Quantitative Analysis of Liver Protein Expression During Hibernation in the Golden-mantled Ground Squirrel*,S

L. Elaine Epperson{ddagger}, Timothy A. Dahl{ddagger} and Sandra L. Martin{ddagger},§

From the {ddagger} Program in Molecular Biology, Department of Cell and Developmental Biology, University of Colorado School of Medicine, P.O. Box 6511, Mail Stop 8108, Aurora, CO 80045


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammals that enter deep hibernation experience extreme reductions in body temperature and in metabolic, respiratory, and heart rates for several weeks at a time. Survival of these extremes likely entails a highly regulated network of tissue- and time-specific gene expression patterns that remain largely unknown. To date, studies to identify differentially-expressed genes have employed a candidate gene approach or in a few cases broader unbiased screens at the RNA level. Here we use a proteomic approach to compare and identify differentially expressed liver proteins from two seasonal stages in the golden-mantled ground squirrel (summer and entrance into torpor) using two-dimensional gels followed by MS/MS. Eighty-four two-dimensional gel spots were found that quantitatively alter with the hibernation season, 68 of which gave unambiguous identifications based on similarity to sequences in the available mammalian database. Based on what is known of these proteins from prior research, they are involved in a variety of cellular processes including protein turnover, detoxification, purine biosynthesis, gluconeogenesis, lipid metabolism and mobility, ketone body formation, cell structure, and redox balance. A number of the enzymes found to change seasonally are known to be either rate-limiting or first enzymes in a metabolic pathway, indicating key roles in metabolic control. Functional roles are proposed to explain the changes seen in protein levels and their potential influence on the phenotype of hibernation.


Mammalian hibernators display the physiological traits of a nonhibernator or homeotherm in the summer months, but in the winter function in a heterothermic manner. They spend most of the winter in a state of deep torpor during which body temperature is as low as –2.9 °C (1), and there are concomitant extreme reductions in heart, respiratory, and metabolic rates (reviewed in Refs. 2 and 3). These "bouts" of torpor last from as few as 5 days to as many as 35 weeks depending on the species. They are separated by arousals to euthermy that are 10–14 h on average for our model species, the golden-mantled ground squirrel, Spermophilus lateralis (unpublished calculations, see also Fig. 1). The arousals to euthermy are driven by endogenous mechanisms of rewarming and appear to be essential for survival, because the animals persist in regular arousals despite their high energetic expense (4).



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FIG. 1. Ground squirrel body temperature trace through an arousal cycle showing sacrifice times for winter (entrance) animals. A single representative interbout arousal is depicted showing the body temperature change (y-axis, °C) versus time (x-axis, 4-h increments). Arrows denote time of sacrifice for entrance animals. See "Experimental Procedures" for dates. Inset, body temperature trace for 90 days from a golden-mantled ground squirrel measured in our environmental chamber. Shown are the fall active period (FA), entrance into torpor (Ent), early torpor (ET), late torpor (LT), arousing (Ar), and interbout aroused (IBA). Such cycles are repeated numerous times (~20) throughout the winter season.

 
As in any organism, homeostasis is maintained by means of the fine-tuning of cellular processes that find their basis in differential patterns of gene expression and protein activity. Gene expression in the summer active liver is expected to resemble that of any euthermic mammalian liver; whereas during the hibernation season this expression pattern could change to reflect the unique biochemistry of hibernation. Such changes may adapt the animal for survival of heterothermy and other aspects of the torpid phenotype. Hibernation research has discovered examples of differential gene expression at the mRNA and protein levels (512). All of these differentially expressed genes are present in the genomes of nonhibernators, and their basic function was elucidated in nonhibernating animals. In the hibernator, the temptation is to search for genes that are unique to hibernators and demonstrate special function at low temperatures, but to date none of these "super genes" have been identified. Instead, current data lend credence to the proposed concept that differential expression of mammalian genes provides the basis for the phenotype of hibernation, rather than newly derived hibernation-specific proteins and enzymes (12). This conservation among mammals is significant in that it suggests it will be possible to utilize new bioinformatics approaches despite the lack of genomic information for ground squirrel, and also that it provides basis for medical applications that derive from an understanding of hibernation (13).

Research on differential gene expression in hibernating mammals has been primarily at the nucleic acid level in the identification of mRNAs whose expression varies seasonally. For more abundant RNAs, as most methods favor, there is a positive correlation between the steady-state levels of proteins and their corresponding mRNAs (14). However, the RNA studies are limited in that they remain at least one step removed from the phenotype. But protein abundance is a step closer to functionality, and technology is advancing such that these studies are now becoming feasible. The application of two-dimensional (2D)1 SDS-PAGE with LC-MS/MS enables a broad spectrum, noncandidate approach to analysis of relative protein expression ("proteomics"). Following these established approaches with newly developed staining and scanning methods results in a powerful means to quantitatively assess several hundred steady-state protein levels in multiple stages of hibernation. However, the use of proteomics in cross-species analysis such as this is still in its infancy; and ground squirrels as rodents are only distantly related to rat and mouse (15), the closest model organisms. This study establishes the effectiveness of proteomic approaches with regard to ground squirrel sequences.

The liver was chosen for this study due to its critical role in a number of processes that are likely to be crucial for survival of hibernation. Ground squirrels store fuel in the form of triacylglycerols in the adipose tissue and rely largely on this stored fat for survival of the winter (Ref. 16 and references therein). The processes of lipid metabolism include the lipolytic enzymes and proteins involved in transport of fatty acids, many of which are synthesized in the liver. The liver is also the site of synthesis for a number of enzymes involved in gluconeogenesis and ketone body formation, both processes required for fuel generation during the hibernation season (1720). Another major function of hepatocytes is the synthesis of bile, which is used by the gut for emulsification of dietary fats. During fasting, the requirements for bile acids may be greatly reduced and the lack of bile would affect cholesterol metabolism and some aspects of lipid mobility. Cholesterol not used for bile acid synthesis might be shunted for use in hormone synthesis, e.g. for use in the adrenal synthesis of corticoids. In addition to liver-specific alterations in protein expression, a number of processes are expected to be affected in most cell types. For example, the cellular machinery required for protein protection, stability, and turnover and for redox balance in the cell is expected to undergo changes with respect to season (3). These are a few of the many processes that could be affected at the protein level in the hibernator.

The goal of this work was to identify liver proteins that are differentially expressed or modified during hibernation. Initially, 2D gel comparisons were made between three stages of the hibernation cycle: summer active (SA), interbout aroused (IBA), and late in torpor (LT) (see Fig. 1). Each group had a sample size of four, and a number of differences were found. However, only seven spots were found that changed with significance (p < 0.05), and SA levels were higher than IBA and/or LT in all of these (21). We noted a great deal of individual variability that precluded determination of many statistical differences without a larger sample size. This variability along with a consistent lack of equivalent resolution in the LT gels (22) led to an experimental redesign. Earlier we proposed that interbout arousals might be essential to restore gene products that are slowly lost during torpor (23). If this is the case, then animals re-entering torpor after a replenishing period of interbout arousal will have fully restored their complement of required proteins; hence, protein samples from nine SA animals were compared with the same from nine entrance (Ent) animals. Ent animals are those that had been euthermic (35–37 °C body temperatures) for a complete arousal, ~10–14 h, and they were sacrificed upon their descent into torpor (body temperatures range from 30 °C to 16 °C, see Fig. 1).

Here we report the results from a comparison of total liver samples from two seasonal stages: SA and winter hibernation, specifically during entrance into torpor (Ent; Fig. 1). We found 84 reproducible 2D gel spots that changed in steady-state level seasonally with statistical significance (p < 0.05). Two-thirds of these were higher in entrance than in summer. Only six spots were not identified using the available databases, 10 others contained more than one protein, and 68 gave unique identifications. The power of this approach lies in the fact that hundreds of protein changes are assessed quantitatively in a single experiment, generating an unbiased and broad view of alterations in the biochemical pathways of hibernation. Collectively, the results from this study significantly enhance our current comprehensive understanding of the biochemical basis of the hibernating phenotype.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Acquisition of Tissue—
Golden-mantled ground squirrels (S. lateralis) to be sacrificed during hibernation were trapped in late August and abdominally surgically implanted with radio telemeters (VM-FH discs; Mini Mitter Co., Inc., Sunriver, OR) prior to the onset of hibernation for precise remote monitoring of body temperatures (10). After healing from surgery (~2 weeks), the animals were moved to an environmental chamber. Each cage housed a single animal and was placed on top of a receiving pad to capture and transmit the radiotelemetry signal of body temperature. The temperature in the chamber was lowered stepwise to 5 °C over 2 weeks, where it was maintained for the hibernation season. To mimic burrow conditions, the animals were kept in constant darkness without food and water while the chamber was at 5 °C. Telemetry data were collected every 10 min on a computer in an adjacent room using Datacol 3 software. The SA animals were trapped in May-July, maintained at 22 °C in a 12-hour light-dark cycle with food and water ad libitum and sacrificed for tissue collection within 12–36 h. Animals were sacrificed according to protocol in either summer (SA), body temperature (Tb) approx. 37 °C or entrance (Ent), Tb ranging from 30 °C to 16 °C; CO2 asphyxiation was used for all animals. Livers were removed, snap frozen in N2 (l), and stored at –80 °C until needed. The animals used for this study were sacrificed on the following dates: SA animals: 30 May (2 animals), 3 June (2), 23 June (2), 25 July (1), 27 July (2); Ent animals: 1 Dec (2), 18 Dec (1), 27 Dec (1), 12 Jan (1), 17 Jan (1), 18 Jan (1), 19 Jan (1), 26 Jan (1). All animal care and use procedures were approved by the University of Colorado Institutional Animal Care and Use Committee.

Tissue Preparation—
For each animal, ~200 mg of frozen liver was removed from –80 °C and homogenized using a Polytron (Brinkmann Instruments, Westbury, NY) in a sucrose buffer containing protease inhibitors, i.e. 0.5 M sucrose, 0.1 M phosphate, 5 mM MgCl2, 1 mM PMSF, 10 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin. Homogenate was passed through a 25-gauge needle 10 times and centrifuged 10 min at 4 °C, 500 x g. The supernatant was divided into small (20-µl) aliquots, snap frozen in liquid nitrogen, and stored again at –80 °C until use. One aliquot was removed to determine total protein content using a BCA Protein Assay Reagent Kit (Pierce, Rockford, IL).

2D Gels—
A methanol/chloroform precipitation was used to remove lipids from 200 µg of total liver protein at room temperature as described (24). The entire precipitate from the 200 µg was solubilized in sample buffer first by repeat pipetting, then shaking in a Thermomixer R (Eppendorf, Westbury, NY) at 30 °C for 30–60 min at 900 rpm (25). This protein solution was used to rehydrate a dried first-dimension strip (Immobiline DryStrip, pH 3–10 nonlinear, 18 cm; Amersham Pharmacia Biotech, Piscataway, NJ) overnight at room temperature under mineral oil. The strips were subjected to IEF (26) using a Multiphor II apparatus (Amersham Pharmacia Biotech) with focusing parameters as follows (in hh:mm): step to 500 V, 00:15; ramp to 3,500 V, 01:30; hold at 3,500 V, 15:00. After IEF, the first-dimension strips were reduced and alkylated in DTT and iodoacetamide, and the proteins were separated by size in the second dimension by SDS-PAGE on a 9–16% gradient gel. Note: each sample was run repeatedly (2–5x) until the same sample yielded two gels that were of identical high quality and useful for individual spot comparison across the whole gel. The gels were stained using SYPRO Ruby (Bio-Rad, Hercules, CA) as follows: all six gels from one set were placed together in 1 liter of fix for 1 h (10% methanol, 7% acetic acid), then overnight in 1 liter of SYPRO Ruby with gentle rocking in a foil-covered plastic container at room temperature. Stain was used repeatedly (5–6x) in separate experiments. The gels were removed into fresh fix solution and left to destain during the day; fix was replaced for another overnight destaining. The gels were scanned on a Typhoon 9400 (Molecular Dynamics, Sunnyvale, CA) using the green or blue laser for excitation. Each gel was scanned and the photomultiplier tube voltage adjusted until the most intense spots were in the linear range, just short of saturating, allowing for subsequent quantitation of all the spots on each gel. A control scanning experiment showed that photobleaching was negligible, as the same region scanned 10 times resulted in the same pixel volume each time, indicating that there was no loss of pixel signal with multiple scans. Gel images were analyzed with Melanie 4 software (GeneBio, Geneva, Switzerland). The group identification (ID), that is, the single number that represents the same protein spot on many gels, was assigned with the automated feature of Melanie 4 software followed by a significant amount of "manual" matching. The t test statistics were determined with the use of Excel software (Microsoft Corporation, Redmond, WA).

In-gel Tryptic Digests—
Gel spots were excised with clean razor blades on a UV light box and subsequently digested with trypsin essentially as described (25); although extraction times were increased for greater peptide recovery to 3x for 1 h each.

MS and Protein Identification—
The dried tryptic fragments from a single spot were resuspended in a small volume (10 µl) of 5% formic acid. This sample was loaded using a pressure bomb onto a narrow-bore (0.150-mm inner diameter) fused silica column that contained C18 reversed-phase packing material (Aqua C18/ODS 5-µm particle size (Phenomenex, Inc., Torrance, CA) from cracked column p/n 00A-4299E0). The sample was eluted from the column into the mass spectrometer (LCQ-Deca; ThermoFinnigan, San Jose, CA) by HPLC (Agilent 1100 series pump; Agilent Technologies, Wilmington, DE) and nanospray using a hydrophobicity gradient over 30 min. The buffers that comprised the gradient were 5% ACN, 0.1% formic acid (buffer A) and 80% ACN, 0.1% formic acid (buffer B). Full and tandem mass spectra were collected for each spot and analyzed using XCalibur and Sequest software (ThermoFinnigan), followed by a stringent filter, DTASelect (27). All of the positive matches are based on sequence identity at the peptide level and sequence homology at the protein level to sequences in the mammalian RefSeq database (downloaded December 2003: "vertebrate_mammalian," ftp.ncbi.nih.gov/refseq/release), because it contains very few ground squirrel sequences. Additionally, many spectra were analyzed against a small golden-mantled ground squirrel database, which was generated by translation of a partial cDNA database available at legr.liv.ac.uk under squirrelBASE. Although most proteins identified were absent from this database, if it happened that a protein was present, the peptide coverage was generally higher than for the mammalian database.

During the spectral collection, the full MS scan was followed by tandem mass spectral (MS/MS) scans of the three highest peaks, with a dynamic exclusion of 2 min. The parameters used for the initial Sequest search were as follows: parent and fragment masses were both set to monoisotopic, low to high mass limits for the precursor were 700–4,000 m/z (about 6 aa minimum), maximum number of internal cleavage sites was 2, peptide mass tolerance was 1.0, fragment ion tolerance was 0.0, trypsin was the enzyme used initially followed by a search with "no enzyme" if an ID was not obtained from the first run, searches were performed for covalently modified (alkylated) cysteines with a mass shift of +57. No statistics were applied. The DTASelect output is a list of proteins comprising virtually unambiguous IDs. Short peptides are filtered out, as are peptides with low cross-correlation scores from Sequest (XCorr) (27). The thresholds for DTASelect were set as follows: minimum XCorr for +1, 1.8; +2, 2.5; +3, 3.5; minimum DeltaCN, 0.08; minimum peptides per locus, 2. This list, for a single spot, often includes human keratin, trypsin, and a single other ID, which is the actual protein ID. However, 6 spots gave no ID and 10 spots gave more than one ID. Conclusions regarding a change in abundance of any of the proteins comprising these 10 spots will require independent measurements.

The accession numbers and species listed in Tables IIII were selected from a typically multi-species list of protein matches (see supplemental table). They represent the best protein ID in that their peptide recovery resulted in either the highest protein coverage found or was equivalent to the high coverage found in the homologous protein from another species. The peptide number as given in Tables I and II were tabulated conservatively due to the cross-species nature of this study. Frequently, multiple peptides are recovered in the mass spectrometer, each with its own fragmentation spectrum, that are found to overlap one another on the protein sequence; often these peptides differ in length by a single amino acid. We have chosen not to count these as separate peptides; rather, if the sequence coverage for one peptide is completely "accounted for" by another recovered peptide, this was counted as a single peptide. However, if two peptides partially overlapped, they were counted separately. For example, for group ID 502, three of the peptides recovered were: N.TVIVKPAEQTPL.T, K.PAEQTPLTALHVASLIK.E, and L.TALHVASLIK.E. The first two overlap by several residues, but each covers unique sequence, so they were counted as two separate peptides. The third peptide was not counted because it is completely contained within the second peptide.


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TABLE I Protein identifications for spots whose entrance levels are greater than summer

Listed are the group identification as assigned by Melanie 4 during analysis (ID); 2-tailed t-test p value, all are p < 0.05; *, p < 0.01; **, p < 0.001; the average fold change in mean % volume Ent/SA (Fold change); number of gels in which a spot is clearly defined (n, SA) (n, Ent); protein identification(s); abbreviation of the protein name (Abbrev.); the molecular weight of the human homolog (MW); the number of peptides recovered from the database (No. of pept.) (Note: for the method for peptide counting, see "Experimental Procedures"); a representative NCBI gi accession number for one of the protein matches (Accession); and the species for the accession number given (Species). If multiple IDs were obtained for a spot, the names were listed in separate rows. For these, the group ID, p value, fold change, and n columns were left blank as they are the same as that of the preceding row. A complete list of peptides and their mass spectral parameters is available in a supplemental table. In No. of pept. column, * indicates the inclusion of ground squirrel peptides in the count.

 

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TABLE III Information about peptides for proteins that were identified from a single peptide

The group ID is given, along with the protein identification, the peptide sequence, the Sequest XCorr and {Delta}CN values, the peptide precursor mass and charge, the proportion of the peptide that was accounted for by recovered b and y ions, the accession number (GI) for the protein of best match, and the species of that accession number.

 

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TABLE II Protein identifications for spots whose summer levels are greater than entrance

Listed are the group identification as assigned by Melanie 4 during analysis (ID); 2-tailed t-test p value, all are p < 0.05; *, p < 0.01; **, p < 0.001; the average fold change in mean % volume SA/Ent (Fold change); number of gels in which a spot is clearly defined (n, SA) (n, Ent); protein identification(s); abbreviation of the protein name (Abbrev.); the molecular weight of the human homolog (MW); the number of peptides recovered from the database (No. of pept.) (Note: for the method for peptide counting, see "Experimental Procedures"); a representative NCBI gi accession number for one of the protein matches (Accession); and the species for the accession number given (Species). If multiple IDs were obtained for a spot, the names were listed in separate rows. For these, the group ID, p value, fold change, and n columns were left blank as they are the same as that of the preceding row. A complete list of peptides and their mass spectral parameters is available in a supplemental table. For ID 349, the MW given is for the rat homolog rather than human. In No. of pept. column, * indicates the inclusion of ground squirrel peptides in the count.

 

    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An individual total liver protein sample was separated by isoelectric point in the first dimension and by size in the second dimension. The samples begin as total protein of the liver, but only the most abundant and soluble are visible on the finished gel. About 900 spots are resolved on each gel, and the liver produces ~10,000 proteins. Some proteins are represented in more than one spot due to posttranslational modifications. From this, we estimate that about 3–5% of the liver protein complement is represented in a quantifiable way on the 2D gels. The percent volume for each spot was found using Melanie 4 software and the comparison made with a two-tailed Student’s t test, two-tailed because for almost all spots it was not known whether to expect an increase or a decrease in winter. A total of 961 groups (i.e. the same protein spot from all gels) were subjected to t tests, and 130 were found to differ with a p < 0.05. Of these, 84 were found to be reproducibly resolved spots on enough gels for statistical analysis, in most cases this was all 18 gels. Fig. 2 shows two of the 18 gels used in this analysis; one is a summer sample, the other a winter (entrance) sample. Protein spots that showed a statistically significant change are indicated. These images demonstrate the relatively high resolution throughout most of the gel, allowing for quantitative assessment and comparison. Of the 84 reproducible and significantly changing groups, 28 had a higher protein steady-state level in summer, and 56 were higher in winter (entrance); that is, two-thirds of the proteins that changed were higher in entrance.



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FIG. 2. Sample 2D gels. Shown are one gel from each of the two stages of comparison, one SA total liver protein sample (left) and one Ent (right). The proteins were separated by IEF in the first dimension, pH 3–10, then by size in the second dimension. Markers for pH are given along the top of the gels; markers for size are on the left (kDa). Spots found to be higher in summer are indicated with their group number on the summer gel, same for entrance.

 
Fig. 3 shows a small section from all 18 gels used in the analysis; all whole-gel images are not shown due to space limitations, but in these details, the reproducibility among gels for this spot is demonstrated. The spot shown, group ID 1139, subsequently identified as the liver isoform of fatty acid-binding protein (L-FABP), demonstrated a 2.8-fold induction in entrance over summer. In initial experiments (n = 4 per group) comparing summer liver to samples from either IBA livers or those of animals sacrificed in LT, the sample to sample variability was too high to discern a significant difference from summer for this protein. However, in using the more synchronized entrance samples and a larger sample size, the winter to summer comparison is highly significant, with a p value of less than 1 x 10–6 for this spot.



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FIG. 3. 2D gel details of L-FABP spot on all 18 gels. To demonstrate gel reproducibility, the region containing L-FABP on each gel is shown here. Upper panels are from the SA gels, lower panels are from Ent. Other protein spots were included for reference.

 
Because the percent volume for each spot results in an internal normalization to each gel, we needed to assess whether total pixel volume and spot numbers differed greatly between summer and entrance gels. If they do differ in a seasonal manner, this could generate artifacts during normalization that do not truly reflect biologically relevant changes. Therefore, in order to do this comparison, the sums of the spot pixel volumes and spot numbers for each gel were found, nine from summer and nine from entrance. These were compared by t test and were found not to differ (p > 0.1 in both cases). Because neither of these parameters differed greatly, it is possible that even a small difference, e.g. 1.12-fold for group ID 596 (small ribosomal protein p40), reflects an authentic seasonal change.

Spots found to be significantly different according to a p < 0.05 were excised, digested in-gel with trypsin, extracted from the gel, and subsequently identified by MS. The names of proteins found, the spot ID, and the fold difference in the mean are shown in Tables I and II. Protein identifications are divided into those that showed an increase in entrance over summer (Table I) and summer over entrance (Table II). The purpose of including the molecular mass of the human homolog is that this is one parameter that was used to validate protein identifications. The molecular mass of a mammalian homolog is generally quite close to that of ground squirrel on the 2D gels. (Compare group IDs in Fig. 2 to group IDs and their corresponding molecular masses from Tables I and II.) The discovery of 84 spots that were found to differ significantly, some of which were small changes (e.g. spot group 783, 1.24-fold change), but still highly significant (p < 0.0005), indicates that this approach can effectively discern small changes in protein levels with good reproducibility, providing strong evidence that such a change has occurred. The cause and effect of the change in protein level remains unknown, however, and will require further experimentation.

A sample of the peptides recovered for one protein using a "mammalia" database of all available mammal protein sequences is shown in Fig. 4. Because the ground squirrel genome sequence is not available at this time, we were forced to rely on homology and sequence conservation to make protein identifications. The ground squirrel peptide sequence typically matched its homolog in a number of different organisms, although the regions of greatest similarity varied from species to species. Peptide coverages increased as sequences from more species were added to the database, and consequently so did the chance of obtaining a positive protein identification. As few as two peptides with quality LC-MS/MS spectra are enough in many cases unambiguously to identify a protein and, in the case of longer peptides, sometimes only one is needed (28). Four proteins in this study were identified with a single peptide match, and the specific information about those peptides is given in Table III. The approach using 2D gels followed by LC-MS/MS clearly can be used with success to identify proteins from an organism whose genome remains largely unsequenced, provided it shares significant sequence similarity with a model organism.



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FIG. 4. Protein sequence alignment of peroxiredoxin 2 mammalian homologs (PRDX2, group ID 1043) showing peptide recovery. Ground squirrel mass spectra that matched the database are depicted in bold. The highest sequence coverage for any one animal was 30% (59/199 amino acids for Chinese hamster); combined coverage was 35% (69/199 amino acids). Note: Although the cow sequence is identical to the others within the last recovered peptide, the preceding residue (T) prevented its being recognized as a theoretical tryptic fragment digestion site during creation of the database, because trypsin cleaves only after K and R.

 
The proteins have been grouped into broad functional categories according to their cellular roles as defined in online databases such as Swiss-Prot (us.expasy.org/sprot) and EMBL Harvester (harvester.embl.de). Their functions fall into a number of anticipated biochemical pathways: energy homeostasis, fuel (lipid and glucose) mobility and use, protein turnover, cellular redox balance and detoxification, among others (Table IV).


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TABLE IV Putative functional groupings of proteins that change in steady-state level with the hibernation season

The functional pathways are in bold. Proteins demonstrating a winter increase are listed in the left column, a winter decrease in the right column. Following each protein name is the fold change for that spot and the statistical significance taken from Tables I and II. All are p < 0.05; *, p < 0.01; **, p < 0.001. If no fold change is given, the corresponding spot contained more than one identified protein. If more than one fold change is given, more than one spot gave the same protein ID.

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we found that it is possible quantitatively to evaluate and compare multiple samples on 2D gels from SA liver and that of animals entering torpor to identify proteins that differ in a state-dependent manner. The fact that our model system is an animal whose genome is mostly unsequenced, while a disadvantage, is not insurmountable, and we were able to obtain unique identifications for most of the proteins that varied by using current methods of LC-MS/MS analysis. The identified proteins have known physiological roles in other systems that could play a plausible role in the hibernating phenotype. A number of the enzymes that were found to alter are rate-limiting and/or first enzymes in a particular pathway. Control over such enzymes often results in control over the entire pathway, implying that only that one enzyme needs to be regulated with the hibernation season (29) and not the other enzymes in that same pathway.

The use of proteomics in cross-species analysis, that is, identifying proteins from an organism whose genome is mostly absent from the database, should be approached with caution, although efforts are being made to find methods that will improve the fidelity of these comparisons (30, 31). In this study, the most comprehensive coverage of the ground squirrel protein sequence, and therefore the greatest confidence in the protein identity based on Sequest analysis, was obtained using a database constructed of all available mammalian protein sequences, although no organisms routinely gave greater coverage than rat and mouse. Ground squirrels are actually quite distant relatives of rat and mouse, sharing their most recent common ancestor ~100 million years ago (15). The success with which we were able to use the mammalian protein sequence database suggests that species with similar divergence times from any model organism will prove equally successful, greatly expanding the utility of the model organism sequence databases for studies of interest to comparative biologists. But beyond sequence availability, even if the sequences share high sequence identity, the function of homologous enzymes in a separate organism, a separate species, or a separate subcellular compartment may not necessarily be the same as in the original. The data from this study should be recognized as hypothesis-generating, i.e. as a guide for testing the involvement of specific biochemical pathways in hibernation using more targeted methods and experiments.

In addition to being quantitative, an advantage of the 2D gel proteomics approach is the potential for visualizing posttranslational modifications. Often a polypeptide that has undergone modification will display itself in a series of spots with each adjacent spot containing, for example, another phosphate group that alters the pI and shifts the size slightly. While it has been proposed that differential posttranslational modification to existing proteins is a major form of biochemical control for protein activities during hibernation (29), we found no such examples in this particular pairwise comparison. But seasonal differences are much more likely to manifest themselves in changes of the steady-state levels of mRNA or proteins; whereas the more finely timed changes during the cycles of torpor and arousal in winter are more likely to be controlled at the level of posttranslational modification, e.g. rapidly reversible phosphorylation (3). In this study, we found that if there was a series of spots on the gel that changed with the season, and there were several, then all of the spots in the series changed in the same direction instead of one spot being elevated and the next reduced. To address the question of the importance of differential phosphorylation or other forms of posttranslational modification in hibernation, proteins from early and late entrance or early and late arousal should be examined, although the arousal analysis could be complicated by individual variation as described for torpor and IBA gels in the introduction.

Some Functional Implications of Seasonal Changes in Protein Levels—
Many proteins changed with respect to season. In an effort to create a more digestible picture of the changes taking place, we limit our discussion to several pathways that are affected and a model of their potential role in the hibernating phenotype. These pathways and their changes will be discussed in the context of the current understanding of hibernation as well as in the biology of nonhibernation systems in which the protein functions were elucidated.

Detoxification—
Several aldehyde dehydrogenases demonstrated a significant change in levels between summer and entrance. All but one of them, aldehyde dehydrogenase 1A1, were increased in entrance over summer. These enzymes are known to play a role in detoxification of xenobiotic aldehyde derivatives resulting from lipid peroxidation, which due to the animals’ fasting state are surprising to find as up-regulated. However, the natural accumulation of conjugated dienes indicates that lipid peroxidation occurs during torpor (32), and this process generates aldehydes that must be processed for maintenance of redox balance and protection from oxidative damage. In contrast, aldehyde dehydrogenase 1A1 is a cytosolic enzyme and a member of the class I aldehyde dehydrogenases. They show a great deal of specificity for retinaldehyde, a derivative of retinol (vitamin A) that is introduced in the diet (33). Because it appears that the main substrate is dietary, this enzyme may be one of many that are down-regulated due to disuse in the fasting state.

Nucleotide Biosynthesis—
Three enzymes were found that are influential in the formation of nucleotides: 10 formyltetrahydrofolate dehydrogenase (FTHFD), which is substantially down-regulated in entrance (3.2- to 7.3-fold depending on the spot), transaldolase 1 (1.6-fold up in entrance), and UMP-CMP kinase (1.5-fold up in entrance). FTHFD demonstrated the largest seasonal change of any of the spots examined, a 7.3-fold reduction in winter for spot 173. Its substrate, 10 formyltetrahydrofolate (10 formyl THF), is the formyl donor in two steps of the pathway for de novo purine biosynthesis, and the presence of active FTHFD enzyme results in depletion of 10 formyl THF available for use in this cellular pathway. The down-regulation of FTHFD has been proposed as a cellular mechanism to enhance cell proliferation, as its expression is reduced at both the mRNA and protein levels in several types of cancer (34). In this case, rather than streamlining gene expression because its function is not required, this may represent a derepression mechanism employed by this hibernator to allow for hyperactive nucleotide and protein synthesis during interbout arousals. We also found that 10 formyl THF synthase is down-regulated in entrance, but to a lesser extent. The balance of these enzymes and their reaction properties requires further elucidation. Transaldolase 1 catalyzes a reversible reaction in the pentose phosphate pathway. Depending on cellular requirements, its up-regulation can result in accumulation of ribose-5-phosphate for use in nucleotide biosynthesis. Another enzyme involved in replenishment of nucleotide stores is UMP-CMP kinase, which specifically phosphorylates the precursors to UDP and CDP to furnish the cell with pyrimidines. Another kinase is required for the succeeding phosphorylation to the triphosphate form.

Protein Turnover—
Protein turnover, that is, the degradation of partially degraded or long-lived proteins and the synthesis of new proteins, is established as a characteristic feature of the hibernator’s interbout arousal (3539). Much of the machinery required for this global turnover has not yet been seen to change in the hibernator, although there are examples in the literature (38, 40). The process of protein turnover can be divided roughly into three parts: protein degradation, protein synthesis, and quality control of proteins, or their folding and transport.

Protein degradation heavily involves the activity of the proteasome of which three components were found to be up-regulated in entrance animals in this study: 26S proteasome ATPase 4 isoform 1, non-ATPase regulatory subunit 9, and ATPase 3. The proteolytic function of the proteasome is extremely sensitive to reductions in temperature, and the accumulation of ubiquitin conjugates in LT may call for the hyperactivation of proteasomal machinery during IBA (reviewed in Ref. 41). We also found cathepsin B to be up-regulated in entrance over summer levels by 1.5-fold; this protease of the endosomal/lysosomal cysteine protease family acts to degrade peptides, proteins, and toxins that enter from either outside the cell or from other cellular compartments (42). Another family member, cathepsin H, is seasonally up-regulated in the winter at the level of transcription (10). Phenylalanine hydroxylase, the first and rate-limiting enzyme in the degradation pathway for phenylalanine and tyrosine, was found to be reduced 1.4-fold in entrance. This reduction may act to preserve functional amino acids for use in reconstructing the protein pool.

The synthesis of new proteins requires functional machinery for translation in addition to the machinery needed for folding and delivery of functional proteins. The small ribosomal protein p40 was slightly up-regulated in entrance, as was the eukaryotic translation initiation factor, 4A-1. The first factor mediates the interaction between an mRNA and the 40S ribosomal subunit. Another interesting component of the translation machinery is the UNR interacting protein (aka MAWD), which has been shown to enhance internal ribosome entry site activity, a means of cap-independent translation (43). Also required for new protein synthesis are the chaperone proteins that act as facilitators of proper folding, some of which are stress-induced. BiP (GRP78) is a resident of the endoplasmic reticulum that assists in proper protein folding, and we found it to be elevated in the entrance samples by about 1.3-fold. Chaperonin {theta} subunit (TCP-1 containing) is another chaperone that is specifically implicated in the proper folding of actin and tubulin, two cell structure proteins that demonstrated elevated levels in entrance over summer. Three other chaperone proteins, HSP60, HSP75, and GRP75, were all found to be at a lower level in entrance than in summer. This may indicate that the animals are not under stress conditions during entrance into hibernation, and it may be that the early arousal period would find higher levels of these proteins. GRPs 75, 78, and 94 demonstrate induction upon arousal from hibernation in the brain of bats (44), GRP75 is elevated seasonally in the intestine of 13-lined ground squirrels (32), and numerous other experiments indicate that expression of the stress-response proteins varies greatly with the tissue assayed and precise time point of measurement (3). Complementing these chaperone proteins, which can dually function as protein folding chaperones and defenders against oxidative damage, several proteins that change in level seasonally are directly involved in management of oxidation/reduction state (see Table IV, redox balance).

Gluconeogenesis—
The process of gluconeogenesis during interbout arousal has long been recognized as a feature of hibernation (45, 46). Glycogen stores in liver and muscle of the arctic ground squirrel are replenished during interbout arousal, and glucose levels in the plasma at high levels appear to be important for the arousal process (17, 19). These glycogen stores must then be regenerated during arousal and interbout arousal. In the current study, we found three key enzymes for the process of gluconeogenesis to be up-regulated in the entrance period. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase 2 (PEPCK) catalyze the conversion of pyruvate to oxaloacetate to phosphoenolpyruvate (PEP), a gluconeogenic substrate. Additionally, depending on the subcellular location of the second and rate-limiting enzyme, PEPCK, the cell may have to employ the malate/aspartate shuttle for transport of oxaloacetate to the cytosol. Malate dehydrogenase is also up-regulated in entrance relative to summer, and this enzyme reduces oxaloacetate to malate for transport out of the mitochondria, because gluconeogenesis occurs in the cytosol. The cytosolic form of malate dehydrogenase then carries out the reverse reaction, and the resulting oxaloacetate can be converted to PEP by PEPCK. PEPCK activity is known to be elevated in the winter in 13-lined ground squirrels (47). The function of all of these enzymes relies on adequate levels of the precursor pyruvate as documented in the arctic ground squirrel (17). The very high levels of lactate released from muscle into the plasma are quickly depleted during the rewarming of arousal, probably being taken up by the liver and kidney, and the doubling of activity for hepatic and renal lactate dehydrogenase during late arousal would result in a large accumulation of pyruvate for use as a substrate for new glucose synthesis during IBA (45).

Another source of substrate for gluconeogenesis that is recognized in hibernators is the glycerol derived from triacylglycerol catabolism. We did not see any changes in the enzymes that catalyze glycerol’s conversion to dihydroxyacetone phosphate for use in this pathway, although this may be simply because these enzymes were not resolved in the 2D gel system, or abundant enough to detect by the methods used. If these enzymes do resolve but do not change in abundance, our study would not have found them, because only those spots that changed in abundance were analyzed by MS to determine their identity. Furthermore, the percentage of glucogenic precursors that are contributed from this source is thought to be substantially lower than that from lactate and amino acids (17).

Lipid Metabolism and Mobility—
No discussion of mammalian hibernation is complete without the mention of lipid metabolism. The shift in fuel source to lipids during deep torpor is a key element for survival of the hibernation season. We found a number of components of this process that change in a seasonal manner. The liver isoform of fatty acid-binding protein, L-FABP, known in other systems for its transport function of long chain fatty acids, showed an induction in entrance of 2.8-fold in one spot and 3.7-fold in another. The liver isoform is unusual in that it binds with high affinity to two long-chain fatty acids in addition to bile salts, heme, and other small hydrophobic moieties (reviewed in Ref. 48). These two spots with the same identification probably represent two different forms via posttranslational modification, both induced in these winter animals. FABP is known to reside in various parts of the cell, and this modification could be some form of membrane tether or other mechanism of localization of the protein. A change in charge state of FABPs has been shown to affect the membrane interaction capabilities (48). Other isoforms of FABP have been reported in heart, brown adipose, and skeletal muscle as induced in the winter season at the nucleic acid and protein levels (49). The higher levels of the long-chain-preferring acyl CoA dehydrogenase and carboxylesterase 2 are consistent with an overall increase in catabolism of long-chain fatty acids, the lipid source derived from triacylglycerols, the main unit of lipid storage in hibernators (16). Acyl CoA dehydrogenase (long chain, LCAD) catalyzes the first and most critical step in the mitochondrial pathway of long-chain fatty acid ß-oxidation, a crucial fuel-providing pathway for hibernators. Carboxylesterase 2, which increases in entrance over summer by 2.6-fold, is able to hydrolyze long-chain fatty acid esters, although to date has been primarily associated with the hydrolysis of xenobiotics. Interestingly, its family member, carboxylesterase 3, which we found to be down-regulated in entrance 1.8-fold, does not have an affinity for fatty acyl esters. The potential function of carboxylesterase 2 in the liver is to assist in the dismemberment of imported triacylglycerols for the formation of long-chain free fatty acyl groups to be used as fuel. ApoA1, although one of 2 IDs in spot 983 (see Table I), was shown previously to be induced at the RNA level in winter (10); its up-regulation is consistent with the increased plasma transport of lipids and the need for their rapid mobilization during interbout arousal from adipose tissue to other tissues. The winter down-regulation of those enzymes involved in ß-oxidation of short and branched chain fatty acids was also reflected in our data, i.e. dihydrolipoamide branched-chain transacylase and two other acyl-CoA dehydrogenases, C2 to C3 short chain and short/branched chain.

When the acyl-CoA dehydrogenase acts on a long-chain fatty acyl group, FAD is reduced to FADH2, and this moiety is subsequently reoxidized by the mitochondrial electron transport transport chain. Two enzymes involved in the relay of electrons were found in this study to be up-regulated in entrance over summer. Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) and NADH coenzyme Q reductase are both elevated 1.5-fold over summer. This process of electron transport results in the generation of two more ATP molecules and may facilitate an increase in energy availability during the subsequent arousal.

Aldo-keto reductase family 1 cytoplasmic 4 (AKR1C4) plays a critical role in the formation of bile because it produces 5ß-chloestane-3{alpha},7{alpha}-diol, a committed precursor of bile acids (50). Down-regulation of this enzyme may simply be a result of the fasting state wherein dietary fats are not being acquired, thereby minimizing the need for their emulsification. Because bile production is a main function of the liver, it is not surprising to see a reduction here if this process is less in demand. Additionally, because AKR1C4 plays a role in cholesterol and fat metabolism, its 1.3-fold reduction from SA to Ent may have a redirecting effect on fatty acid metabolism. For example, cholesterol that would have been used in bile acid biosynthesis could be funneled into pathways for use as precursors to corticosteroids synthesized in the adrenal gland, impacting the balance of glucose and lipid mobility.

Ketone Body Formation—
The ketone bodies are derived from acetyl CoA and are therefore tightly correlated to the process of fatty acid oxidation. They serve as the fuel of choice for heart and skeletal muscle tissue; during starvation when glucose is not available in the plasma, they are fuel for the brain. Elevated levels of ketone bodies during hibernation have been measured in the plasma of Belding’s ground squirrels (19), in black bears (20), and jerboa (51). While we only found a single enzyme of the ketogenic pathway, it is up-regulated in entrance almost 5-fold over summer. This enzyme is hydroxymethylglutaryl-CoA synthase 2 (HMG-CoA synthase 2, mitochondrial), and catalyzes the condensation of acetoacetyl-CoA with another acetyl-CoA to form HMG-CoA, which is then degraded to acetoacetate and acetyl-CoA by HMG-CoA lyase. Acetoacetate, its derivative, D-ß-hydroxybutyrate, and acetone comprise the ketone bodies that serve as fuel for muscle tissue, especially heart.

In summary, the changes seen at the protein level during the hibernation season are consistent with a general replenishment of proteins, lipid components, and carbohydrate stores required for survival of the next bout of torpor (3). A hyperactivation or suppression of specific processes results in a quantitative change in the steady-state levels of about 9% of the total liver protein complement. About 6% were found to be up-regulated in entrance over summer, and about 3% were down-regulated in entrance. Because the gels allowed visualization of only ~3% of the liver proteins, and we were able to measure differences with respect to hibernation stage, then it follows that there remains a great deal of untapped information regarding seasonal protein changes in the liver. Nevertheless, the results of this study provide a major increase in the numbers of known, differentially expressed proteins during hibernation. We hope in the future to increase the depth of the screen in order to recover more changes in less abundant and hydrophobic proteins and in other tissues. A more comprehensive knowledge of the protein level changes in liver and in a number of other organs will assist greatly in implementing practical medical applications of hibernation research.


    ACKNOWLEDGMENTS
 
We thank G. Maniero and C. Carey for assistance with animal care and procurement, N. Ahn for critical technical advice, C. Wu and M. MacCoss for advice and assistance in setting up the LC-MS/MS and analysis, and D. Branciforte, P. Li, K. Howell, and C. Finnigan for helpful discussion.


    FOOTNOTES
 
Received, March 25, 2004, and in revised form, July 14, 2004.

Published, MCP Papers in Press, July 20, 2004, DOI 10.1074/mcp.M400042-MCP200

1 The abbreviations used are: 2D, two-dimensional; SA, summer active; Ent, entrance; ET, early torpor; LT, late torpor; Ar, Arousing; IBA, interbout aroused; ID, identification; FABP, fatty acid-binding protein; FTHFD, 10 formyltetrahydrofolate dehydrogenase; 10 formyl THF, 10 formyltetrahydrofolate; PEPCK, phosphoenolpyruvate carboxykinase 2; PEP, phosphoenolpyruvate. Back

* This work was supported by ARO DAAD19-01-1-0550 and DARPA N66001-02-C-8054. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material. Back

§ To whom correspondence should be addressed: Program in Molecular Biology, Department of Cell and Developmental Biology, University of Colorado School of Medicine, 4200 E. Ninth Avenue, B111, Denver, CO 80262. Tel.: 303-315-6284; Fax: 303-315-4729; E-mail: Sandy.Martin{at}uchsc.edu


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