LIPOGENESIS IN ISOLATED I N T R A M U S C U L A R ADIPOSE TISSUE FROM FOUR BOVINE MUSCLES 1
University of Minnesota, St. Paul 55101
SUMMARY
The incorporation of acetate-1-14C into CO2 and total fatty acids and the activities of NADP-malate dehydrogenase, NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and acetyl CoA carboxylase were studied in intramuscular adipose tissue from the trapezius, longissimus, semimembranosus and pectoralis profundus muscles of 470 kg, 14-month-old Hereford • Angus steers. It was concluded that intramuscular lipid can be synthesized in situ, but at a slower rate than subcutaneous lipid. The rate of synthesis of fatty acids and CO2 from acetate-l-14C was directly related to adipose cell volume and for this reason the intramuscular adipose tissue of the pectoralis profundus muscle was more active than that of the other three muscles. The activities of the four NADPH-generating enzymes were positively related to both adipose cell volume and fatty acid synthesis. NADP-malate dehydrogenase activity was low in intramuscular adipose tissue; however, the hexose monophosphate shunt dehydrogenases and possibly NADP-isocitrate dehydrogenase are capable of supplying NADPH for fatty acid synthesis in bovine intramuscular adipose tissue. (Key Words: Lipogenesis; Intramuscular Adipose Tissue; Bovine Muscles; Adipose Cell Volume.) INTRODUCTION
The deposition of extramuscular fat in meat animals for the most part is undesirable. However, visible intramuscular fat makes a positive contribution to the palatability characteristics
of beef, particularly flavor and juiciness (Blumer, 1963). Because of the organoleptic importance of intramuscular fat and the economic considerations involved in growing meat animals with adequate intramuscular fat, an understanding of the growth and development of this lipid depot is desirable. In studying the enzyme systems regulating lipid synthesis in intramuscular fat, the size of laboratory animals has been a limiting factor in the dissection and isolation of intramuscular adipose tissue. Consequently metabolic data relating to this adipose tissue deposit have not appeared in the literature. However, reports have appeared recently concerning lipogenic enzyme activity in fat fragments from several muscles of the pig (Lee and Kauffman, 1974a,b) and from the longissimus muscle of steers and heifers (Chakrabarty and Romans, 1972). Howarth et al. (1968) have studied the activities of several important enzymes for lipid synthesis in calf skeletal muscle. Recent studies on the cellular development of bovine intramuscular adipose tissue (Hood and Allen, 1973c) confirm earlier observations (Hammond, 1955) that intramuscular adipose tissue is probably the last of the lipid depots to develop. This research was undertaken to determine if intramuscular lipid can be synthesized in situ and to study the inter-relationships among cell size, fatty acid synthesis and NADPH formation in four bovine muscles which contain different quantities of intramuscular lipid. EXPERIMENTAL PROCEDURE
Animals and Tissue Samples. Tissue samples were collected from six 14-month-old Hereford • Angus steers which had been given a diet high in corn silage from 180 to 340 kg live weight and then finished to 470 kg with a high-energy t Paper No. 8352 Scientific Journal Series of the diet rich in ground shelled corn (4-02-861). Minnesota Agricultural Experiment Station. 2CSIRO Division of Food Research, P.O. Box 52, Each steer also consumed daily approximately 454 g of supplement cohtaining 45 g urea, 72 g North Ryde, N.S.W. 2113, Australia. 1626 JOURNAL OF ANIMALSCIENCE,Vol. 46, No. 6, 1978
Downloaded from https://academic.oup.com/jas/article-abstract/46/6/1626/4699275 by Iowa State University user on 19 January 2019
R. L. Hood 2 and C. E. Allen
LIPOGENESIS IN BOVINE MUSCLE
ml of 25% KOH absorbed onto a filter4paper strip was placed in each flask to collect I CO2. Each flask was flushed with oxygen, sealed with a rubber serum cap and incubated with shaking at 37 C for 3 hr in a water bath. The reaction was terminated by the injection of .25 ml of 1M H2SO4 through the serum cap and into the incubation medium. The incorporation of acerate-1J aC into fatty acids and CO2 was determined as described by Hood et al. (1972). Statistics. Differences between treatments were determined by comparing each treatment with all other treatments using an F-test with two treatments (Steel and Torrie, 1960). RESULTS AND DISCUSSION
For the comparison of enzyme activities among adipose tissues containing cells of different sizes an equal number of adipose cells has been shown to be the most suitable reference unit (Hood and Allen, 1973a). The average diameter and volume of intramuscular adipose cells and the lipid content of the four muscles are listed in Table 1. The cellular aspects of the growth and development of bovine intramuscular adipose tissue have been discussed in a previous report (Hood and Allen, 1973c). Intramuscular adipose tissue was observed to develop in a different manner than subcutaneous and perirenal adipose tissue in Hereford x Angus steers. Differences in intramuscular adipose cell size (table 1) can be attributed mainly to the proportion of small cells in the population of adipose cells. The adipose tissue from the semimembranosus and trapezius contained proportionately more small adipose cells than tissue from the longissimus and pectoralis profundus muscles, hence the mean adipose cell volume was greatest in the latter two muscles. In the subcutaneous and perirenal adipose tissue of the pig (Hood and Allen, 1977) and the steer (Hood and Allen, 1973c) the amount of lipid deposited was positively correlated to the size of the adipose cells and not the number of cells. However, the quantity of intramuscular lipid was directly related to the number of adipose cells rather than the size of the cells (Hood and Allen, 1973c). These authors reported that intramuscular adipose tissue in these 14-month-old Hereford x Angus steers developed by both cellular hypertrophy and hyperplasia, while in the subcutaneous and perirenal depots hyperplasia was complete by approximately 8 months of
Downloaded from https://academic.oup.com/jas/article-abstract/46/6/1626/4699275 by Iowa State University user on 19 January 2019
limestone (6-02-632), 2 g elemental sulfur, 3 g vitamin A premix, 5 g diethylstilbesterol premix, 45 g trace mineralized salt and 282 g corn. Intramuscular adipose tissue fragments were isolated by dissection from the semimembranosus, longissimus, trapezius and pectoralis profundus muscles within 30 min of exsanguination of the animal. Each adipose tissue fragment was dissected free of muscle tissue prior to either incubation with ~4C_acetate or homogenization in buffer. Muscle Lipid Content. A cross-section of each muscle was frozen in liquid nitrogen and pulverized in a Waring Blendor in the frozen state to facilitate complete lipid extraction. The intramuscular lipid content of individual muscles was then determined by extracting the oven-dried muscle sample with diethylether in a soxhlet extraction apparatus. Lipid content was expressed on the basis of wet muscle weight. Enzyme Assays. A 5 g portion of adipose tissue fragments which had been isolated by dissection was homogenized at 24 C in 25 ml of .15M KCI in 50 mM tris (CI-) buffer, pH 7.4, with a Polytron PT-IO-12 homogenizer for 20 seconds. The homogenate was then centrifuged at 48,500 • g for 1 hr at 0 C. The supernatant was removed by careful aspiration for enzyme activity determinations. Acetyl CoA carboxylase (CBX) (E.C. 6.4.1.2) and NADP-isocitrate dehydrogenase (ICDH) (E.C. 1.1.1.42) were assayed at 37 C and 30 C respectively as described by Hood and Allen (1973b). Glucose-6-phosphate dehydrogenase (G-6-PDH) (E.C. 1.1.1.49), 6-phosphogluconate dehydrogenase (6-PGDH) (E.C. 1.1.1.44) and NADPmalate dehydrogenase (ME) (E.C.1.1.1.40) were assayed at 30 C as described by Hood and Allen (1973a). Enzyme activities expressed on a cellular basis were determined by dividing the activity per gram of adipose tissue by the number of adipose cells per gram of tissue. A modification (Hood and Allen, 1977) of the Coulter Counter technique described by Hirsch and Gallian (1968) was used to determine the number and size of intramuscular adipose cells. Acetate Metabolism. Sliced fragments (105 to 175 mg) of intramuscular adipose tissue were weighed and placed in a 25 ml Erlenmeyer flask containing 10 /amoles of a c e t a t e - l J 4 C (2 /aCi/10 #mole), 10/amoles of glucose, 1 /amole of propionate and .3 units of insulin (lletin, Eli Lilly Co.) in 3 ml of Ca++-free Krebs Ringer bicarbonate buffer at pH 7.4 (Umbreit et al., 1964). A 10 x 34 mm glass vial containing .1
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University of Minnesota, St. Paul 55101
SUMMARY
The incorporation of acetate-1-14C into CO2 and total fatty acids and the activities of NADP-malate dehydrogenase, NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and acetyl CoA carboxylase were studied in intramuscular adipose tissue from the trapezius, longissimus, semimembranosus and pectoralis profundus muscles of 470 kg, 14-month-old Hereford • Angus steers. It was concluded that intramuscular lipid can be synthesized in situ, but at a slower rate than subcutaneous lipid. The rate of synthesis of fatty acids and CO2 from acetate-l-14C was directly related to adipose cell volume and for this reason the intramuscular adipose tissue of the pectoralis profundus muscle was more active than that of the other three muscles. The activities of the four NADPH-generating enzymes were positively related to both adipose cell volume and fatty acid synthesis. NADP-malate dehydrogenase activity was low in intramuscular adipose tissue; however, the hexose monophosphate shunt dehydrogenases and possibly NADP-isocitrate dehydrogenase are capable of supplying NADPH for fatty acid synthesis in bovine intramuscular adipose tissue. (Key Words: Lipogenesis; Intramuscular Adipose Tissue; Bovine Muscles; Adipose Cell Volume.) INTRODUCTION
The deposition of extramuscular fat in meat animals for the most part is undesirable. However, visible intramuscular fat makes a positive contribution to the palatability characteristics
of beef, particularly flavor and juiciness (Blumer, 1963). Because of the organoleptic importance of intramuscular fat and the economic considerations involved in growing meat animals with adequate intramuscular fat, an understanding of the growth and development of this lipid depot is desirable. In studying the enzyme systems regulating lipid synthesis in intramuscular fat, the size of laboratory animals has been a limiting factor in the dissection and isolation of intramuscular adipose tissue. Consequently metabolic data relating to this adipose tissue deposit have not appeared in the literature. However, reports have appeared recently concerning lipogenic enzyme activity in fat fragments from several muscles of the pig (Lee and Kauffman, 1974a,b) and from the longissimus muscle of steers and heifers (Chakrabarty and Romans, 1972). Howarth et al. (1968) have studied the activities of several important enzymes for lipid synthesis in calf skeletal muscle. Recent studies on the cellular development of bovine intramuscular adipose tissue (Hood and Allen, 1973c) confirm earlier observations (Hammond, 1955) that intramuscular adipose tissue is probably the last of the lipid depots to develop. This research was undertaken to determine if intramuscular lipid can be synthesized in situ and to study the inter-relationships among cell size, fatty acid synthesis and NADPH formation in four bovine muscles which contain different quantities of intramuscular lipid. EXPERIMENTAL PROCEDURE
Animals and Tissue Samples. Tissue samples were collected from six 14-month-old Hereford • Angus steers which had been given a diet high in corn silage from 180 to 340 kg live weight and then finished to 470 kg with a high-energy t Paper No. 8352 Scientific Journal Series of the diet rich in ground shelled corn (4-02-861). Minnesota Agricultural Experiment Station. 2CSIRO Division of Food Research, P.O. Box 52, Each steer also consumed daily approximately 454 g of supplement cohtaining 45 g urea, 72 g North Ryde, N.S.W. 2113, Australia. 1626 JOURNAL OF ANIMALSCIENCE,Vol. 46, No. 6, 1978
Downloaded from https://academic.oup.com/jas/article-abstract/46/6/1626/4699275 by Iowa State University user on 19 January 2019
R. L. Hood 2 and C. E. Allen
LIPOGENESIS IN BOVINE MUSCLE
ml of 25% KOH absorbed onto a filter4paper strip was placed in each flask to collect I CO2. Each flask was flushed with oxygen, sealed with a rubber serum cap and incubated with shaking at 37 C for 3 hr in a water bath. The reaction was terminated by the injection of .25 ml of 1M H2SO4 through the serum cap and into the incubation medium. The incorporation of acerate-1J aC into fatty acids and CO2 was determined as described by Hood et al. (1972). Statistics. Differences between treatments were determined by comparing each treatment with all other treatments using an F-test with two treatments (Steel and Torrie, 1960). RESULTS AND DISCUSSION
For the comparison of enzyme activities among adipose tissues containing cells of different sizes an equal number of adipose cells has been shown to be the most suitable reference unit (Hood and Allen, 1973a). The average diameter and volume of intramuscular adipose cells and the lipid content of the four muscles are listed in Table 1. The cellular aspects of the growth and development of bovine intramuscular adipose tissue have been discussed in a previous report (Hood and Allen, 1973c). Intramuscular adipose tissue was observed to develop in a different manner than subcutaneous and perirenal adipose tissue in Hereford x Angus steers. Differences in intramuscular adipose cell size (table 1) can be attributed mainly to the proportion of small cells in the population of adipose cells. The adipose tissue from the semimembranosus and trapezius contained proportionately more small adipose cells than tissue from the longissimus and pectoralis profundus muscles, hence the mean adipose cell volume was greatest in the latter two muscles. In the subcutaneous and perirenal adipose tissue of the pig (Hood and Allen, 1977) and the steer (Hood and Allen, 1973c) the amount of lipid deposited was positively correlated to the size of the adipose cells and not the number of cells. However, the quantity of intramuscular lipid was directly related to the number of adipose cells rather than the size of the cells (Hood and Allen, 1973c). These authors reported that intramuscular adipose tissue in these 14-month-old Hereford x Angus steers developed by both cellular hypertrophy and hyperplasia, while in the subcutaneous and perirenal depots hyperplasia was complete by approximately 8 months of
Downloaded from https://academic.oup.com/jas/article-abstract/46/6/1626/4699275 by Iowa State University user on 19 January 2019
limestone (6-02-632), 2 g elemental sulfur, 3 g vitamin A premix, 5 g diethylstilbesterol premix, 45 g trace mineralized salt and 282 g corn. Intramuscular adipose tissue fragments were isolated by dissection from the semimembranosus, longissimus, trapezius and pectoralis profundus muscles within 30 min of exsanguination of the animal. Each adipose tissue fragment was dissected free of muscle tissue prior to either incubation with ~4C_acetate or homogenization in buffer. Muscle Lipid Content. A cross-section of each muscle was frozen in liquid nitrogen and pulverized in a Waring Blendor in the frozen state to facilitate complete lipid extraction. The intramuscular lipid content of individual muscles was then determined by extracting the oven-dried muscle sample with diethylether in a soxhlet extraction apparatus. Lipid content was expressed on the basis of wet muscle weight. Enzyme Assays. A 5 g portion of adipose tissue fragments which had been isolated by dissection was homogenized at 24 C in 25 ml of .15M KCI in 50 mM tris (CI-) buffer, pH 7.4, with a Polytron PT-IO-12 homogenizer for 20 seconds. The homogenate was then centrifuged at 48,500 • g for 1 hr at 0 C. The supernatant was removed by careful aspiration for enzyme activity determinations. Acetyl CoA carboxylase (CBX) (E.C. 6.4.1.2) and NADP-isocitrate dehydrogenase (ICDH) (E.C. 1.1.1.42) were assayed at 37 C and 30 C respectively as described by Hood and Allen (1973b). Glucose-6-phosphate dehydrogenase (G-6-PDH) (E.C. 1.1.1.49), 6-phosphogluconate dehydrogenase (6-PGDH) (E.C. 1.1.1.44) and NADPmalate dehydrogenase (ME) (E.C.1.1.1.40) were assayed at 30 C as described by Hood and Allen (1973a). Enzyme activities expressed on a cellular basis were determined by dividing the activity per gram of adipose tissue by the number of adipose cells per gram of tissue. A modification (Hood and Allen, 1977) of the Coulter Counter technique described by Hirsch and Gallian (1968) was used to determine the number and size of intramuscular adipose cells. Acetate Metabolism. Sliced fragments (105 to 175 mg) of intramuscular adipose tissue were weighed and placed in a 25 ml Erlenmeyer flask containing 10 /amoles of a c e t a t e - l J 4 C (2 /aCi/10 #mole), 10/amoles of glucose, 1 /amole of propionate and .3 units of insulin (lletin, Eli Lilly Co.) in 3 ml of Ca++-free Krebs Ringer bicarbonate buffer at pH 7.4 (Umbreit et al., 1964). A 10 x 34 mm glass vial containing .1
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