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Altered tyrosine and tryptophan metabolism during hypothermic hibernation in the 13-lined ground squirrel (Spermophilus tridecemlineatus)
CRYOBIOLOGY
2,
504-512 (1987)
Altered Tyrosine and Tryptophan Metabolism during Hypothermic Hibernation in the 13-Lined Ground Squirrel (Spermophilus
tridecemlineatus)
BRUCE L. WEEKLEY AND H. J. HARLOW Departmentof Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071 (1) Tyrosine and tryptophan metabolism in brain and peripheral tissues were studied in hypothermic hibernating and normothermic nonhibernating 13-lined ground squirrels (Spermophilus tridecemlineatus). (2) In the hypothermic hibernating state, there were significant elevations of brain stem tyrosine, norepinephrine, and dopamine levels; forebrain norepinephrine and dopamine levels; and cerebellum norepinephrine and tyrosine levels. (3) On the other hand, plasma norepinephrine levels were significantly decreased in hypothermic hibernating squirrels while plasma tyrosine levels were increased. Kidney norepinephrine levels were significantly increased in hypothermic hibernating squirrels, while kidney tyrosine levels were decreased. Total plasma tryptophan and free plasma tryptophan were significantly reduced in hypothermic hibernating squirrels. Hepatic tyrosine aminotransferase K,,, and V,,,,, were decreased in hypothermic hibernating squirrels, while tryptophan 2,3-dioxygenase activity was not altered. Plasma and liver albumin were increased in hypothermic hibernating squirrels, while plasma and liver total protein were not altered. (4) These results demonstrate that significant changes in tyrosine and tryptophan metabolism occur in both central and peripheral tissues with concomitant alterations in metabolites during hypothermic hibernation in 13-lined ground squirrels. 6 1987 Academic Press, Inc.
Animals that hibernate exhibit circannual alterations in the regulation of body temperature, food and water intake, and patterns of sleep and wakefulness (28). Indeed, the transition from the homeothermic phase to the heterothermic phase of a hibernator’s circannual cycle requires metabolic adaptations (37) marked by a large reduction in oxygen consumption (41). In addition to cessation of food intake in the ground squirrel and minimal release of substrates from fuel stores (24), considerable data are available which indicate a central nervous system (CNS) site for regulation of these processes in nonhibernating species (2, 19). Furthermore, in hibernating species intrahypothalamic injection of both norepinephrine (NE) and serotonin (5hydroxytryptamine; 5-HT) have been shown to
Received September 22, 1986; accepted June 8, 1987.
stimulate arousal in Spermophilus lateralis (1). On the other hand, hypothalamic lesions in ground squirrels block arousal from hibernation (34). The literature on brain monoamine changes in hibernation does not provide a clear consensus. Some investigators have reported increased (25, 30, 36, 44), decreased (5, 11, 38), or unaltered (48) brain serotonin (5-HT) levels during hiberation. Furtheremore, other studies have reported changes in brain 5-HT and its principal metabolite 5-hydroxyindole-3-acetic acid (5HIAA) during entry into and arousal from hibernation (13, 32). Brain levels of catecholamines have also been reported to decrease (13) or not change (5, 42) during hibernation. Since the CNS plays a key role in regulating both the sympathoadrenal and endocrine systems (39) and since both of the systems undergo marked changes during the hibernation cycle (13, 15, IS), we un-
504 OOll-2240187$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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these experiments. Normothermic nonhibernating animals used in these experiments were completely aroused for a maximum of 24 hr prior to use in the experiment. Preliminary experiments indicated that the 13-lined ground squirrel is capable of arousal within 2-3 hr. These experiments were carried out in March 1983. All animals were left in their cages in the cold room until immediately before death to avoid arousal or excitement in hypothermic hibernating or normothermic nonhibernating squirrels. Normothermic nonhibernating or hypothermic hibernating squirrels were removed from cages immediately prior to death during the 2-hr daily light period, stunned by a sharp blow to the head, and decapitated. Blood MATERIALS AND METHODS was collected from the cervical stump in a Adult male and female 13-lined ground heparinized test tube, cooled to 4”C, and squirrels weighing an average of 178.4 + centrifuged. The plasma was removed and 36.6 (SD) g were acclimated for 4 weeks at a 1.O-ml aliquot was stored in sodium meta4 & 1°C in individual stainless steel cages bisulfite as a preservative for catecholwith a 2:22, 1ight:dark cycle during De- amines and frozen at - 15°C. Permanent cember 1982. Food (Purina Laboratory Rat cannulation for blood collection was not Chow) and tap water were available ad li- used because of problems in maintaining bitum. Squirrels were monitored daily for patency in a long-term study and the proba10 weeks starting in January 1983 prior to bility that implantation of the cannula for a the experiment in order to determine the short-term study would alter normal hibernormal hibernation pattern for each animal. nation patterns and mechanisms regulating Body temperature (rectal) was determined hibernation. daily with a thermistor and a YSI teletherThe brain was removed and dissected on mometer (Model 44TE). All animals used a cold glass plate into cerebellum, forebrain in these experiments demonstrated a well- or cerebral cortex (telencephalon), and developed pattern of hibernation with pe- brain stem, which includes diencephalon, riods of continuous torpor for at least 5 mesencephalon, metencephalon (minus days. Squirrels were considered to be in cerebellum) and myencephalon caudal torpor if they had a rectal temperature of to Cl. 5-8°C and did not respond to handling. The abdominal cavity was opened by a Conversely, squirrels were considered to ventral incision along the linea alba, and be in a completely aroused state if they had the caudate and papillary processes of the a rectal temperature of 33°C or greater and caudate liver lobe were removed for endid respond to handling. As a result of this zyme assays. The remaining lobes were redaily monitoring of hibernation status, all moved and utilized for other assays. Peri13-lined ground squirrels were acclimated renal fat and both kidneys were excised. to handling which served to reduce stress The thoracic cavity was opened by cutting at the time of death. Squirrels with inter- the ribs ventrally and slightly lateral to the mediate body temperature were not used in sternum. The heart was cut free of the dertook this study to monitor changes in central and peripheral monoamines and in their amino acid precursors. It was the aim of this project to investigate the following parameters of the 13-lined ground squirrel (Spermophilus tridecemlineatus) during the hibernation season: first, fluctuations in available tyrosine and tryptophan (the amino acid precursors of NE and 5-HT, respectively), as well as circulating and tissue catecholamines; and second, brain stem tryptophan ?Ghydroxylase, hepatic tyrosine aminotransferase, and hepatic tryptophan 2,3-dioxygenase that are the rate-limiting enzymes in serotonin synthesis and tyrosine and tryptophan degradation, respectively.
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great vessels and removed. The gastrocnemius muscle was also removed from both legs. Tissue tryptophan, serotonin, 5-hydroxyindole-3-acetic acid, tyrosine, norepinephrine, and dopamine were chemically extracted (4) by homogenization of tissues at 4°C in 5.0 ml of analytical grade acidified n-butanol. Following centrifugation (5OOOg for 30 min at 4”C), 4.0 ml of the supernatant was returned to a second set of tubes containing 7.0 ml of analytical grade n-heptane and 0.3 ml of 0.01 N HCl. This mixture was shaken for 10 min and centrifuged (1OOgfor 10 min at 4°C) to separate the organic and aqueous layers. The upper organic layer was removed and washed with 0.3 ml of 0.033 M NaHCO, to extract 5-hydroxyindole-3-acetic acid. An aliquot of the acid extract was assayed for serotonin by the ophthalaldehyde fluorometric procedure (40). Tissue levels of tyrosine and tryptophan were also determined using acid extract (6, 17, 45). Catecholamines were further purified from the acid extract by use of a CG,, ion-exchange resin (H+ form) (27). Tissue levels of epinephrine, norepinephrine, and dopamine were determined fluorometrically (4, 27). Total plasma tryptophan was determined fluorometrically (6). Free tryptophan was determined by treating plasma with dextran charcoal (17) prior to assay, and albuminbound tryptophan was calculated as the difference. Plasma tyrosine and catecholamine levels were determined fluorometritally (4, 27, 45). Brain stem tryptophan 5-hydroxylase was assayed by the method of Gal and Patterson (16) with subsaturating conditions of tryptophan (200 t&f) and dimethyltetrahydrobiopterin (100 t~I4). Hepatic tryptophan 2,3-dioxygenase was assayed spectrophotometrically as described by Knox et al. (23). Tyrosine aminotransferase was measured spectrophotometrically in vitro (8). All in vitro enzyme assays were conducted at 37°C. Total plasma protein was assayed
by the method of Lowry et al. (26). Plasma albumin was determined spectrophotometrically (9) to determine whether changes in circulating albumin levels alter the free/ bound plasma tryptophan ratios. Measurement of total plasma protein allowed determination of whether albumin is specifically altered or whether changes in circulating levels are merely a reflection of changes in all proteins. All data were analyzed by a 2tailed student t test and the null hypothesis was rejected at P < 0.05. RESULTS
Brain and Hepatic Enzyme Activities Brain stem tryptophan hydroxylase activity in low speed supernatants prepared from brain stem of either hypothermic hibernating or normothermic nonhibernating squirrels was not significantly different (data not shown). Hepatic tryptophan 2,3dioxygenase activity assayed in vitro also was not significantly different between hypothermic hibernating and normothermic nonhibernating squirrels when assayed at 37°C. The heme-saturation ratio of the enzyme did not significantly change during the hibernation cycle. The b,,, of tyrosine aminotransferase (2.79 ? 0.28 p,rn L-tyr/mg Plmin in hypothermic hibernating squirrels vs 24.63 & 13.33 km L-tyr/mg P/min in normothermic nonhibernating squirrels) was significantly reduced (P < 0.01) and the K, (335.45 + 56.44 pm L-tyr in hypothermic hibernating squirrels vs 2637.88 +- 130.80 km L-tyr in normothermic nonhibernating squirrels) was significantly reduced (P < 0.01) in liver from hypothermic hibernating squirrels tested at normothermic temperature (37°C). Tryptophan, Tyrosine, and Catecholamine Metabolism Total and free plasma tryptophan were depressed in hypothermic hibernating squirrels (P < 0.01 and P < 0.05, respec-
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TYROSINE AND TRYPTOPHAN METABOLISM TABLE 1 Peripheral Tissue Levels of Tryptophan (TRP) and Tyrosine (TYR) in Normothermic and Hypothermic 13-Lined Ground Squirrels” Tissue
Metabolite
Normothermic
Heart Skeletal muscle Liver Kidney Plasma Skeletal muscle Liver Liver
TYR cLdg TYR I*& TYR w/g TYR v& TYR wk TRP mgig TRP i-&it TRPlorgan kg/liver TRP ds TRP kg/ml TRP kg/ml
224.94 k 69.03 43.34 r 20.71 389.87 r 63.67 216.02 k 75.46 86.5 2 26.70 433.7 2 57.26 0.549 2 0.395 3.136 IT 2.85 1.431 ? 0.47 5.704 -r- 1.389 1.003 2 0.815
TRP &ml
4.702 !I 1.97
Kidney Total plasma Albumin-bound plasma Free plasma
n
Significance
4 4 4 4 5 4 6 6
n.s. ns. ns. P < 0.01 P < 0.001 n.s. n.s. ns.
0.907 ” 0.392 2.549 ” 0.925 0.568 2 0.675
4 9 9
n.s. P < 0.01 n.s.
2.799 * 1.89
9
P < 0.05
Hypothermic 339.64 2 33.64 k 365.04 f 109.89 2 281.52 t 526.49 k 1.113 2 5.85 4
73.1 30.84 44.95 10.78 58.58 105.36 0.914 5.15
(1Each value is presented as the mean t SD.
tively), while albumin-bound tryptophan was not significantly different (Table 1). Plasma norepinephrine levels were significantly (P < 0.001) reduced in hypothermic hibernating squirrels but plasma epinephrine and dopamine levels were unaltered (Table 2). In addition, plasma tyrosine was significantly (P < 0.001) elevated in hypothermic hibernating squirrels (Table 3). Liver, kidney, and skeletal muscle (gastrocnemius) levels of tryptophan did not change during hibernation (Table 1). Concentrations of tyrosine in heart, skeletal muscle, or liver also did not change during
the hibernation cycle (Table 1), although kidney tyrosine levels decreased in hypothermic hibernating squirrels (P < 0.01). On the other hand, liver norepinephrine levels did not change during hypothermic hibernation, while concentrations in the kidney increased (Table 2). In addition, norepinephrine levels in heart and perirenal fat did not change as a function of the state of hibernation (Table 2). Dopamine levels also did not change in either liver or kidney during hypothermia. Brain levels of tryptophan were not altered during torpor, whereas brain stem tyrosine, norepineph-
TABLE 2 Peripheral Tissue Levels of Norepinephrine (NE), Epinepherine (E), and Dopamine (DA) in Normothermic and Hypothermic 13-Lined Ground Squirrels” Tissue
Metabolite
Normothermic
Hypothermic
n
Liver Kidney Heart Plasma Perirenal fat Plasma Liver Kidney Plasma
NE cL&t NE cL& NE ds NE rig/ml NE rig/g E rig/ml DA ngig DA ngig DA ngiml
3.39 2 5.78 k 46.16 ? 110.78 + 89.46 k 28.27 2 96.78 2 78.45 2 17.63 2
5.37 k 9.87 f 34.16 ” 28.24 2 51.08 ? 23.07 k 86.85 ” 85.11 k 14.19 k
5 5 4 5 5 5 4 4 5
a Each value is expressed as the mean 2 SD.
1.15 1.66 5.07 40.71 67.63 2.65 12.28 12.56 1.72
0.57 1.23 7.71 20.90 20.16 3.53 16.18 13.73 1.43
Significance
P <%I5 n.s.
P < 0.0001 ns. n.s. n.s. n.s. n.s.
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rine, and dopamine levels Were significantly increased (Table 3). Cerebellum tyrosine and norepinephrine were also elevated in hypothermic hibernating squirrels, as were forebrain norepinephrine and dopamine. Plasma Albumin
and Total Protein
Plasma albumin was significantly increased (P < 0.05) in hypothermic hibernating squirrels in addition to an increased plasma albumin/total protein ratio. Furthermore, the liver albumin levels and liver albumin/protein ratios were significantly (P < 0.001) increased in hypothermic hibernating squirrels (Table 4). DISCUSSION
From the standpoint of potential mechanisms influencing hibernation, an interesting observation on the 13-lined ground squirrel was the dramatic shift in central and peripheral tyrosine metabolism following entry into the hypothermic phase of hibernation. It seems reasonable to suggest
that this metabolic effect may be involved with the changes in autonomic tone occurring at this time. Indeed, it has been suggested that changes in brain stem monoamines may cause reductions in sympathetic activity (22) in nonhibernating species. Additionally, it is well documented that there is a reduction in sympathetic activity during entrance into hibernation (43). On the other hand, injection of norepinephrine into the brain of a hibernator has been reported to elicit arousal (I), while drugs that block norepinephrine synthesis usually block arousal from hypothermia (12). Draskoczy and Lyman (10) found that norepinephrine turnover is reduced in brown fat, heart, and brain of hibernating ground squirrels, whereas our observations indicate that tissue levels of norepinephrine in brain, kidney, and plasma are significantly increased in hypothermic hibernating squirrels. However Draskoczy and Lyman (10) estimated norepinephrine turnover by intraperitoneal injection of H-NE in aroused 13-lined ground squirrels that were
TABLE 3 Levels of Tryptophan (TRP), Tyrosine (TYR), Serotonin (5HT), 5-Hydroxyindole-3-Acetic Acid (5HIAA), Norepinephrine(NE), and Dopamine (DA) in the Forebrain, Cerebellum, and Hindbrain of Normothermic and Hypothermic 13-Lined Ground Squirrels” Brain region
Metabolite
Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Forebrain Forebrain Forebrain Forebrain Forebrain Forebrain Brain stem Brain stem Brain stem Brain stem Brain stem Brain stem
NE nglg TYR t&g DA nglg TRP wdg 5-HT rig/g 5-HIAA r&g NE ngig TYR (*.dg DA rig/g TN i&g 5-HT ngig 5-HIAA rig/g NE ngig TYR I*& DA nglg TRP t-&g 5-HT rig/g 5-HIAA nak
Normothermic 734.39 ? 1322.39 k 1177.15 2 489.25 k 1666.97 2 740.10 k 546.50 2 1233.64 2 696.54 ” 264.07 2 938.13 k 244.26 f 581.25 k 887.13 2 587.14 ” 333.38 k 509.62 t 232.96 k
206.47 585.06 415.07 215.80 531.45 234.92 96.15 100.77 150.88 92.16 283.91 52.11 131.76 140.66 103.14 70.58 86.83 35.72
a Each value is expressed as the mean 2 SD (N = 5).
Hypothermic 1339.44 f 3218.06 lr 1562.39 t 259.05 2 1745.71 e 613.67 k 1477.69 2 1515.55 t 977.31 i 125.27 k 772.67 -c 221.72 _’ 1023.84 2 1149.3 k 804.66 t 308.83 k 491.71 k 218.52 2
118.58 490.02 206.06 139.60 185.69 104.88 271.73 308.78 126.92 101.27 211.38 24.92 204.02 215.33 277.01 178.7 140.52 26.41
Significance P < 0.01 P < 0.01
n.s. n.s. n.s. n.s. P < 0.01 n.s. P < 0.05
ns. ns. ns. P < 0.01 P < 0.05 P < 0.05
n.s. n.s. n.s.
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TYROSINE AND TRYPTOPHAN METABOLISM
TABLE 4 Tissue Levels of Total Protein and Albumin in Normothermic and Hypothermic 13-Lined Ground Squirrels” Tissue Skeletal muscle total protein (mglg) Plasma total protein (mgig) Liver total protein (mg/g) Plasma albumin (mg%) Plasma albumin/protein ratio (pg albuminimg protein) Liver albumin (pg albumin/g) Liver albumin/protein ratio (pg albumin/mg protein)
Normothermic
Hypothermic
n
Significance
3.53 k 1.16 75.76 5 1.02 4.342 -c 0.596 12.26 re_1.76
3.125 74.98 3.566 20.29
0.34 1.44 0.678 6.04
4 5 6 5
n.s. n.s. ns. (P < 0.05)
0.2704 +- 0.077 11.92 2 3.11
5 6
(P < 0.05) (P < 0.001)
3.394 4 1.097
6
(P < 0.001)
0.163 k 0.0315 1.705 2 1.33 0.304 2 0.277
k 5 t 2
a Each value is presented at the mean ? SD.
then induced to hibernate. We have re- imals were subjected to the same treatcently found (45) that intraperitoneal injec- ment. Dopamine and epinephrine, also intions of 0.9% sodium chloride stimulates creased by stress (3), were not different between hypothermic hibernating and normoarousal in the 13-lined ground squirrel. These observations raise a question as to thermic nonhibernating squirrels. The fact whether it is possible to carry out a turn- that catecholamine and tyrosine levels over study in hibernating squirrels (i.e., hi- were altered in most tissues of hypothermic hibernating squirrels as compared to those bernating without any arousal following drug injection). We elected not to attempt in normothermic nonhibernating squirrels to carry out a turnover study since pre- suggests that some mechanism other than vious experiments have indicated that stress is responsible for these changes. Furvarious drugs known to alter brain mono- thermore, epinephrine, the predominant amines also affect arousal (46) in a differen- adrenal medullary catecholamine in most tial manner. It is possible that merely mea- species studied, would also be expected to suring (pharmacologically) the turnover of increase in normothermic nonhibernating monoamines may change the state of hiber- squirrels if the stress of death were responnation. Sauerbier and Lemmer (35) deter- sible for elevated catecholamine levels. mined that peripheral catecholamine turn- The selective increase in only norepinephover in hedgehogs (Erinaceus euuopaeus) is rine in normothermic nonhibernating occurring at a reduced level during hiber- squirrels suggests that some other mechanation. In contrast, Petrovic et al. (31) nism (e.g., altered release or turnover) may demonstrated that adrenal tyrosine hydrox- be responsible. In addition, we maintain ylase (the rate-limiting enzyme in NE for- that as a result of the precautions taken to mation) activity is increased during cold acclimate the animals to handling, the likeadaptation in ground squirrels. Although lihood of a selective norepinephrine inplasma catecholamine levels in 13-lined crease in normothermic nonhibernating ground squirrels are higher than those re- squirrels in response to the stress of death ported in other species, no investigators was reduced. have previously reported seasonal or periThe mechanism by which brain NE and odic variations in these compounds during DA is increased in hypothermic hibernating the hibernation cycle in this species. Even squirrels was not determined in our study, though stress (e.g., handling) is known to although it could be altered secondarily as increase plasma norepinephrine (7), all an- a result of the increased plasma tyrosine.
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The source of tyrosine in the plasma is presently not known although it clearly cannot be of dietary origin. Of the other organs examined, only the kidney showed significant decreases in tyrosine levels. Proteolysis may be a source of the tyrosine during the anorexia of hypothermic hibernation; however, liver and total plasma protein were unchanged, whereas albumin actually increased in plasma during hypothermic hibernation. Indeed, albumin is known to bind tryptophan and reduce its competition with tyrosine for transport into the brain. This would normally increase NE biosynthesis and reduce 5-HT biosynthesis since the respective rate-limiting enzymes are unsaturated in viva (14, 47). However, we did not observe a significant decrease in brain tryptophan or 5-HT levels during hibernation. The observation that free and total plasma tryptophan are decreased during hibernation while albuminbound tryptophan is unaltered suggests that the increased circulating albumin (Table 4) binds a greater percentage of total tryptophan and reduces tryptophan turnover during hibernation. Such a physiochemical property of this system would serve to provide a buffer pool of tryptophan which may be utilized during arousal. Although our study did not specifically address the mechanisms behind the increase in plasma albumin in hypothermic hibernating squirrels, the fact that absolute albumin levels and the albumin/protein ratio are increased in plasma and liver suggests that albumin synthesis is increased or that albumin clearance is decreased. These results are also not in agreement with those that have reported brain 5-HT turnover to increase dramatically and catecholamine levels to remain unaltered during hibernation in the golden hamster (21). Our results, in contrast, demonstrate no change in brain tryptophan, 5-HT, or 5HIAA during hibernation, while tyrosine and catecholamine levels dramatically increase. Other investigators draw differing
conclusions about the changes in brain norepinephrine (5, 13, 42) and 5-HT (5, 11, 25, 30, 36, 38, 44, 48) during the hibernation process in different species. These differences may be due to species differences in regulation of hibernation and may reflect differences in the way the brain was subdivided for assay of monoamines. The NE/5HT ratio of various critical midbrain regions may provide insight as to the precise role that monoamines play in this process. For example, stimulation of the locus coeruleus (site of NE-containing cell bodies) or dorsal raphe (site of 5-HT-containing cell bodies) causes reductions in NE or 5-HT metabolism, respectively, in other brain regions (20, 33). Those studies on rats suggest that brain stem nuclei may have inhibitory effects through their projections in other regions of the brain. Such a mechanism in a hibernator may be important in that hypothalamic input may consist of brain stem and peripheral information as well as visual information. The hypothalamus then integrates the internal metabolic information with external visual cues and makes appropriate adjustments in autonomic and endocrine functions as well as behavioral changes. On the other hand, we did not observe any change in either brain stem tryptophan hydroxylase activity or liver tryptophan 2,3-dioxygenase during the hibernation cycle, while Novotona and Civin (29) reported a decrease in hepatic tryptophan pyrrolase activity during hibernation in the golden hamster. These apparent discrepancies may be due to species differences or to differences in experimental design. The in vitro assays were conducted at 37°C and it is possible that enzyme activity may be altered at 4°C in viva during hibernation. However, Novotona and Civin (29) also conducted assays at 37°C in vitro and reported a depression in tryptophan pyrrolase activity during hibernation. On the other hand, since brain indoles did not change significantly during hibernation, it
TYROSINE AND TRYPTOPHAN METABOLISM
is possible that turnover of those amines may not be altered in this species. Tyrosine aminotransferase activity plays a key role in systemic metabolism and these results support the view that tyrosine metabolism is significantlv altered during hibernation in the U13-lined*ground sq&rel; although, whether this is a primary event or whether it occurs secondarily in the hibernation process remains to be determined. REFERENCES 1. Beckman, A. L., and Satinoff, E. Arousal from hibernation by intrahypothalamic injections of biogenic amines in ground squirrels. Amer. J. Physiol. 222, 875-879 (1972). 2. Brodbeck, J. R. Regulation of feeding and drinking. In “Handbook of Physiology.” Section 1, Vol. 2, Chap. 47, pp. 1197-1206. Washington, D.C. 1960. 3. Buhler, H. U., Prada, D. A., Haefely, W., and Protti, G. B. Plasma adrenaline, noradrenaline, and dopamine in man and different animal species. J. Physiol. 276, 31l-320 (1978). 4. Ciarlone, A. C. Modification of a spectrophotofluorometric method of analyzing serotonin, norepinephrine, and dopamine in one brain sample. Microchem. J. 21, 349-354 (1976). 5. Conguilhem, B., Kempf, E., Mack, G., and Schmitt, P. Regional studies of brain serotonin and norepinephrine in the hibernating, awakening, or active European hamster (Cricetus cricetus) during winter. Comp. Biochem. Physiol. 57, 175-179 (1977). 6. Denckla, W. D., and Dewey, H. K. The determination of tryptophan in plasma, liver and urine. J. Lab. Clin. Med. 69, 160-169 (1967). 7. Depocas, F., and Behrens, W. A. Effects of handling, decapitation, anesthesia and surgery on plasma norepinephrine levels in white rats. Canad. .I. Physiol. Pharmacol. 55, 212-219 (1977). 8. Diamondstone, T. L. Assay of tyrosine transaminase activity by conversion of p-hydroxyphenyl pyruvate to p-hydroxybenzaldehyde. Anal. Biochem. 16, 395-400 (1966). 9. Doumas, B., Watson, W., and Biggs, H. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta. 31, 87-96 (1971). 10. Draskoczy, P. R., and Lyman, C. P. Turnover of catecholamines in active and hibernating ground squirrels. J. Pharmacol. Exp. Ther. 155, 101-111 (1967). 11. Duncan, R. E., and Tricklebank, M. On the stimu-
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24. Kristofferson, R., and Soumalainen, P. Studies on the physiology of the hibernating hedgehog. II. Changes of body weight of hibernating and non-hibernating animals. Ann. Acad. Sci. Fenn. Ser. A4 76, I-11 (1964). 25. Kudryautseva, N., and Popova, N. Serotonin content in various regions of the brain during hibernation and arousal. Bull. Exp. Biol. Med. USSR 4, 44-47 (1973). 26. Lowry, 0. H., Rosebrough, N., Farr, A. L., and Randall, R. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). 27. Miura, Y., Campese, V., DeQuattro, V., and Maijer, D. Plasma catecholamines via an improved fluorometric assay: Comparison with an enzymatic method. J. Lab. C/in. Med. 89, 421-427 (1977). 28. Mrosvosky, N. Circannual cycles in hibernators. In “Strategies in the Cold: Natural Torpidity and Thermogenesis.” (L. Wong and J. Hudson, Ed.), pp. 21-65. Academic Press, New York, 1978. 29. Novotona R., and Civin, J. The relationship between tryptophan pyrrolase activity and serotonin metabolism in the brain and induction of hibernation. Physiol. Bohemoslav. 2864, 339-346 (1979). 30. Novotona, R., Jansky, L., and Drahota, Z. Effect of hibernation on serotonin metabolism in the brain stem of the golden hamster (Mesocricerus auratus). Gen. Pharmacol. 6, 23-26 (1975). 31. Petrovic, V. M., Janic-Sibalic, V., Aminot, A., and Roffi, J. Adrenal tyrosine hydroxylase activity in the ground squirrel. Effect of cold and arousal from hibernation. Comp. Biochem. Physiol. 61C, 99-101 (1978). 32. Popova, N., and Voitenko, N. Brain serotonin metabolism in hibernation. Pharmacol. Biochem. Behav. 14, 773-777 (1981). 33. Salzman, P. E., and Roth, R. H. Post-stimulation catecholamine synthesis and tyrosine hydroxylase activation in central nor-adrenergic neurons. I. In viva stimulation of the locus coeruleus. J. Pharmacol. Exp. Ther. 212, 64-73 (1980). 34. Satinoff, E. Disruption of hibernation caused by hypothalamic lesions. Science 155, 1031- 1033 (1967). 35. Sauerbier, I., and Lemmer, B. Seasonal variations in the turnover of noradrenalin of active and hibernating hedgehogs. Comp. Biochem. Physiol. 57C, 61-63 (1977). 36. Shaskan, E. Hypothalamic and whole brain monoamine levels in bats: Some aspects of central control of thermoregulation. Diss. Abstr. (1969).
37. South, E E., and House, W. A. Energy metabolism in hibernation. In “Mammalian Hibemation III” (K. Fisher, Ed.), pp. 205-324. Oliver and Boyd, London, 1967. 38. Spafford, D., and Pengelley, E. The influence of the neurohumor serotonin on hibernation in the golden mantled ground squirrel Citellis lateralis. Comp. Biol. Physiol. 38A, 239-250 (1971). 39. Swanson, L., and Sawchenko, P. Paraventricular nucleus: A site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31, 410-417 (1980). 40. Tachiki, K. H., and Aprison, M. H. Fluorometric assay for 5-hydroxytryptophan with sensitivity in the picomole range. Anal. Chem. 47, 7-15 (1975). 41. Tahti, H. Effect of changes in CO, and 0, concentrations in the inspired gas on respiration in the hibernating hedgehog. Ann. Zoo/. Fenn. 12, 183-187 (1975). 42. Twente, J., Cline, W., and Twente, J. Distribution of epinephrine and norepinephrine in the brain of Citellus laterulis during the hibernation cycle. Comp. Gen. Pharmacol. 1, 47-53 (1970). 43. Twente, J. W., and Twente, J. Autonomic regulation of hibernation by Citellus and Eptesicus. In “Strategies in the Cold: Natural Torpidity and Thermogenesis.” (L. Wong and J. Hudson, Eds.), pp. 327-372. Academic Press, New York, 1978. 44. Uuspaa, V. The serotonin content of brain and other organs of the hedgehog (Erinaceus europaeus) during activity and hibernation. Experienfia 19, 156- 158 (1963). 45. Waalkes, T. I?, and Udenfriend, S. A fluorometric method for the estimation of tyrosine in plasma and tissues. J. Lab. C/in. Med. 50, 733-736 (1957). 46. Weekley, B., and Harlow, H. J. Effect of pharmacological manipulation of the renin-angiotensin system on the hibernation cycle of the 13-lined ground squirrel (Spermophilus tridecemlineatus). Physiol. Behav. 34, 147- 149 (1985). 47. Wurtman, R. J., Larin, F., Mostafapour, S., and Fernstrom, J. D. Brain catechol synthesis: Control by brain tyrosine concentration. Science 185, 183-185 (1974). 48. Young, R., Robinson, D., Vagenakis, A., Saauedra, J., Louenberg, W., Krupp, P., and Danforth, E. Brain TRH+, monoamines, tyrosine hydroxylase and tryptophan hydroxylase in the woodchuck Marmota monax during the hibernation season. Comp. Biol. Physiol. 63C, 319-323 (1979).
2,
504-512 (1987)
Altered Tyrosine and Tryptophan Metabolism during Hypothermic Hibernation in the 13-Lined Ground Squirrel (Spermophilus
tridecemlineatus)
BRUCE L. WEEKLEY AND H. J. HARLOW Departmentof Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071 (1) Tyrosine and tryptophan metabolism in brain and peripheral tissues were studied in hypothermic hibernating and normothermic nonhibernating 13-lined ground squirrels (Spermophilus tridecemlineatus). (2) In the hypothermic hibernating state, there were significant elevations of brain stem tyrosine, norepinephrine, and dopamine levels; forebrain norepinephrine and dopamine levels; and cerebellum norepinephrine and tyrosine levels. (3) On the other hand, plasma norepinephrine levels were significantly decreased in hypothermic hibernating squirrels while plasma tyrosine levels were increased. Kidney norepinephrine levels were significantly increased in hypothermic hibernating squirrels, while kidney tyrosine levels were decreased. Total plasma tryptophan and free plasma tryptophan were significantly reduced in hypothermic hibernating squirrels. Hepatic tyrosine aminotransferase K,,, and V,,,,, were decreased in hypothermic hibernating squirrels, while tryptophan 2,3-dioxygenase activity was not altered. Plasma and liver albumin were increased in hypothermic hibernating squirrels, while plasma and liver total protein were not altered. (4) These results demonstrate that significant changes in tyrosine and tryptophan metabolism occur in both central and peripheral tissues with concomitant alterations in metabolites during hypothermic hibernation in 13-lined ground squirrels. 6 1987 Academic Press, Inc.
Animals that hibernate exhibit circannual alterations in the regulation of body temperature, food and water intake, and patterns of sleep and wakefulness (28). Indeed, the transition from the homeothermic phase to the heterothermic phase of a hibernator’s circannual cycle requires metabolic adaptations (37) marked by a large reduction in oxygen consumption (41). In addition to cessation of food intake in the ground squirrel and minimal release of substrates from fuel stores (24), considerable data are available which indicate a central nervous system (CNS) site for regulation of these processes in nonhibernating species (2, 19). Furthermore, in hibernating species intrahypothalamic injection of both norepinephrine (NE) and serotonin (5hydroxytryptamine; 5-HT) have been shown to
Received September 22, 1986; accepted June 8, 1987.
stimulate arousal in Spermophilus lateralis (1). On the other hand, hypothalamic lesions in ground squirrels block arousal from hibernation (34). The literature on brain monoamine changes in hibernation does not provide a clear consensus. Some investigators have reported increased (25, 30, 36, 44), decreased (5, 11, 38), or unaltered (48) brain serotonin (5-HT) levels during hiberation. Furtheremore, other studies have reported changes in brain 5-HT and its principal metabolite 5-hydroxyindole-3-acetic acid (5HIAA) during entry into and arousal from hibernation (13, 32). Brain levels of catecholamines have also been reported to decrease (13) or not change (5, 42) during hibernation. Since the CNS plays a key role in regulating both the sympathoadrenal and endocrine systems (39) and since both of the systems undergo marked changes during the hibernation cycle (13, 15, IS), we un-
504 OOll-2240187$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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these experiments. Normothermic nonhibernating animals used in these experiments were completely aroused for a maximum of 24 hr prior to use in the experiment. Preliminary experiments indicated that the 13-lined ground squirrel is capable of arousal within 2-3 hr. These experiments were carried out in March 1983. All animals were left in their cages in the cold room until immediately before death to avoid arousal or excitement in hypothermic hibernating or normothermic nonhibernating squirrels. Normothermic nonhibernating or hypothermic hibernating squirrels were removed from cages immediately prior to death during the 2-hr daily light period, stunned by a sharp blow to the head, and decapitated. Blood MATERIALS AND METHODS was collected from the cervical stump in a Adult male and female 13-lined ground heparinized test tube, cooled to 4”C, and squirrels weighing an average of 178.4 + centrifuged. The plasma was removed and 36.6 (SD) g were acclimated for 4 weeks at a 1.O-ml aliquot was stored in sodium meta4 & 1°C in individual stainless steel cages bisulfite as a preservative for catecholwith a 2:22, 1ight:dark cycle during De- amines and frozen at - 15°C. Permanent cember 1982. Food (Purina Laboratory Rat cannulation for blood collection was not Chow) and tap water were available ad li- used because of problems in maintaining bitum. Squirrels were monitored daily for patency in a long-term study and the proba10 weeks starting in January 1983 prior to bility that implantation of the cannula for a the experiment in order to determine the short-term study would alter normal hibernormal hibernation pattern for each animal. nation patterns and mechanisms regulating Body temperature (rectal) was determined hibernation. daily with a thermistor and a YSI teletherThe brain was removed and dissected on mometer (Model 44TE). All animals used a cold glass plate into cerebellum, forebrain in these experiments demonstrated a well- or cerebral cortex (telencephalon), and developed pattern of hibernation with pe- brain stem, which includes diencephalon, riods of continuous torpor for at least 5 mesencephalon, metencephalon (minus days. Squirrels were considered to be in cerebellum) and myencephalon caudal torpor if they had a rectal temperature of to Cl. 5-8°C and did not respond to handling. The abdominal cavity was opened by a Conversely, squirrels were considered to ventral incision along the linea alba, and be in a completely aroused state if they had the caudate and papillary processes of the a rectal temperature of 33°C or greater and caudate liver lobe were removed for endid respond to handling. As a result of this zyme assays. The remaining lobes were redaily monitoring of hibernation status, all moved and utilized for other assays. Peri13-lined ground squirrels were acclimated renal fat and both kidneys were excised. to handling which served to reduce stress The thoracic cavity was opened by cutting at the time of death. Squirrels with inter- the ribs ventrally and slightly lateral to the mediate body temperature were not used in sternum. The heart was cut free of the dertook this study to monitor changes in central and peripheral monoamines and in their amino acid precursors. It was the aim of this project to investigate the following parameters of the 13-lined ground squirrel (Spermophilus tridecemlineatus) during the hibernation season: first, fluctuations in available tyrosine and tryptophan (the amino acid precursors of NE and 5-HT, respectively), as well as circulating and tissue catecholamines; and second, brain stem tryptophan ?Ghydroxylase, hepatic tyrosine aminotransferase, and hepatic tryptophan 2,3-dioxygenase that are the rate-limiting enzymes in serotonin synthesis and tyrosine and tryptophan degradation, respectively.
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great vessels and removed. The gastrocnemius muscle was also removed from both legs. Tissue tryptophan, serotonin, 5-hydroxyindole-3-acetic acid, tyrosine, norepinephrine, and dopamine were chemically extracted (4) by homogenization of tissues at 4°C in 5.0 ml of analytical grade acidified n-butanol. Following centrifugation (5OOOg for 30 min at 4”C), 4.0 ml of the supernatant was returned to a second set of tubes containing 7.0 ml of analytical grade n-heptane and 0.3 ml of 0.01 N HCl. This mixture was shaken for 10 min and centrifuged (1OOgfor 10 min at 4°C) to separate the organic and aqueous layers. The upper organic layer was removed and washed with 0.3 ml of 0.033 M NaHCO, to extract 5-hydroxyindole-3-acetic acid. An aliquot of the acid extract was assayed for serotonin by the ophthalaldehyde fluorometric procedure (40). Tissue levels of tyrosine and tryptophan were also determined using acid extract (6, 17, 45). Catecholamines were further purified from the acid extract by use of a CG,, ion-exchange resin (H+ form) (27). Tissue levels of epinephrine, norepinephrine, and dopamine were determined fluorometrically (4, 27). Total plasma tryptophan was determined fluorometrically (6). Free tryptophan was determined by treating plasma with dextran charcoal (17) prior to assay, and albuminbound tryptophan was calculated as the difference. Plasma tyrosine and catecholamine levels were determined fluorometritally (4, 27, 45). Brain stem tryptophan 5-hydroxylase was assayed by the method of Gal and Patterson (16) with subsaturating conditions of tryptophan (200 t&f) and dimethyltetrahydrobiopterin (100 t~I4). Hepatic tryptophan 2,3-dioxygenase was assayed spectrophotometrically as described by Knox et al. (23). Tyrosine aminotransferase was measured spectrophotometrically in vitro (8). All in vitro enzyme assays were conducted at 37°C. Total plasma protein was assayed
by the method of Lowry et al. (26). Plasma albumin was determined spectrophotometrically (9) to determine whether changes in circulating albumin levels alter the free/ bound plasma tryptophan ratios. Measurement of total plasma protein allowed determination of whether albumin is specifically altered or whether changes in circulating levels are merely a reflection of changes in all proteins. All data were analyzed by a 2tailed student t test and the null hypothesis was rejected at P < 0.05. RESULTS
Brain and Hepatic Enzyme Activities Brain stem tryptophan hydroxylase activity in low speed supernatants prepared from brain stem of either hypothermic hibernating or normothermic nonhibernating squirrels was not significantly different (data not shown). Hepatic tryptophan 2,3dioxygenase activity assayed in vitro also was not significantly different between hypothermic hibernating and normothermic nonhibernating squirrels when assayed at 37°C. The heme-saturation ratio of the enzyme did not significantly change during the hibernation cycle. The b,,, of tyrosine aminotransferase (2.79 ? 0.28 p,rn L-tyr/mg Plmin in hypothermic hibernating squirrels vs 24.63 & 13.33 km L-tyr/mg P/min in normothermic nonhibernating squirrels) was significantly reduced (P < 0.01) and the K, (335.45 + 56.44 pm L-tyr in hypothermic hibernating squirrels vs 2637.88 +- 130.80 km L-tyr in normothermic nonhibernating squirrels) was significantly reduced (P < 0.01) in liver from hypothermic hibernating squirrels tested at normothermic temperature (37°C). Tryptophan, Tyrosine, and Catecholamine Metabolism Total and free plasma tryptophan were depressed in hypothermic hibernating squirrels (P < 0.01 and P < 0.05, respec-
507
TYROSINE AND TRYPTOPHAN METABOLISM TABLE 1 Peripheral Tissue Levels of Tryptophan (TRP) and Tyrosine (TYR) in Normothermic and Hypothermic 13-Lined Ground Squirrels” Tissue
Metabolite
Normothermic
Heart Skeletal muscle Liver Kidney Plasma Skeletal muscle Liver Liver
TYR cLdg TYR I*& TYR w/g TYR v& TYR wk TRP mgig TRP i-&it TRPlorgan kg/liver TRP ds TRP kg/ml TRP kg/ml
224.94 k 69.03 43.34 r 20.71 389.87 r 63.67 216.02 k 75.46 86.5 2 26.70 433.7 2 57.26 0.549 2 0.395 3.136 IT 2.85 1.431 ? 0.47 5.704 -r- 1.389 1.003 2 0.815
TRP &ml
4.702 !I 1.97
Kidney Total plasma Albumin-bound plasma Free plasma
n
Significance
4 4 4 4 5 4 6 6
n.s. ns. ns. P < 0.01 P < 0.001 n.s. n.s. ns.
0.907 ” 0.392 2.549 ” 0.925 0.568 2 0.675
4 9 9
n.s. P < 0.01 n.s.
2.799 * 1.89
9
P < 0.05
Hypothermic 339.64 2 33.64 k 365.04 f 109.89 2 281.52 t 526.49 k 1.113 2 5.85 4
73.1 30.84 44.95 10.78 58.58 105.36 0.914 5.15
(1Each value is presented as the mean t SD.
tively), while albumin-bound tryptophan was not significantly different (Table 1). Plasma norepinephrine levels were significantly (P < 0.001) reduced in hypothermic hibernating squirrels but plasma epinephrine and dopamine levels were unaltered (Table 2). In addition, plasma tyrosine was significantly (P < 0.001) elevated in hypothermic hibernating squirrels (Table 3). Liver, kidney, and skeletal muscle (gastrocnemius) levels of tryptophan did not change during hibernation (Table 1). Concentrations of tyrosine in heart, skeletal muscle, or liver also did not change during
the hibernation cycle (Table 1), although kidney tyrosine levels decreased in hypothermic hibernating squirrels (P < 0.01). On the other hand, liver norepinephrine levels did not change during hypothermic hibernation, while concentrations in the kidney increased (Table 2). In addition, norepinephrine levels in heart and perirenal fat did not change as a function of the state of hibernation (Table 2). Dopamine levels also did not change in either liver or kidney during hypothermia. Brain levels of tryptophan were not altered during torpor, whereas brain stem tyrosine, norepineph-
TABLE 2 Peripheral Tissue Levels of Norepinephrine (NE), Epinepherine (E), and Dopamine (DA) in Normothermic and Hypothermic 13-Lined Ground Squirrels” Tissue
Metabolite
Normothermic
Hypothermic
n
Liver Kidney Heart Plasma Perirenal fat Plasma Liver Kidney Plasma
NE cL&t NE cL& NE ds NE rig/ml NE rig/g E rig/ml DA ngig DA ngig DA ngiml
3.39 2 5.78 k 46.16 ? 110.78 + 89.46 k 28.27 2 96.78 2 78.45 2 17.63 2
5.37 k 9.87 f 34.16 ” 28.24 2 51.08 ? 23.07 k 86.85 ” 85.11 k 14.19 k
5 5 4 5 5 5 4 4 5
a Each value is expressed as the mean 2 SD.
1.15 1.66 5.07 40.71 67.63 2.65 12.28 12.56 1.72
0.57 1.23 7.71 20.90 20.16 3.53 16.18 13.73 1.43
Significance
P <%I5 n.s.
P < 0.0001 ns. n.s. n.s. n.s. n.s.
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rine, and dopamine levels Were significantly increased (Table 3). Cerebellum tyrosine and norepinephrine were also elevated in hypothermic hibernating squirrels, as were forebrain norepinephrine and dopamine. Plasma Albumin
and Total Protein
Plasma albumin was significantly increased (P < 0.05) in hypothermic hibernating squirrels in addition to an increased plasma albumin/total protein ratio. Furthermore, the liver albumin levels and liver albumin/protein ratios were significantly (P < 0.001) increased in hypothermic hibernating squirrels (Table 4). DISCUSSION
From the standpoint of potential mechanisms influencing hibernation, an interesting observation on the 13-lined ground squirrel was the dramatic shift in central and peripheral tyrosine metabolism following entry into the hypothermic phase of hibernation. It seems reasonable to suggest
that this metabolic effect may be involved with the changes in autonomic tone occurring at this time. Indeed, it has been suggested that changes in brain stem monoamines may cause reductions in sympathetic activity (22) in nonhibernating species. Additionally, it is well documented that there is a reduction in sympathetic activity during entrance into hibernation (43). On the other hand, injection of norepinephrine into the brain of a hibernator has been reported to elicit arousal (I), while drugs that block norepinephrine synthesis usually block arousal from hypothermia (12). Draskoczy and Lyman (10) found that norepinephrine turnover is reduced in brown fat, heart, and brain of hibernating ground squirrels, whereas our observations indicate that tissue levels of norepinephrine in brain, kidney, and plasma are significantly increased in hypothermic hibernating squirrels. However Draskoczy and Lyman (10) estimated norepinephrine turnover by intraperitoneal injection of H-NE in aroused 13-lined ground squirrels that were
TABLE 3 Levels of Tryptophan (TRP), Tyrosine (TYR), Serotonin (5HT), 5-Hydroxyindole-3-Acetic Acid (5HIAA), Norepinephrine(NE), and Dopamine (DA) in the Forebrain, Cerebellum, and Hindbrain of Normothermic and Hypothermic 13-Lined Ground Squirrels” Brain region
Metabolite
Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Forebrain Forebrain Forebrain Forebrain Forebrain Forebrain Brain stem Brain stem Brain stem Brain stem Brain stem Brain stem
NE nglg TYR t&g DA nglg TRP wdg 5-HT rig/g 5-HIAA r&g NE ngig TYR (*.dg DA rig/g TN i&g 5-HT ngig 5-HIAA rig/g NE ngig TYR I*& DA nglg TRP t-&g 5-HT rig/g 5-HIAA nak
Normothermic 734.39 ? 1322.39 k 1177.15 2 489.25 k 1666.97 2 740.10 k 546.50 2 1233.64 2 696.54 ” 264.07 2 938.13 k 244.26 f 581.25 k 887.13 2 587.14 ” 333.38 k 509.62 t 232.96 k
206.47 585.06 415.07 215.80 531.45 234.92 96.15 100.77 150.88 92.16 283.91 52.11 131.76 140.66 103.14 70.58 86.83 35.72
a Each value is expressed as the mean 2 SD (N = 5).
Hypothermic 1339.44 f 3218.06 lr 1562.39 t 259.05 2 1745.71 e 613.67 k 1477.69 2 1515.55 t 977.31 i 125.27 k 772.67 -c 221.72 _’ 1023.84 2 1149.3 k 804.66 t 308.83 k 491.71 k 218.52 2
118.58 490.02 206.06 139.60 185.69 104.88 271.73 308.78 126.92 101.27 211.38 24.92 204.02 215.33 277.01 178.7 140.52 26.41
Significance P < 0.01 P < 0.01
n.s. n.s. n.s. n.s. P < 0.01 n.s. P < 0.05
ns. ns. ns. P < 0.01 P < 0.05 P < 0.05
n.s. n.s. n.s.
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TABLE 4 Tissue Levels of Total Protein and Albumin in Normothermic and Hypothermic 13-Lined Ground Squirrels” Tissue Skeletal muscle total protein (mglg) Plasma total protein (mgig) Liver total protein (mg/g) Plasma albumin (mg%) Plasma albumin/protein ratio (pg albuminimg protein) Liver albumin (pg albumin/g) Liver albumin/protein ratio (pg albumin/mg protein)
Normothermic
Hypothermic
n
Significance
3.53 k 1.16 75.76 5 1.02 4.342 -c 0.596 12.26 re_1.76
3.125 74.98 3.566 20.29
0.34 1.44 0.678 6.04
4 5 6 5
n.s. n.s. ns. (P < 0.05)
0.2704 +- 0.077 11.92 2 3.11
5 6
(P < 0.05) (P < 0.001)
3.394 4 1.097
6
(P < 0.001)
0.163 k 0.0315 1.705 2 1.33 0.304 2 0.277
k 5 t 2
a Each value is presented at the mean ? SD.
then induced to hibernate. We have re- imals were subjected to the same treatcently found (45) that intraperitoneal injec- ment. Dopamine and epinephrine, also intions of 0.9% sodium chloride stimulates creased by stress (3), were not different between hypothermic hibernating and normoarousal in the 13-lined ground squirrel. These observations raise a question as to thermic nonhibernating squirrels. The fact whether it is possible to carry out a turn- that catecholamine and tyrosine levels over study in hibernating squirrels (i.e., hi- were altered in most tissues of hypothermic hibernating squirrels as compared to those bernating without any arousal following drug injection). We elected not to attempt in normothermic nonhibernating squirrels to carry out a turnover study since pre- suggests that some mechanism other than vious experiments have indicated that stress is responsible for these changes. Furvarious drugs known to alter brain mono- thermore, epinephrine, the predominant amines also affect arousal (46) in a differen- adrenal medullary catecholamine in most tial manner. It is possible that merely mea- species studied, would also be expected to suring (pharmacologically) the turnover of increase in normothermic nonhibernating monoamines may change the state of hiber- squirrels if the stress of death were responnation. Sauerbier and Lemmer (35) deter- sible for elevated catecholamine levels. mined that peripheral catecholamine turn- The selective increase in only norepinephover in hedgehogs (Erinaceus euuopaeus) is rine in normothermic nonhibernating occurring at a reduced level during hiber- squirrels suggests that some other mechanation. In contrast, Petrovic et al. (31) nism (e.g., altered release or turnover) may demonstrated that adrenal tyrosine hydrox- be responsible. In addition, we maintain ylase (the rate-limiting enzyme in NE for- that as a result of the precautions taken to mation) activity is increased during cold acclimate the animals to handling, the likeadaptation in ground squirrels. Although lihood of a selective norepinephrine inplasma catecholamine levels in 13-lined crease in normothermic nonhibernating ground squirrels are higher than those re- squirrels in response to the stress of death ported in other species, no investigators was reduced. have previously reported seasonal or periThe mechanism by which brain NE and odic variations in these compounds during DA is increased in hypothermic hibernating the hibernation cycle in this species. Even squirrels was not determined in our study, though stress (e.g., handling) is known to although it could be altered secondarily as increase plasma norepinephrine (7), all an- a result of the increased plasma tyrosine.
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The source of tyrosine in the plasma is presently not known although it clearly cannot be of dietary origin. Of the other organs examined, only the kidney showed significant decreases in tyrosine levels. Proteolysis may be a source of the tyrosine during the anorexia of hypothermic hibernation; however, liver and total plasma protein were unchanged, whereas albumin actually increased in plasma during hypothermic hibernation. Indeed, albumin is known to bind tryptophan and reduce its competition with tyrosine for transport into the brain. This would normally increase NE biosynthesis and reduce 5-HT biosynthesis since the respective rate-limiting enzymes are unsaturated in viva (14, 47). However, we did not observe a significant decrease in brain tryptophan or 5-HT levels during hibernation. The observation that free and total plasma tryptophan are decreased during hibernation while albuminbound tryptophan is unaltered suggests that the increased circulating albumin (Table 4) binds a greater percentage of total tryptophan and reduces tryptophan turnover during hibernation. Such a physiochemical property of this system would serve to provide a buffer pool of tryptophan which may be utilized during arousal. Although our study did not specifically address the mechanisms behind the increase in plasma albumin in hypothermic hibernating squirrels, the fact that absolute albumin levels and the albumin/protein ratio are increased in plasma and liver suggests that albumin synthesis is increased or that albumin clearance is decreased. These results are also not in agreement with those that have reported brain 5-HT turnover to increase dramatically and catecholamine levels to remain unaltered during hibernation in the golden hamster (21). Our results, in contrast, demonstrate no change in brain tryptophan, 5-HT, or 5HIAA during hibernation, while tyrosine and catecholamine levels dramatically increase. Other investigators draw differing
conclusions about the changes in brain norepinephrine (5, 13, 42) and 5-HT (5, 11, 25, 30, 36, 38, 44, 48) during the hibernation process in different species. These differences may be due to species differences in regulation of hibernation and may reflect differences in the way the brain was subdivided for assay of monoamines. The NE/5HT ratio of various critical midbrain regions may provide insight as to the precise role that monoamines play in this process. For example, stimulation of the locus coeruleus (site of NE-containing cell bodies) or dorsal raphe (site of 5-HT-containing cell bodies) causes reductions in NE or 5-HT metabolism, respectively, in other brain regions (20, 33). Those studies on rats suggest that brain stem nuclei may have inhibitory effects through their projections in other regions of the brain. Such a mechanism in a hibernator may be important in that hypothalamic input may consist of brain stem and peripheral information as well as visual information. The hypothalamus then integrates the internal metabolic information with external visual cues and makes appropriate adjustments in autonomic and endocrine functions as well as behavioral changes. On the other hand, we did not observe any change in either brain stem tryptophan hydroxylase activity or liver tryptophan 2,3-dioxygenase during the hibernation cycle, while Novotona and Civin (29) reported a decrease in hepatic tryptophan pyrrolase activity during hibernation in the golden hamster. These apparent discrepancies may be due to species differences or to differences in experimental design. The in vitro assays were conducted at 37°C and it is possible that enzyme activity may be altered at 4°C in viva during hibernation. However, Novotona and Civin (29) also conducted assays at 37°C in vitro and reported a depression in tryptophan pyrrolase activity during hibernation. On the other hand, since brain indoles did not change significantly during hibernation, it
TYROSINE AND TRYPTOPHAN METABOLISM
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