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Seasonal changes in plasma proteins and eipids in the belding ground squirrel (Spermophilus beldingi beldingi)
(*~¢rIp. lftot'ht*t}l t~hL~tol. 1970. t ~ l 54A. I~I' 2 3 q I," 2 4 3 t','l~hJ~'m* Prt'.~.~. I ' r t n t , ' d tn Gr,'dt llril,ttn
S E A S O N A L C H A N G E S IN PLASMA P R O T E I N S A N D E1PIDS IN THE B E L D I N G G R O U N D S Q U I R R E L
(SPERMOPHILUS BELDINGI BELDINGI) SI'IL:NA NAN-YING HUANG AND MARTIN L. MfIt,'.'I'ON Biolog) Dep;irtmcnt, Occident College, Los Angeles. CA 9(X)41. U.S.A.
(Receired 19 .'hqlust 1975) Abstract--1. Concentrations of plasma lipid and plasma protein, and i~lasma protein composition ~vrc siudied in two populations of Bclding ground squirrels at high ahitude throughoul their season of activity. 2. Plasma lipid was highest in lactating fcm~0cs and tended to vary othcrwisc in direct relationship to total hod 3" lipid. 3. Plasma protein decreased during the first weeks after emergence from hibernation ~md increased thereafter. It is suggested that this was related to regu[alion of circulating crythrocytes. 4. There was some seasonal variation in all protein fr~lctions, with greatest klbility seen in :x2-globulin followed by albumin. Squirrels living at 2130 m exhibited a prcalbutnit~ fraction more frequently than those from 3020 m. 5. Seasonal cycles in all plasma constituents tended to be more pronounced in squirrels living at the higher altitude.
IN'rt~ooucl"ION FOR T1~r~past several d e c a d e s t h e b i o l o g y o f h i b c r n a t o r s h a s b e e n e x t e n s i v e l y investigated. P r i m a r y e m p h a s i s in these s t u d i e s h a s b e e n o n the m o d e s a n d mechanisms of body temperature regulation during p h a s e s o f t o r p o r . Recently, h o w e v e r , i n t e r e s t h a s quickened concerning other aspects of the annual cycle. Increasingly, a t t e m p t s h a v e b e e n m a d e t o relate s e a s o n a l c h a n g e s in f u n c t i o n a l c a p a c i t y t o e n v i r o n m e n t a l events. T h e a i m , of c o u r s e , is to u n d e r s t a n d h o w a d a p t a t i o n to t h e e n v i r o n m e n t ~ i n d u c e d a n d effected. D u e to its u b i q u i t o u s role in b o d y f u n c t i o n a n d a m e n a b i l i t y to study, b l o o d a n d its c o n s t i t u e n t s h a v e b e e n a f a v o r i t e f o c u s for t h e s e i n v e s t i g a t i o n s . In o u r s t u d y we h a v e m e a s u r e d q u a n t i t a t i v e seas o n a l c h a n g e s in b l o o d p l a s m a molecules, specifically p r o t e i n s a n d lipids, o f a h i b e r n a t o r , t h e B e l d i n g g r o u n d s q u i r r e l (Spermophihls beMinoi heMingi), d u r ing its i n t e r h i b e r n a t o r y s e a s o n o f activity a n d n o r m o t h e r m i a . W e h a v e related t h e s e c h a n g e s to t h e major events of summer activity; reproduction and p r e h i b e r n a t o r y fattening.
METHODS
This study was conducted on two pgpulalions of S. b.
anaounti~g to as much as 4 weeks (Morton. 1976) and the schedules shown ill Fig. 1 arc only approximate for a given year. Animals were collected with livetraps b;tited with pear, at butter then held separately with nesting material and water but no food for 24 hr prior to sampling, Sampling was always done in the morning to minimize possible diurn-d rhythmx in blood constituents such as those found by Scheving et ul. (1968) in the rat. Body weights were taken to within 0.1 g ,;raa pan balqnce. At sampling. :miroals were lightly a~esthetizcd with ether and 2-3 ml of blood were obtait~ed via cardiac puncture. Blood was centrifuged at once and total plasma protein concerttration was measured with a refi'aetomctcr. The remaining plasma was stored in sealed tubes at ca. 2'~C for no longer than 24 hr before further analysis. Subsequently, plasma lipid concentration was determined sp¢ctropholometrically using the method of Zollner & Kirzch (1962) except that a standard containing 750 mg lipid/100 mt alcohol (formula 3-A. American Hospital Supply Co., Miami, Florida) was used instead of corn oil. Plasma protein components were analyzed clcclrophorctically using Sepraphore 111 cellulose polyacetate strips at pH 8-8 and ionic strength 0.05 (Gelman Instrument Co.. Miami, Florida). A constant 250 V was applied for 60 rain. Strips were stained with Ponceau S. dcstained with three washes of 5~,~ acetic acid. dehydrated in two baths of absolute methanol, and cleared in 7 : t v/v methanol-acetic acid. After drying, strips were scanned with a densitometer and the curves obtained from protein bands were integrated.
beldingi living in meadows of the Sierra Nevada M o u n t a i n s in M o n o County, California. One population was at about R ESU LTS 2130 m elevation at Big Bend. the other at about 3020 na elevation at Tioga Pass. These populations were only Plasma lipid concentrations I0 km apart but because of the difference in climatic condiN e w l y e m e r g e d a d u l t m a l e s ,at T i o g a Pass in m i d tions associated with altitude, the active season of squirrels M a y h a d a m e a n p l a s m a lipid c o n c e n t r a t i o n o f 598 at Big Bend began a n d ended 6--8 weeks earlier than those m g ~ (Fig. 2, upper). T h e i r lipid t h e n d e c r e a s e d steadat Tioga Pass (Fig. 1). Events within the two study populaily t o a s e a s o n a l low in m i d - J u n e o f 432 m g ~ . T h i s tions for a given active season were similar in sequence and duration. There was interannuai variation, however. low w a s followed by a n i n c r e a s e t h a t was m a i n t a i n e d 239
240
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Fig. I. Major events in the active season of aduh S. h. heldinfli at Big Bend (upper calendar) and at Tioga Pass (lower calendar). near or above 600 mg "//,, until immergence in August, In adult females a seasonal low in mean plasma lipid concentration of 552 nag 'X, occurred soon after emergence. This was followed by a significant increase (P < 0.05) to 810 mg ?/,, in mid-June when most females had begun lactating. Subsequently, plasma lipid decreased in females to about the same concentrations seen in males. Starting in mid-July, plasma lipid began increasing in both sexes; maxima of 825 mg ~,, in females and 700 mg %, in males being attained in mid-August at the very end of their active s~ason.
There was little seasonal variation in plasma lipids in Big Bend animals (Fig. 2, lower), but levels did tend to increase during the last 2 m o n t h s of activity as at Tioga Pass. Highest concentrations were seen in two lactating females in late April. At both locations plasma lipid concentrations tended to be higher in females than in males throughout the season.
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Fig. 3. Seasonal chzmges in mean pk~sma protein concentration of adult S. b. lwldim.ti at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical b~rs show ± 2 SAL and numbers give sample size. Plasma protein cotwentrations Total plasma protein concentrations varied seasonally and in a similar pattern in both study populations (Fig. 3). Protein levels tended to decrease early
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Fig, 2. Seasonal changes in mean plasma lipid concentration of adult S. b. beldhnji at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical bars show + 2 S.E, and numbers give sample size.
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Fig. 4. Seasonal ch,'uages in mean per cent of y-globulin and of fl-globulin of adult S. b. heldinqi at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical bars show ± 2 S,E. and numbers give sample size.
Seasonal changes in plasma proteins in the season then increase slightly thereafter until the onset of hibernation.
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s0;~ N o sexual difference in percentage of plasma proo . . . . J ° . . . . . o . o" ALBUMtN tein c o m p o n e n t s were detected so data for the two sexes were combined for p r e ~ n t a t i o n . o Gamma-#h)h~din. At Tioga Pass the 7-globulin fracP" 4C tO 12 9 tion tended to decrease as the season progressed (Fig. g 3m 4, upper). The first sample taken in M a y had a signifio~, cantly larger fraction of ~,-globulin then the last sample taken in August; I1 vs 7 ~ (P < 0-01). At Big 114 TIOGA PASS Bend y-globulin was comparatively low in April at the beginning of the season (Fig. 4, lower), then was maintained at slightly higher fractions thereafter. MAy' JUNE JULY AUG Beta-ohdmli~,. There was little seasonal variation in //-globulin fraction in either study population (Fig. 40 ~..'" ', P'C(E~.LBUMI R 4). The mean of the tirst sample at Tioga Pass was ~3! BO o / about 16'!-/, and the last was 17/0. The difference was not significant (P > 0-05). At Big Bend the values at the temporal extremes were 19 and 16.%; again not t" LBU•IN z o different statistically. All,ha2-~flobulin. The most d r a m a t i c seasonal oscillations of any protein occurred in the a2-globulinfraction. During the first month of the active season 28 BIG SEND at Tioga Pass it was about 6% of total protein but J ,1 * ....... i, , ......... , increased suddenly and significantly (P < 0-01) in late APR MAY JUNE JULY June to a sustained level of a b o u t 16°/0 (Fig. 5, upper). Fig. 6. Seasonal changes in mean per cent of albumin The seasonal pattern for this fraction was r e m a r k a b l y (solid lines) and in per cent of animals possessing a prealdifferent at Big Bend (Fig. 5, lower). Levels were highbumin fraction (broken lines). Vertical bars show _+ 2 S.E. for albumin and numbers give sample size for both cst curly in the season then dec:eased significantly fractions. (P < 0-01) in curly May. Later, at the same calendar time as ut Tioga Pass but much later in the active Alpha I-~flobulin. The ~tl-globulin fraction increased season schedule, the ~2-globulin fraction increased steadily throughout tile season o f activity in both signilicantly (P < 0"01). study populations (Fig. 5). The first sample in M a y at Tioga Pass (mean of 7 ~ ) was significantly lower (P < 0'01) than the final sample in August (mean of 12~/~). Similarly, the first sample in April at Big Bend (mean of 9%) was significantly lower (P < 0-02) than the final sample in July (mean of t2%). Albumin. The plasma albumin fraction was highest in those animals most recently emerged from hibernation at both study areas (Fig. 6). Levels trended downward throughout the active season at Big Bend but first a n d final samples were not different (P > 0-05). At Tioga Pass, however, albumin decreased from 44 I*d~y dUN[ JULY AU'G to 35Y/o in June (P < 0-0t) and remained consistently at a b o u t that level thereafter. Prealbumin. Unlike other plasma proteins, the 2: BIG BEND prealbumin fraction could "not be detected in all samples. When present, it constituted from 5 to 9% of the total protein. Its presence varied enormously in Tioga Pass samples. O n l y 11~/~ o f the animals samo ,, \ ,o ~ /I .t pled in mid-June had a detectable preal6umin fraction whereas 8 0 ~ had this protein band in late August (Fig. 6, upper), Less variability was noted in ° IC B 1 Big Bend squirrels in that throughout the season 70--100~/o of the samples obtained from them contained prealbumin (Fig. 6, lower).
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Fig. 5. Seasonal changes in mean per cent of ~2-globulin and of ~l-globulin of adult S. b. beldingi at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical bars show -4- ? S.E. and numbers give sample size.
DISCUSSION
Plasma lipid Oscillations in plasma lipid concentration in S. b.
beldinyi a p p e a r to be related in a straightforward way
242
Sm~.~^ Nam-Yl.~(i I-tuA.~¢i^N~) Mar~ll~ L Mo~txoy
to the se,'tsonul rhythm of energy storage. Thi,s is most easily observed in Tioga Pass sqtfirrets, a popukttion whose annual cycle ~f bodily composition has bccn thoroughly investigalcd (Morton, 19761. Following reproduction they undergo extensive hlltening and enter hibernation with intracorporeal lipid stores equivalent Io or exceeding lean, dry body components. Upon emergence, some 8-9 inonths ]filer, they still retain 20 25";~, of this fat. These remaining depots tire nearly totally depleted in the weeks after emergence which encompass the p e r i o d of reproduclion, Animals at Big Band probably have a similar cycle (Morton, 19761. P/asnla lipkl levels in Tioga Pass males seem to follow the samc seasonal trends as the depiction-de position cycle of stored body fat. I n females tim plasma lipid pattern was modilied by an elevation during lactation as they mobilized fat for milk produclion. Ctlptivc thirteen-lined ground squirrels (S. tridecemlim'atus) also show seasonal changes in ~ r u m lipid that :tppear to I~ related d i r e c t l y to fat storage or utilization IGalsler & Morrison, 1966). T o t a l m r u m lipids in another hil~rnator, lhc woodchuck (Martllol~l Illllllil.'V). have been found to follow the same paltcrn as body weights (and therefore degree of obesity) in one study (Wcnberg & Holland, 1973) but not in another (Davis, 19651. In our own" study plasma lipids in Big Bend animals showcd much less seasonal Iluctuation than those tit Tioga Pass. This effect may be related to altitude but we cannot bc sure. l)hl,~lll(I ])l'ogt'ill CoIICtqlD'tltioltS ~tttd UOlIIpl)IIUIIIS Because of its utmost importance in body fluid dynamics one might expect little tolerancc to variation in plasma protein concentration. However, seasonal trends seem to exist in both study populations. Wc have observed seasonal changes in hcmatocrit that seem pertinent here. F o r example, squirrels tit Tioga Pass have a mean hematocrit of about 52~/,, at time of emergence. This decreases to a low of a b o u t 44",~; in mid-June then returns to about 50~; tot the remainder of the active season. This parallels the trend observed in plasma protein concentration. It may bc that dehydration during hibernation causes a decrease in plasma volume with an attdndant increase in ptasnla protein concentration. However, one would expect rehydralion to occur almost immedialely aflcr emergence since snow or free wa!er are readily available. It seems m o r e likely that annual changes in erythropoietic activity and crythrocyte storage associated with hibernation are involved. It is known, for example, that hedgehogs (Erinaceus em'opaeus) have maximum hematocrits during hibernation (Bi6rek et al., 19651 and at e m e r g e n c e due to emptying o f the spleen (Kristoffersson & Suornalainen, 19701. Similarly, S. tridecemlilleattts have their highest hematocrit during hibernation and their lowest during the usual period of reproduction (Galster & Morrison, 19661. If b l o o d volume remains constant, changes in plasma protein concentration could be a consequence of the regulation of circulating erythrocyte numbers. Sealander (1969) found in his vahmble study, of redbacked voles (Ctethrionomys rutikls) that in this nonhibernator there was a pronounced annual cycle in
plasma protein concentration. Summer protein levels were signilkamtly lower than those in winter. Sealander feels that interplay a m o n g a host of environmental factors such as temperature and light, and physiologicad factors such as reproductive condition and nutrional state fire respQnsible for plasma protein tluctuations. In our study five male S. I~. hel~',:~Wi held in laboratory hibernacula at 5~'C were s a m , i : while torpid in January. Their mean plasma protein concentration was 9,2 g",i;; significanlly higher tb.an any sample taken during the active season {P < 0.05). T w o of the protein fractions from these torpid animals were also signiiicantly different from those found in feral, normothcrmic animals. Beta-globulin averaged 24-5~,, higher than that found at any time during the active season (P < 0.05), and 7-globulin was 5-2'?,~;, lower than any of the active season samples. Similar relationships for these fractions were found in hamsters (Me~sovricetus aumaus} by South & Jeffay, 19581. Although a steriod-binding protein which binds both male :md female sex hormones migrates with the fl-globulin fraction [Rosner, 19691, there was no signilicant change in relative quantity of this fraction during tile reproductive season of S. b. beMim fi. The remarkable seasonal changes in ~x2-globulin are difficult to understand but it must be recognized that there is considerable molecular heterogeneity in this fraction. There are at least three major c o m p o n e n t s thai arc unseparated by electrophoretic techniques. One, 0crtdoplasnfin, binds to copper, another, :~2-macroglobulin. is a trypsin stabilizer (James et aL, 19661. and a third, haptoE, Iobin. is a major fraction that binds to hemoglobin preventing loss of this respiratory pignnent into the glomerular filtrate (Janusch, 1970). A p p a r e n l | y haptoglobin is responsible for much of the increase in a2-globulin noted during various diseases (Sutton & Jamieson, t972). Sealander (19691 noted that ~-globulin increases coincided with stressful periods in the life cycle o f C. rutilus. There was a striking difference in the seasonal pattern of ~2-globulin between the two populations of the present study. There were no consistent correlations with life history but a significant increase in this fraction did occur at the same calendar time (late June). Caution seems advisable in interpreting variation in ~2-globulins. Besides their heterogeneity, they are known to be c o n t a m i n a t e d by certain p r e a l b u m i n lipoproteins during temperature changes and prolonged storage (Larsen & T6nder, 19621 and t o have a comparatively rapid turnover rate (Sutton & Jamieson, 19721. An additional confounding effect, due possibly to polymorphism, was noted by us in that the scans of ~-globulin fractions early in the active season often had separate peaks or shoulders. P o l y m o r p h i s m in c~-globulin was also noted by W e n b e r g & H o l l a n d (1972) in M . monax near the end o f and immediately lbllowing the whole hibernation period. Perhaps animals at a r o u s a l are exposed to exogenous stimuli that p r o m p t adjustments in the immune system. The i m p o r t a n c e of albumin in fatty acid transport is well established (Polonovski, 19651. In S. b. beldhrgi albumin tended to vary inversely with lipid concentration during much of the season. A negative correla-
Seasonal changes in plasma proteins and lipids tion was found between our measurements of these two kinds of molecules in Tioga Pass animals (r=-0'45, n - 95, P < 0-01). Low quantities of plasma lipid may induce an increase in plasma albumin as a means of increasing eflMency of fatty acid transport. We have mentioned that protein polymorphism is a troublesome variable in interpreting electrophoresis data. The prealbumin fraction in S. b. beldh~qi represents a good example of this type of gene expression. The percent of animals possessing prealbumin follows nearly the stone seasonal pattern as plasma lipid levels, particularly when effects due to lactation are disregarded. No substantiated function for prealbumin has been determined, but it has been tentatively assigned a role in lipid transport in M. monax (Wenberg & Holland, 1972). Finally, we wish to point out4hree areas that need the concern and energies of investigators using functional systems such as blood to determine the intricacies of environmental adaptation. First, the efficacy of current analytical procedures i s inadequate for resolving some of the key questions. Second, considerable normal variation in blood parameters that has to be accounted for exists between species and probably within populations or individuals: differing results should not be ignored or presumed to represent artifact or error by the investigative parties. Third, more data are needed from animals in their natural setting, uninfluenced by conditions of captivity.
REFERENCES
BIORCKG., JOHANSSONB. & VE1GES. (1965) Some laboratory data on hedgehogs, hibernating and non.hibernating. Acta physiol, stand, 37, 281-294. DAws D. E. (1965) Major fatty acids in blood serum and adipose tissue of woodchuck (Marmota monax). BioL Sci, 15, 749-750.
243
GALSTt~.RW. A. & MORRISON P. (1966) Seasonal changes in ~rum lipids and proteins in the 13-lined ground squirrel. Comp. Biochem. Physiol. Ig, 489-501. JAMES K,, TAYLOR F. B. J. & FUDENIIEP,G H, H. (1966) Trypsin stabilizers in human ~rum. The role of :t2macroglobulin. CIhlica. chhn. Acta 13, 35%368. .l^suso! J. B. (1970) Evolution of serum protein polymorphism. A. Ret,. Gen. 4, 47-68. KRISTO~ERSSON R. & SUOMALAINrr4P. (1970) Stodies on the physiology of the hiN'.rnating hedgehog--9. Alterations in serum proteins in hibernating and non-hibernating hedgehogs. Am~. Acad. Sci.fi, mT. A. IV, 159. I-9. I¢ARSON B. & T6NI)H~ O. (1967) Serum proteins in the hedgehog. Aeta. physiol, scand. 69, 262-269. a MORTON M. L. (19761 Seasonal cycles of body weights and lipids in Belding ground squirrels. Bull. So. Cat![2 Acad. Sci. (In press). POLONOVSKIJ. 11965) Role of plasma proteins in transport of lipids. In Transport Fum'tion of p.~.~tsmaproteitls. Vol. 5. RosNIsrt W. (1969) Interaction of adrenal and gonadal steroids with proteins in human plasma. New Em,tL J. Med. 281,658-665. SCHEVINC~L. E., PAULVJ. E. & TSM T.-H. (19681 Circadian fluctuation in plasma protein of the rat. Am. J. Physiol. 215, 1096-1 I01. S~^LaND~ J. A. (1969) Effect of season on plasma and urinary proteins of the northern red-backed vole Clethrionomys rutilus. Physiol. Zool. 42, 275~287. Sou'm F. E. 2nd. & JErFAV H. (1958) Alterations in serum proteins of hibernating hamsters. Proc. Soc. exp. Biol. Med. 98, 885-887. Sutton H. E. & JAMIESONG. A. (1972) Transferrins, h:q.~toglobin and ceruloplasmin. In Glycoproteins 2nd ,.'-&~ Vol. 5, pp. 653-691. Wt~NrSER~G. M. & HOLt.ANbJ. C. (1972) The circann::al variations on the serum protein fractions of the woodchuck (Marmota monax). Comp. Biochem. Physiol. 42A, 989-997. WI~NaI/RGG. M. & HOt+aND J. C. (1973) The circannual variations in the total serum lipids and cholesterol with respect to body weight in the woodchuck (Marn~ota monax). Comp. Biochem. Physiol. 44A, 577-583. ZOLt.NER N. & Kmscrl K. (1962) The quantitative determination of lipids (micromethod) by means of sulfophospho-vanillin reaction common to man)' natural lipids. Z. ,qes. exp. Med. 135, 545-561.
S E A S O N A L C H A N G E S IN PLASMA P R O T E I N S A N D E1PIDS IN THE B E L D I N G G R O U N D S Q U I R R E L
(SPERMOPHILUS BELDINGI BELDINGI) SI'IL:NA NAN-YING HUANG AND MARTIN L. MfIt,'.'I'ON Biolog) Dep;irtmcnt, Occident College, Los Angeles. CA 9(X)41. U.S.A.
(Receired 19 .'hqlust 1975) Abstract--1. Concentrations of plasma lipid and plasma protein, and i~lasma protein composition ~vrc siudied in two populations of Bclding ground squirrels at high ahitude throughoul their season of activity. 2. Plasma lipid was highest in lactating fcm~0cs and tended to vary othcrwisc in direct relationship to total hod 3" lipid. 3. Plasma protein decreased during the first weeks after emergence from hibernation ~md increased thereafter. It is suggested that this was related to regu[alion of circulating crythrocytes. 4. There was some seasonal variation in all protein fr~lctions, with greatest klbility seen in :x2-globulin followed by albumin. Squirrels living at 2130 m exhibited a prcalbutnit~ fraction more frequently than those from 3020 m. 5. Seasonal cycles in all plasma constituents tended to be more pronounced in squirrels living at the higher altitude.
IN'rt~ooucl"ION FOR T1~r~past several d e c a d e s t h e b i o l o g y o f h i b c r n a t o r s h a s b e e n e x t e n s i v e l y investigated. P r i m a r y e m p h a s i s in these s t u d i e s h a s b e e n o n the m o d e s a n d mechanisms of body temperature regulation during p h a s e s o f t o r p o r . Recently, h o w e v e r , i n t e r e s t h a s quickened concerning other aspects of the annual cycle. Increasingly, a t t e m p t s h a v e b e e n m a d e t o relate s e a s o n a l c h a n g e s in f u n c t i o n a l c a p a c i t y t o e n v i r o n m e n t a l events. T h e a i m , of c o u r s e , is to u n d e r s t a n d h o w a d a p t a t i o n to t h e e n v i r o n m e n t ~ i n d u c e d a n d effected. D u e to its u b i q u i t o u s role in b o d y f u n c t i o n a n d a m e n a b i l i t y to study, b l o o d a n d its c o n s t i t u e n t s h a v e b e e n a f a v o r i t e f o c u s for t h e s e i n v e s t i g a t i o n s . In o u r s t u d y we h a v e m e a s u r e d q u a n t i t a t i v e seas o n a l c h a n g e s in b l o o d p l a s m a molecules, specifically p r o t e i n s a n d lipids, o f a h i b e r n a t o r , t h e B e l d i n g g r o u n d s q u i r r e l (Spermophihls beMinoi heMingi), d u r ing its i n t e r h i b e r n a t o r y s e a s o n o f activity a n d n o r m o t h e r m i a . W e h a v e related t h e s e c h a n g e s to t h e major events of summer activity; reproduction and p r e h i b e r n a t o r y fattening.
METHODS
This study was conducted on two pgpulalions of S. b.
anaounti~g to as much as 4 weeks (Morton. 1976) and the schedules shown ill Fig. 1 arc only approximate for a given year. Animals were collected with livetraps b;tited with pear, at butter then held separately with nesting material and water but no food for 24 hr prior to sampling, Sampling was always done in the morning to minimize possible diurn-d rhythmx in blood constituents such as those found by Scheving et ul. (1968) in the rat. Body weights were taken to within 0.1 g ,;raa pan balqnce. At sampling. :miroals were lightly a~esthetizcd with ether and 2-3 ml of blood were obtait~ed via cardiac puncture. Blood was centrifuged at once and total plasma protein concerttration was measured with a refi'aetomctcr. The remaining plasma was stored in sealed tubes at ca. 2'~C for no longer than 24 hr before further analysis. Subsequently, plasma lipid concentration was determined sp¢ctropholometrically using the method of Zollner & Kirzch (1962) except that a standard containing 750 mg lipid/100 mt alcohol (formula 3-A. American Hospital Supply Co., Miami, Florida) was used instead of corn oil. Plasma protein components were analyzed clcclrophorctically using Sepraphore 111 cellulose polyacetate strips at pH 8-8 and ionic strength 0.05 (Gelman Instrument Co.. Miami, Florida). A constant 250 V was applied for 60 rain. Strips were stained with Ponceau S. dcstained with three washes of 5~,~ acetic acid. dehydrated in two baths of absolute methanol, and cleared in 7 : t v/v methanol-acetic acid. After drying, strips were scanned with a densitometer and the curves obtained from protein bands were integrated.
beldingi living in meadows of the Sierra Nevada M o u n t a i n s in M o n o County, California. One population was at about R ESU LTS 2130 m elevation at Big Bend. the other at about 3020 na elevation at Tioga Pass. These populations were only Plasma lipid concentrations I0 km apart but because of the difference in climatic condiN e w l y e m e r g e d a d u l t m a l e s ,at T i o g a Pass in m i d tions associated with altitude, the active season of squirrels M a y h a d a m e a n p l a s m a lipid c o n c e n t r a t i o n o f 598 at Big Bend began a n d ended 6--8 weeks earlier than those m g ~ (Fig. 2, upper). T h e i r lipid t h e n d e c r e a s e d steadat Tioga Pass (Fig. 1). Events within the two study populaily t o a s e a s o n a l low in m i d - J u n e o f 432 m g ~ . T h i s tions for a given active season were similar in sequence and duration. There was interannuai variation, however. low w a s followed by a n i n c r e a s e t h a t was m a i n t a i n e d 239
240
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Fig. I. Major events in the active season of aduh S. h. heldinfli at Big Bend (upper calendar) and at Tioga Pass (lower calendar). near or above 600 mg "//,, until immergence in August, In adult females a seasonal low in mean plasma lipid concentration of 552 nag 'X, occurred soon after emergence. This was followed by a significant increase (P < 0.05) to 810 mg ?/,, in mid-June when most females had begun lactating. Subsequently, plasma lipid decreased in females to about the same concentrations seen in males. Starting in mid-July, plasma lipid began increasing in both sexes; maxima of 825 mg ~,, in females and 700 mg %, in males being attained in mid-August at the very end of their active s~ason.
There was little seasonal variation in plasma lipids in Big Bend animals (Fig. 2, lower), but levels did tend to increase during the last 2 m o n t h s of activity as at Tioga Pass. Highest concentrations were seen in two lactating females in late April. At both locations plasma lipid concentrations tended to be higher in females than in males throughout the season.
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Fig. 3. Seasonal chzmges in mean pk~sma protein concentration of adult S. b. lwldim.ti at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical b~rs show ± 2 SAL and numbers give sample size. Plasma protein cotwentrations Total plasma protein concentrations varied seasonally and in a similar pattern in both study populations (Fig. 3). Protein levels tended to decrease early
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Fig, 2. Seasonal changes in mean plasma lipid concentration of adult S. b. beldhnji at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical bars show + 2 S.E, and numbers give sample size.
APR
MAY
JUNE
JULY
Fig. 4. Seasonal ch,'uages in mean per cent of y-globulin and of fl-globulin of adult S. b. heldinqi at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical bars show ± 2 S,E. and numbers give sample size.
Seasonal changes in plasma proteins in the season then increase slightly thereafter until the onset of hibernation.
and
lipids
241 tOO
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48
Pktsma proteil~ components
PREALBUMIN
46
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s0;~ N o sexual difference in percentage of plasma proo . . . . J ° . . . . . o . o" ALBUMtN tein c o m p o n e n t s were detected so data for the two sexes were combined for p r e ~ n t a t i o n . o Gamma-#h)h~din. At Tioga Pass the 7-globulin fracP" 4C tO 12 9 tion tended to decrease as the season progressed (Fig. g 3m 4, upper). The first sample taken in M a y had a signifio~, cantly larger fraction of ~,-globulin then the last sample taken in August; I1 vs 7 ~ (P < 0-01). At Big 114 TIOGA PASS Bend y-globulin was comparatively low in April at the beginning of the season (Fig. 4, lower), then was maintained at slightly higher fractions thereafter. MAy' JUNE JULY AUG Beta-ohdmli~,. There was little seasonal variation in //-globulin fraction in either study population (Fig. 40 ~..'" ', P'C(E~.LBUMI R 4). The mean of the tirst sample at Tioga Pass was ~3! BO o / about 16'!-/, and the last was 17/0. The difference was not significant (P > 0-05). At Big Bend the values at the temporal extremes were 19 and 16.%; again not t" LBU•IN z o different statistically. All,ha2-~flobulin. The most d r a m a t i c seasonal oscillations of any protein occurred in the a2-globulinfraction. During the first month of the active season 28 BIG SEND at Tioga Pass it was about 6% of total protein but J ,1 * ....... i, , ......... , increased suddenly and significantly (P < 0-01) in late APR MAY JUNE JULY June to a sustained level of a b o u t 16°/0 (Fig. 5, upper). Fig. 6. Seasonal changes in mean per cent of albumin The seasonal pattern for this fraction was r e m a r k a b l y (solid lines) and in per cent of animals possessing a prealdifferent at Big Bend (Fig. 5, lower). Levels were highbumin fraction (broken lines). Vertical bars show _+ 2 S.E. for albumin and numbers give sample size for both cst curly in the season then dec:eased significantly fractions. (P < 0-01) in curly May. Later, at the same calendar time as ut Tioga Pass but much later in the active Alpha I-~flobulin. The ~tl-globulin fraction increased season schedule, the ~2-globulin fraction increased steadily throughout tile season o f activity in both signilicantly (P < 0"01). study populations (Fig. 5). The first sample in M a y at Tioga Pass (mean of 7 ~ ) was significantly lower (P < 0'01) than the final sample in August (mean of 12~/~). Similarly, the first sample in April at Big Bend (mean of 9%) was significantly lower (P < 0-02) than the final sample in July (mean of t2%). Albumin. The plasma albumin fraction was highest in those animals most recently emerged from hibernation at both study areas (Fig. 6). Levels trended downward throughout the active season at Big Bend but first a n d final samples were not different (P > 0-05). At Tioga Pass, however, albumin decreased from 44 I*d~y dUN[ JULY AU'G to 35Y/o in June (P < 0-0t) and remained consistently at a b o u t that level thereafter. Prealbumin. Unlike other plasma proteins, the 2: BIG BEND prealbumin fraction could "not be detected in all samples. When present, it constituted from 5 to 9% of the total protein. Its presence varied enormously in Tioga Pass samples. O n l y 11~/~ o f the animals samo ,, \ ,o ~ /I .t pled in mid-June had a detectable preal6umin fraction whereas 8 0 ~ had this protein band in late August (Fig. 6, upper), Less variability was noted in ° IC B 1 Big Bend squirrels in that throughout the season 70--100~/o of the samples obtained from them contained prealbumin (Fig. 6, lower).
t!i
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i.
,L
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MAY
JUNE
JULY
Fig. 5. Seasonal changes in mean per cent of ~2-globulin and of ~l-globulin of adult S. b. beldingi at Tioga Pass (upper panel) and at Big Bend (lower panel). Vertical bars show -4- ? S.E. and numbers give sample size.
DISCUSSION
Plasma lipid Oscillations in plasma lipid concentration in S. b.
beldinyi a p p e a r to be related in a straightforward way
242
Sm~.~^ Nam-Yl.~(i I-tuA.~¢i^N~) Mar~ll~ L Mo~txoy
to the se,'tsonul rhythm of energy storage. Thi,s is most easily observed in Tioga Pass sqtfirrets, a popukttion whose annual cycle ~f bodily composition has bccn thoroughly investigalcd (Morton, 19761. Following reproduction they undergo extensive hlltening and enter hibernation with intracorporeal lipid stores equivalent Io or exceeding lean, dry body components. Upon emergence, some 8-9 inonths ]filer, they still retain 20 25";~, of this fat. These remaining depots tire nearly totally depleted in the weeks after emergence which encompass the p e r i o d of reproduclion, Animals at Big Band probably have a similar cycle (Morton, 19761. P/asnla lipkl levels in Tioga Pass males seem to follow the samc seasonal trends as the depiction-de position cycle of stored body fat. I n females tim plasma lipid pattern was modilied by an elevation during lactation as they mobilized fat for milk produclion. Ctlptivc thirteen-lined ground squirrels (S. tridecemlim'atus) also show seasonal changes in ~ r u m lipid that :tppear to I~ related d i r e c t l y to fat storage or utilization IGalsler & Morrison, 1966). T o t a l m r u m lipids in another hil~rnator, lhc woodchuck (Martllol~l Illllllil.'V). have been found to follow the same paltcrn as body weights (and therefore degree of obesity) in one study (Wcnberg & Holland, 1973) but not in another (Davis, 19651. In our own" study plasma lipids in Big Bend animals showcd much less seasonal Iluctuation than those tit Tioga Pass. This effect may be related to altitude but we cannot bc sure. l)hl,~lll(I ])l'ogt'ill CoIICtqlD'tltioltS ~tttd UOlIIpl)IIUIIIS Because of its utmost importance in body fluid dynamics one might expect little tolerancc to variation in plasma protein concentration. However, seasonal trends seem to exist in both study populations. Wc have observed seasonal changes in hcmatocrit that seem pertinent here. F o r example, squirrels tit Tioga Pass have a mean hematocrit of about 52~/,, at time of emergence. This decreases to a low of a b o u t 44",~; in mid-June then returns to about 50~; tot the remainder of the active season. This parallels the trend observed in plasma protein concentration. It may bc that dehydration during hibernation causes a decrease in plasma volume with an attdndant increase in ptasnla protein concentration. However, one would expect rehydralion to occur almost immedialely aflcr emergence since snow or free wa!er are readily available. It seems m o r e likely that annual changes in erythropoietic activity and crythrocyte storage associated with hibernation are involved. It is known, for example, that hedgehogs (Erinaceus em'opaeus) have maximum hematocrits during hibernation (Bi6rek et al., 19651 and at e m e r g e n c e due to emptying o f the spleen (Kristoffersson & Suornalainen, 19701. Similarly, S. tridecemlilleattts have their highest hematocrit during hibernation and their lowest during the usual period of reproduction (Galster & Morrison, 19661. If b l o o d volume remains constant, changes in plasma protein concentration could be a consequence of the regulation of circulating erythrocyte numbers. Sealander (1969) found in his vahmble study, of redbacked voles (Ctethrionomys rutikls) that in this nonhibernator there was a pronounced annual cycle in
plasma protein concentration. Summer protein levels were signilkamtly lower than those in winter. Sealander feels that interplay a m o n g a host of environmental factors such as temperature and light, and physiologicad factors such as reproductive condition and nutrional state fire respQnsible for plasma protein tluctuations. In our study five male S. I~. hel~',:~Wi held in laboratory hibernacula at 5~'C were s a m , i : while torpid in January. Their mean plasma protein concentration was 9,2 g",i;; significanlly higher tb.an any sample taken during the active season {P < 0.05). T w o of the protein fractions from these torpid animals were also signiiicantly different from those found in feral, normothcrmic animals. Beta-globulin averaged 24-5~,, higher than that found at any time during the active season (P < 0.05), and 7-globulin was 5-2'?,~;, lower than any of the active season samples. Similar relationships for these fractions were found in hamsters (Me~sovricetus aumaus} by South & Jeffay, 19581. Although a steriod-binding protein which binds both male :md female sex hormones migrates with the fl-globulin fraction [Rosner, 19691, there was no signilicant change in relative quantity of this fraction during tile reproductive season of S. b. beMim fi. The remarkable seasonal changes in ~x2-globulin are difficult to understand but it must be recognized that there is considerable molecular heterogeneity in this fraction. There are at least three major c o m p o n e n t s thai arc unseparated by electrophoretic techniques. One, 0crtdoplasnfin, binds to copper, another, :~2-macroglobulin. is a trypsin stabilizer (James et aL, 19661. and a third, haptoE, Iobin. is a major fraction that binds to hemoglobin preventing loss of this respiratory pignnent into the glomerular filtrate (Janusch, 1970). A p p a r e n l | y haptoglobin is responsible for much of the increase in a2-globulin noted during various diseases (Sutton & Jamieson, t972). Sealander (19691 noted that ~-globulin increases coincided with stressful periods in the life cycle o f C. rutilus. There was a striking difference in the seasonal pattern of ~2-globulin between the two populations of the present study. There were no consistent correlations with life history but a significant increase in this fraction did occur at the same calendar time (late June). Caution seems advisable in interpreting variation in ~2-globulins. Besides their heterogeneity, they are known to be c o n t a m i n a t e d by certain p r e a l b u m i n lipoproteins during temperature changes and prolonged storage (Larsen & T6nder, 19621 and t o have a comparatively rapid turnover rate (Sutton & Jamieson, 19721. An additional confounding effect, due possibly to polymorphism, was noted by us in that the scans of ~-globulin fractions early in the active season often had separate peaks or shoulders. P o l y m o r p h i s m in c~-globulin was also noted by W e n b e r g & H o l l a n d (1972) in M . monax near the end o f and immediately lbllowing the whole hibernation period. Perhaps animals at a r o u s a l are exposed to exogenous stimuli that p r o m p t adjustments in the immune system. The i m p o r t a n c e of albumin in fatty acid transport is well established (Polonovski, 19651. In S. b. beldhrgi albumin tended to vary inversely with lipid concentration during much of the season. A negative correla-
Seasonal changes in plasma proteins and lipids tion was found between our measurements of these two kinds of molecules in Tioga Pass animals (r=-0'45, n - 95, P < 0-01). Low quantities of plasma lipid may induce an increase in plasma albumin as a means of increasing eflMency of fatty acid transport. We have mentioned that protein polymorphism is a troublesome variable in interpreting electrophoresis data. The prealbumin fraction in S. b. beldh~qi represents a good example of this type of gene expression. The percent of animals possessing prealbumin follows nearly the stone seasonal pattern as plasma lipid levels, particularly when effects due to lactation are disregarded. No substantiated function for prealbumin has been determined, but it has been tentatively assigned a role in lipid transport in M. monax (Wenberg & Holland, 1972). Finally, we wish to point out4hree areas that need the concern and energies of investigators using functional systems such as blood to determine the intricacies of environmental adaptation. First, the efficacy of current analytical procedures i s inadequate for resolving some of the key questions. Second, considerable normal variation in blood parameters that has to be accounted for exists between species and probably within populations or individuals: differing results should not be ignored or presumed to represent artifact or error by the investigative parties. Third, more data are needed from animals in their natural setting, uninfluenced by conditions of captivity.
REFERENCES
BIORCKG., JOHANSSONB. & VE1GES. (1965) Some laboratory data on hedgehogs, hibernating and non.hibernating. Acta physiol, stand, 37, 281-294. DAws D. E. (1965) Major fatty acids in blood serum and adipose tissue of woodchuck (Marmota monax). BioL Sci, 15, 749-750.
243
GALSTt~.RW. A. & MORRISON P. (1966) Seasonal changes in ~rum lipids and proteins in the 13-lined ground squirrel. Comp. Biochem. Physiol. Ig, 489-501. JAMES K,, TAYLOR F. B. J. & FUDENIIEP,G H, H. (1966) Trypsin stabilizers in human ~rum. The role of :t2macroglobulin. CIhlica. chhn. Acta 13, 35%368. .l^suso! J. B. (1970) Evolution of serum protein polymorphism. A. Ret,. Gen. 4, 47-68. KRISTO~ERSSON R. & SUOMALAINrr4P. (1970) Stodies on the physiology of the hiN'.rnating hedgehog--9. Alterations in serum proteins in hibernating and non-hibernating hedgehogs. Am~. Acad. Sci.fi, mT. A. IV, 159. I-9. I¢ARSON B. & T6NI)H~ O. (1967) Serum proteins in the hedgehog. Aeta. physiol, scand. 69, 262-269. a MORTON M. L. (19761 Seasonal cycles of body weights and lipids in Belding ground squirrels. Bull. So. Cat![2 Acad. Sci. (In press). POLONOVSKIJ. 11965) Role of plasma proteins in transport of lipids. In Transport Fum'tion of p.~.~tsmaproteitls. Vol. 5. RosNIsrt W. (1969) Interaction of adrenal and gonadal steroids with proteins in human plasma. New Em,tL J. Med. 281,658-665. SCHEVINC~L. E., PAULVJ. E. & TSM T.-H. (19681 Circadian fluctuation in plasma protein of the rat. Am. J. Physiol. 215, 1096-1 I01. S~^LaND~ J. A. (1969) Effect of season on plasma and urinary proteins of the northern red-backed vole Clethrionomys rutilus. Physiol. Zool. 42, 275~287. Sou'm F. E. 2nd. & JErFAV H. (1958) Alterations in serum proteins of hibernating hamsters. Proc. Soc. exp. Biol. Med. 98, 885-887. Sutton H. E. & JAMIESONG. A. (1972) Transferrins, h:q.~toglobin and ceruloplasmin. In Glycoproteins 2nd ,.'-&~ Vol. 5, pp. 653-691. Wt~NrSER~G. M. & HOLt.ANbJ. C. (1972) The circann::al variations on the serum protein fractions of the woodchuck (Marmota monax). Comp. Biochem. Physiol. 42A, 989-997. WI~NaI/RGG. M. & HOt+aND J. C. (1973) The circannual variations in the total serum lipids and cholesterol with respect to body weight in the woodchuck (Marn~ota monax). Comp. Biochem. Physiol. 44A, 577-583. ZOLt.NER N. & Kmscrl K. (1962) The quantitative determination of lipids (micromethod) by means of sulfophospho-vanillin reaction common to man)' natural lipids. Z. ,qes. exp. Med. 135, 545-561.