Developmental Changes in Ketogenic Enzyme Gene Expression During Sheep Rumen Development M

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Developmental changes in ketogenic enzyme gene expression during sheep rumen development M. A. Lane, R. L. Baldwin, 4th and B. W. Jesse

J Anim Sci 2002. 80:1538-1544.

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Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. Developmental changes in ketogenic enzyme gene expression during sheep rumen development1

M. A. Lane*2, R. L. Baldwin, VI†, and B. W. Jesse*3

*Department of Animal Sciences, Rutgers—The State University of New Jersey, New Brunswick 08903 and †Energy Metabolism Unit, USDA/ARS, Beltsville, MD 20705

ABSTRACT: Ketogenesis is the conversion of acetyl- taryl-CoA synthase, the rate-limiting enzyme in the CoA to the ketone bodies acetoacetate and β-hydroxybu- ketogenic pathway in nonruminant liver, were exam- tyrate (BHBA). In hepatic ketogenesis, which occurs ined. Acetoacetyl-CoA thiolase and 3-hydroxy-3-meth- during fasting in both nonruminant and ruminant ani- ylglutaryl-CoA synthase mRNA concentrations in- mals, the source of acetyl-CoA is the mitochondrial oxi- creased with age independent of diet. 3-Hydroxy-3- dation of predominantly long-chain fatty acids. In the methylglutaryl-CoA synthase mRNA levels in ruminal mature, fed ruminant animal, the ruminal epithelium epithelium obtained from milk-fed lambs were low be- is also capable of producing ketone bodies. In this case, fore 42 d of age, but a marked increase occurred by 42 the source of acetyl-CoA is the mitochondrial oxidation d of age. At 84 d of age, there were no differences in of butyrate produced by the microbial fermentation of acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglu- feed. The purposes of this study were to determine onto- taryl-CoA synthase expression due to diet. The pattern genic and dietary effects on ketogenic enzyme gene ex- of the expression of these genes, in particular, 3-hy- pression in developing lamb ruminal epithelium. droxy-3-methylglutaryl-CoA synthase, parallels the Twenty-seven conventionally reared lambs and twenty- rate of production of BHBA by rumen epithelial cells seven milk-fed lambs were slaughtered between 1 and isolated from the same lambs, which increased to con- 84 d of age. Six additional milk-fed lambs were weaned ventionally reared adult levels at 42 d of age and did (the fed group) or maintained on milk replacer with a not differ with diet. In conclusion, development of the volatile fatty acid gavage (the VFA group) until 84 d of ketogenic capacity of the ruminal epithelium occurs as age. At slaughter, total RNA was extracted from sam- the animal ages, regardless of dietary treatment. Thus, ples of ruminal epithelium. The expression of the genes the expression of the genes encoding the ketogenic en- encoding acetoacetyl-CoA thiolase, the ﬁrst enzyme in zymes are not affected by the presence of VFA in the the ketogenic pathway, and 3-hydroxy-3-methylglu- ruminal lumen.

Key Words: Development, Ketogenesis, Rumen, Sheep, Volatile Fatty Acids

Introduction production from butyrate was not measured by Bald- win and Jesse (1992). Its production is likely to in- The neonatal rumen epithelium is not ketogenic. crease in parallel with BHBA because Giesecke et al. Yet, β-hydroxybutyrate (BHBA) production increases (1979) demonstrated that rumen epithelial slices from sixfold between 42 and 56 d of age in conventionally mature steers produced greater amounts of acetoace- reared lambs (Baldwin and Jesse, 1992). Acetoacetate tate than 14-d-old steers when expressed on a dry tissue weight basis. In milk-fed lambs, BHBA production is minimal before 42 d of age. After 42 d, the rate of BHBA production by rumen epithelial cells isolated 1The authors thank Toshiyuki Fukao of the Gifu University School of Medicine, Gifu, Japan, for the donation of the cDNA probe encoding from milk-fed lambs is similar to that of rumen epithe- rat acetoacetyl-CoA thiolase. The authors also thank Fausto Hegardt lial cells isolated from 56-d-old conventionally reared of the Universidad de Barcelona, Barcelona, Spain, for the donation lambs (Lane et al., 2000). of the cDNA probe encoding human HMG-CoA synthase. Hepatic ketogenesis in nonruminant animals is reg- 2 Present address: Dept. of Human Ecology, University of Texas, ulated by the rate of long-chain fatty acid entry into Austin 78712. the mitochondria (controlled by carnitine-palmityl 3Correspondence: phone: (732)932-8165, ext. 104; fax: (732)932- 6535; E-mail: [email protected]. transferase-I) and 3-hydroxy-3-methylglutaryl-CoA Received July 16, 2001. synthase (HMG-CoA synthase) activity (Quant, Accepted January 4, 2002. 1994). Although ruminal epithelium can oxidize pal-

1538 Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. Ontogeny of rumen ketogenic gene expression 1539 mitate (Jesse et al., 1992), the primary ketogenic sub- The three lambs assigned to the fed group consumed strate is butyrate, which diffuses freely into the mito- milk replacer ad libitum until 49 d of age and were chondria. Thus, carnitine-palmityl transferase-I ac- weaned onto a pelletted lamb starter (330 g/kg corn, tivity is not likely to be a key regulator of ruminal 250 g/kg barley, 167 g/kg 17% protein alfalfa meal, ketogenesis. The activity of HMG-CoA synthase is 167 g/kg soybean meal, and 83 g/kg molasses) between thought to be the primary regulatory step for ketogen- 49 and 77 d of age. All lambs in the fed group were esis as its activity is lower than that of acetoacetyl- slaughtered at 84 d of age. CoA thiolase in both nonruminants (Quant, 1994; Wil- Milk replacer and feed intake were recorded daily, liamson et al., 1968) and mature ruminants (Leighton and body weight was measured weekly. Animal proto- et al., 1983). However, the activities of both acetoace- cols were approved by the Rutgers University and the tyl-CoA thiolase and HMG-CoA synthase parallel he- Beltsville Area Animal Care and Use Committee (pro- patic ketogenesis in growing rats (Lockwood and tocols #88-016 and #94-058, respectively). Baily, 1971; Quant, 1990); thus, both enzymes may At slaughter, the lambs were stunned with a captive regulate flux through this pathway in developing ru- bolt gun and exsanguinated. The rumen was quickly minal epithelium. The objectives of this study were to removed, washed with tap water to remove feed parti- determine the effects of age and diet on acetoacetyl- cles, and the epithelium was separated from the un- CoA thiolase and HMG-CoA synthase mRNA concen- derlying muscle layers. Samples of ventral ruminal tration. epithelium were snap frozen and stored at −70°C for subsequent RNA isolation. Materials and Methods Northern Blot Hybridization Animals Total RNA was isolated from rumen epithelial tissue Twenty-seven conventionally reared lambs were ob- using the method of Chomczynski and Sacchi (1987). tained from the Rutgers University sheep flock. These Total RNA (30 ␮g per lane) was electrophoresed lambs were housed in group pens with free access to through 1.2% denaturing agarose-formamide gels and their dams and feed. All lambs were weaned between transferred onto nylon membranes. The membranes 49 and 56 d of age and thereafter had ad libitum access were hybridized with randomly primed 32P-dCTP-la- to feed. The feed consisted of poor-quality first cutting beled probes (RadPrime DNA Labeling System, Life mixed alfalfa-grass hay and crimped barley supple- mented with vitamin and mineral premixes. Three Technologies, Gaithersburg, MD) encoding the mito- lambs were slaughtered at each of nine ages (0, 4, 7, chondrial isoforms of rat acetoacetyl-CoA thiolase (ob- 14, 28, 42, 49, 56, and 84 d), and ventral ruminal tained from Toshiyuki Fukao at the Gifu University epithelium was collected. School of Medicine, Gifu, Japan) and human HMG- In addition, 33 1-d-old mixed-breed lambs from the CoA synthase (obtained from Fausto Hegardt at the ARS/USDA farm at Beltsville, Maryland, were sepa- Universidad de Barcelona, Barcelona, Spain). All × 6 rated from their dams, trained to suckle milk replacer membranes were hybridized with 2 10 cpm of probe from nipple pails, and housed in individual pens. Milk per milliliter of hybridization buffer. Northern blots replacer (Land O Lakes Ultra Fresh lamb milk re- were hybridized for 18 to 24 h with acetoacetyl CoA ° placer, Land O Lakes, Inc., Fort Dodge, IA) containing thiolase cDNA at 37 C or HMG-CoA synthase cDNA ° × 35% pork fat and 65% dried whey was fed twice daily at 42 C and washed once in 2 SSC (0.3 mM NaCl, and prepared by mixing 185 g of milk replacer powder 0.03 M sodium citrate) plus 1% SDS at room tempera- with1Lofwater. Lambs were randomly divided into ture for 20 min. The second wash for Northern blots three dietary treatment groups: milk-fed (the milk hybridized with acetoacetyl-CoA thiolase cDNA con- × ° group), VFA-infused (the VFA group), and fed (the fed sisted of 2 SSC plus 1% SDS at 37 C for 20 min. The group). Twenty-seven lambs were assigned to the second wash for Northern blots hybridized with HMG- milk group and allowed free access to milk replacer CoA synthase cDNA was conducted at 42°Cin2× SSC until slaughter. Three lambs were slaughtered at each and 1% SDS for 20 min. The final wash for all blots of the following ages: 0, 4, 7, 14, 28, 42, 49, 56, and consisted of 0.5 × SSC for 20 min at room temp. All 84 d. blots were exposed to Hyperfilm (Amersham, Arling- The three lambs assigned to the VFA group con- ton, OH) for 4 d. Autoradiographs and the 28S rRNA sumed milk replacer ad libitum and, beginning at 49 band on the gel photographs were quantified using a d of age, received an intraruminal infusion of a VFA Logitech Scanman scanner (Freemont, CA) and the solution consisting of an acetate:propionate:butyrate software package U-Scan-It (Silk Scientific Corpora- molar ratio of 55.2:36.9:7.2 mmol/100 mmol, respec- tion, Orem UT). The amount of acetoacetyl-CoA thio- tively, at a concentration of 1 g/mL. Volatile fatty acids lase or HMG-CoA synthase mRNA in each lane was were administered via oral gavage three times daily divided by the amount of 28S rRNA in each lane, to to meet 10% of the predicted NEg requirement. All correct for unequal gel loading. The resulting optical lambs in the VFA group were slaughtered at 84 d density was then expressed as a portion of the mRNA of age. expressed by the 84-d sample to determine the effect Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. 1540 Lane et al. of age on acetoacetyl-CoA thiolase or HMG-CoA synthase mRNA levels in milk-fed or conventionally reared lambs. To determine the effect of diet on acetoacetyl-CoA thiolase or HMG-CoA synthase mRNA levels the optical density for 84 d conventionally reared, VFA, or fed lambs was expressed as a portion of the 84-d milk-fed sample.

Statistical Analysis

All data are presented as mean +/− SEM. The gen- eral linear model procedure of SAS (SAS Inst. Inc., Cary, NC) was used to perform one-way ANOVA to test for the effect of age within the milk-fed group and conventionally reared group. One-way ANOVA were also performed to determine the effect of diet between the four treatment groups at 84 d of age. Significant mean differences were determined by a least significant differences test (SAS, Inst. Inc.). Results were considered significant at P < 0.05.

Results and Discussion

Hepatic and ruminal ketogeneses occur at similar rates in the adult, fed, nonlactating, and nonpregnant animal (Heitmann et al., 1987). The production of BHBA from butyrate increases sixfold between 42 and 56 d of age in conventionally reared lambs (Baldwin and Jesse, 1992). Before 42 d of age, in both conventionally reared (Baldwin and Jesse, 1992) and milk- fed lambs (Lane and Jesse, 1996; Lane et al., 2000), Figure 1. Acetoacetyl-CoA thiolase mRNA abundance BHBA production is minimal. Surprisingly, after 42 in ruminal epithelium from conventionally reared lambs d of age, BHBA production rates by rumen epithelial of increasing age. a) Total rumen epithelial RNA was cells isolated from the milk-fed lambs from the current probed with a full-length cDNA construct encoding experiment (Lane et al., 2000) are similar to produc- acetoacetyl-CoA thiolase. The cDNA probe hybridized to tion rates observed when rumen epithelial cells were an mRNA transcript of 1.7 kb in length. b) Messenger isolated from 56-d-old conventionally reared lambs RNA was quantified using a Logitech Scanman scanner (Baldwin and Jesse, 1992). Messenger RNA encoding (Freemont, CA). Arbitrary mRNA optical density (OD) acetoacetyl-CoA thiolase, the first enzyme in the keto- units are expressed as proportion of mRNA expressed genic pathway, increased with age in both convention- by the 84-d-old lamb. Values are mean ± SEM OD (n = ally reared (Figure 1) and milk-fed lambs (Figure 2), 3 lambs per age). Results were considered significant at reflecting the increase in rumen epithelial ketogenesis P < 0.05. regardless of dietary treatment (Lane et al., 2000). The peak in acetoacetyl-CoA thiolase mRNA concentration at 4 d of age in ruminal epithelium from conventionally reared lambs (Figure 2b) was due to a high 49 d of age, HMG-CoA synthase gene expression had degree of variance in the expression of this gene at increased sixfold in ruminal epithelium from milk-fed this age among the three experimental animals. At 84 lambs (Figure 5), and by 84 d of age there were no d of age, expression of the acetoacetyl-CoA thiolase differences in HMG-CoA mRNA concentrations due to gene was similar regardless of dietary treatment (Fig- dietary treatment (Figure 6). Strikingly, the observed ure 3), again reflecting the rate of rumen epithelial increase in HMG-CoA synthase gene expression in ru- ketogenesis (Lane et al., 2000). minal epithelium from milk-fed lambs exactly paral- Expression of the HMG-CoA synthase gene in- lels the increase in BHBA production by rumen epithe- creased with age (P < 0.05) in conventionally reared lial cells isolated from the same lambs at this age lambs (Figure 4). In contrast, the expression of this (Lane et al., 2000). Thus, the rate of ruminal ketogene- mRNA species remained low through 42 d of age in sis, at least in milk-fed lambs, is probably controlled milk-fed lambs (Figure 5). Thus, dietary regimen ap- by HMG-CoA synthase activity and not by acetoacetyl pears to alter the ontogenic expression of the HMG- CoA thiolase, which did not increase sharply at 49 d CoA synthase gene before 42 d of age. However, by of age in milk-fed lambs. Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. Ontogeny of rumen ketogenic gene expression 1541

Figure 2. Acetoacetyl-CoA thiolase mRNA abundance in ruminal epithelium from milk-fed lambs of increasing Figure 3. Acetoacetyl-CoA thiolase mRNA abundance age. a) Total rumen epithelial RNA was probed with in ruminal epithelium from lambs in different dietary a full-length cDNA construct encoding acetoacetyl-CoA treatment groups. a) Total rumen epithelial RNA was thiolase. The cDNA probe hybridized to an mRNA tran- probed with a full-length cDNA construct encoding script 1.7 kb in length. b) Messenger RNA was quantified acetoacetyl-CoA thiolase. The cDNA probe hybridized using a Logitech Scanman scanner (Freemont, CA). Arbi- to an mRNA transcript 1.7 kb in length. Conventionally trary mRNA optical density (OD) units are expressed as reared is abbreviated Conv. b) Messenger RNA was quan- proportion of mRNA expressed by the 84-d-old sample. tified using a Logitech Scanman scanner (Freemont, CA). Values are mean ± SEM OD (n = 3 lambs per age except Arbitrary mRNA optical density (OD) units are expressed for the 42-, 49-, and 56-d time points, where n = 2). Results as proportion of mRNA expressed by the 84-d-old milk- were considered significant at P < 0.05. fed sample. Conventionally reared is abbreviated Conv. Values are mean ± SEM OD (n = 3 lambs per treatment). Results were considered significant at P < 0.05. Expression of the HMG-CoA synthase gene in suck- ling rats parallels hepatic ketogenesis (Thumelin, 1993), and the activity of HMG-CoA synthase is be- HMG-CoA synthase is accomplished at the transcrip- lieved to regulate flux through the intramitochondrial tional level (Quant, 1990; Thumelin, 1993). The abun- ketogenic pathway in rat liver (Quant, 1994; William- dance of the mRNA encoding HMG-CoA synthase (Fig- son et al., 1968). Furthermore, in mature ruminal epi- ure 5) increases in parallel with rumen epithelial keto- thelium, the activity of HMG-CoA synthase is lower genic capacity, thus flux through HMG-CoA synthase than that of acetoacetyl-CoA thiolase but is high and the resultant ketogenesis may be limited by HMG- enough to account for ketogenic rates observed in vitro CoA synthase mRNA production before 49 d of age. (Leighton et al., 1983). Thus, HMG-CoA synthase ac- After 49 d of age, regardless of dietary treatment, tivity may regulate flux through the intramitochon- there were no differences in HMG-CoA synthase drial ketogenic pathway in ruminal epithelium and mRNA concentrations (Figure 6) or rumen epithelial expression of the gene may control the developmental ketogenic rates (Lane et al., 2000). pattern of ketogenesis. Although it is generally believed that intraruminal Short-term regulation of HMG-CoA synthase activ- butyrate stimulates gene transcription and ultimately ity is achieved by succinylation of the enzyme (Lowe rumen maturation, we show here that the develop- and Tubbs, 1985), whereas long-term regulation of ment of ruminal ketogenesis is probably independent Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. 1542 Lane et al.

Figure 5. HMG CoA synthase mRNA abundance in Figure 4. HMG CoA synthase mRNA abundance in ruminal epithelium from milk-fed lambs of increasing ruminal epithelium from conventionally reared lambs of age. a) Total rumen epithelial RNA was probed with a increasing age. a) Total rumen epithelial RNA was probed full-length cDNA construct encoding HMG-CoA syn- with a full-length cDNA construct encoding HMG-CoA thase. The cDNA probe hybridized to an mRNA tran- synthase. The cDNA probe hybridized to an mRNA transcript 2.0 kb in length. b) Messenger RNA was quantified script 2.0 kb in length. b) Messenger RNA was quantified using a Logitech Scanman scanner (Freemont, CA). Arbi- using a Logitech Scanman scanner (Freemont, CA). Arbi- trary mRNA optical density (OD) units are expressed as trary mRNA optical density (OD) units are expressed as proportion of mRNA expressed by the 84-d-old sample. proportion of mRNA expressed by the 84-d-old sample. Values are mean ± SEM OD (n = 3 lambs per age except Values are mean ± SEM OD (n = 3 lambs per age). Results for the 42-, 49-, and 56-d time points, where n = 2). Results were considered significant at P < 0.05. were considered significant at P < 0.05.

of intraruminal butyrate concentrations. A very small significantly larger than rumens from lambs in the amount of butyrate was detected within the rumens milk-fed or VFA treatment groups (unpublished data). of milk-fed lambs (Lane et al., 2000), and we cannot Although there were no differences in the amount of rule out that even this small amount affected keto- ketone bodies produced per million cells (Lane et al., genic gene transcription either directly or indirectly. 2000) or ketogenic enzyme mRNA levels among the To determine whether low concentrations of butyrate different dietary treatments, the larger rumens of the are affecting rumen ketogenic development, the pro- lambs consuming solid feed would produce more ke- duction of ketone bodies from rumen epithelial cells tone bodies simply because there is more tissue. An isolated from gnotobiotic milk-fed lambs could be ex- ontological increase in ketogenesis, combined with an amined. increase in rumen size due to solid feed consumption It is important to consider the effects of dietary would supply the adult ruminant with the large treatment on rumen size when considering the contri- amount of ketone bodies required to fulfill their en- bution of the entire ruminal epithelium to ketogenesis ergy needs. on a whole-animal level. Rumens from conventionally Although this is the first paper showing ontogenic reared lambs as well as lambs in the fed group were regulation of genes involved in rumen metabolism, Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. Ontogeny of rumen ketogenic gene expression 1543 the HMG-CoA synthase gene will lead to the identifi- cation of ontogenic signals regulating the expression of this gene and ruminal ketogenesis. In conclusion, during rumen development, the activities of both acetoacetyl-CoA thiolase and HMG-CoA synthase may be regulated by the abundance of mRNA encoding these enzymes. Because increases in HMG- CoA synthase mRNA level and BHBA production (Lane et al., 2000) both occur from 42 to 49 d of age, we believe that HMG-CoA synthase is the rate-limiting enzyme in ruminal ketogenesis. The expression of these genes increases with animal age, regardless of dietary treatment. This indicates that the presence of VFA within the rumen is not required for the development of the ketogenic capacity of the ruminal epithelium.

Implications

The genes responsible for stimulating rumen development are not known. The development of ketogenic ruminal epithelium is a hallmark of the maturation of ruminal epithelium. It is believed that the volatile fatty acids produced by the fermentation of feed within the rumen stimulate rumen metabolic development, including ketogenesis. This paper shows that the genes encoding the enzymes controlling ketogenesis are expressed independently of intraruminal volatile fatty acid concentrations. We also show that the rate of the production of ketone bodies by the ruminal epithelium parallels the expression of the gene encoding Figure 6. HMG CoA synthase mRNA abundance in the enzyme 3-hydroxy-3-methylglutaryl-CoA syn- ruminal epithelium from lambs in different dietary treat- thase, which controls flux through this metabolic path- ment groups. a) Total rumen epithelial RNA was probed way in nonruminant animals. Future studies in this with a full-length cDNA construct encoding HMG-CoA area will determine which genes regulate other as- synthase. The cDNA probe hybridized to an mRNA tran- pects of rumen development and the effects of volatile script 2.0 kb in length. Conventionally reared is abbrevi- fatty acids on these genes. ated Conv. b) Messenger RNA was quantified using a Logitech Scanman scanner (Freemont, CA). Arbitrary Literature Cited mRNA optical density (OD) units are expressed as proportion of mRNA expressed by the 84-d-old milk-fed Baldwin, VI, R. L., and B. W. Jesse. 1992. Developmental changes sample. Conventionally reared is abbreviated Conv. Val- in glucose and butyrate metabolism by isolated sheep rumen ues are mean ± SEM OD (n = 3 lambs per treatment). epithelial cells. J. Nutr. 122:1149–1153. < Chomczynsk, P., and N. Sacchi. 1987. Single-step method of RNA Results were considered significant at P 0.05. isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 62:156–159. Giesecke, D., U. Beck, S. Wiesmayr, and M. Stangassinger. 1979. ontogenic regulation of gene expression does occur The effect of rumen epithelial development on metabolic activi- during the development of the nonruminant intestinal ties and ketogenesis by tissue in vitro. Comp. Biochem. Physiol. tract. For example, although sucrase levels are af- 62B:459–463. fected by diet, Yen and Holt (1986) showed that the Heitmann, R. N., D. J. Dawes, and S. C. Sensenig. 1987. Hepatic ketogenesis and peripheral ketone body utilization in the rumi- timing of initial sucrase appearance in rodent intes- nant. J. Nutr. 117:1174–1180. tines was independent of luminal contents. When Jesse, B. W., R. K. Solomon, and R. L. Baldwin. 1992. Palmitate grafts of intestine were transplanted into animals 5 metabolism by isolated sheep rumen cells. J. Anim. Sci. d younger than the donors, sucrase appeared earlier 70:2235–2242. in the graft than in the rest of the intestine. This Lane, M. A., R. L. Baldwin VI, and B. W. Jesse. 2000. Sheep rumen suggests that sucrase is induced by a biological clock metabolic development in response to different dietary treatments. J. Anim. Sci. 78:1990–1996. within the intestine itself. The same may be true of Lane, M. A., and B. W. Jesse. 1996. Effect of volatile fatty acid the enzymes involved in rumen ketogenesis. Analysis infusion on development of neonatal sheep rumen epithelium. of the regulatory sequences in the promoter region of J. Dairy Sci. 80:740–746. Downloaded from jas.fass.org at USDA Natl Agricultural Library on March 21, 2008. Copyright © 2002 American Society of Animal Science. All rights reserved. For personal use only. No other uses without permission. 1544 Lane et al.

Leighton, B., A. R. Nicholas, and C. I. Pogson. 1983. The pathway Quant, P. A, P. K. Tubbs, and M. D. Brand. 1990. Glucagon activates of ketogenesis in rumen epithelium of the sheep. Biochem. J. mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in 216:769–772. vivo by decreasing the extent of succinylation of the enzyme. Lockwood, E. A., and E. Bailey. 1971. The course of ketosis and Eur. J. Biochem. 187:169–174. the activity of key enzymes of ketogenesis and ketone-body Thumelin, S., M. Forestier, J. Girard, and J. Pegorier. 1993. Devel- utilization during development of the postnatal rat. Biochem. opmental changes in mitochondrial 3-hydroxy-3-methylglu- J. 124:249–254. taryl-CoA synthase gene expression in rat liver, intestine and Lowe, D. M., and P. K. Tubbs. 1985. 3-Hydroxy-3-methylglutaryl- kidney. J. Biochem. 292:493–496. coenzyme A synthase from ox liver. Biochem. J. 227:591–599. Williamson, D. H., M. W. Bates, and H. A. Krebs. 1968. Activity Quant, P. A. 1990. Activity and expression of hepatic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase during the starved- and intracellular distribution of enzymes of ketone body metab- to-fed transition. Biochem. Soc. Trans. 18:994–995. olism in rat liver. Biochem. J. 108:353–361. Quant, P. A. 1994. The role of mitochondrial HMG-CoA synthase Yeh, K. Y., and P. R. Holt. 1986. Ontogenic timing mechanism in regulation of ketogenesis. In: K. F. Tipton (ed.) Essays in initiates the expression of rat sucrase activity. Gastroenterol- Biochemistry. pp 13–25. Portland Press Ltd., London, U.K. ogy 90:520–526.

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