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The effects of exercise and feeding on the activity of lipoprotein lipase in nine different adipose depots of guinea pigs
Inf. J. Biochem.Vol. 24, No. 11, pp. 1825-1831,1992 Printed in Great Britain. All rights reserved
THE EFFECTS ACTIVITY DIFFERENT
0020-711X/92$5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd
OF EXERCISE AND FEEDING ON THE OF LIPOPROTEIN LIPASE IN NINE ADIPOSE DEPOTS OF GUINEA PIGS
CAROLINEM. POND, CHRISTINEA. MATTACKSand DAWN SADLER Department of Biology, The Open University, Milton Keynes MK7 6AA, U.K. [Tel. (0908) 655077; Fax (0908) 6537441 (Received 9 March 1992) Abstract-l.
The activity of lipoprotein lipase (LPL) was measured in whole adipose tissue from 9 identified adipose depots of sedentary, fasting adult guinea pigs and following 30min of exercise or voluntary ingestion of chow, and in adipocyte and stromal-vascular fractions from exercised specimens. 2 In sedentary, fasting specimens, LPL activity was up to 4 times higher in the small intermuscular depots than in the p&renal and epididymal depot (Table 1). 3. LPL activity increased significantly after feeding only in the large superficial depot, groin, and in the perirenal depot. LPL activity decreased after exercise only in the 2 intermuscular depots and in small anterior superficial depots. These effects of exercise were consistently greater in males than in females (Table 3). 4. Followng exercise, there was up to twice as much LPL in the adipocytes as in the stromal-vascular fraction of the intermuscular depots, about 50% more in adipocytes from the minor superficial depots and about equal quantities in the 2 fractions of the intra-abdominal and groin depots (Table 2). 5. The data demonstrate the physiological inhomogeneity of both superficial and internal adipose depots, and are consistent with the hypothesis that LPL originating from adipose tissue may enter the circulation.
MATERIALS AND
INTRODUClTON Lipoprotein lipase (LPL) is the principal enzyme mediating the uptake of lipids from circulating lipoprotein, and is synthesized by many extra-hepatic tissues in mammals (Bensadoun, 1991). Most of the extensive information (Olivecrona and BengtssonOlivecrona, 1990) about its molecular structure and genetic basis come from the epididymal or parametrial depots of the rat and a few superficial depots in humans (Pouliot et al., 1991). In these adipose depots, LPL activity is modulated by feeding (Bensadoun, 1991; Nilsson-Ehle et al., 1975; Doolittle et al., 1990), but the effects of exercise are minimal (LaDu et al., 199la), which is surprising in view of the importance of this enzyme in regulating the concentration of circulating triacylglycerol. Site-specific differences in adipocyte volume (Pond et al., 1986), maximum glycolytic capacity (Mattacks et al., 1987), fatty acid-triacylglycerol cycling (Mattacks and Pond, 1988) and response to insulin and noradrenaline (Pond and Mattacks, 1991), have been described with many of the most atypical properties occurring
in small, rarely studied depots (Marchington and Pond, 1990). Guinea pigs are the smallest convenient laboratory animal in which there is sufficient tissue in these minor depots for accurate assay of LPL. In this paper, we report on the effects of feeding and a brief period of exercise on LPL activity in 9 depots that together comprise almost all the adipose mass.
METHODS
Animals and dissection procedure The guinea pigs used for assays of LPL in whole tissue (groups l-3) were Bolivians that were born and raised at the Open University in standard cages (area 0.45 m*) in which breeding-grade guinea pig chow and water with added vitamin C (0.1 mg ml-‘) were available ad Zibitum. They were also given cabbage, carrot and apple on 5 days per week and hay every day. They were transferred to permanent, single-sex groups at weaning and used as mature adults, aged 615 months (mean 11.1 f 0.54), body mass 7OCN175 g (mean 965 f 18.5). Those used for assays of fractionated adipose tissue (group 4) were commercially obtained Dunkin Hartleys. They were rested and fed the same diet as the Bolivians for at least a month before being used, at the age of about 11 months, mean body mass 1152 f 14.4 g. Each experimental group consisted of 5 males and 5 females. All specimens were fasted for 18 hr before being killed, with an IP injection of pentabarbitone dissolved in phosphate buffered saline. The sedentary specimens (group 1) were isolated in small (area 0.19 m2) cages for 18 hr before the experiment and handled very gently before being killed. In nearly all cases, this procedure was completed without any squeaking or struggling. Pilot experiments showed that previously ad libitm-fed guinea pigs would not reliably eat significant quantites of chow after a single 18 hr overnight fast. Therefore, group 2 specimens were placed in separate cages and given food for only 5 hr per day for the 2 days before the experiment. A known mass of chow was placed in the cage at 0800 and the animals were killed at 0930,
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CAROLINE M. POND et al.
provided they had eaten at least 3 g. The exercised specimens (groups 3 and 4) remained in the larger cages (area 0.45 m2) in single sex groups of 26 animals. Guinea pigs do not exercise spontaneously in running wheels as murid rodents do, so an exercise pen, consisting of two concentric squares of “walls” 30 cm high placed on a rough mat that formed an enclosed path 30 cm wide and approx 7 m long was used. They were chased around this apparatus for 30 mm immediately before being killed by a similar procedure. In a few specimens, running was accompanied by squeaking and other signs of general excitement, and most were close to exhaustion by the end of the exercise period. Immediately after death, l-2 samples of adipose tissue were taken from the following sites: mesenteric and omental (MES + OME), around kidney and dorsal wall of abdomen plus epididymal (DWA), in front of forelimb and shoulder (S), behind forelimb (BA), side and ventral groin (= inguinal), interscapular (HUMP), medial to trapezius muscle of neck (UMN), plus the whole of the popliteal (POP) depot and all dissectible adipose tissue from around the heart (Cardiac). These depots, which, in guinea pigs, comprise almost all the dissectible adipose tissue, are exactly homologous with those studied previously (Pond et al., 1986; Mattacks ef al., 1987; Pond and Mattacks 1991). The rest of the adipose tissue in each depot was later dissected out and weighed. Total fatness is the sum of the masses of these adipose depots expressed as a percentage of the live body mass. In some previous studies (Pond et al., 1986; Mattacks ef al., 1987) in front of forelimb and in front of shoulder depots, and perirenal and epididymal have been studied separately, but since they have similar properties in guinea pigs, they were combined for this project. Because of the small quantities of cardiac adipose tissue, no attempt was made to separate this depot into adipocytes and extracellular fractions. Fractionation
and drying of tissue and LPL assay
LPL was assayed in acetone/ether dried samples of whole
or fractionated adipose tissue using PH]triolein as a substrate, following the method developed by Nilsson-Ehle er al. (1972). For experiments 1-3, preweighed samples of about 0.5 g (in the case of the cardiac depot, all available tissue) of whole tissue were homogenized in cold acetone on ice. For experiment 4, the tissue was fractionated into adipocytes and stromal-vascular material using established methods (Rodbell, 1964): about 1 g from each sample was placed in warm freshly gassed Krebs-Hensliet buffer and chopped into pieces of about 100 mg. After further washing, the tissues were incubated with 5 mg’ml-’ collagenase (Sigma type lA-C-9891) for 25min at 37°C in a shaking waterbath. The resulting suspension was filtered through fine gauze, and centrifuged at 1000 rpm for about 5 min and the isolated cells collected and placed in cold acetone. The extracellular material was spun down at 2OOOrpm for 10min and decanted into cold acetone. All samples were centrifuged at 1000 rpm for IO min, the acetone decanted off and centrifuged again with more cold acetone. The procedure was repeated with ether and any remaining ether was evaporated in an atmosphere of nitrogen. The dried tissue was weighed out as 10mg samples and stored frozen at -15°C for up to 1 week. To start the assay, 1 ml of 0.05 M Tri-HCl with 1 M ethylene glycol (pH 8.0) was added to each sample, the mixture was homogenized on ice and centrifuged at 15,000 rpm for 10 min and the supematant collected. The substrate was prepared from 1.5ml heptane, 5OpCi
(1.85 mBq) [‘Hltriolein and 21 mg unlabelled triolein and 1.2 mg lysophosphatidyl choline, mixed thoroughly. The heptane was evaporated in an atmosphere of nitrogen. 7.5 ml of 0.2 M Tri-HCl (PH 8.1) was added and the cooled preparation was sonicated for 8 bursts lasting 30 set each, and cooled on ice between bursts. 0.9ml of 4% BSA and 0.6ml of serum prepared from a fasted guinea pig were added and mixed well, and 0.1 ml of the supernatant was incubated with 0.1 ml of substrate in a shaking waterbath for 1 hr at 37°C. The reaction was stopped with 3.25 ml methanol:chloroform: heptane (2.3: 2.5: 0.8) and 1.05ml of 0.1 M potassium carbonate was added to each vial and the mixtures were vortexed for 15 set and centrifuged at 2000 rpm for 15 min. 0.25 ml samples of the upper phase were counted in a Beckman 1701 scintillation counter for 10min. Total protein was measured in the pellet residue from the sample preparation using the Bradford (1976) method. Student’s r-test was used to compare values from homologous depots from differently treated animals, and except where otherwise stated, values indicating P < 0.05 were taken as significant.
RESULTS
The Bolivians (mean fatness 6.0% f 0.11) were slightly but significantly (t = 2.904) fatter than the Dunkin Hartleys (mean fatness 5.5% f 0.13), although the latter were significantly larger than the Bolivians (t = 7.99). As expected, the females were significantly smaller (mean BM 961 &-27 g) but fatter (mean fatness 6.1% f 0.14) than the males (mean BM 1061 f 23 g; mean fatness 5.7% f 0.10). The measures of enzyme activity did not correlate significantly with age, body mass or fatness any more frequently than would be expected by chance. The relative masses of the depots were similar to measurements reported previously (Pond et al., 1986; Pond and Mattacks, 1991). The quantity of cardiac adipose tissue is always very variable (Marchington and Pond, 1990), and there was sufficient adipose tissue in this depot for analysis in only 10 males and 12 females; the mean quantity in the latter was 0.35 + 0.03 g, representing 0.62% of the total adipose tissue, compared to 0.31 + 0.03 g (0.56% of the total) in males. LPL activities in samples of whole adipose tissue from nine different depots in the 4 treatment groups are listed in Table 1. The average quantity of food eaten by the group 2 animals was 5.8 f 1.15 g, and the sexes did not differ in this respect. There were large site-specific differences in LPL activity at rest, with that in the intermuscular depots (POP and UMN) being up to 3.8 times higher than that of the much larger DWA depot. LPL activities in the 2 most commonly studied depots (DWA and Groin) were not significantly different from each other in resting, fasting animals. Following feeding (group 2), LPL activity increased in these 2 large depots, that together accounted for 39% of all the adipose tissue, and in the much smaller cardiac depot. In all the other depots, increases were barely significant or
Site-specificdifferences in LPL activity
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Table 1. Mean and standard deviation of the activity of lipoprotein lipase and approximate relative mass (mass of depot as % total dissectible adipose tissue) in 9 adipose depots of guinea pigs treated in 3 different ways (n = 10, 5 male, 5 female, except for cardiac depot, for which n = 8 for group 2 and n = 7 for groups 1 and 3) I Sedentary, fasting
Group number 2 3 Sedentary, fed Exercised, fasting
4 Exercised, fasting
Mean
SD
Mean
SD
Mean
SD
9 17 16
0.48 0.38 0.51 0.26
0.08 0.05 0.11 0.04
0.56 0.48. 0.60 0.3911
0.14 0.13 0.15 0.10
0.35** 0.31. 0.38* 0.25
0.06 0.07 0.11 0.05
0.29 0.23 0.28 0.20
0.06 0.06 0.06 0.04
21 23
0.41 0.19
0.12 0.04
0.53 0.33”
0.14 0.09
0.30. 0.20
0.07 0.02
0.27 0.19
0.06 0.04
3 5 0.6
0.74 0.63 0.24
0.21 0.13 0.09
0.71 0.67 0.42..
0.17 0.18 0.13
0.44.. 0.41.. 0.28
0.08 0.07 0.05
0.35 0.10 0.32 0.08 not measured
Relative mass % total AT Superficial S
BA HUMP Groin
6
SD
Mean
Intro-abdominal
MES + OME DWA Intermuscular
POP UMN Cardiac
Groups l-3 were Bolivian strain; Group 4 was Dunkin Hartley strain. All units are nmol fatty acid/min/mg dry weight of tissue. *Significantly different from homologous depot in group 1 (sedentary, fasting) at P < 0.05. **Significantly different from homologous depot in group 1 (sedentary, fasting) at P < 0.01.
insignificant and were vanishingly small in the intermuscular depots. The effect of these changes was to make the LPL activity per g of tissue more nearly equal in all the depots studied: the ratio of its activity in POP to that in DWA fell from 3.8 in sedentary, fasting specimens, to 2.1 in the fed group, and 2.2 and 1.9 in the exercised groups 3 and 4 respectively. Analysis of variance showed that there were no significant sex differences in the relative masses of the depots or in their site-specific LPL activity in either sedentary group. Some of the site-specific differences in LPL activity per of tissue can be eliminated by taking account of differences in adipocyte volume. Combining the data on Table 1 with the mean site-specific adipocyte volumes of similarly maintained guinea pigs of the same age and strain (Pond et al., 1986) shows that, in the sedentary fasting animals, the LPL activities per adipocyte were not significantly different in the 4 superficial depots (0.24-0.30 nmol fatty acid/min/106 adipocytes). However, LPL activity in POP adipocytes was substantially higher (0.75 mnol fatty acid/min/106 adipocytes) and that in the cardiac and DWA depots lower (0.11 and 0.17 nmol fatty
acid/min/106 adipocytes respectively) than in the other depots. LPL activity in UMN (0.32) and MES + OME (0.35) were slightly higher than that in the superficial depots. Following the brief bout of exercise (group 3), LPL activity per g of tissue was unchanged in DWA and groin, dropped slightly in BA, HUMP and MES + OME and substantially in the 2 intermuscular depots and the superficial depot S, which together comprise only 14% of the adipose tissue, with the greatest changes (40% lower than the controls) being in the intermuscular depots. These effects made the LPL activity in all depots studied more nearly equal than under resting condition, but the capacity for lipid uptake in the intermuscular depots was still more than twice that of DWA. Following exercise, all site-specific differences in LPL activity per adipocyte became insignificant except for those of POP and the cardiac depot. In fasting, sedentary animals, the activities in the MES + OME depot did not correlate with the values measured from any other depot of the same specimens, values for groin and DWA correlated significantly (P < 0.01) only with each other, the 2 intermuscular depots also correlated only with
Table 2. Means and standard errors of the activity of lipoprotein lipase in isolated adipocytes (A) and stromal-vascular tissue of 9 adioose deoots of exercised Dunkin Hartley guinea pigs Females (n = 5)
Males (n = 5) Adipocytes Mean + SE
Stromal-vascular Mean k SE
Ratio A/SV Mean
Superficial s
0.95 + 0.07
0.58 f 0.07
BA HUMP Groin
0.79 ; 0.08 0.80 * 0.05 0.65 + 0.06
0.64 IO.07 0.63 f 0.06 0.57 f 0.04
1.68 1.37 1.31 I.15
0.98 0.64 0.86 0.55
Adipocytes Mean f SE
0.87 k 0.09 0.59 f 0.08
0.74 f 0.05 0.56 f 0.05
1.18 1.05
0.98 + 0.04 0.99 + 0.04
0.56 k 0.06 0.51 f 0.06
1.83 2.08
f + f f
0.09 0.03 0.10 0.03
Stromal-vascular Mean + SE 0.67 + 0.68 * 0.74 f 0.63 f
Ratio A/SV Mean
0.03 0.03 0.07 0.01
0.95 1.16 0.88
0.66 + 0.04 0.57 * 0.03
0.64 * 0.02 0.62 f 0.02
1.04 0.92
0.84 + 0.03 0.91 f 0.05
0.61 + 0.03 0.62 f 0.03
1.37 1.48
Intra-abdominal
MES + OME DWA Intermuscular
POP UMN
All units are nmol fatty acid/min/mg protein.
(SV)
CAROLINE M. POND et al.
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Table 3. Sex differences in the activity of lipoprotein lipase (nmol fatty acid/min/mg dry weight of tissue in 9 adipose depots of sedentary, fasting guinea pigs and following 0.5 hr of exercise
I (n = 5) Sedentary
Superficial IFS BA HUMP Groin Irma-abdominal MES + OME DWA Inrermuxular POP UMN
males
Group 3 (n = 5) Exercised males
number 1 (n = 5) Sedentary females
3 (n = 5) Exercised females
Mean
SD
Mean
SD
Mean
SD
Mean
SD
0.48 0.40 0.48 0.25
0.09 0.06 0.10 0.04
0.30** 0.26** 0.32* 0.22
0.05 0.05 0.07 0.05
0.48 0.36 0.54 0.26
0.07 0.05 0.12 0.03
0.39’ 0.36 0.45 0.25
0.04 0.03 0.12 0.05
0.37 0.20
0.14 0.04
0.27 0.19
0.06 0.06
0.45 0.19
0.10 0.05
0.33 0.21
0.07 0.09
0.66 0.56
0.06 0.10
0.41” 0.371’
0.09 0.07
0.81 0.66
0.26 0.16
0.48* 0.45
0.05 0.04
lSignificantly different from the homologous **Significantly different from the homologous
depot in the sedentary specimens of the same sex at P < 0.05. depot in the sedentary specimens of the same sex at P < 0.01.
each other and UMN with HUMP. Following exercise, values from more depots correlated significantly: HUMP, S, BA and the 2 intermuscular depots correlated with 4 or 5 other depots, and values from the MES +OME with 3 others. the DWA and groin depots remained isolated, correlating only with each other and, weakly, with MES + OME. Table 2 shows the quantities of LPL activity in, or firmly bound to, the adipocytes and that in the stromal-vascular tissue. In both sexes, a greater proportion of the LPL was found in the extra-adipocyte fraction of the depots that responded most to exercise (Table 1). In other depots, there were about equal amounts of LPL in each fraction. There were significant sex differences in the effects of exercise on LPL activity in whole tissue (Table 3): except in the case of the BA depot, the pattern of changes in the females was similar to but less pronounced than that of the males. Similar sex differences were detectable in the data from the Dunkin Hartleys (group 4) but these data are not included in Table 3 because of the confounding effects of breed differences. The quantity of LPL found in the stromalvascular fraction (Table 2) was about the same in all depots, particularly in the females, but there were site-specific differences in the amount remaining in or on the adipocytes. There was also a consistent trend towards a higher ratio of LPL activity in the adipocytes to that in the stromalvascular fraction in the males than in the females, and these data faithfully reflect the pattern of sex differences in the response to exercise shown on Table 2. DISCUSSION
Methods
The handling protocols were designed to emphasise the contrasts between the 3 states. In experiment 2, the animals were allowed to feed spontaneously, not force fed, as has been done previously (Enerblick et al., 1988). Although such procedures produce
greater variation in the quantity of food consumed, they also eliminate the stress generated by handling which may mimic the effects of exercise and confound the distinction between the 2 states. Semb and Olivecrona (1986) preferred an alternative LPL assay procedure for use on guinea pig tissues. The method we used requires only small quantities of tissue and is easier to manage when assaying numerous samples (16 for experiment 4) simultaneously. We found the method to be satisfactory for guinea pigs, possibly because our specimens were older than those used previously (Semb and Olivecrona, 1986); adipose tissue of mature adult rats aged 15 months produces 2-3 times more LPL than that of 3 month old specimens (Ursini et al., 1991). This method is therefore more appropriate to this project, which is aimed at comparing the relative rates of LPL activity in different depots under different conditions rather than quantifying maximum enzyme activity. Unfortunately, LPL activity in whole tissue was consistently lower in the Dunkin Hartley specimens (group 4) than in similarly treated Bolivians (group 3). Previous investigators (Semb and Olivecrona, 1986) have also reported finding large, unexplained differences between different batches of guinea pigs. Site-specific differences in adipocyte volume are remarkably consistent in guinea pigs and do not change significantly with age or strain (Pond et al., 1986). Site-specific responses to exercise and feeding
LPL activity per g of tissue is far from equal, with depots consisting of small adipocytes, such as UMN, S and HUMP (Pond et al., 1986), having greater capacity for lipid uptake per g of tissue than largeadipocyte depots such as groin. Site-specific differences in adipocyte volume account for many of these differences between depots, but LPL activity per adipocyte is not constant in all depots, with the intermuscular depot POP again standing out as having atypical properties (Cryer and Jones, 1979; Pond et al., 1986, Mattacks and Pond 1988; Pond and Mattacks, 1991).
Site-specific differences in LPL activity
The data on Table 1 are also consistent with previous observations on the effects of feeding and fasting on LPL activity (LaDu et al., 1991b; Tan et al., 1977), but by examining more than twice as many depots as these studies, we demonstrate a much wider range of site-specific differences which show that it is not accurate to estimate the total adipose tissue LPL production from the average activity measured in a few large depots (Deshaies er al., 1990). LPL activity increases significantly following spontaneous feeding only in the 2 depots, groin and DWA, that are homologous to those usually selected for study in rodents (Semb and Olivecrona, 1986; Deshaies et al., 1990; Savard et al., 1986; PradinesFigueres et al., 1990) and following exercise, it decreases most in the 2 intermuscular depots and small anterior superficial depots that have rarely been studied. Groin and DWA together account for about 40% of the total adipose mass, and an increase in LPL activity in them would raise substantially the rate at which lipids could be removed from the circulation. The increase in LPL activity in the cardiac depot following feeding is consistent with the hypothesis that this specialized mass of adipose tissue contributes to the supply and regulation of fuel for the myocardium (Marchington and Pond, 1990). Because its mass is so small, the increased LPL activity in the cardiac depot would make very little difference to the overall rate of removal of circulating lipid. There are trends towards increased LPL activity after feeding in all the other superficial and intraabdominal depots, but not the intermuscular depots, suggesting that their capacity for lipid uptake may also rise significantly following ingestion of an unnaturally large meal, such as might happen with forcible feeding (Enerback et al., 1988). Apparently, the response of the intermuscular depots to feeding is minimal. Most rodents, including guinea pigs, are nibblers, taking numerous small meals. Because the animals were allowed to control their own food intake, we believe that our data describe the natural, physiologically regulated situation more accurately. In normal meals, LPL activity increases in a few depots in which it was low during fasting, thereby making the capacity for lipid uptake per g of tissue more nearly equal in all the depots. Very large or very lipid-rich meals may stimulate greater LPL production in more of the depots, thereby increasing the differences between the sites. The effects of exercise (Table 1) are consistent with the conclusions from studies on rats (Deshaies et al., 1990; Savard et al., 1986), but they demonstrate that in the intermuscular and some minor superficial depots, much larger changes in LPL activity can occur after much briefer periods of exercise than previously reported. Interestingly, no trend towards lower LPL in groin and DWA was observed in the group 3 (exercised) specimens corresponding to that observed in some superficial and intra-abdominal
1829
depots in fed specimens, suggesting that the activity of the enzyme is never modulated by exercise in these two large depots. There is close correspondence between site-specific differences in the effect of noradrenaline on the rate of glycerol release and LPL activity, both processes being smallest in adipocytes isolated from groin and DWA, and greatest in adipocytes from the 2 intermuscular depots (POP and UMN) and in the superficial depot S, particularly in tissue taken from animals that have been exercised under a similar regime to that used here (Pond and Mattacks, 1991). These experiments point to the conclusion that, as least in these nibbling rodents, site-specific differences in LPL activity are maximized by fasting and inactivity. If such properties lead to differential expansion of depots, and if similar mechanisms operate in humans, they could help to explain why frequent exercise and regular, moderate-sized meals maintain the normal body shape while prolonged lack of exercise and irregular meals often lead to selective enlargement of some depots (Borkan and Norris, 1977). There was very little LPL activity in the stromalvascular fractions prepared by Rodbell (1964) but subsequent studies have led to the conclusion that the enzyme is secreted by adipocytes and attaches to endothelial tissue (Ohvecrona and BengtssonOlivecrona, 1990). The data in Tables 1 and 2 indicate that site-specific differences in LPL activity in whole tissue arise mainly from differences in the quantity remaining in or on the adipocytes, rather than in the stromal-vascular tissue, although these measurements do not exclude the possibility of large differences in the rate of turnover of LPL within the stromal-vascular fraction. There is a strong association between the decrease in total tissue LPL following exercise and the increase in the proportion of the enzyme that is found in the stromal-vascular fraction. These observations are consistent with the conclusion that exercise raises both the rate of secretion of enzyme into the microvasculature, and the rate at which it was washed out of the tissue of origin, probably by the faster flowing blood. The ultimate fate of bloodborne LPL is breakdown in the liver (Olivecrona and Bengtsson-Olivecrona, 1990; Wallinder et al., 1984) but it may spread to other tissues and modulate their physiological functions during its sojorn in the bloodstream (Olivecrona and Bengtsson-Olivecrona, 1990; Camps et al., 1990). There is increasing circumstantial evidence for interchange of metabolites between intermuscular adipose tissue and skeletal muscle (Mattacks et al., 1987; Mattacks and Pond, 1988, 1991) and in resting hamsters, blood flow through POP and UMN is significantly higher than that in perirenal, epididymal or any superficial depot (Mattacks and Pond, 1991). LPL originating in intermuscular depots may migrate into adjacent muscles where it could facilitate the uptake of circulating lipids into exercising muscles, thereby promoting fatty acid utilization and maxi-
1830
CAROLINEM. POND et al.
mizing glycogen sparing (Newsholme, 1977; Rett et al., 1986). The fall in total tissue LPL in these depots may reduce the capacity of the adipose tissue to compete with the muscles for substrates. LaDu et al. (199 1b) also proposed migration of LPL from adipose tissue to muscle to explain their observations on changes in the activity of the enzyme in certain limb muscles and the epididymal depot of exercised rats. The fact that total LPL production is highest in the intermuscular depots, and in other depots in which the response to noradrenaline is subst~tiaIiy modified by exercise (Pond and Mattacks, 1991), is also consistent with this concept. The involvement of intermuscular depots in exercise, and their apparent lack of response to feeding, may help to explain why, in humans, changes in size of the portion of the POP depot over the gastrocnemius muscle correlate very poorly with those in the other superficial depots (Borkan and Norris, 1977). Modulation of lymphocyte activities by exogenous LPL in the circulation may also contribute to the syndrome of disturbances caused by prolonged, strenuous physical exercise (Keast et al., 1988). In contrast to the conclusions reached from comparing adipose tissue from rats of different ages (Ursini et al., 1991; Fried and DiGirolamo, 1986), when numerous adipose depots of mature adult guinea pigs are compared, site-specific adipocyte volume per se proves to be irrelevant to the site-specific rate of LPL production and secretion, or its response to feeding and exercise, as it has for several other biochemical properties (Mattacks and Pond, 1988; Pond and Mattacks, 1991). LPL activity also does not correlate closely with adipocyte volume in massively obese men and women (Fried and Kral, 1987), or in cattle (Chilliard and Robelin, 1985). A consequence of the site-specific differences in LPL production is that, in spite of their small size, the 2 small intermuscular depots produce 14% of the total adipose tissue LPL in resting, fasting guinea pigs, 24% comes from MES + OME, but the 2 most fr~uently studied depots, groin and DWA (which includes epididymal) contribute only 11 and 13% respectively. Sex d@erences The sex differences (Tables 2 and 3), which were not anticipated in the original design of the experiments, indicate that the response of adipose tissue to exercise is stronger in males than in females. The females ran just as well as the males in the apparatus used, and, with the exception of BA, the procedure modified LPL activity in the same depots, but the effects were smaller in females. A greater proportion of the enzyme was secreted into the stromal-vascular fraction in femaIes than in males, suggesting that the rate of turnover of LPL is higher in this sex, or that adipose tissue, particularly in intermuscular depots, contributes more LPL to other tissues in females.
Sex differences in the rate of fatty acid/ triacylglycerol cycling in homologous adipose depots have been reported in dwarf hamsters (Mattacks and Pond, 1988): pa~icularly in the superficial depots, FA/TAG cycling was slower in males than in females at rest, but increased more in males following an hour of moderate exercise. The biggest differences between the sexes were measured from BA and groin depots, and, as in the data presented here, involvement of the intra-a~ominal depots in the response to exercise was mi~mal. It is difficult to assess the relevance of these data to the human condition: LPL activity has been measured in only a few depots in humans (Pouliot et al., 1991; Fried and Kral, 1987), which are usually chosen for their accessibility, rather than because they are known to have physiologically significant properties. One of the most widely studied biopsy sites, the superficial abdominal “paunch”, has no exact homologue in rodents (Pond, 1991). The sex differences are consistent with mechanisms that might, over a long period, lead to many of the observed differences in dist~bution of adipose tissue in normal human adults but no such differences in site-specific adipocyte volume or in the relative mass of the depots can be demonstrated in guinea pigs (Pond et al., 1986) or in most other mammals (Keast et al., 1988). We suggest that there are additional factors that redress the effects of those demonstrated here, but which are reduced or suppressed in humans. For example, LPL activity measured from human biopsy samples does not correlate with the tissues’ capacity to take up labelled fatty acids (M&in et al., 1990). CONCLUSIONS
In sedentary, fasting adults of both sexes, there are large site-specific differences in LPL activity per unit mass of adipose tissue, being highest in 2 small intermuscular depots and lowest in the large superficial depot, groin, and in DWA (perirenal and epididymal). LPL increases after a spontaneously ingested meal only in these large, frequently studied depots, and fails to decrease after 30 min of exercise in most adipose depots except groin or DWA. The effects of exercise are greatest in intermuscular depots. Total tissue LPL changes more in males than in homologous depots of females, but in males, a greater porportion of the total tissue enzyme activity is associated with the adipocytes than with the stromalvascular fraction. The data demonstrate the physiological inhomogeneity of adipose tissue, and are consistent with the hypothesis that LPL, particularly that originating from the intermuscular and small superficial depots, may enter the circulation and modulate the metabolism of other tissues. These sex differences in physiology could, in the long-term, generate some of the sex differences in the distribution of adipose tissue observed in humans, but no such sex differences in gross anatomy can be detected in guinea pigs.
Site-specific differences in LPL activity Acknowledgement-We
thank Dr Rhys Evans (Metabolic Research Unit, Oxford) for technical advice. REFERENCES
Bensadoun A. (1991) Lipoprotein lipase. A. Rev. Nutr. 11, 217-237. Borkan G. A. and Norris A. H. (1977) Fat redistribution and the changing body of the adult male. Hum. ISof. 49, 495-514.
Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.
Camps L., Reina M., Llobera M., Vilaro S. and Olivecrona T. (1990) Lipoprotein lipase: cellular origin and functional distribution. Am. J. Physiol. 258, C673C681. Chilliard, Y. and Robelin J. (1985) Activid lipoprodinelipasique de differents depots adipeux et ses relations avec la taille des adipocytes chez la vache tarie en tours d’engraissement ou en debut de lactation. Reprod. Nutr. Dev. 25, 287-293.
Cryer A. and Jones H. M. (1979) The early development of white adpose tissue. Biochem. J. 178, 711-724. Deshaies Y., Lortie G. and Richard D. (1990) Lipoprotein lipase activity in white adipose tissue of rats subject to exercise-rest cycles. Can. J. Physiol. Pharmac. 68, 157-163.
Doolittle M. H., Ben-Zeev O., Elovson J., Martin D. and Kirchgessner T. G. (1990) The response of lipoprotein lipase to feeding and fasting. Evidence for post-translational regulation. J. biol. Chem. 265, 45704577. Enerback S., Semb H., Tavemier J., Bjursell G. and Olivecrona T. (1988) Tissue-specific regulation of guinea pig lipoprotein lipase: effect of nutritional state and tumour necrosis factor on mRNA levels in adipose tissue, heart and liver. Gene 64, 97-106. Fried S. K. and DiGirolamo M. (1986) Lipoprotein lipase from isolated rat fat cells of different sizes. Life Sci. 39, 2111-2119. Fried S. K. and Kral J. G. (1987) Sex differences in regional distribution of fat cell size and lipoprotein lipase activity in morbidly obese patients. Int. J. Obesity 11, 129-140. Keast D., Cameron K. and Morton A. R. (1988) Exercise and the immune response. Sports Med. 5, 248-267. LaDu M. J., Kapsas H. and Palmer W. K. (199la) Regulation of lipoprotein lipase in muscles and adipose tissue during exercise. J. appl. Physiol. 71, 404409. LaDu M. J., Schultz C. J., Essig D. A. and Palmer W. K. (1991b) Regulation of lipoprotein lipase in adipose and muscle tissues during fasting. Am. J. Physiol. 260, R953-R959. Marchington J. M. and Pond C. M. (1990) Site-specific properties of pericardial and epicardial adipose tissue: the effects of insulin and high-fat feeding on lipogenesis and the incorporation of fatty acids in vitro. Inr. J. Obesity 14, 1013-1022. MBrin P., Rebuff&Strive M. and Bjiimtorp P. (1990) Uptake of triglyceride fatty acids in adipose tissue in uivo in man. Eur. J. clin. Invest. 20, 158-165. Mattacks C. A. and Pond C. M. (1988) Site-specific and sex differences in the rates of fatty acid/triacylglycerol substrate cycling in adipose tissue and muscle of sedentary and exercised dwarf hamsters (Phodopus sungorus). Inf. J. Obesity 12, 585-597.
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Mattacks C. A. and Pond C M. (1991) Site-specific differences in the uptake in vivo of 2-deoxyglucose by adipose tissue in the Djungarian hamster, PhodopuF sungorus. Nufr. Res. 11, 1083-1086. Mattacks C. A., Sadler D. and Pond C. M. (1987) The effects of exercise on the activities of hexokinase and phosphofructokinase in superficial, intra-abdominal and intermuscular adipose tissue of guinea pigs. Comp. Biothem. Physiol. 87B, 533-542.
Newsholme E. A. (1977) The regulation of intracellular and extracellular fuel supply during sustained exercise. Ann. N. Y. Acud. Sci. 301, 81-91. Nilsson-Ehle P., Carlstrom S. and Belfrage P. (1975) Rapid effect on lipoprotein lipase activity in adipose tissue of humans after carbohydrate and lipid intake. Scund. J. clin. Lab. Invest. 35, 373-378. Nilsson-Ehle P., Tornqvist H. and Belfrage P. (1972) Rapid determination of lipoprotein lipase in human adipose tissue. Clin. chim. Acta 42, 383-390. Olivecrona T. and Bengtsson-Olivecrona G. (1990) Lipoprotein lipase and hepatic lipase. Curr. Opinion Lipidol. 1, 222-230.
Pond C. M. (1991) Adipose tissue in human evolution. In The Aquatic Ape: Fact or Fiction? (Edited by Roede M., Wind J., Patrick J. M. and Reynolds V.), pp. 193-220. Souvenir Press, London. Pond C. M. and Mattacks C. A. (1991) The effects of noradrenaline and insulin on lipolysis in adipocytes isolated from nine different adipose depots of guinea pigs. Int. J. Obesity 15, 609618. Pond C. M., Mattacks C. A., Thompson M. C. and Sadler D. (1986) The effects of age, dietary restriction, exercise and maternity on the abundance and volume of adipocytes in twelve adipose depots of adult guinea pigs. Brit. J. Nutr. 56, 2948.
Pouliot M. C., Desprts J.-P., Moorjani S., Lupien P. J., Tremblay A., Nadeau A. and Bouchard C. (1991) Regional variation in adipose tissue lipoprotein lipase activity-association with plasma high density lipoprotein levels. Eur. J. c/in. Invest. 21, 398405. Pradines-Figueres A., Vannier C. and Ailhaud G. (1990) Lipoprotein lipase stored in adipocytes and muscle cells is a cryptic enzyme. J. Lipid Res. 31, 1467-1476. Rett K., Wicklmayr M., Dietze G., Mehnert H., Wolfram G. and Hailer S. (1986) Inhibition of muscular glucose uptake by lipid infusion in man. C/in. Nutr. 5, 187-192. Rodbell M. (1964) Localization of lipoprotein lipase in fat cells of rat adipose tissue. J. biol. Chem. 239, 753-755. Savard R., Palmer J. E. and Greenwood M. R. C. (1986) Effects of exercise training on regional adipose tissue metabolism in pregnant rats. Am. J. Physiol. 250, R837-R844. Semb H. and Olivecrona T. (1986) Nutritional regulation of lipoprotein lipase in guinea pig tissues. Biochem. biophys. Acra 876, 249-255.
Tan M. H., Tsunako S. and Have1 R. J. (1977) The significance of lipoprotein lipase in rat skeletal muscles. J. Lipid Res. 18, 363-370. Ursini F., Vugman M., Femandes L. C., Curi C. M. 0. N. and Curi R. (1991) Metabolic changes of several adipose depots as caused by aging. Physiol. Behav. SO, 317-321. Wallinder L., Peterson J., Olivecrona T. and BengtssonOlivecrona G. (1984) Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase. Biochem. biophys. Acta 795, 513-524.
THE EFFECTS ACTIVITY DIFFERENT
0020-711X/92$5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd
OF EXERCISE AND FEEDING ON THE OF LIPOPROTEIN LIPASE IN NINE ADIPOSE DEPOTS OF GUINEA PIGS
CAROLINEM. POND, CHRISTINEA. MATTACKSand DAWN SADLER Department of Biology, The Open University, Milton Keynes MK7 6AA, U.K. [Tel. (0908) 655077; Fax (0908) 6537441 (Received 9 March 1992) Abstract-l.
The activity of lipoprotein lipase (LPL) was measured in whole adipose tissue from 9 identified adipose depots of sedentary, fasting adult guinea pigs and following 30min of exercise or voluntary ingestion of chow, and in adipocyte and stromal-vascular fractions from exercised specimens. 2 In sedentary, fasting specimens, LPL activity was up to 4 times higher in the small intermuscular depots than in the p&renal and epididymal depot (Table 1). 3. LPL activity increased significantly after feeding only in the large superficial depot, groin, and in the perirenal depot. LPL activity decreased after exercise only in the 2 intermuscular depots and in small anterior superficial depots. These effects of exercise were consistently greater in males than in females (Table 3). 4. Followng exercise, there was up to twice as much LPL in the adipocytes as in the stromal-vascular fraction of the intermuscular depots, about 50% more in adipocytes from the minor superficial depots and about equal quantities in the 2 fractions of the intra-abdominal and groin depots (Table 2). 5. The data demonstrate the physiological inhomogeneity of both superficial and internal adipose depots, and are consistent with the hypothesis that LPL originating from adipose tissue may enter the circulation.
MATERIALS AND
INTRODUClTON Lipoprotein lipase (LPL) is the principal enzyme mediating the uptake of lipids from circulating lipoprotein, and is synthesized by many extra-hepatic tissues in mammals (Bensadoun, 1991). Most of the extensive information (Olivecrona and BengtssonOlivecrona, 1990) about its molecular structure and genetic basis come from the epididymal or parametrial depots of the rat and a few superficial depots in humans (Pouliot et al., 1991). In these adipose depots, LPL activity is modulated by feeding (Bensadoun, 1991; Nilsson-Ehle et al., 1975; Doolittle et al., 1990), but the effects of exercise are minimal (LaDu et al., 199la), which is surprising in view of the importance of this enzyme in regulating the concentration of circulating triacylglycerol. Site-specific differences in adipocyte volume (Pond et al., 1986), maximum glycolytic capacity (Mattacks et al., 1987), fatty acid-triacylglycerol cycling (Mattacks and Pond, 1988) and response to insulin and noradrenaline (Pond and Mattacks, 1991), have been described with many of the most atypical properties occurring
in small, rarely studied depots (Marchington and Pond, 1990). Guinea pigs are the smallest convenient laboratory animal in which there is sufficient tissue in these minor depots for accurate assay of LPL. In this paper, we report on the effects of feeding and a brief period of exercise on LPL activity in 9 depots that together comprise almost all the adipose mass.
METHODS
Animals and dissection procedure The guinea pigs used for assays of LPL in whole tissue (groups l-3) were Bolivians that were born and raised at the Open University in standard cages (area 0.45 m*) in which breeding-grade guinea pig chow and water with added vitamin C (0.1 mg ml-‘) were available ad Zibitum. They were also given cabbage, carrot and apple on 5 days per week and hay every day. They were transferred to permanent, single-sex groups at weaning and used as mature adults, aged 615 months (mean 11.1 f 0.54), body mass 7OCN175 g (mean 965 f 18.5). Those used for assays of fractionated adipose tissue (group 4) were commercially obtained Dunkin Hartleys. They were rested and fed the same diet as the Bolivians for at least a month before being used, at the age of about 11 months, mean body mass 1152 f 14.4 g. Each experimental group consisted of 5 males and 5 females. All specimens were fasted for 18 hr before being killed, with an IP injection of pentabarbitone dissolved in phosphate buffered saline. The sedentary specimens (group 1) were isolated in small (area 0.19 m2) cages for 18 hr before the experiment and handled very gently before being killed. In nearly all cases, this procedure was completed without any squeaking or struggling. Pilot experiments showed that previously ad libitm-fed guinea pigs would not reliably eat significant quantites of chow after a single 18 hr overnight fast. Therefore, group 2 specimens were placed in separate cages and given food for only 5 hr per day for the 2 days before the experiment. A known mass of chow was placed in the cage at 0800 and the animals were killed at 0930,
1825
1826
CAROLINE M. POND et al.
provided they had eaten at least 3 g. The exercised specimens (groups 3 and 4) remained in the larger cages (area 0.45 m2) in single sex groups of 26 animals. Guinea pigs do not exercise spontaneously in running wheels as murid rodents do, so an exercise pen, consisting of two concentric squares of “walls” 30 cm high placed on a rough mat that formed an enclosed path 30 cm wide and approx 7 m long was used. They were chased around this apparatus for 30 mm immediately before being killed by a similar procedure. In a few specimens, running was accompanied by squeaking and other signs of general excitement, and most were close to exhaustion by the end of the exercise period. Immediately after death, l-2 samples of adipose tissue were taken from the following sites: mesenteric and omental (MES + OME), around kidney and dorsal wall of abdomen plus epididymal (DWA), in front of forelimb and shoulder (S), behind forelimb (BA), side and ventral groin (= inguinal), interscapular (HUMP), medial to trapezius muscle of neck (UMN), plus the whole of the popliteal (POP) depot and all dissectible adipose tissue from around the heart (Cardiac). These depots, which, in guinea pigs, comprise almost all the dissectible adipose tissue, are exactly homologous with those studied previously (Pond et al., 1986; Mattacks ef al., 1987; Pond and Mattacks 1991). The rest of the adipose tissue in each depot was later dissected out and weighed. Total fatness is the sum of the masses of these adipose depots expressed as a percentage of the live body mass. In some previous studies (Pond et al., 1986; Mattacks ef al., 1987) in front of forelimb and in front of shoulder depots, and perirenal and epididymal have been studied separately, but since they have similar properties in guinea pigs, they were combined for this project. Because of the small quantities of cardiac adipose tissue, no attempt was made to separate this depot into adipocytes and extracellular fractions. Fractionation
and drying of tissue and LPL assay
LPL was assayed in acetone/ether dried samples of whole
or fractionated adipose tissue using PH]triolein as a substrate, following the method developed by Nilsson-Ehle er al. (1972). For experiments 1-3, preweighed samples of about 0.5 g (in the case of the cardiac depot, all available tissue) of whole tissue were homogenized in cold acetone on ice. For experiment 4, the tissue was fractionated into adipocytes and stromal-vascular material using established methods (Rodbell, 1964): about 1 g from each sample was placed in warm freshly gassed Krebs-Hensliet buffer and chopped into pieces of about 100 mg. After further washing, the tissues were incubated with 5 mg’ml-’ collagenase (Sigma type lA-C-9891) for 25min at 37°C in a shaking waterbath. The resulting suspension was filtered through fine gauze, and centrifuged at 1000 rpm for about 5 min and the isolated cells collected and placed in cold acetone. The extracellular material was spun down at 2OOOrpm for 10min and decanted into cold acetone. All samples were centrifuged at 1000 rpm for IO min, the acetone decanted off and centrifuged again with more cold acetone. The procedure was repeated with ether and any remaining ether was evaporated in an atmosphere of nitrogen. The dried tissue was weighed out as 10mg samples and stored frozen at -15°C for up to 1 week. To start the assay, 1 ml of 0.05 M Tri-HCl with 1 M ethylene glycol (pH 8.0) was added to each sample, the mixture was homogenized on ice and centrifuged at 15,000 rpm for 10 min and the supematant collected. The substrate was prepared from 1.5ml heptane, 5OpCi
(1.85 mBq) [‘Hltriolein and 21 mg unlabelled triolein and 1.2 mg lysophosphatidyl choline, mixed thoroughly. The heptane was evaporated in an atmosphere of nitrogen. 7.5 ml of 0.2 M Tri-HCl (PH 8.1) was added and the cooled preparation was sonicated for 8 bursts lasting 30 set each, and cooled on ice between bursts. 0.9ml of 4% BSA and 0.6ml of serum prepared from a fasted guinea pig were added and mixed well, and 0.1 ml of the supernatant was incubated with 0.1 ml of substrate in a shaking waterbath for 1 hr at 37°C. The reaction was stopped with 3.25 ml methanol:chloroform: heptane (2.3: 2.5: 0.8) and 1.05ml of 0.1 M potassium carbonate was added to each vial and the mixtures were vortexed for 15 set and centrifuged at 2000 rpm for 15 min. 0.25 ml samples of the upper phase were counted in a Beckman 1701 scintillation counter for 10min. Total protein was measured in the pellet residue from the sample preparation using the Bradford (1976) method. Student’s r-test was used to compare values from homologous depots from differently treated animals, and except where otherwise stated, values indicating P < 0.05 were taken as significant.
RESULTS
The Bolivians (mean fatness 6.0% f 0.11) were slightly but significantly (t = 2.904) fatter than the Dunkin Hartleys (mean fatness 5.5% f 0.13), although the latter were significantly larger than the Bolivians (t = 7.99). As expected, the females were significantly smaller (mean BM 961 &-27 g) but fatter (mean fatness 6.1% f 0.14) than the males (mean BM 1061 f 23 g; mean fatness 5.7% f 0.10). The measures of enzyme activity did not correlate significantly with age, body mass or fatness any more frequently than would be expected by chance. The relative masses of the depots were similar to measurements reported previously (Pond et al., 1986; Pond and Mattacks, 1991). The quantity of cardiac adipose tissue is always very variable (Marchington and Pond, 1990), and there was sufficient adipose tissue in this depot for analysis in only 10 males and 12 females; the mean quantity in the latter was 0.35 + 0.03 g, representing 0.62% of the total adipose tissue, compared to 0.31 + 0.03 g (0.56% of the total) in males. LPL activities in samples of whole adipose tissue from nine different depots in the 4 treatment groups are listed in Table 1. The average quantity of food eaten by the group 2 animals was 5.8 f 1.15 g, and the sexes did not differ in this respect. There were large site-specific differences in LPL activity at rest, with that in the intermuscular depots (POP and UMN) being up to 3.8 times higher than that of the much larger DWA depot. LPL activities in the 2 most commonly studied depots (DWA and Groin) were not significantly different from each other in resting, fasting animals. Following feeding (group 2), LPL activity increased in these 2 large depots, that together accounted for 39% of all the adipose tissue, and in the much smaller cardiac depot. In all the other depots, increases were barely significant or
Site-specificdifferences in LPL activity
1827
Table 1. Mean and standard deviation of the activity of lipoprotein lipase and approximate relative mass (mass of depot as % total dissectible adipose tissue) in 9 adipose depots of guinea pigs treated in 3 different ways (n = 10, 5 male, 5 female, except for cardiac depot, for which n = 8 for group 2 and n = 7 for groups 1 and 3) I Sedentary, fasting
Group number 2 3 Sedentary, fed Exercised, fasting
4 Exercised, fasting
Mean
SD
Mean
SD
Mean
SD
9 17 16
0.48 0.38 0.51 0.26
0.08 0.05 0.11 0.04
0.56 0.48. 0.60 0.3911
0.14 0.13 0.15 0.10
0.35** 0.31. 0.38* 0.25
0.06 0.07 0.11 0.05
0.29 0.23 0.28 0.20
0.06 0.06 0.06 0.04
21 23
0.41 0.19
0.12 0.04
0.53 0.33”
0.14 0.09
0.30. 0.20
0.07 0.02
0.27 0.19
0.06 0.04
3 5 0.6
0.74 0.63 0.24
0.21 0.13 0.09
0.71 0.67 0.42..
0.17 0.18 0.13
0.44.. 0.41.. 0.28
0.08 0.07 0.05
0.35 0.10 0.32 0.08 not measured
Relative mass % total AT Superficial S
BA HUMP Groin
6
SD
Mean
Intro-abdominal
MES + OME DWA Intermuscular
POP UMN Cardiac
Groups l-3 were Bolivian strain; Group 4 was Dunkin Hartley strain. All units are nmol fatty acid/min/mg dry weight of tissue. *Significantly different from homologous depot in group 1 (sedentary, fasting) at P < 0.05. **Significantly different from homologous depot in group 1 (sedentary, fasting) at P < 0.01.
insignificant and were vanishingly small in the intermuscular depots. The effect of these changes was to make the LPL activity per g of tissue more nearly equal in all the depots studied: the ratio of its activity in POP to that in DWA fell from 3.8 in sedentary, fasting specimens, to 2.1 in the fed group, and 2.2 and 1.9 in the exercised groups 3 and 4 respectively. Analysis of variance showed that there were no significant sex differences in the relative masses of the depots or in their site-specific LPL activity in either sedentary group. Some of the site-specific differences in LPL activity per of tissue can be eliminated by taking account of differences in adipocyte volume. Combining the data on Table 1 with the mean site-specific adipocyte volumes of similarly maintained guinea pigs of the same age and strain (Pond et al., 1986) shows that, in the sedentary fasting animals, the LPL activities per adipocyte were not significantly different in the 4 superficial depots (0.24-0.30 nmol fatty acid/min/106 adipocytes). However, LPL activity in POP adipocytes was substantially higher (0.75 mnol fatty acid/min/106 adipocytes) and that in the cardiac and DWA depots lower (0.11 and 0.17 nmol fatty
acid/min/106 adipocytes respectively) than in the other depots. LPL activity in UMN (0.32) and MES + OME (0.35) were slightly higher than that in the superficial depots. Following the brief bout of exercise (group 3), LPL activity per g of tissue was unchanged in DWA and groin, dropped slightly in BA, HUMP and MES + OME and substantially in the 2 intermuscular depots and the superficial depot S, which together comprise only 14% of the adipose tissue, with the greatest changes (40% lower than the controls) being in the intermuscular depots. These effects made the LPL activity in all depots studied more nearly equal than under resting condition, but the capacity for lipid uptake in the intermuscular depots was still more than twice that of DWA. Following exercise, all site-specific differences in LPL activity per adipocyte became insignificant except for those of POP and the cardiac depot. In fasting, sedentary animals, the activities in the MES + OME depot did not correlate with the values measured from any other depot of the same specimens, values for groin and DWA correlated significantly (P < 0.01) only with each other, the 2 intermuscular depots also correlated only with
Table 2. Means and standard errors of the activity of lipoprotein lipase in isolated adipocytes (A) and stromal-vascular tissue of 9 adioose deoots of exercised Dunkin Hartley guinea pigs Females (n = 5)
Males (n = 5) Adipocytes Mean + SE
Stromal-vascular Mean k SE
Ratio A/SV Mean
Superficial s
0.95 + 0.07
0.58 f 0.07
BA HUMP Groin
0.79 ; 0.08 0.80 * 0.05 0.65 + 0.06
0.64 IO.07 0.63 f 0.06 0.57 f 0.04
1.68 1.37 1.31 I.15
0.98 0.64 0.86 0.55
Adipocytes Mean f SE
0.87 k 0.09 0.59 f 0.08
0.74 f 0.05 0.56 f 0.05
1.18 1.05
0.98 + 0.04 0.99 + 0.04
0.56 k 0.06 0.51 f 0.06
1.83 2.08
f + f f
0.09 0.03 0.10 0.03
Stromal-vascular Mean + SE 0.67 + 0.68 * 0.74 f 0.63 f
Ratio A/SV Mean
0.03 0.03 0.07 0.01
0.95 1.16 0.88
0.66 + 0.04 0.57 * 0.03
0.64 * 0.02 0.62 f 0.02
1.04 0.92
0.84 + 0.03 0.91 f 0.05
0.61 + 0.03 0.62 f 0.03
1.37 1.48
Intra-abdominal
MES + OME DWA Intermuscular
POP UMN
All units are nmol fatty acid/min/mg protein.
(SV)
CAROLINE M. POND et al.
1828
Table 3. Sex differences in the activity of lipoprotein lipase (nmol fatty acid/min/mg dry weight of tissue in 9 adipose depots of sedentary, fasting guinea pigs and following 0.5 hr of exercise
I (n = 5) Sedentary
Superficial IFS BA HUMP Groin Irma-abdominal MES + OME DWA Inrermuxular POP UMN
males
Group 3 (n = 5) Exercised males
number 1 (n = 5) Sedentary females
3 (n = 5) Exercised females
Mean
SD
Mean
SD
Mean
SD
Mean
SD
0.48 0.40 0.48 0.25
0.09 0.06 0.10 0.04
0.30** 0.26** 0.32* 0.22
0.05 0.05 0.07 0.05
0.48 0.36 0.54 0.26
0.07 0.05 0.12 0.03
0.39’ 0.36 0.45 0.25
0.04 0.03 0.12 0.05
0.37 0.20
0.14 0.04
0.27 0.19
0.06 0.06
0.45 0.19
0.10 0.05
0.33 0.21
0.07 0.09
0.66 0.56
0.06 0.10
0.41” 0.371’
0.09 0.07
0.81 0.66
0.26 0.16
0.48* 0.45
0.05 0.04
lSignificantly different from the homologous **Significantly different from the homologous
depot in the sedentary specimens of the same sex at P < 0.05. depot in the sedentary specimens of the same sex at P < 0.01.
each other and UMN with HUMP. Following exercise, values from more depots correlated significantly: HUMP, S, BA and the 2 intermuscular depots correlated with 4 or 5 other depots, and values from the MES +OME with 3 others. the DWA and groin depots remained isolated, correlating only with each other and, weakly, with MES + OME. Table 2 shows the quantities of LPL activity in, or firmly bound to, the adipocytes and that in the stromal-vascular tissue. In both sexes, a greater proportion of the LPL was found in the extra-adipocyte fraction of the depots that responded most to exercise (Table 1). In other depots, there were about equal amounts of LPL in each fraction. There were significant sex differences in the effects of exercise on LPL activity in whole tissue (Table 3): except in the case of the BA depot, the pattern of changes in the females was similar to but less pronounced than that of the males. Similar sex differences were detectable in the data from the Dunkin Hartleys (group 4) but these data are not included in Table 3 because of the confounding effects of breed differences. The quantity of LPL found in the stromalvascular fraction (Table 2) was about the same in all depots, particularly in the females, but there were site-specific differences in the amount remaining in or on the adipocytes. There was also a consistent trend towards a higher ratio of LPL activity in the adipocytes to that in the stromalvascular fraction in the males than in the females, and these data faithfully reflect the pattern of sex differences in the response to exercise shown on Table 2. DISCUSSION
Methods
The handling protocols were designed to emphasise the contrasts between the 3 states. In experiment 2, the animals were allowed to feed spontaneously, not force fed, as has been done previously (Enerblick et al., 1988). Although such procedures produce
greater variation in the quantity of food consumed, they also eliminate the stress generated by handling which may mimic the effects of exercise and confound the distinction between the 2 states. Semb and Olivecrona (1986) preferred an alternative LPL assay procedure for use on guinea pig tissues. The method we used requires only small quantities of tissue and is easier to manage when assaying numerous samples (16 for experiment 4) simultaneously. We found the method to be satisfactory for guinea pigs, possibly because our specimens were older than those used previously (Semb and Olivecrona, 1986); adipose tissue of mature adult rats aged 15 months produces 2-3 times more LPL than that of 3 month old specimens (Ursini et al., 1991). This method is therefore more appropriate to this project, which is aimed at comparing the relative rates of LPL activity in different depots under different conditions rather than quantifying maximum enzyme activity. Unfortunately, LPL activity in whole tissue was consistently lower in the Dunkin Hartley specimens (group 4) than in similarly treated Bolivians (group 3). Previous investigators (Semb and Olivecrona, 1986) have also reported finding large, unexplained differences between different batches of guinea pigs. Site-specific differences in adipocyte volume are remarkably consistent in guinea pigs and do not change significantly with age or strain (Pond et al., 1986). Site-specific responses to exercise and feeding
LPL activity per g of tissue is far from equal, with depots consisting of small adipocytes, such as UMN, S and HUMP (Pond et al., 1986), having greater capacity for lipid uptake per g of tissue than largeadipocyte depots such as groin. Site-specific differences in adipocyte volume account for many of these differences between depots, but LPL activity per adipocyte is not constant in all depots, with the intermuscular depot POP again standing out as having atypical properties (Cryer and Jones, 1979; Pond et al., 1986, Mattacks and Pond 1988; Pond and Mattacks, 1991).
Site-specific differences in LPL activity
The data on Table 1 are also consistent with previous observations on the effects of feeding and fasting on LPL activity (LaDu et al., 1991b; Tan et al., 1977), but by examining more than twice as many depots as these studies, we demonstrate a much wider range of site-specific differences which show that it is not accurate to estimate the total adipose tissue LPL production from the average activity measured in a few large depots (Deshaies er al., 1990). LPL activity increases significantly following spontaneous feeding only in the 2 depots, groin and DWA, that are homologous to those usually selected for study in rodents (Semb and Olivecrona, 1986; Deshaies et al., 1990; Savard et al., 1986; PradinesFigueres et al., 1990) and following exercise, it decreases most in the 2 intermuscular depots and small anterior superficial depots that have rarely been studied. Groin and DWA together account for about 40% of the total adipose mass, and an increase in LPL activity in them would raise substantially the rate at which lipids could be removed from the circulation. The increase in LPL activity in the cardiac depot following feeding is consistent with the hypothesis that this specialized mass of adipose tissue contributes to the supply and regulation of fuel for the myocardium (Marchington and Pond, 1990). Because its mass is so small, the increased LPL activity in the cardiac depot would make very little difference to the overall rate of removal of circulating lipid. There are trends towards increased LPL activity after feeding in all the other superficial and intraabdominal depots, but not the intermuscular depots, suggesting that their capacity for lipid uptake may also rise significantly following ingestion of an unnaturally large meal, such as might happen with forcible feeding (Enerback et al., 1988). Apparently, the response of the intermuscular depots to feeding is minimal. Most rodents, including guinea pigs, are nibblers, taking numerous small meals. Because the animals were allowed to control their own food intake, we believe that our data describe the natural, physiologically regulated situation more accurately. In normal meals, LPL activity increases in a few depots in which it was low during fasting, thereby making the capacity for lipid uptake per g of tissue more nearly equal in all the depots. Very large or very lipid-rich meals may stimulate greater LPL production in more of the depots, thereby increasing the differences between the sites. The effects of exercise (Table 1) are consistent with the conclusions from studies on rats (Deshaies et al., 1990; Savard et al., 1986), but they demonstrate that in the intermuscular and some minor superficial depots, much larger changes in LPL activity can occur after much briefer periods of exercise than previously reported. Interestingly, no trend towards lower LPL in groin and DWA was observed in the group 3 (exercised) specimens corresponding to that observed in some superficial and intra-abdominal
1829
depots in fed specimens, suggesting that the activity of the enzyme is never modulated by exercise in these two large depots. There is close correspondence between site-specific differences in the effect of noradrenaline on the rate of glycerol release and LPL activity, both processes being smallest in adipocytes isolated from groin and DWA, and greatest in adipocytes from the 2 intermuscular depots (POP and UMN) and in the superficial depot S, particularly in tissue taken from animals that have been exercised under a similar regime to that used here (Pond and Mattacks, 1991). These experiments point to the conclusion that, as least in these nibbling rodents, site-specific differences in LPL activity are maximized by fasting and inactivity. If such properties lead to differential expansion of depots, and if similar mechanisms operate in humans, they could help to explain why frequent exercise and regular, moderate-sized meals maintain the normal body shape while prolonged lack of exercise and irregular meals often lead to selective enlargement of some depots (Borkan and Norris, 1977). There was very little LPL activity in the stromalvascular fractions prepared by Rodbell (1964) but subsequent studies have led to the conclusion that the enzyme is secreted by adipocytes and attaches to endothelial tissue (Ohvecrona and BengtssonOlivecrona, 1990). The data in Tables 1 and 2 indicate that site-specific differences in LPL activity in whole tissue arise mainly from differences in the quantity remaining in or on the adipocytes, rather than in the stromal-vascular tissue, although these measurements do not exclude the possibility of large differences in the rate of turnover of LPL within the stromal-vascular fraction. There is a strong association between the decrease in total tissue LPL following exercise and the increase in the proportion of the enzyme that is found in the stromal-vascular fraction. These observations are consistent with the conclusion that exercise raises both the rate of secretion of enzyme into the microvasculature, and the rate at which it was washed out of the tissue of origin, probably by the faster flowing blood. The ultimate fate of bloodborne LPL is breakdown in the liver (Olivecrona and Bengtsson-Olivecrona, 1990; Wallinder et al., 1984) but it may spread to other tissues and modulate their physiological functions during its sojorn in the bloodstream (Olivecrona and Bengtsson-Olivecrona, 1990; Camps et al., 1990). There is increasing circumstantial evidence for interchange of metabolites between intermuscular adipose tissue and skeletal muscle (Mattacks et al., 1987; Mattacks and Pond, 1988, 1991) and in resting hamsters, blood flow through POP and UMN is significantly higher than that in perirenal, epididymal or any superficial depot (Mattacks and Pond, 1991). LPL originating in intermuscular depots may migrate into adjacent muscles where it could facilitate the uptake of circulating lipids into exercising muscles, thereby promoting fatty acid utilization and maxi-
1830
CAROLINEM. POND et al.
mizing glycogen sparing (Newsholme, 1977; Rett et al., 1986). The fall in total tissue LPL in these depots may reduce the capacity of the adipose tissue to compete with the muscles for substrates. LaDu et al. (199 1b) also proposed migration of LPL from adipose tissue to muscle to explain their observations on changes in the activity of the enzyme in certain limb muscles and the epididymal depot of exercised rats. The fact that total LPL production is highest in the intermuscular depots, and in other depots in which the response to noradrenaline is subst~tiaIiy modified by exercise (Pond and Mattacks, 1991), is also consistent with this concept. The involvement of intermuscular depots in exercise, and their apparent lack of response to feeding, may help to explain why, in humans, changes in size of the portion of the POP depot over the gastrocnemius muscle correlate very poorly with those in the other superficial depots (Borkan and Norris, 1977). Modulation of lymphocyte activities by exogenous LPL in the circulation may also contribute to the syndrome of disturbances caused by prolonged, strenuous physical exercise (Keast et al., 1988). In contrast to the conclusions reached from comparing adipose tissue from rats of different ages (Ursini et al., 1991; Fried and DiGirolamo, 1986), when numerous adipose depots of mature adult guinea pigs are compared, site-specific adipocyte volume per se proves to be irrelevant to the site-specific rate of LPL production and secretion, or its response to feeding and exercise, as it has for several other biochemical properties (Mattacks and Pond, 1988; Pond and Mattacks, 1991). LPL activity also does not correlate closely with adipocyte volume in massively obese men and women (Fried and Kral, 1987), or in cattle (Chilliard and Robelin, 1985). A consequence of the site-specific differences in LPL production is that, in spite of their small size, the 2 small intermuscular depots produce 14% of the total adipose tissue LPL in resting, fasting guinea pigs, 24% comes from MES + OME, but the 2 most fr~uently studied depots, groin and DWA (which includes epididymal) contribute only 11 and 13% respectively. Sex d@erences The sex differences (Tables 2 and 3), which were not anticipated in the original design of the experiments, indicate that the response of adipose tissue to exercise is stronger in males than in females. The females ran just as well as the males in the apparatus used, and, with the exception of BA, the procedure modified LPL activity in the same depots, but the effects were smaller in females. A greater proportion of the enzyme was secreted into the stromal-vascular fraction in femaIes than in males, suggesting that the rate of turnover of LPL is higher in this sex, or that adipose tissue, particularly in intermuscular depots, contributes more LPL to other tissues in females.
Sex differences in the rate of fatty acid/ triacylglycerol cycling in homologous adipose depots have been reported in dwarf hamsters (Mattacks and Pond, 1988): pa~icularly in the superficial depots, FA/TAG cycling was slower in males than in females at rest, but increased more in males following an hour of moderate exercise. The biggest differences between the sexes were measured from BA and groin depots, and, as in the data presented here, involvement of the intra-a~ominal depots in the response to exercise was mi~mal. It is difficult to assess the relevance of these data to the human condition: LPL activity has been measured in only a few depots in humans (Pouliot et al., 1991; Fried and Kral, 1987), which are usually chosen for their accessibility, rather than because they are known to have physiologically significant properties. One of the most widely studied biopsy sites, the superficial abdominal “paunch”, has no exact homologue in rodents (Pond, 1991). The sex differences are consistent with mechanisms that might, over a long period, lead to many of the observed differences in dist~bution of adipose tissue in normal human adults but no such differences in site-specific adipocyte volume or in the relative mass of the depots can be demonstrated in guinea pigs (Pond et al., 1986) or in most other mammals (Keast et al., 1988). We suggest that there are additional factors that redress the effects of those demonstrated here, but which are reduced or suppressed in humans. For example, LPL activity measured from human biopsy samples does not correlate with the tissues’ capacity to take up labelled fatty acids (M&in et al., 1990). CONCLUSIONS
In sedentary, fasting adults of both sexes, there are large site-specific differences in LPL activity per unit mass of adipose tissue, being highest in 2 small intermuscular depots and lowest in the large superficial depot, groin, and in DWA (perirenal and epididymal). LPL increases after a spontaneously ingested meal only in these large, frequently studied depots, and fails to decrease after 30 min of exercise in most adipose depots except groin or DWA. The effects of exercise are greatest in intermuscular depots. Total tissue LPL changes more in males than in homologous depots of females, but in males, a greater porportion of the total tissue enzyme activity is associated with the adipocytes than with the stromalvascular fraction. The data demonstrate the physiological inhomogeneity of adipose tissue, and are consistent with the hypothesis that LPL, particularly that originating from the intermuscular and small superficial depots, may enter the circulation and modulate the metabolism of other tissues. These sex differences in physiology could, in the long-term, generate some of the sex differences in the distribution of adipose tissue observed in humans, but no such sex differences in gross anatomy can be detected in guinea pigs.
Site-specific differences in LPL activity Acknowledgement-We
thank Dr Rhys Evans (Metabolic Research Unit, Oxford) for technical advice. REFERENCES
Bensadoun A. (1991) Lipoprotein lipase. A. Rev. Nutr. 11, 217-237. Borkan G. A. and Norris A. H. (1977) Fat redistribution and the changing body of the adult male. Hum. ISof. 49, 495-514.
Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.
Camps L., Reina M., Llobera M., Vilaro S. and Olivecrona T. (1990) Lipoprotein lipase: cellular origin and functional distribution. Am. J. Physiol. 258, C673C681. Chilliard, Y. and Robelin J. (1985) Activid lipoprodinelipasique de differents depots adipeux et ses relations avec la taille des adipocytes chez la vache tarie en tours d’engraissement ou en debut de lactation. Reprod. Nutr. Dev. 25, 287-293.
Cryer A. and Jones H. M. (1979) The early development of white adpose tissue. Biochem. J. 178, 711-724. Deshaies Y., Lortie G. and Richard D. (1990) Lipoprotein lipase activity in white adipose tissue of rats subject to exercise-rest cycles. Can. J. Physiol. Pharmac. 68, 157-163.
Doolittle M. H., Ben-Zeev O., Elovson J., Martin D. and Kirchgessner T. G. (1990) The response of lipoprotein lipase to feeding and fasting. Evidence for post-translational regulation. J. biol. Chem. 265, 45704577. Enerback S., Semb H., Tavemier J., Bjursell G. and Olivecrona T. (1988) Tissue-specific regulation of guinea pig lipoprotein lipase: effect of nutritional state and tumour necrosis factor on mRNA levels in adipose tissue, heart and liver. Gene 64, 97-106. Fried S. K. and DiGirolamo M. (1986) Lipoprotein lipase from isolated rat fat cells of different sizes. Life Sci. 39, 2111-2119. Fried S. K. and Kral J. G. (1987) Sex differences in regional distribution of fat cell size and lipoprotein lipase activity in morbidly obese patients. Int. J. Obesity 11, 129-140. Keast D., Cameron K. and Morton A. R. (1988) Exercise and the immune response. Sports Med. 5, 248-267. LaDu M. J., Kapsas H. and Palmer W. K. (199la) Regulation of lipoprotein lipase in muscles and adipose tissue during exercise. J. appl. Physiol. 71, 404409. LaDu M. J., Schultz C. J., Essig D. A. and Palmer W. K. (1991b) Regulation of lipoprotein lipase in adipose and muscle tissues during fasting. Am. J. Physiol. 260, R953-R959. Marchington J. M. and Pond C. M. (1990) Site-specific properties of pericardial and epicardial adipose tissue: the effects of insulin and high-fat feeding on lipogenesis and the incorporation of fatty acids in vitro. Inr. J. Obesity 14, 1013-1022. MBrin P., Rebuff&Strive M. and Bjiimtorp P. (1990) Uptake of triglyceride fatty acids in adipose tissue in uivo in man. Eur. J. clin. Invest. 20, 158-165. Mattacks C. A. and Pond C. M. (1988) Site-specific and sex differences in the rates of fatty acid/triacylglycerol substrate cycling in adipose tissue and muscle of sedentary and exercised dwarf hamsters (Phodopus sungorus). Inf. J. Obesity 12, 585-597.
1831
Mattacks C. A. and Pond C M. (1991) Site-specific differences in the uptake in vivo of 2-deoxyglucose by adipose tissue in the Djungarian hamster, PhodopuF sungorus. Nufr. Res. 11, 1083-1086. Mattacks C. A., Sadler D. and Pond C. M. (1987) The effects of exercise on the activities of hexokinase and phosphofructokinase in superficial, intra-abdominal and intermuscular adipose tissue of guinea pigs. Comp. Biothem. Physiol. 87B, 533-542.
Newsholme E. A. (1977) The regulation of intracellular and extracellular fuel supply during sustained exercise. Ann. N. Y. Acud. Sci. 301, 81-91. Nilsson-Ehle P., Carlstrom S. and Belfrage P. (1975) Rapid effect on lipoprotein lipase activity in adipose tissue of humans after carbohydrate and lipid intake. Scund. J. clin. Lab. Invest. 35, 373-378. Nilsson-Ehle P., Tornqvist H. and Belfrage P. (1972) Rapid determination of lipoprotein lipase in human adipose tissue. Clin. chim. Acta 42, 383-390. Olivecrona T. and Bengtsson-Olivecrona G. (1990) Lipoprotein lipase and hepatic lipase. Curr. Opinion Lipidol. 1, 222-230.
Pond C. M. (1991) Adipose tissue in human evolution. In The Aquatic Ape: Fact or Fiction? (Edited by Roede M., Wind J., Patrick J. M. and Reynolds V.), pp. 193-220. Souvenir Press, London. Pond C. M. and Mattacks C. A. (1991) The effects of noradrenaline and insulin on lipolysis in adipocytes isolated from nine different adipose depots of guinea pigs. Int. J. Obesity 15, 609618. Pond C. M., Mattacks C. A., Thompson M. C. and Sadler D. (1986) The effects of age, dietary restriction, exercise and maternity on the abundance and volume of adipocytes in twelve adipose depots of adult guinea pigs. Brit. J. Nutr. 56, 2948.
Pouliot M. C., Desprts J.-P., Moorjani S., Lupien P. J., Tremblay A., Nadeau A. and Bouchard C. (1991) Regional variation in adipose tissue lipoprotein lipase activity-association with plasma high density lipoprotein levels. Eur. J. c/in. Invest. 21, 398405. Pradines-Figueres A., Vannier C. and Ailhaud G. (1990) Lipoprotein lipase stored in adipocytes and muscle cells is a cryptic enzyme. J. Lipid Res. 31, 1467-1476. Rett K., Wicklmayr M., Dietze G., Mehnert H., Wolfram G. and Hailer S. (1986) Inhibition of muscular glucose uptake by lipid infusion in man. C/in. Nutr. 5, 187-192. Rodbell M. (1964) Localization of lipoprotein lipase in fat cells of rat adipose tissue. J. biol. Chem. 239, 753-755. Savard R., Palmer J. E. and Greenwood M. R. C. (1986) Effects of exercise training on regional adipose tissue metabolism in pregnant rats. Am. J. Physiol. 250, R837-R844. Semb H. and Olivecrona T. (1986) Nutritional regulation of lipoprotein lipase in guinea pig tissues. Biochem. biophys. Acra 876, 249-255.
Tan M. H., Tsunako S. and Have1 R. J. (1977) The significance of lipoprotein lipase in rat skeletal muscles. J. Lipid Res. 18, 363-370. Ursini F., Vugman M., Femandes L. C., Curi C. M. 0. N. and Curi R. (1991) Metabolic changes of several adipose depots as caused by aging. Physiol. Behav. SO, 317-321. Wallinder L., Peterson J., Olivecrona T. and BengtssonOlivecrona G. (1984) Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase. Biochem. biophys. Acta 795, 513-524.