Flight muscle catabolism during overnight fasting in a passerine bird, Eremophila alpestris

J Comp Physiol B (1992) 162:383-392 Journal of Comparat ive ~,~,omlo,"~emlo~"

and Environ-

Physiology B mental Physiology

�9 Springer-gerlag 1992

Flight muscle catabolism during overnight fasting in a passerine bird, Eremophila Mpestris Steve D. Swain*

Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA

Accepted October 17, 1991

Summary. Brood-rearing passerine birds often have sparse lipid reserves coupled with potentially high energy demands. This may necessitate increased fasting protein catabolism; however, the largest source of protein, flight muscle, must be maintained. This problem was examined in the horned lark (Eremophila alpestris), a 28-g passerine. Overnight fasting caused significant depletion of protein in flight muscle and liver, but not in other muscle groups. Proteolytic enzyme activity of the flight muscle doubled during fasting. Biochemical and ultrastructural studies revealed that protein was depleted disproportionately from the sarcoplasm of flight muscle cells. Fast- ing caused a reduction in the protein-specific glycolytic capacity of flight muscle tissue. Oxidative capacity of the flight muscle, as measured by both in vivo and in vitro assays, was not significantly affected. The disproportionate catabolism of flight muscle sarcoplasmic protein may be due to a greater susceptibility to proteolysis, and not necessarily because it represents a source of redun- dant storage protein.

Key words: Muscle - Protein - Catabolism - Fasting - Passerine birds, Eremophila alpestris

Introduction

Protein is primarily a structural and catalytic component of animal tissue, but under certain circumstances it must also serve as a metabolic fuel (Munro 1964). Although lipid is normally a major endogenous energy store, when those reserves are inadequate protein is catabolized to provide energy (e.g., Cherel et al. 1987). Even when lipid reserves are adequate, protein may be catabolized to

Abbreviations: BMR, basal metabolic rate; GPDH, alpha- glycerophosphate dehydrogenase; mATPase, myofibrillar adenosine triphosphatase; SDH, succinate dehydrogenase; STPD, standard temperature pressure dry

*Present address and address for correspondence: 43-B Cloverview Dr., Helena, MT 59601, USA

provide gluconeogenic amino acids (Felig and Wahren 1974) or anaplerotic substrates (Lee and Davis 1979).

Protein can be catabolized from many tissues, but the largest single source is skeletal muscle, which normally constitutes 40M5 % of body mass in birds and mammals (Calder 1984). Birds are unique in that 40-60% of their skeletal muscle is contained in the large flight muscles, the pectoralis major and supracoracoideus (Greenewalt 1975). For these reasons, flight muscle is a likely source of protein for catabolism in birds.

Catabolism of avian flight muscle is seen in times of intense energy demands, such as migration (Evans 1969), moult (Baggott 1975), severe weather (Davidson 1981), and breeding (Jones and Ward 1976). Flight muscle catabolism is also common during extended periods of food deprivation in chickens (e.g., Grammeltvedt 1978). In small passerines, substantial flight muscle protein loss can occur in less than 1 day of fasting (Kendall et al. 1973; Jones 1980). Voluntary flightlessness can also cause marked flight muscle atrophy even when energy demands are not limiting (Gaunt et al. 1990).

Despite strong evidence for the occurrence of flight muscle protein degradation in fasting birds, the consequences of this phenomenon are poorly known. It is uncommon in animals that protein serves exclusively as an energy store, since its suitability as an energy storage molecule is much lower than that of lipid (Pond 1981). Therefore, one would predict that some type of functional consequence would result from even moderate protein catabolism in avian flight muscle.

The question arises whether the protein catabolism that actually occurs in wild birds results in minimal functional impairment, given that animal's environmen- tal demands; in other words, can protein catabolism in wild birds occur in an adaptive manner? For example, muscles used in essential locomotion could be spared from protein catabolism relative to other muscles. This type of preferential catabolism may occur in fasting geese (Le Maho et al. 1981).

Another possibility is that a specific subset of muscle protein is preferentially degraded, thus sparing proteins whose functions are more immediately important. Ken-

384 S.D. Swain: Flight muscle catabolism in a passerine

dall et al. (1973) have suggested that flight muscles of birds con t a in a "labile pro te in reserve" tha t is readily recrui ted for ca tabol ism, bu t tha t the deple t ion o f this p ro te in does n o t impa i r muscle funct ion .

In this study, p ro te in ca tabo l i sm resul t ing f rom short periods of fast ing is examined in a small (28-g) passerine, the ho rned lark (Eremophila alpestris). D u r i n g b rood- rear ing horned larks have marg ina l l ipid reserves (Swain 1991) and m a i n t a i n high b lood glucose concen t ra t ions even while glycogen reserves are depleted (Swain 1989). Hence, p ro te in ca tabo l i sm migh t be expected, even dur ing short overn ight fasts, to supply energy substrates and g luconeogenic precursors. However , ho rned larks are actively foraging and feeding nest l ings for 10-12 h per day dur ing b r o o d rear ing (Boyd 1976). F o r tha t reason, any deple t ion of flight muscle capabil i t ies could be det r imenta l , and any adapt ive type of p ro te in ca tabolism that spared flight performance m a y be advantageous. As is repor ted here, however, overn ight fast ing in ho rned larks causes significant flight muscle ca tabol ism. More- over, this ca tabo l i sm d i spropor t iona te ly affects flight muscle and results in changes to its s t ruc tura l and func- t ional characteristics.

Materials and methods

Animals. A total of 18 horned larks were used in this study. All birds were captured in Albany county, Wyoming, USA, during the season of brood-rearing for this population. Capture, care, and treatment of these birds was as previously described (Swain 1991). One group of ten birds was used over a 3-week period for in vivo measurement of summit metabolism. Later, those birds were sacrificed by de- capitation and tissue samples were taken for the following procedures (described below): measurement of tissue weights and protein fractions of flight muscle, histochemistry of pectoral muscle, in vitro oxidative capacity of flight muscle, in vitro glycolytic capacity of flight muscle tissue, and cathepsin D activity of flight muscle tissue. A second group of eight birds were sacrificed by pentobar- bital injection within 10 days of capture, and tissue samples taken for the following procedures: measurement of tissue weights and protein fractions of flight muscle, tissue protein concentration, and electron microscopy of pectoral muscle tissue. Birds were sampled in either the fed or the fasted state. Fed larks were sacrificed 1-3 h after having food replenished in their cages, either mid-morning or late afternoon. Fasted larks had food removed in the evening and were sacrificed 20 h later, after an extended overnight fast.

Summit metabolism. In order to assess the summit, or maximum sustainable metabolic output of these birds in vivo, measurements were made of their oxygen consumption at - 9 to - 15 ~ in an atmosphere of 80% helium and 20% oxygen after the method of Rosenmann and Morrison (1974). Under these conditions, an en- dotherm will normally attain its maximum rate of shivering thermogenesis in a short time and at temperatures that are easily achieved with normal laboratory equipment. As the flight muscles are the major contributors to shivering thermogenesis in birds (West 1965; Calder and King 1974), this test was considered to be an assessment of in vivo flight muscle metabolic capacity.

Birds were placed in a 750-ml polyethylene chamber, the bottom of which was lined with cloth to prevent the bird's feet from freez- ing. The inlet was connected to a regulated tank of 80 % He and 20 % 02. At the altitude of Laramie this amounts to an oxygen partial pressure of approximately 122 mm Hg, as against 160 mm Hg at sea level. Excurrent gas from the chamber passed through a Matheson mass flow meter (East Rutherford, N J, USA), a column of drierite, and a Selwomex 57A oxygen analyzer (Fullerton, CA, USA). The

oxygen concentration of excurrent gas and the total flow rate were recorded continually, and chamber temperature was recorded every 30 s, using a copper-constantan thermocouple. Flow rate of the He- t2 mixture was 5 1 �9 min - 1 to assure that hypoxic conditions did not occur, and also to generate a rapid response time in the 02 measurement system. The chamber was then submerged in an iso- propanol bath cooled to approximately - 30 ~ with dry ice. Cham- ber temperature dropped to -- 10 to - 15 ~ within 8-10 min, and the bird's oxygen consumption typically reached a plateau within 6 8 min. The tests were terminated after 12-14 min, and the lark was removed and warmed. The rate of oxygen consumption for each test was calculated using a N 2 bleed calibration technique (Fedak et al. 1981). Nine larks were tested four times each, twice in the fed state, and twice in the fasted state, over a period of 3 weeks.

Tissue weights and protein. Immediately upon sacrifice, the birds were quickly dissected and specific tissues were weighed to the nearest milligram. Visceral organs removed and weighed were liver, heart, brain, and kidney. The muscles of each bird were divided into five compartments: flight muscle (defined as pectoral plus supracoracoideus muscles), wing, leg, back, and neck. Some facial muscles of the birds were not removed, so total muscle mass values are slight underestimates of actual values. Tissue weights were obtained from a total of 18 birds, 9 fed and 9 fasted.

Total protein content of several tissues was estimated in fed and fasted larks. At the time of dissection, 50- to 100-mg samples &three muscle types (flight muscle, leg, and wing) and liver were frozen in liquid nitrogen and kept at - 60 ~ until analysis (within 2 weeks). The specific sites of the muscle samples used were as follows: flight muscle, from the belly of the pectoralis major; leg, mixed flexors of leg; and wing, all muscles of one wing. Tissue samples were homogenized in ice-cold distilled water by hand using a ground-glass homogenizer. The homogenates were then dissolved in four volumes of 0.3 M KOH. Aliquots of these mixtures were assayed for protein concentration using a modified Lowry procedure (Sigma Chemical Co., St. Louis, MO, USA, Procedure No. P5656) and compared to standards of bovine serum albumin. The protein concentration of each sample was then multiplied by the appropriate tissue weight (e.g., leg protein x total leg muscle mass) to obtain a value for total tissue protein of each tissue.

Cathepsin D assay. The activity of cathepsin D, a major lysosomal enzyme, was measured in flight muscle samples according to a variation of the method of Barrett (1972). Tissue samples were frozen in liquid nitrogen at the time of dissection, and kept at - 6 0 ~ until analysis (within 2 weeks). Homogenates were made of the tissue samples using the procedure of Berg and Steen (1976) by homogenizing in a solution of 0.15 M NaC1 plus 0.1% Triton X-100 using a Polytron homogenizer. Final tissue concentration of the homogenates was 0.5%. The inclusion of Triton X-100 allows for the measurement of total enzyme activity; i.e., it includes the latent fraction of cathepsin D (Bird and Carter 1980). Duplicate 0.5-ml aliquots of each homogenate were incubated for 45 rain at 40 ~ with 0_25 ml 1.0 M formate buffer (pH 3.0) and 0.25 ml 8% hemoglobin. In order to subtract endogenous peptides in the homogenate from those released by proteolytic activity, a second set of tubes was mixed with the same components at the end of the incubation period. At this time 5 ml of 3 % trichloroacetic acid was added to all the tubes to stop the reaction and precipitate proteins. The samples were filtered and peptide products of proteolysis in the filtrates were quantified using the Folin-Lowry reaction (Barrett 1972). Optical density at 700 nm was compared to standard solu- tions of tyrosine, and protein concentrations of the homogenates were determined using a modified Lowry procedure. Activities of cathepsin D were expressed as micromoles of tyrosine equivalents released per minute per milligram of non-collagen protein. Samples from ten birds, five fed and five fasted, were assayed using this procedure.

Protein fractions. A variation of the method of Helander (1957) was used to measure the content of myofibrillar, sarcoplasmic, and

S.D. Swain: Flight muscle catabolism in a passerine 385

stromal proteins. Samples of flight muscle tissue (0.50-0.75 g) were dissected from birds, frozen in liquid nitrogen, and kept at - 6 0 ~ until analysis, up to 3 months later. Samples were thawed, minced, weighed, and homogenized in ten volumes of 0.03 M potassium phosphate buffer (pH 7.4) using a Polytron homogenizer. The homogenates were kept at 4 ~ for 3 h; sarcoplasmic proteins are extracted in this low-salt buffer. The samples were then centrifuged for 20 min at 1400 xg, the supernatants decanted, and the pellets extracted a second time in low-salt buffer. The two sets of supernatants were combined as the sarcoplasmic fraction. The pellets were then suspended in a high-salt buffer (0.1 M potassium phosphate, pH 7.4, and 1.1 M KI) to extract myofibrillar proteins. The first high-salt extraction was done overnight, after which the samples were centrifuged for 20 min at 1400 x 9 and the supematants decanted. The pellets were then extracted a second time in high-salt buffer for 3 h. The two supernatants from these extractions were combined as the myofibrillar fraction. The remaining pellets were suspended in ten volumes of high-salt buffer as the stromal fraction.

The protein concentration of each fraction was analyzed using a modified Lowry procedure. Results were expressed as the percentage of the total protein that each protein type represented, and as the ratio of sarcoplasmic to myofibrillar protein.

Aliquots of the original crude homogenates were reserved for analysis of total free alpha-amino nitrogen concentration. Proteins and peptides were precipitated with an equal volume of 16 % trichloroacetic acid. The resultant supernatants were then neutralized with one half-volume of 0.7 M KOH, chilled on ice for 5 min, centrifuged for 10 min, and the supernatants were assayed for alpha-amino nitrogen by the method of Lee and Takahashi (1966).

In-vitro glycoIytic capacity. The procedure used to assess the relative glycolytic capacity of pectoral tissue in fed and fasted Horned Larks is a modification of a procedure used for ascites tumor cells by Wu and Racker (1959). Dissected pieces of pectoral tissue (approximately 0.5 g) were removed, minced, weighed and homogenized with a Polytron homogenizer in 9 ml of buffer to 1 g of tissue. The buffer contained 40 m M TRIS, 80 m M NaC1, 20 m M KC1, and 4 m M K2HPO 4, adjusted to a pH of 7.4. Quadruplicate 1-ml aliquots of each homogenate were added to Gilson respirometer flasks, and an additional I ml of buffer containing 40 m M glucose was added to each flask. Potassium hydroxide (15%, 0.2 ml) and a filter paper wick were added to the center well of each flask to absorb CO,. The flasks were connected to a Gilson differential respirometer, gassed with 100% N 2, sealed, then agitated for 10 min at 30 ~ Two flasks of each tissue were then removed, 2 ml of ice cold 0.7 N perchloric acid was added to each, and they were left on ice; these flasks served as blanks. After an additional 30 min the remaining flasks were removed, and 2 ml of cold HC104 was added to each. The contents of each flask were transferred to tubes and centrifuged for 10 rain. Aliquots of each supernatant were removed and kept at - 20 ~ for up to 1 week for analysis of lactate concentration using a Cal- biochem-Behring Rapid Lactate kit (La Jolla, CA, USA, catalog no. 869218). The amount of lactate produced by the homogenate over 30 min was calculated as the average amount of lactate in flasks incubated for the entire period, minus the average amount of lactate in the appropriate blank flasks. This correction was necessary to subtract endogenous lactate, as well as lactate produced during the equilibration period. The protein concentration of the homogenates was determined using a modified Lowry procedure. Glycolytic capacity of the tissue was expressed as nanomoles of lactate produced per hour and per milligram of non-collagen protein (under anaerobic conditions).

In-vitro oxidative capacity. In order to assess the protein-specific oxidative capacity of horned lark flight muscle tissue, an in-vitro assay of succinic oxidase activity was performed on pectoral tissue from fed and fasted birds. The method used was a modification of that described by Carey et al. (1978). Pectoral tissue samples of approximately 0.5 g were taken at the time of dissection, minced, weighed, and homogenized in 0.01 M phosphate buffer (pH 7.4) at a ratio of 29 ml buffer per gram of tissue, using a Polytron homoge-

nizer. Duplicate 0.5-ml aliquots of the homogenates were added to 2.3 ml reaction medium in glass flasks. The reaction mixture consisted of 7.5 ml 4 m M A1C13, 7.5 ml 4 m M CaC12, 12.5 ml 0.2 M phosphate buffer (pH 7.4), 2.4 ml 0.24 m M cytochrome c, 27.5 ml distilled water, and 1.01 g sodium succinate. Duplicate blank flasks were made by adding 0.5 ml 0.01 M phosphate buffer (pH 7.4), instead of tissue homogenates, to 2.3 ml of reaction mixture in separate flasks. The center wells of all flasks contained 0.2 ml 15 % KOH and a filter paper wick to absorb COz. All flasks were connected to a Gilson differential respirometer and equilibrated, open to room air, for 10 min. The system was then closed, and the volume micrometer for each flask was read every 10 min for a total of 40 min. Oxygen consumption of the homogenates was initially erratic, but was linear over the period of 10-40 min of incubation. The rates of oxygen consumption of the homogenates during this period were corrected for any non-specific volume changes that occurred in the blank flasks (always less than 1% of the homogenate oxygen consumption). Aliquots of the original homogenates were kept at - 60 ~ for up to 2 weeks, when the protein concentration of each sample was determined using a modified Lowry procedure. Oxygen consumption rates were finally expressed as microliters of 02 STPD consumed per minute per milligram of non-collagen protein.

Histochemistry. Samples of muscle approximately 5 8 mm in diam- eter and 3 4 mm thick were taken from the pectoral muscle of ten horned larks (fed and fasted) for histochemical staining of indicator enzymes. These samples were taken from the same area of the pectoral on all birds, and included superficial and deeper fibers. The tissue was trimmed and frozen in embedding medium (Fischer Histo Prep, Pittsburgh, PA, USA) by immersion in isopentane cooled with liquid nitrogen. Sections 10 ~m thick were cut from each sample and stained for three enzymes: myofibrillar adenosine triphosphatase (mATPase), succinate dehydrogenase (SDH) and alpha- glycerophosphate dehydrogenase (GPDH). Sections to be stained for SDH were air-dried for 5 rain and processed according to the method of Brumback and Leech (1984). Sections stained for G P D H were air-dried overnight and processed according to Brumback and Leech (1984). Sections stained for mATPase were air-dried overnight and subjected to alkaline preincubation (0.1 M amino-n- propanoI, pH 10.4), which selectively inactivates slow-twitch fibers so that only fast-twitch fibers are stained (Guth and Samaha 1969). The stained sections were analyzed using the following criteria: if a fiber was darkly stained using the mATPase procedure it was classified as a fast-twitch fiber; if it was not stained it was classified as a slow-twitch fiber. Fibers that were moderately to darkly stained by the SDH procedure were classified as oxidative. Fibers that were moderately to darkly stained by the G P D H procedure were classified as glycolytic. Under these criteria, each fiber was assigned a combination of the above classifications, such as "fast and glycolytic" or "fast, glycolytic, and oxidative". At least 500 fibers were visible in each section.

Electron microscopy and stereology. Pectoral muscle samples were taken from the same sites as for histochemical procedures. The muscle was cut into small strips (about 3 mm wide) and fixed in 5 % glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4) for 6 weeks at 4 ~ At that time the tissue was cut into small cubes (1-3 mm), rinsed in fresh cacodylate buffer, and post fixed for 2 h in 1% OsO4 (cacodylate buffered at pH 7.4). The tissue samples were finally rinsed, dehydrated in ascending concentrations of ethanol and propylene oxide, and embedded in Araldite.

Transverse sections were cut from the blocks at a thickness of 60 100 nm and placed on parlodion-coated 200-square-hole mesh grids. The grids were stained in uranyl acetate and lead citrate, and examined with an RCA E M U ~ C transmission electron micro- scope.

In order to have sufficient images to conduct stereological analysis (Weibel 1979) the following regimen was followed: samples from eight birds were used, four fed and four fasted. Two blocks from each bird were sectioned, and six micrographs were taken in each


section at a magnification of 4 • 100. The micrographs of each section were taken from predetermined locations on the grid to prevent bias. For stereological analysis, the micrographs were projected onto a 100-point overlay. Half of each micrograph covered the overlay so that each micrograph was sampled at a total of 200 points.

The micrographs were evaluated in the following manner: the cellular structure at each overlay point was determined and tab- ulated. The categories used were: myofibrils, mitochondria (subdivided into intermyofibrillar and subsarcolemmal), sarcoplasm (including sarcoplasmic reticulum and other electron-dense material situated between myofibrils), lipid droplets, lysosome-like structures, and "other" (mostly nuclei and sarcolemma). The formulas of Weibel (1979) were used to calculate the volume percent (volume density in Weibel's notation) of each structure, i.e., the average percent of the total muscle cell volume occupied by that structure.

Statistics. In all cases statistical comparisons were made between mean values for fed horned larks and mean values for fasted horned larks for all parameters tested. Both t-tests and non-parametric Wilcoxon rank sum tests were performed to determine whether group means were significantly different. In most cases the results of the two tests were similar. When this was not the case, the distribution of the data sets involved was examined. If any of these sets were significantly non-normally distributed, then the non- parametric test alone was used as the indicator of significance.

also significantly lower in fasted larks. The relative loss of flight muscle mass (14.87%) is similar to that of postural muscles (14.53 %), but greater than that of leg muscles (5.38%). However, the relative loss of liver mass (18.46%) is greater than that of any muscle group.

Total flight muscle protein content was significantly lower in fasted than in fed horned larks (Table 2). Al- though there was a significant decrease in the total protein content of liver and possibly in wing muscle, the absolute amounts of protein involved (26 mg and 74 mg, respectively) were small compared with the amount con- tributed by the flight muscle (238 mg). The total protein content of leg muscle was identical in fed and fasted horned larks.

Flight muscle protein also changed in a qualitative manner in fasted horned larks. The amount of sarcoplasmic protein (including soluble cytoplasmic and mitochondrial proteins), relative to the amount of myofibrillar protein, was significantly decreased in food-deprived birds (Table 3).

Ultrastructural examination revealed that fasting caused a qualitative change in flight muscle cellular structure, specifically a reduction in the volume percent of sarcoplasm (Table 4). There was no significant change in

Results

Flight muscle was the largest single contributor to mass lost during fasting in horned larks (Table 1). Masses of postural muscles (back, wing, and neck) and liver were

Table 1. Weights of selected tissues from fed and fasted horned larks

Tissue Weights (g) P

Fed Fasted

Total weight 28.504-0.95 (9) 25.694- 1.05 (9) 0.005 Total muscle 12.36• (8) 10.65• (9) 0.027 Flight muscle 7.81 • (9) 6.65• (9) 0.044 Leg muscle 1.53 • 0.060 (9) 1.45 • 0.097 (9) 0.241 Other muscles a 2.994-0.101 (8) 2.444-0.152 (9) 0.011 Liver 0.72• (9) 0.59• (9) 0.020 Brain 0.704-0.023 (9) 0.74• (9) 0.090 Heart 0.494-0.013 (9) 0.454-0.020 (9) 0.060 Kidney 0.20 • 0.005 (4) 0.20-4- 0.007 (5) 0.250

Values are means• SEM. Number in parentheses is sample size. P is the probability that fed and fasted values are equal " From wings, back, and neck

Table 2. Total protein content of selected tissues in fed and fasted horned larks

Tissue Total tissue protein (g) P

Fed Fasted

Flight muscle 2.01 • (4) 1.77• (4) 0.042 Leg muscle 0.33 • 0.014 (4) 0.33 -4- 0.027 (3) 0.480 Wing muscle 0.37 • 0.025 (4) 0.29 • 0.041 (4) 0.089 Liver 0.134-0.006 (4) 0.10• (4) 0.036

Values are means• SEM. Numbers in parentheses show sample sizes. P is the probability that fed and fasted values are equal

Table 3. Relative protein content of the major fractions of flight muscle in fed and fasted horned larks

Fraction Percentage of total protein (%) P

Fed Fasted

Sarcoplasmic 45.1 _+0.65 (9) 43.8 +0.81 (7) 0.06 Myofibrillar 53.8 _+0.65 (9) 55.3 ___0.83 (7) 0.04 Stromal 0.9 __0.16 (9) 1.2 _+0.16 (7) 0.11

Ratio % sarcoplasmic 0.84_+ 0.022 (9) 0.79_+ 0.028 (7) 0.04 % myofibrillar

Values are means _+ SEM. Numbers in parentheses are sample sizes. P is the probability that fed and fasted values are equal using a Wilcoxon rank-sum test

Table 4. Volume percent of major organelle fractions in flight muscle of fed and fasted horned larks

Organelle Volume percent (%) P

Fed Fasted

Myofibrils 53.4+ 1.49 54.2:t: 1.49 0.365

Mitochondria: Total 28.1 4- 1.56 25.64- 0.55 0.124 SS a 11.1 • 1.40 9.1 4- 1.31 0.170 IM u 17.0• 16.54- 1.34 0.380

Sarcoplasm 15.5 • 0.59 13.3 4- 0.79 0.035 Lipid droplets 0.1 • 3.24-0.45 0.010 Lysosome 0.7• 1.0• 0.127 Other 2.2• 2.7• 0.148

Values are means• SEM, expressed as the percentage of total cell volume; n= 4 for each group; P is the probability that fed and fasted values are equal a Subsarcolemmal fraction of mitochondria b Intermyofibrillar fraction of mitochondria


Fig. 1A, B. Selected electron micrographs of pectoral muscle tissue in fed (A) and fasted (B) horned larks. Visible changes in fasted tissue include less distinct intra- mitochondrial membranes (large arrow), and numerous lipid droplets (arrow- head). Also visible is a possible autophagic vacuole containing part of a myofibril (small arrow). 6150 x ; bar, 1 gm

the volume percent of myofibrils or of mitochondria (whether examined as a whole or subdivided into subsarcolemmal and intermyofibrillar fractions). An alternative method of expressing the change in muscle fiber composition is seen in Table 5. These figures represent estimates of the total volume of the cellular structures in the entire flight muscle of a bird, obtained by multiplying the

Table 5. Total organelle volume of flight muscle in fed and fasted horned larks

Organelle Total organelle volume (cm 3)

Fed Fasted P

Myofibrils 3.65+0.168 3.19i0.072 0.022 Mitochondria 1.92 + 0.087 1.51 • 0.087 0.009 Sarcoplasm 1.06 + 0.037 0.79 4- 0.063 0.005 Lipid droplets 0.01 + 0.008 0.19 4- 0.032 0.001 Lysosome 0.05 + 0.004 0.06 4- 0.012 0.224 Other 0.15=1:0.017 0.164-0.024 0.360

Values represent volume % of an organelle multiplied by total flight muscle mass, expressed as mean + SEM. n = 4 for each group; P is the probability that fed and fasted values are equal

Table 6. Activities of cathepsin D and concentration of or-amino nitrogen in the flight muscle of fed and fasted horned larks

Fed Fasted P

Cathepsin D a 13.9:t:0.54 (5) 27.0:t: 1.97 (5) 0.0001 c~-amino nitrogen b 9.6=t: 1.05 (4) 18.24- 1.60 (4) 0.002

Values are means =k SEM. Number in parentheses is sample size. P is the probability that fed and fasted values are equal " Activity expressed as nanomoles tyrosine equivalents released per minute per milligram of non-collagen protein b Concentration expressed as micromoles of ninhydrin-reactive material per gram of weight of tissue

volume percent of a given cell fraction by the flight muscle mass, and assuming a density of 1 g . cm-3. This emphasizes the point that some depletion occurs f rom all major organelle fractions (myofibrils, mitochondria, and sarcoplasm). However, the magnitude of loss was greatest f rom the sarcoplasm (which accounts for its reduced propor t ion in fasted tissue), moderate f rom mitochondria, and least f rom myofibrils.

The volume percent of lipid droplets was significantly increased in the muscle fibers of fasted horned larks. Some of these ultrastructural differences can be seen in Fig. 1. The abundance of lipid droplets is readily apparent in the fasted lark; also visible in Fig. 1 (small arrow) is a possible autophagic vacuole containing a fragment of myofibril. There is the subjective appearance of less sarcoplasmic material between adjacent myofibrils in the cells of fasted larks than in cells of fed larks, which is supported by the stereological analysis. Furthermore, the appearance of the mitochondria in micrographs of pectoral tissue f rom fasted larks is subjectively different f rom those of fed larks; the mitochondrial contents seem more amorphous and the cristae are less distinct. It is possible that the different appearance of the mitochondria of fasted birds is due to a systematic artifact of fixation. During fasting, horned larks have high circulating ketone body concentrations (Swain 1989), which may alter the pH of their intracellular fluid, and fixation with buffer o f a standard p H could lead to artifacts in those structures.

The activity of the lysosomal enzyme cathepsin D in flight muscle tissue of fasted horned larks was almost twice that of fed birds (Table 6). There is also an approximately two-fold increase in the concentration of free a-amino nitrogen in the flight muscle tissue of fasted birds, as compared to fed larks (Table 6).

In vivo summit metabolism, as measured through max imum shivering thermogenesis, was not significantly


Table 7. Metabolic assessments of fed and fasted horned larks at the whole animal and tissue (flight muscle) levels

Fed Fasted P

Summit metabolism a 23.7_+ 0.37 (9) (whole animal)

In vitro oxidative 1.3___ 0.06 (5) capacity 2

In vitro glycolytic 124.0 _+ 20.22 (5) capacity c

22.6_+ 0.50 (9) 0.10

1.4_+ 0.17 (4) 0.30

53.6_+23.86 (5) 0.03

All values are means__+ SEM. Number in parentheses is sample size. The P value is the probability that fed and fasted values are equal a Expressed as millilitres Oz per gram body mass per hour b Expressed as microlitres 02 (STPD) per milligram non-collagen protein per minute c Expressed as nanomoles of lactate produced per milligram of non-collagen protein per hour

different between fed and fasted horned larks (Table 7). The metabolic rates expressed here represent an ap- proximate 9.4-fold increase over basal metabolic rate for these birds (Swain 1991). This figure is slightly lower than reported values for the aerobic scope of birds (Bar- tholomew 1982); therefore, these values may un- derestimate the maximum sustained oxygen consumption for these birds.

There was no significant difference in succinic oxidase activity between flight muscle tissue of fed and fasted horned larks (Table 7). Because these activities are expressed in a protein-specific form, the indication is that the loss of protein that occurs during fasting does not have a disproportionate effect on the oxidative machin- ery of the muscle tissue.

The rate of protein-specific lactate production by crude flight muscle homogenates under anaerobic conditions was significantly higher in tissue from fed horned larks (Table 7). These results indicate that glycolytic capacity of flight muscle was disproportionately reduced in fasted larks.

Histochemical staining revealed a homogeneity of muscle fiber types in the pectoral muscle of horned larks, according to the standard criteria outlined above. All fibers were darkly stained with the alkaline preincubation mATPase procedure, darkly stained for the SDH reaction, and moderately stained for the GPDH reaction. Therefore, all fibers were classified as fast-oxidative- glycolytic fibers.

Discussion

Catabolism of flight muscle. It is apparent that flight muscle protein is not spared from catabolism during fasting in horned larks, despite the importance of this muscle in locomotion. Indeed, its contribution to protein catabolism during fasting was proportionately greater than that of any other tissues. It was surprising that other groups of muscle, such as leg and postural muscles, did not contribute significant amounts of protein during the

same fasting period over which flight muscle lost 12% of its original protein content. Visceral tissue, as represented by liver, also made a significant contribution to protein depleted during fasting. This is similar to the response seen during fasting in rats (Addis et al. 1936). Other tissues, such as reproductive organs (e.g., Gram- meltvedt 1978) and the digestive tract, may undergo protein catabolism during fasting in birds, but these were not measured in this study.

The rates of mass and protein loss of horned larks during fasting are similar to, or greater than, those seen in other birds. For example, over a 4-day fast the loss of flight muscle mass in willow grouse constituted 34 % of total mass lost (Grammeltvedt 1978), whereas in horned larks loss of flight muscle mass over a 20-h fast was 41% of the total mass lost. However, the rate of protein catabolism in larger birds changes over the course of fasting; it is initially high, decreases as protein-sparing mechanisms take effect, then increases as fat reserves become depleted (Le Maho et al. 1981). These phases may not be as distinct in the horned lark, because of its low fat reserves, short fasting survival time, and rapid onset of protein-sparing mechanisms (Swain 1989, 1991). A more appropriate comparison can be made with a bird of similar size, the house sparrow. During an overnight fast, the flight muscles of house sparrows lost lean dry material at the rate of 12.5 mg �9 h -1 (Jones 1980). This is almost identical to the rate of 11.9 mg. h- 1 in horned larks, especially since a portion of the lean dry mass is glycogen.

The energy content of the protein lost by the flight muscle of horned larks during fasting is considerable. If protein is oxidized completely to uric acid, the energy available from an average 0.238 g of protein would be 4.28" 103 J. Using the conservative estimate that these birds would be operating at their basal metabolic rate over 20 h of fasting, this could supply roughly 15% of their energetic needs during that period. However, it is unlikely that all the degraded protein is oxidized completely. The maintenance of high blood glucose levels in these birds (Swain 1989) would create a demand for gluconeogenesis, perhaps from amino acids (Langslow 1978). Amino acids from muscle proteins could also be used for the synthesis of other essential proteins [discussed in King and Murphy (1985)], or for the anaplerotic generation of citric acid cycle intermediates (Lee and Davis 1979). So, while the uses of protein catabolized from horned lark flight muscle are probably varied, it is likely that this material is important in fuel homeostasis during fasting in these birds.

One benefit of protein catabolism that is often over- looked is the liberation of bound water. For some mammals that are both fasted and water-deprived, this protein-associated water is believed to be crucial to the animal's overall water balance (e.g., Bintz and Strand 1983). During overnight fasting, it is likely that horned larks are also water-deprived, at least during darkness. Therefore, besides supplying caloric needs and gluconeogenic precursors, protein catabolism may also have a role in supplying water during the period of overnight fasting in horned larks.


Proteolytic activity in flight muscle tissue. The increase in cathepsin D activity and the elevated concentration of free amino acids in flight muscle tissue of fasted horned larks provide evidence that proteolytic activity increases in this tissue during fasting. Cathepsin D is a proteolytic enzyme of primarily lysosomal origin that is implicated in the degradation of both sarcoplasmic and myofibrillar proteins of muscle (Bird and Carter 1980). An increase in muscle cathepsin D activity coincides with increased protein degradation in many instances in mammals, such as fasting, diabetes, denervation [review: Bird and Carter (1980)], and after exhaustive exercise (Vihko et al. 1978). Free amino acids, as the products of proteolysis, also increase concomitantly with increased levels of proteolytic enzymes, such as under conditions of exhaustive exercise (Mohan et al. 1985).

The high absolute values of cathepsin D in the flight muscle of horned larks, even in the fed state, are surprising. Mammalian muscle normally has low activities of lysosomal enzymes compared with other tissues (Bird and Carter 1980). Horned lark flight muscle cathepsin D activities are approximately 10-20 times those reported for several mammals (Pearson and Kar 1979). Part of this discrepancy may be due to the assay technique; i.e., whether or not it is measuring total cathepsin D activity, such as after treatment with Triton X-100. Li et al. (1979) report that the total activity of cathepsin D in two rat muscles (psoas and gastrocnemius) is 5-10 times the activity measured without detergent treatment. Horned lark flight muscle cathepsin D activities are within the range of other bird species; they are only about 70 % of that seen in pectoral tissue of the willow ptarmigan (Berg and Steen 1976) but approximately three times those of chicken pectoral muscle. In general, it would appear that horned larks have a high capacity for proteolysis in their flight muscles, which may allow for the rapid recruitment of protein from this tissue under fasting conditions.

Origins of protein lost from flight muscle. Although myofibrils, mitochondria, and sarcoplasm all showed some decrease in absolute amounts of protein, sarcoplasm was disproportionately depleted, to the extent that it occupied significantly less of the relative volume of the muscle cells. This pattern of protein loss is similar to that seen in the red-billed quelea (Kendall et al. 1973). Some mammals may also exhibit this type of protein loss; rats that have undergone short-term exhaustive exercise undergo disproportionate depletion of sarcoplasmic proteins (Mohan et al. 1985). Food-deprived rats also show a greater relative loss of sarcoplasmic proteins, but only during the initial stage of food deprivation; in longer- term fasting loss of sarcoplasmic and myofibrillar proteins is roughly equal (Millward 1970). This is probably due to an increase in the rate of myofibrillar protein degradation, perhaps to maintain a stable proportion of these two components (Millward 1970; Bates and Mill- ward 1983). It is not known if avian species undergo these changes of muscle protein catabolism during fasting. House sparrows exhibit a loss of pectoral protein during the period of egg laying, but in this instance the myofibrillar fraction is disproportionately depleted (Jones

1991). However, the birds in that study were captured and sacrificed in the late afternoon and had presumably been eating. The protein loss in those birds may represent catabolism of specific proteins to supply specific needs of egg production. What this suggests is that catabolic processes in avian flight muscle may be differentially regulated according to nutritional demands.

The specific identity of the sarcoplasmic proteins that are lost from avian flight muscle during fasting is not clear. Kendall et al. (1973) suggest that myoglobin is a component. A possibility suggested by this study is that glycolytic enzymes in the sarcoplasm are degraded (discussed below). More elaborate biochemical studies of flight muscle samples from fed and fasted birds would yield valuable clues to the identity of the proteins that are depleted from this tissue during fasting.

Although their relative composition in muscle cells remained the same after fasting, the absolute volume of mitochondria was decreased, and possibly their ultrastructural appearance. The fact that the decrease in the absolute volume of mitochondria was less than sarcoplasm but more than myofibrils suggests that they are intermediate in susceptibility to catabolism at this level of fasting. Should fasting of greater intensity be encoun- tered, it is possible that the relative proportion of mitochondria would be significantly reduced. Although not directly related to protein catabolism, an increase was seen in the volume percent of lipid droplets in the pectoral tissue of fasted horned larks, as seen in other avian species. Vallyathan et al. (1970) report an increase in both intercellular and intracellular lipid in the pectoral tissue of fasted pigeons. They attribute this to the possibility that mobilization of lipid from adipose tissue is proceeding at a greater rate than it is being oxidized by the muscle.

Consequences of flight muscle catabolism. The question remains as to the consequences of protein catabolism in the flight muscle of these birds. At a gross level, loss of flight muscle mass may result in decreased maximum lift production if mass is lost from the flight muscle at a greater rate than from the body as a whole, thus reducing the ratio of flight muscle mass to body mass (Marden 1987). This was the case for horned larks, although the decrease in flight muscle ratio was very small (0.273-0.257). This type of decrement of flight performance would be most evident in rapid takeoffs, and so may have some bearing on a bird's ability to escape predators. There is some empirical evidence that this occurs; woodpigeons that were caught by goshawks, usually while flushing from vegetation, had significantly lower supracoracoideus muscle dry masses than a ran- dom sample of woodpigeons from the same site (Ken- ward 1978).

If the types of protein in flight muscle tissue were not uniformly degraded during fasting (which seems to be the case in horned larks), then it is possible that certain functions of the muscle tissue will be disproportionately affected by that protein catabolism. As the sarcoplasm is disproportionately degraded, followed next by mitochondria, and the myofibrils are least affected, then


the ratio of mitochondria to myofibrils could decrease. Pennycuick and Rezende (1984) have theorized that the capacity for sustained power output of a muscle dimin- ishes as this ratio decreases. In theory, continued catabolism, as described here, would result in birds becoming increasingly limited to very short bursts of flight. This is supported by anecdotal observations of starving birds in the wild that fly short distances when flushed, but are rapidly exhausted (e.g., Spencer 1980).

The possibility of more specific consequences of flight muscle catabolism was examined in this study by measuring the protein-specific oxidative and glycolytic capaci- ties of flight muscle from fed and fasted birds. Oxidative capacity of the flight muscle in horned larks was not significantly reduced during fasting, as indicated by both in vivo and in vitro measurements. It should be noted that the in vivo test of summit Oz metabolism may not accurately reflect the total oxidative capacity of the horned lark flight muscle. Marsh and Dawson (1989) point out that the type of isometric muscular contrac- tions occurring during shivering thermogenesis may be limiting blood flow as compared to flight conditions, so O2 delivery may not be optimal under these conditions. In fact, these authors reviewed measurements of maximum O2 consumption as tested by cold exposure and concluded that those values are lower than measured and/or predicted 02 consumption during flight. How- ever, the elevation of O2 consumption (relative to BMR) was higher in this study (9.4-fold) than those reviewed for other passerines (4.7- to 6.4-fold) by Marsh and Dawson (1989). The fact that both in vivo and in vitro estimates of maximal Oz consumption and the relative fraction of muscle mitochondria are unchanged after this fasting period suggests that oxidative capacity is not disproportionately decreased by moderate protein catabolism.

However, the glycolytic capacity of flight muscle tissue does seem to be disproportionately reduced during fasting. This may be due to metabolic adjustments occurring within the muscle during fasting, such as those mediated by changes in the concentrations of allosteric modifiers (Rennie and Edwards 1981). Oxidative muscle of mammals is known to decrease its use of carbohydrate during fasting (Issad et al. 1987). An alternative possibility is that glycolytic enzymes are among the proteins that are being catabolized within the sarcoplasm. Both decreased activities of glycolytic enzymes (Moon and John- ston 1980) and disruption of the binding of these enzymes to myofibrillar components (Lowery et al. 1987) occur in fasting fish. In either case, if glycolysis is im- paired in horned lark flight muscle during fasting then it is conceivable that flight performance will be affected in some way. Lipid is the major fuel of sustained flight in birds (Rothe et al. 1987); however, carbohydrate is necessary to fuel flight muscle at times of very high power output (e.g., Suarez et al. 1986).

How the metabolic changes described above would translate to changes in flight capabilities is not totally clear. However, it seems, for reasons discussed above, that burst-flight capabilities would suffer most with moderate protein catabolism, and sustainable power output would be decreased as catabolism continued. For a

ground-foraging bird such as the horned lark, while foraging ability may not be greatly affected the ability to escape predators could be compromised.

Is protein catabolism in avian flight muscle adaptive? There are some compelling arguments for suggesting that flight muscle degradation occurs in an adaptive manner. For example, salmon lose protein from white muscle to a greater extent than from red muscle during prolonged periods of food deprivation (Mommsen et al. 1980). This has been interpreted as adaptive in that the white muscle is only used for high-speed burst-swimming, so that its depletion spares the red muscle used for normal locomotion (Lowery et al. 1987). While that is a valid argument, it is difficult to propose that preferential loss of flight muscle protein in a small bird could be adaptive. In small passerines the pectoral muscle is homogeneous with re- spect to fiber type (Rosser and George 1986), and the same muscle type must supply both burst activity and sustained locomotion. It could be proposed, for the sake of argument, that the depletion of flight muscle protein in horned larks is sparing protein in the leg muscles, since horned larks are ground-foragers (Pickwell 1931).

A more reasonable explanation at this point is that protein is more readily recruited from the flight muscle of birds because the flight muscle is better equipped mechanistically to supply it. Flight muscles that have uniformly red fibers have higher metabolic capacity (as indicated by activities of key enzymes) than leg muscles of mixed fiber type (Kiessting 1977). The flight muscles of horned larks (this study) and willow ptarmigan (Berg and Steen 1976) have relatively high levels of proteolytic enzymes. Finally, red muscles typically have higher turnover rates than white or mixed muscle in mammals (Mill- ward 1970) and chickens (Laurent et al. 1978). For these reasons, it is likely that the flight muscle would be more readily responsive to increased catabolic demands. A comparative study using a variety of bird species with different demands on flying and walking might be of interest. If the contribution to protein catabolism, and the proteolytic potential of various muscle were inversely related to the importance of that muscle group in the activity of that species, then support would be given to the idea that protein catabolism of different muscle groups is adaptive in birds.

The same type of questions can be asked about avian muscle protein catabolism at the cellular level. The im- plication of Kendall et al. (1973)is that a labile protein reserve would be adaptive in that it is easily recruited and its depletion does not impair muscle performance. How- ever, in horned larks there is evidence that depiction of sarcoplasmic proteins may impair glycolytic capacity of the flight muscle. The lability of this type of protein may simply reflect the proteolytic mechanism of muscle tissue, as already discussed. If the depletion of a labile protein has no functional consequence then that would imply that it either has no important function in muscle cells, or that it is stored in surplus quantities. However, in some cases the storage of soluble protein in muscle cells could result in volume changes which would affect 02 flow into the cell and represent a constraint on muscle


funct ion . K e n d a l l et al. (1973) suggest t ha t the s to rage p r o t e in m a y be m y o g l o b i n . In tha t case, the decrease in Oz c o n d u c t a n c e caused b y vo lume increase due to p ro - tein s to rage m a y be offset b y the abi l i ty o f m y o g l o b i n to increase the Oz u p t a k e o f the cell. However , there is no t enough i n f o r m a t i o n at this t ime to accept o r reject this poss ibi l i ty .

In s u m m a r y , the f l ight musc le o f h o r n e d la rks is the m a j o r c o n t r i b u t o r o f p ro t e in ca t abo l i zed dur ing fast ing. This is due in p a r t to an increase o f the a l r e ad y high p ro t eo ly t i c ac t iv i ty o f this tissue. Sa rcop la smic and perhaps m i t o c h o n d r i a l p ro t e ins a re dep le ted to a grea ter ex ten t t han myof ib r i l l a r pro te ins . This p ro t e in dep le t ion is a c c o m p a n i e d b y dec reased glycolyt ic , b u t no t ox ida - tive, c apac i ty o f the fl ight muscle. The p a t t e r n o f p ro t e in ca t a bo l i sm tha t is seen in these b i rds is p r o b a b l y a reflec- t ion o f the mechan i s t i c capabi l i t i es o f the tissue, and is n o t necessar i ly a d a p t i v e in na ture . I t is a lso ev ident t ha t even t h o u g h h o r n e d la rks e m p l o y r a p i d i m p l e m e n t a t i o n o f p r o t e i n - s p a r i n g mechan i sms du r ing fas t ing (Swain 1989), these mechan i sms are no t sufficient to e l iminate the need for p ro t e in ca t abo l i sm.

Acknowledgements. This work represents part of a dissertation submitted in partial fulfillment of the requirement for the degree of Ph.D. at the University of Wyoming. Financial support for part of this work came from the Department of Zoology and Physiolbgy at the University of Wyoming. I am indebted to the following people for their help and advice with this project: S.L. Lindstedt, H. Harlow, W. Gern, R. Lewis, S. Anderson, R. Jenkins, D. Bagby, D.J. Wells, J. Hokanson, and L. Swain.

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Flight muscle catabolism during overnight fasting in a passerine bird, Eremophila alpestris