Hibernating black bears (Ursus americanus) experience skeletal muscle protein balance during winter anorexia

Comparative Biochemistry and Physiology, Part B 147 (2007) 20 – 28 www.elsevier.com/locate/cbpb

Hibernating black bears (Ursus americanus) experience skeletal muscle protein balance during winter anorexia T.D. Lohuis a,⁎, H.J. Harlow a , T.D.I. Beck b a

Deparment of Zoology and Physiology, University of Wyoming, Laramie, WY, USA 82071 b Colorado Division of Wildlife, Fort Collins, CO, USA 80536

Received 10 June 2005; received in revised form 4 October 2005; accepted 5 December 2006 Available online 30 January 2007

Abstract Black bears spend four to seven months every winter confined to their den and anorexic. Despite potential for skeletal muscle atrophy and protein loss, bears appear to retain muscle integrity throughout winter dormancy. Other authors have suggested that bears are capable of net protein anabolism during this time. The present study was performed to test this hypothesis by directly measuring skeletal muscle protein metabolism during the summer, as well as early and late hibernation periods. Muscle biopsies were taken from the vastus lateralis of six free-ranging bears in the summer, and from six others early in hibernation and again in late winter. Protein synthesis and breakdown were measured on biopsies using 14 C-phenylalanine as a tracer. Muscle protein, nitrogen, and nucleic acid content, as well as nitrogen stable isotope enrichment, were also measured. Protein synthesis was greater than breakdown in summer bears, suggesting that they accumulate muscle protein during periods of seasonal food availability. Protein synthesis and breakdown were both lower in winter compared to summer but were equal during both early and late denning, indicating that bears are in protein balance during hibernation. Protein and nitrogen content, nucleic acid, and stable isotope enrichment measurements of the biopsies support this conclusion. © 2007 Elsevier Inc. All rights reserved. Keywords: Bears; Hibernation; Fasting; Immobility; Protein synthesis and breakdown; Protein sparing

1. Introduction Inactivity due to confinement, or starvation, usually results in loss of skeletal muscle and whole body protein. In most cases, muscle function is compromised as a result of protein loss (Gamrin et al., 1998). The American black bear (Ursus americanus) may be an exception to this paradigm. The black bear spends four to seven months each winter hibernating in its den (Beck, 1991) without eating, drinking, urinating or defecating (Nelson, 1973; Nelson et al., 1975). However, if disturbed, the hibernating bear is capable of a rapid response and surprisingly high mobility with sustained walking and even running through heavy snow. This locomotor activity would simply not be possible if the bear had the same profile of skeletal ⁎ Corresponding author. Current address: Kenai Moose Research Center, Alaska Department of Fish and Game, Suite B, 43961 K-Beach Road, Soldotna AK 99669, USA. Tel.: +1 907 260 2927; fax: +1 907 262 4709. E-mail address: [email protected] (T.D. Lohuis). 1096-4959/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2006.12.020

muscle loss that usually accompanies prolonged inactivity and starvation. The bear relies on endogenous lipid reserves for its major energy requirements (Nelson, 1973; Nelson et al., 1983; LeBlanc et al., 2001; Harlow et al., 2002), but there is a metabolic need for protein catabolism to provide water, Krebs cycle intermediates, and gluconeogenic amino acids (Bintz et al., 1979; Barboza et al., 1997; Harlow et al., 2002). In spite of this, the net loss of protein by overwintering bears is far below the predicted value based upon body mass, temperature, and metabolic demands (Koebel et al., 1991; Tinker et al., 1998, Barboza et al., 1997). Hibernating bears are an apparent contrast to both immobilized humans and small mammal hibernators. For example, in humans, loss of skeletal muscle mass and nitrogen continues over longterm confinement by enforced bedrest, with a concomitant decrease in strength, even on a nutritionally adequate diet (LeBlanc et al., 1992; Alkner and Tesch, 2004). Similarly, hibernating small mammals including golden-mantled ground squirrels (Spermophilus lateralis) and big brown bats (Eptesicus

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fuscus) lose between 20 and 40% of muscle protein content (Yacoe, 1983; Steffen et al., 1991; Wickler et al., 1991), and show elevated levels of protein metabolites including urea (Kristofferson, 1963; Yacoe, 1983). On the other hand, hibernating bears have been shown to lose only 4–11% of muscle protein content (Tinker et al., 1998) and do not exhibit elevated plasma levels of urea or ammonia (Nelson et al., 1975; Barboza et al., 1997). This may be a result of the hibernating bear's ability to hydrolyze almost 100% of the urea produced from protein catabolism (Barboza et al., 1997). It is believed that, in hibernating bears, urea, and thus nitrogen, is recycled and resynthesized into skeletal muscle and other body proteins (Barboza et al., 1997; Hissa et al., 1998), which may contribute to their protein sparing ability. Knowledge of the changes in the rates of skeletal muscle protein synthesis and breakdown that accompany hibernation is critical to understanding the metabolic strategy used by the bear to maintain skeletal muscle protein, and therefore strength, during several months of winter confinement and anorexia. Here we encounter a primary question: What changes occur in skeletal muscle protein metabolism as the bear transitions from the summer active period through early and then late hibernation? The results from previous research are contradictory. Lundberg et al. (1976) reported that rates of whole-body protein synthesis and breakdown were elevated 3- to 5-fold in hibernating bears compared to bears tested prior to hibernation. It has also been suggested that de novo synthesis of essential amino acids and overall net protein anabolism is possible by the hibernating bear (Wolfe et al., 1982a,b; Ahlquist et al., 1984; Nelson, 1987, 1989). And, Koebel et al. (1991) concluded that skeletal muscle protein synthesis might be elevated in hibernating bears relative to active animals. However, several studies contradict this assertion. For example, the work by Hissa et al. (1994;1998) demonstrated that hibernating European brown bears (U. arctos) display changes in plasma levels of essential amino acids and protein metabolites indicative of protein catabolism during hibernation, and suggested that elevation of essential amino acids could be due to muscle or connective tissue breakdown rather than de novo synthesis. Additionally, hibernating bears have been reported to have a moderate loss of lean body mass and skeletal muscle protein over the winter hibernation period (Farley and Robbins, 1995; Barboza et al., 1997; Tinker et al., 1998; Harlow et al., 2002), perhaps as a result of elevated whole-body protein catabolism without simultaneous increase in protein synthesis (Barboza et al., 1997). Since confusion exists as to the direction and magnitude of muscle protein turnover, and to the sources of nitrogen for protein synthesis in the hibernating bear without an exogenous nitrogen source during winter denning, we designed this study to measure rates of protein synthesis and breakdown in muscle biopsies taken from free-ranging bears during their summer active period, and also in early and late winter. This study was conducted by making in vitro measurements of protein synthesis and breakdown using 14C phenylalanine as a tracer in concert with measurement of muscle biopsy protein, nitrogen, RNA and DNA content, and nitrogen stable isotope enrichment, which are accepted as indices of protein metabo-

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lism. The ultimate questions addressed in this study were whether protein metabolism in hibernating black bears is characteristic of other mammalian models for muscle atrophy, long-term fasting, protein limitation, or malnutrition, and how specific changes in the rates of muscle protein synthesis and breakdown would affect protein conservation, ultimately affecting muscle function in the hibernating bear. 2. Materials and methods 2.1. Bear capture and handling Six adult bears (U. americanus) (5 female, 1 male) were captured in Middle Park, Colorado (105W 59′ × 40N 05′; 2580– 3550 M elevation) during summer 1999 in 1 × 1 × 2 m woven metal box traps with a spring door activated by a foot treadle. Bears were determined to be adult based on degree of tooth wear and staining. These bears were anesthetized with 7.0 mg/kg tiletamine-zolezapem (Telazol®) administered with a 1 m spring-operated jab pole. Juvenile bears were released without being anesthetized. After anesthesia, bears were placed on a Therm-a rest® pad and their rectal body temperature continuously monitored with a digital thermometer. Radio telemetry collars (ATS, Isanti, MN, USA) were placed around the neck of each animal. Bears were located and sampled in their dens in early winter just after den entry (median date November 28) and again in late winter (median date March 15), just before their anticipated emergence. The mean interval between den visits was 110 days. After early winter sampling, two (both female) of the original six bears left their dens and were unable to be located. Thus, we substituted two bears (one female, one male) of comparable age and body mass that had not been previously sampled as part of the late winter sample set. All bears were anesthetized in their dens using 7.0 mg/kg tiletamine-zolezapem (Telazol®) administered by a 1 m spring operated jab pole. After anesthesia, bears were removed from their den, placed on a Therm-a rest® pad, covered with a wool blanket to prevent abnormal hypothermia, and muscle biopsies taken. According to the original study design, bears were fitted with remote capture collars (ATS) after sampling in late winter of 2000. We had anticipated recapturing and resampling these bears during June and July of 2000, allowing a comparison of data from the same bears for all three seasons. However, the capture collars did not operate and a target trapping effort was made. As a result, six different bears (1 female, 5 males) were trapped which represented our summer data set for comparison with the early and late winter hibernating groups. While the original intent was to repeatedly sample the same bears three times during the year, we were unable to do so. However, studies on protein synthesis and muscle function in humans do not differentiate between adult male and female subjects and pool data from both sexes (Hortobagyi et al., 2000; Rasmussen et al., 2000; Tipton et al., 2003). Therefore, for the purposes of our investigation, we assumed that muscle protein metabolism and muscle function would not be substantially different between male and female subjects.

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2.2. Muscle biopsy and surgical procedures An area of approximately 8 cm × 10 cm was shaved over the vastus lateralis of the quadriceps, and sterilized using Betadine®. A sterile drape was placed over the bear's leg and a muscle biopsy (approximately 1.0 g) was removed from the vastus lateralis of the quadriceps using aseptic surgical techniques. At the conclusion of surgery, a topical antibiotic powder (Nitrofurazone NFZ puffer®, AgriLabs, St. Joseph, MO, USA) was applied, and the incision sutured with Braun #0 non-absorbable suture. The biopsy was cut longitudinally in an effort to maximize the number of complete muscle fibers. Approximately half the biopsy was placed into a heated (22 °C) oxygenated (95% O2, 5% CO2, premixed gas bottle, Great Western Airgas, Cheyenne, WY, USA) pH 7.4 Krebs–Henseleit buffer (Sigma) in a custom-built tissue transport incubator (UW machine and electrical shops) with electric power supplied by a 12 V deep cycle battery. The incubator was built on a backpack frame for transport of the live tissue from the den to the field laboratory for in vitro measurement of protein synthesis and breakdown. Return to the field lab took varying lengths of time, which were always less than 2 h. However, all samples were tested at 2 h after removal from the bear in order to maintain consistency of comparison within bears and between seasons. The other half of the biopsy was immediately placed into a portable liquid nitrogen dewar at the den site for transport to the field lab, where it was placed in a large 35 L dewar of liquid nitrogen for transport back to the University of Wyoming for analysis of protein content, nitrogen content, RNA: DNA ratios, and stable isotope analysis. All analyses were performed in triplicate to ensure consistency. After surgery, hibernating bears were placed back into their den and the entrance covered with pine boughs and snow in an attempt to encourage them to remain in their original den sites. Summer bears were placed in a sternal recumbent position and monitored until they aroused. All handling and surgical procedures were approved by the Colorado Division of Wildlife and University of Wyoming animal care and use committees. 2.3. In vitro measurement of protein synthesis and breakdown Once at the field lab, the biopsy was sectioned into several muscle bundles and cleaned of connective tissue using a dissecting microscope and micro dissection tools. Single muscle bundles of equivalent size (approximately 0.5 mm in diameter and 2.0 cm in length) were obtained from each biopsy in order to ensure consistent nutrient supply to and oxygenation of tissue to facilitate comparison from different biopsies. The small cross-sectional diameter allowed substantial surface area to volume for uniform perfusion of oxygen and nutrients supplied via the incubation medium. We were able to verify that these muscle strips maintained viability because strips from the same biopsy maintained under identical condition were used in a simultaneous companion study (Lohuis et al. 2007) on muscle contractile strength. In all cases, these strips maintained contractility and viability for more than 4 h after removal from the bear. The cut ends of muscle strips have been shown to

reseal and reestablish membrane potential (Lehmann-Horn and Iaizzo, 1989) after a period of 2 h (our standard incubation interval), which ensured that the interior of the muscle cells containing protein and free amino acids would not come into contact with the incubation medium. Protein synthesis and degradation were measured simultaneously (Waalkes and Udenfriend, 1957; modified by Fulks et al., 1975 and further by Tischler et al., 1982; Tawa and Goldberg, 1992; Dardevet et al., 1995). Muscle tissue does not synthesize or break down tyrosine, but tyrosine is released when it is degraded. Protein breakdown was determined by measuring tyrosine released from the muscle biopsy. The rate of protein synthesis was expressed in tyrosine equivalents, calculated as a multiple of the amount of 14-C labeled phenylalanine incorporated per milligram tissue per hour following the method used by Tischler et al. (1982); Tawa and Goldberg (1992), and Dardevet et al. (1995). The muscle sample was preincubated for 30 min at 34 °C (the average deep core body temperature of hibernating bears) in 10 mL of oxygenated (95% O2, 5% CO2) Krebs–Henseleit buffer. This buffer was pH buffered to 7.4 and contained 120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl, 1.2 mM KH2PO4, and 1.2 mM MgSO4, 5.0 mM glucose, 5.0 mM HEPES, 0.1% BSA, 0.17 mM leucine, 0.20 mM valine, and 0.10 mM isoleucine. After preincubation, the muscle sample was transferred to a vial of fresh, oxygenated Krebs–Henseleit buffer at 34 °C containing the same concentrations of NaCl, KCl, NaHCO3, CaCl, KH2PO4, and MgSO4, and glucose, HEPES buffer, BSA, and leucine, valine, and isoleucine as the preincubation medium, but also containing 15.0 μmol 14-C labeled phenylalanine (New England Nuclear, NEC-284E, lot #3380067, 505 mCi/mmol) as a tracer. After 60 min incubation time, the vial was boiled for 4 min to kill the tissue and halt phenylalanine uptake and tyrosine release. After boiling, the vial and contents were frozen for return to the laboratory at the University of Wyoming. Upon return, the tissue was thawed, removed from the vial, and blotted dry, then freeze-dried and weighed to 0.001 mg (Sartorius M2P, Gottingen, Germany). The tissue was homogenized and washed 3 times in 0.5 mL 10% TCA, then solubilized in 4.0 mL BTS-450 quaternary ammonium base tissue solubilizer. A cocktail of 1.0 mL solubilized tissue homogenate and 4.0 mL Beckman Ready Organic scintillation fluid was mixed for determination of labeled phenylalanine uptake on a Beckman LS-5000 liquid scintillation counter. No evidence of chemiluminescence was found using a cocktail of 1.0 mL BTS 450 and 4.0 mL Beckman Ready Organic scintillation cocktail. Quench correction was conducted using a six-point quench curve and a fourth-order polynomial regression equation. Tyrosine release was measured spectrofluorometrically from an aliquot of the incubation solution, which initially contained no tyrosine. Thus, all tyrosine in solution was released from the muscle tissue and as such was representative of protein breakdown. Protein synthesis and breakdown were expressed in nanomoles of tyrosine per mg tissue per hour using the methods used by Tischler et al. (1982), Tawa and Goldberg (1992), and Dardevet et al. (1995).

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2.4. Skeletal muscle protein concentration A section of the frozen muscle biopsy (≅100 mg wet mass) was freeze-dried and ground under liquid nitrogen. Dry samples were weighed to .001 mg (Sartorius M2P). Protein concentration of each muscle biopsy was measured using a modified Bradford colorimetric assay of peptide bonds (Bio-Rad laboratories, Hercules, CA, USA). Total protein concentration was expressed as μg protein/mg dry mass of tissue. 2.5. Skeletal muscle nitrogen content A small piece (≅50 mg wet mass) of the muscle biopsy was freeze-dried and ground under liquid nitrogen. Dry samples were weighed to 0.001 mg and packaged in aluminum foil. Total nitrogen content was assessed using a Fisons CHNS analyzer model EA 1108, and expressed as percent nitrogen per dry mass of muscle.

Fig. 2. Changes in skeletal muscle protein concentration (μg/mg− 1 dry mass) measured by the Bio-Rad assay of peptide bonds from summer (dotted bar) compared to early denning (bar with horizontal marks) and late denning (bar with vertical marks). An asterisk depicts a significantly greater protein concentration compared to biopsies from denning bears. Values are means of six bears. Vertical lines represent ± one standard error.

2.6. Nucleic acid assays RNA and DNA were extracted and purified from a ≅50 mg wet mass portion of muscle biopsy using TRI-REAGENT® (Molecular Research Center, Cincinnati, OH, USA). Nucleic acids were quantified and tested for contamination spectrophotometrically, and results were expressed in μg nucleic acid/mg dry mass of tissue (Ausubel et al., 1990; Chomczynski, 1993; TRI-REAGENT® protocol, 1995). The TRI-REAGENT® protocol precludes the use of dry tissue to extract nucleic acids. In order to calculate ribosomal efficiency, and RNA:DNA and DNA: protein ratios, all values were based on the dry mass of homogenized muscle tissue. 2.7. Nitrogen stable isotope ratio analysis A final portion of the biopsy was analyzed for nitrogen stable isotope signature using an isotope ratio mass spectrometer for

stable isotope analysis (Coastal Science Laboratories, Austin, TX, USA). δ15N values are expressed relative to an atmospheric nitrogen standard. 2.8. Statistical analyses Two bears sampled during late denning were substitutes for bears of the same size and age class that were only sampled during early denning. However, the summer cohort was composed of six different animals from either the early or late denning groups. As a result, standard unpaired Student's t-tests were performed using Sigmastat (Systat Software, Point Richmond, CA, USA) to assess significant differences in the mean values of the measured parameter between early denning and summer, and late denning and summer groups, as well as between early and late denning on all six animals (Zar, 1999). Alpha levels were set at 0.05. In addition, a paired t-test was also performed using Sigmastat to assess significant differences between seasons on only the four animals that were repeatedly tested during both early and late denning (Zar, 1999). Alpha levels were again set to 0.05. The standard and paired t-tests provided similar statistical conclusions. Consequently, results and P values are reported for the means of six bears. Numbers in parentheses are standard errors of the mean. 3. Results 3.1. In vitro measurement of protein synthesis and breakdown

Fig. 1. Rates of protein synthesis (dark bars) and breakdown (light bars) measured by the rate of tyrosine uptake and release (nmol tyrosine⁎mg tissue − 1⁎h− 1) in biopsies from the vastus lateralis from summer active bears (left columns), bears in early denning (center columns), and bears in late denning (right columns). Different letters depict values that are significantly different from one another. Values are means of six bears. Vertical lines represent ± one standard error.

Protein synthesis was significantly greater than breakdown (P = 0.033) in muscle biopsies from summer active bears (Fig. 1). Synthesis was also significantly higher in biopsies from bears tested during the summer active period compared to bears tested during both early (P = 0.001) and late (P = 0.001) winter hibernation (Fig. 1). Similarly, protein breakdown was significantly greater in biopsies from summer active animals

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Fig. 3. Percent nitrogen content in biopsies from summer active bears (dotted bars), bears in early denning (bar with horizontal marks) and late denning (bar with vertical marks). Values are means of six bears. Vertical lines represent ± one standard error.

than from those taken from bears during early (P = 0.008) and late (P = 0.023) hibernation. Interestingly, both protein synthesis (P = 0.42), and protein breakdown (P = 0.17) were not significantly different between early and late denning. Additionally, synthesis was not significantly different than breakdown in either early (P = 0.16) or late (P = 0.48) denning bears (Fig. 1). 3.2. Skeletal muscle protein concentration Protein content of muscle samples, assessed by the Bradford assay of peptide bonds, was higher in summer active bears compared to early (P = 0.03) or late (P = 0.04) denning bears. However, there was no significant difference (P = 0.65) between the protein content of samples taken during early and late winter (Fig. 2).

Fig. 4. δ15N values for biopsies taken from summer active bears (dotted bar) early denning (bar with horizontal marks) and late denning (bar with vertical marks) compared to an atmospheric nitrogen standard. An asterisk depicts a δ15N signature that is significantly enriched compared to biopsies from summer active bears. Values are means of six bears. Vertical lines represent ± one standard error.

(Fig. 3). There was no alteration in the nitrogen content of biopsies from early and late winter (P = 0.54). 3.4. Nucleic acid assays: RNA and DNA content, RNA: DNA ratios, ribosomal efficiency, and DNA: protein ratios RNA content, RNA: DNA ratios, and protein synthesis per RNA (ribosomal efficiency) were higher in summer bears compared to early (P = 0.037, 0.043, and 0.003, respectively) and late (P = 0.019, 0.045, 0.003, respectively) winter hibernating bears. There were no changes (P = 0.309, 0.801, and 0.54, respectively) in these parameters between early and late winter (Table 1). There were no alterations in DNA content, or protein: DNA ratio between summer and early (P = 0.89, and 0.18, respectively) or summer and late (P = 0.08, and 0.79, respectively) denned animals, nor did these parameters change between early and late (P = 0.15 and 0.281, respectively) denning (Table 1).

3.3. Skeletal muscle nitrogen content 3.5. Nitrogen stable isotope ratio analysis The nitrogen content of biopsies taken from summer active bears was not significantly different from that of biopsies taken from early denning (P = 0.5) or late denning (P = 0.81) animals Table 1 Nucleic acid content Summer

During early and late denning, the δ15N signature (the ratio of N to 14N in the sample compared to the ratio in a known standard) was greater compared to biopsies from summer animals (P = 0.031, and 0.026, respectively) but was not different (P = 0.92) between the two denning seasons (Fig. 4). 15

Early denning Late denning

1

4.51 (0.35) 4.40 (0.44) 4.02 (0.22) DNA 2.47 (0.32) 1.74 (0.17)⁎ 1.52 (0.09)⁎ RNA1 (ribosomal capacity) RNA: DNA ratio 0.517 (0.05) 0.378 (0.01)⁎ 0.385 (0.02)⁎ Synthesis/RNA2 (ribosomal 3.28 (0.51) 1.38 (0.26)⁎ 1.56 (0.23)⁎ efficiency) 122.79 (11.8) 111.03 (9.70) 118.83 (9.10) Protein/DNA3 Values are means of six bears (±SE), an asterisk indicates a mean significantly different from that calculated for summer active bears. Superscript 1 indicates μg nucleic acid/mg tissue dry mass. Superscript 2 indicates [nanomoles of tyrosine incorporated/μg nucleic acid]/mg tissue dry mass. Superscript 3 indicates μg protein/μg DNA.

4. Discussion 4.1. In vitro measurement of protein synthesis and breakdown Bears underwent a period of net muscle protein deposition during the summer, as synthesis rates were some 1.4-times higher than degradation (Fig. 1). During denning, rates of synthesis and breakdown decreased by 60–70% but bears were apparently in protein balance, as rates of synthesis and breakdown were the same. Accordingly, muscle samples taken from the summer cohort in this study had approximately 20% greater protein

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content than those obtained from winter denning bears. This suggests that either bears were in a state of summer body growth or protein accretion. All bears were adults and therefore, not in a state of growth. We interpret the higher ratio of muscle protein synthesis to catabolism in summer bears as representing the regeneration of lean body mass lost between the previous summer and early hibernation. This may be similar to a condition in human athletes where turnover is enhanced by ingestion of carbohydrates and amino acids, resulting in sarcoplasmic and myofibrillar proteins synthesis to replace utilized tissue, leading to net muscle deposition (Biolo et al., 1995; Narici et al., 1996; Rasmussen et al., 2000; Tipton et al., 2001). Skeletal muscle protein synthesis (Fig. 1) and content (Fig. 2) decreased in bears between summer and early denning. Prior to hibernation, bears have no special adaptation to imposed fasting, as they readily catabolize skeletal muscle to meet nutritional requirements and do not effectively hydrolyze urea, as indicated by high urea: creatinine (U:C) ratios if food deprived (Nelson et al., 1975; Barboza et al., 1997). However, hibernation is not immediate. There is a transition phase that can be of variable length depending on food availability and environmental conditions (Nelson et al., 1984a,b). Once bears attain the hibernating state, they have very low urea: creatinine (U: C) ratios (Nelson et al., 1973, 1983), and exhibit dramatic protein sparing during prolonged winter food deprivation. As a result, the protein loss by bears from summer to the early denning period observed in this study may reflect a transition state characteristic of early fasting (e.g. phase 1, Cahill, 1970) by humans. However, once overwintering bears attained hibernation, protein synthesis and degradation rates balanced, and robust protein conservation occurred. Muscle protein synthesis and degradation rates did not significantly change as the winter progressed (Fig. 1), and protein was not lost from muscle tissue during dormancy (Fig. 2). This has important adaptive consequences for hibernating bears, which appear dissimilar to malnourished, fasting, or immobilized humans. If bears lost muscle tissue in a manner characteristic of the typical mammalian response to food deprivation over hibernation, it would be predicted that protein synthesis would decrease and protein catabolism elevate, resulting in a dramatic loss of protein (Waterlow, 1984; Medina et al., 1991; Mitch and Goldberg, 1996). If skeletal muscle was lost in accordance with a disuse atrophy model, rather than a food-deprived proteolytic model, it would be evidenced by a decreased protein synthesis rate, accompanied by only slightly increased muscle protein catabolism (Goldspink et al., 1986; Gibson et al., 1987; Medina et al., 1995; Ferrando et al., 1996), also resulting in a reduction in muscle protein content over the winter. Alternatively, if the bear responded to hibernation like the protein, but not energylimited human, skeletal muscle protein synthesis would be maintained with a concomitant decrease in catabolism in an effort to maintain skeletal muscle protein (Tawa et al., 1992; Tawa and Goldberg, 1992; Lecker et al., 1999). Muscle protein synthesis and breakdown by bears in the present study did not fit standard models describing immobility, starvation, or malnutrition. Rates of synthesis and degradation simultaneously decreased from the summer fed state, suggesting

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that these animals may have attained protein balance by the early denning period. They then maintained protein balance, with synthesis equal to degradation, without loss of protein from the vastus lateralis during the remaining 110 days of winter anorexia and confinement. Additional support for this assertion is gained from the constant level of protein (Fig. 2) and nitrogen (Fig. 3) content we measured in muscle biopsies from these bears throughout the winter. In contrast to the hibernating bear, immobilized or chronically food deprived humans lose nitrogen at a steady rate (LeBlanc et al., 1992; Ferrando et al., 1996). The bear's ability to recycle 100% of the urea (Barboza et al., 1997), and thus nitrogen, liberated from its low levels of skeletal muscle protein catabolism may allow it to be far more efficient at protein sparing during the winter than the immobilized, starved, or malnourished human. As a result, skeletal muscle protein and nitrogen is largely conserved, ultimately leading to minimal loss of strength and maintenance of muscle function by overwintering bears (Harlow et al., 2002; Lohuis et al., 2007). Whole-body in vivo measurements of protein synthesis and degradation (Barboza et al., 1997) showed that captive bears gained protein during summer and were in nitrogen balance during both hyperphagia and denning. These investigators also reported that rates of whole body synthesis and breakdown were equal during denning, supporting our findings in this study on muscle protein measured in free-ranging bears in their natural dens. However, they report that rates of whole-body protein catabolism increased significantly during winter denning compared to fall hyperphagia, whereas we found a decrease in protein catabolism in muscle biopsies during winter compared to summer. These studies are not in contradiction. We believe that to have an increase in whole body catabolism (Barboza et al., 1997) without an increase in muscle catabolism (current study) suggests that protein reserves other than skeletal muscle are being used to meet metabolic demands for protein during dormancy. In the active, fed bear, high turnover and associated protein deposition during summertime may result in the accumulation of protein reserves. Indeed, animals that experience long-term fasting (e.g. bats (Yacoe, 1983), penguins (Cherel et al., 1994, Groscolas and Robin, 2001), incubating waterfowl (LeMaho et al., 1981)) accumulate body protein stores during times of food availability, then catabolize these reserves during fasting or migration. This may also be the case in the bear. 4.2. Indirect measurements of synthesis and breakdown: skeletal muscle protein and nitrogen content, nucleic acid assays, and nitrogen stable isotope ratio analysis Data from these indirect measurements of protein breakdown and synthesis support our conclusions drawn from in vitro radioisotope studies on protein turnover. 4.3. Skeletal muscle protein and nitrogen content There was a 20% decrease in protein content from the vastus lateralis between summer and early denning animals measured using the Bio-Rad assay (Fig. 2). However, the CHN analysis for nitrogen content showed no difference in the percentage of

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nitrogen in muscle biopsies during this interval (Fig. 3). The BioRad assay only measures peptide bonds, indicative of protein content, while results from the nitrogen assay represent additional nitrogen in the form of organic acids, amino acids, urea, and creatinine. A drop in protein content from summer to early denning without a concomitant drop in total nitrogen suggests that there is a larger non-protein component during the denning period compared to summer. A large portion of this component could include urea, which is hydrolyzed by hibernating but not summer active bears. A drop in protein content from summer to early denning could represent the lag in capacity to recycle urea nitrogen. Once bears entered hibernation, both muscle protein and total nitrogen remained stable throughout the winter, indicative of balanced degradation and synthesis. Protein loss and rates of synthesis and breakdown may vary between muscle groups depending on size and function (Yacoe, 1983; LeBlanc et al., 1992). As the current study investigated only one muscle, it could be suggested that the results reported here cannot be generalized to whole body protein metabolism in the hibernating bear. However, changes in protein content of biopsies from vastus lateralis (this study) are supported by results from other muscles tested in captive (Koebel et al., 1991), or free-ranging (Tinker et al., 1998) bears. Koebel et al. (1991) reported a small (approximately 10%) but not significant decrease in protein concentration in biopsies from the gastrocnemius and extensor hallucis longus muscles between the summer active period and hibernation, with stable protein content during hibernation. In agreement with our work, Tinker et al. (1998) also reported only marginal losses of 4–10% of skeletal muscle protein in biopsies from the biceps femoris and gastrocnemius of bears during hibernation. Interestingly, six of the seven bears studied by Tinker et al. (1998) were lactating females. Only one of six hibernating bears and none of the six summer active bears in the present study were lactating females. Considering the additional metabolic demands of gestation and lactation (Farley and Robbins, 1995; Harlow et al., 2002), it is expected that bears in the present study would require even less nitrogen and thus would show minimal or no loss of skeletal muscle protein over the course of hibernation as we report here. 4.4. Nucleic acid assays: RNA and DNA content, RNA: DNA ratios, ribosomal efficiency and DNA: protein ratios The amount of nucleic acid present in the form of RNA and DNA has been used to assess changes in protein in skeletal muscle and organs. Since DNA is a constant chromosomal component, its concentration reflects the number of cells present in a muscle biopsy (Steffen and Mussachia, 1984). Assessment of the number of cells and the degree of hypertrophy or atrophy can be made by comparing both the total DNA concentration and the ratio of protein content to DNA (Cheek et al., 1971; Steffen and Mussachia, 1984) between time periods. As the amount of DNA/weight of tissue remained constant throughout the course of hibernation (Table 1), it appears that the total number of muscle cells remained the same, suggesting no muscle atrophy, from summer, through the entire denning period. The DNA: protein ratio remained unchanged between summer, early denning, and

late denning. However, as total protein content decreased by 20%, it should be expected that the DNA: protein ratio should parallel this change. This unexpected result is not consistent with the remainder of the data from this work and may be an artifact. Approximately 80% of RNA is ribosomal, and, as such, measurement of RNA content provides an indication of the potential capacity of a tissue for protein synthesis (Millward et al., 1973). RNA content decreases in tissues that have atrophied (Steffen and Mussachia, 1984), been nutrient restricted, or starved (Li and Goldberg, 1976; Li et al., 1979). Ribosomal efficiency is defined as the actual amount of protein synthesis per unit weight of tissue and is expressed by the ratio of protein synthesis to RNA content measured in tissue. In fasted or immobilized animals, ribosomal efficiency is reported to initially decrease, followed by a decrease in the RNA content. The ratio of RNA to DNA is used to provide an index of synthesis per cell number (Millward et al., 1973; Steffen and Mussachia, 1984). In the present study, there was a 60%, 40%, and 25% decrease in RNA content, ribosomal efficiency, and RNA: DNA ratio, respectively, between summer active and early denning bears (Table 1). These measures reflect the decrease in protein content and synthetic rates measured in vitro using 14C phenylalanine during the transition between summer and early denning. We measured no difference between the RNA content, ribosomal efficiency, or RNA: DNA ratios of muscle biopsies sampled in early and late winter (Table 1). These data confirm our conclusions from the in vitro measurement of protein synthesis and breakdown that bears do maintain protein balance over the winter denning period. 4.5. Nitrogen stable isotope ratio analysis As an animal fasts and loses protein the heavy isotope of nitrogen (15N) becomes fixed, while the light isotope (14N) is preferentially metabolized and eliminated (Minigawa and Wada, 1984; Macko et al., 1986). This process is called fractionation, which is expressed by the δ15N value, representing the change in the isotope ratio composition of the sample relative to the ratio in a nitrogen standard. Nitrogen fractionation in liver and skeletal muscle has been shown to accompany protein restriction or fasting in birds and mammals (Hobson et al., 1993; Polishuck et al., 2001). In the present study on bears there was enrichment in the biopsied muscle (18%) at a similar magnitude as the decrease observed in protein content (20%) from summer to early denning. This supports our previous conclusions of increased protein breakdown during the transition period prior to denning. The δ15N signature was then unchanged during denning (Fig. 4). This is also in agreement with our conclusions that protein and nitrogen content remains stable and protein synthesis and breakdown rates are in balance over the winter. 5. Conclusions Black bears accumulate protein in the summer, when food is available and the animals are mobile. A period of protein loss occurs as the bears make the transition from summer to hibernation and metabolic protein sparing. Very little protein is

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lost during the protracted period of confinement and food deprivation of denning, unlike that predicted for inactive, though fed humans (LeBlanc et al., 1992; Berg and Tesch, 1996; Berg et al., 1997; Ferretti et al., 2001), and other animal models (Li and Goldberg, 1976; Steffen and Mussachia, 1984; review, Thomason and Booth, 1990). The decrease in skeletal muscle protein synthesis and breakdown during winter denning leads to preservation of protein mass as well as muscle fiber integrity by overwintering bears (Tinker et al., 1998; Lohuis et al., 2007). Summer accretion and winter conservation of skeletal muscle protein through balanced breakdown and synthesis during hibernation inactivity and fasting are adaptive mechanisms for winter survival in bears. Retention of protein and muscular integrity allows remarkable conservation of skeletal muscle. In addition, periodic muscle contractions throughout the winter (Harlow et al., 2004) may enhance strength retention by bears and the maintenance of a sustained locomotor response and ability to travel upon emergence (Harlow et al., 2001). However, as in the malnourished human, there is a metabolic requirement for nitrogen. Several other species that are welladapted to long-term fasting, including penguins (Cherel et al., 1994), seals (Worthy and Lavigne, 1983; Oritsland et al., 1985; Worthy and Lavigne, 1987), hibernating golden-mantled ground squirrels (Steffen et al., 1991), and bats (Yacoe, 1983), all preferentially utilize some body reserves while sparing others. Bears also evolved to endure long periods of anorexia, and may utilize other labile protein reserves to meet metabolic needs and thus spare crucial skeletal muscle. However, the sources of that nitrogen are as yet unknown. In order to fully understand the protein partitioning and kinetics in the hibernating bear, a comparison of protein synthesis and breakdown in several organs and tissues is required. Acknowledgements This research was funded primarily by NSF grant IBN 9808785 to HJH. We thank the Colorado Division of Wildlife and the Wyoming Game and Fish department for their cooperation and logistical support during this study. This project could not have been successful without expert technicians Lyle Willmarth, Joe Koloski, Mike Hooker, and Craig Jamison. Many thanks and a cold beer are extended to the many volunteers, particularly Mark Murphy, Kevin McDonough, and Todd and John Perdue, whom, along with many others, provided assistance with den locating and load carrying in subzero, snowy conditions. References Ahlquist, D.A., Nelson, R.A., Steiger, D.L., Jones, J.D., Ellefson, R.D., 1984. Glycerol metabolism in the hibernating black bear. J. Comp. Physiol. 155B, 75–79. Alkner, B.A., Tesch, P.A., 2004. Knee extensor and plantar flexor muscle size and function following 90 days of bed rest with or without resistance exercise. Eur. J. Appl. Physiol. 93, 294–305. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1990. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York, NY. V.2, p.A.1.5.

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