A prominent example of seasonal phenotypic ﬂexibility is the winter increase in thermogenic capacity (¼summit metabolism, M ) in small birds, which is often accompanied by increases in sum pectoralis muscle mass and lipid catabolic capacity. Temperature or photoperiod may be drivers of the winter phenotype, but their relative impacts on muscle remodeling or lipid transport pathways are little known. We examined photoperiod and temperature effects on pectoralis muscle expres- sion of myostatin, a muscle growth inhibitor, and its tolloid-like protein activators (TLL-1 and TLL- 2), and sarcolemmal and intracellular lipid transporters in dark-eyed juncos Junco hyemalis.We acclimated winter juncos to four temperature (3 Cor24 C) and photoperiod [short-day (SD) ¼ 8L:16D; long-day (LD) ¼ 16L:8D] treatments. We found that myostatin, TLL-1, TLL-2, and lipid transporter mRNA expression and myostatin protein expression did not differ among treatments, but treatments interacted to inﬂuence lipid transporter protein expression. Fatty acid translocase (FAT/CD36) levels were higher for cold SD than for other treatments. Membrane-bound fatty acid binding protein (FABPpm) levels, however, were higher for the cold LD treatment than for cold SD and warm LD treatments. Cytosolic fatty acid binding protein (FABP ) levels were higher on LD than on SD at 3 C, but higher on SD than on LD at 24 C. Cold temperature groups showed upregu- lation of these lipid transporters, which could contribute to elevated M compared to warm sum groups on the same photoperiod. However, interactions of temperature or photoperiod effects on muscle remodeling and lipid transport pathways suggest that these effects are context-dependent. Key words: birds, FABPpm, FABPc, FAT/CD36, myostatin, pectoralis, phenotypic ﬂexibility, photoperiod, temperature. Seasonal phenotypes in small birds are one well-known example of (M ; maximum cold-induced metabolism) metabolic rates relative sum reversible phenotypic flexibility and allow birds to better match to summer (Dubois et al. 2016). Such increments of metabolic rates, their phenotypes to seasonal climates (Swanson 2010; Swanson and especially M , are positively correlated with cold tolerance sum Ve ´ zina 2015). Small birds experience high thermoregulatory de- (Swanson, 2001; Swanson and Liknes 2006). As a result, M is sum mands in cold climates, and due to their high surface area-to-volume commonly interpreted as an indicator of a bird’s ability to endure ratios, they are expected to undergo substantial variation in meta- cold environments. bolic phenotypes between seasons. The winter phenotype in small Adjustments in M throughout the annual cycle in birds may sum birds is distinguished by improved cold tolerance and elevated be mediated through variation in skeletal muscle (Marsh 1981; basal (BMR; minimum maintenance metabolism) and summit Evans et al. 1992; Liknes and Swanson 2011b) and heart (Piersma V C The Author (2017). Published by Oxford University Press. 23 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/23/3091112 by Ed 'DeepDyve' Gillespie user on 16 March 2018 24 Current Zoology, 2018, Vol. 64, No. 1 1998; Battley et al. 2001; Swanson et al. 2014b) masses and/or cel- shivering thermogenesis in birds is almost exclusively fueled by lular metabolic intensities (Liknes and Swanson 2011a; Zheng et al. exogenous lipids from adipose tissues (Swanson 2010). As a result, 2014) coupled with enhanced substrate transport (Liknes et al. capacities for non-esterified fatty acid (NEFA) uptake into the myo- 2014a). Previous studies indicated that flight muscle hypertrophy cyte and their subsequent transport to mitochondrial membranes are contributed to increases in organismal metabolic rates (Marsh 1981, potential regulatory steps for lipid catabolic capacity in support of 1984; Evans and Rose 1988; Dietz et al. 1999; Petit and Ve ´ zina shivering (Swanson 2010; Zhang et al. 2015a). Once circulating 2014), especially for winter phenotypes in birds (Swanson et al. NEFAs reach the myocyte, plasma membrane-bound fatty acid 2009, 2013; Liknes and Swanson 2011b; Petit et al. 2014). Because binding protein (FABPpm) and fatty acid translocase (FAT/CD36) seasonal increases in pectoralis muscle and heart masses consistently cooperate to transport NEFAs across the endothelium, the intersti- contribute to the winter phenotype in small birds, the mechanisms tial space, and the sarcolemma to enter muscle cells (Kiens 2006; regulating seasonal muscle remodeling are an important research Glatz et al. 2010). The effects of winter acclimatization or migration target for understanding the flexible metabolic phenotypes in birds. on skeletal muscle FAT/CD36 and FABPpm levels are incompletely One candidate for the flexible regulation of skeletal muscle mass is resolved. Some studies showed elevated FAT/CD36 and FABPpm myostatin, which belongs to the TGF-b family of growth factors and levels during migration compared to non-migrants (McFarlan et al. is an autocrine/paracrine inhibitor of muscle growth in birds and 2009; Zhang et al. 2015c), but others indicated stable FAT/CD36 mammals (Lee 2004). Myostatin acts to inhibit skeletal muscle and FABPpm during migration or wintering (Zhang et al. 2015c, growth and reduces muscle mass in birds and mammals, thereby reg- 2015d). Moreover, results from exercise and cold training studies sug- ulating muscle remodeling in adults (Lee 2004; Rodgers and gest that responses of FAT/CD36 and/or FABPpm levels to increasing Garikipati 2008). Several studies have examined variation in expres- energy demands also vary among species (Price et al. 2010; Zhang sion of myostatin in birds during migration and winter acclimatiza- et al. 2015a). Intramyocyte fatty acid transport in skeletal muscles is tion accompanied by variation in muscle mass (Swanson et al. 2009, mediated by cytosolic fatty acid binding protein (FABP )(Kiens 2006; 2014a; King et al. 2015). These studies offer variable support for a Guglielmo 2010). FABP also acts as an intramyocyte fatty acid recep- role for myostatin in regulating flexible muscle masses throughout tor, acting to regulate and potentially limit fatty acid uptake (Glatz the annual cycle in birds, with some finding reduced expression of et al. 2010). Most studies of migration (Guglielmo et al. 2002; either mRNA or protein for myostatin in pectoralis muscle McFarlan et al. 2009), cold acclimation (Stager et al. 2015), or winter (Swanson et al. 2009), or no changes in other species for migratory acclimatization (Liknes et al. 2014; Zhang et al. 2015d)show thatin- or wintering phenotypes (Swanson et al. 2014a; King et al. 2015). creases in pectoralis FABP levels are consistent correlates of enhanced However, white-throated sparrows Zonotrichia albicollis under mi- flight and shivering performance in birds. gratory photoperiod exhibited the opposite effect, with elevated The regulation of winter phenotypes in small resident birds in mRNA expression of myostatin compared to the winter photo- temperate-zone climates could potentially be driven by environmen- period, but European starlings Sturnus vulgaris did not show any tal cues such as photoperiod (Carey and Dawson 1999) and tem- changes in myostatin mRNA expression with exercise training (Price perature (Swanson and Olmstead 1999). To examine mechanistic et al. 2011). In contrast, exercise- and cold-trained house sparrows bases for photoperiod and temperature-induced variation in organis- Passer domesticus showed significantly reduced myostatin protein mal metabolic capacities, we studied dark-eyed juncos Junco hyema- levels associated with increased pectoralis mass compared to their lis exposed to different photoperiod [long-day (LD) and short-day controls (Zhang et al. 2015b). (SD)] and temperature (cold and warm) treatments. Dark-eyed jun- Myostatin is secreted as an inactive form and requires cleavage cos are a common winter resident in South Dakota and resident by metalloproteinases, including the tolloid-like proteins TLL-1 and populations in western North America show seasonal variation in TLL-2, to form the active C-terminal dimer myostatin (Huet et al. pectoralis mass and M (Swanson 1990, 1991). Moreover, a previ- sum 2001). Thus, upstream of myostatin receptors, regulation of muscle ous study using the same individual birds indicated that cold expos- mass through myostatin can involve both synthesis of myostatin and ure, but not short photoperiod, elevated M compared to warm sum activation of the protein via cleavage (Lee 2004, 2008). Myostatin exposed birds (Swanson et al. 2014c). This study also demonstrated and TLL-1 gene expression were both downregulated in winter that even though pectoralis muscle mass did not vary significantly house sparrows relative to summer sparrows (Swanson et al. 2009). among acclimation treatments, cellular metabolic intensity in the Moreover, mRNA expression for either or both TLL-1 and TLL-2 pectoralis was generally higher for LD and cold groups (Swanson were downregulated in winter relative to summer for American et al. 2014c). In addition, Stager et al. (2015) conducted a study of goldfinches (Spinus tristis) and black-capped chickadees (Poecile genome-wide transcriptional profiles on pectoralis muscle samples atricapillus, Swanson et al. 2014a). In contrast, Price et al. (2011) from the same individual birds and found that mRNA expression found that TLL-1 mRNA expression did not differ from controls for for muscle hypertrophy, angiogenesis and lipid transport and oxida- exercise-trained European starlings. Moreover, exercise- and cold- tion pathways were generally upregulated by cold exposure. Several trained house sparrows showed only non-significant variation in studies, however, have documented that mRNA and protein expres- TLL mRNA expression in trained groups compared to their controls sion in pectoralis muscle during migration and winter acclimatiza- (Zhang et al. 2015b). Thus, although some evidence suggests that tion in small birds are sometimes not correlated for myostatin or lipid transport pathways (e.g., Swanson et al. 2014a, 2017; King modulation of myostatin and myostatin processing capacity may regulate phenotypic flexibility of metabolic capacities in birds in re- et al. 2015; Zhang et al. 2015d). Consequently, the present study sponse to changing energy demands, this modulation does not ap- builds upon these previous studies by evaluating, in pectoralis pear to be universal for birds under all such conditions. muscle (the main thermogenic organ in birds), both gene and protein In addition to flight muscle hypertrophy, winter and migratory expression for myostatin, gene expression for myostatin activators enhancement of lipid transport (Liknes et al. 2014; Zhang et al. TLL-1 and TLL-2, and gene and protein expression for the lipid 2015d) and catabolism (Marsh and Dawson 1989; Liknes and transporters, FABPpm, FABP (protein only) and FAT/CD36 for Swanson 2011a; Corder et al. 2016) also occurs in birds. Prolonged these same individual juncos, as well as by examining correlations Downloaded from https://academic.oup.com/cz/article-abstract/64/1/23/3091112 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhang et al. Temperature and photoperiod effects on myostatin and lipid transport 25 among these factors and with M and pectoralis muscle mass. We 15 min, then 95 C for 10 min, followed by 40 cycles at 95 C for sum hypothesize that cold exposure will reduce expression of the myosta- 15 s and 60 C for 1 min. We optimized protocols for all six genes to tin system and increase lipid transport capacities, but that photo- verify efficiencies for these probe and primer sets for dark-eyed jun- cos. The FABP probe and primer sets failed to amplify mRNA using period will have limited impacts on these systems. These analyses c the StepOnePlus Real-Time PCR System, so we were unable to will identify the relative variation in pectoralis gene and protein ex- quantify gene expression for FABP . Slopes and efficiencies for each pression for the myostatin and lipid transport systems in response to gene were: GAPDH (3.35, 98.8%), Myostatin (3.5, 93.1%), temperature and photoperiod in small birds and how such variation TLL-1 (3.42, 96.1%), TLL-2 (3.49, 93.4%), FABPpm (3.45, might relate to seasonal metabolic flexibility. 94.9%), and FAT/CD36 (3.36, 98.4%). We quantified changes in DDCT mRNA expression using the 2 method (Livak and Schmittgen, Materials and Methods 2001). We used the mean value for all acclimation treatments for each gene as the reference sample and set the value for this reference Bird collection and acclimation treatments sample equal to 1. We then normalized mRNA expression All procedures reported herein were approved by the University of to this reference sample to determine relative amounts of mRNA South Dakota Institutional Animal Care and Use Committee expression (relative to GAPDH expression) for all other samples for (Protocol 79-01-11-14C). Details of bird collection and acclimation the same tissue and species (e.g., Zhang et al. 2015a, 2015b, 2015c, treatments are provided in Swanson et al. (2014b). Briefly, dark- 2015d). eyed juncos were captured near Vermillion, South Dakota (approxi- mately 42 47’ N, 97 W) during mid-December under appropriate Western blots state (11-7, 12-2) and federal (MB758442) scientific collecting per- We measured pectoralis protein levels for myostatin, FAT/CD36, mits. After capture, birds were individually housed in FABP , and FABPpm using Western blots with glyceraldehyde phos- 59 45 36 cm stainless-steel cages with controlled temperature phate dehydrogenase (GAPDH) as a housekeeping protein (6 2 C) and photoperiod with ad libitum mixed seed, protein sup- (Zhang et al. 2015a 2015b, 2015c, 2015d). We were unable to ac- plement (mixture of homogenized dog food and hard-boiled egg quire functional antibodies for TLL-1 and TLL-2, so we did not con- with seed) and vitamin-enriched (Wild Harvest Multi-Drops vitamin duct Western blots for TLL-1 and TLL-2. For Western blot assays, supplement for all birds, United Pet Group, Inc., Cincinnati, OH, we removed pectoralis samples from 80 C storage and homogen- USA) water. Birds were acclimated to captive conditions, at room ized small samples by sonication on ice with a Cole-Parmer temperature (23 C) and natural photoperiod (9 h:15 h, light:dark), (Chicago, IL, USA) 4710 Series Ultrasonic homogenizer for three for at least two weeks. We randomly assigned birds into four 10 s bursts, with 30 s in between bursts to disrupt membranes and temperature–photoperiod treatments: 24 C, 8 h:16 h light:dark separate membrane proteins from phospholipids. The homogenizing (warm SD); 24 C, 16 h:8 h light:dark (warm LD); 3 C, 8 h:16 h buffer contained 50 mM Tris, pH 7; 100 mM NaCl; 2% SDS. light:dark (cold SD); and 3 C, 16 h:8 h light:dark (cold LD). We then separated soluble and phospholipid fractions by centrifuga- We included 12 birds in each treatment and acclimation treatments tion, and collected the soluble fraction for analysis. We determined lasted for 6 weeks. protein concentrations on the soluble fraction using a modified DC After the 6-week acclimation treatments, we euthanized birds by Lowry improved protein assay and we used 10 lg of protein for ana- cervical dislocation and quickly excised the pectoralis muscles on lysis via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ice. We weighed the left pectoralis muscle to the nearest 0.1 mg and R R We ran all samples on NuPAGEV NovexV 4–12% Bis-Tris protein divided it into two sub-samples, one of which was placed in gels with the same random sample included on every gel to serve as RNAlater (Ambion, Grand Island, NY, USA) for real-time quantita- a standard for detecting gel-to-gel variation. After transferring pro- tive reverse transcription PCR (qRT–PCR) and one flash-frozen in teins onto membranes, we probed blots with antibodies against liquid nitrogen for Western blot analysis. Both sub-samples were myostatin (goat polyclonal; R&D Systems, Minneapolis, MN, USA; stored frozen at 80 C until later assays. 1:100 dilution), FAT/CD36 (rabbit polyclonal; Novus Biologicals, Littleton, CO, USA; 1:1,000 dilution), FABPpm (rabbit polyclonal, Quantitative real-time RT–PCR from Christopher G. Guglielmo; 1:10,000 dilution), FABP (rabbit We measured pectoralis mRNA expression for myostatin, TLL-1, polyclonal, from Christopher G. Guglielmo; 1:8,000 dilution) TLL-2, FABPpm, and FAT/CD36 by qRT–PCR, as described in (McFarlan et al. 2009), and GAPDH (chicken polyclonal; Millipore, Swanson et al. (2014a) and Zhang et al. (2015a, 2015b, 2015c, Temecula, CA, USA; 1:8,000 dilution). We washed membranes with 2015d), using glyceraldehyde-3-phosphate dehydrogenase TBS-T and incubated them with horseradish peroxidase-conjugated (GAPDH) as a housekeeping gene. For these assays, we first ex- secondary antibodies: anti-rabbit (1:1,000; Santa Cruz tracted total RNA with b-mercaptoethanol and the RNeasy Fibrous Biotechnology, Dallas, TX, USA) for FAT/CD36, FABPpm, and Tissue Mini kit (QIAGEN, Valencia, CA, USA). We then quantified FABP , anti-chicken (1:1,500; Abcam, Cambridge, MA, USA) for extracted RNA with an Agilent 2100 Bioanalyzer (Agilent GAPDH, and anti-Goat (1:1,000 dilution; Santa Cruz Technologies, Santa Clara, CA, USA) and used 50 ng of purified Biotechnology) for myostatin. For analysis, we visualized blots with RNA for qRT–PCR reactions with a TaqMan RNA-to-CT 1-Step the ECL Plus Western Blotting Detection System (GE Healthcare, Kit (Applied Biosystems, Carlsbad, CA, USA) and a Step-One-Plus Buckinghamshire, UK) and captured chemiluminescent images with Real-Time PCR System (Applied Biosystems). For all genes, we used a VersaDoc 3000 Molecular Imager (Bio-Rad, Hercules, CA, USA). the custom qRT–PCR probe and primer sets (Applied Biosystems) We analyzed images with Quantity One software (Bio-Rad containing the sequences listed in Swanson et al (2014a, see their Laboratories), normalizing each protein level by dividing by Table 1), which were derived from partial cDNA sequences from GAPDH protein levels for the same tissue sample (Zhang et al. house sparrows (Swanson et al. 2009; GenBank Accession Numbers 2015b). We used these normalized protein levels for subsequent KP337454–KP337456). We conducted the qRT–PCR at 48 C for statistical comparisons. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/23/3091112 by Ed 'DeepDyve' Gillespie user on 16 March 2018 26 Current Zoology, 2018, Vol. 64, No. 1 Table 1. Correlations among mass-independent summit metabolic rate residuals (from regressions of metabolic rate versus M Pearson correlation coefficient M residual PEC residual Myostatin mRNA level TLL-1 mRNA level TLL-2 mRNA level Myostatin protein level sum M residual 0.028 0.201 0297 0.2 0.277 sum PEC residual 0.063 0.095 0.06 0.187 mRNA expression Myostain 0.119 0.127 0.044 TLL-1 0.738** 0.375* TLL-2 0.251 Swanson et al. (2014b), pectoralis muscle mass (PEC), myostatin mRNA level, tolloid-like protein 1 (TLL-1) mRNA level, tolloid-like protein 2 (TLL-2) mRNA level, protein levels of myostatin in pectoralis muscles of dark-eyed junco. *P< 0.05; **P< 0.01. 2.0 Statistical analyses Cold SD We present data as means6 SE, unless otherwise noted. We com- Cold LD Warm SD pared mean values of mRNA and protein expression among different Warm LD treatments with two-way ANOVA. If parametric assumptions of nor- 1.5 mal distribution (Kolmogorov–Smirnov test) or homogenous vari- ances (Levene’s test) were violated, we log -transformed data prior to comparisons. If significant differences were detected by two-way 1.0 ANOVA, we used Tukey tests to identify which means differed sig- nificantly. We further calculated Pearson correlation coefficients to examine relationships among separate components for each pathway. 0.5 We obtained values for M ,body mass(M ), pectoralis mass, and sum b pectoralis activities of carnitine palmitoyl transferase (CPT, an indica- tor of fatty acid transport across the mitochondrial membrane), cit- 0.0 Myostatin TLL-1 TLL-2 FAT/CD36 FABPpm rate synthase (CS, a key regulatory enzyme of the Krebs cycle), and b-hydroxyacyl Co-A dehydrogenase (HOAD, a key enzyme regulating Figure 1. Temperature and photoperiod effects on relative mRNA expression fat oxidation capacity) for the same individual birds from Swanson levels from qRT-PCR for myostatin, tolloid-like protein 1 (TLL-1), tolloid-like et al. (2014c). We tested for correlations for M ,body mass(M ), sum b protein 2 (TLL-2), plasma membrane-bound fatty acid binding protein and pectoralis mass with myostatin mRNA and protein expression, (FABPpm), and fatty acyl translocase (FAT/CD36) in pectoralis muscles of dark-eyed junco (Junco hyemalis). Error bars represent SE. Sample sizes for and with mRNA expression for the TLLs to examine the muscle re- the different treatment groups were: cold short day (Cold SD), n ¼ 11; cold modeling pathway. We tested for correlations for M and enzyme sum long day (Cold LD), n ¼ 10; warm short day (warm SD), n ¼ 10; warm long day activities with fatty acid transporter mRNA and protein expression to (warm LD), n ¼ 10. examine lipid transport and metabolism pathways. To remove the ef- fects of M from analyses of relationships for pectoralis mass and M , we calculated residuals from allometric regressions with M , sum b and then used least squares linear regression of residuals to test for Cold SD correlations with these variables. All statistical analyses were con- Cold LD ab Warm SD ducted with SigmaStat Version 3.5 (Systat, Point Richmond, CA, Warm LD USA). We accepted statistical significance for all tests at P< 0.05. b b Results Myostatin and TLLs Both GAPDH mRNA expression and protein levels did not differ bb significantly among groups, so GAPDH should serve and effective housekeeping role in this study. No significant differences were de- tected in pectoralis mRNA expression for myostatin or the TLLs Myostatin FAT/CD36 FABPpm FABPc among acclimation treatments (Figure 1). We detected a single band for the myostatin antibody in our Western blots, which correspond Figure 2. Temperature and photoperiod effects on relative protein levels from to the 52 kDa unprocessed latent form of myostatin. As for mRNA western blot for myostatin, tolloid-like protein 1 (TLL-1), tolloid-like protein 2 expression, protein expression of myostatin did not vary signifi- (TLL-2), plasma membrane-bound fatty acid binding protein (FABPpm), and fatty acyl translocase (FAT/CD36) in pectoralis muscles of dark-eyed junco cantly among acclimation groups (Figure 2). (Junco hyemalis). Error bars represent SE. Sample sizes for the different treatment groups were: cold short day (Cold SD), n ¼ 11; cold long day (Cold Trans-sarcolemmal and intramyocyte lipid transport LD), n ¼ 10; warm short day (warm SD), n ¼ 10; warm long day (warm LD), No significant differences among acclimation treatments were detected n ¼ 10. Different letters denote signiﬁcant differences between treatment for pectoralis mRNA expression of FAT/CD36 or FABPpm (Figure 1). groups. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/23/3091112 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Relative mRNA Expression Relative Protein Level Zhang et al. Temperature and photoperiod effects on myostatin and lipid transport 27 Table 2. Correlations among mass-independent metabolic rate residuals (from regressions of metabolic rate versus M Pearson correlation coefficient M FAT/CD36 FABPpm FAT/CD36 FABPpm FABPc CPT activity CS activity HOAD sum residual mRNA level mRNA level Protein level Protein level Protein level activity M Residuals 0.307 0.179 0.167 0.092 0.127 0.278 0.144 0.185 sum mRNA expression FAT/CD36 0.783** 0.054 0.051 0.255 0.187 0.245 0.279 FABPpm 0.087 0.099 0.315* 0.116 0.338* 0.364* Protein level FAT/CD36 0.13 0.026 0.108 0.02 0.172 FABPpm 0.763** 0.103 0.253 0.196 FABP 0.116 0.218 0.256 Enzyme activities CPT 0.345* 0.353* CS 0.557** Swanson et al. (2014b), activities of carnitine palmitoyl transferase (CPT), citrate synthase (CS), b-hydroxyacyl CoA-dehydrogenase (HOAD), protein levels of cytosolic fatty acid binding protein (FABP ), and protein levels and mRNA expression of fatty acyl translocase (FAT/CD36) and plasma membrane-bound fatty acid binding protein (FABPpm) in pectoralis muscles of dark-eyed junco. *P< 0.05; **P< 0.01. Pectoralis protein expression, however, showed more variation, with FABPc). Both of these responses could be interpreted as occurring to significant differences among acclimation treatments for all three fat match to the higher energetic demands associated with cold and/or transporters (Figure 2; FAT/CD36: F ¼ 11.734, P¼ 0.002; migratory status, the latter promoted by exposure of winter-collected 1,40 FABPpm: F ¼ 6.053, P¼ 0.019; FABP : F ¼ 13.741, P< 0.001). birds to long days. Previous studies of these same juncos detected 1,40 c 1,40 FAT/CD36 protein expression was highest in the cold SD treatment upregulation in genes and enzyme activities involved in fatty acid me- and was significantly higher than for the other acclimation treatments. tabolism (Swanson et al. 2014b, Stager et al. 2015), but, importantly, FABPpm protein expression was highest in the cold LD acclimation this study extended these previous findings to protein expression in treatment and was significantly higher than cold SD and warm LD the pectoralis. Swanson et al. (2014c) previously documented for treatments. FABP protein levels were significantly higher in the cold these same individual juncos that cold temperatures increased M , c sum LD and warm SD than in the cold SD and warm LD groups. but this was not associated with increased pectoralis muscle mass. In addition, CS and HOAD activities in these same juncos generally increased on long days, but activities were highest on the cold LD Correlations treatment and lowest on the warm SD treatment, potentially due to Neither pectoralis muscle mass residuals, myostatin mRNA or pro- stimulation of the migratory disposition by long days (Swanson et al. tein expression, fatty acid transporter mRNA or protein expression, 2014c). Stager et al. (2015) built upon these findings with analyses of nor cellular catabolic enzyme activities were significantly correlated genome-wide patterns of mRNA expression, documenting cold- with M residuals (Tables 1 and 2). In the muscle remodeling sum induced increases in transcripts for muscle growth (although not myo- pathway, mRNA expression of TLL-1 and TLL-2 were significantly statin), angiogenesis, and lipid transport and catabolic pathways. positively correlated. In addition, myostatin protein expression was We found little evidence in this study for upregulation of gene or significantly positively correlated with TLL-1 mRNA expression protein expression of the myostatin system in pectoralis muscle in (Table 1). In the fatty acid transport and catabolism pathway, response to cold or short days. The absence of such an upregulation mRNA expression of FAT/CD36 and FABPpm were strongly posi- is consistent with the lack of an increase in pectoralis muscle mass in tively correlated. Protein expression of FABP was also positively response to cold or short days (Swanson et al. 2014c) as well as the correlated with both mRNA and protein expression of FABPpm. In absence of a transcriptomic signature for the myostatin pathway addition, FABPpm mRNA expression was significantly positively (Stager et al. 2015) for these same juncos. Surprisingly, muscle re- correlated with both CS and HOAD activities. Finally, CS, CPT, modeling mechanisms leading to hypertrophy, at least as mediated and HOAD enzyme activities were significantly positively correlated through the myostatin pathway, appear less important to with each other (Table 2). temperature-induced variation in organismal metabolic capacities for dark-eyed juncos than is typical for the winter phenotype in birds Discussion (Swanson 2010). Collectively, these results suggests that enhanced lipid transport and catabolism capacities, rather than muscle hyper- Results from the present study suggest that regulation of lipid trans- trophy, are one of the primary drivers of enhanced thermogenic cap- porter protein levels in pectoralis muscle is complex, with variation in acity during cold acclimation for dark-eyed juncos. either temperatures or photoperiod potentially resulting in changes to lipid transport capacities. For example, high protein expression for all three lipid transporters occurred in cold temperatures, but high ex- Myostatin and TLLs pression in the cold was dependent on photoperiod, with some trans- Myostatin, TLL-1 and TLL-2 mRNA expression and myostatin pro- porter protein levels reaching maximum levels under cold and short tein levels did not differ among acclimation treatments in this study. days (FAT/CD36) and others under cold and long days (FABPpm and The qRT-PCR and Western blot analyses in the present study Downloaded from https://academic.oup.com/cz/article-abstract/64/1/23/3091112 by Ed 'DeepDyve' Gillespie user on 16 March 2018 28 Current Zoology, 2018, Vol. 64, No. 1 validate the absence of variation in myostatin expression docu- FAT/CD36 protein expression with incresing energy demands mented via transcriptomics in these same individual birds (Stager (Zhang et al. 2015a, 2015d). In addition, black-capped chickadees et al. 2015). Because pectoralis muscle mass also did not vary signifi- increased pectoralis FABPpm protein expression during winter com- cantly among treatment groups for these birds (Swanson et al. pared to summer (Zhang et al. 2015d). Migratory birds may also in- 2014c), our results do not preclude a general role for the myostatin crease pectoralis FABPpm protein levels during migration compared system in mediating seasonal muscle remodeling in birds. Even to non-migratory periods (McFarlan et al. 2009). Moreover, pector- though increases in pectoralis muscle mass are a common compo- alis FAT/CD36 protein expression increased during migration rela- nent of phenotypic flexibility associated with winter or cold accli- tive to summer for warbling vireos Vireo gilvus and yellow warblers mation in many small birds (Ve ´ zina et al. 2006, 2007; Swanson Setophaga petechia and increased during spring relative to fall mi- 2010; Liknes and Swanson 2011b), a few previous studies have also gration for yellow-rumped warblers (Zhang et al. 2015c). Pectoralis documented an absence of pectoralis muscle mass changes for win- FABPpm protein levels also showed a similar seasonal pattern of tering (Swanson et al. 2014a) or migratory (King et al. 2015) variation for yellow and yellow-rumped warblers, but not for war- phenotypes. bling vireos (Zhang et al. 2015c). Collectively, these studies suggest that pectoralis levels of the two sarcolemmal lipid transporters often increase in concert with increasing energy demands, but this is not Trans-sarcolemmal lipid transport always the case. Neither temperature nor photoperiod significantly altered pectoralis Reasons for the inconsistency between patterns of FAT/CD36 FAT/CD36 and FABPpm mRNA expression in this study. Winter or and FABPpm protein expression in the present study are unclear. migratory status in most bird species also fail to promote changes in Some studies have, however, documented different responses be- mRNA expression for these two sarcolemmal lipid transporters tween these two fat transporters in skeletal muscles after contraction (Zhang et al. 2015c, 2015d) and Stager et al. (2015) found no sig- or insulin-treatment in mammals (Chabowski et al. 2004). These nificant variation in mRNA expression of pectoralis FAT/CD36 two proteins often act in conjunction at the plasma membrane, but among temperature and photoperiod treatments for these same jun- translocation of FAT/CD36 and FABPpm to other locations in the cos. Moreover, cold and exercise training in house sparrows also did myocyte under conditions of altered energy demand may differ not change mRNA expression for FAT/CD36 or FABPpm (Zhang (Chabowski et al. 2004; Han et al. 2007). Such differences in trans- et al. 2015a). In contrast, pectoralis muscle mRNA expression of location could lead to different patterns of protein expression for FAT/CD36 was upregulated for captive white-throated sparrows the two sarcolemmal transporters, as documented in the present photostimulated to migratory condition (Zajac et al. 2011) and study. yellow-rumped warblers Setophaga coronata showed higher FAT/ CD36 and FABPpm mRNA expression during spring migration than during fall migration (Zhang et al. 2015c). The present study Intramyocyte lipid transport detected a strong positive correlation in pectoralis mRNA, but not Variation in FABP protein expression among acclimation treat- protein, expression between FAT/CD36 and FABPpm, suggesting ments in this study was difficult to interpret, as levels increased on co-expression of these genes in response to acclimation treatments. cold LD compared to cold SD and on warm SD compared to warm Regarding the difference in mRNA and protein expression with ac- LD. Pectoralis intramyocyte lipid transport is consistently an im- climation, some studies in mammals have also failed to detect correl- portant target of upregulation for migratory (Guglielmo et al. 2002; ations between mRNA or protein expression and the rate of fatty McFarlan et al. 2009) and winter (Liknes et al. 2014; Zhang et al. acid transport into myocytes (Luiken et al. 2003; Chabowski et al. 2015d) phenotypes in birds. In addition, photo-stimulated migratory 2006). In contrast, some recent studies document that FAT/CD36 white-throated sparrows (Zajac et al. 2011) and cold- and exercise- and FABPpm are ubiquitously expressed (Nickerson et al. 2009), trained house sparrows (Zhang et al. 2015a) also showed increases suggesting that the rate of trans-sarcolemmal fatty acid uptake de- in FABP expression. These data suggest that intracellular lipid pends on a cooperative role for the two fatty acid transporters transport is an important target of adjustment underlying elevation (Chabowski et al. 2007). The positive correlation between pectoralis of metabolic capacities with increasing energy demands. The in- mRNA expression for FAT/CD36 and FABPpm in this study pro- crease in FABP on LD under cold treatment in this study supports vided support for a cooperative role between these two trans- the migratory disposition hypothesis, as exposure to a LD photo- sarcolemmal transporters in birds. period for winter-collected birds could induce the spring migratory In contrast to results for mRNA expression in this study, tem- phenotype. On the other hand, within the warm treatment, SD birds perature and photoperiod treatments in the present study both had significantly higher FABP levels than LD birds, possibly to induced changes in pectoralis protein expression for both FAT/ maintain thermogenic function on a winter photoperiod. CD36 and FABPpm, with cold exposure effects being modified by Complicating this interpretation further, Stager et al. (2015) docu- photoperiod treatments. Pectoralis protein expression for these two mented cold-induced increases in pectoralis FABP mRNA expres- transporters had different responses to photoperiod, with FAT/ sion for these same individual juncos. We were unable to amplify CD36 increasing on cold SD treatments and FABPpm increasing on FABP mRNA in the present study, so we were unable to validate cold LD treatments. Because protein levels of these sarcolemmal this finding. lipid transporters are more directly indicative of fatty acid transport Given the inconsistent results in the present study, it is, perhaps, capacities than mRNA expression, post-transcriptional processing important to note that FABP serves not only as an intramyocyte of FABPpm and FAT/CD36 may alter protein levels (Bonen et al. fatty acid transporter, but also as a fatty acid receptor and may be 1999). As a consequence, fatty acid transporter protein expression co-regulated with fatty acid binding proteins on the cell membrane might be expected to show more variation than mRNA expression (Luiken et al. 2003). The membrane-bound and cytosolic forms of (Glatz et al. 2010) with varying energy demands, as detected in the FABP in pectoralis were positively correlated in the present study. present study. Previous studies of wintering American goldfinches FABP may also be more important to overall lipid transport and cold-trained house sparrows both showed elevated pectoralis capacity than membrane-associated lipid transporters because very Downloaded from https://academic.oup.com/cz/article-abstract/64/1/23/3091112 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhang et al. Temperature and photoperiod effects on myostatin and lipid transport 29 little fatty acid exists as free or unbound molecules inside muscle membrane-bound FABPpm. Similar correlations between these cells (Kiens 2006), whereas at least some fatty acid transport across FABPs have also been observed for exercise- and acute cold-trained membranes occurs by simple diffusion (Hamilton et al. 2002). house sparrows (Zhang et al. 2015a). Together with the positive Intramyocyte lipid transport capacity in birds may be especially im- correlations of pectoralis CS with CPT and HOAD, the data suggest portant to organismal metabolic capacities because birds rely almost parallel variation and integration of the various steps in fatty acid exclusively on exogenous lipids to fuel prolonged muscular activity transport and catabolism pathways in dark-eyed juncos in this (Jenni-Eiermann et al. 2002; Guglielmo 2010). In contrast, mamma- study. Such relationships are consistent with the concept of symmor- lian aerobic muscular activity is mainly fueled by carbohydrates and phosis and suggest that correlated variation is important for accli- lipid droplets inside the myocyte (Guglielmo 2010). Despite the im- matization to temperature and photoperiod in birds (Suarez, 1998, portance of FABPc to overall lipid transport in birds during elevated Swanson, 2010). Similar correlations among these pathways were energy demands, protein expression was not consistently correlated also observed in exercise- and cold-trained house sparrows (Zhang with elevated energy demands for juncos in this study, suggesting et al. 2015a) and during migration in white-throated sparrows complex interactions between temperature and photoperiod in the (McFarlan et al. 2009), but were not observed during migration or regulation of lipid transport in these birds. winter acclimatization for other birds (Zhang et al. 2015c, 2015d). Taken together, cold exposure, at least under some photoperiod As a result, evidence for the concept of symmorphosis of metabolic conditions, increased all three fat transporters, suggesting that pathways under conditions of increased energy demands is still in- changes in this pathway could contribute to elevated M under sum conclusive, but is seemingly more consistent for experimental ma- cold exposure in these same individual birds (Swanson et al. 2014c). nipulation under standardized laboratory conditions than under However, photoperiod did not consistently alter either muscle mass, natural conditions requiring elevated energy demands. the myostatin system, or fatty acid transport pathways. This result In conclusion, along with the data of Swanson et al. (2014c) and is, perhaps, not surprising given that the transcriptomic study of Stager et al. (2015), these data suggest that regulation of flight Stager et al. (2015) on these same birds demonstrated increased con- muscle mass, the myostatin system, and lipid transport capacities certed gene expression of the citric acid cycle and oxidative phos- are common methods for regulating metabolic capacities of birds in phorylation pathways on SD, but increased concerted gene general. Effects of cold acclimation or winter acclimatization, mi- expression in fatty acid metabolism pathways on LD. Because mi- gratory status, or photoperiod on these pathways, however, might gratory phenotypes can be stimulated by either or both photoperiod be species- or context-specific, so firm generalizations about regula- or exercise (Price et al. 2011), it is possible that fuel transport and tion of these pathways under different conditions promoting catabolism capacities did not change in the same direction due to increased energy demands are currently difficult to delineate. photoperiod alone. Exercise might also be important for inducing changes in metabolic pathways and physiological responses for the Acknowledgments migratory phenotype. Indeed, exercise training for house sparrows resulted in increases in pectoralis muscle mass, trans-sarcolemmal We thank Sol Redlin, Kathie Rasmussen, and Bob Garner for access to their and intramyocyte lipid transport capacities and cellular metabolic property or helps on collection of juncos. We thank Ming Liu, Kyle Kirby, intensities on constant photoperiods (Zhang et al. 2015a). Stephanie Owens, and Will Culver III for technical assistance in the labora- tory and ﬁeld. We also thank Jianqiu Zou and Yi-Fan Li for their advice on Western blots. Two anonymous reviewers provided helpful comments on a Correlations previous version of this article and we thank them for their efforts. In this study, neither pectoralis muscle mass, fat transport capacity, the myostatin system, nor regulatory enzymes for cellular metabolic intensity were significantly correlated with M . Positive correl- sum Funding ations of pectoralis muscle mass or cellular metabolic intensity with This research was funded by NSF IOS-1021218 to D.L.S. M have been observed in previous avian studies (Petit and Ve ´ zina sum 2014; Swanson et al. 2013, 2014b). We observed significant positive correlations between protein expression of the active form of myo- References statin and mRNA expression of TLL-1, but not TLL-2, which sug- Battley PF, Dekinga A, Dietz MW, Piersma T, Tang S et al., 2001. Basal meta- gests that TLL-1 serves as the major activator for myostatin in bolic rate declines during long-distance migratory ﬂight in great knots. juncos. Prominent changes in mRNA expression of TLL-1 but not Condor 103:838–845. 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Current Zoology – Oxford University Press
Published: Feb 1, 2018
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