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Fasting-induced, selective loss of fatty acids from muscle triacylglycerols
Nutrition Research 23 (2003) 205–213 www.elsevier.com/locate/nutres
Fasting-induced, selective loss of fatty acids from muscle triacylglycerols Gene R. Herzberg*, Brian Farrell Department of Biochemistry, Memorial University of Newfoundland St. John’s, Newfoundland A1B 3X9 Canada Received 15 April 2002; received in revised form 24 September 2002; accepted 25 September 2002
Abstract We studied the loss of fatty acids from soleus muscle triacylglycerol (TAG) in rats fasted for 48 hours. During the 48 h fast the TAG content of the muscle decreased from 2.432 ⫾ 0.632 mg to 1.376 ⫾ 0.354 mg (X ⫾ s.d, n⫽8.) although the muscle weight was unchanged (0.255 ⫾ 0.016 vs. 0.273 ⫾ 0.032 g). Fatty acid mobilization was calculated as the percentage of the mass of a given fatty acid lost during the fast. The average mobilization for all fatty acids was 43.2%. The mobilization of individual fatty acids, varied from 30% for 20:1, the least mobilized fatty acid to 78.5% for EPA (20:5 n3) the most mobilized fatty acid. The polyunsaturated fatty acids were mobilized to a significantly greater extent than the monounsaturated and saturated fatty acids. This resulted in an enrichment in monounsaturated and saturated fatty acids in the muscle TAGs following the fast. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Muscle; Triacylglycerol; Fatty acids; Selectivity
1. Introduction The fatty acid composition of triacylglycerols (TAGs) in adipose tissue, brown adipose tissue and skeletal muscle is broadly reflective of the fatty acid composition of the diet [1– 4]. However, there are differences in the storage and release of dietary fatty acids that lead to differences between the fatty acid composition of the diet and that in the tissue [1,5,6]. It has been determined that the structure of the fatty acids in the tissue TAGs determines their net
* Corresponding author. Tel.: ⫹1-709-737-8800; fax: ⫹1-709-737-2422. E-mail address: [email protected] (G.R. Herzberg). 0271-5317/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 5 1 5 - 8
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Table 1 Modified AIN 93G Diet Component
g/Kg
AIN 93 Mineral Mix AIN 93 Vitamin Mix L-Cystine Choline Bitartrate Tert Butyl Hydroquinone Casein Fibre (Alphacel) Corn Starch Dextrinized Corn Starch Sucrose Fat*
35 10 3 2.5 0.014 200 50 252.5 83.5 63.5 300
* The fat was made up of 40 g soybean oil, 60 g seal blubber oil and 200 g fish oil.
storage in both white and brown adipose tissue but even in these two tissues there are significant differences in the specificity of net storage. It has previously been shown that certain long-chain n3 fatty acids are underrepresented in adipose tissue and skeletal muscle compared to other fatty acids and relative to their proportion in the diet [3]. The fatty acid composition of tissue TAG is modulated by both diet and the selectivity of the loss of fatty acids from TAG. In particular, in WAT loss of fatty acids from TAG is dependent on both chain length and number of double bonds. Specifically, loss increases with number of double bonds and decreases with increasing chain length. The same general rules apply to BAT with the exception that linoleic acid 18:2 n6 is selectively retained in BAT TAG in comparison to WAT TAG. The relationship between fatty acid structure and loss from TAG in skeletal muscle has not previously been reported. We hypothesized that the retention of 18:2 n6 in BAT compared to WAT was due to it playing a specific role in BAT, perhaps related to the special role of fatty acid oxidation in BAT. To see if this retention of 18:2n6 was related to the oxidative function of BAT, we have examined the loss of fatty acids from soleus muscle, an oxidative muscle, during starvation to see if it also selectively retains linoleic acid. As part of this study we were also able to determine the pattern of fatty acid loss from muscle TAG as it relates to fatty acid structure.
2. Methods 2.1. Animals and diets Sixteen Sprague-Dawley rats weighing approximately 300g were obtained from Animal Care Services of Memorial University. The rats were housed two-per cage in a temperature and humidity controlled room, with a 12 hour light/dark cycle. The animals were placed on a Modified AIN 93G Diet (30% fat wt/wt, Table 1) for 2 weeks, with water provided ad libitum. The fatty acid composition of the diet is given in Table 2. The purpose of using a mixture of three fat sources was to enrich the TAG stores of the rats with a wide variety of
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Table 2 Diet Fatty Acid Composition (weight % ⫾ S.D.) Fatty Acid
Mean ⫾ SD
14:0 14:1 15:0 15:1 16:0 16:1 16:2 16:3 17:1 18:0 18:1 19:0 18:2 18:3 18:3 18:3 20:1 18:4 21:0 20:4 20:4 22:1 20:5 24:1 22:5 22:6
6.95 ⫾ 1.08 0.34 ⫾ 0.19 0.45 ⫾ 0.23 0.19 ⫾ 0.09 13.63 ⫾ 0.79 10.97 ⫾ 0.70 0.40 ⫾ 0.02 1.47 ⫾ 0.04 0.98 ⫾ 0.10 3.42 ⫾ 0.05 12.58 ⫾ 1.38 0.13 ⫾ 0.14 7.66 ⫾ 0.17 0.19 ⫾ 0.04 0.18 ⫾ 0.01 1.72 ⫾ 0.04 1.80 ⫾ 0.79 3.20 ⫾ 0.37 0.17 ⫾ 0.06 0.67 ⫾ 0.05 1.35 ⫾ 0.23 0.12 ⫾ 0.10 13.07 ⫾ 1.09 0.24 ⫾ 0.04 1.92 ⫾ 0.19 8.15 ⫾ 0.80
n7 n4 n4 n9 n6 n6 n4 n3 n9 n3 n6 n3 n9 n3 n3 n3
fatty acids, including long-chain polyunsaturates. The diet was prepared at the beginning of the study, divided into 50g portions and stored at ⫺70°C until used. Each morning, a new feed tray with a fresh 50g portion of feed was placed in the cage and the old feed tray was removed. At the end of the 14 day feeding period 1 rat from each cage was weighed and killed, and both soleus muscles were removed and weighed prior to lipid analysis, serving as the (fed) control group. The remaining 8 rats were then fasted for 48 hours, weighed and had their muscles removed for analysis. 2.2. Tissue sampling and lipid extraction Rats were anaesthetized with an I.P. injection of 40 mg Ketalean and 10 mg Xylazine per kilogram of body weight (MTC Pharmaceuticals). The soleus muscles were removed from both legs, weighed and placed in a 1:1 mixture of chloroform:methanol until they were extracted. The muscles were minced with scissors and extracted in chloroform/methanol (2:1) according to the method of Folch [7], adding 1 mg of triheptadecanoin per gram of muscle tissue as an internal standard. The Folch extracts were dried under N2, then dissolved in chloroform and stored under nitrogen at ⫺20°C until analysis.
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2.3. Lipid analysis Triacylglycerols were separated from other lipid fractions by thin-layer chromatography on silica gel G, using hexane-diethyl ether-glacial acetic acid (80:20:2 v/v/v) as the mobile phase. The TAG fraction was visualized by developing the plate in iodine and identified by comparison with known standards. The TAG spot was scraped off and extracted into chloroform, and the dried eluate was transmethylated in a 94% methanol/6% HCL mixture (8). The resultant fatty acid methyl esters were extracted from the transmethylation fluid into hexane, washed with distilled water and placed at ⫺10°C overnight allowing any left over water to be removed as ice crystals. The hexane was evaporated with N2, the methyl esters were resuspended in carbon disulfide and placed in GC vials with limited volume inserts (Hewlett Packard). Fatty acid methyl esters were separated by gas-chromatography using a Supelco SP-2330 fused silica capillary column (30m x 0.32 mm ID, 0.20m film thickness) in a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionization detector. Separation was performed isothermally at 180°C, with He as the carrier gas at a flow rate of 1mL/min. The split ratio was 100:1. Fatty acids were identified by comparing retention times with known standards, and were quantified by comparing peak areas with that of the C-17 internal standard. 2.4. Statistical analysis All statistics were determined using Graphpad Prism version 2.0. Percent fatty acid composition data were transformed prior to statistical analysis according to [9]by taking the arcsin square root of each weight percentage. 2.5. Chemicals Potassium Chloride and hydroquinone were purchased from Sigma. All gases, including Helium, Hydrogen, Nitrogen and Medical Air were supplied by Canadian Liquid Air Ltd. All other products were purchased from Fisher Scientific. Diet components were purchased from ICN Biomedicals Inc. Seal blubber oil was a gift from Dr. P. Davis, Biochemistry Department, Memorial University of Newfoundland. Max EPA was a gift of R.P. Scherer.
3. Results There was no change in the soleus muscle weight during the 48 h fast. However, the muscle TAG content was reduced by 43% (Table 3). The fatty acid composition of the muscle TAG was altered by the fast. The alterations were related to the structure of the fatty acids (Table 4). In general, those fatty acids whose percentage composition declined were polyunsaturated while those whose percentage increased were saturated. While the overall loss of muscle TAG was 43%, there was considerable variation in the loss of individual fatty acids (Figure 1). The loss of individual fatty acids varied from 30%
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209
Table 3 Solens muscle weight and triacyglycerol content from fed and 48 hour fasted rats
Muscle weight (g) Muscle triacylglycerol (mg)
Fed
Fasted
0.255 ⫾ 0.016 2.432 ⫾ 0.632
0.273 ⫾ 0.032 1.376 ⫾ 0.354*
Muscle weight and triacylglycerol are for the combined muscles from each rat. * Significantly different p⬍0.06. Values are mean ⫾ s.d. for rats.
for 22:1 n11, the fatty acid lost the least to 78.9% for 20:5 n3, the fatty acid lost to the greatest extent. An examination of the pattern of loss of fatty acids shows that fatty acids are not randomly lost but rather the loss depends on the structure of the individual fatty acids. As seen in Figure 1, the polyunsaturated fatty acids are lost to the greatest extent while the saturated and monounsaturated fatty acids are lost to a lesser extent. This difference in the mobilization of the polyunsaturated compared to the saturated and monounsaturated fatty acids was significant,(p⬍ 0.001 Mann-Whitney test). Figure 2 illustrates the selectivity of fatty acid loss during the fast. In this figure the results are presented as the ratio of the percent of a given fatty acid in the soleus muscle of fasted to fed rats. A value less than one indicates that that fatty acid makes up a lower percentage of total fatty acids in the fasted than the fed state. A value greater than one indicates that the Table 4 Fatty Acid Composition of Triacylglycerols from Soleus Muscle of Fed and Fasted Rats (weight % ⫾ S.D. for rats.) Fatty Acid
Fed
Fasted
12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:1 18:2 18:3 18:4 20:1 20:3 20:4 20:4 20:5 22:1 22:4 22:5 22:6
0.266 ⫾ 0.039 4.56 ⫾ 0.23 0.25 ⫾ 0.025 23.09 ⫾ 0.88 7.95 ⫾ 0.63 4.87 ⫾ 0.42 21.75 ⫾ 1.25 3.95 ⫾ 0.62 11.66 ⫾ 0.76 0.98 ⫾ 0.045 0.50 ⫾ 0.12 1.66 ⫾ 0.21 0.10 ⫾ 0.035 0.42 ⫾ 0.045 0.38 ⫾ 0.036 1.37 ⫾ 0.19 0.082 ⫾ 0.021 0.097 ⫾ 0.027 0.72 ⫾ 0.083 1.85 ⫾ 0.27
0.251 ⫾ 0.041 5.076 ⫾ 0.70 0.21 ⫾ 0.017* 28.25 ⫾ 2.77* 6.36 ⫾ 0.73* 5.69 ⫾ 0.67* 21.15 ⫾ 2.35 4.63 ⫾ 0.048 7.92 ⫾ 1.40* 0.51 ⫾ 0.12* 0.33 ⫾ 0.07* 1.48 ⫾ 0.29 0.11 ⫾ 0.037 0.25 ⫾ 0.036* 0.22 ⫾ 0.041* 0.52 ⫾ 0.15* 0.10 ⫾ 0.007 0.074 ⫾ 0.014* 0.49 ⫾ 0.15* 1.33 ⫾ 0.21*
n5 n7 n9 n7 n6 n3 n3 n9 n6 n3 n6 n3 n11 n6 n3 n3
* Indicates a significant difference between fed and fasted samples (p ⬍ 0.05).
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Fig. 1. Fractional mobilization of fatty acids from soleus muscle triacylglycerol. Fractional mobilization is the percentage of the mass of a given fatty acid that is lost from soleus muscle during the 48 hour fast. Values are mean ⫾ s.d for 8 pairs of rats. Polyunsaturated fatty acids are shown in black, saturated and monounsaturated fatty acids are shown in grey. The values are arranged around the value of 43% which represents the average loss of all fatty acids. Values above the line have a greater mobilization and those below the line a smaller mobilization than average. Significant differences from the average are indicated by an *.
fatty acid makes up a higher percentage of total fatty acids in the fasted than the fed state. It can be seen than the PUFA generally have a ratio less than one which is consistent with their greater absolute loss as seen in Figure 1. The relationship between the loss of individual fatty acids from muscle TAG and that from either WAT or BAT is shown in Figure 3. It can be seen that the pattern of loss from muscle is generally similar to both WAT and BAT. However, it can be seen that the loss of linoleic acid from muscle is more like that from WAT than BAT.
4. Discussion The loss of fatty acids from adipose tissue triacylglycerol during fasting depends on the structure of the fatty acids and is not merely a function of the relative abundance of the individual fatty acids [1,5]. Specifically, the loss increases with increasing unsaturation at a given chain length and for a given number of double bonds, loss decreases with increasing chain length. The general pattern of loss is similar in both white and brown adipose tissue with the striking exception of linoleic acid which is specifically retained in BAT TAGs during food deprivation. We had hypothesized that this might be due to the special role of fatty acid oxidation in BAT compared to WAT.
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Fig. 2. Ratio of the percent of a given fatty acid in the soleus muscle of fasted to fed rats. Polyunsaturated fatty acids are shown in black, saturated and monounsaturated fatty acids are shown in grey. A value less than one indicates that that fatty acid makes up a lower percentage of total fatty acids in the fasted than the fed state. A value greater than one indicates that the fatty acid makes up a higher percentage of total fatty acids in the fasted than the fed state.
To test this we examined the loss of fatty acids during food deprivation from soleus muscle TAGs. The pattern of loss of fatty acids from muscle TAG during fasting has not previously been examined. It is known that muscle TAGs are metabolically active and that they are used for energy in the muscle during exercise [10,11] and that they also decline in muscle during starvation ([12] and Table 3). We hypothesized that if the selective retention of linoleic acid in BAT were related to a specific role in fatty acid oxidation for energy, then we should see a similar selective retention in soleus muscle TAGs. This would appear to not be the case since, as can be seen in Figure 2, the loss of linoleate is similar in both soleus muscle and WAT. Thus, it appears that the selective retention of linoleate in BAT must be related to some function of the tissue other than fatty acid oxidation. This is the first detailed study of the specific utilization of muscle TAG fatty acids. Our findings that PUFA are preferentially used during fasting raises interesting possibilities. If the same pattern of use is observed during exercise, as seems likely since the same biochemical mechanisms are involved in hydrolysis and oxidation, then it may be possible to improve muscle performance by enriching the TAG fatty acid pool with PUFA. It is known that during exercise muscle TAG fatty acids provide a significant portion of the fatty acids oxidized [13,14]. One of the limiting factors in the endurance performance of skeletal muscle is the preservation of muscle glycogen by use of fat as a fuel source [14]. It is possible that enriching the muscle TAG pool with PUFA will spare glycogen better than if the pool were enriched with saturated or monounsaturated fatty acids. Thus, a diet enriched in PUFA,
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Fig. 3. Relationship between the fractional mobilization of fatty acids from soleus muscle and that from white adipose tissue or brown adipose tissue. The mobilizations from soleus muscle are taken from the present study. The mobilizations from white adipose tissue are taken from [5] and the mobilizations from brown adipose tissue are taken from [1]. The solid line is the regression line for BAT and the dashed line is the regression line for WAT. The data points for linoleic acid are identified.
which will enrich the muscle TAGs with PUFA, may improve endurance performance. This hypothesis can easily be tested both with in vitro working muscle preparations and in vivo In conclusion, we have shown that the loss of fatty acids from muscle TAGs during short term starvation depends on the fatty acid structure of the component TAGs with PUFA being preferentially lost. The pattern of loss is similar to that in white adipose tissue but different from BAT in the specific case of linoleic acid which is preferentially retained in BAT but not in WAT or muscle. This leads us to conclude that the retention of linoleate in BAT is not related to the specific role of fatty acid oxidation in BAT. The preferential use of PUFA by muscle raises the possibility of improving muscle endurance performance by enriching the TAG pool with PUFA.
Acknowledgments Supported by a grant to GRH from the Natural Sciences and Engineering Research Council (Canada).
References [1] Groscolas R, Herzberg GR. Fasting-induced selective mobilization of brown adipose tissue fatty acids. J Lipid Res 1997;38:228 –38.
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[2] Field CJ, Clandinin MT. Modulation of adipose tissue fat composition by diet: a review. Nutr Res 1984;4:743–55. [3] Sheppard K, Herzberg GR. Triacylglycerol composition of adipose tissue, muscle and liver of rats fed diets containing fish oil or corn oil. Nutr Res 1992;12:1405–18. [4] Raclot T, Groscolas R, Leray C. Composition and structure and triacylglycerols in brown adipose tissue of rats fed fish oil. Lipids 1994;29:759 – 64. [5] Raclot T, Groscolas R. Differential mobilization of white adipose tissue fatty acids according to chain length, unsaturation, and positional isomerism. J Lipid Res 1993;34:1515–26. [6] Herzberg GR. The 1990 Borden Award Lecture. Dietary regulation of fatty acid and triglyceride metabolism. Can J Physiol Pharmacol 1991;69:1637– 47. [7] Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497–509. [8] Chernenko GA, Barrowman JA, Kean KT, Herzberg GR, Keough KMW. Intestinal absorption and lymphatic transport of fish oil (MaxEPA) in the rat. Biochim Biophys Acta 1989;1003:95–102. [9] Zar JH. Biostatistical Analysis. 2nd ed. Englewood Cliffs, NJ: Prentice-Hall, Inc., 1984. [10] Boesch C, De´ combaz J, Slotboom J, Kreis R. Observation of intramyocellular lipids by means of 1H magnetic resonance spectroscopy. Proc Nut Soc 1999;58:841–50. [11] Gorski J. Muscle triglyceride metabolism during exercise. Can J Physiol Pharmacol 1992;70:123–31. [12] Jaromowska M, Gorski J. Effect of fasting on skeletal muscle triglyceride content. Experientia 1985;41: 357– 8. [13] Romijn JA, Coyle EF, Sidossis LS, et al. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am J Physiol 1993:E380 –91. [14] Horowitz JF, Klein S. Lipid metabolism during endurance exercise. Am J Clin Nutr 2000;72:558A– 63S.
Fasting-induced, selective loss of fatty acids from muscle triacylglycerols Gene R. Herzberg*, Brian Farrell Department of Biochemistry, Memorial University of Newfoundland St. John’s, Newfoundland A1B 3X9 Canada Received 15 April 2002; received in revised form 24 September 2002; accepted 25 September 2002
Abstract We studied the loss of fatty acids from soleus muscle triacylglycerol (TAG) in rats fasted for 48 hours. During the 48 h fast the TAG content of the muscle decreased from 2.432 ⫾ 0.632 mg to 1.376 ⫾ 0.354 mg (X ⫾ s.d, n⫽8.) although the muscle weight was unchanged (0.255 ⫾ 0.016 vs. 0.273 ⫾ 0.032 g). Fatty acid mobilization was calculated as the percentage of the mass of a given fatty acid lost during the fast. The average mobilization for all fatty acids was 43.2%. The mobilization of individual fatty acids, varied from 30% for 20:1, the least mobilized fatty acid to 78.5% for EPA (20:5 n3) the most mobilized fatty acid. The polyunsaturated fatty acids were mobilized to a significantly greater extent than the monounsaturated and saturated fatty acids. This resulted in an enrichment in monounsaturated and saturated fatty acids in the muscle TAGs following the fast. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Muscle; Triacylglycerol; Fatty acids; Selectivity
1. Introduction The fatty acid composition of triacylglycerols (TAGs) in adipose tissue, brown adipose tissue and skeletal muscle is broadly reflective of the fatty acid composition of the diet [1– 4]. However, there are differences in the storage and release of dietary fatty acids that lead to differences between the fatty acid composition of the diet and that in the tissue [1,5,6]. It has been determined that the structure of the fatty acids in the tissue TAGs determines their net
* Corresponding author. Tel.: ⫹1-709-737-8800; fax: ⫹1-709-737-2422. E-mail address: [email protected] (G.R. Herzberg). 0271-5317/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 5 1 5 - 8
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Table 1 Modified AIN 93G Diet Component
g/Kg
AIN 93 Mineral Mix AIN 93 Vitamin Mix L-Cystine Choline Bitartrate Tert Butyl Hydroquinone Casein Fibre (Alphacel) Corn Starch Dextrinized Corn Starch Sucrose Fat*
35 10 3 2.5 0.014 200 50 252.5 83.5 63.5 300
* The fat was made up of 40 g soybean oil, 60 g seal blubber oil and 200 g fish oil.
storage in both white and brown adipose tissue but even in these two tissues there are significant differences in the specificity of net storage. It has previously been shown that certain long-chain n3 fatty acids are underrepresented in adipose tissue and skeletal muscle compared to other fatty acids and relative to their proportion in the diet [3]. The fatty acid composition of tissue TAG is modulated by both diet and the selectivity of the loss of fatty acids from TAG. In particular, in WAT loss of fatty acids from TAG is dependent on both chain length and number of double bonds. Specifically, loss increases with number of double bonds and decreases with increasing chain length. The same general rules apply to BAT with the exception that linoleic acid 18:2 n6 is selectively retained in BAT TAG in comparison to WAT TAG. The relationship between fatty acid structure and loss from TAG in skeletal muscle has not previously been reported. We hypothesized that the retention of 18:2 n6 in BAT compared to WAT was due to it playing a specific role in BAT, perhaps related to the special role of fatty acid oxidation in BAT. To see if this retention of 18:2n6 was related to the oxidative function of BAT, we have examined the loss of fatty acids from soleus muscle, an oxidative muscle, during starvation to see if it also selectively retains linoleic acid. As part of this study we were also able to determine the pattern of fatty acid loss from muscle TAG as it relates to fatty acid structure.
2. Methods 2.1. Animals and diets Sixteen Sprague-Dawley rats weighing approximately 300g were obtained from Animal Care Services of Memorial University. The rats were housed two-per cage in a temperature and humidity controlled room, with a 12 hour light/dark cycle. The animals were placed on a Modified AIN 93G Diet (30% fat wt/wt, Table 1) for 2 weeks, with water provided ad libitum. The fatty acid composition of the diet is given in Table 2. The purpose of using a mixture of three fat sources was to enrich the TAG stores of the rats with a wide variety of
G.R. Herzberg, B. Farrell / Nutrition Research 23 (2003) 205–213
207
Table 2 Diet Fatty Acid Composition (weight % ⫾ S.D.) Fatty Acid
Mean ⫾ SD
14:0 14:1 15:0 15:1 16:0 16:1 16:2 16:3 17:1 18:0 18:1 19:0 18:2 18:3 18:3 18:3 20:1 18:4 21:0 20:4 20:4 22:1 20:5 24:1 22:5 22:6
6.95 ⫾ 1.08 0.34 ⫾ 0.19 0.45 ⫾ 0.23 0.19 ⫾ 0.09 13.63 ⫾ 0.79 10.97 ⫾ 0.70 0.40 ⫾ 0.02 1.47 ⫾ 0.04 0.98 ⫾ 0.10 3.42 ⫾ 0.05 12.58 ⫾ 1.38 0.13 ⫾ 0.14 7.66 ⫾ 0.17 0.19 ⫾ 0.04 0.18 ⫾ 0.01 1.72 ⫾ 0.04 1.80 ⫾ 0.79 3.20 ⫾ 0.37 0.17 ⫾ 0.06 0.67 ⫾ 0.05 1.35 ⫾ 0.23 0.12 ⫾ 0.10 13.07 ⫾ 1.09 0.24 ⫾ 0.04 1.92 ⫾ 0.19 8.15 ⫾ 0.80
n7 n4 n4 n9 n6 n6 n4 n3 n9 n3 n6 n3 n9 n3 n3 n3
fatty acids, including long-chain polyunsaturates. The diet was prepared at the beginning of the study, divided into 50g portions and stored at ⫺70°C until used. Each morning, a new feed tray with a fresh 50g portion of feed was placed in the cage and the old feed tray was removed. At the end of the 14 day feeding period 1 rat from each cage was weighed and killed, and both soleus muscles were removed and weighed prior to lipid analysis, serving as the (fed) control group. The remaining 8 rats were then fasted for 48 hours, weighed and had their muscles removed for analysis. 2.2. Tissue sampling and lipid extraction Rats were anaesthetized with an I.P. injection of 40 mg Ketalean and 10 mg Xylazine per kilogram of body weight (MTC Pharmaceuticals). The soleus muscles were removed from both legs, weighed and placed in a 1:1 mixture of chloroform:methanol until they were extracted. The muscles were minced with scissors and extracted in chloroform/methanol (2:1) according to the method of Folch [7], adding 1 mg of triheptadecanoin per gram of muscle tissue as an internal standard. The Folch extracts were dried under N2, then dissolved in chloroform and stored under nitrogen at ⫺20°C until analysis.
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2.3. Lipid analysis Triacylglycerols were separated from other lipid fractions by thin-layer chromatography on silica gel G, using hexane-diethyl ether-glacial acetic acid (80:20:2 v/v/v) as the mobile phase. The TAG fraction was visualized by developing the plate in iodine and identified by comparison with known standards. The TAG spot was scraped off and extracted into chloroform, and the dried eluate was transmethylated in a 94% methanol/6% HCL mixture (8). The resultant fatty acid methyl esters were extracted from the transmethylation fluid into hexane, washed with distilled water and placed at ⫺10°C overnight allowing any left over water to be removed as ice crystals. The hexane was evaporated with N2, the methyl esters were resuspended in carbon disulfide and placed in GC vials with limited volume inserts (Hewlett Packard). Fatty acid methyl esters were separated by gas-chromatography using a Supelco SP-2330 fused silica capillary column (30m x 0.32 mm ID, 0.20m film thickness) in a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionization detector. Separation was performed isothermally at 180°C, with He as the carrier gas at a flow rate of 1mL/min. The split ratio was 100:1. Fatty acids were identified by comparing retention times with known standards, and were quantified by comparing peak areas with that of the C-17 internal standard. 2.4. Statistical analysis All statistics were determined using Graphpad Prism version 2.0. Percent fatty acid composition data were transformed prior to statistical analysis according to [9]by taking the arcsin square root of each weight percentage. 2.5. Chemicals Potassium Chloride and hydroquinone were purchased from Sigma. All gases, including Helium, Hydrogen, Nitrogen and Medical Air were supplied by Canadian Liquid Air Ltd. All other products were purchased from Fisher Scientific. Diet components were purchased from ICN Biomedicals Inc. Seal blubber oil was a gift from Dr. P. Davis, Biochemistry Department, Memorial University of Newfoundland. Max EPA was a gift of R.P. Scherer.
3. Results There was no change in the soleus muscle weight during the 48 h fast. However, the muscle TAG content was reduced by 43% (Table 3). The fatty acid composition of the muscle TAG was altered by the fast. The alterations were related to the structure of the fatty acids (Table 4). In general, those fatty acids whose percentage composition declined were polyunsaturated while those whose percentage increased were saturated. While the overall loss of muscle TAG was 43%, there was considerable variation in the loss of individual fatty acids (Figure 1). The loss of individual fatty acids varied from 30%
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Table 3 Solens muscle weight and triacyglycerol content from fed and 48 hour fasted rats
Muscle weight (g) Muscle triacylglycerol (mg)
Fed
Fasted
0.255 ⫾ 0.016 2.432 ⫾ 0.632
0.273 ⫾ 0.032 1.376 ⫾ 0.354*
Muscle weight and triacylglycerol are for the combined muscles from each rat. * Significantly different p⬍0.06. Values are mean ⫾ s.d. for rats.
for 22:1 n11, the fatty acid lost the least to 78.9% for 20:5 n3, the fatty acid lost to the greatest extent. An examination of the pattern of loss of fatty acids shows that fatty acids are not randomly lost but rather the loss depends on the structure of the individual fatty acids. As seen in Figure 1, the polyunsaturated fatty acids are lost to the greatest extent while the saturated and monounsaturated fatty acids are lost to a lesser extent. This difference in the mobilization of the polyunsaturated compared to the saturated and monounsaturated fatty acids was significant,(p⬍ 0.001 Mann-Whitney test). Figure 2 illustrates the selectivity of fatty acid loss during the fast. In this figure the results are presented as the ratio of the percent of a given fatty acid in the soleus muscle of fasted to fed rats. A value less than one indicates that that fatty acid makes up a lower percentage of total fatty acids in the fasted than the fed state. A value greater than one indicates that the Table 4 Fatty Acid Composition of Triacylglycerols from Soleus Muscle of Fed and Fasted Rats (weight % ⫾ S.D. for rats.) Fatty Acid
Fed
Fasted
12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:1 18:2 18:3 18:4 20:1 20:3 20:4 20:4 20:5 22:1 22:4 22:5 22:6
0.266 ⫾ 0.039 4.56 ⫾ 0.23 0.25 ⫾ 0.025 23.09 ⫾ 0.88 7.95 ⫾ 0.63 4.87 ⫾ 0.42 21.75 ⫾ 1.25 3.95 ⫾ 0.62 11.66 ⫾ 0.76 0.98 ⫾ 0.045 0.50 ⫾ 0.12 1.66 ⫾ 0.21 0.10 ⫾ 0.035 0.42 ⫾ 0.045 0.38 ⫾ 0.036 1.37 ⫾ 0.19 0.082 ⫾ 0.021 0.097 ⫾ 0.027 0.72 ⫾ 0.083 1.85 ⫾ 0.27
0.251 ⫾ 0.041 5.076 ⫾ 0.70 0.21 ⫾ 0.017* 28.25 ⫾ 2.77* 6.36 ⫾ 0.73* 5.69 ⫾ 0.67* 21.15 ⫾ 2.35 4.63 ⫾ 0.048 7.92 ⫾ 1.40* 0.51 ⫾ 0.12* 0.33 ⫾ 0.07* 1.48 ⫾ 0.29 0.11 ⫾ 0.037 0.25 ⫾ 0.036* 0.22 ⫾ 0.041* 0.52 ⫾ 0.15* 0.10 ⫾ 0.007 0.074 ⫾ 0.014* 0.49 ⫾ 0.15* 1.33 ⫾ 0.21*
n5 n7 n9 n7 n6 n3 n3 n9 n6 n3 n6 n3 n11 n6 n3 n3
* Indicates a significant difference between fed and fasted samples (p ⬍ 0.05).
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Fig. 1. Fractional mobilization of fatty acids from soleus muscle triacylglycerol. Fractional mobilization is the percentage of the mass of a given fatty acid that is lost from soleus muscle during the 48 hour fast. Values are mean ⫾ s.d for 8 pairs of rats. Polyunsaturated fatty acids are shown in black, saturated and monounsaturated fatty acids are shown in grey. The values are arranged around the value of 43% which represents the average loss of all fatty acids. Values above the line have a greater mobilization and those below the line a smaller mobilization than average. Significant differences from the average are indicated by an *.
fatty acid makes up a higher percentage of total fatty acids in the fasted than the fed state. It can be seen than the PUFA generally have a ratio less than one which is consistent with their greater absolute loss as seen in Figure 1. The relationship between the loss of individual fatty acids from muscle TAG and that from either WAT or BAT is shown in Figure 3. It can be seen that the pattern of loss from muscle is generally similar to both WAT and BAT. However, it can be seen that the loss of linoleic acid from muscle is more like that from WAT than BAT.
4. Discussion The loss of fatty acids from adipose tissue triacylglycerol during fasting depends on the structure of the fatty acids and is not merely a function of the relative abundance of the individual fatty acids [1,5]. Specifically, the loss increases with increasing unsaturation at a given chain length and for a given number of double bonds, loss decreases with increasing chain length. The general pattern of loss is similar in both white and brown adipose tissue with the striking exception of linoleic acid which is specifically retained in BAT TAGs during food deprivation. We had hypothesized that this might be due to the special role of fatty acid oxidation in BAT compared to WAT.
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Fig. 2. Ratio of the percent of a given fatty acid in the soleus muscle of fasted to fed rats. Polyunsaturated fatty acids are shown in black, saturated and monounsaturated fatty acids are shown in grey. A value less than one indicates that that fatty acid makes up a lower percentage of total fatty acids in the fasted than the fed state. A value greater than one indicates that the fatty acid makes up a higher percentage of total fatty acids in the fasted than the fed state.
To test this we examined the loss of fatty acids during food deprivation from soleus muscle TAGs. The pattern of loss of fatty acids from muscle TAG during fasting has not previously been examined. It is known that muscle TAGs are metabolically active and that they are used for energy in the muscle during exercise [10,11] and that they also decline in muscle during starvation ([12] and Table 3). We hypothesized that if the selective retention of linoleic acid in BAT were related to a specific role in fatty acid oxidation for energy, then we should see a similar selective retention in soleus muscle TAGs. This would appear to not be the case since, as can be seen in Figure 2, the loss of linoleate is similar in both soleus muscle and WAT. Thus, it appears that the selective retention of linoleate in BAT must be related to some function of the tissue other than fatty acid oxidation. This is the first detailed study of the specific utilization of muscle TAG fatty acids. Our findings that PUFA are preferentially used during fasting raises interesting possibilities. If the same pattern of use is observed during exercise, as seems likely since the same biochemical mechanisms are involved in hydrolysis and oxidation, then it may be possible to improve muscle performance by enriching the TAG fatty acid pool with PUFA. It is known that during exercise muscle TAG fatty acids provide a significant portion of the fatty acids oxidized [13,14]. One of the limiting factors in the endurance performance of skeletal muscle is the preservation of muscle glycogen by use of fat as a fuel source [14]. It is possible that enriching the muscle TAG pool with PUFA will spare glycogen better than if the pool were enriched with saturated or monounsaturated fatty acids. Thus, a diet enriched in PUFA,
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Fig. 3. Relationship between the fractional mobilization of fatty acids from soleus muscle and that from white adipose tissue or brown adipose tissue. The mobilizations from soleus muscle are taken from the present study. The mobilizations from white adipose tissue are taken from [5] and the mobilizations from brown adipose tissue are taken from [1]. The solid line is the regression line for BAT and the dashed line is the regression line for WAT. The data points for linoleic acid are identified.
which will enrich the muscle TAGs with PUFA, may improve endurance performance. This hypothesis can easily be tested both with in vitro working muscle preparations and in vivo In conclusion, we have shown that the loss of fatty acids from muscle TAGs during short term starvation depends on the fatty acid structure of the component TAGs with PUFA being preferentially lost. The pattern of loss is similar to that in white adipose tissue but different from BAT in the specific case of linoleic acid which is preferentially retained in BAT but not in WAT or muscle. This leads us to conclude that the retention of linoleate in BAT is not related to the specific role of fatty acid oxidation in BAT. The preferential use of PUFA by muscle raises the possibility of improving muscle endurance performance by enriching the TAG pool with PUFA.
Acknowledgments Supported by a grant to GRH from the Natural Sciences and Engineering Research Council (Canada).
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