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The body mass cycle of the migratory garden warbler (Sylvia borin) is associated with changes of basal plasma metabolite levels
Comparative Biochemistry and Physiology Part A 121 (1998) 127 – 133
The body mass cycle of the migratory garden warbler (Syl6ia borin) is associated with changes of basal plasma metabolite levels Uwe Totzke *, Franz Bairlein Institute for A6ian Research ‘Vogelwarte Helgoland’, An der Vogelwarte 21, 26386 Wilhelmsha6en, Germany Received 8 August 1998; accepted 13 August 1998
Abstract Garden warblers show pronounced seasonal changes in body mass in relation to migration. In order to reveal if fattening and defattening is associated with changes in plasma levels of metabolic key factors, body mass, food intake, molt, visible fat, and plasma metabolite, and electrolyte levels were measured under constant indoor conditions for the non-reproductive period (September to May). During the phase of high body mass and high fat loads, the plasma levels of glucose, triglycerides, cholesterol and free fatty acids were higher, sodium concentration and glucose tolerance lower than in the phase of low body mass and molt. Urea, uric acid, b-hydroxybutyrate and potassium levels showed no or only small variations throughout the year with no significant relationship to the body mass cycle. In preparation for migration a metabolic change in preferring fat as substrate for energy metabolism is assumed. This perhaps results in constraints as predicted by the glucose fatty-acid cycle, which appear to be similar to those in mammalian obesity. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Bird migration; Glucose fatty-acid cycle; Glucose tolerance; Hyperglycemia; Hyperlipemia; Migratory fattening; Obesity
1. Introduction Prior to migration, many birds accumulate large amounts of fat as the main fuel for the migratory flights [14,34]. This is particularly pronounced in species crossing mountains, oceans or deserts where feeding opportunities are scarce [11]. For several species fattening in preparation for migration has been shown to rely on an endogenous time program [6,13] and to be associated with increases in food intake [10] and utilization efficiency [2,24] and with changes in food preference [7]. Garden warblers (Syl6ia borin) breeding in Europe and migrating in the Afrotropics are a prominent example [4], which has been addressed also in respect of Abbre6iations: BM, body mass; GLUC, glucose; TG, triglycerides; CHOL, cholesterol; HDLC, high density lipoprotein-cholesterol; HBA, b-hydroxybutyrate; URIC, uric acid. * Corresponding author. Tel.: +49 4421 96890; fax: + 49 4421 968955; e-mail: [email protected]
metabolic adjustments involved in long-distance migration. On fall migration garden warblers landing in the Sahara showed significantly higher plasma levels of glucose and triglycerides concomitantly with larger amounts of depot fat than at the breeding grounds before embarking [8]. At a site in central Europe, birds resting for an early migratory stop-over or for postbreeding molt, both still lean, did not differ in plasma metabolite levels [28]. However, garden warblers caught during active flight on fall migration at an Alpine pass also showed higher triglyceride levels than post-exercise, after a one-night fast or postprandially, but decreased glucose levels [25]. The increase in triglycerides is regarded as a special adaptation of small birds to maintain energy supply during endurance migratory flights [26]. However, in view of the close relationship of some plasma metabolite levels to the amount of visible fat in migrating individuals [8] it is unclear, whether the changes are exclusively due to exercise or whether endogenous changes are also involved.
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In mammals, obesity is often associated with increases in plasma glucose and lipid levels, insulin resistance and decreased glucose utilization (e.g. [15]). In the garden warbler endogenous seasonal preparations for migration [6], including fattening as the most prominent one, are well established. However, the extent of accompanying metabolic changes is unknown, although an increase of the plasma glucose level [1], and for some other species an increase in the capacity for lipid oxidation prior to migration has been shown [30 – 32]. This study aimed to investigate whether in the garden warbler, a species with pronounced fattening, blood chemistry and glucose utilization change with body fat loads as known from mammalian obesity. Because of the complex situation in the field, where implications of different resorptive/fasting states, exercise, and ambient temperatures could not be evaluated, the study was carried out under standardized laboratory conditions.
2. Materials and methods 20 garden warblers, mostly caught as yearlings around Wilhelmshaven (53°3 N, 8°1 E), were caged under constant indoor conditions (20°C, LD 12:12, standard insect food [3] ad libitum) for the non-reproductive period (September to May). At night, rooms were totally dark to suppress zugunruhe [17], which was registered in 10 birds with hopping perches. During the breeding season (June to August), the birds were kept in outdoor aviaries. Body mass and food intake were recorded daily in the morning, visible fat (9 grade scale [29]) and molt intensity (0, none; 1, less than 1/3; 2, up to 2/3; 3, more than 2/3 of 12 parts of plumage with growing feathers; modified method of [12]) were scored weekly. For each bird body mass changes were calculated per day. Food intake was calculated as dry matter mass after correction for evaporative losses according to F= Fin
Cdry C −Fout dry Cin Cout
F, net food intake by the bird (g dm); Fin, wet food mass offered to the bird; Fout, mass of the food left by the bird at the next day; Cdry, dry mass of a control food sample; Cin, wet mass of a control food sample; Cout, mass of the control sample the next day. At 2-week intervals, the birds were bled in the morning before feeding by puncturing the wing vein and collecting the blood with heparinized capillaries. The blood was centrifuged (15 min; 8900× g; 4°C) and plasma analyzed for glucose (GLUC), triglycerides (TG), cholesterol (CHOL), high density lipoproteincholesterol (HDLC), urea (UREA), uric acid (URIC), Na and K with the EKTACHEM DT II analyzing
system (Kodak, Stuttgart, Germany) [8]. Triglyceride measurements included amounts of free glycerol. Free fatty acids (FFA) and b-hydroxybutyrate (HBA) were measured using standard kits obtained from Boehringer (Mannheim, Germany) and Sigma (St. Louis, USA), respectively, following the producers’ instructions. Most assays were based on enzymatic detection. Electrolytes were quantified by potentiometry. Interassay variance was between 1.7% (URIC) and 5.3% (UREA) for the EKTACHEM DT II parameters (for each n= 45–50), 2.2% for HBA (n= 24), and 13.2% for FFA (n= 29). Intraassay variance was always below 5%. Glucose utilization was measured in the same individuals once in the fat phase and once lean thereafter prior to prenuptial molt. Birds were orally administered approximately 80 mg glucose, dissolved in 100 ml of water, and at 0, 1.5 and 3 h afterwards plasma glucose levels were determined. For several reasons, the number of measurements in most cases was markedly below the number of birds: (a) The collected blood amount was not always sufficient to measure all parameters, (b) nine birds had died until the end of the study and (c) when body mass was below 16 g, birds were assumed to be to weak for repetitive blood sampling every 2 weeks. For similar reasons, the number of measurements each bird contributed per month ranged from none to three. Therefore, in order to avoid weighting prior to further statistical evaluation, monthly averages for each bird were calculated. These values were used for the calculation of monthly means for all birds. The relationships among parameters were investigated with the original data by calculation of correlation coefficients or by analysis of covariance controlling for individual birds.
3. Results Body mass showed considerable seasonal variation with maximum values during November to January and minimum values in March (Fig. 1(a)). Monthly averages for body mass changed with those of fat amounts throughout the study. Both correlated closely among each other with significant correlation coefficients for individual birds between 0.80 and 0.98 (mean 9 S.D.: r= 0.8990.04; test of homogeneity: x 2 =1.0; n= 20; p\ 0.05). The pattern of molt was opposite (Fig. 1), also reflected by a negative correlation between fat and molt levels with significant correlation coefficients for individual birds between − 0.35 and −0.90 (r= − 0.549 0.04; x 2 = 7.0; n= 15; p\ 0.05). Food intake was highest from September to December (Fig. 1(b)) followed by a minimum in January and a continuous increase afterwards. For most birds, it correlated with daily body mass changes with significant correlation coefficients for individual birds between 0.39 and
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0.79 (r= 0.6590.02; x 2 =18.5; n =16; p \ 0.05). Zugunruhe varied among the ten birds registered, but ranged from November to March with monthly averages always below 1 h night − 1. Nevertheless, in five birds there was a significant negative correlation with daily body mass change (r = − 0.35 90.04; x 2 = 5.6; n= 5; p \0.05) and food intake (r = − 0.51 9 0.05; x 2 = 4.5; n= 5; p \0.05). However, no significant relationship with zugunruhe resulted for any of the plasma parameters (each p \0.1). Plasma levels of triglycerides, cholesterol and glucose showed considerable seasonal variation with highest levels from September to January and lowest values between February and April, when most birds were
Fig. 2. Seasonal variations of plasma (a) triglycerides and cholesterol and (b) free fatty acids and b-hydroxybutyrate. Values are means 9S.E., numbers of birds above and beneath the curves; p-values result from one-way ANOVA.
Fig. 1. Seasonal variations of (a) body mass and depot fat and (b) food intake and molt. Values are means 9S.E., numbers of birds above and beneath the curves; p-values from one-way ANOVA revealing (significant) seasonal variations. Differences in sample sizes between months are due to the varying number of birds from which blood samples could be taken. Since it was intended to investigate a link of plasma parameters and the body mass cycle, only data from those birds on the particular blood sampling days are shown.
molting (Fig. 2(a), Fig. 4(a)). Total cholesterol consisted almost completely of HDL-cholesterol, the average percentages varying not significantly between 80 and 96% throughout the year. For FFA and HBA, the variances were much greater and differences between months were not significant (Fig. 2(b)). However, FFA levels were highest during the months of maximum body mass and fat (Fig. 2(b)) and correlated significantly with both (Table 1). Sodium levels during and shortly after fat deposition were significantly lower than in spring (Fig. 3(b); mean values 9 S.E.: 115.991.3 mmol l − 1 (November to January)/119.3 91.4 mmol l − 1 (March to May), paired t-test: p= 0.028, n=8), while potassium remained fairly constant. Plasma uric acid and urea levels varied throughout the year, but without any apparent relationship to the annual body mass cycle (Fig. 3(a)).
+0.59 (186) −0.36 (179) +0.61 (168) — (138) −0.37 (176) +0.30 (185) TG
+0.32 (230) — (220) +0.26 (211) — (165) — (219)
GLUC
TG
+0.44 (172) +0.72 (172) CHOL
+0.60 (173) −0.30 (166) +0.71 (156) — (126) −0.35 (164)
CHOL
— (68) +0.74 (68) +0.91 (68) HDLC
+0.57 (68) — (66) +0.62 (67) — (54) — (68)
HDLC
— (181) +0.67 (154) +0.55 (149) +0.61 (62) FFA
+0.40 (182) −0.28 (177) +0.50 (168) −0.32 (139) — (176)
FFA
— (128) — (124) +0.39 (123) +0.44 (63) +0.40 (123) HBA
— (129) −0.44 (125) — (116) −0.44 (104) — (124)
HBA
— (194) — (176) — (169) — (67) — (161) −0.51 (122) URIC
— (195) — (188) — (176) +0.37 (141) — (185)
URIC
(68) (65) (57) (46) (61) +0.44 (67) — (67) — (68) — (28) — (58) — (52) — (68) UREA
— — — — —
UREA
— — — — — — — — K
— — — — —
K
(104) (104) (103) (49) (94) (86) (102) (56)
(104) (100) (93) (79) (96)
(110) (108) (101) (79) (104) — (110) −0.33 (110) −0.36 (109) — (55) — (100) — (90) — (109) — (58) — (93)
— — — — —
Na
r-values when correlations were still significant after sequential Bonferroni adjustment [36] on a table-wide significance level of 0.05; if not significant number of pairs calculated (in parentheses).
BM DBM FAT FOOD INTAKE MOLT
GLUC
Table 1 Relationships of blood parameters to body mass, body mass changes, visible fat, molt and food intake and among each other calculated by an analysis of covariance controlling for individual birds
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Triglycerides, cholesterol, FFA and HBA were closely related to each other as well as to body mass and fat (Table 1), which applied also to glucose, though to a lesser degree. However, correlation of plasma lipids with food intake on the preceding day was negative, most strikingly for FFA and HBA. HBA did not change with body mass and fat, but showed a negative relationship to body mass changes and food intake. Uric acid levels were positively related to food intake, and negatively to HBA. The relationships of urea with body mass, fat, triglycerides and cholesterol were opposite to those of uric acid, except with glucose. However, none was still significant after Bonferroni-adjustment, neither was the relationship between the plasma levels of urea and uric acid (Table 1; Fig. 3(a)). Glucose tolerance was poorer in fat birds (BM: 27.59 0.6 g, n = 5) than in lean birds (BM: 17.690.8
Fig. 4. (a) Seasonal variations of plasma glucose and (b) variations after an oral glucose load on identical birds in lean (open symbols) and fat (filled symbols) condition. Glucose tolerance tests were carried out in January/February prior to and in March/April shortly after defattening; one bird was still fat in April and is therefore missing in the lean group. Values are means 9 S.E., number of birds beneath the curve; p-value from one-way ANOVA, ** significantly different from both the initial level and at 3 h in the lean state (paired t-test: p B0.05). The high starting levels in the glucose tolerance test may have resulted from the selection of birds showing very pronounced fat deposition, while in the annual cycle also birds with moderate fat deposition are included.
g, n= 4, paired t-test: pB0.001) with significantly increased plasma levels 3 h after glucose administration in the fat birds (Fig. 4(b); paired t-test: 0–3 h (fat): pB0.05, n= 5; fat–lean (3 h): pB0.05, n= 4).
Fig. 3. Seasonal variations of plasma (a) uric acid and urea and (b) electrolytes. Values are means 9 S.E. (for K often smaller than symbols), numbers of birds above and beneath the curves; p-values from one-way ANOVA.
4. Discussion The garden warblers showed seasonal variations in body mass associated with changes in the amounts of
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subcutaneous fat. In migratory bird species such seasonal changes in body/fat mass are well-known to be due to a fattening in preparation for migration, which in the garden warbler is controlled by an endogenous circannual rhythm [6,13,22]. In this study it was demonstrated that birds show concomitantly seasonal variations in plasma composition, although the influence of prolonged exercise and fasting as experienced during active migration were almost completely excluded by keeping birds under constant conditions and measuring them in the same postresorptive state. There was no significant relationship of plasma parameters with the amounts of zugunruhe and two single birds with normal body mass cycles, but lacking the subsequent molt, showed changes of plasma metabolites similar to birds with molt. Therefore, changes of plasma concentrations were most likely due to changes in body mass and concomitant effects of non-suppressible zugunruhe and molt on plasma concentrations may have been minor. Increases in plasma glucose and lipid levels and a decrease in sodium in fat birds were in accordance with measurements in migrating garden warblers crossing the Sahara [8] and with results on other small migratory species kept in captivity, such as the white-crowned sparrow Zonotrichia leucophrys gambelii [18] and the red-headed bunting Emberiza bruniceps [38]. Strong correlations of these plasma metabolite levels with body mass changes have previously been reported for the garden warbler [27], whereas in still relatively lean free-living individuals which were already migrating, plasma metabolite levels hardly changed in comparison to postbreeding and molting birds [28]. However, birds in this study showed high plasma levels of glucose, triglycerides and cholesterol in September and October, when body mass and fat were still low. This may be an artefact raised by settling-in birds from the wild or from outdoor aviaries, because birds then normally reduce food intake and lose weight for several days. In herring gulls, triglyceride and cholesterol levels increase due to fasting and afterwards remain high even after refeeding [42]. In fat garden warblers overnight-fasted triglyceride and cholesterol levels also increase initially in response to food restriction [41]. The high levels during September and October could otherwise reflect a high rate of lipogenesis preceding fattening and the body mass increase. However, the reason for the lack of correlation between plasma metabolites and body mass at the beginning of our investigation is still unclear. This also applies to the extent by which triglyceride levels may be overestimated due to unquantified amounts of free glycerol. However, other studies have shown free glycerol to vary closely with FFA and for the garden warbler within one quarter of the range presented for triglycerides during this study [25–28]. Therefore, free glycerol may have increased triglyceride variations but is unlikely to have
influenced the seasonal pattern. It was assumed that high plasma triglycerides in small birds are a necessity for maintaining fuel supply during endurance flights according to the high mass-specific metabolic rates of migrating passerines [26]. The described changes in plasma lipids and glucose are similar to those in mammalian obesity and Type II-diabetes [15]. These metabolic disorders are mostly due to insulin resistance reflected by transient hyperinsulinemia, which, however, is not shown by the garden warbler [40]. Another key mechanism shown for mammals in vitro and also discussed for Type II-diabetes associated with obesity is the glucose fatty-acid cycle [35,39], which is based on the mutual inhibition of glucose and fatty acid oxidation [33,35]. In humans, the oxidation of fatty acids in tissues corresponds well with the plasma fatty acid level (cf. [33]). Therefore, the trend for an increase of plasma FFA in fat birds may be further evidence for an increasing oxidation rate of lipids in preparation for migration, which is also indicated by the premigratory increase of the lipid oxidizing capacity in flight muscles [30–32]. An inhibition of glucose oxidation by fatty acids and acetyl-CoA due to the glucose fatty-acid cycle [33,35] would explain the seasonal variations in the plasma glucose level which were first described for garden warblers by Bairlein [1], and would also account for the poorer glucose tolerance of fat birds compared to lean birds shown in the present study. The lower plasma sodium levels and the consistency of high levels of HBA during the phases of high fat loads in both, caged birds and free-living birds on migration (sodium [8]), are in agreement with the implications of hyperglycemia on water balance [21] and with the resistance to ketoacidosis in Type II-diabetes [20]. The lack of an increase in HBA could be the result of either a minor ketogenic effect of glucagon, as assumed in human Type II-diabetes [20] and indeed proven for the pigeon [9], or of a preference for catabolism of fatty substrates during the phase of fat deposition [5,34]. This may be confirmed by the obviously exogenously caused decrease of overnight-fasted HBA levels in still lean garden warblers on fall migration in central Europe [28]. In contrast to the pathological disorders in humans [15], the fat deposition of migratory birds and its simultaneous changes in metabolism are normal seasonal events and, therefore, apparently well regulated. For example, migratory fattening in the garden warbler appears not to be associated with hyperinsulinemia [40]. However, the physiological importance of pancreatic hormones in birds seems to be different from that in mammals [23], and the regulatory mechanisms of food intake and body mass are less clear [19]. In addition, the existence and the role of the recently described mammalian leptin [16,37] in the regulation of body fat in birds are still unknown. The physiological mecha-
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nisms involved during fat deposition in migratory birds could be used to learn more about human metabolic disorders or vice versa. Acknowledgements The work was supported by the Minister of Science of Lower Saxony. The authors thank R. Gnann and C. Mlingwa for their help with the manuscript and unknown referees for their comments. References [1] Bairlein F. Seasonal variations of serum glucose levels in a migratory songbird, Syl6ia borin. Comp Biochem Physiol 1983;76A:397 – 9. [2] Bairlein F. Efficiency of food utilization during fat deposition in the long-distance migratory garden warbler, Syl6ia borin. Oecologia 1985;68:118 – 25. [3] Bairlein F. Ein standardisiertes Futter fu¨r Erna¨hrungsuntersuchungen an omnivoren Kleinvo¨geln. J Ornithol 1986;127:338 – 40. [4] Bairlein F. The migratory strategy of the garden warbler: a survey of field and laboratory data. Ringing Migration 1987;8:59 – 72. [5] Bairlein F. Nutrition and food selection in migratory birds. In: Gwinner E, editor. Bird Migration. Berlin: Springer, 1990:198 – 213. [6] Bairlein F, Gwinner E. Nutritional mechanisms and temporal control of migratory energy accumulation in birds. Ann Rev Nutr 1994;14:187 – 215. [7] Bairlein F, Simons D. Nutritional adaptations in migrating birds. Isr J Zool 1995;41:357–67. [8] Bairlein F, Totzke U. New aspects on migratory physiology of trans-Saharan passerine migrants. Ornis Scand 1992;23:244 – 50. [9] Ballantyne JS, John TM, George JC. The effects of glucagon on hepatic mitochondrial metabolism in the pigeon, Columba li6ia. Gen Comp Endocrinol 1988;72:130–5. [10] Berthold P. Migration: Control and metabolic physiology. In: Farner DS, King JR, editors. Avian Biology, vol. 5. New York: Academic Press, 1975:77–128. [11] Berthold, P. Bird Migration—A General Survey. Oxford: Oxford University Press, 1993. [12] Berthold P, Gwinner E, Klein H. Vergleichende Untersuchung der Jugendentwicklung eines ausgepra¨gten Zugvogels, Syl6ia borin, und eines weniger ausgepra¨gten Zugvogels, S. atricapilla. Vogelwarte 1970;25:297 – 331. [13] Berthold P, Gwinner E, Klein H. Circannuale Periodik bei Grasmu¨cken: I. Periodik des Ko¨rpergewichtes, der Mauser und der Nachtunruhe bei Syl6ia atricapilla und S. borin unter verschiedenen konstanten Bedingungen. J Ornithol 1972;113:170 – 90. [14] Blem CR. The energetics of migration. In: Gauthreaux SA, editor. Animal Migration, Orientation, and Navigation. New York: Academic Press, 1980:175–224. [15] Bray GA, Bouchard C, James WPT, editors. Handbook of Obesity. New York: Marcel Dekker, 1997. [16] Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS. Role of leptin in fat regulation. Nature 1996;380:677. [17] Czeschlik D. Der Einfluß der Beleuchtungssta¨rke auf die Zugunruhe von Garten- und Mo¨nchsgrasmu¨cken (Syl6ia borin und S. atricapilla). J Ornithol, 1977;118:268–281. [18] DeGraw WA, Kern MD, King JR. Seasonal changes in the blood composition of captive and free-living white-crowned sparrows. J Comp Physiol 1979;129:151–62.
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[19] Denbow DM. Food intake control in birds. Neurosci Biobehav Rev 1985;9:223 – 32. [20] Foster DW. Diabetes mellitus. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, editors. The Metabolic Basis of Inherited Disease. 5th ed. New York: McGraw-Hill, 1983:99 – 117. [21] Foster DW, McGarry JD. Acute complications of diabetes: ketoacidosis, hyperosmolar coma, lactic acidosis. In: DeGroot LJ, editors. Endocrinology, vol. 2. 2nd ed. Philadelphia: Saunders, 1989:1439-53. [22] Gwinner, E. Circannual Rhythms. Berlin: Springer, 1986. [23] Hazelwood RL. Carbohydrate metabolism. In: Sturkie PD, editor. Avian Physiology. New York: Springer, 1986:303 – 325. [24] Hume ID, Biebach H. Digestive tract function in the long-distance migratory garden warbler, Syl6ia borin. J Comp Physiol B 1996;166:388 – 95. [25] Jenni-Eiermann S, Jenni L. Metabolic responses to flight and fasting in night migrating passerines. J Comp Physiol B 1991;161:465 – 74. [26] Jenni-Eiermann S, Jenni L. High plasma triglyceride levels in small birds during migratory flight: a new pathway for fuel supply during endurance locomotion at very high mass-specific metabolic rates? Physiol Zool 1992;65:112 – 23. [27] Jenni-Eiermann S, Jenni L. Plasma metabolite levels predict individual body-mass changes in a small long-distance migrant, the garden warbler. Auk 1994;111:888 – 99. [28] Jenni-Eiermann S, Jenni L. Metabolic differences between the postbreeding, molting and migratory periods in feeding and fasting passerine birds. Funct Ecol 1996;10:62 – 72. [29] Kaiser A. A new multi-category classification of subcutaneous fat deposits of songbirds. J Field Ornithol 1993;64:246 – 55. [30] Lundgren BO, Kiessling K-H. Seasonal variation in catabolic enzyme activities in breast muscle of some migratory birds. Oecologia 1985;66:468 – 71. [31] Lundgren BO, Kiessling K-H. Catabolic enzyme activities in the pectoralis muscle of premigratory and migratory juvenile reed warblers Acrocephalus scirpaceus (Herm.). Oecologia 1986;68:529 – 32. [32] Marsh RL. Catabolic enzyme activities in relation to premigratory fattening and muscle hypertrophy in the gray catbird (Dumetella carolinensis). J Comp Physiol B 1981;141:417 – 23. [33] Newsholme EA, Start C. Regulation in Metabolism. London: Wiley, 1973. [34] Ramenofsky M. Fat storage and fat metabolism in relation to migration. In: Gwinner E, editor. Bird Migration. Berlin: Springer, 1990:214 – 231. [35] Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1:785 – 9. [36] Rice WR. Analyzing tables of statistical tests. Evolution 1989;43:223 – 5. [37] Scott J. New chapter for the fat controller. Nature 1996;379:113– 4. [38] Singh VB, Lal P, Thapliyal JP. Role of thyroid in photoperiodically induced lipid metabolism of the migratory redheaded bunting Emberiza bruniceps (Brandt). Ind J Exp Biol 1993;31:422–5. [39] Sugden MC, Holness MJ. Substrate interactions in the development of insulin resistance in type II diabetes and obesity. J Endocrinol 1990;127:187 – 90. [40] Totzke U, Hu¨binger A, Bairlein F. A role for pancreatic hormones in the regulation of autumnal fat deposition of the garden warbler (Syl6ia borin)? Gen Comp Endocrinol 1997;107:166 – 71. [41] Totzke U. Klinisch-chemische Charakterisierung und Befunde zur endokrinen Regulation der Zugzeltlichen Fettdeposition Gartengrasmu¨cke (Syl6ia borin). Ph.D. thesis University of Cologne, 1996. [42] Totzke U, Fenske M, Hu¨ppop O, Raabe H, Schach N. The influence of fasting on blood and plasma composition of herring gulls (Larus argentatus). Physiol Zool, submitted.
The body mass cycle of the migratory garden warbler (Syl6ia borin) is associated with changes of basal plasma metabolite levels Uwe Totzke *, Franz Bairlein Institute for A6ian Research ‘Vogelwarte Helgoland’, An der Vogelwarte 21, 26386 Wilhelmsha6en, Germany Received 8 August 1998; accepted 13 August 1998
Abstract Garden warblers show pronounced seasonal changes in body mass in relation to migration. In order to reveal if fattening and defattening is associated with changes in plasma levels of metabolic key factors, body mass, food intake, molt, visible fat, and plasma metabolite, and electrolyte levels were measured under constant indoor conditions for the non-reproductive period (September to May). During the phase of high body mass and high fat loads, the plasma levels of glucose, triglycerides, cholesterol and free fatty acids were higher, sodium concentration and glucose tolerance lower than in the phase of low body mass and molt. Urea, uric acid, b-hydroxybutyrate and potassium levels showed no or only small variations throughout the year with no significant relationship to the body mass cycle. In preparation for migration a metabolic change in preferring fat as substrate for energy metabolism is assumed. This perhaps results in constraints as predicted by the glucose fatty-acid cycle, which appear to be similar to those in mammalian obesity. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Bird migration; Glucose fatty-acid cycle; Glucose tolerance; Hyperglycemia; Hyperlipemia; Migratory fattening; Obesity
1. Introduction Prior to migration, many birds accumulate large amounts of fat as the main fuel for the migratory flights [14,34]. This is particularly pronounced in species crossing mountains, oceans or deserts where feeding opportunities are scarce [11]. For several species fattening in preparation for migration has been shown to rely on an endogenous time program [6,13] and to be associated with increases in food intake [10] and utilization efficiency [2,24] and with changes in food preference [7]. Garden warblers (Syl6ia borin) breeding in Europe and migrating in the Afrotropics are a prominent example [4], which has been addressed also in respect of Abbre6iations: BM, body mass; GLUC, glucose; TG, triglycerides; CHOL, cholesterol; HDLC, high density lipoprotein-cholesterol; HBA, b-hydroxybutyrate; URIC, uric acid. * Corresponding author. Tel.: +49 4421 96890; fax: + 49 4421 968955; e-mail: [email protected]
metabolic adjustments involved in long-distance migration. On fall migration garden warblers landing in the Sahara showed significantly higher plasma levels of glucose and triglycerides concomitantly with larger amounts of depot fat than at the breeding grounds before embarking [8]. At a site in central Europe, birds resting for an early migratory stop-over or for postbreeding molt, both still lean, did not differ in plasma metabolite levels [28]. However, garden warblers caught during active flight on fall migration at an Alpine pass also showed higher triglyceride levels than post-exercise, after a one-night fast or postprandially, but decreased glucose levels [25]. The increase in triglycerides is regarded as a special adaptation of small birds to maintain energy supply during endurance migratory flights [26]. However, in view of the close relationship of some plasma metabolite levels to the amount of visible fat in migrating individuals [8] it is unclear, whether the changes are exclusively due to exercise or whether endogenous changes are also involved.
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In mammals, obesity is often associated with increases in plasma glucose and lipid levels, insulin resistance and decreased glucose utilization (e.g. [15]). In the garden warbler endogenous seasonal preparations for migration [6], including fattening as the most prominent one, are well established. However, the extent of accompanying metabolic changes is unknown, although an increase of the plasma glucose level [1], and for some other species an increase in the capacity for lipid oxidation prior to migration has been shown [30 – 32]. This study aimed to investigate whether in the garden warbler, a species with pronounced fattening, blood chemistry and glucose utilization change with body fat loads as known from mammalian obesity. Because of the complex situation in the field, where implications of different resorptive/fasting states, exercise, and ambient temperatures could not be evaluated, the study was carried out under standardized laboratory conditions.
2. Materials and methods 20 garden warblers, mostly caught as yearlings around Wilhelmshaven (53°3 N, 8°1 E), were caged under constant indoor conditions (20°C, LD 12:12, standard insect food [3] ad libitum) for the non-reproductive period (September to May). At night, rooms were totally dark to suppress zugunruhe [17], which was registered in 10 birds with hopping perches. During the breeding season (June to August), the birds were kept in outdoor aviaries. Body mass and food intake were recorded daily in the morning, visible fat (9 grade scale [29]) and molt intensity (0, none; 1, less than 1/3; 2, up to 2/3; 3, more than 2/3 of 12 parts of plumage with growing feathers; modified method of [12]) were scored weekly. For each bird body mass changes were calculated per day. Food intake was calculated as dry matter mass after correction for evaporative losses according to F= Fin
Cdry C −Fout dry Cin Cout
F, net food intake by the bird (g dm); Fin, wet food mass offered to the bird; Fout, mass of the food left by the bird at the next day; Cdry, dry mass of a control food sample; Cin, wet mass of a control food sample; Cout, mass of the control sample the next day. At 2-week intervals, the birds were bled in the morning before feeding by puncturing the wing vein and collecting the blood with heparinized capillaries. The blood was centrifuged (15 min; 8900× g; 4°C) and plasma analyzed for glucose (GLUC), triglycerides (TG), cholesterol (CHOL), high density lipoproteincholesterol (HDLC), urea (UREA), uric acid (URIC), Na and K with the EKTACHEM DT II analyzing
system (Kodak, Stuttgart, Germany) [8]. Triglyceride measurements included amounts of free glycerol. Free fatty acids (FFA) and b-hydroxybutyrate (HBA) were measured using standard kits obtained from Boehringer (Mannheim, Germany) and Sigma (St. Louis, USA), respectively, following the producers’ instructions. Most assays were based on enzymatic detection. Electrolytes were quantified by potentiometry. Interassay variance was between 1.7% (URIC) and 5.3% (UREA) for the EKTACHEM DT II parameters (for each n= 45–50), 2.2% for HBA (n= 24), and 13.2% for FFA (n= 29). Intraassay variance was always below 5%. Glucose utilization was measured in the same individuals once in the fat phase and once lean thereafter prior to prenuptial molt. Birds were orally administered approximately 80 mg glucose, dissolved in 100 ml of water, and at 0, 1.5 and 3 h afterwards plasma glucose levels were determined. For several reasons, the number of measurements in most cases was markedly below the number of birds: (a) The collected blood amount was not always sufficient to measure all parameters, (b) nine birds had died until the end of the study and (c) when body mass was below 16 g, birds were assumed to be to weak for repetitive blood sampling every 2 weeks. For similar reasons, the number of measurements each bird contributed per month ranged from none to three. Therefore, in order to avoid weighting prior to further statistical evaluation, monthly averages for each bird were calculated. These values were used for the calculation of monthly means for all birds. The relationships among parameters were investigated with the original data by calculation of correlation coefficients or by analysis of covariance controlling for individual birds.
3. Results Body mass showed considerable seasonal variation with maximum values during November to January and minimum values in March (Fig. 1(a)). Monthly averages for body mass changed with those of fat amounts throughout the study. Both correlated closely among each other with significant correlation coefficients for individual birds between 0.80 and 0.98 (mean 9 S.D.: r= 0.8990.04; test of homogeneity: x 2 =1.0; n= 20; p\ 0.05). The pattern of molt was opposite (Fig. 1), also reflected by a negative correlation between fat and molt levels with significant correlation coefficients for individual birds between − 0.35 and −0.90 (r= − 0.549 0.04; x 2 = 7.0; n= 15; p\ 0.05). Food intake was highest from September to December (Fig. 1(b)) followed by a minimum in January and a continuous increase afterwards. For most birds, it correlated with daily body mass changes with significant correlation coefficients for individual birds between 0.39 and
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0.79 (r= 0.6590.02; x 2 =18.5; n =16; p \ 0.05). Zugunruhe varied among the ten birds registered, but ranged from November to March with monthly averages always below 1 h night − 1. Nevertheless, in five birds there was a significant negative correlation with daily body mass change (r = − 0.35 90.04; x 2 = 5.6; n= 5; p \0.05) and food intake (r = − 0.51 9 0.05; x 2 = 4.5; n= 5; p \0.05). However, no significant relationship with zugunruhe resulted for any of the plasma parameters (each p \0.1). Plasma levels of triglycerides, cholesterol and glucose showed considerable seasonal variation with highest levels from September to January and lowest values between February and April, when most birds were
Fig. 2. Seasonal variations of plasma (a) triglycerides and cholesterol and (b) free fatty acids and b-hydroxybutyrate. Values are means 9S.E., numbers of birds above and beneath the curves; p-values result from one-way ANOVA.
Fig. 1. Seasonal variations of (a) body mass and depot fat and (b) food intake and molt. Values are means 9S.E., numbers of birds above and beneath the curves; p-values from one-way ANOVA revealing (significant) seasonal variations. Differences in sample sizes between months are due to the varying number of birds from which blood samples could be taken. Since it was intended to investigate a link of plasma parameters and the body mass cycle, only data from those birds on the particular blood sampling days are shown.
molting (Fig. 2(a), Fig. 4(a)). Total cholesterol consisted almost completely of HDL-cholesterol, the average percentages varying not significantly between 80 and 96% throughout the year. For FFA and HBA, the variances were much greater and differences between months were not significant (Fig. 2(b)). However, FFA levels were highest during the months of maximum body mass and fat (Fig. 2(b)) and correlated significantly with both (Table 1). Sodium levels during and shortly after fat deposition were significantly lower than in spring (Fig. 3(b); mean values 9 S.E.: 115.991.3 mmol l − 1 (November to January)/119.3 91.4 mmol l − 1 (March to May), paired t-test: p= 0.028, n=8), while potassium remained fairly constant. Plasma uric acid and urea levels varied throughout the year, but without any apparent relationship to the annual body mass cycle (Fig. 3(a)).
+0.59 (186) −0.36 (179) +0.61 (168) — (138) −0.37 (176) +0.30 (185) TG
+0.32 (230) — (220) +0.26 (211) — (165) — (219)
GLUC
TG
+0.44 (172) +0.72 (172) CHOL
+0.60 (173) −0.30 (166) +0.71 (156) — (126) −0.35 (164)
CHOL
— (68) +0.74 (68) +0.91 (68) HDLC
+0.57 (68) — (66) +0.62 (67) — (54) — (68)
HDLC
— (181) +0.67 (154) +0.55 (149) +0.61 (62) FFA
+0.40 (182) −0.28 (177) +0.50 (168) −0.32 (139) — (176)
FFA
— (128) — (124) +0.39 (123) +0.44 (63) +0.40 (123) HBA
— (129) −0.44 (125) — (116) −0.44 (104) — (124)
HBA
— (194) — (176) — (169) — (67) — (161) −0.51 (122) URIC
— (195) — (188) — (176) +0.37 (141) — (185)
URIC
(68) (65) (57) (46) (61) +0.44 (67) — (67) — (68) — (28) — (58) — (52) — (68) UREA
— — — — —
UREA
— — — — — — — — K
— — — — —
K
(104) (104) (103) (49) (94) (86) (102) (56)
(104) (100) (93) (79) (96)
(110) (108) (101) (79) (104) — (110) −0.33 (110) −0.36 (109) — (55) — (100) — (90) — (109) — (58) — (93)
— — — — —
Na
r-values when correlations were still significant after sequential Bonferroni adjustment [36] on a table-wide significance level of 0.05; if not significant number of pairs calculated (in parentheses).
BM DBM FAT FOOD INTAKE MOLT
GLUC
Table 1 Relationships of blood parameters to body mass, body mass changes, visible fat, molt and food intake and among each other calculated by an analysis of covariance controlling for individual birds
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Triglycerides, cholesterol, FFA and HBA were closely related to each other as well as to body mass and fat (Table 1), which applied also to glucose, though to a lesser degree. However, correlation of plasma lipids with food intake on the preceding day was negative, most strikingly for FFA and HBA. HBA did not change with body mass and fat, but showed a negative relationship to body mass changes and food intake. Uric acid levels were positively related to food intake, and negatively to HBA. The relationships of urea with body mass, fat, triglycerides and cholesterol were opposite to those of uric acid, except with glucose. However, none was still significant after Bonferroni-adjustment, neither was the relationship between the plasma levels of urea and uric acid (Table 1; Fig. 3(a)). Glucose tolerance was poorer in fat birds (BM: 27.59 0.6 g, n = 5) than in lean birds (BM: 17.690.8
Fig. 4. (a) Seasonal variations of plasma glucose and (b) variations after an oral glucose load on identical birds in lean (open symbols) and fat (filled symbols) condition. Glucose tolerance tests were carried out in January/February prior to and in March/April shortly after defattening; one bird was still fat in April and is therefore missing in the lean group. Values are means 9 S.E., number of birds beneath the curve; p-value from one-way ANOVA, ** significantly different from both the initial level and at 3 h in the lean state (paired t-test: p B0.05). The high starting levels in the glucose tolerance test may have resulted from the selection of birds showing very pronounced fat deposition, while in the annual cycle also birds with moderate fat deposition are included.
g, n= 4, paired t-test: pB0.001) with significantly increased plasma levels 3 h after glucose administration in the fat birds (Fig. 4(b); paired t-test: 0–3 h (fat): pB0.05, n= 5; fat–lean (3 h): pB0.05, n= 4).
Fig. 3. Seasonal variations of plasma (a) uric acid and urea and (b) electrolytes. Values are means 9 S.E. (for K often smaller than symbols), numbers of birds above and beneath the curves; p-values from one-way ANOVA.
4. Discussion The garden warblers showed seasonal variations in body mass associated with changes in the amounts of
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subcutaneous fat. In migratory bird species such seasonal changes in body/fat mass are well-known to be due to a fattening in preparation for migration, which in the garden warbler is controlled by an endogenous circannual rhythm [6,13,22]. In this study it was demonstrated that birds show concomitantly seasonal variations in plasma composition, although the influence of prolonged exercise and fasting as experienced during active migration were almost completely excluded by keeping birds under constant conditions and measuring them in the same postresorptive state. There was no significant relationship of plasma parameters with the amounts of zugunruhe and two single birds with normal body mass cycles, but lacking the subsequent molt, showed changes of plasma metabolites similar to birds with molt. Therefore, changes of plasma concentrations were most likely due to changes in body mass and concomitant effects of non-suppressible zugunruhe and molt on plasma concentrations may have been minor. Increases in plasma glucose and lipid levels and a decrease in sodium in fat birds were in accordance with measurements in migrating garden warblers crossing the Sahara [8] and with results on other small migratory species kept in captivity, such as the white-crowned sparrow Zonotrichia leucophrys gambelii [18] and the red-headed bunting Emberiza bruniceps [38]. Strong correlations of these plasma metabolite levels with body mass changes have previously been reported for the garden warbler [27], whereas in still relatively lean free-living individuals which were already migrating, plasma metabolite levels hardly changed in comparison to postbreeding and molting birds [28]. However, birds in this study showed high plasma levels of glucose, triglycerides and cholesterol in September and October, when body mass and fat were still low. This may be an artefact raised by settling-in birds from the wild or from outdoor aviaries, because birds then normally reduce food intake and lose weight for several days. In herring gulls, triglyceride and cholesterol levels increase due to fasting and afterwards remain high even after refeeding [42]. In fat garden warblers overnight-fasted triglyceride and cholesterol levels also increase initially in response to food restriction [41]. The high levels during September and October could otherwise reflect a high rate of lipogenesis preceding fattening and the body mass increase. However, the reason for the lack of correlation between plasma metabolites and body mass at the beginning of our investigation is still unclear. This also applies to the extent by which triglyceride levels may be overestimated due to unquantified amounts of free glycerol. However, other studies have shown free glycerol to vary closely with FFA and for the garden warbler within one quarter of the range presented for triglycerides during this study [25–28]. Therefore, free glycerol may have increased triglyceride variations but is unlikely to have
influenced the seasonal pattern. It was assumed that high plasma triglycerides in small birds are a necessity for maintaining fuel supply during endurance flights according to the high mass-specific metabolic rates of migrating passerines [26]. The described changes in plasma lipids and glucose are similar to those in mammalian obesity and Type II-diabetes [15]. These metabolic disorders are mostly due to insulin resistance reflected by transient hyperinsulinemia, which, however, is not shown by the garden warbler [40]. Another key mechanism shown for mammals in vitro and also discussed for Type II-diabetes associated with obesity is the glucose fatty-acid cycle [35,39], which is based on the mutual inhibition of glucose and fatty acid oxidation [33,35]. In humans, the oxidation of fatty acids in tissues corresponds well with the plasma fatty acid level (cf. [33]). Therefore, the trend for an increase of plasma FFA in fat birds may be further evidence for an increasing oxidation rate of lipids in preparation for migration, which is also indicated by the premigratory increase of the lipid oxidizing capacity in flight muscles [30–32]. An inhibition of glucose oxidation by fatty acids and acetyl-CoA due to the glucose fatty-acid cycle [33,35] would explain the seasonal variations in the plasma glucose level which were first described for garden warblers by Bairlein [1], and would also account for the poorer glucose tolerance of fat birds compared to lean birds shown in the present study. The lower plasma sodium levels and the consistency of high levels of HBA during the phases of high fat loads in both, caged birds and free-living birds on migration (sodium [8]), are in agreement with the implications of hyperglycemia on water balance [21] and with the resistance to ketoacidosis in Type II-diabetes [20]. The lack of an increase in HBA could be the result of either a minor ketogenic effect of glucagon, as assumed in human Type II-diabetes [20] and indeed proven for the pigeon [9], or of a preference for catabolism of fatty substrates during the phase of fat deposition [5,34]. This may be confirmed by the obviously exogenously caused decrease of overnight-fasted HBA levels in still lean garden warblers on fall migration in central Europe [28]. In contrast to the pathological disorders in humans [15], the fat deposition of migratory birds and its simultaneous changes in metabolism are normal seasonal events and, therefore, apparently well regulated. For example, migratory fattening in the garden warbler appears not to be associated with hyperinsulinemia [40]. However, the physiological importance of pancreatic hormones in birds seems to be different from that in mammals [23], and the regulatory mechanisms of food intake and body mass are less clear [19]. In addition, the existence and the role of the recently described mammalian leptin [16,37] in the regulation of body fat in birds are still unknown. The physiological mecha-
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