effect on total glucose utilization in adipose tissue from cattle. Acetate provided ... (adipocytes versus tissue slices) or substrate ... experimental techniques.
Comparisons of Lipogenesis and Glucose Metabolism Between Ovine and Bovine Adipose Tissues1 STEPHEN B. SMITH2 ANDRONALD L. PRIOR3 Texas A ir M University College Station, TX and R. L. Hruska U.S. Meat Animal Research Center, P.O. Box 166, Clay Center, NE
Investigations of lipogenesis or glucose metabolism in ruminant adipose tissues typi cally have involved the utilization of tissues from either sheep (1-5) or cattle (6-14), but seldom both. While numerous metabolic similarities exist between ovine and bovine adipose tissues, enough conflicting results have been obtained to cast doubt on the wis dom of extending the results based on studies from one ruminant species to ruminants in general. Robertson et al. (2) determined that, at physiological concentrations of acetate, lactate and glucose, the contributions of lac tate and glucose to fatty acid biosynthesis in
adipose tissue from adult sheep were only 3 and 2%, respectively. However, Smith and Grouse (11)demonstrated that, in bovine sub cutaneous tissue slices incubated with 5 mM acetate, lactate and glucose, the contribution of lactate to lipogenesis was 15-30%, depend ing on the age and diet of the cattle. Studies of glucose metabolism in ovine adipocytes ©1986 American Institute of Nutrition. Received for publication: 31 May 1985. Accepted for publication: 19 February 1986. 'Technical article no. 21201, Texas Agricultural Experiment Station. 'Animal Science Department, TexasA&M University, College Station, TX 77843. 'Current address: RFD 2, Box 108, Hastings, NE 68901.
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ABSTRACT Studies were initiated to compare glucose and lipid metabolism in vitro in subcutaneous adipose tissue of mature sheep and cattle. Mean adipocyte volume was significantly less in subcutaneous adipose tissue of sheep than in adipose tissue from cattle. The presence of acetate and lactate in the incubation medium in creased total glucose utilization two- to three-fold in ovine adipose tissue, but had no effect on total glucose utilization in adipose tissue from cattle. Acetate provided 72-82 % of the acetyl units to lipogenesis, depending on species and substrate concen tration. There were no significant (P > 0.05) differences in the contribution of the pentose cycle to the provision of reducing equivalents to fatty acid biosynthesis, based on the incorporation of label from [3-3H]glucose into fatty acids. In ovine adipose tis sue, acetyl-CoA carboxylase appeared to be rate-limiting to lipogenesis, while in bo vine subcutaneous adipose tissue, the activity of fatty acid synthetase may have been the limiting step in lipogenesis. In addition, the low activity of ATP-citrate lyase, espe cially relative to aconitate hydratase, probably limited the conversion of lactate to fatty acids in ovine adipose tissue. It is unlikely that ATP-citrate lyase activity was rate-limiting to lipogenesis from lactate in bovine adipose tissue. The data indicate that extending the results obtained from adipose tissue from one species to lipid metabolism in ruminants in general may not be valid. J. Nutr. 116: 1279-1286, 1986. INDEXING KEY WORDS ruminant adipose tissue •lipogenesis •glucose metabolism •pentose cycle
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(9). Lipids were extracted from the adipose tissue slices by the method of Folch et al. (15), modified as described by Smith (9). The glyceride fatty acids were saponified by the method of Hood et al. (16), by utilizing sodium methoxide. The recovery of 14Cfrom [U-14C]glucose in lactate was performed as described previously (9). Enzyme activities. Fresh samples of adi pose tissue were homogenized and centrifu gal fractions were prepared as described previously (7). The soluble enzyme fraction was utilized to determine the activities of fatty acid synthetase (17), acetyl-CoA carboxylase (EC 6.4.1.2) (7), ATP-citrate lyase (EC 4.1.3.8) (18), NADP-malate dehydrogenase (EC 1.1.1.40) (19), NADP-isocitrate dehydrogenase (EC 1.1.1.42) (20), aconitate hydratase (EC 4.2.1.3) (7), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (21). Adipose cell size determinations. Adipose tissue samples were fixed with osmium MATERIALS AND METHODS tetroxide by the method of Etherton et al. Animals. Subcutaneous adipose tissue (22), modified as described by Prior (6). Par samples were obtained at slaughter from ticle cell size distributions were determined mature Angus x Hereford crossbred steers with a Coulter Counter, Model ZBI coupled and Columbia- and Suffolk-sired wether with a logarithmic range expander and (Coulter Electronics, Inc., lambs (six animals per group). Animals were channelyzer fed a high energy, ground corn/corn silage Hialeah, FL). Only those cells with di diet (11) for 200 d (steers) or 100 d (sheep). ameters greater than 30 ¿imwere included in the calculation of major peak diameters. Subcutaneous adipose tissue was obtained 10-20 cm to the left of the dorsal midline, The number of adipose cells per gram of tis sue was calculated from the mean cell between the 8th and 12th thoracic ver volume and the lipid content of the adipose tebrae Adipose tissue lipogenesis. Adipose tissue tissues (23). Analysis of data. Data were evaluated slices (75 to 125 mg) were incubated in tripli cate in 3 ml Krebs-Henseleit Caz*-free buffer statistically by analysis of variance (24). The (pH 7.35-7.40) plus 3 mM glucose. Some statistical model tested species as the main flasks also contained 1 mM acetate and 1 mM effect. The effects of substrate concentra tions on lipogenesis and glucose utilization lactate, or 10 mM acetate and 10 mM lactate were evaluated by paired f-test. All flasks contained 33 mU/ml bovine insu Source of chemicals. Radioisotopes were lin (10) (Calbiochem, San Diego, CA). Each flask contained 1 ¿iCi of [U-u]glucose plus 1 purchased from Amersham Corporation liC'\ [3-3H]glucose, or 1 /tCi of either (Arlington Heights, IL). Approximately [U-uC]lactate or [U-14C]acetate Flasks were 91% of the 3H in [3-3H]glucose was located gassed for 30 s with 95% O2/5% CO2 and on the 3-carbon of glucose, as determined stoppered and incubated for 3 h at 37 °Cand by NMR spectroscopy by the manufacturer. Chemicals were purchased from Fisher at 90 oscillations per min. Scientific Company (Fairlawn, NJ) and bioSeparation of lipid components. Reac tions were terminated after 3 h by injecting chemicals obtained from P-L Biochemicals (Milwaukee, WI) or Sigma Chemical Com 0.5 ml l N H2SO4 into the medium. The col lection of UCO2 was as described previously pany (St. Louis, MO).
indicated that the addition of acetate to in cubation media stimulated total glucose utili zation two- to three-fold (5), yet the magni tude of glucose utilization by bovine adipose tissue slices was not affected by the presence of acetate (9). Comparison of results obtained from different investigators is difficult in that differences in the experimental systems (adipocytes versus tissue slices) or substrate concentrations may have a significant impact on the interpretation of the results. A number of studies from this laboratory have dealt with the importance of fatty acid biosynthesis from lactate (6-8, 11) and the utilization of glucose (9, 12) in modulating overall lipid metabolism in ruminants. The current study was undertaken to establish whether the inconsistencies between results from this laboratory and others (2, 5, 9) were due to fundamental differences be tween sheep and cattle, or to differences in experimental techniques.
OVINE AND BOVINE LIPOGENESIS
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listed in table 2. Under both incubation con ditions, glucose provided less, and lactate more, acetyl units to lipogenesis (P < 0.05) Age, live weight, carcass weight and sub cutaneous adipose tissue cell volume and in adipose tissue from cattle relative to that cells per gram are listed in table 1. The from sheep when data were pooled across substrate concentrations. Acetate provided weights for the cattle were typical slaughter weights for these animals, while the weights less, and glucose more, carbon to lipogenesis for the sheep were somewhat heavier than at 1 mM acetate and lactate than at 10 mM in ovine adipose tissue market weight. Mean adipocyte volume was concentrations (P < 0.05; paired i-test). The same trend 25% greater (P < 0.05) in bovine sub cutaneous adipose tissue than in adipose tis was noted in bovine adipose tissue, espe sue from sheep. Due to this difference in cell cially for glucose. The percentage contribu tion of lactate to fatty acid synthesis was un volume and lipid content, ovine adipose tis sue contained approximately 40% more affected by substrate concentrations. In ovine adipose tissue, total glucose utili cells per gram than bovine adipose tissue (table 1; 23). Data in subsequent tables are zation increased significantly (P < 0.05; expressed as per gram tissue rather than per paired i-test) upon addition of acetate plus number of adipose cells per gram, since the lactate to the incubation media (table 3). latter values are based on estimations of total Glucose utilization in bovine adipose tissue cells per gram, rather than actual values. was unaffected by the presence of acetate Expressing the data in either manner did and lactate. Total glucose utilization is de not alter the statistical interpretation of the fined as the sum of the recovery of carbon from [U-14C]glucose in CO2, fatty acids, data. Total acetyl unit incorporation into fatty glyceride-glycerol, and lactate (9). The re acids at both concentrations of substrates (1 coveries of glucose carbon in each of these mM or 10 mM acetate and lactate and 3 mM products, expressed as percentages of total glucose) was significantly greater (P < 0.05) glucose utilization (specific yields; 25), also in adipose tissue from sheep than in tissue are listed in table 3. The production of CO2 from steers (table 2). Total acetyl unit incor accounted for the major proportion of uti poration is defined as the sum of the incor lized glucose. The presence of acetate and poration of acetyl units from U-14C-labeled lactate in the incubation media resulted in a acetate, lactate, and glucose into fatty acids greater percentage of glucose carbon being (9). Acetyl unit incorporation into fatty recovered as CO2, and a lesser percentage in acids was significantly greater (P < 0.05; fatty acids in adipose tissue from both paired i-test) at 10 mM substrates than at 1 species. The recovery of glucose carbon in lactate mM substrates. The contributions of each precursor to fatty acid synthesis also are (table 3) was significantly greater in adipose RESULTS
Age, live weight and hot carcass weight at slaughter and adipose cell volume and number of cells per gram adipose tissue from sheep and cattle1 Species ItemAge at slaughter, d Live weight at slaughter, kg Hot carcass weight, kg Mean volume, firn3 x JO"5 Cells per gram,2 x 10JCattle430
463.0 286.4 116.6 1.23
'Values are means ± SEMof six animals per species.
±8° ±5.3a ±4.4* ±0.8° ±0.05'Sheep168
60.1 31.6 93.5 1.69
±6b ±2.8b ±2.1b ±7.8" ±0.29b
2Based on an adipose cell density of 0.915 (23) and
0.88 ±0.03 and 0.79 ±0.08 g lipid/g adipose tissue for cattle and sheep, respectively. Means within a row with similar superscripts are not different (P > 0.05).
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TABLE 1
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TABLE 2 Contributions of glucose, lai-tati- and acetate to fatty acid synthesis in ovine and bovine adipose tissue1 contributionLactate%16
Cs concentration2mu110110Total incorporation3nmol/(min SpeciesOvineBovineAcetate/lactate g)65.8
• 4*4± 2b7±
6.1b220.5 ± 56.2C23.5 ±
2'14± 3"21±
4"82± 4"72±
2b1± 4'21± 6'b79 ± 6.6"45.0 ± ±11.6bGlucose12 ±OcRelative ±4a4Acetate72 ±4'b Species difference5
NS
genase. One mole of glucose was assumed to yield 2 mol C2 units. Percentage contribution of each substrate to total acetyl unit incorporation into fatty acids. 'Indicates significance between species (P < 0.05) when data were pooled across substrate concentrations. S, significant; NS, not significant. Values within a column not shar ing a similar superscript are significantly different (P < 0.05).
tissue from cattle than in adipose tissue from sheep. Conversely, the percentage re covery of glucose carbon in fatty acids was significantly greater in adipose tissue from sheep than that from cattle, as was total glu cose utilization, when the data were pooled across substrate concentrations. There was a significant effect of media acetate and lactate on lactate yields at the higher substrate
concentration (10 mM) in ovine adipose tis sue (P < 0.05). As observed for total acetyl unit incorpo ration into fatty acids (table 2), the recovery of label from [3-3H]glucose in fatty acids was greater in adipose tissue from sheep than that from cattle (table 4). Since the in corporation of label from [3-3H]glucose into fatty acids requires the involvement of
TABLE 3 Total glucose utilization and specific yields in ovine and bovine adipose tissue1 yields4C0237 glucose concentration2mM01100110Total utilization3nmol/(min SpeciesOvineBovineSpecies g)8
acids%33
•
T19± 3b25± 4b9±
2"11± 2"12± 2"SSpecific ±
4"55± 6b48± 7bc41 ±
6'12± 3"12± 3'12±
6"15± 5b10± 4cd18 ±
2'18± 3'30± 7b29±
2"54± 1'13± 4C5 ± 6b28± 3b55± 2*13± ld1± 4b32± 5bNSGlycerol16 ± 2'NSFatty ± ± 0eSLactate14 6bS ±
difference5Acetate/lactate 'Values are means ± SEMof adipose tissue from six animals per species. 2A11flasks contained 3 mM glucose and 33 mu/ml insulin. 'Total glucose utilization is the sum of recovery of isotope from [U-uC]glucose in CO2, glyceride-glycerol, fatty acids and lactate. Percentage of total glucose utilization for each product. 'Indicates significance between species (P < 0.05) when data were pooled across substrate concentrations. S, significant; NS, not significant. Values within a column not sharing a similar superscript are significantly different (P < 0.05).
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'Values are means ± SEMof adipose tissue from six animals per species. 2All flasks contained 3 mM glucose and 33 mu/ml insulin. 3Total C2 units calculated as sum of U-'4C-labeled glucose, láclateand acetate incor poration into fatty acids. Lactate and glucose incorporation was corrected for loss of 14Cat pyruvate dehydro-
OVINE
AND BOVINE
TABLE
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LIPOGENESIS
4
Incorporation of tritium from [3-*H]glucose into fatty acids and contribution of the peritose cycle to fatty acid synthesis1 SpeciesOvineAcetate/lactate
concentration2mu1
incorporation3nmol/(min
g)12.8
103H
35.5
Bovine 1 10
4.0 6.3
cycle contribution4%67
•
± 1.4* ±8.9bPentose
1.3° 1.7"
±6' 58 ±61*
61 ± 10ab 51 ±
Species difference5
NS
data were pooled across substrate concentrations. S, significant; NS, not significant. sharing similar superscripts are significantly different (P < 0.05).
6-phosphogluconate dehydrogenase, this in corporation can be used to calculate the per centage of reducing equivalents required for fatty acid synthesis that was supplied by the pentose cycle (table 4) (9, 26). Although sig nificant species-related differences in the in corporation of label from [3-3H]glucose in fatty acids were observed, there were no sig nificant differences in the percentage con tribution of the pentose cycle to lipogenesis. The pentose cycle contributed a lesser per centage (P < 0.05; paired f-test) of the reducing equivalents to lipogenesis in the presence of 10 mM acetate and lactate than in the presence of 1 mM acetate and lactate in adipose tissue from both species (table 4). The activities of several lipogenic en zymes are listed in table 5. The activities of acetyl-CoA carboxylase, fatty acid synthetase, aconitate hydratase and 6-phosphoglu conate dehydrogenase were significantly greater in ovine adipose tissue than in bo vine adipose tissue (P < 0.05) (table 5). Conversely, ATP-citrate lyase activity was greater in adipose tissue from steers. AcetylCoA carboxylase activity was greater than fatty acid synthetase, and ATP-citrate lyase activity greater than aconitate hydratase, in bovine adipose tissue (P < 0.05; paired ttest). The reverse was true for ovine adipose tissue.
Values within a column
not
DISCUSSION Some of the most pronounced differences between ovine and bovine adipose tissues were related to glucose metabolism. Total glucose utilization by ovine adipose tissue was stimulated two- to threefold by the addition of acetate and lactate to the incubation me dia, reflected primarily in large increases in COa production. In bovine adipose tissue, acetate and lactate had no effect on the mag nitude of glucose utilization, although these lipogenic precursors did influence the over all pattern of glucose metabolism. These results corroborate the findings of Yang et al. (5), in which isolated ovine adipocyte prepa rations were utilized, and Smith (9), who uti lized a bovine adipose tissue slice system iden tical to that used in the present study. The results indicate that discrepancies between the effects of lipogenic precursors on glucose utilization in ovine (5) and bovine (9, 12) adi pose tissues were based on species differences, and not differences in preparations. The percentage contributions of glucose, lactate and acetate to lipogenesis observed in this study differed substantially from values reported by Robertson et al. (2). Glucose and lactate provided substantially more acetyl units to fatty acid biosynthesis in ovine adi pose tissue than the 2-3 % reported previously
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'Values are means ±SEMof adipose tissue from six animals per species. 2A11flasks contained 3 mM glucose plus 33 mu/ml insulin. 'Incorporation of 3H from [3-3H]glucose into fatty acids. 'Percentage NADPH re quired for lipogenesis that was supplied by the pentose cycle calculated as: 6 x (3H from [3-3H]glucose in fatty acids)/(14/8 x total C2 incorporated into fatty acids). 'Indicates significance between species (P < 0.05) when
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