment of each isotopologue is relative to the mass distribution of all the isotopologues in the metabolite (molar percent) and is on a scale of 0 to 100%. Graphs are presented as highest level of significant or tendency for significant interaction, and associated P-values can be found in . Time effects are presented at the bottom of each graph with a letter in order of 0.5, 15, and 60 min, and different letters define significant difference from each other (P ≤ 0.05). Treatment by time interactions significantly different from each other at each time point are denoted by * (P ≤ 0.05) and tendencies are denoted by † (P ≤ 0.10). 13C% enrichment of hepatic [M+3]glucose was transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.

      DISCUSSION

      Experiment 1: DNPME

      Although no treatment by time effects were detected, DNPME increased DNP and inorganic phosphate concentrations. An increase in inorganic phosphate was expected because DNP is known to liberate phosphate (
      • Lardy H.A.
      • Wellman H.
      The catalytic effect of 2, 4-dinitrophenol on adenosine triphosphate hydrolysis by cell particles and soluble enzymes.
      ;
      • Katyare S.S.
      • Fatterpaker P.
      • Sreenivasan A.
      Effect of 2, 4-dinitrophenol (DNP) on oxidative phosphorylation in rat liver mitochondria.
      ). As such, an overall increase in inorganic phosphate and DNP concentration compared with CON supports that DNP was utilized by the explants to some extent.
      An increase in 13C% enrichment of all measured metabolites and isotopologues over time, except for acetyl CoA and [M+1]acetyl CoA, confirmed that propionate was extracted and metabolized by the liver explants. Furthermore, we detected an increase in 13C enrichment in TCA cycle intermediates over time, suggesting that the tissue was not dying nor the nutrient supply diminishing over the sampling time. However, DNPME had little effect on metabolism of propionate in this experiment because no significant treatment or treatment by time interactions were observed.
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      reported no significant differences among the TCA cycle intermediates in liver samples biopsied 4 h after lactating dairy cows were given an intrajugular injection of 10 mg/kg of BW0.75 of DNPME compared with the control, which agrees with our results. Furthermore, the lack of treatment effect on 13C% enrichment of acetyl CoA suggests that DNPME does not divert propionate toward acetyl CoA production to a greater extent than CON.

      Experiment 2: SAL

      The increase in salicylate concentration over time by SAL compared with CON2 showed that salicylate was extracted by the tissue. However, SAL treatment had no effect on inorganic phosphate compared with CON2, which contrasts with
      • Brody T.M.
      • Fouts W.
      Action of sodium salicylate and related compounds on tissue metabolism in vitro.
      , who measured a decrease in inorganic phosphate uptake and increase in oxygen uptake, resulting in a decreased P:O ratio in rats. Others have also reported a decrease in the P:O ratio with a concentration of 5 mM sodium salicylate in rat liver mitochondria in vitro (
      • Jeffrey S.W.
      • Smith M.J.H.
      Some effects of salicylate on mitochondria from rat liver.
      ;
      • Charnock J.S.
      • Opit L.J.
      • Hetzel B.S.
      An evaluation of the effect of salicylate on oxidative phosphorylation in rat-liver mitochondria.
      ).
      A significant increase in total 13C% enrichment of measured metabolites, except for acetyl CoA (tendency), and select isotopologues over time, except for [M+1]acetyl CoA (no effect), confirmed that propionate was extracted and metabolized by the liver explants. Furthermore, we also detected an increase in 13C enrichment in TCA cycle intermediates over time, suggesting that the tissue was not dying nor nutrient supply diminishing over the sampling time.
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      reported an increase in acetyl CoA concentration with a 50 mg/kg of BW intrajugular injection of SAL compared with a control. However, the cows in the
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      study were in a lipolytic state, and thus the acetyl CoA pool was likely primarily from β-oxidation of free fatty acids. Our results supported that the acetyl CoA pool was likely primarily derived from β-oxidation of free fatty acids during the lipolytic state because total 13C% enrichment of acetyl CoA did not differ between SAL and CON, suggesting that propionate is not diverted toward acetyl CoA production with SAL, similar to the DNPME treatment in Exp. 1.
      An increase in total 13C% enrichment of succinate and tendency for a decrease in total 13C% enrichment of fumarate with SAL supported that a time delay occurred within the propionate metabolism pathway. Because succinate and fumarate are the reactant and product, respectively, of the succinate dehydrogenase reaction, salicylate likely negatively affects this enzyme, as has been previously reported (
      • Kaplan E.H.
      • Kennedy J.
      • Davis J.
      Effects of salicylate and other benzoates on oxidative enzymes of the tricarboxylic acid cycle in rat tissue homogenates.
      ;
      • Miyahara J.T.
      • Karler R.
      Effect of salicylate on oxidative phosphorylation and respiration of mitochondrial fragments.
      ). The decrease in total 13C% enrichment of malate at 15 min was likely a direct result of the decrease in total 13C% enrichment of fumarate at 15 min. Consequently, the decrease in total 13C% enrichment of malate may also have caused the decrease and tendency for a decrease in the 13C% enrichment of [M+3]citrate and [M+4]citrate, respectively, detected with SAL compared with CON2. Although no difference in succinate concentration was noted with salicylate treatment by
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      , salicylate has a short half-life (50 min in cattle;
      • Short C.R.
      • Neff-Davis C.A.
      • Hsieh L.C.
      • Koritz G.D.
      • Malbrough M.S.
      • Barker S.A.
      • Davis L.E.
      Pharmacokinetics and elimination of salicylic acid in rabbits.
      ), and sampling occurred 4 h posttreatment; however, this study focused on metabolism within an hour. Although other enzymatic reactions within the TCA cycle, such as the succinyl CoA to succinate reaction, may be influenced by salicylate and contribute to observed labeling patterns, a kinetic model by
      • Mogilevskaya E.
      • Demin O.
      • Goryanin I.
      Kinetic model of mitochondrial Krebs cycle: Unraveling the mechanism of salicylate hepatotoxic effects.
      that examined the effects of salicylate on energy metabolism in hepatic mitochondria suggests that inhibition of succinate dehydrogenase and α-ketoglutarate dehydrogenase by salicylate are the principal contributors to disrupting oxidation in the TCA cycle. Our results support that succinate dehydrogenase may be impaired by salicylate in bovine liver as well.
      Although we detected no differences in total 13C% enrichment of glucose, a decrease in [M+2]glucose and [M+3]glucose enrichment with SAL may suggest that synthesis of glucose from propionate is impaired by salicylate. However, our results are limited in scope as it is likely that the majority of synthesized glucose is exported from the explant into the media, which was not measured in this experiment because of dilution from the [12C]glucose present in the media. Yet, impairment of gluconeogenesis by salicylate through an unknown mechanism is consistent with results reported by
      • Farney J.K.
      • Mamedova L.K.
      • Coetzee J.F.
      • KuKanich B.
      • Sordillo L.M.
      • Stoakes S.K.
      • Minton J.E.
      • Hollis L.C.
      • Bradford B.J.
      Anti-inflammatory salicylate treatment alters the metabolic adaptations to lactation in dairy cattle.
      , who reported a decrease in plasma glucose concentration at 7 d postpartum with oral administration of salicylate in drinking water to dairy cows for the first 7 DIM.
      • Bertoni G.
      • Trevisi E.
      • Piccioli-Cappelli F.
      Effects of acetyl-salicylate used in post-calving of dairy cows.
      also reported a decrease in plasma glucose concentration over the first week of lactation when a daily intramuscular injection of lysine acetylsalicylate was administered to dairy cows in the first 5 DIM. However, a single pulse dose of salicylate had no effect on plasma glucose concentration in
      • Kennedy K.M.
      • Allen M.S.
      The effect of uncouplers of oxidative phosphorylation on the feeding behavior of lactating dairy cows.
      , possibly because changes in gluconeogenesis were too minute to detect in the general circulation. Recently,
      • Montgomery S.R.
      • Mamedova L.K.
      • Zachut M.
      • Kra G.
      • Häussler S.
      • Vaughn M.
      • Gonzalez J.
      • Bradford B.J.
      Effects of sodium salicylate on glucose kinetics and insulin signaling in postpartum dairy cows.
      reported that glucose turnover rate tended to decrease with administration of salicylate in drinking water to dairy cows for the first 7 DIM, but no change in transcription abundance of pyruvate carboxylase or glucose-6-phosphatase in the liver was detected. Further experiments need to be conducted to determine if salicylate negatively affects gluconeogenesis in ruminants and to identify potential biological mechanisms.

      CONCLUSIONS

      The DNPME did not alter metabolism of propionate compared with CON1. The SAL treatment increased total 13C% enrichment of succinate and tended to decrease or decreased total 13C% enrichment of fumarate and malate, respectively, compared with CON2. However, we did not observe an increase in total 13C% enrichment of TCA cycle intermediates, as hypothesized, for either experiment. Further research is needed to determine the effects of salicylate and DNP on OXPHOS in the liver of dairy cows, with particular focus on measuring oxidation and energy charge (ATP, ADP, and AMP). Additional research is also necessary to determine how parity of dairy cows could potentially be affected by uncouplers of OXPHOS. Finally, more research is needed to further examine the effects of salicylate on succinate dehydrogenase and gluconeogenesis, particularly in early postpartum animals when milk production and energy demands increase.

      ACKNOWLEDGMENTS

      This work was supported by Agriculture and Food Research Initiative National Institute for Food and Agriculture (AFRI NIFA) Fellowships Grant Program (2017-67011-26042/1010850) from USDA NIFA (Washington, DC) and Michigan Alliance of Animal Agriculture (M AA18-009; MI). We also thank H. Brunengraber (Case Western Reserve University, Cleveland, OH) and D. G. Main, R. Albornoz, G. Maldini, A. Meade, and R. West (all from Michigan State University, East Lansing, MI), as well as the staff of the Michigan State University Dairy Cattle Field Laboratory (East Lansing) and Michigan State University Mass Spectrometry and Metabolomics Core (East Lansing) for their assistance in this experiment. The authors have not stated any conflicts of interest.

      Appendix

      Figure thumbnail gr5
      Figure A1Total 13C% enrichment of hepatic propionyl CoA (A), succinate (B), fumarate (C), malate (D), citrate (E), glutamate (F), pyruvate (G), lactate (H), and glucose (I) from liver explants from dairy cows in the postpartum period for experiment 1. Total 13C% enrichment is the percent increase of 13C within each metabolite above the natural abundance. Explants were incubated in [13C3]propionate with treatments of either 10 μM 2,4-dinitrophenol methyl ether or propylene carbonate (control) and sampled at baseline (no enrichment, 0), 0.5, 15, and 60 min. Time effects are presented at the bottom of each graph with a letter in order of baseline, 0.5, 15, and 60 min, and different letters define significant differences from each other (P ≤ 0.05). Total 13C% enrichment of hepatic succinate and glutamate were transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation purposes. Total 13C% enrichment of hepatic citrate and lactate were transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      Figure thumbnail gr6
      Figure A213C% enrichment of hepatic [M+3]propionyl CoA (A), [M+2]pyruvate (B), [M+2]acetyl CoA (C), [M+3]citrate (D), [M+4]citrate (E), [M+5]citrate (F), [M+2]glutamate (G), [M+3]glutamate (H), [M+2]glucose (I), and [M+3]glucose (J) from liver explants from dairy cows in the postpartum period for experiment 1. The enrichment of each isotopologue is relative to the mass distribution of all the isotopologues in the metabolite (molar percent) and is on a scale of 0 to 100%. Explants were incubated in [13C3]propionate with treatments of either 10 μM 2,4-dinitrophenol methyl ether or propylene carbonate (control) and sampled at 0.5, 15, and 60 min. Time effects are presented at the bottom of each graph with a letter in order of 0.5, 15, and 60 min, and different letters define significant differences from each other (P ≤ 0.05). 13C% enrichment of hepatic [M+2]acetyl CoA was transformed using square root (n + 10) for statistical analysis and back-transformed for interpretation. 13C% enrichment of hepatic [M+3]citrate and [M+4]citrate were transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation. 13C% enrichment of hepatic [M+5]citrate and [M+3]glutamate were transformed using inverse square root (n + 1) for statistical analysis and back-transformed for interpretation. 13C% enrichment of hepatic [M+2]glutamate and [M+2]glucose were transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.
      Figure thumbnail gr7
      Figure A3Total 13C% enrichment of hepatic propionyl CoA (A), citrate (B), glutamate (C), pyruvate (D), lactate (E), glucose (F), and acetyl CoA (G) from liver explants from dairy cows in the postpartum period for experiment 2. Total 13C% enrichment is the percent increase of 13C within each metabolite above the natural abundance. Explants were incubated in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control) and sampled at baseline (no enrichment, 0), 0.5, 15, and 60 min. Time effects are presented at the bottom of each graph with a letter in order of baseline, 0.5, 15, and 60 min and different letters define significant differences from each other (P ≤ 0.05). Total 13C% enrichment of hepatic citrate and glutamate were transformed using inverse square root (n + 1) for statistical analysis and back-transformed for interpretation purposes. Total 13C% enrichment of hepatic pyruvate and lactate were transformed using square root (n + 1) for statistical analysis and back-transformed for interpretation purposes.
      Figure thumbnail gr8
      Figure A413C% enrichment of hepatic [M+3]propionyl CoA (A), [M+2]pyruvate (B), [M+2]acetyl CoA (C), [M+3]citrate (D), [M+4]citrate (E), [M+5]citrate (F), [M+2]glutamate (G), and [M+3]glutamate (H) from liver explants from dairy cows in the postpartum period for experiment 2. The enrichment of each isotopologue is relative to the mass distribution of all the isotopologues in the metabolite (molar percent) and is on a scale of 0 to 100%. Explants were incubated in [13C3]propionate with treatments of either 5 mM sodium salicylate or no treatment (control) and sampled at 0.5, 15, and 60 min. Time effects are presented at the bottom of each graph with a letter in order of baseline, 0.5, 15, and 60 min, and different letters define significant differences from each other (P ≤ 0.05). 13C% enrichment of hepatic [M+4]citrate and [M+2]glutamate were transformed using natural log (n + 1) for statistical analysis and back-transformed for interpretation. 13C% enrichment of hepatic [M+3]glutamate was transformed using inverse (n + 1) for statistical analysis and back-transformed for interpretation.

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