and J. H. Clark T. R. Overton, J. K. Drackley, C. J. Ottemann-Abbamonte, A. D. Beaulieu, L. S. Emmert demand in ruminants Substrate utilization for hepatic gluconeogenesis is altered by increased glucose 1999, 77:1940-1951. J ANIM SCI http://jas.fass.org/content/77/7/1940 the World Wide Web at: The online version of this article, along with updated information and services, is located on www.asas.org by guest on July 14, 2011 jas.fass.org Downloaded from
Transcript
and J. H. ClarkT. R. Overton, J. K. Drackley, C. J. Ottemann-Abbamonte, A. D. Beaulieu, L. S. Emmert
demand in ruminantsSubstrate utilization for hepatic gluconeogenesis is altered by increased glucose
1999, 77:1940-1951.J ANIM SCI
http://jas.fass.org/content/77/7/1940the World Wide Web at:
The online version of this article, along with updated information and services, is located on
www.asas.org
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1Supported in part by the Univ. of Illinois Agric. Exp. Sta.(projects 35-0352 and 35-342).
2Current address: Dept. of Anim. Sci., Cornell Univ., Ithaca, NY14853.
3To whom correspondence should be addressed: 260 AnimalSciences Laboratory, 1207 W. Gregory Drive (phone (217)244-3157; fax: (217) 333-7088; E-mail: [email protected]).
Received October 27, 1998.Accepted March 18, 1999.
Substrate Utilization for Hepatic Gluconeogenesis Is Altered byIncreased Glucose Demand in Ruminants1
Thomas R. Overton2, James K. Drackley3, Cynthia J. Ottemann-Abbamonte,A. Denise Beaulieu, Laurel S. Emmert, and Jimmy H. Clark
Department of Animal Sciences, University of Illinois, Urbana 61801
ABSTRACT: Hepatocytes isolated from 10 Dorsetwethers that were treated with excipient or 1.0 g/d ofphlorizin for 72 h were used to determine the effects ofincreased glucose demand on utilization of [1-14C]propionate and [1-14C]alanine for oxidativemetabolism and gluconeogenesis. Control andphlorizin-treated wethers excreted 0 and 62.8 g/d ofglucose into the urine, respectively. Phlorizin treat-ment tended to increase conversion of propionate andalanine to CO2. A phlorizin × substrate interaction forconversion to glucose indicated that conversion ofalanine to glucose was increased more by phlorizintreatment than was conversion of propionate (285 vs166% of controls). Phlorizin treatment did not affectestimated Ks for conversion of substrates to eitherCO2 or glucose; however, phlorizin increased esti-mated Vmax for conversion of substrates to CO2 andtended to increase estimated Vmax for conversion ofsubstrates to glucose. Phlorizin treatment slightly
increased the ratio of conversion of propionate toglucose compared with CO2 and slightly decreased theratio of conversion of alanine to glucose compared withCO2. In vitro addition of 2.5 mM NH4Cl decreasedconversion of propionate to CO2 and glucose but hadlittle effect on conversion of alanine to CO2 andglucose. Estimated Ks and Vmax for conversion ofsubstrates to CO2, Ks for conversion of substrates toglucose, and Vmax for conversion of alanine to glucosewere not affected by NH4Cl; however, Vmax forconversion of propionate to glucose was decreased byNH4Cl. These data indicate that although utilizationof propionate for gluconeogenesis is extensive, aminoacids have the potential to increase in importance asgluconeogenic substrates when glucose demand isincreased substantially. Furthermore, excess ammo-nia decreases the capacity of hepatocytes to utilizepropionate for oxidation and gluconeogenesis.
1999 American Society of Animal Science. All rights reserved. J. Anim. Sci. 1999. 77:1940–1951
Introduction
Glucose metabolism in ruminants is characterizedby a pronounced ability to adapt to changinghomeorhetic state (Bauman and Currie, 1980). Mam-mary demand for glucose increases dramaticallyfollowing parturition (Bickerstaffe et al., 1974). Thisdemand is met in part by increased glucose entry rate(Bergman and Hogue, 1967; Bennink et al., 1972;Wilson et al., 1983) and decreased whole-body oxida-tion of glucose (Bennink et al., 1972; Wilson et al.,1983).
Ruminants depend on hepatic gluconeogenesis tomeet much of their metabolic demand for glucose(Reynolds et al., 1994). However, adaptations inutilization of substrates for hepatic gluconeogenesis inresponse to increased glucose demand have not beenwell characterized. Nutrient intake lags nutrientdemand during early lactation, especially in dairycows (Bell, 1995); therefore, ruminal supply ofpropionate, the primary substrate for gluconeogenesis,may not be sufficient.
Overton et al. (1998) determined that wethersinjected with phlorizin excreted approximately 50% oftheir probable glucose entry rate into the urine andhad increased concentrations of urea N in plasma,which suggested increased catabolism of amino acids.We concluded that this model system should besuitable for further investigation of adaptations ofhepatic gluconeogenesis to sudden increases of glucosedemand, such as occur upon initiation of lactation.Therefore, our primary objective was to determinewhether adaptations occur in metabolism of two majorgluconeogenic substrates (propionate and alanine) by
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hepatocytes isolated from control and phlorizin-treated wethers. Intraruminal infusions of ureadecreased glucose production rates in steers (Spiresand Clark, 1979). Furthermore, catabolism of aminoacids results in production of ammonia, which isdetoxified to urea. Accordingly, our second objectivewas to determine the impact of ammonia on utiliza-tion of substrates for gluconeogenesis by hepatocytes.
Materials and Methods
Reagents
The sodium salt of [1-14C]propionate, [1-3H]L-glucose, and [1-14C]L-alanine were obtainedfrom American Radiolabeled Chemicals (St. Louis,MO). All chemicals were cell-culture-tested or of thehighest purity available from Sigma Chemical Co. (St.Louis, MO). The perfusion and wash media were thesame as those used by Donkin and Armentano (1993),with the exception that the BSA (bovine albuminCohn fraction-V, charcoal treated, dialyzed) was notdialyzed again before use. The incubation medium(pH 7.4) was Krebs-Ringer bicarbonate ( KRB) buffer(Laser, 1961) containing 5 mg/L phenol red and 2.0mM CaCl2·H2O. Part of the NaCl (25 mM) wasreplaced with sodium HEPES (6.5 g/L). Before use,the perfusion media was filter-sterilized through asterile .22-mm Millex-GV filter (Millipore Corp., Bed-ford, MA).
Animals, Experimental Design, and Management
All procedures involving animals were approved bythe University of Illinois Laboratory Animal CareAdvisory Committee. Ten Dorset wethers averaging34.2 ± 6.6 kg of BW were housed individually instainless steel metabolism cages in a room maintainedat 25°C. Wethers were assigned randomly to twogroups of five lambs each that were injected eitherwith an excipient (7.5 mL/d of propylene glycol) orphlorizin (Sigma; 1.0 g/d dissolved in 7.5 mL/d ofpropylene glycol). This phlorizin dose was determinedto cause near-maximal responses in glucosemetabolism in wethers during a previous experiment(Overton et al., 1998). Wethers were fed a pelleteddiet at 2.9% (DM basis) of BW; the diet had the sameingredient composition (Table 1) as that fed duringour previous experiment (Overton et al., 1998). Asample of the pelleted diet was dried at 55°C for 72 h,ground through a 1-mm screen in a Wiley mill(Arthur H. Thomas, Philadelphia, PA), and analyzedfor DM (105°C for 24 h), CP (AOAC, 1990), ADF,and NDF (Van Soest et al., 1991). Wethers wereallowed 7 d for adaptation to metabolism cages andthe experimental diet before beginning the treatmentperiod.
Each treatment period consisted of 72 h duringwhich wethers were injected subcutaneously in the
scapular region at 0800, 1600, and 2400 with one-third of the appropriate daily treatment dose. Urinaryexcretion of glucose was determined by total collectionof urine during each treatment period. Plastic contain-ers used to collect urine contained sufficient amountsof concentrated HCl to maintain urine at a pH < 2 topreserve the glucose. Containers were replaced at8-h intervals, urine volume was measured, andapproximately 100 mL was frozen at −20°C untilanalysis. Urine was thawed and analyzed for glucoseconcentration using the glucose oxidase method (kit315, Sigma).
Isolation of Hepatocytes
At 72 h of treatment, hepatocytes were isolatedfrom the liver of each wether using the procedures ofDonkin and Armentano (1993) and Cremin et al.(1995) with minor modifications. Wethers were givenheparin (30,000 U; Steris Laboratories, Phoenix, AZ)intrajugularly, and anesthesia was induced by in-trajugular injection of sodium pentobarbital (Nembu-tal, Abbott Laboratories, Chicago, IL; 30 mg/kg ofBW). An incision was made in the right side of theabdominal wall and the liver was isolated manually.The caudate process was excised and the animal thenwas euthanatized immediately by intrajugular injec-tion of .22 mL/kg of concentrated sodium pentobarbital(Sleepaway II; Ft. Dodge Laboratories, Ft. Dodge,IA). Immediately after excision, the caudate processwas flushed with 120 mL of perfusion mediumsaturated with 95% O2:5% CO2 and supplementedwith 4,000 U of sodium heparin. The perfusionmedium was maintained at 39°C and was deliveredvia two 60-mL catheter-tip syringes inserted into oneof the two major exposed blood vessels. The outflowfrom this wash was discarded. The caudate processwas transferred to a chamber at 39°C and connected to
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a perfusion apparatus. The caudate process wasflushed with another 150 mL of perfusion medium,and the outflow was discarded. The caudate processwas gravity-perfused with 300 mL of perfusionmedium in a recirculating system at a flow rate of 50to 60 mL/min. After 5 min of recirculating perfusion,collagenase (Type IV, Sigma) was added to theperfusion medium (.5 mg/mL). The perfusion wascontinued for another 5 min, at which time CaCl2 wasadded to a final concentration of .5 mM to activatecollagenase. The perfusion was continued for approxi-mately 10 min until the caudate process lost defini-tion. The caudate process was removed from theapparatus and immersed in 100 mL of the previouslyrecirculated perfusion medium containing deox-yribonuclease I (30,000 U; Sigma). Blunt scissorswere used to gently tease cells away from bloodvessels and Gilman’s capsule and into the medium.The tissue suspension was filtered through nylonmesh (250 mm pore size) and poured into125-mL flasks, which were gassed continuously with95% O2:5% CO2 and shaken for 15 min in anoscillating water bath at 39°C. The suspension thenwas filtered again through nylon mesh (250 mm poresize) into a beaker on ice, which contained 150 mL ofKRB wash buffer, 3% BSA (wt/vol), and 45.6 mg ofEGTA (ethylene glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, tetrasodium salt). Thesupernatant was discarded, and the cells wereresuspended and washed two times in wash mediumcontaining 1% BSA (wt/vol) and then washed once inKRB incubation medium at 4°C. The liver cellsuspension was prepared in KRB incubation mediumat a packed cell volume of approximately 2%, gassedwith 95% O2:5% CO2, stirred slowly in a beaker on ice,and used immediately. Throughout the cell isolationprocedure, media were gassed continuously with 95%O2:5% CO2 to prevent cell hypoxia. The suspensionwas examined for cellular debris using lightmicroscopy. Viability of cells was estimated usingtrypan blue staining after initiation of incubations andwas found to be greater than 80% for all isolations.
Incubations
Hepatocytes were used to determine the oxidationand conversion to glucose of increasing concentrationsof propionate and alanine. Concentrations of propi-onate and alanine were .625, 1.25, 2.5, 5.0, and 10.0mM. Approximately 1 mCi/flask of [1-14C]Na-propi-onate or [1-14C]L-alanine was added to the appropri-ate medium and 2.4 mL of the appropriate KRBincubation medium was added to duplicate flasks foreach treatment. The actual amount of radioactivityadded to each flask was determined by measuring analiquot (100 mL) of each medium by liquid scintilla-tion spectroscopy (Model LS6000IC; Beckman Instru-ments, Fullerton, CA) following addition of 10 mL ofscintillation cocktail (ScintiSafe Econo 2; Fisher
Scientific, Pittsburgh, PA). To assess the effects ofaddition of 2.5 mM NH4Cl on metabolism of propi-onate and alanine by hepatocytes, 100 mL of eitherKRB or KRB plus NH4Cl was added to duplicateflasks for each substrate and concentration. To assessthe effects of addition of propionate on metabolism ofalanine and addition of alanine on metabolism ofpropionate by hepatocytes, 100 mL of KRB containingsufficient alanine or propionate to result in a finalflask concentration of 10 mM for each substrate wasadded to duplicate flasks.
Aliquots (.5 mL) of the hepatocyte suspension werepipetted into 25-mL Erlenmeyer flasks containing 2.5mL of KRB incubation medium; flasks were capped,gassed for 20 s with 95% O2:5% CO2, and placed in anoscillating water bath at 39°C for 90 min. Four blankflasks were prepared for each radioisotope in a similarmanner, except they were terminated at zero time andplaced into a shaking ice-water bath for 1 h. Incuba-tions were terminated by addition of .5 mL of a 1.5 NH2SO4 solution to the media. Carbon dioxide wastrapped by addition of .1 mL of 30% NaOH (wt/vol) toa filter paper in a glass well suspended in each flaskand subsequent incubation of the flask in a shakingice-water bath for 1 h. The CO2 well was removedfrom the flask, and the filter paper was transferred toa scintillation vial. The well was rinsed twice withdeionized distilled water, and the washes were addedto the scintillation vial, which then was dried over-night under moving air. Radioactivity in CO2 wasmeasured by liquid scintillation spectroscopy asdescribed above.
Following removal of the CO2 well, the media wereprocessed as follows. An internal standard (.11 mCi of[1-3H]glucose) was added to each flask. An equivalentamount (100 mL) of the [1-3H]glucose stock solutionwas pipetted into a scintillation vial at the time ofaddition to the incubation flasks, and radioactivitywas measured as described below to determine theactual amount of 3H added to each flask. Flaskcontents were transferred into polypropylene testtubes containing 50 mL of pH indicator (UniversalIndicator; Fisher Scientific, Pittsburgh, PA). Mediawere neutralized and deproteinized by additions of asaturated solution of Ba(OH)2 in H2O. Tubes werecentrifuged at 700 × g to remove the precipitate; thesupernatant was poured into scintillation vials andfrozen at −20°C until glucose was isolated.
Assays
Glucose was isolated by ion-exchange chromatogra-phy using the three-column procedure of Mills et al.(1981) with [1-3H]glucose as the internal standard.Radioactivity in glucose was measured by dual-labelliquid scintillation spectroscopy (Model LS6000IC;Beckman Instruments). The DM content of triplicate1-mL aliquots of hepatocyte suspension and incuba-tion media was determined by drying the aliquots
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Figure 1. Least squares means for cumulative excre-tion of glucose in urine by wethers injected withexcipient (ÿ) or 1.0 (⁄) g/d of phlorizin (SEM = 4.5).
overnight in a forced-air oven at 70°C. The DM in theincubation media was subtracted from the DM in thehepatocyte suspension to calculate the amount ofhepatocyte DM added to each flask. Rates of conver-sion of radiolabeled propionate and alanine to glucoseand CO2 were expressed as micromoles converted perhour per gram of hepatocyte DM.
Statistical Analysis
Quadruplicate values for blanks and duplicatevalues for flasks containing live suspensions wereaveraged before statistical analysis. After correctionfor blanks, rates of conversion of substrates to CO2and glucose were subjected to ANOVA using the GLMprocedure of SAS (1992). The model contained theeffects of treatment; wether within treatment (used asthe error term to test the effect of treatment in vivo);the in vitro effects of substrate, concentration ofsubstrate, and addition of NH4Cl to the incubationmedia; and interactions of treatment with in vitroeffects. In vitro effects, interactions among in vitroeffects, and interactions of in vitro effects withtreatment were tested using the residual error. Thelowest P-value for three- and four-way interactionstested was .42; therefore, these interactions are notdiscussed.
Estimates of Ks and Vmax for conversion of [1-14C]propionate and [1-14C]alanine to CO2 and glucosewere derived by nonlinear regression analysis usingthe Gauss-Newton method within PROC NLIN (SAS,1992) applied to the Michaelis-Menten equation. Allmodels converged. The derived values for Ks and Vmaxwere subjected to statistical analysis using the modeldescribed above, except that the effects of substrateconcentration and its interactions with other effectswere not included. Cumulative excretion of glucoseinto the urine was analyzed using the GLM procedureof SAS (1992). Terms included in the model weretreatment, hour of treatment, and the treatment ×hour of treatment interaction; wether within treat-ment was used to test the effect of treatment.Additions of alanine and propionate (10 mM) to flaskscontaining 10 mM [1-14C]propionate and [1-14C]alanine, respectively, were analyzed using amodel containing the effects of treatment, wetherwithin treatment (i.e., whole-plot error), substrate,addition of second substrate, and two-way and three-way interactions of treatment, substrate, and additionof second substrate. Incomplete data were availablefor assessment of addition of a second substrate (n = 2for control wethers and n = 4 for phlorizin-treatedwethers). Probability values of ≤ .05 indicate asignificant difference and those between .05 and .15are discussed as trends toward a significant difference.
Results
Urinary excretion of glucose by wethers injectedwith 0 or 1.0 g/d of phlorizin averaged 0 and 62.8 g/d.
Wethers injected with 1.0 g/d of phlorizin excreted atotal of 188.4 g of glucose during the 72-h treatmentperiod (Figure 1).
Conversion of propionate and alanine to CO2 inisolated hepatocytes tended ( P < .12) to be increasedby phlorizin injection and rates of conversion to CO2were greater ( P < .001) for propionate than foralanine (Figure 2A). Addition of 2.5 mM NH4Cl to theincubation media resulted in a substrate × NH4Clinteraction ( P < .001), because NH4Cl additiondecreased rates of conversion of propionate to CO2 butdid not affect conversion of alanine to CO2 (Figure2B).
Injecting wethers with phlorizin did not affect theestimated substrate concentration for half-maximalrates of conversion of substrates to CO2 (Ks) inhepatocytes isolated from these wethers; however,phlorizin injection increased ( P < .02) the estimatedmaximal rates (Vmax) of conversion of both propi-onate and alanine to CO2 (Table 2). The Ks forconversion of alanine to CO2 was greater ( P < .001)than that for propionate. A treatment × substrateinteraction ( P < .05) indicated that phlorizin injectionincreased the Ks for conversion of alanine but did notaffect Ks of propionate. The Vmax for conversion ofpropionate to CO2 tended ( P < .14) to be greater thanthat for alanine. A trend ( P < .11) for a treatment ×substrate interaction was detected because an in-crease in the Vmax for conversion of alanine to CO2was responsible for most of the overall increase inVmax for conversion of substrates to CO2 whenphlorizin was given. In vitro addition of 2.5 mMNH4Cl did not affect Ks or Vmax for conversion ofsubstrates to CO2.
Rates of conversion of propionate to glucose weregreater ( P < .001) than those of alanine (Figure 3A);however, a treatment × substrate interaction ( P < .05)was present because phlorizin injection slightly in-creased rates of conversion of propionate to glucose
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Figure 2. Least squares means and standard errors formain effects of conversion of substrates to CO2. (A)Conversion of [1-14C]propionate and [1-14C]alanine toCO2 in control and phlorizin-treated wethers. (B)Conversion of [1-14C]propionate and [1-14C]alanine toCO2 as affected by addition of NH4Cl.
Figure 3. Least squares means and standard errors formain effects of conversion of substrates to glucose. (A)Conversion of [1-14C]propionate and [1-14C]alanine toglucose in control and phlorizin-treated wethers. (B)Conversion of [1-14C]propionate and [1-14C]alanine toglucose as affected by addition of NH4Cl.
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Figure 4. Least squares means and standard errors formain effects of the ratio of conversion of substrates toglucose and CO2. (A) Ratio of conversion of [1-14C]propionate and [1-14C]alanine to glucose and CO2 incontrol and phlorizin-treated wethers. (B) Ratio ofconversion of [1-14C]propionate and [1-14C]alanine toglucose as affected by addition of NH4Cl. (C) Ratio ofconversion of radiolabeled substrates to glucose andCO2 in control and phlorizin-treated wethers as affectedby addition of NH4Cl.
but more than doubled rates of conversion of alanineto glucose. A substrate × NH4Cl interaction ( P < .001)occurred because rates of conversion of propionate toglucose were substantially decreased by in vitroaddition of 2.5 mM NH4Cl, but rates of conversion ofalanine were decreased only slightly (Figure 3B).This interaction was similar to that existing forconversion of substrates to CO2 (Figure 2B).
Similar to results for CO2, injecting wethers withphlorizin did not affect estimated Ks for conversion ofsubstrates to glucose by hepatocytes isolated fromthese wethers (Table 3); however, the estimated Vmaxtended ( P < .09) to be increased by phlorizin injection.The Ks for conversion of alanine to glucose was greater( P < .001) than that for propionate, but Vmax forconversion of alanine to glucose was lower than thatfor propionate. The Ks for conversion of substrates toglucose was not affected by NH4Cl addition in vitro.However, a substrate × NH4Cl interaction ( P < .02)was present for estimated Vmax, because addition ofNH4Cl to the incubation media substantiallydecreased the estimated Vmax for conversion of propi-onate to glucose but only slightly decreased Vmax forconversion of alanine to glucose.
Figures 4A, 4B, and 4C show the ratio of theconversions of radiolabel from substrates to glucoseand CO2 as affected by treatment, substrate, andaddition of NH4Cl to the incubation media. These dataserve merely as an index of partitioning of radiola-beled substrates. A treatment × substrate interactionexisted ( P < .001), because injection of wethers withphlorizin slightly increased the ratio of conversion ofradiolabel from propionate to glucose compared withCO2 and slightly decreased the ratio of conversion ofradiolabel from alanine to glucose (Figure 4A).Addition of NH4Cl in vitro decreased ( P < .001) theratio of conversion of radiolabel to glucose comparedwith CO2 (Figure 4B). A treatment × NH4Cl interac-tion ( P < .006) indicated that hepatocytes isolatedfrom wethers injected with phlorizin had greaterratios of conversion of radiolabeled substrate toglucose compared with CO2 when NH4Cl was added tothe incubation media and had decreased ratios ofconversion of radiolabeled substrate to glucose com-pared with CO2 when NH4Cl was not added to theincubation media, compared with control wethers(Figure 4C).
Limited data were available to evaluate the effectsof addition of a second substrate on metabolism of theradiolabeled substrate as affected by phlorizin treat-ment (Table 4). The three-way interactions amongtreatment, substrate, and addition of a second sub-strate for conversion to CO2 and glucose had P-valuesof .16 and .18, respectively. Although these interac-tions were not significant, interpretation of maineffects is difficult. Addition of a second substrate hadlittle effect on the ratio of conversion of substrates toglucose compared with CO2.
Discussion
Our overall hypothesis was that the liver increasesutilization of amino acids for gluconeogenesis inresponse to metabolic situations in which whole-bodyglucose demand is increased suddenly. Such informa-tion would be valuable for determining how ruminantssuch as dairy cows adapt hepatic gluconeogenesis tothe markedly increased glucose demand upon initia-tion of lactation. In a previous experiment, wecharacterized metabolism of phlorizin-treated wethersand determined that injection of 1.0 g/d of phlorizinresulted in near-maximal response of urinary glucoseexcretion to phlorizin administration (Overton et al.,1998). This model results in a situation in whichapproximately 50% or more of estimated basal glucoseentry rate is excreted suddenly into the urine (Over-ton et al., 1998). We sought to use such a model to
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Table 4. Least squares means and standard errors for effect of addition of a second substrate on conversionof [1-14C]propionate and [1-14C]alanine to CO2 and glucose and the ratio of conversion of substrates to
glucose and CO2 by hepatocytes isolated from control- and phlorizin-treated wethers
elucidate the importance of amino acids for gluconeo-genesis in ruminants subjected to suddenly increasedglucose demand before attempting to examine thisbiology in periparturient dairy cows.
The amount of glucose excreted into the urine when1.0 g/d of phlorizin was administered was less (62.8vs. 97.9 g/d) in this experiment than in our previousexperiment (Overton et al., 1998). Wethers in thisexperiment averaged 34.2 kg of BW, whereas theaverage BW of wethers over the course of our previous
experiment was 48.4 kg. This difference in BW ofwethers between experiments, together with theproportionately decreased amount of feed consumed bywethers in this experiment, probably accounts formuch of the difference in urinary glucose excretionbetween experiments (1.84 vs 2.02 g/kg of BW,respectively). Glucose metabolism should have beenperturbed to a similar extent by injection of 1.0 g/d ofphlorizin in this experiment, as in our previousexperiment.
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Hepatocytes isolated from phlorizin-treated wethersin this experiment tended to have increased rates ofconversion of both propionate and alanine to CO2.Furthermore, a treatment × substrate interaction forconversion of these two substrates to glucose occurred(Figure 3A); conversion of propionate to glucose wasincreased slightly (166% of controls; 23.2 vs 14.0mmol/[h·g cell DM]) by phlorizin injection, but that ofalanine was more than doubled (285% of controls; 6.9vs 2.4 mmol/[h·g cell DM]). Maximal rates of conver-sion of propionate to CO2 and glucose were greaterthan those of alanine regardless of phlorizin treat-ment; however, alanine clearly increased in impor-tance as a gluconeogenic substrate when glucosedemand was increased suddenly.
Rates of conversion of [1-14C]propionate to glucoseand CO2 in our experiment were approximately 40 to50% of those reported by Faulkner and Pollock(1986); however, rates of conversion of [1-14C]alanineto glucose and CO2 were approximately 25 timesgreater than those reported by Mutsvangwa et al.(1996, 1997). Rates of conversion of [2-14C]propionateto glucose and CO2 reported by Faulkner and Pollock(1986) were slightly greater than those reported byother investigators (Aiello and Armentano, 1987;Cremin et al., 1995); rates reported by Faulkner andPollock (1986) for conversion of [2-14C]propionate toglucose were twice those noted by Aiello and Armen-tano (1988) and 10 times greater than those reportedby Armentano et al. (1991). All investigators reportedhigh (> 80%) viability of cells as measured byexclusion of trypan blue dye. Therefore, the substan-tial variation in rates among experiments and labora-tories probably is attributable to differences amonglaboratories and personnel, timing of removal of thecaudate process relative to initiation of the perfusionand hepatocyte isolation procedure (i.e., excision ofthe lobe under anesthesia before euthanasia comparedwith excision of the lobe following stunning andexsanguination in an abbattoir), differences in theexperimental animals used (species, sex, and age)and diets fed, and differences in the composition ofincubation media used and procedures used in differ-ent laboratories for incubating hepatocytes. Thesedifferences suggest that it is more appropriate tocompare rates of conversion of substrates to productsas affected by treatments within a single experimentthan to compare actual rates among experiments.
Differences in metabolism of propionate acrossphysiological states in ruminants have been reasona-bly well characterized. Wiltrout and Satter (1972)determined that propionate accounted for a minimumof 32 and 45% of the glucose entry rate in nonlactatingand early lactation dairy cows, respectively, and amaximum of 61% of the glucose entry rate in earlylactation cows after correction for crossover of labeledcarbon from [2-14C]propionate. Mean daily glucoseentry rates for nonlactating and lactating cows were
7.6 and 14.0 mol/d and were in proportion to increasedfeed intake measured during the lactating period(Wiltrout and Satter, 1972). Emmison et al. (1991)determined that the rate of conversion of [2-14C]propionate to glucose was doubled in hepatocytesisolated from lactating ewes compared with nonlactat-ing, nonpregnant ewes. Similarly, hepatocytes isolatedfrom lactating does and incubated in the absence ofCa+2 had increased rates of conversion of [2-14C]propionate to glucose compared with those fromwethers, but differences were smaller at 2.0 mM Ca+2
(Aiello and Armentano, 1987) as used in our experi-ment. Conversion of [1-14C]propionate to CO2 andglucose in liver slices biopsied from dairy cows wasgreater at d 30 than at d 60, 90, or 180 of lactation(Aiello et al., 1984).
We are aware of only one experiment that assessedthe conversion of several substrates to glucose by liverduring the physiological state targeted by this experi-ment. Mills et al. (1986) measured conversion of [2-14C]propionate, [U-14C]lactate, [U-14C]alanine, [U-14C]aspartate, and [U-14C]glutamate to glucose andCO2 by liver slices from dairy cows during the last 2wk prepartum, 2 wk postpartum, and then duringinduction of and recovery from an experimentalketosis protocol. Although differences were not signifi-cant, rates of conversion of propionate to glucose weredecreased by more than 50% at 2 wk postpartumcompared with rates prepartum. Rates of conversion oflactate, aspartate, and glutamate to glucose weresimilar between prepartal and postpartal stages;however, conversion of alanine to glucose was slightlyincreased. Conversion of all four substrates to CO2was similar between prepartal and postpartal stages.The researchers used only four cows in that experi-ment, which made it difficult to obtain statisticallysignificant differences between physiological stages.
To our knowledge, only one experiment has exa-mined gluconeogenesis in sheep injected withphlorizin. Egan et al. (1983) infused either .23 or .43g/d of phlorizin into the jugular vein of wethers anddetermined that glucose irreversible loss rate in-creased from an average of 63.1 to 80.4 g/d and from77.2 to 100.6 g/d, respectively. Wethers administered.23 and .43 g/d of phlorizin excreted an average of 24.8and 27.6 g/d of glucose into their urine. Egan et al.(1983) also investigated the conversion of carbon fromthreonine to glucose as an index of the contribution ofamino acid carbon to glucose. The transfer quotient forconversion of threonine carbon into glucose was notincreased appreciably by phlorizin treatment andirreversible loss of threonine was not affected bytreatment. Therefore, the flux of carbon from threo-nine to glucose was not increased significantly, butthese researchers employed a dose of phlorizin sub-stantially less than that demonstrated to cause near-maximal excretion of glucose into the urine of wethers(Overton et al., 1998). Furthermore, the carbon
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skeleton of threonine may enter the tricarboxylic acidcycle for gluconeogenesis either at pyruvate or suc-cinyl-CoA. Carbon from threonine also forms acetyl-CoA, which results in no net synthesis of glucose.
We chose [1-14C]alanine to provide an index ofcarbon flux from amino acids to glucose in partbecause it is converted directly to pyruvate aftertransamination. In addition, increased flux of [1-14C]alanine to glucose requires increased flux throughpyruvate carboxylase because the radiolabel will bedecarboxylated and released as 14CO2 if [1-14C]alanineis metabolized through pyruvate dehydrogenase. Wealso chose alanine because carbon from catabolism ofcysteine, glycine, serine, threonine, and tryptophanforms pyruvate, and flux from pyruvate to glucoseentails a substantially different pathway to oxaloace-tate compared with that for propionate. Although theydid not examine gluconeogenesis directly, Holteniusand Holtenius (1997) determined that peroral ad-ministration of alanine to lactating ewes treated withphlorizin slightly increased concentrations of glucosein plasma. Similar to Egan et al. (1983), Holteniusand Holtenius (1997) used a smaller dose ( ∼ .35 g/d)of phlorizin and estimated that urinary glucose outputwas only 20 to 30 g/d, approximately 15% of estimatedglucose entry, which is much lower than we achievedin our experiment.
The lack of a larger increase in conversion ofpropionate to glucose in this experiment is not entirelysurprising given that intake of the pelleted diet, and,therefore, propionate supply was constant through thetreatment period. Data from several experimentssuggest that propionate supply and the capacity of theliver to convert propionate to glucose may be related.Armentano et al. (1991) found that hepatocytesisolated from lactating goats after 4 d of feeddeprivation, except for 233 g/d of ovalbumin ad-ministered by stomach tube, had decreased rates ofconversion of propionate to glucose compared withthose from lactating goats in positive energy balance.Armentano et al. (1991) attributed this decrease inapparent gluconeogenic capacity to increased concen-trations of lipid in liver from goats in negative energybalance, although propionate supply probably also wasdecreased. Lomax et al. (1986) determined thatstarvation of sheep for 4 d decreased synthesis ofglucose from propionate by approximately 37%.
In addition to the likelihood of a constant supply ofpropionate between control and phlorizin-treatedwethers in our experiment, intake and absorption ofamino acids from the intestine probably also wasconstant through the treatment period. Therefore,increased fractional conversion of alanine to glucosecompared with that of propionate caused by phlorizininjection indicates that phlorizin-treated wethers hadincreased capacity to utilize catabolized amino acids tomeet the increased demand for glucose. These data areconsistent with the chronically increased concentra-
tions of urea N in plasma of wethers injected withincreasing doses of phlorizin measured previously(Overton et al., 1998). In support of this apparentcapacity of liver to increase synthesis of glucose fromamino acids to meet increased metabolic demands forglucose, Nolan and Leng (1970) reported (based onurea entry rates) that the possible amount of glucosesynthesized from amino acid carbon was similar forewes fed on a high plane of nutrition regardless ofpregnancy status (e.g., 0, 1, or 2 fetuses); however,the possible amount of glucose synthesized from aminoacid carbon in nonpregnant ewes was decreased by45% after 4 d of undernutrition. Increasing thenumber of fetuses supported by undernourished ewesincreased the urea entry rate and accordingly theamount of glucose that could have been synthesizedfrom amino acid carbon. Heitmann and Bergman(1980) reported that, compared with fed sheep, liverof sheep deprived of feed for 3 d removed greateramounts of alanine from the portal circulation.
The mechanism underlying the increased capacityof hepatocytes to convert alanine to glucose duringincreased glucose demand in our experiment isuncertain, but several possibilities exist. Smith andWalsh (1982) determined that activity of pyruvatecarboxylase was increased five- to sixfold in sheepduring late gestation and early lactation comparedwith nonpregnant control sheep. Physiological statehad little effect on activity of phosphoenolpyruvatecarboxykinase in their experiment, and activity ofpyruvate kinase was unaffected by lactation anddecreased during late gestation. Mesbah and Baldwin(1983) reported that lactating dairy cows had higherhepatic phosphoenolpyruvate carboxykinase activitiesthan did nonlactating or nonlactating, pregnant cows.Recently, Greenfield et al. (1998) reported thathepatic expression of phosphoenolpyruvate carbox-ykinase was not different at 1 d postpartum in dairycows compared with 14 and 28 d prepartum, but thatpyruvate carboxylase expression was significantlyhigher at 1 d postpartum compared with 14 and 28 dprepartum. These data suggest that potential changesin phosphoenolpyruvate carboxykinase activity withsuddenly increased glucose demand are small, butthat activity of pyruvate carboxylase may be modu-lated substantially and may alter the contribution ofsubstrates entering the gluconeogenic pathwaythrough pyruvate to total glucose synthesis duringincreased glucose demand.
During our experiment to establish phlorizin-treated wethers as a model to examine hepaticadaptation to suddenly increased glucose demand(Overton et al., 1998), wethers injected with phlorizinhad decreased ratios of insulin to glucagon in plasmaduring the first 24 h after initiation of the treatmentprotocol. Glucagon concentrations tended to be chroni-cally elevated during the entire 72-h period ofphlorizin treatment. Brockman and Manns (1974)
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determined that sheep administered a low dosage ofglucagon had increased activity of hepatic pyruvatecarboxylase, but not phosphoenolpyruvate carbox-ykinase or glucose-6-phosphatase, suggesting that theproportion of pyruvate that is converted to oxaloace-tate vs acetyl-CoA may be regulated by glucagon.Infusion of glucagon into an in situ perfused liversystem slightly increased conversion of [1-14C]propionate to CO2 and glucose and slightlyincreased conversion of [U-14C]threonine to CO2, but itdid not affect conversion of [U-14C]threonine to glucose(Gill et al., 1985). Similar to the experiment of Eganet al. (1983), differences in conversion of [U-14C]threonine to glucose may have been difficult todetect because of dilution of label by entry at severalsites in the tricarboxylic acid cycle, together with thesubstantial conversion of radiolabel from a uniformlylabeled compound to CO2. Total hepatic glucoseproduction tended to be increased by glucagon infusion(Gill et al., 1985). Similarly, glucagon addition tohepatocyte monolayer cultures increased gluconeogen-esis from propionate in ruminating calves but did notaffect conversion of lactate to glucose (Donkin andArmentano, 1995). Nevertheless, intraportal infusionof glucagon into sheep increased net hepatic uptake ofalanine, glycine, glutamine, arginine, asparagine,threonine, serine, and lactate and doubled concentra-tions of arterial glucose (Wolff et al., 1972), whichsuggests that glucagon potentially can increase thecontribution of amino acids to gluconeogenesis.
The estimated Ks for conversion of propionate toCO2 and glucose was not affected by treatment in ourexperiment. These estimates were not different fromzero ( P < .10) and had large standard errors, in partbecause concentrations of substrates chosen for thisexperiment probably were too high to obtain accurateestimates of Ks. Mesbah and Baldwin (1983) reporteda Ks of 1.8 mM for conversion of propionate to glucose,and Looney et al. (1987) reported a Ks of 1 mM forconversion of propionate to glucose in hepatocytesisolated from lambs. The treatment × substrateinteraction for Ks and the trend for a treatment ×substrate interaction for Vmax for conversion ofsubstrates to CO2, such that each was increased morefor alanine than for propionate, suggest that hepato-cytes from phlorizin-treated wethers had substantiallyincreased capacity to oxidize alanine compared withpropionate. The estimated Ks for conversion of alanineto glucose was not affected by treatment; however,phlorizin tended to increase Vmax for conversion ofboth propionate and alanine to glucose, furtherindicating the increased capacity of hepatocytes iso-lated from phlorizin-treated wethers to synthesizeglucose.
Intraruminal infusions of urea have been demon-strated to decrease glucose production rates in steers(Spires and Clark, 1979). Furthermore, bovinehepatocytes previously loaded with triglycerides in
vitro as a model for bovine fatty liver demonstrateddecreased ureagenic capacity (Strang et al., 1998). Invivo, increased triglyceride content of liver tissue fromdairy cows at 2 d postpartum was associated withincreased concentrations of ammonia in plasma withconcomitantly unchanged concentrations of plasmaurea (Zhu et al., 1998). Furthermore, catabolism ofamino acids results initially in the production ofammonia. Accordingly, the implications of increasedammonia concentrations on hepatic capacity tometabolize gluconeogenic substrates are of relevance.Addition of 2.5 mM NH4Cl to the incubation media inour experiment decreased conversion of propionate toCO2 and substantially decreased its conversion toglucose. The estimated Vmax for conversion of propi-onate to CO2 was not decreased significantly byaddition of NH4Cl; however, estimated Vmax forconversion to glucose was decreased substantially.These data are similar to those of Weekes et al.(1978), who determined that increasing NH4Cl con-centrations above 1 mM in vitro decreased glucoseproduction from propionate to rates not significantlygreater than rates of glucose production in the absenceof propionate or in the presence of 2 mM NH4Clwithout propionate. Demigne et al. (1991) found thataddition of 1.25 mM ammonia had little effect, butaddition of 2.5 or 5 mM ammonia decreased conver-sion of [2-14C]propionate to glucose. Although conver-sion of [2-14C]propionate to CO2 was increased slightlyby NH4Cl addition in hepatocytes isolated from goats,conversion of [2-14C]propionate to glucose was in-hibited by addition of NH4Cl (Aiello and Armentano,1987).
Addition of 2.5 mM NH4Cl to the incubation mediain this experiment had little effect on conversion ofalanine to CO2 and only slightly decreased conversionof alanine to glucose. Accordingly, NH4Cl addition didnot affect estimates of Ks or Vmax for conversion ofalanine to either CO2 or glucose. In contrast, Muts-vangwa et al. (1996) found that increasing concentra-tions of NH4Cl from 0 to 5 mM in vitro decreased ratesof conversion of [1-14C]alanine to CO2 and glucose. Ina second experiment, Mutsvangwa et al. (1997)determined that increasing in vitro concentrations ofNH4Cl from 0 to 1.25 mM decreased conversion of [1-14C]alanine to CO2 and glucose. Aiello and Armentano(1987) reported that conversion of [U-14C]lactate,which also enters the gluconeogenic pathway atpyruvate, to glucose was enhanced by NH4Cl addition.Reasons for differences among experiments are notevident; however, results suggest that increasedammonia concentrations are more detrimental toutilization of propionate than compounds entering thegluconeogenic pathway at pyruvate.
Phlorizin-treated wethers in this experiment had aslightly increased ratio for conversion of [1-14C]propionate to glucose relative to conversion to CO2and a decreased ratio of conversion of [1-14C]alanine to
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glucose relative to conversion to CO2. Knapp et al.(1992) suggested that applying this ratio tometabolism of [1-14C]propionate provided an index ofthe theoretical efficiency of gluconeogenesis. Becauseof randomization of label from [1-14C]propionate in thetricarboxylic acid cycle, for every mole of labeled CO2produced in the conversion of oxaloacetate to phos-phoenolpyruvate, one mole of labeled glucose shouldbe produced. This would yield a theoretical efficiencyof 1 for the ratio of glucose to CO2 if every molecule oflabeled propionate that forms oxaloacetate is shunteddirectly into the gluconeogenic pathway. Although thisconcept does not apply to metabolism of [1-14C]alanine, the ratio still is useful because the flux of[1-14C]alanine through pyruvate dehydrogenase (viapyruvate) results in oxidative decarboxylation of theradiolabel and release as 14CO2, and, if [1-14C]alanineis converted to oxaloacetate through pyruvate andpyruvate carboxylase, the radiolabel either proceeds toglucose through phosphoenolpyruvate carboxykinaseor is released as 14CO2 if it proceeds through citrateand the tricarboxylic acid cycle. Data from thisexperiment imply that hepatocytes from phlorizin-treated wethers utilized propionate with slightlygreater efficiency than hepatocytes from controlwethers. The slightly decreased efficiency of utiliza-tion of alanine by hepatocytes from phlorizin-treatedwethers reflects greater conversion of radiolabel fromalanine to CO2, possibly as a result of increasedmetabolic rate of hepatocytes from phlorizin-treatedwethers. Addition of NH4Cl decreased efficiency ofutilization of propionate and alanine for gluconeogene-sis; however, the decrease was not as substantial inphlorizin-treated wethers as in control wethers. Rea-sons for this difference are not known but may berelated to possible reprioritizing of metabolic processesto support increased gluconeogenesis in hepatocytesisolated from phlorizin-treated wethers, which maypartially override the negative effect of NH4Cl addi-tion.
The three-way interaction of treatment × substrate× addition of a second substrate for conversion ofradiolabeled substrates to CO2 and glucose approxi-mated a trend despite limited replication. This inter-action occurred because addition of propionate toflasks containing [1-14C]alanine did not affect conver-sion to CO2 and glucose by hepatocytes from eithercontrol or phlorizin-treated wethers and addition ofalanine to flasks containing [1-14C]propionate did notaffect conversion to CO2 and glucose by hepatocytesfrom control wethers, but addition of alanine to flaskscontaining [1-14C]propionate substantially increasedconversion of propionate to CO2 and glucose byhepatocytes from phlorizin-treated wethers. Our datafor phlorizin-treated wethers are similar to those ofSmith and Osborne-White (1971), who demonstratedthat addition of pyruvate increased conversion ofcarbon from propionate to citrate in mitochondria from
ovine liver. Our data for control wethers support thoseof Demigne et al. (1991), who demonstrated thataddition of pyruvate did not affect metabolism ofpropionate by ovine hepatocytes. In contrast to ourdata for the control and phlorizin-treated wethers,Demigne et al. (1991) reported that addition ofpropionate decreased utilization of pyruvate. Further-more, addition of propionate up to 1.25 mM (Muts-vangwa et al., 1997) or 5 mM (Mutsvangwa et al.,1996) decreased conversion of [1-14C]alanine to CO2but increased conversion of [1-14C]alanine to glucose.Differences between our experiment and those ofMutsvangwa et al. (1996, 1997) may be related todifferences in concentrations of substrates comparedwith the maximal capacity of hepatocytes to convertsubstrates to glucose and CO2.
Implications
Ruminant liver utilizes propionate for oxidativemetabolism and gluconeogenesis at much greaterrates than alanine. Subjecting ruminants to a suddenincrease in glucose demand slightly increases thepotential to utilize propionate but greatly increasesthe potential to utilize alanine. Increased utilization ofamino acids for glucose synthesis may be a primaryadaptation to a suddenly increased metabolic demandfor glucose, such as that which occurs at parturition indairy cows. Furthermore, large excesses of ammoniafrom excess dietary nitrogen intake, imbalances inruminally degradable and nondegradable proteins, orimbalances in amino acids, along with metabolicsituations (e.g., fatty liver) that may compromise theability of liver to synthesize urea, may decrease theability of liver to synthesize glucose from propionate.These findings may affect current models of aminoacid nutrition and utilization of body protein forgluconeogenesis in periparturient ruminants.
Literature Cited
Aiello, R. J., and L. E. Armentano. 1987. Gluconeogenesis in goathepatocytes is affected by calcium, ammonia and other keymetabolites but not primarily through cytosolic redox state.Comp. Biochem. Physiol. 88B:193−201.
Aiello, R. J., and L. E. Armentano. 1988. Fatty acid effects ongluconeogenesis in goat, calf, and guinea pig hepatocytes.Comp. Biochem. Physiol. 91B:339−344.
Aiello, R. J., T. M. Kenna, and J. H. Herbein. 1984. Hepaticgluconeogenic and ketogenic interrelationships in the lactatingcow. J. Dairy Sci. 67:1707−1715.
AOAC. 1990. Official Methods of Analysis (15th Ed.). Association ofOfficial Analytical Chemists, Arlington, VA.
Armentano, L. E., R. R. Grummer, S. J. Bertics, T. C. Skaar, and S.S. Donkin. 1991. Effects of energy balance on hepatic capacityfor oleate and propionate metabolism and triglyceride secre-tion. J. Dairy Sci. 74:132−139.
Bauman, D. E., and W. B. Currie. 1980. Partitioning of nutrientsduring pregnancy and lactation: A review of mechanisms in-volving homeostasis and homeorhesis. J. Dairy Sci. 63:1514−1529.
by guest on July 14, 2011jas.fass.orgDownloaded from
Bell, A. W. 1995. Regulation of organic nutrient metabolism duringtransition from late pregnancy to early lactation. J. Anim. Sci.73:2804−2819.
Bennink, M. R., R. W. Mellenberger, R. A. Frobish, and D. E.Bauman. 1972. Glucose oxidation and entry rate as affected bythe initiation of lactation. J. Dairy Sci. 55:712 (Abstr.).
Bergman, E. N., and D. E. Hogue. 1967. Glucose turnover andoxidation rates in lactating sheep. Am. J. Physiol. 213:1378−1384.
Bickerstaffe, R., E. F. Annison, and J. L. Linzell. 1974. Themetabolism of glucose, acetate, lipids, and amino acids in lac-tating dairy cows. J. Agric. Sci. 82:71−85.
Brockman, R. P., and J. G. Manns. 1974. Effects of glucagon onactivities of hepatic enzymes in sheep. Cornell Vet. 64:217−224.
Cremin, J. D., Jr., J. K. Drackley, L. R. Hansen, D. E. Grum, J. Odle,and G. C. Fahey, Jr. 1995. Effects of glycine and bovine serumalbumin on inhibition of propionate metabolism in ovinehepatocytes caused by reduced phenolic monomers. J. Anim.Sci. 73:3009−3021.
Demigne, C., C. Yacoub, C. Morand, and C. Remesy. 1991. Interac-tions between propionate and amino acid metabolism in iso-lated sheep hepatocytes. Br. J. Nutr. 65:301−317.
Donkin, S. S., and L. E. Armentano. 1993. Preparation of extendedin vitro cultures of bovine hepatocytes that are hormonallyresponsive. J. Anim. Sci. 71:2218−2227.
Donkin, S. S., and L. E. Armentano. 1995. Insulin and glucagonregulation of gluconeogenesis in preruminating and ruminatingbovine. J. Anim. Sci. 73:546−551.
Egan, A. R., J. C. MacRae, and C. S. Lamb. 1983. Threoninemetabolism in sheep. 1. Threonine catabolism and gluconeogen-esis in mature blackface wethers given poor quality hill her-bage. Br. J. Nutr. 49:373−383.
Emmison, N., L. Agius, and V. A. Zammit. 1991. Regulation of fattyacid metabolism and gluconeogenesis by growth hormone andinsulin in sheep hepatocyte cultures. Biochem. J. 274:21−26.
Faulkner, A., and H. T. Pollock. 1986. Propionate metabolism andits regulation by fatty acids in ovine hepatocytes. Comp. Bi-ochem. Physiol. 84B:559−563.
Gill, W., G. E. Mitchell, Jr., J. A. Boling, R. E. Tucker, G. T.Schelling, and R. M. DeGregorio. 1985. Glucagon influence ongluconeogenesis and oxidation of propionic acid and threonineby perfused ovine liver. J. Dairy Sci. 68:2886−2894.
Greenfield, R., S. S. Donkin, and M. J. Cecava. 1998. Pyruvatecarboxylase and phosphoenolpyruvate carboxykinase expres-sion in the transition dairy cow. J. Dairy Sci. 81(Suppl. 1):320(Abstr.).
Heitmann, R. N., and E. N. Bergman. 1980. Integration of aminoacid metabolism in sheep: Effects of fasting and acidosis. Am. J.Physiol. 239:E248−E254.
Holtenius, K., and P. Holtenius. 1997. Effects of peroral alanineadministration in lactating ewes with decreased availability ofglucose. Br. J. Nutr. 78:805−813.
Knapp, J. R., H. C. Freetly, B. L. Reis, C. C. Calvert, and R. L.Baldwin. 1992. Effects of somatotropin and substrates on pat-terns of liver metabolism in lactating dairy cattle. J. Dairy Sci.75:1025−1035.
Laser, H. 1961. Salt solutions. In: C. Long (Ed.) Biochemist’sHandbook. p 58. Van Nostrand, Princeton, NJ.
Lomax, M. A., I. A. Donaldson, and C. J. Pogson. 1986. The effect offatty acids and starvation on the metabolism of gluconeogenicprecursors by isolated sheep liver cells. Biochem. J. 240:277−280.
Looney, M. C., R. L. Baldwin, and C. C. Calvert. 1987. Gluconeogen-esis in isolated lamb hepatocytes. J. Anim. Sci. 64:283−294.
Mesbah, M. M., and R. L. Baldwin. 1983. Effects of diet, pregnancy,and lactation on enzyme activities and gluconeogenesis inruminant liver. J. Dairy Sci. 66:783−788.
Mills, S. E., L. E. Armentano, R. W. Russell, and J. W. Young. 1981.Rapid and specific isolation of radioactive glucose from biologi-cal samples. J. Dairy Sci. 64:1719−1723.
Mills, S. E., D. C. Beitz, and J. W. Young. 1986. Evidence forimpaired metabolism in liver during induced lactation ketosisof dairy cows. J. Dairy Sci. 69:362−370.
Mutsvangwa, T., J. G. Buchanan-Smith, and B. W. McBride. 1996.Interactions between ruminal degradable nitrogen intake andin vitro addition of substrates on patterns of amino acidmetabolism in isolated ovine hepatocytes. J. Nutr. 126:209−218.
Mutsvangwa, T., J. G. Buchanan-Smith, and B. W. McBride. 1997.Effects of ruminally degradable nitrogen intake and in vitroaddition of ammonia and propionate on the metabolic fate of L-[1-14C]alanine and L-[15N]alanine in isolated sheep hepato-cytes. J. Anim. Sci. 75:1149−1159.
Nolan, J. V., and R. A. Leng. 1970. Metabolism of urea in latepregnancy and the possible contribution of amino acid carbon toglucose synthesis in sheep. Br. J. Nutr. 24:905−915.
NRC. 1985. Nutrient Requirements of Sheep (6th Rev. Ed.). Na-tional Academy Press, Washington, DC.
Overton, T. R., J. K. Drackley, C. J. Ottemann-Abbamonte, A. D.Beaulieu, and J. H. Clark. 1998. Metabolic adaptation to ex-perimentally increased glucose demand in ruminants. J. Anim.Sci. 76:2938−2946.
Reynolds, C. K., D. L. Harmon, and M. J. Cecava. 1994. Absorptionand delivery of nutrients for milk protein synthesis by portal-drained viscera. J. Dairy Sci. 77:2787−2808.
SAS. 1992. SAS User’s Guide: Statistics (Release 6.03). SAS Inst.Inc., Cary, NC.
Smith, R. M., and W. S. Osborne-White. 1971. Synthesis of phos-phoenolpyruvate from propionate in sheep liver. Biochem. J.124:867−876.
Smith, R. W., and A. Walsh. 1982. Effects of pregnancy and lactationon the activities in sheep liver of some enzymes of glucosemetabolism. J. Agric. Sci. 98:563−565.
Spires, H. R., and J. H. Clark. 1979. Effect of intraruminal ureaadministration on glucose metabolism in dairy steers. J. Nutr.109:1438−1447.
Strang, B. D., S. J. Bertics, R. R. Grummer, and L. E. Armentano.1998. Effect of long-chain fatty acids on triglyceride accumula-tion, gluconeogenesis, and ureagenesis in bovine hepatocytes. J.Dairy Sci. 81:728−739.
Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods fordietary fiber, neutral detergent fiber, and nonstarch polysac-charides in relation to animal nutrition. J. Dairy Sci. 74:3583.
Weekes, T.E.C., R. I. Richardson, and N. Geddes. 1978. The effect ofammonia on gluconeogenesis by isolated sheep liver cells. Proc.Nutr. Soc. 38:3A (Abstr.).
Wilson, S., J. C. MacRae, and P. J. Buttery. 1983. Glucose produc-tion and utilization in non-pregnant, pregnant, and lactatingewes. Br. J. Nutr. 50:303−316.
Wiltrout, D. W., and L. D. Satter. 1972. Contribution of propionateto glucose synthesis in the lactating and nonlactating cow. J.Dairy Sci. 55:307−317.
Wolff, J. E., E. N. Bergman, and H. H. Williams. 1972. Netmetabolism of plasma amino acids by liver and portal-drainedviscera of fed sheep. Am. J. Physiol. 223:438−446.
Zhu, L. H., L. E. Armentano, D. R. Bremmer, R. R. Grummer, and S.J. Bertics. 1998. Plasma concentration of urea, ammonia, gluta-mine (gln) and glutamate (glu) around calving and theirrelation to liver triglyceride (TG) and plasma Ca. J. Dairy Sci.81(Suppl. 1):321 (Abstr.).
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