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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal Science, Texas A&M University, College Station 77843;
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Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan; and
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Texas A&M University McGregor Research Center, McGregor 76657
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: adipose tissue • bovine • lipogenesis • stearoyl-CoA desaturase
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; May et al., 1993; Chung et al., 2006). In steers fed identical, high-roughage diets to the same physiological maturity, Wagyu s.c. adipose tissue lipids had a greater MUFA:SFA ratio than Angus adipose tissue lipids (May et al., 1993). However, stearoyl-CoA desaturase (SCD) enzyme activity and gene expression were similar in the s.c. adipose tissues of the Wagyu and Angus steers (Cameron et al., 1994). Therefore, the greater MUFA:SFA ratio the Wagyu steers may have been caused by greater SCD activity in Wagyu adipose tissue at some point earlier in production. To match physiological maturity, the Wagyu steers of May et al. (1993) were approximately 8 mo older than the Angus steers (Lunt et al., 1993), so the differences in the MUFA:SFA ratio reported by May et al. (1993) may have been caused by disparities in age rather than genetic differences between the Wagyu and Angus steers.
Expression of the SCD gene tripled from 5 to 12 mo of age in s.c. adipose tissue of Angus steer calves (Martin et al., 1999). From 12 to 18 mo of age, while the steers were fed a corn-based finishing diet, de novo lipogenesis increased nearly 3-fold, but SCD mRNA levels declined somewhat. Lipogenesis in s.c. adipose tissue declined in British-cross cattle after 19 mo of age (Smith et al., 1984). Therefore, we hypothesized that any changes in SCD gene expression and activity would precede similar changes in adipose tissue lipogenesis between short- and long-fed endpoints. Furthermore, changes in SCD activity and gene expression between production endpoints would differ between corn- and hay-fed steers and between Wagyu and Angus steers.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and are summarized briefly here. Care, handling, and sampling of the steers were approved by the Texas A&M University Institutional Animal Care and Use Committee. Sixteen Angus and 16 American Wagyu steers were purchased as weanling calves (approximately 8 mo of age) and assigned to 1 of 2 dietary treatments: a high-energy, corn-based finishing diet designed to support 1.36 kg of ADG/d; and a medium-energy, coastal Bermudagrass hay-based diet designed to support 0.9 kg of ADG/d (Table 1). One Angus steer from the corn-fed group escaped the holding pen before slaughter and had to be removed from the investigation.
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For the corn-fed steers, actual ADG for the Angus and Wagyu steers were 1.25 and 1.03 kg, respectively (short-fed), and 0.93 and 0.84 kg, respectively (long-fed). For the hay-fed steers, actual ADG for the Angus and Wagyu steers were 0.88 and 0.84 kg, respectively (short-fed) and 0.78 and 0.73 kg, respectively (long-fed; Lunt et al., 2005). The average initial BW for Angus and Wagyu steers were 210 and 174 kg, respectively, and the diets were designed so that hay-fed and corn-fed steers within a breed type achieved a similar BW at sampling. The short-fed Angus and Wagyu steers weighed 527 and 454 kg, respectively, at sampling. The long-fed Angus and Wagyu steers weighed 663 and 588 kg, respectively, at sampling (Lunt et al., 2005).
After being fed for their respective time periods, the steers in each group were slaughtered on 2 consecutive days. All steers were slaughtered at the Rosenthal Meat Science and Technology Center on the Texas A&M University campus. Immediately postexsanguination, a portion of the LM from the fifth to eighth thoracic vertebrae region (with adhering s.c. adipose tissue) was removed for sampling of adipose tissue for lipogenesis and cellularity (s.c. and i.m.), or SCD enzyme activity and gene expression (s.c. only).
In Vitro Lipogenesis
Two-hour in vitro incubations were performed with fresh i.m. and s.c. adipose tissues as described previously (May et al., 1994). Acetate incorporation into neutral lipids was measured by incubating adipose tissue (50 to 100 mg) in Krebs-Henseleit bicarbonate buffer (pH 7.4), 5 mM sodium acetate, 5 mM glucose, 10 mM hepes buffer, and 1 µCi [U-14C]acetate (Amersham, Arlington Heights, IL) in 3 mL of total volume. The vials were gassed for 1 min with 95% O2:5% CO2, capped, and incubated in a shaking water bath (37°C) for 2 h. The reactions were terminated by adding 3 mL of 5% trichloroacetic acid to each vial.
Lipids were extracted according to published procedures (Folch et al., 1957; May et al., 1994). Lipids were evaporated to dryness with a MultiVap evaporator (Associates Inc., South Berlin, MA) in a 40°C water bath. The lipids were resuspended with 10 mL of Econo-Safe scintillation fluid (Research Products International Corp., Mount Prospect, IL) and counted on a Beckman LS-3800 scintillation counter (Beckman Instruments, Palo Alto, CA). The incorporation rate of acetate into neutral lipid is expressed as nmol·2 h–1·105 cells–1.
Cellularity
The procedure of Etherton et al. (1977), as modified by Prior (1983), was used to estimate the number of adipocytes per gram of adipose tissue. Frozen i.m. and s.c. adipose tissues were sectioned (1-mm thick), fixed with 3% osmium tetraoxide, and digested with 8 M urea. The fixed cells were filtered through 240-, 64-, and 20-µm nylon mesh screens with 0.01% Triton in 0.154 M NaCl. Cell fractions collected from the 64 and 20-µm mesh screens were used to determine cells per gram of adipose tissue with a Coulter Counter [Model ZM equipped with a channelizer (Model Z56), Coulter Electronics, Hialeah, GA].
Microsome Extraction and SCD Enzyme Activity
Subcutaneous adipose tissue was homogenized in 3 volumes (wt/vol) of buffer with a Polytron (The Virtis Company Inc., Gardiner, NY) for 60 s. The buffer (pH = 7.4) was composed of 0.25 M sucrose, 0.01 M potassium phosphate, 1 mM EDTA, and 1 mM dithioerythritol. The homogenate was centrifuged at 5,000 x g for 15 min. The supernate was decanted into another tube, and the pellet and fat cake were discarded. The supernate was centrifuged at 17,300 x g for 30 min and decanted into another tube, which was centrifuged at 104,000 x g for 2 h. The supernate was discarded, and the microsomes were resuspended in 100 mM Tris-HCl buffer (pH = 7.4), snap-frozen in liquid nitrogen, and stored at –80°C until further analysis.
Stearoyl-CoA desaturase enzyme activity was determined as described by St. John et al. (1991) and as modified by Archibeque et al. (2005). The assay system was composed of 100 mM Tris-HCl (pH 7.4), 2 mM NADPH, 25 µM palmitoyl-CoA, and 0.025 µCi of [1-14C]palmitoyl-CoA in 1.5 mL of total volume. The reactions were begun by addition of 0.1 mg of protein and were incubated in a 37°C water bath for 7 min. The reaction was stopped by adding 1 mL of 12% KOH in ethanol, followed by heating for 1 h at 80°C. After acidification by addition of 9 mL of 3 N HCl, fatty acids were washed with 9 mL of n-pentane. The pentane phases were evaporated under nitrogen and methylated by the addition of 14% BF3 in methanol. Methyl esters were separated by thin layer chromatography on a 10% AgNO3-impregnated, silica gel plate in a petroleum ether:diethylether (97:3) solvent system. Plates were sprayed with 0.2% dichlorofluoroscein in ethanol, and spots containing palmitate methyl ester and palmitoleate methyl ester were scraped and counted using a liquid scintillation spectrometer. The ratio of palmitate methyl ester:palmitoleate methyl ester was used to quantify nanomoles of palmitic acid converted to palmitoleic acid·7 min–1·mg of microsomal protein–1.
Northern Blot Analysis
The cDNA fragment of the bovine SCD gene (662 bp, GenBank accession number AB075020) was cloned into the vector of pCRII-TOPO (Invitrogen, Carlsbad, CA) using reverse transcription-PCR (RT-PCR) and conventional recombinant DNA techniques. The primers used for RT-PCR were: forward primer, 5'-CCTGTGGAGTCACCGAACC-3', and reverse primer, 5'-CCTTGGATACTTTCTTCCGGTC-3'. The cDNA insert was confirmed by sequencing. The digoxigenin (DIG)-labeled anti-sense RNA probe was generated with DIG RNA Labeling Kit (SP6/T7, Roche Diagnostics, Mannheim, Germany).
Total RNA samples (2 µg) were separated with a 1% agarose gels containing 6.7% formaldehyde and transferred to a Hybond N+ membrane (Amersham). The membrane was hybridized with the DIG-labeled RNA probes. The bands corresponding to SCD mRNA were detected using DIG Luminescent Detection Kit for Nucleic Acids (Roche Diagnostics). Northern blotting was analyzed by ImageQuant, version 5.2 software (Molecular Dynamics, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The SCD bands were normalized to the 28S ribosomal RNA bands.
Statistical Analyses
Data were analyzed using the GLM procedure (SAS Inst. Inc., Cary, NC) as a 3-factor design. Data for cellularity, lipogenesis, SCD enzyme activity, and SCD gene expression were evaluated for the main effects of breed type, diet, and end point (short- vs. long-fed), and the model tested all 2- and 3-way interactions against their appropriate error terms in the F-test. Interaction means were separated using the probability statement of GLM (P < 0.05).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). There was a breed type x end point interaction (P = 0.03); short-fed Angus steers had fewer adipocytes/100 mg of s.c. adipose tissue than long-fed Angus steers, whereas the reverse was true for Wagyu steers.
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). The greatest lipogenic activity was observed in adipose tissues of short-fed cattle and was over 90% less than in long-fed cattle consuming the corn-based diet. Conversely, lipogenesis in i.m. and s.c. adipose tissues was greater in long-fed steers than in short-fed steers consuming the hay-based diet. Lipogenesis tended to be greater (P = 0.06) in Wagyu s.c. adipose tissue than in Angus s.c. adipose tissue, primarily because of the greater rates in samples from the short-fed Wagyu steers consuming the corn-based diet. Lipogenesis was greater (P < 0.01) in s.c. adipose tissue of corn-fed steers than in hay-fed steers.
Stearoyl-CoA Desaturase Enzyme Activity and Gene Expression
There was no effect of breed type (P = 0.99) or diet (P = 0.40) on SCD enzyme activity (Figure 2). There was greater SCD activity in s.c. adipose tissue from long-fed steers than in short-fed steers (P < 0.01), and there tended to be diet x end point (P = 0.08) and breed x diet x end point (P = 0.08) interactions. The diet x end point interaction was caused by a greater increase in SCD activity over time in corn-fed steers than in hay-fed steers (Figure 1). The 3-way interaction indicated that SCD activity was greater in long-fed than in short-fed Angus steers consuming the corn-based diet; SCD activity was similar in short- and long-fed Angus steers consuming the hay-based diet, and the increase in SCD activity over time was similar in corn-and hay-fed Wagyu steers.
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). The northern blot indicated that SCD gene expression was very low in adipose tissue from the short-fed steers fed the corn-based diet. For this reason, there was a diet x end point interaction (P = 0.05), in that SCD gene expression was less in corn-fed, short-fed steers than in the any of the other production groups.
Expression of the SCD gene tended (P = 0.06) to be greater in hay-fed steers than in corn-fed steers and tended (P = 0.06) to be greater in long-fed cattle than in short-fed cattle (Figure 2). The northern blots indicated that the greatest degree of SCD hybridization was observed in Wagyu adipose tissue, especially in samples from long-fed Wagyu steers fed the corn-based diet. There was no main effect of breed type on SCD gene expression (P = 0.34), but there was a breed x end point interaction (P < 0.01); SCD gene expression was less in adipose tissue of short-fed than in long-fed Wagyu steers but was greater in adipose tissue of short-fed than in long-fed Angus steers.
There was a significant relationship between the 16:1n-7/18:0 ratio and SCD activity (R2 = 0.37; P = 0.02; Figure 3A) and between the 16:1n-7/18:0 ratio and SCD gene expression (R2 = 0.27; P = 0.05; Figure 3B), but only for the corn-fed steers. There was no relationship between the 16:1n-7/18:0 ratio and SCD activity or gene expression for the hay-fed steers (R2 = 0.05, P = 0.43; and R2 = 0.05, P = 0.42, respectively).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and are summarized briefly in Table 3. Angus steers had greater carcass weights, adjusted fat thickness, and yield grades than Wagyu steers. Long-fed steers had greater carcass weights, marbling scores, adjusted fat thickness, and yield grades than short-fed steers. Corn-fed steers had greater marbling scores than hay-fed steers. There was a much greater increase in yield grades between the short- and long-fed endpoints in Angus steers than in Wagyu steers.
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We previously demonstrated that acetate incorporation into fatty acids in s.c. adipose tissue of Angus x Hereford and Red Poll steers increased approximately 9-fold between 10 and 16 mo of age and then decreased by 50% with additional time on feed (to 19 mo of age; Smith et al., 1984). A similar pattern was observed for activities of the lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthetase, ATP-citrate lyase, and NADP-malate dehydrogenase (Smith et al., 1984). In the current study, the depressed rate of lipogenesis in adipose tissues of long-fed steers fed the corn-based diet was similar to the loss of lipogenic capacity reported in our early study. Expression of the SCD gene tripled from 5 to 12 mo of age in s.c. adipose tissue of Angus steer calves (Martin et al., 1999). From 12 mo of age, the calves were fed a corn-based finishing diet to 18 mo of age. During this latter period, the rate of de novo lipogenesis nearly tripled, but SCD mRNA levels declined somewhat. Taken together, the results of our previous experiments suggested that increases in SCD gene expression preceded proportional increases in lipogenesis in s.c. adipose tissue. Therefore, we hypothesized that any changes in SCD activity that occurred between production endpoints would be reflected in parallel changes in lipogenesis. This was true for hay-fed steers of the current study, which exhibited slightly greater rates of SCD activity and lipogenesis in long-fed cattle than in short-fed cattle. In corn-fed steers, however, there was over a 2-fold increase in SCD activity between endpoints, whereas lipogenesis declined sharply at some time between endpoints. This suggests that SCD gene expression and the expression of genes contributing to de novo fatty acid biosynthesis were regulated independently in s.c. adipose tissue of the corn-fed cattle.
The results of this study confirmed our final hypothesis that the patterns of change over time for SCD activity and gene expression would differ between breed types. The long-fed, hay-fed Angus and Wagyu steers represented the group most similar to the steers of our previous reports (Lunt et al., 1993; May et al., 1993; Cameron et al., 1994). In the current study, SCD activity was 20% greater, and SCD mRNA was 70% greater, in Wagyu than in Angus adipose tissue. Cameron et al. (1994) reported that SCD activity was 20% less (not greater) and SCD mRNA was 45% greater in Wagyu than in Angus adipose tissue (n = 5 per breed type), although neither difference was significant. The Wagyu steers of Cameron et al. (1994) were over 32 mo of age, whereas the Angus steers were 26 mo of age; and SCD gene expression and catalytic activity may have been declining in the Wagyu steers by the time the cattle were sampled.
Lee et al. (2005) reported a nearly 3-fold increase in SCD gene expression from 6 to 12 mo of age in LM of Korean Hanwoo steers; there was a slight decline in SCD mRNA levels between 18 and 30 mo of age. The Hanwoo steers of Lee et al. (2005) were fed a commercial concentrate to 12 mo of age, followed by free choice access to concentrates to 30 mo of age. Details of the diets were not provided by Lee et al. (2005), but probably the diet was intermediate in concentrates between our corn- and hay-based diets. The small decline in SCD mRNA levels between 18 and 30 mo of age reported by Lee et al. (2005) was similar to the results observed for the short- and long-fed Angus steers of this study. Therefore, the pattern of SCD gene expression over time in the Hanwoo steers more closely resembled that of the Angus steers than the Wagyu steers.
Lee et al. (2005) also demonstrated that the increase in SCD gene expression in Hanwoo adipose tissue preceded an increase in a 
9 desaturase index (the ratio of 18:1n-9/18:0). To confirm the relationship between SCD activity and gene expression and adipose tissue fatty acid composition in the current study, s.c. adipose tissue 16:1n-7/18:0 ratios were plotted as a function of SCD activity and gene expression. Although there was a significant relationship between the 16:1n-7/18:0 ratio and SCD activity and between the 16:1n-7/18:0 ratio and SCD gene expression, this was only for the corn-fed steers. We have argued against the use of fatty acid ratios to predict SCD activity (Archibeque et al., 2005). The 16:1n-7/18:0 ratio may be predictive of SCD activity under some conditions, such as the corn-fed steers of this study, but certainly failed to predict SCD activity in the hay-fed steers.
There is a remarkably consistent relationship between the concentrations 16:1n-7 and 18:0 in adipose tissues across studies. For adipose tissue lipids from these steers, the relationship between 16:1n-7 and 18:0 was y = 0.015x2 – 0.796x + 12.32; R2 = 0.93 (Chung et al., 2006). The relationship across 4 independent studies from our laboratory was 0.016x2 – 0.835x + 12.04; R2 = 0.91 (reviewed in Smith et al., 2004). Unlike 18:1n-9, there is little 16:1n-7 present in the diet, so any 16:1n-7 occurring in adipose must arise from desaturation of 16:0. The high correlation between 16:1n-7 and 18:0 further indicates that, as 16:1n-7 concentrations increase, 18:0 concentrations decrease, arguing strongly that SCD activity regulates the 16:1n-7/18:0 ratio. However, the lack of correlation between the 16:1n-7/18:0 ratio and SCD activity of the hay-fed steers indicates that SCD activity did not solely dictate the concentrations of these fatty acids.
The plasma 16:1n-7/18:0 ratio was over 50% less in hay-fed steers than in corn-fed steers (Chung et al., 2006). We previously reported SCD activity in bovine liver and duodenal mucosal cells (Chang et al., 1992; Archibeque et al., 2005) and suggested that some portion of the MUFA in adipose tissues may arise from hepatic and mucosal desaturation of dietary SFA. The lesser 16:1n-7/18:0 ratio in plasma of hay-fed steers suggests depressed SCD activity in liver cells, intestinal mucosal cells, or both, relative to corn-fed steers. Thus, the uptake and deposition of plasma fatty acids may strongly influence adipose tissue fatty acid composition, especially under conditions in which endogenous rates of fatty acid biosynthesis are depressed (i.e., as in the adipose tissues of the hay-fed steers).
Chung et al. (2006) confirmed our previous observation (May et al., 1993) that Wagyu adipose tissue has a greater MUFA:SFA ratio than Angus adipose tissue and that this is true even when cattle are sampled at the same age. Previous studies reported contrasts in SCD activity between breed types (Cameron et al., 1994; Siebert et al., 2003), whereas others reported developmental changes in SCD gene expression (Martin et al., 1999; Lee et al., 2005). This is the first study to demonstrate the interactions between breed type, age, and diet on adipose tissue lipogenesis and SCD activity and gene expression.
1 Corresponding author: sbsmith{at}tamu.edu
Received for publication February 15, 2006. Accepted for publication July 18, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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9 desaturase activity between Jersey- and Limousin-sired cattle. Lipids 38:539–543.[CrossRef][Medline]This article has been cited by other articles:
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  K. Y. Chung and B. J. Johnson Application of cellular mechanisms to growth and development of food producing animals J Anim Sci, April 1, 2008; 86(14_suppl): E226 - E235. [Abstract] [Full Text] [PDF]
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S. B. Smith, A. A. Chapman, D. K. Lunt, J. J. Harris, and J. W. Savell Adiposity of calf- and yearling-fed Brangus steers raised to constant-age and constant-body weight endpoints J Anim Sci, May 1, 2007; 85(5): 1136 - 1143. [Abstract] [Full Text] [PDF]
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