The Utilization by Rabbit Aorta of Carbohydrates, Fatty Acids, Ketone Bodies, and Amino Acids as Substrates for Energy Production

Transcription

1 85 CIRCULATION RESEARCH VOL. 48, No. 6, JUNE 1981 Shanbour LL, Jacobsen ED, Brobmann GF, Hinshaw LB (1971) Effec of ouabain on splanchnic hemodynamics in he rhesus monkey. Am Hear J 81: Sark JJ, Sanders CA, Powell WJ (1972) Neurally mediaed and direc effecs of aceyl srophanhidin on canine skeleal muscle vascular resisance. Circ Res 3: Swain JA, Heyndricks GR, Boecher DH, Vaner SF (1975) Prosaglandin conrol of renal circulaion in he unanesheized dog and baboon. Am J Physiol 229: Trea E, Ulano HB, Jacobsen ED (1971) Effecs of inra-arerial ouabain on meseneric and caroid hemodynamics. J Pharmacol Exp Ther 179: Waldhausen JA, Herendeen T (1964) Direc effecs of digialis on renal blood flow. Surgery 56: The Uilizaion by Rabbi Aora of Carbohydraes, Fay Acids, Keone Bodies, and Amino Acids as Subsraes for Energy Producion KENNETH V. CHACE A RICHARD ODESSEY Downloaded from hp://ahajournals.org by on November 5, 218 SUMMARY The abiliy of rabbi aora o oxidize various subsraes was sudied o deermine which of hese compounds may be energy subsraes for vascular smooh muscle (VSM). Glucose, keone bodies, medium-chain lengh fay acids, branched-chain amino acids, and gluamine all are oxidized a comparable raes on a molar basis. Some oher amino acids, long chain fay acids, pyruvae and glycerol also are oxidized, bu a lower raes. The oxidaion of 6 amino acids could no be deeced. VSM was found o release keone bodies when incubaed in leucine /J-hydroxybuyrae or ocanoae. This suggess ha he aceoaceyl CoA and/or aceoaceae derived from hese subsraes is no compleely oxidized. The oxidaion rae of several subsraes when measured individually is inhibied by 5-8% by he presence of a combinaion of oher subsraes in he medium. Under hese condiions, glucose is a minor subsrae for oxidaive meabolism accouning for only 5% of O 2 consumpion. The oxidaion rae of all he exogenous subsraes ogeher is calculaed o accoun for less han half of he oxygen consumpion; his finding indicaes ha an endogenous subsrae mus also be uilized. Circ Res 48: ,1981 SEVERAL invesigaors have examined he meabolism of vascular smooh muscle (VSM) by sudying he effec of simulaion (Lundholm and Mohme- Lundholm, 1962; Peerson and Paul, 1974) or he effec of age or disease (Morrison e al., 1972b; Daly, 1976) on is meabolism. They have generally assumed ha glucose is he major energy source for VSM, and have no aemped o examine he meabolism of oher possible energy sources. One reason for his assumpion is ha VSM has a respiraory quoien of.99 (Kirk e al., 1954, Kosan and Buron, 1966). However, he sudies ha esablished his figure were performed wih 11 mm glucose as he only subsrae. Since VSM can oxidize fay acids (Hashimoo and Dayon, 1971; Mor- From he Deparmen of Physiology, School of Medicine, Universiy of Virginia, Charloesville, Virginia. This work was suppored by Gran HL from he Hear, Lung, and Blood Insiue of he Naional Insiues of Healh. A preliminary repor of his work has appeared previously in absrac form. Address for reprins: Dr. Richard Odessey, Deparmen of Physiology, LSU Medical Cener, 1542 Tulane Avenue, New Orleans, Louisianna Received March 21, 198; acceped for publicaion November 25,198. rison, e al., 1974) and amino acids (Morrison e al., 1976b), i is quie possible ha incubaion of VSM in a physiological mixure of all possible energy subsraes presen in he blood would reduce he respiraory quoien. Glucose also was hough o be he major energy source for VSM because carbohydrae was more effecive han oher subsraes in resoring conraciliy o subsrae-depleed issue (Coe e al., 1968). In his sudy, i was assumed ha issue could no conrac afer incubaion wihou subsrae because he high-energy phosphaes in he issue were exhaused. Measuremens were no made o deermine wheher he inabiliy of subsrae-depleed issue o conrac was due o is low-energy sae, or wheher subsraes ha resored is abiliy o conrac improved is energy sae. Since he resoraion of he abiliy of VSM o conrac could be caused by means oher han he regeneraion of high-energy phosphae, (Edwards e al., 1977; Robers e al., 1979, Piman and Quinn, 1979), his sudy did no really deermine wha subsraes are mos effecive a generaing high-energy phosphae. The quesion of which subsraes are mos effec-

2 FUEL UTILIZATION BY RABBIT AORTA/Chace and Odessey 851 Downloaded from hp://ahajournals.org by on November 5, 218 ive and which are acually used under physiological condiions can be approached by learning which poenial subsraes can be oxidized, and heir relaive uilizaion by VSM under physiological condiions. The deerminaion of he soichiomery beween oxygen consumpion and exogenous subsrae oxidaion would indicae wheher endogenous subsraes are also being oxidized by he issue. These sudies would also furnish some informaion abou wha meabolic pahways are presen in VSM. The following experimens were designed o address hese quesions. Mehods Male New Zealand Whie rabbis weighing kg were killed and heir horacic aoras removed. The aoras were superfused wih Ham's F12 nurien medium (Grand Island Biological Company) equilibraed wih 95% oxygen, 5% carbon dioxide while exerior fa was removed. In some cases, inima-media srips were peeled from he aora (Wolinsky and Daly, 197). Aoric rings or srips weighing 1-3 mg or inima-media srips weighing 5-2 mg were incubaed a 37 C in Krebs-Ringer bicarbonae (KRB) medium equilibraed wih 95% oxygen, 5% carbon dioxide. The oxidaion rae of subsraes was deermined by measuring he producion of 14 C-labeled carbon dioxide (Odessey and Goldberg, 1972). The procedure was modified by using phenehylamine o collec he carbon dioxide and by injecing 1 N perchloric acid ino he medium o drive off he carbon dioxide. Vials conaining subsrae bu no issue were used as blanks in hese experimens. Radiochemicals and phenehylamine were purchased from New England Nuclear. The subsrae concenraions used were 5 mm for glucose and.5 mm for all subsraes excep /?-hydroxybuyrae. A racemic mixure of cold carrier /8-hydroxybuyrae (Calbiochem) was used a a concenraion of 1. mm, whereas he labeled /S-hydroxybuyrae consised of only he D isomer. (The common biological sereoisomer was used for all oher subsraes.) Following he collecion of carbon dioxide from he medium, he medium was adjused o ph 6-8 wih neuralizing soluion (3 N poassium hydroxide conaining.25 M 2-(iV-morpholinoehanesulfonic acid (MES) (Calbiochem) and.25 M 3- (./V-morpholino)propanesulfonic acid (MOPS) (Calbiochem). MES and MOPS buffers were included in he neuralizing soluion, since hese buffers have a pk value near neuraliy (6.15 for MES, 7.2 for MOPS). The neuralized medium was sored a -8 C. Afer he incubaion period, he issue was removed from he medium and was frozen immediaely in liquid nirogen. The issue hen was weighed and homogenized in eiher 3 N perchloric acid (PCA) or, when lipid oxidaion was being sudied, in a mixure of ehanol and eher (1:1, vol/vol). The we weigh obained here was used for all calculaions. The emperaure of he homogenizaion soluion was mainained a 1 C. Afer he homogenae had been cenrifuged a 1 g for 1 minues, he ph of he perchloric acid supernaan was adjused o ph 6-8 wih neuralizing soluion, and he neuralized supernaan was sored a 8 C. Lacae, pyruvae, aceoaceae, and /8-hydroxybuyrae released ino he medium were assayed by fluoromeric, enzymaic echniques (Lowry and Passoneau, 1972; Bergmeyer, 1974). Adenine nucleoides, creaine, and creaine phosphae in he issue homogenae also were assayed by fluoromeric enzymaic echniques (Lowry and Passoneau, 1972) wih a fluoromeer manufacured by Farrand Opical Co., Inc. Enzymes used in hese assays were purchased from Boehringer-Mannheim or Sigma Biochemicals. Oher biochemicals used in hese assays were purchased from Sigma Biochemicals or Calbiochem. All chemicals were of he highes puriy available commercially. Aceoaceae is decarboxylaed during incubaion in PCA (Bergmeyer, 1974). Tess wih sandards of a known amoun of aceoaceae showed ha 33.4 ± 2.% of he aceoaceae in he medium was los during he PCA incubaion prior o aceoaceae deerminaion. Our repored aceoaceae values have been correced o reflec his fac. Some of he labeled carbon dioxide produced during he oxidaion of subsraes ha produce aceoaceae migh derive from he PCA-induced decarboxylaion of aceoaceae. To calculae how much of he labeled carbon dioxide was derived from his source, i was assumed ha all he aceoaceae released ino he medium was a meabolic produc of he exogenous subsrae. Leucine[ H C(U)] and ocanoae[l- 14 C] are he only subsraes sudied which are likely o release significan amouns of aceoaceae labeled in he C-l carbon aom. Therefore, he oxidaion raes of leucine[ l4 C(U)] and ocanoae[l- U C] have been correced using he following formula. R = Ro - A-D/(l - D), where R = rae of subsrae oxidaion, Ro = observed oxidaion rae, A = observed rae of aceoaceae producion, and D= fracion of aceoaceae decarboxylaed. The exracellular fluid volume of he issue was deermined by incubaion in KRB medium conaining.5 mm sucrose[ H C(U)] (New England Nuclear). Toal fluid volume was deermined by incubaing he issue in KRB medium conaining riiaed waer (New England Nuclear), and he inracellular fluid volume was deermined by subracing he exracellular volume from he oal volume. The inracellular concenraions of he subsrae and is meabolies were deermined by measuring he amoun of 14 C label in he homogenae supernaan and subracing he amoun of label calculaed o have been in he exracellular space of he issue. The specific aciviy of he medium and he inracellular volume were used o calculae he concenraion of subsrae and is meabolies. For his calculaion, he assumpion was made ha he spe-

3 852 CIRCULATION RESEARCH VOL. 48, No. 6, JUNE 1981 TABLE 1 Oxidaion of Subsraes by VSM Rae of subsrae oxidaion o U CO 2 Subsrae (/imol/g we w per hr) Glucose[' 4 C(U)] Palmiae[l- l4 C] Oleae[l- 14 C] Ocanoae[l- l4 C] /S-hydroxy buyrae-[3- U C] Aceoaceae[3-'"C] Glycerol[3- l4 C] Pyruvae[l-' 4 C].21 ± ±.3.38 ± ± ±.4.26 ±.28.5 ±.1.53 ±.7 (24) (7) (7) Inracellular subsrae and meabolies (DIM) 6.69 ± ± ± ± ± ±.26.6 ±.5.27 ±.2 Pieces of rabbi aora were incubaed 5 hours a 37 C in KRB medium conaining 5. mm glucose wih oher subsraes presen, where indicaed in he able, a.5 mm. All values are given as mean ± SE, wih he number of pieces used o deermine he value shown in parenheses. (21) Downloaded from hp://ahajournals.org by on November 5, 218 cific aciviy of he subsrae and is meabolies was he same as he specific aciviy of he subsrae in he medium. The amoun of 14 C-labeled fay acids eserified o various lipid classes was deermined by hin layer chromaography on silica gel-g plaes (Morrison e al., 1974). Fay acids [1-14 C] were purified before use (Harper and Saggerson, 1976) and were added o he medium as a complex wih bovine serum albumen wih a molar raio of fay acid o proein of 5:1 (Evans and Mueller, 1963). Labeled aceoaceae was prepared from is ehyl eser (Krebs e al., 1966). Alanine[l- 14 C] and valine[l- I4 C] were purified before use on a Dowex-5 (Bio-Rad Laboraories) column. When oxygen consumpion was measured, 3- o 6-mg pieces of aora were used and all medium was equilibraed wih 95% air, 5% carbon dioxide. The oxygen consumpion was measured using a micro-cahode oxygen elecrode (Insrumenaion Laboraories). The issue was incubaed a 37 C in KRB medium which was sirred consanly inside a waer-jackeed chamber (Gilson). The change over ime in oxygen concenraion in he medium wih issue presen was compared wih he change wih no issue presen o obain consumpion values. Readings were obained over a 5- o 1-minue period. Resuls Deerminaion of Energy Sae To show ha he aoric issue was viable and sable, aoric rings were incubaed in KRB medium conaining (1) glucose, leucine, /Miydroxybuyrae, glucose and leucine, glucose and /8-hydroxybuyrae, or in he absence of any subsrae. (Glucose concenraion was 5. mm, leucine and /?- hydroxybuyrae concenraions were.5 mm.) Adenine nucleoide, creaine phosphae, and creaine levels were measured afer incubaion of rabbi aora in KRB medium for 3-5 hours. The subsrae used in he medium did no significanly affec he adenine nucleoide, creaine phosphae, or creaine levels. The pooled daa show ha phosphocreaine (.43 ±.3 /umol/g) and ATP (.6 ±.3 /xmol/g) are higher han hose ohers have repored for rabbi aora (Needleman and Blehm, 197; Namm and Zucker; 1973). The creaine phosphae: creaine raio is probably a more accurae reflecion of he energy sae han is he ATP:ADP raio, since much of he ADP is bound ighly o acin and herefore no in equilibrium wih he free nucleoide pool (McGilvery and Murray, 1974). We found a phosphocreaine:creaine raio of (1.1 ±.1) which is higher han repored for oher VSM issue (Daemers-Lamber, 1964). These measuremens sugges ha rabbi aoric VSM was sable and viable during he incubaion period. When he energy sae of inima-media srips was compared wih ha of he whole aora, he ATP: ADP raio for he inima-media srips was significanly lower han he raio for he whole aora (daa no shown). I was also found ha he oxidaion rae of leucine (bu no glucose or /8-hydroxybuyrae) dropped significanly over an incubaion period of 5 hours when inima-media were used, bu remained consan when aoric rings were used. For hese reasons, aoric rings or srips were used for he experimens described below. Inracellular and Exracellular Volume The incubaion of aoric rings in he riiaed waer indicaed ha he oal fluid volume of he aora was.74 ±.1 ml/g we weigh. Incubaion in sucrose indicaed an exracellular volume of.38 ±.2 ml/g we weigh. These values are close o previously repored values for VSM (Villamil e al., 1968). Oxidaion Raes The oxidaion raes for poenial subsraes were obained by incubaing pieces of rabbi aora in KRB medium conaining labeled subsrae and glucose and measuring he 14 C-labeled carbon dioxide produced. The inracellular concenraions of subsrae and is meabolies also were measured in mos cases o deermine wheher he subsrae enered he cells. These daa are presened in Table 1 for glucose, fay acids, keone bodies, pyruvae,

4 FUEL UTILIZATION BY RABBIT AORTA/Chace and Odessey 853 Downloaded from hp://ahajournals.org by on November 5, 218 TABLE 2 Oxidaion of Amino Acids by VSM Subsrae Leucine[l-' 4 C] Leucine[' 4 C(U)] Valine[l' 4 C] Valine[ 14 C(U)] Isoleucine[ l4 C(U)] Gluamae[' 4 C(U)] Gluamine[ l4 C(U)] Asparae[' 4 C(U)] Asparagine[ 14 C(U)] Lysine[ 14 C(U)] Arginine[ 14 C(U)] Threonine[' 4 C(U)] Glycine[ 14 C(U)] Alanine[l-' 4 C] Hae of subsrae oxidaion o '"COs (/imol/g we w per ir) ± ± ± ±.5.69 ±.3.15 ±.1.78 ±.3.9 ±.4.5 ±.2.16 ±.2.11 ±.4.6 ±.2.5 ±.1.1 ±.4 and glycerol. The oxidaion raes for all subsraes bu palmiae were consan from 1 o 5 hours afer he sar of incubaion. The oxidaion rae for palmiae rose over he 5-hour incubaion period; he rae shown in Table 1 was measured from 4 o 5 hours afer he sar of he incubaion. When here is no glucose in he medium, he oxidaion rae of ys-hydroxybuyrae rises wih ime. When issue was incubaed in medium conaining 5mM glucose, 1.1 ±.9 jiimol/g per hour of lacae and.8 ±.1 /nmol/ g we weigh per hour of pyruvae were released ino he medium. This rae of release also was consan from 1 o 5 hours afer he sar of incubaion. Chromaography of he inracellular meabolies following incubaion of VSM in medium conaining long-chain fay acids shows ha 9-1% of he 14 C fay acids are found incorporaed ino esers: 15-2% of he label is incorporaed ino phospholipids, and 75-8% of he label is incorporaed ino di- or riglycerides. The oxidaion of amino acids by VSM was sudied also. Table 2 compares he oxidaion rae of 18 amino acids in he presence of glucose. All amino acids bu asparagine were oxidized a a consan rae from 1 o 5 hours afer he sar of he incubaion period. The oxidaion rae of asparagine increased hroughou he incubaion period. The rae shown in Table 2 was obained from 4 o 5 hours afer he sar of he incubaion. From Tables 1 and 2, i is apparen ha, on a molar basis, he medium-chain lengh fay acid, ocanoae, he keone bodies, and he branchedchain amino acids are oxidized a raes approaching or exceeding he oxidaion rae of glucose. Gluamine and asparagine are oxidized a one-half o onequarer he rae of glucose, while long-chain fay acids, pyruvae, glycerol, and he oher amino acids are oxidized a a much lower rae. Of he compounds esed, all bu glycerol and gluamae clearly were aken up by he issue. I is possible, herefore, ha membrane ranspor may limi he degradaion by VSM of hese blood-borne subsraes. A comparison of he producion of labeled carbon dioxide from leucine[l- 14 C] and valine[l- 14 C] wih is producion from uniformly labeled leucine and valine shows ha he oxidaion of he C-l carbon of hese amino acids accouns for more han onehalf of he carbon dioxide formed from hese amino acids (Table 2). The release of he C-l carbon as carbon dioxide occurs afer he firs wo seps of leucine or valine oxidaion. The resuls above indicae ha he oxidaion of he remaining carbon aoms may yield some final produc(s) oher han carbon dioxide. In fac, he inracellular meabolie levels of he uniformly labeled amino acids are higher han ha of C-1-labeled amino acids (Table 2). However, he difference beween he oxidaion raes of he C-l carbon and he res of he carbon aoms should lead o a greaer difference in inracellular meabolie level han is acually seen. I is likely, hen, ha some produc of leucine and valine meabolism, in addiion o carbon dioxide, is released from he cell. Leucine is oxidized o aceoaceae and aceyl- CoA which can hen be oxidized o carbon dioxide. The mos likely leucine meabolies o be released from he cell are keone bodies. Table 3 shows ha significan amouns of keone bodies are released (31) (21) (6) (8) (12) Inracellular subsrae and meabolies (mm).44 ± ± ± ± ±.2 < ± ± ± (14) (9) (6) (1) Cyseine[ M C(U)] Hisidine[ 14 C(U)] Phenylalanine[' 4 C(U)] Proline[ 14 C(U)] Serine[ l4 C(U)] Tyrosine[ 14 C(U)] (1) 1.2 ± ± Pieces of rabbi aora were incubaed 5 hours a 37 C in KRB medium conaining 5. mm glucose wih oher subsraes presen, where indicaed in he able a.5 mm. All values are as mean ± SE, wih he number of pieces used o deermine he value shown in parenheses. indicaes ha he value was no deermined. (1)

5 854 CIRCULATION RESEARCH VOL. 48, No. 6, JUNE 1981 TABLE 3 Producion of Keone Bodies by VSM Subsrae None Glucose Ocanoae Palmiae Leucine Gluamine Isoleucine /?-Hydroxybuyrae Combinaion Aceoaceae (/zmol/g we w per hr).3 ±.2. ±.1.51 ±.6.3 ±.2.9 ±.2. ±.2.3 ±.2.21 ±.2.35 ±.3 (8) (16) (21) /?-hydroxybuyrae (/imol/g we w per hr).3 ±.1. ±.1.12 ±.3.4 ±.1.13 ±.3 Pieces of rabbi aora were incubaed 1 hour a 37 C in KRB medium supplemened as shown. The issue hen was ransferred o fresh medium and incubaed an addiional 2-3 hours. A he end of incubaion, he medium was acidified wih 1 N PCA. Afer neuralizaion of he medium, he keone bodies were assayed enzymaically. Glucose concenraion in he medium was 5. mm, and he concenraion of all oher subsraes was.5 mm. The combinaion of subsraes conained a mixure of all he subsraes lised above bu ocanoae. All values are expressed as mean ± SE, wih he number of pieces used o deermine he value shown in parenheses. indicaes ha value was no deermined. (7) (7) Downloaded from hp://ahajournals.org by on November 5, 218 when leucine is presen in he medium. Ocanoae and /?-hydroxybuyrae also are oxidized o aceoaceae and/or aceyl-coa, and keone bodies also are released when hey are presen in he medium (Table 3). No keone bodies are released when here is no subsrae in he medium or when glucose, isoleucine, or gluamine is he only subsrae presen. The amoun of keone bodies released ino he medium when VSM is incubaed in medium conaining leucine is grea enough o indicae ha keone bodies and carbon dioxide are likely o be he only major oxidaive producs of he carbon chain of leucine released. Afer he C-1 aom of valine is oxidized o carbon dioxide, he remaining carbon aoms are oxidized o succinyl-coa. Succinyl-CoA is glycogenic, and lacae or gluamine (Chang and Goldberg, 1978) would be expeced o be released when issue is incubaed in valine if VSM resembles skeleal muscle. We have no ye deermined wheher his occurs. The oxidaion raes presened in Tables 1 and 2 were obained wih issue incubaed in he presence of 5 mm glucose and he labeled subsrae. Glucose oxidaion was measured wih no oher subsrae TABLE 4 Inhibiion of Subsrae Oxidaion by Oher Subsraes Subsrae oxidized Glucose[ 14 C(U)] Leucine['"C(U)] Isoleucine[' 4 C(U)] Gluamine[ 14 C(U)] Palmiae[l- l4 C] Ocanoae[- l4 C] /?-Hydroxybuyrae- [3-14 C] Glucose 48* Leucine 53 Isoleucine 48 presen in he medium. In vivo, all he subsraes are presen, and he oxidaion raes of each subsrae may be affeced by he presence of oher subsraes. To es wheher one subsrae affecs he oxidaion rae of oher subsraes, issue was incubaed in he presence of one labeled subsrae, or in he presence of ha subsrae and anoher (unlabeled) subsrae. The amoun of inhibiion of he oxidaion rae of one subsrae caused by he presence of a second subsrae is presened in Table 4. Of he subsraes sudied, ocanoae has he greaes effec in inhibiing he oxidaion of oher subsraes, whereas is oxidaion is no affeced by he presence of oher subsraes esed. /8-Hydroxybuyrae inhibis he oxidaion of hree oher subsraes, while he only subsrae ha significanly inhibied is oxidaion was ocanoae. On he oher hand, glucose oxidaion is inhibied more han 5% by eiher leucine, /?-hydroxybuyrae, or ocanoae, whereas isoleucine oxidaion is inhibied significanly only by glucose. Glycolysis, as indicaed by lacae and pyruvae released, is no affeced by he presence of he oher subsraes in he medium (Table 5). To see wha effec a combinaion of subsraes Percen inhibiion by Gluamine Palmiae 45* Ocanoae 67H 77[ i 631 /8-Hydroxybuyrae Pieces of rabbi aora were incubaed a 37 C in KRB medium conaining one labeled subsrae and addiional subsrae as shown. Glucose concenraion was 5 mm; all oher subsrae concenraions were.5 mm. '*CO 2 released ino he medium 1-3 hours afer he sar of incubaion was measured o deermine oxidaion raes. Confidence levels were deermined using Suden (-es. P <.5; P >.5; HP <.25; P <.1; P <.1. 52* 49* 31

6 FUEL UTILIZATION BY RABBIT AORTA/Chace and Odessey 855 TABLE 5 Effec of Subsraes on Lacae and Pyruvae Release during incubaion in he Presence of Glucose Addiional subsrae None Palmiae Ocanoae Leucine /?-Hydroxybuyrae Combinaion Lacae (/imol/g we w per hr) ± ±.4 1. ± ± ± 1. Pyruvae (/imol/g we w per hr) ±.1.9 ± ± ± ±.1 Pieces of rabbi aora were incubaed a 37 C in KRB medium conaining 5. mm glucose wih addiional subsrae presen a.5 mm where indicaed. The combinaion of subsraes conained 5. mm glucose,.5 mm plamiae,.5 mm leucine,.5 mm isoleucine,.5 mm gluamine, and.5 mm /S-hydroxybuyrae. Lacae and pyruvae released ino he medium 1-3 hours afer he sar of incubaion were measured. Oher deails were as described in Table 4. The values are presened as mean ± SE, wih he number of pieces used o obain he value shown in parenheses. Downloaded from hp://ahajournals.org by on November 5, 218 has on he oxidaion of one subsrae, issue was incubaed in glucose, palmiae, /S-hydroxybuyrae, isoleucine, leucine, and gluamine wih one subsrae labeled. Ocanoae was no used in his sudy since i is no a physiological subsrae (Linscheer e al., 1966). The oxidaion raes obained from his sudy, compared o raes obained in separae experimens wih issue incubaed in only one subsrae, are shown in Table 6. The combinaion of subsraes inhibied he oxidaion of each subsrae by 52-77%. The oxidaion of each subsrae is inhibied more by he combinaion of subsraes han i is by any one of he subsraes in he combinaion. Even js-hydroxybuyrae and gluamine, whose oxidaion raes are no inhibied significanly by any oher individual subsraes in he combinaion, have heir oxidaion raes inhibied by he combinaion. The sudy of he muual inhibiion of he oxidaion of he subsraes leads o he following conclusions. Alhough ocanoae is no presen in he blood, i seems o be oxidized preferenially o any oher subsrae when VSM is incubaed in medium conaining only wo subsraes. Nex o ocanoae, /S-hydroxybuyrae seems o be mos preferred when esed wih only one oher subsrae, bu is oxidaion is inhibied markedly by a combinaion of oher subsraes. No clear paern is seen ha would enable he deerminaion of he order of preference for he oxidaion of he oher subsraes. Alhough glucose oxidaion is inhibied by oher subsraes, glycolysis is no (Table 5). Oxygen Consumpion The oxidaion raes of he subsraes, he release of pyruvae and keone bodies ino he medium, and he known meabolic pahways for oxidaion of he subsraes were used o calculae he oxygen consumpion due o oxidaion of exogenous subsraes when VSM is incubaed in he combinaion of subsraes used in Table 6. For leucine, he producion of carbon dioxide from boh [1- I4 C] and [ 14 C(U)] leucine were used o deermine how much leucine is oxidized compleely o carbon dioxide, and how much is oxidized o carbon dioxide and aceoaceae. These calculaed values are presened in Table 7, where hey are also expressed as a percenage of he oxygen consumpion measured 1.5 hours afer he sar of he incubaion (Table 8). The oal calculaed oxygen consumpion is 32% of he measured value; hese wo values are significanly differen (P <.1) using he Behrens-Fisher es (Bliss, 1957). The calculaed oxygen consumpion increased slighly (2%) wih ime (no shown) (due o increases in he oxidaion rae of gluamine, TABLE 6 Oxidaion Raes in he Presence (+) and Absence ( ) of Oher Subsraes Subsrae Glucose[ 14 C(U)] Palmiae[l- u C] /3-Hydroxybuyrae[3-' 4 C] Leucine[ M C(U)] Isoleucine[' 4 C(U)] Gluamine[ l4 C(U)] Rae of subsrae oxidaion o M CO 2 (/imol/g we w per hr) (+).47 ±.8.11 ±.2.13 ±.5.38 ±.2.61 ±.2.42 ±.6 (-).21 ± ± ± ± ± ±.14 (24) (11) (6) (6) inhibiion Pieces of rabbi aora were incubaed a 37 C in KRB medium conaining only he subsrae whose oxidaion was measured ( ) or a combinaion of subsraes (+). The combinaion conained 5. mm glucose,.5 mm palmiae,.5 mm /3-hydroxybuyrae,.5 mm leucine,.5 mm isoleucine, and.5 mm gluamine. When only one subsrae was presen, i was presen a he same concenraion as i was in he combinaion of subsraes. All values are presened as mean ± SE, wih he number of pieces used o deermine he values shown in parenheses

7 856 CIRCULATION RESEARCH VOL. 48, No. 6, JUNE 1981 TABLE 7 Calculaed Oxygen Consumpion by VSM Subsrae Glucose -» CO 2 Palmiae -> CO 2 /?-Hydroxybuyrae > CO2 Leucine aceoaceae + CO2 Isoleucine > CO 2 Gluamine» CO2 Subsrae» aceoaceae Toal Calculaed oxygen consumpion (/imol/g we w per hr).28 ±.5.26 ±.5.46 ±.2.21 ±.5.36 ±.1.17 ±.2.8 ±.O ±.1 Percen of measured oxygen consumpion Pieces of rabbi aora were incubaed in KRB medium a 37 C wih he same combinaion of subsraes presen in he medium as was described in Table 6. Afer incubaion for 1.5 hour, oxygen consumpion was measured using an oxygen elecrode. The pieces hen were ransferred ino fresh medium in which one of he subsraes was labeled. Afer a.5-hour preincubaion, he issues were ransferred o fresh labeled medium for 1 hour. H CO 2, pyruvae, and keone bodies were colleced from his medium as described in mehods, and oxygen consumpion raes were calculaed using hese oxidaion raes. Values are expressed as mean ± SE, wih he number of pieces of issue used o deermine he values shown in parenheses Downloaded from hp://ahajournals.org by on November 5, 218 palmiae, and /?-hydroxybuyrae), while he measured oxygen consumpion decreased slighly (15%) wih ime so ha he calculaed value rises o 45% of he measured value 4.5 hours afer he sar of incubaion. However, he difference beween he values is sill significan (P <.1) using he Behrens-Fisher es. Oxygen consumpion was measured also when issue was incubaed wihou subsrae in he medium. Oxygen was consumed a a higher rae boh a 1.5 hours and 4.5 hours afer he sar of incubaion wihou subsrae in he medium han when he combinaion of subsraes was presen (Table 8). Discussion For our experimens, aoric rings were used raher han he inima-media srips used by oher invesigaors. Values for glucose oxidaion (Morrison e al., 1976a) lacae release (Morrison e al., 1972a), and oxygen consumpion (Morrison, e al., 1972a) obained by oher invesigaors using inimamedia srips of rabbi aora are similar o he values we found using aoric rings. This suggess ha he meabolic raes of he adveniial issue are no significanly differen from he raes found in he inima-media and ha he primary meabolically acive cell is VSM. TABLE 8 Oxygen Consumpion by VSM Time afer sar of incubaion (hr) subsraes 5.67 ± ±.71 Oxygen consumpion (/imoles/g we w per hr) (19) subsraes 8.66 ± 1.24* 6.63 ±.91 (8) (8) Pieces of rabbi aora were incubaed in KRB medium a 37 C. The medium conained eiher he combinaion of subsraes ( + ) described in Table 6 or conained no subsraes ( ). The daa are expressed as mean ± SE, wih he number of pieces used o deermine he values shown in parenheses. 'P <.5 when compared o oxygen consumpion in he presence of subsraes a 1.5 hours using Suden's Mes. The presen experimens demonsrae ha VSM, like oher muscle issues, can use a specrum of subsraes as energy sources. Glucose, amino acids, fay acids, and keone bodies all are oxidized, bu he oxidaion of no one subsrae can accoun for more han 8% of he oal oxygen consumpion when VSM is incubaed in medium conaining a mixure of subsraes (Table 7). The oxidaion rae of subsraes is inhibied by he presence of oher subsraes in he medium (Table 6). From hese daa i appears likely ha, in vivo, VSM may oxidize a number of differen subsraes o generae energy, while he proporion of energy derived from he oxidaion of a paricular subsrae may change as is concenraion, and he concenraion of oher subsraes in he blood, vary. The concenraions of he various subsraes used in hese experimens are wihin he range of values repored in he lieraure (Long, 1961; Scharf and Wool, 1966), and hus i is likely ha our measuremens (Table 7) reflec he relaive uilizaion of fuels in vivo by quiescen VSM. The muual inhibiion of subsrae oxidaion probably comes abou as a consequence of he compeiion by various subsraes for he limied capaciy of elecron ranspor in quiescen VSM. In he resing sae, he rae of flux hrough he ciric acid cycle is limied by he high phosphorylaion sae and he low availabiliy of phosphae accepor (ADP) for elecron ranspor (Klingenberg, 1961). In he presence of excess subsrae(s), a major raelimiing sep will be he disposal of aceyl-coa, a common inermediae in he oxidaion of mos issue fuels. This should lead o a reduced miochondrial oxidaion-reducion (redox) sae, [as refleced by he high lacae: pyruvae raio (Table 5)] and an increase in acyl- and/or aceyl-coa. The build up of acyl-coa in he cell will also deplee he free CoA pool, required for acivaion of various subsraes. These condiions are inhibiory for he meabolism of many subsraes. For example, since aceyl-coa and NADH inhibi pyruvae dehydro-

8 FUEL UTILIZATION BY RABBIT AORTA/Chace and Odessey 857 Downloaded from hp://ahajournals.org by on November 5, 218 genase (Hansford, 1976), i is no surprising ha glucose oxidaion is impaired severely in he presence of oher subsraes. The inhibiion of leucine oxidaion may be due o a compeiion for CoA and inhibiion of he branched chain keoacid dehydrogenase by he reduced miochondrial redox poenial (Odessey, 1979). Diminished oxidaion of keone bodies by ocanoae also may be due o a high level of miochondrial aceyl-coa which would shif he equilibrium for aceoaceae acivaion o he lef (Williamson and Hems, 197). Inhibiion of he acivaing enzymes or a decrease in he succinyl CoA:succinae raio also may occur. The inabiliy of palmiae o inhibi he oxidaion of oher subsraes may be due o he inabiliy of added long chain fay acid o increase endogenously derived aceyl-coa. As discussed below, excess aceyl-coa generaion by hese subsraes probably is limied a he level of miochondrial upake by a slow carniine acylransferase sep and also a diluion of he added subsrae by a large endogenous pool of fay acids. Since he capaciy o oxidize aceyl-coa is limied, excess aceyl-coa may be funneled ino he formaion of keone bodies as seen in he incubaions of rabbi aora wih ocanoae. Aceoaceae also is formed direcly from leucine and /3-hydroxybuyrae. Again, he presence of high levels of aceyl-coa will inhibi is subsequen oxidaion leading o is release from he cell as observed (Table 3). From hese experimens, i is apparen ha he meabolism of exogenous glucose by resing VSM plays a much smaller role in he mainenance of energy balance han has been previously supposed. When oher subsraes are presen a physiological levels, glucose accouns for only 5% of he oxygen consumpion (Table 7). The amoun of oxygen consumpion due o glucose meabolism may be somewha higher, depending on he fae of soichiomeric amouns of NADH generaed a he glyceraldehyde-3-p dehydrogenase sep when pyruvae is released from he cell (hus avoiding he reoxidaion of NADH by conversion o lacae). If all of his NADH is oxidized by he ciric acid cycle, i could accoun for 11% of he oxygen consumpion. However, considering he acive synhesis of riglyceride and fay acid ha occurs in VSM (Howard, 1971; Morrison e al., 1974) i is quie plausible ha mos or all of he NADH is used for reducive sorage of fa. In his case, he conribuion of glycolyically produced NADH o issue 2 consumpion would be zero. Glycolyically generaed lacae and pyruvae (Table 5) may accoun for 23% of he oal ATP producion. However, his pahway is no essenial since he creaine phosphae: creaine raio and ATP levels are well mainained during subsraefree incubaions where lile or no lacae (<.1 mol/g per hr) is released. Hellsrand e al., (1977) also have shown ha conracile force can be mainained for several hours in he absence of subsrae. We oo have observed a significan increase in oxygen consumpion by VSM in he absence of subsrae (Table 8). This observaion suggess ha he cell may compensae for he lack of glycolyically produced ATP by increasing he oxidaion of some endogenous subsrae. I also has been shown ha dissecion of VSM in he presence of high albumin concenraions leads o a decrease in lacae producion and an increase in oxygen consumpion (Morrison e al., 1976a). A major conclusion of our sudies is ha noncarbohydrae subsraes are imporan fuels for resing VSM. We have observed for he firs ime he oxidaion of keone bodies by VSM which can accoun for 8-16% (Tables 1 and 7) of he issue O 2 consumpion. Our resuls (Tables 3 and 9) also indicae ha branched-chain amino acids are oxidized avidly and migh accoun for as much as 1% of he oxygen consumpion. The oxidaion of amino acids by he swine aora has been repored previously (Morrison e al., 1976b). Alhough ha repor does no presen acual oxidaion raes, i did repor ha gluamine, serine, asparae, and alanine all are oxidized a higher raes han are he branchedchain amino acids. I is no clear wheher he difference beween our resuls and heirs reflecs a species difference, or some difference in mehodology. The medium-chain fay acid, ocanoae, is he mos avidly oxidized of all he subsraes esed, accouning for up o 45% of he 2 consumpion. The exogenously added fay acids of longer chain lengh (Cie, Cis) are oxidized o a much smaller exen (Table 1). These findings confirm he sudies of Hashimoo and Dayon (1971) in he ra aora. Since long-chain fay acids can ener he miochondria only as he carniine eser, whereas medium-chain lengh fay acids can ener as he free acid (Friz e al., 1962; Bode and Klingenberg, 1964), he oxidaion rae of long-chain fay acids by VSM may be limied by he rae of formaion of he carniine eser, or is enry ino he miochondria. However, calculaion of long-chain fay acid oxidaion by VSM based on he specific aciviy in he medium may underesimae markedly he overall conribuion of his fuel, since i ignores he conribuion of endogenous fay acids. In fac, Morrison e al. (1974) have shown ha inracellular specific aciviy of labeled palmiae in rabbi aora is significanly lower han ha of he medium. These observaions sugges ha oxidaion of endogenous fay acids may accoun for he 5% of he O 2 consumpion no derived from exogenous subsraes (unpublished observaion). A presen, i is no possible o exclude enirely some conribuion from he degradaion glycogen or amino acids derived from proein breakdown. In summary, hese experimens indicae ha

9 858 CIRCULATION RESEARCH VOL. 48, No. 6, JUNE 1981 Downloaded from hp://ahajournals.org by on November 5, 218 VSM can uilize a wide variey of subsraes from energy producion. An examinaion of he muual inhibiion by compeing subsraes has revealed ha glucose accouns for only a minor fracion of he oxygen consumpion by VSM in he resuling sae. Non-carbohydrae subsraes, including fay acids keone bodies and he branched-chain amino acids, make a major conribuion o he issue ATP producion. However, in he resing sae, flux hrough he ciric acid cycle is limied, and less han 5% of he oxygen consumpion is derived from exogenous fuels. Work in progress suggess ha oxidaion of endogenous fay acids may accoun for he remainder of he oxygen consumpion. In any case, furher work is required o elucidae he naure of he sored fuel, is ulimae source, and how fuel uilizaion is conrolled in alered meabolic saes. References Bergmeyer HV (1974) Mehods of Enzymaic Analysis, ed 2. 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