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ANIMAL NUTRITION |
Department of Animal Science, Texas Agricultural Experiment Station, Texas A&M University, College Station 77843
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: acetate • adipose tissue • cattle • glucose • insulin
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Smith and Crouse (1984)
demonstrated that i.m. adipocytes utilize glucose, whereas s.c. adipocytes utilize acetate as primary substrates for fatty acid synthesis, and diets greater in starch promote i.m. fat deposition relative to s.c. deposition (Choat et al., 2003). Insulin stimulates peripheral tissue uptake of glucose and increases lipogenesis or reduces lipolysis; also, plasma insulin concentration is positively correlated with carcass adiposity (Trenkle and Topel, 1978). However, negative correlations between plasma glucose concentrations and carcass adiposity have been reported (Matsuzaki et al., 1997; Schoonmaker et al., 2003). Variation in insulin sensitivity may affect caloric partitioning among tissues and tissue development. Gilbert et al. (2003) suggested that i.m. adipose tissue is more sensitive to insulin than s.c. tissue. Propionate is both glucogenic and insulinogenic in ruminants (Sano et al., 1995), which may enhance i.m. deposition. Acetate loading may increase glucose demand (Cronje et al., 1991) or increase ketone load. Ketone body accumulation (Tardif et al., 2001) and diet (Waterman et al., 2006) affect insulin sensitivity and thus energy partitioning in cattle. We hypothesized that dietary energy source may alter tissue sensitivity to insulin and the subsequent uptake of glucose. Due to the substrate preferences of adipose tissues, such alterations would result in differential accumulation of energy among fat depots and may provide a mechanism for manipulating nutrient partitioning. Objectives of this study were to evaluate effects of energy source on in vitro metabolic function and insulin sensitivity in bovine muscle and s.c. and i.m. adipose tissues.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sample Collection
After the feeding periods, steers from each group were slaughtered on 2 consecutive days at the Rosenthal Meat Science and Technology Center, Texas A&M University. A section of the LM between the fifth and eighth thoracic ribs was removed immediately after hide removal (approximately 20 min postmortem). The LM section and associated adipose tissues were immediately placed in oxygenated Krebs-Henseleit bicarbonate (KHB) buffer (pH = 7.4; 37°C) with 5 mM glucose and transported to the laboratory for excision of muscle and s.c. and i.m. adipose tissue samples. Upon arrival of the LM sections at the laboratory, dissection of muscle and s.c. and i.m. adipose tissue samples from individual LM sections were completed within 5 min by several trained personnel. All dissections were completed within 20 min after LM collection. After dissection of an individual sample, tissues were immediately placed into separate weigh boats containing fresh buffer (oxygenated KHB with glucose) and were maintained at 37°C until all dissected tissue could be placed into an incubation flask. Dissected tissues were carefully evaluated for contamination with nontarget tissue particles, and tissue samples were not incubated if contamination was apparent. Five grams of each tissue type was immersed in liquid N for analysis of glycolytic intermediate concentration, another 50 to 100 mg of each tissue type was used immediately for measurement of glucose metabolism in vitro, and another 50 to 100 mg of s.c. and i.m. adipose was used for measurement of lipogenesis in vitro.
Source of Chemicals
All chemicals and biochemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich Chemical Co. (St. Louis, MO). Additionally, [U-14C]glucose and [U-14C]acetate were purchased from Amersham (Arlington Heights, IL).
Substrate Concentrations
Glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P) were measured in frozen LM, s.c., and i.m. samples from each animal using assay systems described by Bergmeyer (1974), with modifications as described by Rhoades et al. (2005). Briefly, a buffer system containing 0.9 mM NADP+ and 1 mM ATP was added to each cuvette (total vol = 1.0 mL) along with 0.1 mL of extract for muscle tissue and 0.5 mL of extract for adipose tissue samples. Glucose-6-phosphate dehydrogenase was added to each cuvette, catalyzing G-6-P to 6-phosphogluconate, and the change in absorbance was measured using a Beckman DU-7400 spectrophotometer (Beckman, Palo Alto, CA) set at 339 nm. In the same cuvette, glucose was converted to G-6-P by the addition of hexokinase, and the change in absorbance was measured at 339 nm. Subsequently, phosphoglucose isomerase was added to each cuvette to convert F-6-P to G-6-P, and the change in absorbance was measured at 339 nm.
Glucose Metabolism In Vitro
Glucose metabolism in vitro was measured using fresh muscle and s.c. and i.m. adipose tissues from each animal, as described by Espinal et al. (1983). Briefly, 50 to 100 mg of each tissue was incubated in 3 mL of 10 mM glucose, KHB, and 20 mM HEPES buffer and 1 µCi of [U-14C]glucose. Flasks were gassed for 1 min with 95% O2:5% CO2, capped with hanging center wells, and incubated in a shaking water bath for 2 h at 37°C. Bovine insulin (0 or 500 ng/mL; insulin from bovine pancreas, 27 USP units/mg, Sigma-Aldrich Chemical Corp., St. Louis, MO) was added to flasks replicated within each tissue type, from each animal, at each level of insulin addition. Adding 3 mL of 5% trichloroacetic acid to incubation media stopped all reactions. A control flask was created within each tissue type, from each animal, at each level of insulin by adding trichloroacetic acid to the media before the tissues were placed in the flask for incubation, thus preventing any reactions from occurring. Determination of [U-14C]glucose C conversion to CO2 trapped in hanging center wells was performed according to Smith (1983), and the glucose C from incubation media was recovered as lactate and determined according to Smith and Freeland (1981). This method of lactate recovery also traps pyruvate and other carboxylic acids. The remaining glucose C in the form of fatty acids and glyceride-glycerol was extracted from the incubated tissue (adipose only) portion, and the fatty acids synthesized from glucose were separated into the glyceride-fatty acids and glyceride-glycerol fractions by the saponification methods described by Hood et al. (1972). All measures of radioactivity were counted using a Beckman liquid scintillation spectrometer.
Lipogenesis In Vitro
Lipogenesis from acetate was measured in fresh s.c. and i.m. adipose tissue from each animal according to Page et al. (1997). Briefly, 50- to 100-mg samples of adipose tissue were incubated in 3 mL of 10 mM Na acetate, 10 mM glucose, KBH, 20 mM HEPES buffer (pH 7.40; 37°C), and 1 µCi of [1-14C]acetate for 2 h. Flasks were replicated within tissue type from each animal. Adding 3 mL of 5% trichloroacetic acid to the incubation media stopped all reactions. A control for each tissue type was created by adding trichloroacetic acid to the flask before tissue insertion, such that no reactions could occur. Measurement of [1-14C]acetate incorporation into fatty acids was conducted as described by Page et al. (1997). Radioactivity was counted using a Beckman liquid scintillation spectrometer.
Cellularity
Adipocyte number per gram of tissue was determined by the method of Etherton et al. (1977), as modified by Smith et al. (1996). Adipose tissue samples were sliced into 1 mm-thick sections, placed into 20-mL scintillation vials, and then processed as described by Smith et al. (1996). The fixed cells were filtered through 250-, 64-, and 20-µm mesh screens, with 0.01% Triton X-100 in 0.154 M NaCl. Cell fractions from the 64- and 20-µm screens were used to determine the number of adipocytes per gram of s.c. and i.m. adipose tissue using a Coulter Counter (model ZM and Coulter Channelyzer 256, Beckman Coulter, Miami, FL).
Calculations involving glucose and acetate utilization measures among adipose tissue depots were expressed on a per-cell basis. Diet had no effect on cellularity pooled across depots (corn = 2.12 x 105 vs. hay = 1.78 x 105 cells per 100 mg; P = 0.44). However, tissue type did influence adipocyte cellularity (s.c. = 1.67 x 105 vs. i.m. = 2.22 x 105 cells per 100 mg; P = 0.03), but no diet x tissue interaction was present.
Statistical Analysis
All analyses were performed using the GLM procedure (SAS Inst. Inc., Cary, NC). Data were analyzed as a split-plot design. Diet, breed, and their interaction served as main plot effects and were tested using animal nested within breed x diet as the error term. For response variables related to substrate concentration and acetate utilization (G-6-P, F-6-P, and acetate incorporation into fatty acids), tissue type and diet x tissue served as subplot effects and were tested using the residual mean square as the error term. For response variables related to glucose utilization (glucose conversion into CO2, lactate, and fatty acids), insulin level (0 or 500 ng/mL), tissue x insulin, diet x insulin, diet x tissue, and diet x insulin x tissue were the subplot effects and were tested with residual mean square as the error term. Breed was considered a whole-plot blocking factor and thus was not included in the subplot interactions (Kuehl, 1994). Least squares means and estimates of variability are consistent with respect to appropriate error terms. When the overall F-tests were significant, means were separated using Fisher’s protected LSD test.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; P = 0.05). The concentration of G-6-P was greater in LM than in adipose tissue depots and was greater in LM tissue of steers fed a hay-based diet compared with that of steers fed a corn-based diet. The F-6-P concentrations were similar between diets, but greater F-6-P concentrations were observed in LM than in i.m. or s.c. adipose tissues (Table 1; P = 0.01).
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; P = 0.03) in s.c. compared with that of i.m. adipose tissue and was not influenced by diet (P = 0.86) or interactions between diet and tissue (P = 0.41). Glucose oxidation to CO2 differed among tissues (Table 2; P < 0.01). Muscle converted 88% more glucose to CO2 per 100 mg of tissue than did adipose tissues, which were similar. Both dietary energy source (P = 0.04) and insulin addition to culture media (P < 0.01) influenced rate of glucose conversion to CO2 per 100 mg of tissue (pooled across LM, and s.c. and i.m. adipose tissues). However, the effect of insulin addition on glucose conversion to CO2 depended upon dietary energy source (Table 2; P < 0.01). When the hay-based diet was fed, insulin addition failed to stimulate an increase in glucose conversion to CO2; however, when the corn-based diet was fed, insulin additions resulted in a 116% increase in rate of conversion to CO2 per 100 mg of tissue.
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; P < 0.01) glucose conversion to lactate per 100 mg of tissue, with tissues from steers fed the corn-based diet converting 190% more glucose to lactate than tissues from steers fed the hay-based diet. Differences in lactate synthesis from glucose among tissues depended on diet (Table 2; diet x tissue, P < 0.01). This diet x tissue interaction was primarily due to the large increase in lactate production seen in adipose tissue depots when steers were fed the corn-based diet; the magnitude of difference in lactate yield from LM tissue was smaller between diets.
Tissues from steers fed the hay-based diet tended to incorporate more glucose into fatty acids than did those from steers fed the corn-based diet (P = 0.14); however, the rate of incorporation of labeled glucose into the glyceride-fatty acid fraction in response to insulin additions was dependent upon tissue type and diet when expressed per 100 mg of adipose tissue (Table 2; diet x tissue x insulin, P = 0.04). Insulin addition to either s.c. or i.m. adipose tissues from steers fed the hay-based diet had no effect on incorporation rates. Insulin additions to s.c. adipose tissue from steers fed the corn-based diet also failed to stimulate glucose incorporation into fatty acids; however, exposing i.m. adipose tissue from steers fed the corn-based diet to insulin resulted in a 165% increase in glucose incorporation into fatty acids.
The effect of insulin on the conversion of glucose to glyceride-glycerol depended on dietary energy source (Table 2; diet x insulin, P < 0.01). When expressed per 100 mg of adipose tissue, insulin decreased glucose incorporation into glyceride-glycerol in adipose tissues from steers fed the hay-based diet. However, adding insulin to tissues from steers fed the corn-based diet increased glyceride-glycerol synthesis from glucose to the level similar to that observed in steers fed the hay-based diet without insulin.
When responses were analyzed only from adipose tissues, expressed per 105 cells, tissue type influenced the rate of acetate incorporation into fatty acids (Table 3; P = 0.02), with s.c. adipose tissue incorporating over 290% more acetate into fatty acids than did i.m. adipose tissues.
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; P = 0.04). When the hay-based diet was fed, insulin caused no significant increase in CO2 production, but insulin increased glucose conversion to CO2 by 103% when the corn-based diet was fed (diet x insulin, P = 0.02).
Glucose conversion to lactate per 105 cells was nearly 4-fold greater in adipose tissues from steers fed the corn-based diet than in adipose tissues from steers fed the hay-based diet (Table 3; P < 0.01); because LM was not included in this evaluation, the interaction between diet and tissue was not significant (P = 0.88). Glucose conversion to lactate was accelerated by at least 50% with addition of insulin to tissues from steers fed the corn-based diet but was not affected by insulin in adipose tissues from steers fed the hay-based diet.
When glucose incorporation into fatty acids was expressed on a cellular basis, the diet x tissue x insulin interaction approached significance (Table 3; P = 0.12). Although the magnitude and direction of separation were similar regardless of basis of expression, greater variation when expressed on a cellular basis resulted in a less sensitive test.
Diet x insulin interaction effects were observed (Table 3; P < 0.01) when glyceride-glycerol synthesis from glucose was expressed per 105 cells, and the effect was similar in both magnitude and direction as when expressed per 100 mg of tissue.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), whereas greater concentrations of G-6-P and F-6-P in bovine muscle tissue were reported by Rhoades et al. (2005). Activities of hexokinase and phosphofructokinase (PFK) are low in bovine adipose tissue (Smith and Prior, 1981; Miller et al., 1991), and PFK limits glycolytic flux in bovine s.c. adipose tissue (Smith, 1984). Similar concentrations of F-6-P between adipose tissues in this study suggested that pathway flux was not constrained by PFK. Miller et al. (1991) reported that the activities of hexokinase and PFK were twice as high in i.m. adipose tissue as in s.c. bovine adipose tissue. Similar metabolite concentrations in adipose tissues in this study suggest that pathway constraints were similar, and, therefore, pathway flux is expected to be similar among tissues. This was confirmed by the measurement of radiolabeled glucose utilization. Total glucose utilization was calculated as the amount of radiolabeled glucose C converted into CO2, lactate, and total lipid. Total glucose utilization was similar across tissues, indicating that glucose uptake and metabolism were not greater in i.m. adipose tissue than in s.c. adipose tissue. Rhoades et al. (2005) reported similar glucose oxidation rates in bovine pars sternomandibularis muscle (14.7 nmol·h–1·100 mg–1) as in the current study. Smith (1983) demonstrated that 55% of metabolized glucose was converted to lactate in s.c. adipose tissue when steers were fed a high-energy diet. In our study, at least 85% of the glucose metabolized resulted in the production of lactate from either diet, but this was similar across tissues.
On a per-cell basis, s.c. adipose tissue produced more CO2 than i.m. adipose tissue. In adipose tissues, up to 25% of CO2 from glucose is generated through the pentose cycle (Smith, 1983; Smith and Prior, 1986). The activities of G-6-P dehydrogenase and 6-phosphogluconate dehydrogenase are greater in s.c. than in i.m. adipose tissue (Miller et al., 1991), and the greater rate of CO2 production by s.c. adipose tissue is consistent with this earlier report. Others have demonstrated differences between adipose tissues in rates of CO2 production. Baldwin et al. (1973) reported that s.c. adipose tissue was 6 times more active in converting glucose to CO2 than perirenal adipose tissue, and Smith and Crouse (1984) demonstrated that s.c. adipose tissue oxidized more glucose than i.m. adipose tissue in the presence of acetate in the incubation media. Also, early in vitro studies demonstrated that the oxidation of glucose was influenced by insulin. Yang and Baldwin (1973) showed that the addition of insulin to culture media increased glucose oxidation in s.c. adipose tissue from corn-fed cattle by up to 45%. Similarly, Baldwin et al. (1973) showed a progressive increase in insulin stimulation with increasing amounts of concentrate in the diet. In our study, these previous findings are synthesized, because s.c. tissue oxidized more glucose, and increasing concentrate level in our diets resulted in an increased response of glucose oxidation to insulin stimulation.
Utilization of glucose C for de novo lipid synthesis was quantified by the incorporation of labeled glucose into glyceride-glycerol and glyceride-fatty acid fractions. Similar results were reported by Gilbert et al. (2003), in which glucose incorporation into glyceride-glycerol was 50% lower in i.m. adipose tissue than in s.c. adipose tissue. Gilbert et al. (2003) also demonstrated that insulin stimulated glucose conversion to glyceride-glycerol in i.m. adipose tissue but not in s.c. adipose tissue. The cattle sampled by Gilbert et al. (2003) were grain-fed, and the results of the current study are similar in that in the corn-fed steers, insulin stimulated glucose conversion to glyceride-fatty acid and glyceride-glycerol but only in i.m. adipose tissue.
Glucose was not limiting in the culture media for tissues from steers fed either diet. Thus, it is unlikely that the greater rate of fatty acid synthesis from glucose observed in vitro in the steers fed the hay-based diet would be observed in vivo. This observation suggests that the capacity for fatty acid synthesis in tissue from steers fed the hay-based diet was not compromised, and, thus, reduced adiposity in these steers was a function of reduced substrate availability and uptake. This inference is consistent with the lesser insulin sensitivity of adipose tissues from steers fed the hay-based diet. Our hypotheses were that diet would influence insulin sensitivity and that i.m tissue would be more sensitive to insulin. These results confirm our hypothesis.
Collectively, these data provide substantial evidence that dietary energy source alters insulin sensitivity, because tissues became insulin-resistant when cattle consumed a hay-based diet. A mechanism for this effect was provided by Tardif et al. (2001), who demonstrated that accumulation of ketones interrupted insulin signal transduction and reduced GLUT-4 migration to cell surfaces. This reduction in the insulin-sensitive glucose transporter translocation would reduce insulin-stimulated glucose uptake and thus would limit the rate of glucose metabolism. Ketone bodies may accumulate under acetate loading, particularly when glucose is limiting (Herdt et al., 1981), and, thus, the greater acetate loads anticipated with the hay-based diet may have affected this response. Schoonmaker et al. (2003) found greater concentrations of insulin in steers fed a high-concentrate diet than in steers fed a high-forage diet during the growing phase. This observation coupled with our results suggests that LM and adipose tissues in steers fed high concentrate diets would not only be exposed to greater circulating insulin but would also be more sensitive to its effects on glucose uptake and subsequent utilization.
Because s.c. adipose tissue converted more glucose into CO2 than i.m. adipose tissue, most likely via the pentose shunt, greater amounts of reducing equivalents (NADPH) would be available to support fatty acid synthesis in this tissue. Past studies have suggested that rates of acetate incorporation into fatty acids are increased when glucose is added to the culture media, due to the additional NADPH available for fatty acid synthesis (Hanson and Ballard, 1967; Smith, 1983). Our results confirm previous findings in which s.c. adipose tissue exhibited greater utilization of acetate than i.m adipose tissue (Smith and Crouse, 1984). Due to the apparently low utilization by i.m. adipose tissue of acetate as a substrate for de novo fatty acid synthesis and the similarities between tissues for glucose incorporation into lipids, the inference can be made that a limitation in glucose supply or uptake would have a more profound effect on rate of lipogenesis in i.m than in s.c. adipose tissue.
Overall, our results suggest that feeding the hay-based diet limited tissues capacity to increase glucose utilization in response to insulin without altering acetate conversion to fatty acids. Recent studies (Schoonmaker et al., 2003, 2004a,b) have reported no differences in performance due to changes in source and amount of energy, yet differences in carcass adiposity have been present, suggesting that dietary source influences partitioning of energy into different fat depots. Because s.c. adipose tissue has been shown to consistently utilize more acetate and oxidize more glucose than i.m. adipose tissue, these results suggest that feeding a hay-based diet may alter i.m. adipose tissue metabolism with less effect on s.c. adipose tissue accretion. These results also support the hypothesis that high-concentrate diets enhance glucose metabolism and increase insulin effects in muscle and adipose tissues. High-concentrate feedstuffs produce a greater proportion of propionic acid than do forage diets (Orskov et al., 1991), and propionic acid is a preferred glucogenic substrate. Conversely, a roughage-based diet produces greater concentrations of acetate. Results from this study confirm previous reports (Smith and Crouse, 1984) that acetate is much more effectively utilized for fatty acid synthesis by s.c. adipose tissue than by i.m. adipose tissue. Previous literature has also suggested that roughage feeding inhibits insulin action. Smith et al. (1983) found that when steers were fed an alfalfa diet, insulin failed to stimulate measures of lipogenesis. However, several examples with concentrate-fed cattle have demonstrated insulin effects on adipose metabolism and that this activity has increased with amount of concentrate in the diet (Baldwin et al., 1973; Miller et al., 1989, 1991). In this study, LM and adipose tissue from steers fed the hay-based diet were not responsive to additional insulin, whereas insulin had profound effects on muscle and adipose tissue glucose utilization rates from steers fed the corn-based diet. These differences could lead to a divergent partitioning of energetic substrate in adipose tissue depots from steers fed different diets, in which feeding a corn-based diet enhances glucose uptake in i.m. adipose tissue, whereas feeding a hay-based diet reduces insulin action without altering acetate incorporation in fatty acids. Because s.c. adipose tissue used acetate more effectively than i.m. adipose tissue, feeding a hay-based diet would promote s.c. adipose tissue deposition over i.m. adipose tissue accretion. Reports exist that are consistent with this hypothesis. Choat et al. (2003) reported increased i.m. adipose tissue deposition in steers fed a concentrate diet that generated 39.3% greater propionate. Schaake et al. (1993) found that feeding grain or high-concentrate diets increased i.m. adipose tissue content relative to forage feeding. These findings, especially in light of our observation about diet effects on insulin sensitivity, correspond with increased accretion of i.m. adipose tissue lipid from glucose carbon (Smith and Crouse, 1984).
In conclusion, the results of this experiment demonstrate diet-mediated differences in insulin sensitivity of muscle and adipose tissues in steers. Apparent differences in s.c. vs. i.m. adipose tissue metabolism, and their interaction with diet, provide the foundation for a hypothesis regarding diet-mediated regulation of differential adipose tissue metabolism. Validation of these hypotheses could generate nutritional strategies that alter the rate and site of adipose deposition.
1 Corresponding author: j-sawyer{at}tamu.edu
Received for publication July 24, 2006. Accepted for publication February 26, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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