Influence of moderate exercise on adipocyte metabolism and hormonal responsiveness.

Influence of moderate exercise on adipocyte metabolism and hormonal responsiveness J. L. OWENS, E. 0. FULLER, D. 0. NUTTER, AND M. DIGIROLAMO Departments of Medicine and Physiology, Emory University School of Medicine, Atlanta, Georgia 30303

OWENS, J. L., E. 0. FULLER, D. 0. NUTTER, AND M. DIInfluence of moderate exercise on adipocyte metabolism and hormonal responsiveness. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 43(3): 425430, 1977. -The effects of a moderate exercise program on several body parameters and on adipocyte metabolic functions were studied in male rats trained to run on a treadmill. At the end of a 12-wk program no significant changes in body weight, food consumption, or the weight of several organs (liver, kidneys, spleen, testes) were observed in trained rats as compared to sedentary animals. The trained rats, however, presented a slight decrease in w-eight of the epididymal fat pads which was due to a minor change in adipocyte size, without any change in adipocyte number. In spite of these minimal effects on adipocyte morphology, exercise training affected several parameters of adipocyte metabolism: it significantly reduced basal lipolysis per cell (P < 0.05) and basal glucose metabolism to CO, and triglyceride (P < 0.02); the insulin effect on stimulation of glucose metabolism was unchanged, whereas the epinephrine effect on it was reduced (P < 0.001); epinephrine produced a significantly greater (P < 0.05) acceleration of lipolysis; the intracellular free fatty acid levels were higher (P < 0.025) in the basal state and also after insulin and epinephrine (P < 0.05 for both). These findings indicate that exercise training in animals leads to modifications of adipocyte metabolism and hormonal responsiveness even when a modest degree of exercise, unable to affect body weight or adipocyte morphology, is applied. GIROLAMO.

adipose mass; fat-cell size; lipolysis; glucose metabolism; cellular free fatty acids; insulin; epinephrine effects

intra-

TISSUE has a central role in the energy metabolism of the body, synthesizing and storing fuel in the form of triglyceride, and releasing it in the form of free fatty acids (FFA) and glycerol, in response to metabolic demands. The importance of adipose tissue to the exercised subject has been emphasized in recent studies which have compared the lipolytic (3, 4, 6, 15, 21, 22, 26, 27) and lipogenic (2, 5, 25) capacities of adipose tissue in exercised animals and in sedentary controls. An increased capacity of the fat cells from trained rats to release free fatty acids in response to epinephrine has been reported by several laboratories (3, 4, 6, 15, 26, 27) with some exceptions (21, 22). The capacity of adipose cells to synthesize lipid from glucose has also received attention (2, 25). But the evaluation of this metabolic function, as affected by exercise, is rendered difficult by the observation that changes in adipocyte size are proADIPOSE

duced by exercise (8, 24), and that adipocyte size can influence glucose metabolism and insulin response (10, 11, 16, 18, 29). For these reasons, in the present report, we have employed a moderate exercise program which would result in minimal changes in body composition and in fat-cell size. In this manner we could separate the effect of cell size from that of training and determine what effect, if any, the latter would have on adipocyte metabolism. We have focused our attention on a comparison of lipolysis, glucose metabolism, intracellular free fatty acid levels, and the responses to insulin and epinephrine, in isolated adipocytes from sedentary controls and from exercise-trained rats. METHODS

Animals. Male Long Evans rats, 3 mo in age and weighing approximately 300 g, were purchased from Charles River Laboratory. The animals were housed in individual cages in a constant-temperature room (23°C)) exposed to 12-h intervals of light and darkness, and were fed ad libitum Purina laboratory chow (ground to granular form), available in a food cup inside the cage. The amount of chow consumed daily by the rats was measured. Water was available at all times. A group of animals (group A), maintained for 1 wk in our animal room, was killed at the beginning of the experiment. A second group of control animals (group B) was allowed to lead a sedentary life for 12 wk. A third group of animals (group C) was subjected to progressive training on a motor-driven treadmill 5 days/wk, for 12 wk prior to death. Throughout the initial experimental period (6 wk), the duration and intensity of the exercise was progressively increased. In the later phase of the experiment (6-12 wk), the animals were running 45 min/day at a speed of 22 m/min (0.62 mph) at a 6” (9%) grade. On the final day of the experiment, the rats were lightly anesthetized with pentobarbital (50 mg/kg body wt) and killed in the fed state. The trained rats were killed at least 24 h after the last bout of forced exercise. Hormones and reagents. The following hormones and reagents were used: bovine insulin (Eli Lilly and Co., lot PJ 4069, 23.8 U/mg); Z-epinephrine bitartrate (Mann Research Laboratory); bovine serum albumin, fatty acid-poor, 1 mmol containing 0.35-0.4 meq FFA (Pentex Inc., Kankakee, Ill.); [14C]glucose, uniformly labeled, 5 mCi/mmol (New England Nuclear); collagenase from

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426 Clostridium histolyticum, 150-175 U/mg (Worthington Biochemical Corp.). Conditions of the experiments and analytic techniques. Immediately after death, the epididymal fat pads were removed, weighed, and incubated in collagenase (10 mg/g tissue) in Krebs-Ringer bicarbonatealbumin medium, at pH 7.4. The liver, testes, kidneys, and spleen were also removed, carefully trimmed of visible fat, blotted, and weighed. Isolated fat cells were prepared according to the method of Rodbell (28), as previously used in this laboratory (10). Aliquots of the fat-cell suspension were incubated in 2 ml Krebs-Ringer bicarbonate (KRB) buffer with 4% albumin, containing 6 mM glucose and 0.5 ,&i/ml [14C] glucose, at pH 7.4. KRB medium or hormones in specified concentrations were added, in 0.1 ml volume, to control or experimental samples. Details of the incubation procedure, and of recovery of radioactivity into C02, glyceride-glycerol 9 and glyceride-fatty acids were as previously described (10). Glycerol release was measured by the method of Eggstein and Kreutz (14), and the intracellular FFA concentration was determined by the method of Angel et al. ( 1). In each experimem, two aliquots of the free fat-cell suspension were taken to determine the triglyceride content (10); another aliquot was stained with methylene blue and the diameters of 300 fat cells were measured by optical sizing to provide a measure of mean fat-cell diameter and volume (12). The number of fat cells in the cell suspension was estimated by dividing the triglyceride content by the mean fat-cell triglyceride (12). The metabolic activity of the cells was expressed on the basis of fat-cell number and in relation to mean fatcell volume. s tatistical evaluation of data. Means, standard deviations (SD), and standard errors of the mean (SE) were calculated in the usual way, as described el.sewhere (9)e The signi .ficance of differences between two group means was analyzed using the Student t-test. Values for P co.05 were taken to indicate significance. RESULTS

Initially, to demonstrate the effect of age and the influence of the exercise program on the animals’ development, we have compared body weight and other parameters of group A rats to those of groups B and C. Subsequently, we have focused on a comparison of findings in groups B and C (animals of the same age) to investigate the modifications in metabolic activities of adipose tissue brought about ingroup C by the exercise program applied. Table 1 shows that the 3 mo elapsed between the death of rats of group A and those of group B produced in the latter group additional increases in body weight -and in the size of the liver, kidneys, testes, and epididyma1 fat pads; this indicates that the animals ofgroups B and C were still actively growing during the experimental period. When rats from group B are compared to those of group C, Table 1 shows that the moderate exercise program to which animals of group C were subjected did not result in any significant changes in body weight or in the weight of the organs measured.

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1. Comparison of body weight and weight organs in sedentary and trained rats at beginning and end of experimental protocol --___ ________ I___--_-~ ---___l-----TABLE

of body Animal Group

A (n = 7) 63 (n = 9) c (n = 8)

Body

wt, g

346 +9.3 534 d2.8 541 +8.8

Liver,

13.26 20.32 18.99 20.78 18.63 k0.44

g

Kidneys,

2.51 to.13 3.52 kO.18 3.46 Iko.12

g

Testes’, g

2.78 20.20 3.32 kO.18 3.73 kO.13

‘pleen7

0.77 a0.05 0.76 +0.04 0.80 20.04

g

Epididymal Fat Pads, g

2.72 to.12 7.61 to.76 6.64 ~0.52

Group A were 3 mo old, sedentary, killed at the beginning of experiment. Group B were 6 mo old, sedentary, killed at the end of the experiment. Group C were 6 mo old, trained rats, killed after 12 wk of exercise program. n = number of animals in each group. Values shown are means + SE of n observations in each group of animals.

Only with regard to the epididymal fat pads it was observed that the trained animals of group C had smaller fat pads than the animals of group B, but this difference was not statistically significant (P > 0.05). Figure 1 shows a comparison of the sedentary rats of group B with the trained rats ofgroup C for the entire duration of the experiment. The lower panel shows the training regimen: after progressive adaptive training for the initial 6 wk, a plateau of training was reached that remained unchanged for the remaining 6 wk. The middle panel shows that, in spite of increased exercise in group C rats, body weights-of the two groups did not show significant differences throughout the 12 wk of the protocol. Similarly, when food consumption was measured daily and averaged for the week (top panel), no differences in food consumption were observed between the two groups at any point during the experiment. The differences in mass and cellular characteristics of adipose tissue between group B (sedentary) and group C (trained) are shown in Fig. 2. The small difference in weight of the epididymal fat pads was mostly accounted for by the smaller fat-cell volume in these animals since the fat-cell number in the pads was unchanged. But even the observed difference in cell volumes between the two groups (mean of 324 vs. 388 pl) was rather small and did not reach statistical significance. Table 2 shows a comparison of glucose metabolism and hormonal responses by isolated adipocytes from sedentary and trained rats, at the beginning and the end of the experimental protocol. When sedentary animals of group B (6 mo old) are compared to sedentary animals of group A (3 mo old), certain differences in metabolic capacities are apparent: group B adipocytes present a greater rate of basal glucose metabolism (P < 0.005) and a greater response to epinephrine on glucose metabolism (P < 0.001). The insulin-stimulated glucose metabolism was not different in the two groups. The differences observed probably represent the effects of aging and/or increasing adipocyte size (10, 13, 29). Since rats from groups B and C were identical in age and stage of development but differed only in the exercise program, we then proceeded to analyze the metabolic differences shown by the adipocytes of these two groups. In absence of insulin, fat cells from trained rats


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TABLE 2. Effects of insulin and epinephrine on glucose metabolism by adipocytes from sedentary and trained rats at beginning and end of experimental protocol ____-

CONSUMPTION

CT---XI A-

SO

SEDENTARY TRA/NED

- --A

Animal GI-CNIp

RATS !?A73

Glucose

Converted

to

Basal

+ Insulin

+ Epinephrine

jmaol1107 fat cells per h

BODY

,’

TRAINING REGIMEN . . . . . . ..--..o ,_.. _,_... So9 22 m/min,

FAT

0 45

CELL

min.

FAT CELL NUMBER

VOLUME (pII

( x 106)

PI

x IO6

20

400

300

200

100

Sedentary

TraIned

+ it -t +

0.05 0.09 0.01 0.12

1.69 2.75 0.27 4.71

+ +t -t

0.09 0.14 0.04 0.20

1.58 2.69 0.07 4.31

+ t -t I?

0.09 0.11 0.01 0.14

B (n = 9)

co* Glyceride-glycerol Fatty acids Total

0.96 2.38 0.07 3.41

+IL + +-

0.07 0.14 0.01 0.22

1.60 3.20 0.24 5.07

-+ 0.12 f 0.18 I: 0.03 + 0.33

1.82 3.94 0.04 5.80

t I? ?I +

0.11 0.21 0.00 0.32

c (I2 = 8)

co* Glyceride-glycerol Fatty acids Total

0.77 1.94 0.02 2.74

It -+ 2 -t

0.05 0.08 0.00 0.12

1.38 2.93 0.10 4.41

2 + t It-

1.33 2.81 0.01 4,14

~fr 0.08 + 0.10 _t 0.00 + 0.18

0.08 0.15 0.02 0.22

Group A were 3 mo old, sedentary, killed at the beginning of the experiment. Group B were 6 mo old, sedentary, killed at the end of the experiment. Group C were 6 mo old, trained rats, killed after 12 wk of the exercise program. n = number of animals in each group. Values shown are means + SE of triplicate observations in n animals for each group. Insulin was present at 1 mu/ml, epinephrine at 1 pg,/ml.

TABLE 3. Effects of insulin and epinephrine on lipolysis and intracellular free fatty acids in adipocytes of sedentary and trained rats -~-

I

Net Glycerol Release, pmol/107 fat cells/h

I_ Medium

FFA,

reqiml

-~

1 lntrace$?l~~t

“,F&’

Pm”’

0

I

Sedentary

Trained

0

Groups A4 medium (60 - 0 measured at the observations for

are the same as in Table 2. Net glycerol release is glycerol released into the min), calculated per lo7 fat cells. Medium FFA and intracellular FFA were end of the incubation (1 h). Values shown are means * SE of duplicate n animals in each group. Ins = insulin at 1 mu/ml; Epi = epinephrine at 1

pg/ml.

~ 4

0.81 1.65 0.06 2.50

MEAN

WEIGHT OF EPIDIDYMAL FAT PADS (9)

6

co2 Glyceride-glycerol Fatty acids Total

WEIGt-iT

FIG. 1. Effects of a training regimen (bottom) on body weight (mid&) and food consumption (top) of a group of rats (A- - -A, trained rats) in comparison to controls (o-o, sedentary rats). Training program was progressively increased in first 6 wk of training and then applied at steady level (6” grade, 22 m/min, 45 min/day) for last 6 wk. Values shown for body weight are means of weekly measurements. Values shown for food consumption are means of daily measurements averaged for the week. See text for details.

8

A (n = 7)

Sedentary

Trained

2. Comparison of weight of epididymal fat pads, mean fatcell volume and fat-cell number in the pads from the trained animals and sedentary controls” Meight of columns represents the mean of 8-9 observations. Vertical line is + 1 SE. See text for statistical significance of the comparison. FIG.

(group C) metabolized significantly less (P < 0.02) glucose than those from sedentary rats (group B). This difference between the two groups was due to a significant decrease in all three products of glucose, namely CO,, glyceride-glycerol and glyceride-fatty acids, and no variations in the pattern of glucose metabolism were observed. Insulin, at 1 mu/ml, stimulated glucose metabolism in both groups of animals to reach maximal levels that were not statistically different. The effect of epinephrine on glucose metabolism was more evident in

adipocytes from the sedentary animals than in those of the trained rats and the difference between these groups was significant at P < 0.001. Thus, adipocytes of trained rats presented a rate of glucose metabolism which was lower in basal conditions, comparable after insulin, and lower in response to epinephrine than adipocytes from sedentary rats. Table 3 shows that the net basal glycerol release by adipocytes was significantly lower (P < 0.05) in the trained group of rats than in the sedentary animals. Addition of insulin had a negligible effect on lipolysis. The effect of epinephrine on glycerol release was marked in both groups, but the magnitude of the hormonal response in the trained rats was significantly higher (P < 0.05) than that in the sedentary animals. Thus, in spite of a lower basal glycerol release, the fat cells from trained animals were capable of responding to a greater extent to the stimulating effect of epinephrine on lipolysis. Table 3 also shows a comparison of intracellular free fatty acid levels in the adipocytes of the two groups. Trained rats had in their adipocytes significantly higher levels of intracellular free fatty acids than the sedentary


428

rats, in absence of hormones and also after the addition of insulin (P < 0.05) or epinephrine (P < 0.05). The marked increase in intracellular free fatty acids produced by epinephrine was also reflected in the effect of this hormone on the medium free fatty acid concentration. Thus, levels of intracellular free fatty acids were higher in the adipocytes of the trained rats under all experimental conditions, whether in the basal state or following addition of insulin or epinephrine. DISCUSSION

The present experiments confirm and extend the results of previous*studies (2-6, 25-27) on the effects of exercise on adipose tissue metabolism and hormonal responsiven .ess in th .e rat. Furthermore by selecting a moderate exercise program which produced no changes in body weight, food consumption, or weight of several organs, and only minimal changes in adipose mass and fat-cell volume, we have also been able to show that the modifications in adipocyte metabolic activity observed probably result from an adaptation to the exercise program rather than from a significant change in fat-cell size per se (10, 11, 16, 18, 29). In studies by Askew et al. (5), the insulin-stimulated glucose metabolism to lipid, expressed on a per cell basis, was found to be similar in adipocytes from trained and untrained rats. We have confirmed this finding and have found, in addition, that adipocytes from trained rats possessa lower rate of basal glucose metabolism than cells from sedentary rats (see Table 2). Palmer and Tipton (25) had reported similar findings for glucose oxidation but not for glucose conversion to lipid. A possible source of the discrepancy between our results and theirs may be the very low (3 nmol/ml) glucose concentration used in their study. We have also found that the basal (unstimulated) rate of lipolysis is significantly lower in the adipocytes of trained rats when compared to those of sedentary controls. In recent studies by Askew et al. (4, 6), these authors have also observed that the epinephrine-stimulated lipolysis was significantly greater, on a per cell basis, in adipocytes from trained rats than in those of untrained controls. Parizkova et al. (26, 27) and others (3, 15) had earlier obtained similar results, but the comparison between adipose tissues of trained and untrained animals had been done on the basis of tissue wet weight rather than on a per fat cell basis. Our results (see Table 3), expressed on a per fat cell basis and comparing populations of cells of similar size, support the studies showing that epinephrine-stimulated lipolysis is significantly greater for adipocytes from trained rats. Other authors, however, have found (21, 22) no difference or only some regional differences in hormone-stimulated lipolysis, expressed on a per cell basis, between adipocytes of trained and untrained rats. Since the latter two studies were carried out with tissue slices rather than with isolated fat cells, it is not known whether this or other methodological differences, or other variables (e.g., animals’ age, nutrition, adipocyte size, degree of exercise) can account for the variable results reported from different laboratories.

OWENS,

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Our observation that the epinephrine effect on glucose metabolism is significantly lower in adipocytes from trained rats is of interest, particularly because it indicates an uncoupling of the lipolytic effect of epinephrine from its effect on glucose metabolism in these animals. Thus, the emerging picture in adipocytes of exercise-trained rats is one of a reduced resting or basal metabolic rate (both with regard to glucose metabolism and rate of lipolysis), but one of variable responsiveness to hormonal stimulation (e.g., preserved for insulin on glucose metabolism, increased for epinephrine on lipolysis, and decreased for epinephrine on glucose metabolism). The possible implications of these findings in the overall fuel economy of the trained rats will be discussed below. An additional novel finding is the observation that adipocytes of trained rats contain elevated levels of intracellular free fatty acids. This was to a degree unexpected since we had previously observed that, in sedentary rats, the level of intracellular free fatty acids was directly related to fat-cell size (10); in the present observations, the fat-cell size was, if anything, slightly smaller in the trained rats than in the sedentary controls. The elevated levels of intracellular free fatty acids seen in the adipocytes of trained rats may reflect a cellular adaptation to the exercise training program and represent a preparatory stage of the adipocytes to mobilize greater amounts of FFA in response to increased metabolic demands and to lipolytic stimulation. Further studies will be necessary to clarify the mechanisms responsible for altered IFFA accumulation and FFA release by adipocytes of trained rats. The present observations, carried out at least 24 h after the active phase of exercise, probably represent certain adaptive effects of the exercise program on adipocyte metabolism. These observations, as well as the previous ones by other investigators, suggest that the adipose organ participates in the general adaptation to exercise and in the increased metabolic demands of this condition. The limited observations available, and the possible pitfalls of extrapolating from in vitro to in vivo conditions, indicate to us that it may be premature to try and assess the full metabolic role of the adipose organ in the adaptation to the various components of exercise (such as stress, increased fuel requirements, changes in plasma concentration of hormones). Nevertheless, the information available provides some specific indications, and exploration of their significance may lead to certain tentative hypotheses and to better defined future studies. For instance, the lower basal rate of glucose metabolism in adipose tissue may reflect a reduced uptake and storage of fuel in adipose tissue, and thus may facilitate glucose uptake and metabolism in other tissues. It is of interest to note that other investigators (7, 19, 20) have reported an increase in the capacity to oxidize carbohydrates in skeletal muscle of trained animals. Also, stores of glycogen in liver and triglyceride and glycogen in muscle of trained animals have been found to be increased (17,23,30, 31). These data indicate that adaptation to training may lead to increased glucose utilization in some organs (e.g., muscle and liver) and reduced


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METABOLISM

utilization of glucose in others (e.g., adipose tissue) in which glucose may not be as necessary. Similarly, a reduced basal rate of lipolysis (as measured by glycerol release) may also indicate an attempt of adipose tissue from the trained rats to preserve lipids during a basal state. The response to epinephrine on acceleration of lipolysis, markedly greater in trained rats than in sedentary rats, may reflect a greater capacity of adipose tissue to contribute fuel in the form of free fatty acids and glycerol during periods of stress, such as exercise, in which the plasma levels of a number of lipolytic hormones are known to be elevated. Previous studies on the effects of exercise on body weight, food consumption and body composition have shown variable results (2, 6, 8, 24, 26, 27). A general observation has been that the more strenuous the exercise and the earlier the onset of exercise (in relation to age and growth stage of the animals) the greater the effect in limiting increases in body weight, relative adipose mass, and adipocyte size. Since the rats ofgroup C were subjected to a moderate exercise program only for 45 min/day and for 5 days/wk, it is not therefore surprising that no changes in body weight or food consumption were observed. This is consonant with other observations (8, 26, 27) in which a moderate exercise

program also produced no major changes. The extra source of fuel for the increased caloric expenditure of exercise in our rats probably derived from mobilization of lipid stored in adipose tissue since a small reduction in fat mass was observed. In addition, we cannot exclude the possibility that there also was a small reduction in spontaneous locomotor activity of the trained rats during the remaining 23 h, to compensate for the increased caloric expenditure of the acute phase of exercise. Further studies will be necessary to evaluate this possibility. Finally, even though this study and previous observations have indicated that significant and important metabolic alterations in adipocyte functions take place during exercise training, it is apparent that additional work needs to be done in these and other areas to fully assessthe role played by adipose tissue in the metabolic adaptations to exercise and the role played by exercise training in the growth and function of the adipose organ. This study was supported in part by Grants AM-13940 and AM17403 from the National Institutes of Health, a grant from the Noble Foundation, and a grant-in-aid of the Georgia Heart Association. Received

for publication

15 November

1976.

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Influence to high-intensity intermittent and moderate-intensity continuous exercise on indices of cardio-inflammatory health in men.

Effect of moderate exercise on urinary MHPG in depressed patients.

Effect of mild-to-moderate airflow limitation on exercise capacity.

Effect of moderate exercise on rat T-cells.

The influence of pre-operative hormonal stimulation on hypospadias repair.

Influence of exercise hyperthermia on exercise breathing pattern.

Hormonal responsiveness of adenylate cyclase activity in cartilage.

[Hormonal contraception, tryptophan metabolism and depression].

Influence of ocular tropicamide on exercise testing.

Influence of exercise-induced bronchoconstriction on refractoriness.

Heterogeneity of Muscle Blood Flow and Metabolism: Influence of Exercise, Aging, and Disease States.