Endurance Exercise Training Causes Adrenal Medullary Hypertrophy in Young and Old Fischer 344 Rats Kathryn N. Schmidt, L. E. Gosselin and W. C. Stanley
Summary The purpose of the present investigation was to determine the effects of endurance exercise training on adrenal medullary volume and epinephrine content in young (5 month) and old (23 month) female Fischer 344 rats. Animals from each group underwent 10 weeks of treadmill running (60 minutes per day, 5 days per week). 72 hours following the last training session animals were killed and the adrenal glands removed for subsequent analysis. Plantaris muscle citrate synthase activity increased with training in both young and old animals (39.8% young; 36.4% old). Trained animals had larger adrenal medullary volumes (48 % increase in young, and 18 % in old) than untrained controls. Trained animals also had higher total adrenal medullary epinephrine content (36% increase in young, and 2 4 % in old). There were no differences in adrenal medullary epinephrine or norepinephrine concentration (Hg/.l medulla). It was concluded that the training-induced increase in adrenal epinephrine content is due to an increase in the size of the medulla, and not to a greater medullary epinephrine concentration. Furthermore, similar responses to training occur in both old and young animals. Key words Adrenal Gland - Aging - Catecholamines Epinephrine - Exertion - Norepinephrine - Sympathetic Nervous System
Introduction Exercise training induces numerous adaptations in the sympathoadrenal system. It has been well documented that endurance exercise training results in a lower plasma epinephrine concentration at a given absolute submaximal exercise intensity (Galbo, Richter, Hoist and Christensen 1977; Winder, Hagberg, Hickson, Ehsani and McLane 1978; Winder, Beattie and Holman 1982). However, recent work by Kjaer and Galbo (Kjaer, Mikines, Christensen, Tronier, Vinten, Sonne, Richter and Galbo 1984; Kjaer, Christensen, Sonne, Richter and Galbo 1985; Kjaer, Farrell, Christensen and Galbo 1986; Kjaer and Galbo 1988) suggests that the epinephrine response to various physiologic stresses is enhanced in trained male endurance athletes compared to sedentary subjects. Horm. metab. Res. 24 (1992) 511-515 © Georg Thieme Verlag Stuttgart • New York
Plasma epinephrine concentration was measured in response to maximal and supramaximal exercise (Kjaer et al. 1986), an intravenous injection of glucagon ( l m g / 7 0 k g body weight) (Kjaer and Galbo 1988), acute insulin-induced hypoglycemia (Kjaer et al. 1984), acute hypercapnia (inspired CO2 = 70%) {Kjaer and Galbo 1988), and acute hypoxia (inspired PC2 = 87 mmHg) (Kjaer and Galbo 1988). In all cases, they found a significantly greater plasma epinephrine response in trained men. These cross-sectional data suggest that physical training may induce an adaptation of the adrenal medulla which results in an increased capacity for epinephrine secretion. The adrenal medulla is the major source of circulating epinephrine. In fact, Scheurink et al. were unable to detect epinephrine in the plasma of adrenomedullated rats before, during or after exhaustive exercise (Scheurink, Steffens, Bouritius, Dreteler, Bruntink, Remie and Zaagsma 1989). It is therefore relevant to examine changes which may occur in the adrenal gland in response to endurance exercise training. Song et al. (Song, Ianuzzo, Saubert and Gollnick 1973) observed that male rats subjected to a 10 week running program had 4 0 % greater adrenal gland weight than cage confined animals. A similar increase in adrenal weight was observed in thyroidectomized and thyroidectomized-thyroxine (T3) treated rats, suggesting that exercise induced adrenal hypertrophy is not dependent on T3. Adrenal gland DNA content (mg/gland) and concentration (mg/g gland) were both higher in trained animals, which suggests the adrenal enlargement was due to both cell hypertrophy and hyperplasia. No attempt was made to delineate adaptations in the adrenal medulla from those in the cortex. Ostman and Sjostrand (1971) observed that total adrenal epinephrine content was higher in trained than in untrained rats, although the adrenal epinephrine concentration (epinephrine content/adrenal gland weight) was not significantly different. Stallknecht et al. (Stallknecht, Kjaer, Mikines, Maroun, Ploug, Ohkuwa, Vinten and Galbo 1990) recently observed that ten weeks of swimming six hours per day resulted in hypertrophy of the adrenal medulla and an increase in adrenal epinephrine content in young rats. In contrast to these results, Mazzeo et al. (Mazzeo, Colburn and Horvath 1986) saw no change in adrenal gland weight or epinephrine concentration in either young or old female Fischer 344 rats in response to a 12 week program of treadmill running. It is unclear whether the increase in adrenal epinephrine content sometimes seen with moderate treadmill exercise training is due to hypertrophy of the adrenal medulla, or an increase in medullary epinephrine concentration. This Received: 2 July 1991
Accepted: 7 Feb. 1992 after revision
Downloaded by: University of Illinois. Copyrighted material.
Biodynamics Laboratory, University of Wisconsin, Madison, Wisconsin, U. S. A.
Horm. metab. Res. 24 (1992)
Kathryn N. Schmidt, L. U. Gosselin and W. C. Stanley
study, therefore, examines the effects of a 10 week treadmill exercise training program on adrenal medullary volume and epinephrine content. Both young (2 month) and old (20 month) female Fischer 344 rats were studied to assess whether adrenal hypertrophy was dependent on growth. Our hypothesis was that exercise training results in adrenal medullary hypertrophy in both young and old animals. Methods Forty female Fischer 344 rats, obtained from the National Institute on Aging, were received at the ages of 2 (young; n = 20) and 20 (old; n = 20) months. All animals were individually housed in climate-controlled quarters (22 °C, 12 hour light-dark cycle). Animals received Purina rodent chow and water ad libitum. Before experimentation, animals were allowed 2 weeks to become adjusted to the new environment and familiarized with investigator handling to minimize stress. At the end of this period animals were weight-matched and randomly assigned to either the control or the experimental group. For the remainder of the study the control animals were handled and placed on the stationary treadmill for 10 min twice each week. Exercise
protocol
All animals were exercised in the two week acclimation period. This consisted of treadmill walking at 5-10 m/min on the level. Subsequent training sessions for the exercise groups conformed to protocols based on previous work done by Mazzeo et al. (Mazzeo, Brooks and Horvath 1984). The training protocol for young animals consisted of treadmill running at a 15 % incline for 10 weeks. The first two weeks were at 12 and 15 meters/min for 10 and 25 minutes, respectively; thereafter all training sessions were 60 min in duration. The treadmill speed was progressively increased from 14 meters/min during week three to 29 meters/min during week eight. It remained at 29 meters/min during the final three weeks of training. The old animals also ran at a 15 % incline for 10 weeks. The first two weeks were at 8 and 10 meters/min for 10 and 21 min respectively. All subsequent sessions were 60 min in duration. The treadmill speed was progressively increased during weeks three to eight from 9 to 15 meters/min, and held constant at 15 meters/min over the last three weeks of training. From the data of Mazzeo et al. (Mazzeo, Brooks and Horvath 1984), it was estimated that the intensity of exercise during the final three weeks of training was approximately 75 % of VO2max for both old and young animals. Animals were motivated to run by means of a horizontal shock grid placed at the back of the treadmill (Quinton, Seattle). Adrenal gland
preparation
Animals were sacrificed 72 hours after the final training session. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbitol (50 mg/kg body weight). Adrenal glands were removed, blotted dry on a clean gauze square and weighed. Previous work in our laboratory indicated no significant difference between left and right adrenal weights for 9-month or for 27-month old rats (unpublished observation). However, in order to correct for possible differences between the left and right adrenal glands within the same rat, adrenal glands from alternating sides were selected for each of the following two procedures: one adrenal gland was quickly frozen in isopentane which had been cooled in liquid nitrogen and stored at 80 °C for biochemical analysis. The remaining adrenal gland from each rat was placed in Bouin's fixative (Sheehan and Hrapchak 1980) for 24 hours at 25 °C and processed for paraffin sectioning. At this point samples were renumbered and subsequent procedures were performed in a blinded fashion to prevent biasing of the results obtained in structural analysis. Paraffin sections were cut on a Reichert-Jung microtome (Reichert-Jung, Heidelberg) at a thickness of 10 micrometers, with every tenth section retained for analysis. Shrinkage of the tissue occurred as a consequence of processing and embedment. However, Reaven and co-workers were unable to detect any age-related differences
in the diameter change of adrenal hemisections resulting from tissue preparation (Reaven, Kostrna, Ramachandran and Azhar 1988). Morphological
Analysis
Tissue sections were stained with hematoxylin and counterstained with eosin to give appropriate contrast. This stain is recommended by Sheehan and Hrapchak (1980), and was successfully used on rat adrenal tissue by Reaven et al. (1988). After staining, slides were fitted with cover slips using 1 - 2 drops Permount mounting medium. Total cross-sectional area and medullary area were measured for each section using a computerized planimeter (Bioquant, R & M Biometrics, Nashville). Cortical area was calculated as the difference between total cross-sectional area and medullary area. As every tenth section was retained for analysis, the volume of each section was estimated by multiplying the cross-sectional area by 100 micrometers, and the total volume was estimated by the sum of the estimated sectional volumes for all sections. The coefficients of variation for eight analyses of one gland were 1.96%, 1.93% and 2.01% for total, cortical and medullary volumes respectively. It is important to note that this approach to morphological analysis rests on the assumption that the shape of the adrenal gland is consistent with a spherical model. Reaven et al. (1988) investigated the accuracy of this assumption, and found that the ratios between longest and shortest observed diameters for both 5 month- and 18 month-old animals fell within the limits set for spheres in morphometry.
Determination of Catecholamine Content Levels of epinephrine and norepinephrine were determined by high pressure liquid chromatography (HPLC) (C18 reverse phase column, 3 micron diameter, microsorb 80,200, Perkin Elmer) with electrochemical detection. Tissue samples were homogenized in 0.5 ml of 0.1 M perchloric acid (PCA) and the vials rinsed twice with 0.5 ml of 0.1M PCA to give a total volume of 1.5 ml of homogenate. Three 400 microliter aliquots of the homogenate were stored at - 80 °C for processing as described by Mazzeo et al. (Mazzeo, Colburn and Horvath 1986).
Citrate Synthase Assay The maximal activity of citrate synthase was measured using the method of Srere (1969). The plantaris muscle was cleaned of connective tissue and minced with scissors at 4 °C. The minced tissue samples were mixed with homogenizing medium (50 mM potassium phosphate (pH 7.4), 1 mM EDTA, 2 mM MgCl2, 2 mM ADP, and 0.5 mM dithiothreitol). Citrate synthase activity was measured spectrophotometrically at a wavelength of 412nm, at 31 °C. Statistical
Analysis
Differences between trained and untrained groups were evaluated nonparametrically using the Mann-Whitney rank test. No comparisons were made between young and old groups. Tests were accepted as significant at p