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. The online version of this article, along with updated information and services, is located on the World Wide Web at: http://jas.fass.org www.asas.org 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 2002 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2002. 80:1538–1544 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 produc- tion 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.