Cynthia Carey1, Richard L. Marsh2, Anne Bekoff1,. Rebecca r1. Johnston1 ... their introduction into New York in 1940 (see Dawson et al. 1983a;. Leek, 1987).
ENZYME ACTIVITIES IN MUSCLES OF SEASONALLY ACCLIMATIZED HOUSE FINCHES
Cynthia Carey1, Richard L. Marsh2, Anne Bekoff1, Rebecca r1. Johnston1, and Ann M. Olin1 1Department of EPO Biology, University of Colorado Boulder, CO 80309, USA and 2Department of Biology Northeastern University, Boston, MA 02115, USA INTRODUCTION Members of the avian subfamily Carduelinae have served as subjects of numerous investigations on adaptations of small birds to cold winters (Salt, 1952; Dawson and Tordoff, 1964; West, 1972; Dawson and Carey, 1976; Carey et al. 1978; Weathers et al. 1980; Marsh et al. 1984; Reinertsen, 1986; Yacoe and Dawson, 1983; and others). These birds, including goldfinches, pine siskins, house finches, grosbeaks, crossbills, and redpolls, are particularly well suited for such study because of their principally northern distribution and small body sizes. Their success in cold climates appears to be linked in part to a type of metabolic acclimatization, involving enhanced capabilities for sustained elevated production of heat for prolonged periods (Dawson and Carey, 1976; Dawson et al. 1983a,b; Dawson and Marsh, 1988). Intensive study of two cardueline finches, American goldfinches (Carduelis tristis) and house finches (Carpodacus mexicanus) has revealed differences between these species in degree of seasonal adjustment to cold. Winter goldfinches exhibit considerable enhancement of endurance during cold stress that is correlated with seasonal accumulation of lipid stores, shifts in activities of S-oxidative and glycolytic enzymes, and increased emphasis on lipid utilization and conservation of glucose during shivering (Dawson and Carey, 1976; Carey et al. 1978; }!arsh and Dawson, 1982; Yacoe and Dawson, 1983; Marsh and Dawson 1988a). In contrast, winter house finches exhibit only a modest improvement in thermogenic capacity and a slight increase in levels of stored lipid (Dawson et al. 1983a). Use of carbohydrate during shivering is similar in summer and winter (Marsh et al. 1984). House finches are plentiful in areas of the western United States in which cold winters are common. They have also recently extended their range throughout the Northeast and northern Midwest following their introduction into New York in 1940 (see Dawson et al. 1983a; Leek, 1987). Their persistence in cold climates is noteworthy considering their apparently limited capacity for seasonal enhancement of thermogenic endurance. In this study we attempted to extend our understanding of the mechanisms by which house finches survive cold winters by addressing the question of whether activities of catabolic enzymes in skeletal muscle vary seasonally. C. Bech et al. (eds.), Physiology of Cold Adaptation in Birds © Springer Science+Business Media New York 1989
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MATERIALS AND METHODS Enzyme Activities House finches were trapped between 0630 and 0900 hrs in December, 1987 (winter), May 1988 (spring), and July 1988 (summer), in Boulder, Boulder County, Colorado (40°01'N, 105°16'W). They were taken immediately to the laboratory where they were weighed to the nearest 0.01 g on a Mettler top-loading balance and then sacrificed by cervical dislocation. The pectoralis muscles and the muscles from both legs were rapidly removed, weighed, and minced on a cold surface. The minced leg and pectoralis muscles were then divided into four groups each. One sample of each tissue was weighed and frozen for analysis of muscle water content by lyophilization. Since water content of house finches carcasses varies seasonally (Dawson et al., 1983a), this measurement was necessary to ensure that seasonal variation in enzyme activity per gram wet mass was not caused by variation in muscle water content. The other three aliquots were weighed and placed in the appropriate buffer for analysis of activities of hexokinase (HK, EC 2.7.1.1.), phosphofructokinase (PFK, EC 2.4.1.1.), B-hydroxyacyl-CoA dehydrogenase, (ROAD, EC 1.1.1.35), and citrate synthase (CS, EC 4.1.3.7). Pectoralis and leg muscles were homogenized in 10 and 20 volumes of buffer, respectively, in hand-held glass-glass homogenizers on ice. The homogenizing buffer for analysis of HK contained 50 mM triethanolamine-HCl, 7.5 mM MgCl2, 1 mM EDTA, 100 mM glucose, and 5 mM mercaptoethanol at pH 7.5. Muscles to be analyzed for PFK, CS and ROAD activity were homogenized, sonicated and stored with methods similar to Marsh (1981). Activities of the four enzymes in the crude homogenates were measured spectrophotometrically at 340 nm (HK, PFK, ROAD) or 413 nm (CS) with a Gilford Model 250 spectrophotometer in conjunction with an Apple lie microcomputer. All assays were done at 25°C in a final volume of 1 ml. Hexokinase was assayed by a modification of the method of Crabtree and Newsholme (1972). The assay medium contained 75 mM Tris-HCl, 35 mM glucose, 10 mM MgCl2, 1 mM EDTA, 1.5 mM KCl, 5 mM mercaptoethanol, 0.4 mM NADP+, 2.5 mM ATP, 10 mM creatine phosphate, 10 units creatine phosphokinase, and 1 unit glucose-6-phosphate dehydrogenase at pH 7.5. Activities of the other enzymes were measured according to methods outlined by Marsh (1981) with the exception of the following: 1) the assay medium for ROAD contained 50 mM triethanolamine, 2) the medium for PFK contained 0.3 mM KCN, 1.5 mM NADH2, approximately 0.8 units aldolase, 0.7 units glucose-6-phosphate dehydrogenase, and 7.0 units triose phosphate isomerase, and 3) the pH of the CS assay was 8.2. Control assays were run for each of the enzymes in the absence of substrate. Duplicate assays were performed for each enzyme and the averaged results are presented as ~moles substrate used (min • g wet mass)-1. Mean values for enzyme activity of the two muscle types and other muscle characteristics are presented + S.E.M. Comparisons between averages from.the three seasons were made with one-way ANOVA. In cases where significant differences were found, the Tukey Honest Sum Difference test was used to identify homogeneous subsets of means that were statistically indistinguishable. Calculation of a regression line by the method of least-squares was used to test for a significant relation between nocturnal low ambient temperature and enzyme activity. These temperatures were provided by the U.S. National Weather Service office in Boulder.
96
Table l.
Hean (.:!:_ SEH) body mass and muscle mass from one side (g) of house finches captured in different seasons. Sample sizes are in parentheses. Results of analysis of variance for seasonal differences among means is at the bottom of each column. Pectoralis
Season
Body Hass
Winter
21.24 + 0.39
l. 75 + .042
0.25 + 0.013
Spring
20.32 + 0.29 (9)
1.73 + .032
0.29 + 0.013
Summer
20.48 + 0.37 (6)
1.75 + 0.062
0.30 + 0.002
£-ratio
2.04
0.12
3.19
.15
.89
.06
(ll)
£-probability
(il)
(9)
(6)
Leg
(9)
(9)
(6)
RESULTS Enzyme Activities Body mass, pectoralis mass and the total mass of the lower leg muscles of house finches did not vary seasonally (Table 1). The water content of the pectoralis and leg muscles averaged 69.9% and 72.0%, respectively in winter. These values did not differ significantly from values in spring (70.8% and 71.5%, respectively) or summer (71.0% and 72.2%, respectively). No significant variation in the activities of HK, PFK, or CS was evident in the pectoralis of winter, spring or summer birds (Table 2), but the activity of !lOAD in spring and summer pectoralis was significantly lower than that in winter (P < 0.001; Table 2). Activities of HK and PFK in leg muscles did not vary seasonally, but CS of leg muscle in summer was significantly reduced below activity levels in other seasons. HOAD activity in winter leg muscles was significantly higher than in the other two seasons (Table 2). In each season, HK activity levels were significantly (P < 0.01 greater in leg than in pectoralis muscle. Levels of CS, HOAD, and PFK were significantly (P < 0.01) greater in pectoralis than in leg in all seasons. No significant relations existed between enzyme activities and the nocturnal low temperature preceding the morning on which a specific bird was captured. However, levels of hexokinase activity in winter leg muscles were inversely correlated with the average low temperature on the three nights previous to the capture of the birds (Fig. 1). The slope of the regression line defining the relation between the average low of the three previous nights and hexokinase activity was significant (p= 0.008). DISCUSSION Compared to American goldfinches, house finches exhibit relatively modest seasonal increases in the capacity to produce heat under conditions of severe cold (Dawson and Carey, 1976; Dawson et al.
97
Table 2.
Mean (+ SEM) enzyme activities [~mol substrate (g wet mass. min)-1] of house finch pectoralis muscles in winter (\J), spring (Sp), and summer (S). Sample sizes are in parentheses. HK = hexokinase; PFK = phosphofructokinase; HOAD = 6-hydroxyacyl-CoA dehydrogenase; CS = citrate synthase. Results from analysis of variance for seasonal differences among the means is at the bottom of each column.
Tissue (Season)
HK
PFK
HOAD
cs
Pectoralis (W)
1.19 + 0.10 (ll)
23.1 + 1.1
19.6 + 1.2
119.7 + 4.8
Pectoralis ( Sp)
1.12 + 0.11
23.3 + 1.3
10.7 + 0.51
113.4 + 6.3
Pectoralis ( S)
0.92 + 0.07
22.4 + 2.7
12 7 + 1.0
100.6 + 1.8
f-ratio f-probability
Table 3.
(9)
(6)
(8)
(9)
(6)
1.60
0.08
.22
.93
(9)
(9)
0
(6)
27.13
(9)
(9)
(6)
2.60
.0001
.10
Mean (+ SEM) enzyme activities of house finch leg muscles in winter-(W), spring (Sp), and summer (S). Sample sizes are in parentheses. Units, symbols, f-ratios, and probabilities are the same as in Table 2.
Tissue (Season)
HK
PFK
HOAD
cs
Leg (W)
2.08 + 0.16 (ll)
9.9 + 1.2
7.8 + 0.54
49.1 + 2.7
Leg (Sp)
2.18 + 0.14
8.0 + 1.2
6.0 + 0.45
46.9 + 1.8
Leg (S)
1.72 + 0.16
9.3 + 1.8
5.5 + 0.19
36.3 + 1.3
f-ratio
1.77
0.64
6.98
f-probability
0.19
0.54
(9)
(6)
(i)
(8)
(6)
(9)
(9)
(6)
.005
(9)
(9) (6)
8.40 .002
1983a), The thermogenic response of house finches differs from that of goldfinches primarily in the ability to sustain high levels of heat production, rather than the degree of metabolic expansibility: during exposure to temperatures as low as -70 C, the peak metabolic rates of goldfinches and house finches were 5.5- and 6.4-times standard levels, respectively. Winter goldfinches remain homeothermic for more than 6-8 hr below -60 C, whereas winter house finches can tolerate similar temperatures for no more than 90 min (Dawson and Carey, 1976; Dawson et al. 1983a). The ability to increase endurance in muscular thermogenesis would have obvious survival benefits for small birds in winter, since ambient temperatures are far below thermoneutral ranges for long periods and since capacities for non-shivering thermogenesis are absent or minimal in adult birds (Marsh and Dawson, 1988b).
98
In our attempt to understand the mechanisms underlying these seasonal shifts in thermogenic endurance, it has been of interest to determine the extent to which biochemical adjustments associated with seasonal increases in thermogenic endurance reflect those made by skeletal muscle in response to endurance exercise training. Hhen mammalian muscle is subjected to aerobic endurance training, overall aerobic capacity of the muscle increases (see Holloszy and Booth, 197 6, for review). Underlying these changes are increases in activities of most mitochondrial enzymes (Holloszy and Booth, 1976). Endurance training either doesn't affect or causes small decreases in most glycolytic enzymes. However, the activity of hexokinase, the enzyme responsible for phosphorylation of glucose absorbed from the blood is augmented considerably by this training; HK activity can increase significantly after even a single bout of exercise (Lamb et al. 1969; Barnard and Peter, 1969). Mammalian muscle adapted for endurance exercise derives more of its ATP from fatty acids and less from carbohydrate than untrained muscle (Holloszy and Booth, 1976). Glucose-sparing is thought to increase endurance, since fatigue is correlated with the depletion of muscle glycogen (Newsholme and Leech, 1983). Glucose-sparing in vertebrate muscle has been thought to result from a reduction in the rate of glycolysis due to inhibition of PFK by citrate (Randle, 1981).
>f-
3.0
HOUSE FINCH Winter, leg
>
f=
u
