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Seasonal changes in serum lipids and proteins in the 13-lined ground squirrel
Comp. Biochem. Physiol., 1966, Vol. 18 ,pp. 489 to 501. Pergamon Press Ltd. Printed in Great Britain
SEASONAL CHANGES IN SERUM L I P I D S AND P R O T E I N S IN T H E 13-LINED G R O U N D SQUIRREL* W I L L I A M A. G A L S T E R and P E T E R M O R R I S O N Departments of Zoology and Physiology, University of Wisconsin, Madison, and Laboratory of Zoophysiologyt, Institute of Arctic Biology, University of Alaska, College, Alaska (Received 3 ffanuary 1966)
Almtract--1. Blood lipid, protein and hematocrit levels in Sperraophilus tridecemlineatus foUow yearly cycles. Total lipid, a-lipid, fl-lipid and chylomicrons were minimal in late spring (7.0, 2"5, 2"3 and 1"0 g/l) and maximal in late fall (27.0, 5.8, 9"8 and 6"0 g/l). Secondary maxima or plateaux were observed in late summer in all, and in the spring for a-lipid. 2. All proteins were minimal in late spring except//-globulin which was minimal in January. Albumin concentration increased to threefold (from 18 g/l) by midsummer, and then to fourfold in early fall, a level maintained through the winter (av. 65 g/l) until early spring when levels decrease sharply. All globulin components increased briefly at midsummer. Brief maxima also occurred in January and March for fl-globulin and in February and May for aand 7-globulin.
INTRODUCTION HIBERNATION reviews cite many comparisons of blood constituents from active and hibernating mammals but few studies have encompassed the whole year (Lyman, 1955; Kayser, 1961). Circulatory lipids have received little consideration except for the work of Bragdon (1954) on the Columbian ground squirrel and Bitrck et al. (1956) on the hedgehog. Plasma proteins have received attention from Suomalainen & Karpannen (1956) and Bi6rck et al. (1956) on the hedgehog, and from South & Jeffray (1958) on the hamster. T h e RBC concentration appears not to have been followed through the year except in the Tien-Shan marmot (Bibikov & Thirnova, 1956). Because of the special nutritional importance of lipicls in the hibernators' yearly cycle the present study examines the levels of blood lipids and proteins throughout the year, attempting to relate these both to each other and to the several activity phases. * These studies were assisted by grants from the National Science Foundation (G24039) and the Kansas Heart Association. Manuscript preparation and publication were assisted by the National Institutes of Health (No. GM10402). ? Present address. These studies are Publication No. 25. 489
490
WILLIAM A. GALSTER AND PETER MORRISON MATERIALS AND M E T H O D S
Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) were collected near Madison, Wisconsin (1960-62), and Atchison, Kansas (1963), and housed in individual cages. T h e temperature was maintained at 20°C and the light cycle at 12 hr of daylight. Animals were fed Purina Rat Chow (50 per cent carbohydrate, 24 per cent protein, 4 per cent fat) and given water ad libitum. Squirrels that became torpid under these conditions (body temperature near ambient) were referred to as "room-temperature hibernators". Hibernation in season was brought about by transferring the animals in early November to a dark hibernaculum where the temperature was maintained at 9 + 2 ° C . Food and water consumption, urination and visual observations were used to identify inactive periods during the hibernating season, Blood samples were obtained by decapitation or by cardiac puncture following a 24 hr fast. Hematocrit values were measured in heparinized capillary tubes and the remainder of the sample allowed to clot for serum separation. Turbidity of serum was estimated on a scale from 0 to 3. Total serum lipids were estimated using the turbidometric method of De la Huerga (1953)modified for 0-1 ml sample. Samples with excess lipid were diluted. Serum proteins were separated electrophoretically on paper strips in a Durrum cell at pH of 8.6 using veronal buffer of ionic strength 0.075. Protein components were stained with bromphenol blue after migration at 2"5 mA for 18 hr and quantitared with the Beckman "Analatrol". A representative separation is compared to that in man in Fig. 1 to show the somewhat different spacing of the components.
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FIG. 1. Representative separation of ground squirrel serum by paper electrophoresis compared to human standard. Dashed line represents fipid staining.
Lipoproteins were stained with oil red O according to Jencks et al. (1955) after migration at 10 mA for 4 hr. T h e chylomicron fraction, separable by centrifugation at 106g rain, showed no mobility. T h e three lipid fractions were quantitated as proportions of t h e total color referred to the total lipid analysis. T h e lipid-beating protein was identified among the several electrophoretic components by staining
SV2~SONAL CH~qOgS IN 1 3 - L I ~
OROUND SQUIRREL
491
two halves of a single strip (Fig. 1). The B-llpid corresponded directly to a/3globulin but the s-lipid fell between the ~l-globulins. RESULTS All lipid components exhibited yearly cycles in concentration with maxima in late fall and minima in late spring. Total serum lipid rose through the summer to more than twice the June concentration in September (6-2-+ 13.5 g/I~ (Fig. 2). In
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MONTH FIG. 2. Total serum lipid concentration by months with mean, standard error, range and sample size indicated. Single points in summer represent animals which were "forced" into hibernation. Single points in spring are from replacement animals that were placed in hibernaculum in February. Shading and crosshatching signify percentage of "deep" and "room-temperature" hibernators.
November the value quiekly doubled again (to 26.2 g/l) and then declined more or less exponentially through the winter and spring. Forced summer hibernators (2 weeks at 5°C in July) and room-temperature hibernators (Sept.-Oct.) had concentrations similar to the November group. Values from three ground squirrels not exposed to the cold during the hibernating season fell within the range of the December values. A wide variance in the total lipid concentration in serum was
4-92
WILLIAM A. GALSTERA N D PETER MORI~ISON
exhibited but there was no correlation with hematocrit, sex, body temperature or body weight. ~- and/3-Lipid and chylomicrons followed the general yearly cycle in total lipid but all exhibited individual differences, s-Lipid rose in early summer (2.3 -+ 5-3 g/l) and then doubled again in November-December (to 10.5 g/l) (Fig. 3). The
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concentration fell somewhat during the winter and then sharply in March. A brief peak was seen,in April-May prior to the June minimum. E-Lipid also rose during summer but fell sharply in September, almost to the June low (Fig. 4), prior to an almost threefold increase in November and December (3.1 -> 8.7 g/l). Values fell through the winter and remained low through the spring. Chylomicrons showed abrief concentration peak in November which had fallen to the average value by the end of December (Fig. 5). Turbidity was observed in most samples with m o r e t h a n 0-1 g/1 of chylomicmns but a third of the clear samples had from 0-1 to 0.5 g/1 of chylomicrons. Turbidity was observed in
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494
WILLIAMA. GALST~AN])PETERMORRISON
82 per cent of the serums from room-temperature hibernators and in only 10 per cent of the serums from deep hibernators. All protein components were minimal in late spring but 0nly albumin was maximal in late fall. Values for serum albumin exhibited a yearly cycle similar to serum lipid (Fig. 6) but was minimal in May (18 g/l), not June, and maintained 80
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their winter elevation until the end of March. The precipitous drop from 60 to 20 g/l during two months of spring was striking. All globulin components increased briefly during midsummer. E-globulins increased threefold by late summer, returned nearly to the June minimum by early fall and increased through winter to the maximum near the end of hibernation. ~-, ~ - and y-Globulins remained unchanged through fall and early winter. The ~- and y-globulins increased briefly during late winter and spring. Hematocrit values exhibited a pronounced yearly cycle (Fig. 7) with low values through late winter and spring (36-0 per cent), intermediate values in the summer (42 per cent) and an October peak (53 per cent).
495
SEASONAL CHANOES I N 1 3 - L I N E D OROUND SQUIRREL
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DISCUSSION
Columbian ground squirrels (Bragdon, 1954) and 13-lined ground squirrels both show concentrations of serum lipid that are seven times those found in the white rat (Boyd, 1942). This striking difference in the hibernators suggests greater capacity for carrying lipid in the blood. Bragdon did not consider seasonal changes in serum lipids and did not give sampling dates. His measurements on non-fasted Columbian ground squirrels were higher than our measurements on the 13,lined ground squirrel made in early fall (16.9 g/l).
Yearly pattern As shown in Fig. 8, the several phases of serum lipid fluctuation corresponded in considerable degree to the yearly activities of the 13-1ined ground squirrel as observed by Landau & Dawe (1960) and Rongstad (1965). First, following the prehibernation maxima, all lipid components declined progressively during hibernation (Dec.-Apr.). Except for a brief rise in a- and//-lipid, this decline continued through the spring breeding and gestational period (Apr.-May), a time of great activity during which the hibernator may still be largely dependent on fat reserve for energy. The minimum in all lipid levels (late May or early June)
496
WILLIAM A. GALSTER AND PETER MORRISON
corresponded to the reappearance of high quality nutrient for the diet. With this food supply, the second phase of growth was accompanied by a steady increase in the level of all serum lipids to a plateau in early September. Rongstad (1965)
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7
8
9
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Serum lipid, protein and hematocrit cycles in relation to activity phases through the year.
observed that some animals may enter hibernation at this time, having completed their preparation in August, but others especially young of the year may delay until early October depending on conditions. About 25 per cent of our animals "hibernated" at room temperature during the fall since they were not placed in the hibernaculum until the first week of November. This could represent some distortion of the natural pattern, but a striking independence of the yearly hibernation cycle of conditions of light and temperature has recently been demonstrated in this species (Morris & Morrison, 1964). This delay (Sept.-Nov.) may perhaps be regarded as an extension of a variable but much shorter natural delay period (Sept.) following the completion of nutritional preparation for hibernation. In the jumping mouse (Zapus) an extended delay period of this sort has been observed with fatal consequences (Morrison & Ryser, 1962). It is interesting to note that the two late summer ground squirrels when exposed to 0°C for 3 weeks had a November
5-7 4-8~-6 ±2"2 6"8 5"~7-5 0"7
3"4 2.4--4.5 ±0"6
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Range S.D.
Range S.D.
Chylomicrons Range S.D. 1"5 0"2-2"4 1"1
13"2 9"0-15"8 ±2-4
11"7 8"8-14-8 ±2"8
Total Range S.D.
Lipid (g/l)
0"8
2"7
0"8 0-2"1 1"0
4"9 2"94"7 1"7
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0
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Month
1-1 0-1"6 0"3
8"4 5"9-12"0 3"9
5"2 2"~9"9 2-7
14"9 10"~23"6 ±4"6
6"6
5-5 2"3-8"2 2"4
9"2 4-2-18"5 5-8
8-4 3-2-12-0 2"7
23"5 18"~30"0 ±3"5
0
5"1 1"3-10-3 3-4
14"1 11-3-16"7 2"3
9-9 5-9-13"2 3"3
26-6 15"~34"0 ±4"5
5 0-8
November
9"4 7"2-10"3 1-4
7-9 4"0-14,8 4"3
9"9 9"1-11"2 0"3
22"4 12"1-26"4 ±6"0
2"7
1"4 0-2.6 + 1"0
1"9 0-3"8 + 0"4
2.6 1-4-4"6 1-0
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6 10-20
11"4 7"5-17"4 + 6"2
8 1-8
January-February
T A B L E 1 - - E F F E C T S OF STARVATION ON SERUM LIPIDS OF ACTIVE AND HIBERNATING 1 3 - L I N E GROUND SQUIRRELS
4~
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WILLIAM A. GALSTER AND PETER MORRISON
498
serum lipid pattern. Hibernation was then immediately preceded by a sharp rise in all lipid components (Nov.) which, together with the delay period, may be taken as the third phase in the yearly cycle of lipids. Although it is rash to speculate on the implications of changes in dynamic, steady-state levels, some comments may be made. The progressive increase in all blood lipids during summer may reasonably be associated with the fat deposition essential for hibernation. According to Olson & Vester (1960) ~-lipoprotein carries lipid from the intestine to the liver and the fat stores, while fl-lipoprotein carries lipid from the liver to the fat stores. And, indeed, ~- and//-lipid levels rose steadily through August and September, the period when fat deposition and lipogenesis would normally be most intense, and fell somewhat in September when fat deposition would normally have been completed. However, since intestinal absorption should have been complete (in 24 hr fasted animals) the elevated level of ~-lipid may indicate that this component also was taking some role in liver-to-fat depot transport in this species. Even with an increase in the fasting period from 1 day to 6½ days total lipid and lipoprotein fractions failed to decline (Table 1), TABLE 2--SERUM LIPID LEVELS IN DORMICE
Yearly light cycle Normal (Boreal)
Reversed (Austral)
Lipid (g/l)
Torpor* (%)
Lipid
Date
(g/l)
Torpor (%)
10/61 12/61 4-5/62 6/62
10-9 (4) 2.6 (7) 6-7 (10) 7.3 (10)
42 60 35 40
10-1 (6) 5.0 (9) 6.1 (9) 4.5 (9)
35 222 60 80
* Percentage of individuals entering torpor when exposed for a 4 day test period at 5°C. Parenthesized values indicate sample size. suggesting that a high blood lipid level may be normally maintained in this species. In the dormouse (Glis glis), another hibernator but a more "permissive" one, individual serum lipid values range from 2.4 to 20 g/1 as compared to 5-0 to 36 g/1 in the 13-lined ground squirrel and the mean values are also about half those in the latter species (6"5 versus 12.6 g/l). T h e dormouse also differed in showing no yearly pattern in the lipid level (Table 2). In the fall, lipid levels rose dramatically just prior to hibernation and chylomicrons increased. These increases were preceded (not accompanied) by a 23 per cent increase in the hematocrit (Fig. 7), indicating that lipid changes were not caused by hemoconcentration. This prehibernation increase in ~- and//-lipid during late fall might reflect a final burst of lipogenesis, but alternatively might equally reflect some adjustment in the dynamic equilibrium between liver, blood
SEASONAL CHANGBS I N
13-LrNED GROUND
SQUIRREL
499
and depot fat. In the rat, French & Morris (1957) observed a half-life of only 12 min but after 24 hr fasting chylomicrons persisted in ground squirrels. The increase in chylomicrons may represent an overloading of the lipid transport mechanism. Animals "hibernating" at room temperature during this time showed increases in serum chylomicrons and turbidity, and may indicate a need for low temperature to facilitate clearing action. Fasting for 2.7 days during this period produced little change in total lipid or the a-fraction (Table 1). The //-lipid increased initially and then decreased, and chylomicrons increased during fasting. Lipid components During the winter and spring, encompassing the hibernation season and the spring breeding period, values for all lipids decreased from the yearly maximum to the yearly minimum a-Lipid remained high through the hibernation season although the function of intestine-to-liver transport proposed by Olson & Vester (1960) does not seem appropriate in this case. a-Lipid increased sharply at awakening and rose during the breeding season before declining to the minimum in early June. //-Lipid and chylomicron fractions and hematocrits declined through the early hibernating season and then remained constant until late spring. There was no correlation between the periodic awakenings during hibernation and the level of the lipid fractions (Table 1). Further, the caloric values at the maximum serum lipidlevel were only sufficient to have maintained the hibernating squirrel for less than 2 days. Therefore these lipids are either maintained or are not being used during torpor and their replenishment appears not to be a cause for periodic awakening. .
Protein components Seasonal changes in plasma protein fractions followed a varied pattern and did not reflect the lipid fraction changes. Limits for a-lipoprotein, located between the s-globulin and the albumin protein bands, were estimated from a-lipid values and the composition data of Olson & Vester (1960). There was no correlation with the values for %-globulin (Fig. 3). Changes in albumin do correlate with changes in s-lipid. The //-lipid appeared on the strip with the E-globulin. Because/glipoprotein is only one of several E-globulins, estimates of its concentrations from the//-lipid values can only account for a fraction of the E-globulin (Fig. 4). The marked reduction in y-globulin during hibernation (Dec.-Jan.) was similar to that observed by South & Jeffray (1958) in the hamster. The early recovery of this fraction in February and its continued rise during the spring may reflect a reconstitution of immune function before awakening. The hematocrit varied through the summer and this may be attributed to the introduction of young squirrels. The 23 per cent increase in early fall similar to the increased erythrocyte count observed by Bibikov & Zhirnova (1956) in Tien-Shan marmots may relate to increased erythropoiesis giving squirrels reserve RBC's to last through hibernation. Raths (1953) and Lyman et al. (1957) measured marked
500
WILLIAMA.
GALSTER AND PETER MORRISON
reductions in erythropoietic activity during hibernation in woodchuck and hamsters, but this could b e paralleled by a like reduction in erythrocyte destruction. The reduction in hematocrit concentration during hibernation parallels that observed by Woodward & Condrin (1945) in Tamias striatus and Stuckey & Coco (1942) in 13-lined ground squirrels. It may be important to re-emphasize the stability of the yearly physiological cycle in the 13-lined ground squirrel without which a coherent pattern could easily have been lost during long.term maintenance under artificial conditions of captivity. Morris & Morrison (1964) have shown that even with maintenance under continuous warm conditions (25°C) and with a reversed yearly light cycle (austral) there was no suppression or shift in the yearly cycles in body temperature, hibernation, tendency or reproductive activity. This need not be the case since in the dormouse (Glis glis) a reversal in light cycle is quickly followed by a reversal in the physiological response. SUMMARY T h e 13'lined ground squirrel exhibits well-defined yearly cycles for the lipid and protein components of serum. The average lipid concentration is very high but the protein concentration is normal for mammals. T h e number of lipid and protein electrophoretic components is usual. Total lipid, s-lipid, r-lipid and chylomicrons were minimal in late spring and maximal in late fall. Secondary maxima or plateaux were observed in late summer in all lipids and in the spring for c~-lipid. All protein components were minimal in late spring except r-globulin which was minimal in January. Albumin concentration increased to threefold by midsummer, and then to fourfold in early fall, a level maintained through the winter until early spring when levels decreased sharply. All globulin components increased briefly in midsummer. Brief maxima also occurred in January and March for r-globulin and in F~bruary and May for ~- and y-globulin. These changes in lipid and protein concentration correspond in considerable degree to the changing functional periods throughout the year--growth and fattening in summer, prehibernation delay and preparation in the fall, hibernation during the winter and reproductive activity in the springmhowever, the direct significance of the changing levels is yet to be established. REFERENCES BIBIKOV D. I. & ZHIRNOVAN. M. (1956) Seasonal modifications of some ecologicallyphysiological peculiarities of Marmota barbacine in the Tien-Shan. Zh. Zool. 35, 1565-1573. BIORCK G. J., JOHANSSONB. & VEIGE S. (1956) Some laboratory data on hedgehogs, hibernators and nonhibernators. Acta physiol. Scand. 37, 281-294. BOYDE. M. (1942) Species variation in normal plasma lipids estimated by oxidative micromethods..7, biol. Chem. 143, 131-132. BRAGDONJ. H. (1954) Hyperlipemla and atheromatosis in a hibernator, Citeilus columbianus. Circulation Res. 2, 520-524.
SEASONAL CHANGES IN 13-LINED GROUND SQUIRREL
501
DE LA HUERGA,YESINICKY. C. & HOPPER H. (1953) Estimation of total serum lipids by a turbidometric method. Am. 07. din. Path. 23, 1163-1168. FRENCH J. E. & MORRIS B. (1957) Removal of C 14 labelled chylomicrons from the circulation in rats. 07. Physiol. 138, 326-337. JENCKS W. R., DURRUM E. L. & JETTON M. R. (1955) Paper electrophoresis as a quantitative method for lipoproteins. 07. din. Invest. 34, 1437-1448. KAYSER C. (1961) The Physiology of Natural Hibernation. Pergamon Press, New York. LANDAU B. & DAWE A. R. (1960) Observations on a colony of captive ground squirrels throughout the year. Mus. comp. Zool. Bull. 124, 123-189. LYMAN C. P. (1955) Physiology of hibernation in mammals. Physiol Rev. 33, 403-425. LYMAN C. P., WEISS L. P., O'BRIEN R. C. & BARREAUA. A. (1957) The effects of hibernation on the replacement of blood in the golden hamster. 07. exp. Zool. 136, 471-485. MORRIS L. & MoRRISON P. (1964) Cyclic responses in dormice (Glis glis) and ground squirrels (Spermophillus trideceralineatus) exposed to normal and reversed yearly light schedules. Proc. 15th Alaskan Sci. Conf., College, Alaska, p. 40. MORRISON P. & RYSER F. A. (1962) Metabolism and body temperature in a small hibernator, the meadow jumping mouse. 07. cell. comp. Physiol. 60, 169-180. OLSON R. E. & VESTERJ. W. (1960) Nutritional endocrine interrelationships in the control of fat transport in man. Physiol. Rev. 40, 677-733. RATHS P. (1953) Untersuchungen fiber die Blutzusammensetzung und ihre Beziehungen zur vegetativien Tonuslage beim Hamster. Z. Biol. 106, 109-123. RONOSTAD O. (1965) A life history of 13-lined ground squirrels in southern Wisconsin. 07. Mammal. 46, 76-87. SOUTH F. C., JR. & JEFFRAYH. (1958) Alterations in serum proteins of hibernating hamsters. Proc. Soc. exp. Biol. Med. 98, 885-887. STUCKEYJ. & Coco R. M. (1942) A comparison of the blood picture of active and hibernating ground squirrels. Am. 07. Physiol. 137, 431-435. SUOMALAINEN P. & KARPPANEN E. (1956) Einfluss des Winterschlafes anf das AlbuminGlobulin Inverhaltnis des Igelserums. Sonderabdr. Suom. Kemistilehti B 29, 74-75. WOODWARD A. E. & CONDRIN J. i . (1945) Physiological studies on hibernation in the chipmunk. Physiol. Zool. 18, 162-167.
SEASONAL CHANGES IN SERUM L I P I D S AND P R O T E I N S IN T H E 13-LINED G R O U N D SQUIRREL* W I L L I A M A. G A L S T E R and P E T E R M O R R I S O N Departments of Zoology and Physiology, University of Wisconsin, Madison, and Laboratory of Zoophysiologyt, Institute of Arctic Biology, University of Alaska, College, Alaska (Received 3 ffanuary 1966)
Almtract--1. Blood lipid, protein and hematocrit levels in Sperraophilus tridecemlineatus foUow yearly cycles. Total lipid, a-lipid, fl-lipid and chylomicrons were minimal in late spring (7.0, 2"5, 2"3 and 1"0 g/l) and maximal in late fall (27.0, 5.8, 9"8 and 6"0 g/l). Secondary maxima or plateaux were observed in late summer in all, and in the spring for a-lipid. 2. All proteins were minimal in late spring except//-globulin which was minimal in January. Albumin concentration increased to threefold (from 18 g/l) by midsummer, and then to fourfold in early fall, a level maintained through the winter (av. 65 g/l) until early spring when levels decrease sharply. All globulin components increased briefly at midsummer. Brief maxima also occurred in January and March for fl-globulin and in February and May for aand 7-globulin.
INTRODUCTION HIBERNATION reviews cite many comparisons of blood constituents from active and hibernating mammals but few studies have encompassed the whole year (Lyman, 1955; Kayser, 1961). Circulatory lipids have received little consideration except for the work of Bragdon (1954) on the Columbian ground squirrel and Bitrck et al. (1956) on the hedgehog. Plasma proteins have received attention from Suomalainen & Karpannen (1956) and Bi6rck et al. (1956) on the hedgehog, and from South & Jeffray (1958) on the hamster. T h e RBC concentration appears not to have been followed through the year except in the Tien-Shan marmot (Bibikov & Thirnova, 1956). Because of the special nutritional importance of lipicls in the hibernators' yearly cycle the present study examines the levels of blood lipids and proteins throughout the year, attempting to relate these both to each other and to the several activity phases. * These studies were assisted by grants from the National Science Foundation (G24039) and the Kansas Heart Association. Manuscript preparation and publication were assisted by the National Institutes of Health (No. GM10402). ? Present address. These studies are Publication No. 25. 489
490
WILLIAM A. GALSTER AND PETER MORRISON MATERIALS AND M E T H O D S
Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) were collected near Madison, Wisconsin (1960-62), and Atchison, Kansas (1963), and housed in individual cages. T h e temperature was maintained at 20°C and the light cycle at 12 hr of daylight. Animals were fed Purina Rat Chow (50 per cent carbohydrate, 24 per cent protein, 4 per cent fat) and given water ad libitum. Squirrels that became torpid under these conditions (body temperature near ambient) were referred to as "room-temperature hibernators". Hibernation in season was brought about by transferring the animals in early November to a dark hibernaculum where the temperature was maintained at 9 + 2 ° C . Food and water consumption, urination and visual observations were used to identify inactive periods during the hibernating season, Blood samples were obtained by decapitation or by cardiac puncture following a 24 hr fast. Hematocrit values were measured in heparinized capillary tubes and the remainder of the sample allowed to clot for serum separation. Turbidity of serum was estimated on a scale from 0 to 3. Total serum lipids were estimated using the turbidometric method of De la Huerga (1953)modified for 0-1 ml sample. Samples with excess lipid were diluted. Serum proteins were separated electrophoretically on paper strips in a Durrum cell at pH of 8.6 using veronal buffer of ionic strength 0.075. Protein components were stained with bromphenol blue after migration at 2"5 mA for 18 hr and quantitared with the Beckman "Analatrol". A representative separation is compared to that in man in Fig. 1 to show the somewhat different spacing of the components.
a i
7"
f,,
~
=2
¢==
; 7"
HUMAN
ALBUMIN
/ ~Z
~I
=~,
=t
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=1 ALBUMIN
FIG. 1. Representative separation of ground squirrel serum by paper electrophoresis compared to human standard. Dashed line represents fipid staining.
Lipoproteins were stained with oil red O according to Jencks et al. (1955) after migration at 10 mA for 4 hr. T h e chylomicron fraction, separable by centrifugation at 106g rain, showed no mobility. T h e three lipid fractions were quantitated as proportions of t h e total color referred to the total lipid analysis. T h e lipid-beating protein was identified among the several electrophoretic components by staining
SV2~SONAL CH~qOgS IN 1 3 - L I ~
OROUND SQUIRREL
491
two halves of a single strip (Fig. 1). The B-llpid corresponded directly to a/3globulin but the s-lipid fell between the ~l-globulins. RESULTS All lipid components exhibited yearly cycles in concentration with maxima in late fall and minima in late spring. Total serum lipid rose through the summer to more than twice the June concentration in September (6-2-+ 13.5 g/I~ (Fig. 2). In
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MONTH FIG. 2. Total serum lipid concentration by months with mean, standard error, range and sample size indicated. Single points in summer represent animals which were "forced" into hibernation. Single points in spring are from replacement animals that were placed in hibernaculum in February. Shading and crosshatching signify percentage of "deep" and "room-temperature" hibernators.
November the value quiekly doubled again (to 26.2 g/l) and then declined more or less exponentially through the winter and spring. Forced summer hibernators (2 weeks at 5°C in July) and room-temperature hibernators (Sept.-Oct.) had concentrations similar to the November group. Values from three ground squirrels not exposed to the cold during the hibernating season fell within the range of the December values. A wide variance in the total lipid concentration in serum was
4-92
WILLIAM A. GALSTERA N D PETER MORI~ISON
exhibited but there was no correlation with hematocrit, sex, body temperature or body weight. ~- and/3-Lipid and chylomicrons followed the general yearly cycle in total lipid but all exhibited individual differences, s-Lipid rose in early summer (2.3 -+ 5-3 g/l) and then doubled again in November-December (to 10.5 g/l) (Fig. 3). The
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mON~4 Fxc. 3. c~-Lipid concentration by months with mean, standard error and range indicated. Broken curve compares =x-glgbulin. Light curves indicate the range of values to be expected for =-lipoprotein protein following the observations of Olson & Vester (1960) on man.
concentration fell somewhat during the winter and then sharply in March. A brief peak was seen,in April-May prior to the June minimum. E-Lipid also rose during summer but fell sharply in September, almost to the June low (Fig. 4), prior to an almost threefold increase in November and December (3.1 -> 8.7 g/l). Values fell through the winter and remained low through the spring. Chylomicrons showed abrief concentration peak in November which had fallen to the average value by the end of December (Fig. 5). Turbidity was observed in most samples with m o r e t h a n 0-1 g/1 of chylomicmns but a third of the clear samples had from 0-1 to 0.5 g/1 of chylomicrons. Turbidity was observed in
493"
SEASONAL CHANGES IN 13-LINED GROUND SQUIRREL 7 I # # rl
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494
WILLIAMA. GALST~AN])PETERMORRISON
82 per cent of the serums from room-temperature hibernators and in only 10 per cent of the serums from deep hibernators. All protein components were minimal in late spring but 0nly albumin was maximal in late fall. Values for serum albumin exhibited a yearly cycle similar to serum lipid (Fig. 6) but was minimal in May (18 g/l), not June, and maintained 80
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their winter elevation until the end of March. The precipitous drop from 60 to 20 g/l during two months of spring was striking. All globulin components increased briefly during midsummer. E-globulins increased threefold by late summer, returned nearly to the June minimum by early fall and increased through winter to the maximum near the end of hibernation. ~-, ~ - and y-Globulins remained unchanged through fall and early winter. The ~- and y-globulins increased briefly during late winter and spring. Hematocrit values exhibited a pronounced yearly cycle (Fig. 7) with low values through late winter and spring (36-0 per cent), intermediate values in the summer (42 per cent) and an October peak (53 per cent).
495
SEASONAL CHANOES I N 1 3 - L I N E D OROUND SQUIRREL
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7
9
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DISCUSSION
Columbian ground squirrels (Bragdon, 1954) and 13-lined ground squirrels both show concentrations of serum lipid that are seven times those found in the white rat (Boyd, 1942). This striking difference in the hibernators suggests greater capacity for carrying lipid in the blood. Bragdon did not consider seasonal changes in serum lipids and did not give sampling dates. His measurements on non-fasted Columbian ground squirrels were higher than our measurements on the 13,lined ground squirrel made in early fall (16.9 g/l).
Yearly pattern As shown in Fig. 8, the several phases of serum lipid fluctuation corresponded in considerable degree to the yearly activities of the 13-1ined ground squirrel as observed by Landau & Dawe (1960) and Rongstad (1965). First, following the prehibernation maxima, all lipid components declined progressively during hibernation (Dec.-Apr.). Except for a brief rise in a- and//-lipid, this decline continued through the spring breeding and gestational period (Apr.-May), a time of great activity during which the hibernator may still be largely dependent on fat reserve for energy. The minimum in all lipid levels (late May or early June)
496
WILLIAM A. GALSTER AND PETER MORRISON
corresponded to the reappearance of high quality nutrient for the diet. With this food supply, the second phase of growth was accompanied by a steady increase in the level of all serum lipids to a plateau in early September. Rongstad (1965)
60
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7
8
9
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3
4
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Serum lipid, protein and hematocrit cycles in relation to activity phases through the year.
observed that some animals may enter hibernation at this time, having completed their preparation in August, but others especially young of the year may delay until early October depending on conditions. About 25 per cent of our animals "hibernated" at room temperature during the fall since they were not placed in the hibernaculum until the first week of November. This could represent some distortion of the natural pattern, but a striking independence of the yearly hibernation cycle of conditions of light and temperature has recently been demonstrated in this species (Morris & Morrison, 1964). This delay (Sept.-Nov.) may perhaps be regarded as an extension of a variable but much shorter natural delay period (Sept.) following the completion of nutritional preparation for hibernation. In the jumping mouse (Zapus) an extended delay period of this sort has been observed with fatal consequences (Morrison & Ryser, 1962). It is interesting to note that the two late summer ground squirrels when exposed to 0°C for 3 weeks had a November
5-7 4-8~-6 ±2"2 6"8 5"~7-5 0"7
3"4 2.4--4.5 ±0"6
5"8 3"2-8"0 1"7
2"2 1"54"1 1"2
Range S.D.
Range S.D.
Chylomicrons Range S.D. 1"5 0"2-2"4 1"1
13"2 9"0-15"8 ±2-4
11"7 8"8-14-8 ±2"8
Total Range S.D.
Lipid (g/l)
0"8
2"7
0"8 0-2"1 1"0
4"9 2"94"7 1"7
4"8 2"9-9"7 ±2"5
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6
Number Days
0
August
Month
1-1 0-1"6 0"3
8"4 5"9-12"0 3"9
5"2 2"~9"9 2-7
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6"6
5-5 2"3-8"2 2"4
9"2 4-2-18"5 5-8
8-4 3-2-12-0 2"7
23"5 18"~30"0 ±3"5
0
5"1 1"3-10-3 3-4
14"1 11-3-16"7 2"3
9-9 5-9-13"2 3"3
26-6 15"~34"0 ±4"5
5 0-8
November
9"4 7"2-10"3 1-4
7-9 4"0-14,8 4"3
9"9 9"1-11"2 0"3
22"4 12"1-26"4 ±6"0
2"7
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1"9 0-3"8 + 0"4
2.6 1-4-4"6 1-0
8.7 4-6-14.2 3-5
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13"4 9"3-18"0 + 3"0
6 10-20
11"4 7"5-17"4 + 6"2
8 1-8
January-February
T A B L E 1 - - E F F E C T S OF STARVATION ON SERUM LIPIDS OF ACTIVE AND HIBERNATING 1 3 - L I N E GROUND SQUIRRELS
4~
O
WILLIAM A. GALSTER AND PETER MORRISON
498
serum lipid pattern. Hibernation was then immediately preceded by a sharp rise in all lipid components (Nov.) which, together with the delay period, may be taken as the third phase in the yearly cycle of lipids. Although it is rash to speculate on the implications of changes in dynamic, steady-state levels, some comments may be made. The progressive increase in all blood lipids during summer may reasonably be associated with the fat deposition essential for hibernation. According to Olson & Vester (1960) ~-lipoprotein carries lipid from the intestine to the liver and the fat stores, while fl-lipoprotein carries lipid from the liver to the fat stores. And, indeed, ~- and//-lipid levels rose steadily through August and September, the period when fat deposition and lipogenesis would normally be most intense, and fell somewhat in September when fat deposition would normally have been completed. However, since intestinal absorption should have been complete (in 24 hr fasted animals) the elevated level of ~-lipid may indicate that this component also was taking some role in liver-to-fat depot transport in this species. Even with an increase in the fasting period from 1 day to 6½ days total lipid and lipoprotein fractions failed to decline (Table 1), TABLE 2--SERUM LIPID LEVELS IN DORMICE
Yearly light cycle Normal (Boreal)
Reversed (Austral)
Lipid (g/l)
Torpor* (%)
Lipid
Date
(g/l)
Torpor (%)
10/61 12/61 4-5/62 6/62
10-9 (4) 2.6 (7) 6-7 (10) 7.3 (10)
42 60 35 40
10-1 (6) 5.0 (9) 6.1 (9) 4.5 (9)
35 222 60 80
* Percentage of individuals entering torpor when exposed for a 4 day test period at 5°C. Parenthesized values indicate sample size. suggesting that a high blood lipid level may be normally maintained in this species. In the dormouse (Glis glis), another hibernator but a more "permissive" one, individual serum lipid values range from 2.4 to 20 g/1 as compared to 5-0 to 36 g/1 in the 13-lined ground squirrel and the mean values are also about half those in the latter species (6"5 versus 12.6 g/l). T h e dormouse also differed in showing no yearly pattern in the lipid level (Table 2). In the fall, lipid levels rose dramatically just prior to hibernation and chylomicrons increased. These increases were preceded (not accompanied) by a 23 per cent increase in the hematocrit (Fig. 7), indicating that lipid changes were not caused by hemoconcentration. This prehibernation increase in ~- and//-lipid during late fall might reflect a final burst of lipogenesis, but alternatively might equally reflect some adjustment in the dynamic equilibrium between liver, blood
SEASONAL CHANGBS I N
13-LrNED GROUND
SQUIRREL
499
and depot fat. In the rat, French & Morris (1957) observed a half-life of only 12 min but after 24 hr fasting chylomicrons persisted in ground squirrels. The increase in chylomicrons may represent an overloading of the lipid transport mechanism. Animals "hibernating" at room temperature during this time showed increases in serum chylomicrons and turbidity, and may indicate a need for low temperature to facilitate clearing action. Fasting for 2.7 days during this period produced little change in total lipid or the a-fraction (Table 1). The //-lipid increased initially and then decreased, and chylomicrons increased during fasting. Lipid components During the winter and spring, encompassing the hibernation season and the spring breeding period, values for all lipids decreased from the yearly maximum to the yearly minimum a-Lipid remained high through the hibernation season although the function of intestine-to-liver transport proposed by Olson & Vester (1960) does not seem appropriate in this case. a-Lipid increased sharply at awakening and rose during the breeding season before declining to the minimum in early June. //-Lipid and chylomicron fractions and hematocrits declined through the early hibernating season and then remained constant until late spring. There was no correlation between the periodic awakenings during hibernation and the level of the lipid fractions (Table 1). Further, the caloric values at the maximum serum lipidlevel were only sufficient to have maintained the hibernating squirrel for less than 2 days. Therefore these lipids are either maintained or are not being used during torpor and their replenishment appears not to be a cause for periodic awakening. .
Protein components Seasonal changes in plasma protein fractions followed a varied pattern and did not reflect the lipid fraction changes. Limits for a-lipoprotein, located between the s-globulin and the albumin protein bands, were estimated from a-lipid values and the composition data of Olson & Vester (1960). There was no correlation with the values for %-globulin (Fig. 3). Changes in albumin do correlate with changes in s-lipid. The //-lipid appeared on the strip with the E-globulin. Because/glipoprotein is only one of several E-globulins, estimates of its concentrations from the//-lipid values can only account for a fraction of the E-globulin (Fig. 4). The marked reduction in y-globulin during hibernation (Dec.-Jan.) was similar to that observed by South & Jeffray (1958) in the hamster. The early recovery of this fraction in February and its continued rise during the spring may reflect a reconstitution of immune function before awakening. The hematocrit varied through the summer and this may be attributed to the introduction of young squirrels. The 23 per cent increase in early fall similar to the increased erythrocyte count observed by Bibikov & Zhirnova (1956) in Tien-Shan marmots may relate to increased erythropoiesis giving squirrels reserve RBC's to last through hibernation. Raths (1953) and Lyman et al. (1957) measured marked
500
WILLIAMA.
GALSTER AND PETER MORRISON
reductions in erythropoietic activity during hibernation in woodchuck and hamsters, but this could b e paralleled by a like reduction in erythrocyte destruction. The reduction in hematocrit concentration during hibernation parallels that observed by Woodward & Condrin (1945) in Tamias striatus and Stuckey & Coco (1942) in 13-lined ground squirrels. It may be important to re-emphasize the stability of the yearly physiological cycle in the 13-lined ground squirrel without which a coherent pattern could easily have been lost during long.term maintenance under artificial conditions of captivity. Morris & Morrison (1964) have shown that even with maintenance under continuous warm conditions (25°C) and with a reversed yearly light cycle (austral) there was no suppression or shift in the yearly cycles in body temperature, hibernation, tendency or reproductive activity. This need not be the case since in the dormouse (Glis glis) a reversal in light cycle is quickly followed by a reversal in the physiological response. SUMMARY T h e 13'lined ground squirrel exhibits well-defined yearly cycles for the lipid and protein components of serum. The average lipid concentration is very high but the protein concentration is normal for mammals. T h e number of lipid and protein electrophoretic components is usual. Total lipid, s-lipid, r-lipid and chylomicrons were minimal in late spring and maximal in late fall. Secondary maxima or plateaux were observed in late summer in all lipids and in the spring for c~-lipid. All protein components were minimal in late spring except r-globulin which was minimal in January. Albumin concentration increased to threefold by midsummer, and then to fourfold in early fall, a level maintained through the winter until early spring when levels decreased sharply. All globulin components increased briefly in midsummer. Brief maxima also occurred in January and March for r-globulin and in F~bruary and May for ~- and y-globulin. These changes in lipid and protein concentration correspond in considerable degree to the changing functional periods throughout the year--growth and fattening in summer, prehibernation delay and preparation in the fall, hibernation during the winter and reproductive activity in the springmhowever, the direct significance of the changing levels is yet to be established. REFERENCES BIBIKOV D. I. & ZHIRNOVAN. M. (1956) Seasonal modifications of some ecologicallyphysiological peculiarities of Marmota barbacine in the Tien-Shan. Zh. Zool. 35, 1565-1573. BIORCK G. J., JOHANSSONB. & VEIGE S. (1956) Some laboratory data on hedgehogs, hibernators and nonhibernators. Acta physiol. Scand. 37, 281-294. BOYDE. M. (1942) Species variation in normal plasma lipids estimated by oxidative micromethods..7, biol. Chem. 143, 131-132. BRAGDONJ. H. (1954) Hyperlipemla and atheromatosis in a hibernator, Citeilus columbianus. Circulation Res. 2, 520-524.
SEASONAL CHANGES IN 13-LINED GROUND SQUIRREL
501
DE LA HUERGA,YESINICKY. C. & HOPPER H. (1953) Estimation of total serum lipids by a turbidometric method. Am. 07. din. Path. 23, 1163-1168. FRENCH J. E. & MORRIS B. (1957) Removal of C 14 labelled chylomicrons from the circulation in rats. 07. Physiol. 138, 326-337. JENCKS W. R., DURRUM E. L. & JETTON M. R. (1955) Paper electrophoresis as a quantitative method for lipoproteins. 07. din. Invest. 34, 1437-1448. KAYSER C. (1961) The Physiology of Natural Hibernation. Pergamon Press, New York. LANDAU B. & DAWE A. R. (1960) Observations on a colony of captive ground squirrels throughout the year. Mus. comp. Zool. Bull. 124, 123-189. LYMAN C. P. (1955) Physiology of hibernation in mammals. Physiol Rev. 33, 403-425. LYMAN C. P., WEISS L. P., O'BRIEN R. C. & BARREAUA. A. (1957) The effects of hibernation on the replacement of blood in the golden hamster. 07. exp. Zool. 136, 471-485. MORRIS L. & MoRRISON P. (1964) Cyclic responses in dormice (Glis glis) and ground squirrels (Spermophillus trideceralineatus) exposed to normal and reversed yearly light schedules. Proc. 15th Alaskan Sci. Conf., College, Alaska, p. 40. MORRISON P. & RYSER F. A. (1962) Metabolism and body temperature in a small hibernator, the meadow jumping mouse. 07. cell. comp. Physiol. 60, 169-180. OLSON R. E. & VESTERJ. W. (1960) Nutritional endocrine interrelationships in the control of fat transport in man. Physiol. Rev. 40, 677-733. RATHS P. (1953) Untersuchungen fiber die Blutzusammensetzung und ihre Beziehungen zur vegetativien Tonuslage beim Hamster. Z. Biol. 106, 109-123. RONOSTAD O. (1965) A life history of 13-lined ground squirrels in southern Wisconsin. 07. Mammal. 46, 76-87. SOUTH F. C., JR. & JEFFRAYH. (1958) Alterations in serum proteins of hibernating hamsters. Proc. Soc. exp. Biol. Med. 98, 885-887. STUCKEYJ. & Coco R. M. (1942) A comparison of the blood picture of active and hibernating ground squirrels. Am. 07. Physiol. 137, 431-435. SUOMALAINEN P. & KARPPANEN E. (1956) Einfluss des Winterschlafes anf das AlbuminGlobulin Inverhaltnis des Igelserums. Sonderabdr. Suom. Kemistilehti B 29, 74-75. WOODWARD A. E. & CONDRIN J. i . (1945) Physiological studies on hibernation in the chipmunk. Physiol. Zool. 18, 162-167.