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Raptor injury-induced and post-feeding hypoglycemia: A rare phenomenon in the American kestrel, Falco sparverius
RAPTOR rNJ~RY-IN~~~ED AND POST-FEEDING HYPOGLYCEMIA: A RARE PHENOMENON IN THE AMERICAN KESTREL, FALCO SPAR VERI US MERLYN C. MINKK Raptor Research and Rehabilitation Laboratory, Department of Biology. Eastern Michigan University, Ypsilanti. MI 48197. U.S.A.
Post-feeding hypoglycemia, an unknown phenomenon in wild raptors, was studied in a central nervous system (CNS) damaged American kestrel, Falco sparcerius Linnaeus. 2. Beef chunk ingestion (200 g/kg BW) produced symptomatic hypoglycemia (45-60 min) which was accompanied by prolonged decreases (240 min) in plasma glucose and increased plasma levels (60 mm) of an immunoreactive component (ICl believed to be insulin. 3. Beef chunk-induced hypoglycemia and increases in plasma If levels were blocked by atropine (0.02 mg/kg) pre-treatment. 4. Oral glucose loading (I g/kg) also produced symptomatic hypoglycemia (3@40 mitt), but of shorter duration (90 min). with corresponding changes in plasma glucose and IC levels but these responses were not blocked by atropine. 5. Data suggest that insulin secretion was stimulated via indirect (vagal) and direct (blood glucose) routes and that post-feeding hypoglycemia resulted primarily from deranged CNS-associated mechanisms (hypothalamic possibly pituitary) which control catabolic bormone release, endogenous glucose production and consequently, plasma glucose levels.
Abstract-l.
INTRODUCTION
in raptors has not been described. In wildlife, this phenomenon is not conducive to survival but has been observed in injured raptors which receive life-sustaining support via veterinary procedures. ~ypogly~mia is not a disease but is indicative of deranged overall utilization of carbohydrate in which glucose is removed from blood faster than it can be replenished. Thus, blood glucose levels decrease to abnormally low levels and this condition usuafiy leads to various disorders one of which is convulsions (Corm & Seltzer, i955). Mechanisms through which hypogIycemia in raptors are manifest are unknown but have been associated (two cases in this laboratory) with central nervous system (CNS) damage. This communication describes the symptoms+ blood-chemjstry changes and possible mechanisms which induce post-feeding hypoglycemia in a CNSdamaged (one remaining) American kestrel, F&o sparverius Cinnaeus. Blood-chemistry changes are also described for five normal (non-injured) kestrels for comparative purposes. Severe or convulsive
hypo~yce~ia
The injured male falcon (age, < I yr) was transported (within 6 hr) to this laboratory by a concerned citizen (Wayne County, MI) after having collided with an automobile while pursuing road-side prey. Obvious injuries consisted of right intraocular edema (eye protrusion from orbit), right cranial lacerations and concussion. However, during the first 7 weeks of convalescence, h~~glycemic symptoms resembling those of insulin-treated mammals appeared 45-6Omin following ingestion of raw beef, liver or whole mice. Symptoms were comprised of debility, complete disorganization (vertigo), shivering, collapse, feather
erection, spastic muscle twitching (convulsions) and pupil dilation (contralateral) all of which were relieved within 5-10min by intra~ritoneally-administered glucose. After the seventh week, the number and severity of hypoglycemic episodes decreased and eventually disappeared altogether. Five normal (non-injured) kestrels (two females, three males), which were also less than 1.yr of age, were obtained from various sources (at earlier dates) and were utilized for comparative purposes. The birds were collected over varying lengths of time (l-2yr) and were maintained in captivity (2-3 months) prior to experimental use. Throughout this study, the falcons (120-135g) were maintained in 4 x 4 x 4 ft cages (1 x 2 in. wire mesh) with drinking and bathing water. Room temperature was stabilized at 24.0 & 1.2”C (x & SD.) and the photoperiod was standardized (12 hr light: 12 hr dark) commencing at 8:OO a.m. Feeding occurred once per day (noon) and the diet for each consisted of one Swiss-Webster mouse, Mus tnn.sculus, (45-50 g) which was sufficient to maintain consistent body weights. Food which was not assimilated by 4:00 p.m. was removed. However, the mouse was usuallydevoured within 20 min. All studies were initiated between S:OO and 9:00 a.m. following 2Ohr of fasting for pre-testing stabilization of blood chemistries. Anesthesia was not utilized during any phase of this study. Whole blood (20_3Opl-) was carefully obtained from wing veins with heparinized @a*) %-gauge needles (O.f in.) and TB syringes or heparinized mi~rohematocrit tubes. Following immediate cooling (4°C) and subsequent centrifugation (IOOOg), the unhemolyzed plasma was isolated and stored (-27’C) until the time of glucose assay. Plasma glucose levels in the injured bird were determined in four different studies: (A) the effect of beef-chunk (nitrite free) ingestion (2OOg/kg BW) in the non-treated or (B) atropin~-treated (0.02 mg/kg) bird and (C) the effect of orally-administered glucose (I g/kg) in the non-treated or (D) atropine-treated bird (Fig. 1)~Twenty-four experimental procedures were performed (six/study). Experimental procedures which were performed (l-2 yr
543
Fig. 1. The effects of beef-chunk Ingestion (200g/kg BW) and glucose loading (I g,‘kg) upon plasma glucose levels in the injured non-treated or atropine-treated (0.02 mgikgl kestrel. --A ~. beef-chunk in the non-treated bird: ---A---beef-chunk in the atropine-treated bird: --a--. glucose loading in the non-treated bird: -+-, glucose loading foilawing atropine pre-treatment. Each point (except time 01 represents the mean k SD. of six determinations. At time 0. .V = 23.
earlier) in the five normal (non-injured) birds consisted of determinations of plasma glucose levels in two studies: (A) the effect of beef-chunk ingestion (2OOg/kg) and (B) oral glucose loading (1 g/kg) but without atropille pre-treatment in either study (Fig. 3). Ten experimental procedures were performed (one,Grd per study). Plasma glucose was determined in duplicate by a
manual and uftramicro-adapted GOD-Perid method ia glucose nxidase. 2. 2’-azinodiethgTbenzthiazofine sulfuni~ acid,
peroxidasc system) (Boehringer Mannheim Corporation. NY). us a plasma lmmunoreactive component (IC) was found to compete with [‘2S11-iodoinsulin for binding sites in
Frrsring p/u.srt~uy/wost~ /~w/.s. Prior tn and throughout convalescence and durmg all experimental procedures. fasting (time (1) ptasma ~~UOOSO lcvcls ranged front I ?? to 2 10 mg. 10 ml (.V = 341 with :i mean of IX7 _t $43 (x k S.t3.) {Fig. [) sod were not signif& cnntly di&rerent (P > QO5) from those found in normal (non-injured) birds (Fig. 3). Fasting hypoglycemia (symptomrtic or chemical) was not observed. EfeHhcro~‘beeflchunk ingesfion. Following beef-chunk ingestion j?OO g/kg BWI which was completed in S--8 min, plasma glucose did not significantly change from fasting levels for 30 min (Fig. I) but thereafter sigqificantiy decreased (P < 0.01) by 45 min (4X1*,,)
i
:SD
OlUCOSf
Bffm3lUNK
LOADfNO
lwoIstlow
TIMf,
min
Fig. 2. The effects of beef-chunk ingestion (200 g/kg BW) and glucose loading (1 &kg) upon relative plasma IC levels (0 and 60min) in the injured non-treated or atropinetreated (0.02 mg/kg)s kestrel. Shaded bars represent experimental fevcls whereas ctear bars represent k’%Sasting levels Each bar &pi& the mean + SD. of six determinations.
MlNUIfS
Fig. 3. The etiects of beef-chunk ingestion (200 g!kg LIWl and glucose loading (1 g,‘kg) upon plasma glucose levels in five normal (non-injured1 kestrels. ^ -e -, beef-chunk ingestion: ---t. glucose loading. Each point (except time 0) repreSents the mean k S.D. of Eve determinations
Raptor injury-induced and post-feeding hypoglycemia
545
reaching the lowest levels of 58.4 + 23.0 mg/lOOml slowly increased (14.3”/,) by 30 min and reached significantly elevated levels (P < 0.01) of 241 k 16.4 mg/ (68.9”” decrease) 120 min post-feeding. Hypoglycemic 100 ml (19.87; increase) at 45 min and 238 f. 18.0 mg/ symptoms appeared 45-60 min post-feeding and perIOOml (17.8% increase) at 60 min post-feeding. sisted throughout the evaluation period (240 min) during which fasting plasma glucose levels were not Plasma glucose levels then slowly returned to and re-established. remained at fasting levels from 90 to I80 min. Intramuscular (pectoral) atropine administration E&t of glucose loading. Following glucose loading (0.02 mg,/kg) prior to feeding (30 min) markedly de(I g/kg), plasma glucose levels at 5 min increased creased the rate of glucose level descent and salivation sharply (32.1%) to 267 f 39.5 mg/lOO ml (P < 0.01) which accompanied feeding. The lowest glucose levels and thereafter rapidly decreased to time 0 levels in attained (156 k 17.5 mg/lOO ml) following atropine I5 min (Fig. 3). Although glucose levels decreased treatment (16.7”” decrease) also occurred 120 min further to 189 + 20.0mg/100ml (90min), all mean post-feeding and were not significantly different from glucose levels from 15 to 180 min post-glucose adminfasting levels (P > 0.05). Symptomatic hypoglycemia istration were not significantly different from fasting was absent. levels (P > 0.05). Eflect of glucose loading. Following glucose intubation (1 g/kg BW), plasma glucose levels remained unDISCUSSION changed from fasting levels for 15min but thereafter significantly decreased (41.2%) by 30min (P < 0.01) To attest to the rarity of convulsions in raptors reaching the lowest levels of 65.8 + 20.5 mg/lOO ml (injured or intact), the author has handled raptors (65.22, decrease) 45 min post-glucose administration for many years (since 1947) and, until recently, had (Fig. I). By 90min, glucose levels increased and never observed such an occurrence. In addition, this returned to normal levels by 180-240 min. Hypoglycephenomenon has not been described in the literature. mic symptoms appeared 3&40min post-glucose adHowever, during the last decade, two kestrels (males) ministration but subsided by 9glOOmin. Thus, the with head injuries were serendipitously acquired and time required to induce and alleviate hypoglycemia both exhibited periodic convulsions. The first (1968) via glucose loading was of a shorter duration than which convulsed mildly and unpredictably, later expired from unknown causes before serious physiothat attained by beef-chunk ingestion. Atropine administration (0.02 mg/kg) prior to glulogically-oriented investigations into its difficulty cose loading (30 min) did not appear to significantly could be initiated. The second (1976) still lives (withalter the rate of glucose level descent from fasting out support) and has been a most valuable source levels (Fig. I). The greatest decrease (49.2%) at 45 min for study as it provided important information and was not significantly different (P > 0.05) from that a unique experimental model which could never be (65.2”;) obtained in the non-treated bird nor were duplicated in the laboratory. For even though convulhypoglycemic symptoms absent. sions in wild raptors are unknown, metabolic conditions which produce them in the injured are of Changes in plasma IC levels. Plasma IC levels at physiological interest and may contribute to the time 0 (fasting), relative to insulin standards, ranged of raptor physiology. from 268 to 440pg/ml with a mean of 354 _t 75.8 understanding (?? + SD.) (Fig. 2). From the beginning, it was clear that convulsions, Following beef-chunk ingestion (60 min), relative without exception, followed ingestion of meals, parIC levels significantly increased (P < 0.01) from time ticularly heavy ones. Because of symptomatic similari0 (126”;) to 800 + 167 pg/‘ml. After atropine pre-treatties to mammalian hypoglycemia, plasma glucose and ment, relative IC levels at 60 min increased (15.29/o) IC (probably insulin) levels prior to and during convulsive states were studied while utilizing standardto 408 + 83.5 pgiml but were not significantly different from fasting levels (P > 0.05). ized weights of beef-chunks and oral glucose loads. Following glucose loading (60min), relative IC Data (Fig. 1) support the hypothesis that convulsions levels significantly increased (P < 0.001) from time 0 arose from post-feeding hypoglycemia. Symptomatic hypoglycemia coincided with predicted times (96.63,) to 696 f 42.3 pgjml (Fig. 2). In the presence of atropine, IC levels at 60 min significantly increased (45-60 min) while esophageal distension was still prominent and appeared at higher plasma glucose (P < 0.02) from time 0 (82.50,/,) to 647 f 93.4pg/ml levels (9c-110 mg/lOO ml) than usually observed in but were not significantly different (P > 0.05) from mammals (Conn & Seltzer, 1955; Minick, 1970) or those attained with glucose alone. fowl (Shin-ichi & Ono, 1962; Langslow & Hales, The fact that IC is antigenically similar to insulin, 1971). This observation may be misleading, however, increases with a decrease in plasma glucose and the as peripheral (wing) blood was utilized for the deterinduction of symptomatic hypoglycemia, lends cremination of plasma glucose and not that which bathes dence to the hypothesis that IC is insulin. the CNS. Therefore, these levels may not be indicative (h) Non-injured raprors of the glucose levels which directly initiate convulsions. Although symptomatic hypoglycemia, during Fasting plasrna glucose /euels. Fasting (time 0) beef-chunk ingestion, did not occur until 45-60min, plasma glucose levels (Fig. 3) ranged from 190 to plasma glucose levels were affected as early as 221 mg/lOOml (N = 10) with a mean of 202 f 10.3 15-30min after feeding (Fig. 1). Even though data (x f S.D.). at this time interval were not acquired, plasma IC Eflect qfbeefchunk ingestion. Following beef-chunk levels were probably elevated above fasting levels. ingestion (2OOg/kg BW) which was completed in Plasma IC levels at 60 min (Fig. 2) were significantly 5510 min, plasma glucose remained unchanged from increased (126%) and the increases appeared to persist fasting levels for 15 min (Fig. 3). Glucose levels then
546
MERLYN C. MINICK
as plasma glucose remained low for 240 min. Following oral glucose loading. plasma glucose levels decreased and returned to the normal range more precipitously than observed after beef-chunk ingestion (Fig. I). This may have resulted from differences in stimuli magnitudes and/or their time courses. Fasting (time 0) plasma glucose levels were within previouslyreported ranges for raptors (Balasch et a/., 1976) and those depicted in Fig. 3. The mode through which beef-chunk-induced hypoglycemia was produced appeared to resemble the “vagal” mechanism found in mammals (Kaneto et al.. 1967: Porte et al., 1973; Bergman & Miller, 1973) as the hypoglycemic response was significantly depressed by atropine (Fig. 1). Atropine blockade of bolus-induced hypoglycemia suggests that cholinergic stimulation (via the parasympathetics) of insulin secretion occurred (Malaisse et a/.. 1967) prior to and during early phases of convulsive episodes and that elevated blood levels of insulin were responsible for the hypoglycemia. That a vagal mechanism may have been operative is also supported by the demonstration that plasma IC levels were elevated in the non-treated bird and failed to increase after atropine treatment (Fig. 2). It is also conceivable, however. that atropine may have blocked the release of gastric secretions in the proventriculus (Hill. 1971; Ziswiler & Farner. 1972) thereby preventing proteolytic-enzyme hydrolysis of the bolus in the posterior muscular stomach. Such a digestive impairment would have prevented elevations in plasma amino acid levels which are known to stimulate insulin release (Edgar et l/l., 1969) and thus. the hypoglycemic response would have been depressed. Salivation itself was strikingly reduced. This explanation is not satisfactory. however. as plasma IC levels were increased. if the assumption that IC is insulin can be made. In addition. if bolus hydrolysis was not impaired and plasma amino acid levels were increased via the gastrin-like effects of avian pancreatic polypeptide (APP) which also stimulates gastric secretion but in the absence of vagal mediation (Hazelwood er ul.. 1973), an atropine blockade of the hypoglycemic response would not be expected. Furthermore. if plasma APP levels were elevated and subsequent inhibition of insulin secretion had occurred (because of its gastrin-like effects). the hypoglycemic responses in both the nontreated or atropine-treated bird would have been abolished. Overall, considerable inquiry is still needed to clarify the above observations and to substantiate the presence of a “vagal” mechanism. Inasmuch as an avian nervous control of pancreatic insulin secretion has not been described as well as evidence of direct innervation of islets (Hazelwood. 1973, 1976~). such a mechanism may exist in raptors which. contrary to that of graniverous birds. may require a more flexible energy-storage mechanism (more insulin dependent) to cope with frequent and prolonged periods of fasting. The mechanism through which glucose-induced hypoglycemia was produced appeared to resemble that which involves direct stimulation of IC release by glucose (Coore & Randle, 1964; Zawalich et al., 1977). Orally administered glucose appeared to stimulate IC release as hypoglycemia quickly ensued (Fig. 1) being significant by 30min post-glucose adminis-
tration. Plasma IC lebcls by 60 mm were elevated (96.6?,,). These observations support those involving other avian species in which glucose administratlon produced elevated plasma insulin levels (Hazelwood. 1973; Langslow & Hales, 1971). Furthermore. atropine had little influence in suppressing glucose stimulation of IC release and therefore. the hypoglycemic response (Fig. 2). It is of interest that early plasma glucose levels (5. IO and I5 min) following glucose loading did not rise significantly above fasting levels as was observed in non-injured subjects (Fig. 3). The absence of a substantial “peak” has been observed during glucose tolerance tests in other vertebrates having rapid rates of glucose absorption (Mlnick. 1970: Louis c’t trl., 1966) which may have occurred in this study (Bogner. 1966). Rapid glucose absorption occasionally leads to “missed” peaks which in this study were probably between time 0 and 5 min postglucose administration. The underlying defect which uas responsible for the bird’s inability to physiologically cope with beefchunk or glucose loads was related to CNS damage which resulted from head injuries. From the foregoing discussion arises the hypothesis that the damage was expressed in the livin_r bird as an inability to marshal rapidly and sustain high levels of insulin antagonism after being challenged with beef-chunk or glucose loads. Thus. when plasma insulinlcatabolic hormone (catecholamines. glucagonl ratios (Unger. I97! ) were abruptly increased, thereby facoring hypoglycemia. the bird was unable to compensate satisfactorily by increasing endogenous glucose production. This was particularly evident after beef-chunk ingestlon (Fig. I). The inability to compensate (decrease insulin,catabolic hormone ratios) may have been manifest chiefly by damage-induced decreases In hypothalamusmediated production of appropriate blood levels ol glucagon and/or catecholamines although pituitarymediated production of adrenocortlcotropic hormone (ACTH) (glucocorticoids), growth hormone (GH) or prolactin may also have been deficient but these hormones would be of less importance in the time sense. That the avian hypothalamo-pituitary axis doe> Influence endogenous glucose production is supported by the observation that hypophysectomy precipitates severe hypoglycemia (Langslow & Hales. 1971). Hypothalamus and or pituitary shut-down would curtail hormone-induced glucose production \,a decreasing rates of glycogenolysis and/or pluconeogenesis (Unger. 1971: Frankel. 1970: Migliorini c’t cl/.. 1973) and thereby greatly increase the bird’s sensitivity to endogenous insulin. Unfortunately, as plasma was limited. blood levels of glucagon. catecholamlnes or those hormones mentioned earlier. could not be determined nor could the hypothesis be evaluated. However. studies involving other avian species lend support. Catecholamines are glycogenolytic and hyperglycemic agents in many birds Including owls (Assenmacher. 1972) but have no substantial effect upon plasma free fatty acid (FFA) levels (Langslow & Hales. 1971). Glucagon is a potent hyperglycemic, glycogenolytic and lipolytic hormone (Hazelwood, 1973. 1976h) and its absence (via alpha-islet pancreatectomy) has been implicated in the production of experimental hypoglycemia in fowl (Shin-ichi & Ono.
547
Raptor injury-induced and post-feeding hypoglycemia 1962). Giucagon may also accelerate protein and amino acid catabolism ultimately providing free glucose (Eisenstein et al., 1974). Furthermore, exogenous glucocorticoids (mainly corticosterone and cortisoi) are known to increase plasma glucose levels in avian species via increased liver giuconeogenesis utilizing nitrogenous energy sources (Assenmacher, 1972; Hazelwood, 19766) but not FFA (Langslow & Hales, 1971). Although gastrin-like APP has been isolated from pancreatic tissues of several avian species including raptors (Langsiow et al., 1973) and is a potent glycogenoiytic agent (Hazeiwood et al., 1973), plasma glucose levels are apparently not affected. Inasmuch as avian ACTH, GH or prolactin have yet to be isolated, their effects upon endogenous glucose production are unknown. Mammalian preparations, at physiolo~cal blood levels, are apparently without effect (Hazelwood, 1976b; Sturkie, 1976). That the hypothalamus may regulate insulin and giucagon secretion from the endocrine pancreas has been proposed for many years. Early investigations demonstrated that hypothalamic polypeptides such as vasotocin and oxytocin are hyperglycemic in fowl (Langslow & Hates, 1971). More recent mammalian studies have shown that electrical stimulation of the ventro-medical hypothalamus (VMH) produces decreased insulin and increased giucagon levels in plasma (Frohman 8~ Bernardis, 1971). The poiypeptide somatostatin inhibits both insulin and giucagon secretion (Johnson et ai., 1975). Neurotensin and substance P (Brown & Vale, 1976) and other unidentified blood-borne substances which are contained and released from VMH inhibit insulin release coincident with augmented glucagon release (Moitz et al., 1977). Thus, the vertebrate hypothalamus may regulate insulin and giucagon secretion via direct neural innervation and the secretion of releasing or inhibiting factors which reach the pancreas via the peripheral circulation. Whether similar control mechanisms 01 pancreatic secretion exist in avian species remains to be determined.
COOREH. G. & RANDLEP. J. (1964) Regulation of insuhn
secretion studied with pieces of rabbit pancreas incubated in vitro. Biochem. J. 93, 6678. EDGAR P., RABINOW~TZ D. & MERIMEET. I. (1969) EfIects of amino acids on insulin release from excised rabbit pancreas. Endocrino/ogy 84, 835-843. E~SENSTEIN A. B., STRACKI. & STEINERA. (1974) Glucagon stimulation of hepatic gluconeogenesis in rats fed a high-protein, carbohydrate-free diet. Metahobm 23, 1S-23. FRANKELA. I. (1970) Neurohumoral control of avian adrenal: a review. Pouitr.r &I’. 49, 8699885. FROHMANL. A. & BERNARDIS L. L. (1971) Effect of hypothalamic stimulation on plasma glucose, insulin and glucagon levels. Am. J. Physiol. 221. 1596-I 603. HAZELWOODR. L. (1973) The avian endocrine pancreas. Am. Zool. 13, 699-709. HAZEL.WOOD R. L., TURNERS. D.. KIWMELJ. R. & POLLOCK H. G. (1973) Spectrum effects of a new polypeptide (third hormone?) isolated from the chicken pancreas. Cen. camp. Endocr. 21, 485491. HA~ELW~~DR. L. (1976~) Pancreas. In Acian Ph~siolog) (Edited by STURKIEP. D.). 3rd edition. pp. 384-388. Springer-Verlag. New York. HAZELWWD R. L. (19766) Carbohydrate 7irtabi;Xjtii. In A&n ~~~sjo~og~(Edited by STU&F P. D.) 3rd edition. no. 21 l-232. Soringer-Verlag. New York. HENDERSON J. R.‘(lP?O) Serum insuhn or plasma msulin’t Lancer
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Acknowledyemenr-The author is indebted to Dr Herbert H. Caswell Jr. Head. Department of Biology. for his much appreciated and continuous encouragement and for providing an appropriate environment for raptor research and rehabilitation.
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Post-feeding hypoglycemia, an unknown phenomenon in wild raptors, was studied in a central nervous system (CNS) damaged American kestrel, Falco sparcerius Linnaeus. 2. Beef chunk ingestion (200 g/kg BW) produced symptomatic hypoglycemia (45-60 min) which was accompanied by prolonged decreases (240 min) in plasma glucose and increased plasma levels (60 mm) of an immunoreactive component (ICl believed to be insulin. 3. Beef chunk-induced hypoglycemia and increases in plasma If levels were blocked by atropine (0.02 mg/kg) pre-treatment. 4. Oral glucose loading (I g/kg) also produced symptomatic hypoglycemia (3@40 mitt), but of shorter duration (90 min). with corresponding changes in plasma glucose and IC levels but these responses were not blocked by atropine. 5. Data suggest that insulin secretion was stimulated via indirect (vagal) and direct (blood glucose) routes and that post-feeding hypoglycemia resulted primarily from deranged CNS-associated mechanisms (hypothalamic possibly pituitary) which control catabolic bormone release, endogenous glucose production and consequently, plasma glucose levels.
Abstract-l.
INTRODUCTION
in raptors has not been described. In wildlife, this phenomenon is not conducive to survival but has been observed in injured raptors which receive life-sustaining support via veterinary procedures. ~ypogly~mia is not a disease but is indicative of deranged overall utilization of carbohydrate in which glucose is removed from blood faster than it can be replenished. Thus, blood glucose levels decrease to abnormally low levels and this condition usuafiy leads to various disorders one of which is convulsions (Corm & Seltzer, i955). Mechanisms through which hypogIycemia in raptors are manifest are unknown but have been associated (two cases in this laboratory) with central nervous system (CNS) damage. This communication describes the symptoms+ blood-chemjstry changes and possible mechanisms which induce post-feeding hypoglycemia in a CNSdamaged (one remaining) American kestrel, F&o sparverius Cinnaeus. Blood-chemistry changes are also described for five normal (non-injured) kestrels for comparative purposes. Severe or convulsive
hypo~yce~ia
The injured male falcon (age, < I yr) was transported (within 6 hr) to this laboratory by a concerned citizen (Wayne County, MI) after having collided with an automobile while pursuing road-side prey. Obvious injuries consisted of right intraocular edema (eye protrusion from orbit), right cranial lacerations and concussion. However, during the first 7 weeks of convalescence, h~~glycemic symptoms resembling those of insulin-treated mammals appeared 45-6Omin following ingestion of raw beef, liver or whole mice. Symptoms were comprised of debility, complete disorganization (vertigo), shivering, collapse, feather
erection, spastic muscle twitching (convulsions) and pupil dilation (contralateral) all of which were relieved within 5-10min by intra~ritoneally-administered glucose. After the seventh week, the number and severity of hypoglycemic episodes decreased and eventually disappeared altogether. Five normal (non-injured) kestrels (two females, three males), which were also less than 1.yr of age, were obtained from various sources (at earlier dates) and were utilized for comparative purposes. The birds were collected over varying lengths of time (l-2yr) and were maintained in captivity (2-3 months) prior to experimental use. Throughout this study, the falcons (120-135g) were maintained in 4 x 4 x 4 ft cages (1 x 2 in. wire mesh) with drinking and bathing water. Room temperature was stabilized at 24.0 & 1.2”C (x & SD.) and the photoperiod was standardized (12 hr light: 12 hr dark) commencing at 8:OO a.m. Feeding occurred once per day (noon) and the diet for each consisted of one Swiss-Webster mouse, Mus tnn.sculus, (45-50 g) which was sufficient to maintain consistent body weights. Food which was not assimilated by 4:00 p.m. was removed. However, the mouse was usuallydevoured within 20 min. All studies were initiated between S:OO and 9:00 a.m. following 2Ohr of fasting for pre-testing stabilization of blood chemistries. Anesthesia was not utilized during any phase of this study. Whole blood (20_3Opl-) was carefully obtained from wing veins with heparinized @a*) %-gauge needles (O.f in.) and TB syringes or heparinized mi~rohematocrit tubes. Following immediate cooling (4°C) and subsequent centrifugation (IOOOg), the unhemolyzed plasma was isolated and stored (-27’C) until the time of glucose assay. Plasma glucose levels in the injured bird were determined in four different studies: (A) the effect of beef-chunk (nitrite free) ingestion (2OOg/kg BW) in the non-treated or (B) atropin~-treated (0.02 mg/kg) bird and (C) the effect of orally-administered glucose (I g/kg) in the non-treated or (D) atropine-treated bird (Fig. 1)~Twenty-four experimental procedures were performed (six/study). Experimental procedures which were performed (l-2 yr
543
Fig. 1. The effects of beef-chunk Ingestion (200g/kg BW) and glucose loading (I g,‘kg) upon plasma glucose levels in the injured non-treated or atropine-treated (0.02 mgikgl kestrel. --A ~. beef-chunk in the non-treated bird: ---A---beef-chunk in the atropine-treated bird: --a--. glucose loading in the non-treated bird: -+-, glucose loading foilawing atropine pre-treatment. Each point (except time 01 represents the mean k SD. of six determinations. At time 0. .V = 23.
earlier) in the five normal (non-injured) birds consisted of determinations of plasma glucose levels in two studies: (A) the effect of beef-chunk ingestion (2OOg/kg) and (B) oral glucose loading (1 g/kg) but without atropille pre-treatment in either study (Fig. 3). Ten experimental procedures were performed (one,Grd per study). Plasma glucose was determined in duplicate by a
manual and uftramicro-adapted GOD-Perid method ia glucose nxidase. 2. 2’-azinodiethgTbenzthiazofine sulfuni~ acid,
peroxidasc system) (Boehringer Mannheim Corporation. NY). us a plasma lmmunoreactive component (IC) was found to compete with [‘2S11-iodoinsulin for binding sites in
Frrsring p/u.srt~uy/wost~ /~w/.s. Prior tn and throughout convalescence and durmg all experimental procedures. fasting (time (1) ptasma ~~UOOSO lcvcls ranged front I ?? to 2 10 mg. 10 ml (.V = 341 with :i mean of IX7 _t $43 (x k S.t3.) {Fig. [) sod were not signif& cnntly di&rerent (P > QO5) from those found in normal (non-injured) birds (Fig. 3). Fasting hypoglycemia (symptomrtic or chemical) was not observed. EfeHhcro~‘beeflchunk ingesfion. Following beef-chunk ingestion j?OO g/kg BWI which was completed in S--8 min, plasma glucose did not significantly change from fasting levels for 30 min (Fig. I) but thereafter sigqificantiy decreased (P < 0.01) by 45 min (4X1*,,)
i
:SD
OlUCOSf
Bffm3lUNK
LOADfNO
lwoIstlow
TIMf,
min
Fig. 2. The effects of beef-chunk ingestion (200 g/kg BW) and glucose loading (1 &kg) upon relative plasma IC levels (0 and 60min) in the injured non-treated or atropinetreated (0.02 mg/kg)s kestrel. Shaded bars represent experimental fevcls whereas ctear bars represent k’%Sasting levels Each bar &pi& the mean + SD. of six determinations.
MlNUIfS
Fig. 3. The etiects of beef-chunk ingestion (200 g!kg LIWl and glucose loading (1 g,‘kg) upon plasma glucose levels in five normal (non-injured1 kestrels. ^ -e -, beef-chunk ingestion: ---t. glucose loading. Each point (except time 0) repreSents the mean k S.D. of Eve determinations
Raptor injury-induced and post-feeding hypoglycemia
545
reaching the lowest levels of 58.4 + 23.0 mg/lOOml slowly increased (14.3”/,) by 30 min and reached significantly elevated levels (P < 0.01) of 241 k 16.4 mg/ (68.9”” decrease) 120 min post-feeding. Hypoglycemic 100 ml (19.87; increase) at 45 min and 238 f. 18.0 mg/ symptoms appeared 45-60 min post-feeding and perIOOml (17.8% increase) at 60 min post-feeding. sisted throughout the evaluation period (240 min) during which fasting plasma glucose levels were not Plasma glucose levels then slowly returned to and re-established. remained at fasting levels from 90 to I80 min. Intramuscular (pectoral) atropine administration E&t of glucose loading. Following glucose loading (0.02 mg,/kg) prior to feeding (30 min) markedly de(I g/kg), plasma glucose levels at 5 min increased creased the rate of glucose level descent and salivation sharply (32.1%) to 267 f 39.5 mg/lOO ml (P < 0.01) which accompanied feeding. The lowest glucose levels and thereafter rapidly decreased to time 0 levels in attained (156 k 17.5 mg/lOO ml) following atropine I5 min (Fig. 3). Although glucose levels decreased treatment (16.7”” decrease) also occurred 120 min further to 189 + 20.0mg/100ml (90min), all mean post-feeding and were not significantly different from glucose levels from 15 to 180 min post-glucose adminfasting levels (P > 0.05). Symptomatic hypoglycemia istration were not significantly different from fasting was absent. levels (P > 0.05). Eflect of glucose loading. Following glucose intubation (1 g/kg BW), plasma glucose levels remained unDISCUSSION changed from fasting levels for 15min but thereafter significantly decreased (41.2%) by 30min (P < 0.01) To attest to the rarity of convulsions in raptors reaching the lowest levels of 65.8 + 20.5 mg/lOO ml (injured or intact), the author has handled raptors (65.22, decrease) 45 min post-glucose administration for many years (since 1947) and, until recently, had (Fig. I). By 90min, glucose levels increased and never observed such an occurrence. In addition, this returned to normal levels by 180-240 min. Hypoglycephenomenon has not been described in the literature. mic symptoms appeared 3&40min post-glucose adHowever, during the last decade, two kestrels (males) ministration but subsided by 9glOOmin. Thus, the with head injuries were serendipitously acquired and time required to induce and alleviate hypoglycemia both exhibited periodic convulsions. The first (1968) via glucose loading was of a shorter duration than which convulsed mildly and unpredictably, later expired from unknown causes before serious physiothat attained by beef-chunk ingestion. Atropine administration (0.02 mg/kg) prior to glulogically-oriented investigations into its difficulty cose loading (30 min) did not appear to significantly could be initiated. The second (1976) still lives (withalter the rate of glucose level descent from fasting out support) and has been a most valuable source levels (Fig. I). The greatest decrease (49.2%) at 45 min for study as it provided important information and was not significantly different (P > 0.05) from that a unique experimental model which could never be (65.2”;) obtained in the non-treated bird nor were duplicated in the laboratory. For even though convulhypoglycemic symptoms absent. sions in wild raptors are unknown, metabolic conditions which produce them in the injured are of Changes in plasma IC levels. Plasma IC levels at physiological interest and may contribute to the time 0 (fasting), relative to insulin standards, ranged of raptor physiology. from 268 to 440pg/ml with a mean of 354 _t 75.8 understanding (?? + SD.) (Fig. 2). From the beginning, it was clear that convulsions, Following beef-chunk ingestion (60 min), relative without exception, followed ingestion of meals, parIC levels significantly increased (P < 0.01) from time ticularly heavy ones. Because of symptomatic similari0 (126”;) to 800 + 167 pg/‘ml. After atropine pre-treatties to mammalian hypoglycemia, plasma glucose and ment, relative IC levels at 60 min increased (15.29/o) IC (probably insulin) levels prior to and during convulsive states were studied while utilizing standardto 408 + 83.5 pgiml but were not significantly different from fasting levels (P > 0.05). ized weights of beef-chunks and oral glucose loads. Following glucose loading (60min), relative IC Data (Fig. 1) support the hypothesis that convulsions levels significantly increased (P < 0.001) from time 0 arose from post-feeding hypoglycemia. Symptomatic hypoglycemia coincided with predicted times (96.63,) to 696 f 42.3 pgjml (Fig. 2). In the presence of atropine, IC levels at 60 min significantly increased (45-60 min) while esophageal distension was still prominent and appeared at higher plasma glucose (P < 0.02) from time 0 (82.50,/,) to 647 f 93.4pg/ml levels (9c-110 mg/lOO ml) than usually observed in but were not significantly different (P > 0.05) from mammals (Conn & Seltzer, 1955; Minick, 1970) or those attained with glucose alone. fowl (Shin-ichi & Ono, 1962; Langslow & Hales, The fact that IC is antigenically similar to insulin, 1971). This observation may be misleading, however, increases with a decrease in plasma glucose and the as peripheral (wing) blood was utilized for the deterinduction of symptomatic hypoglycemia, lends cremination of plasma glucose and not that which bathes dence to the hypothesis that IC is insulin. the CNS. Therefore, these levels may not be indicative (h) Non-injured raprors of the glucose levels which directly initiate convulsions. Although symptomatic hypoglycemia, during Fasting plasrna glucose /euels. Fasting (time 0) beef-chunk ingestion, did not occur until 45-60min, plasma glucose levels (Fig. 3) ranged from 190 to plasma glucose levels were affected as early as 221 mg/lOOml (N = 10) with a mean of 202 f 10.3 15-30min after feeding (Fig. 1). Even though data (x f S.D.). at this time interval were not acquired, plasma IC Eflect qfbeefchunk ingestion. Following beef-chunk levels were probably elevated above fasting levels. ingestion (2OOg/kg BW) which was completed in Plasma IC levels at 60 min (Fig. 2) were significantly 5510 min, plasma glucose remained unchanged from increased (126%) and the increases appeared to persist fasting levels for 15 min (Fig. 3). Glucose levels then
546
MERLYN C. MINICK
as plasma glucose remained low for 240 min. Following oral glucose loading. plasma glucose levels decreased and returned to the normal range more precipitously than observed after beef-chunk ingestion (Fig. I). This may have resulted from differences in stimuli magnitudes and/or their time courses. Fasting (time 0) plasma glucose levels were within previouslyreported ranges for raptors (Balasch et a/., 1976) and those depicted in Fig. 3. The mode through which beef-chunk-induced hypoglycemia was produced appeared to resemble the “vagal” mechanism found in mammals (Kaneto et al.. 1967: Porte et al., 1973; Bergman & Miller, 1973) as the hypoglycemic response was significantly depressed by atropine (Fig. 1). Atropine blockade of bolus-induced hypoglycemia suggests that cholinergic stimulation (via the parasympathetics) of insulin secretion occurred (Malaisse et a/.. 1967) prior to and during early phases of convulsive episodes and that elevated blood levels of insulin were responsible for the hypoglycemia. That a vagal mechanism may have been operative is also supported by the demonstration that plasma IC levels were elevated in the non-treated bird and failed to increase after atropine treatment (Fig. 2). It is also conceivable, however. that atropine may have blocked the release of gastric secretions in the proventriculus (Hill. 1971; Ziswiler & Farner. 1972) thereby preventing proteolytic-enzyme hydrolysis of the bolus in the posterior muscular stomach. Such a digestive impairment would have prevented elevations in plasma amino acid levels which are known to stimulate insulin release (Edgar et l/l., 1969) and thus. the hypoglycemic response would have been depressed. Salivation itself was strikingly reduced. This explanation is not satisfactory. however. as plasma IC levels were increased. if the assumption that IC is insulin can be made. In addition. if bolus hydrolysis was not impaired and plasma amino acid levels were increased via the gastrin-like effects of avian pancreatic polypeptide (APP) which also stimulates gastric secretion but in the absence of vagal mediation (Hazelwood er ul.. 1973), an atropine blockade of the hypoglycemic response would not be expected. Furthermore. if plasma APP levels were elevated and subsequent inhibition of insulin secretion had occurred (because of its gastrin-like effects). the hypoglycemic responses in both the nontreated or atropine-treated bird would have been abolished. Overall, considerable inquiry is still needed to clarify the above observations and to substantiate the presence of a “vagal” mechanism. Inasmuch as an avian nervous control of pancreatic insulin secretion has not been described as well as evidence of direct innervation of islets (Hazelwood. 1973, 1976~). such a mechanism may exist in raptors which. contrary to that of graniverous birds. may require a more flexible energy-storage mechanism (more insulin dependent) to cope with frequent and prolonged periods of fasting. The mechanism through which glucose-induced hypoglycemia was produced appeared to resemble that which involves direct stimulation of IC release by glucose (Coore & Randle, 1964; Zawalich et al., 1977). Orally administered glucose appeared to stimulate IC release as hypoglycemia quickly ensued (Fig. 1) being significant by 30min post-glucose adminis-
tration. Plasma IC lebcls by 60 mm were elevated (96.6?,,). These observations support those involving other avian species in which glucose administratlon produced elevated plasma insulin levels (Hazelwood. 1973; Langslow & Hales, 1971). Furthermore. atropine had little influence in suppressing glucose stimulation of IC release and therefore. the hypoglycemic response (Fig. 2). It is of interest that early plasma glucose levels (5. IO and I5 min) following glucose loading did not rise significantly above fasting levels as was observed in non-injured subjects (Fig. 3). The absence of a substantial “peak” has been observed during glucose tolerance tests in other vertebrates having rapid rates of glucose absorption (Mlnick. 1970: Louis c’t trl., 1966) which may have occurred in this study (Bogner. 1966). Rapid glucose absorption occasionally leads to “missed” peaks which in this study were probably between time 0 and 5 min postglucose administration. The underlying defect which uas responsible for the bird’s inability to physiologically cope with beefchunk or glucose loads was related to CNS damage which resulted from head injuries. From the foregoing discussion arises the hypothesis that the damage was expressed in the livin_r bird as an inability to marshal rapidly and sustain high levels of insulin antagonism after being challenged with beef-chunk or glucose loads. Thus. when plasma insulinlcatabolic hormone (catecholamines. glucagonl ratios (Unger. I97! ) were abruptly increased, thereby facoring hypoglycemia. the bird was unable to compensate satisfactorily by increasing endogenous glucose production. This was particularly evident after beef-chunk ingestlon (Fig. I). The inability to compensate (decrease insulin,catabolic hormone ratios) may have been manifest chiefly by damage-induced decreases In hypothalamusmediated production of appropriate blood levels ol glucagon and/or catecholamines although pituitarymediated production of adrenocortlcotropic hormone (ACTH) (glucocorticoids), growth hormone (GH) or prolactin may also have been deficient but these hormones would be of less importance in the time sense. That the avian hypothalamo-pituitary axis doe> Influence endogenous glucose production is supported by the observation that hypophysectomy precipitates severe hypoglycemia (Langslow & Hales. 1971). Hypothalamus and or pituitary shut-down would curtail hormone-induced glucose production \,a decreasing rates of glycogenolysis and/or pluconeogenesis (Unger. 1971: Frankel. 1970: Migliorini c’t cl/.. 1973) and thereby greatly increase the bird’s sensitivity to endogenous insulin. Unfortunately, as plasma was limited. blood levels of glucagon. catecholamlnes or those hormones mentioned earlier. could not be determined nor could the hypothesis be evaluated. However. studies involving other avian species lend support. Catecholamines are glycogenolytic and hyperglycemic agents in many birds Including owls (Assenmacher. 1972) but have no substantial effect upon plasma free fatty acid (FFA) levels (Langslow & Hales. 1971). Glucagon is a potent hyperglycemic, glycogenolytic and lipolytic hormone (Hazelwood, 1973. 1976h) and its absence (via alpha-islet pancreatectomy) has been implicated in the production of experimental hypoglycemia in fowl (Shin-ichi & Ono.
547
Raptor injury-induced and post-feeding hypoglycemia 1962). Giucagon may also accelerate protein and amino acid catabolism ultimately providing free glucose (Eisenstein et al., 1974). Furthermore, exogenous glucocorticoids (mainly corticosterone and cortisoi) are known to increase plasma glucose levels in avian species via increased liver giuconeogenesis utilizing nitrogenous energy sources (Assenmacher, 1972; Hazelwood, 19766) but not FFA (Langslow & Hales, 1971). Although gastrin-like APP has been isolated from pancreatic tissues of several avian species including raptors (Langsiow et al., 1973) and is a potent glycogenoiytic agent (Hazeiwood et al., 1973), plasma glucose levels are apparently not affected. Inasmuch as avian ACTH, GH or prolactin have yet to be isolated, their effects upon endogenous glucose production are unknown. Mammalian preparations, at physiolo~cal blood levels, are apparently without effect (Hazelwood, 1976b; Sturkie, 1976). That the hypothalamus may regulate insulin and giucagon secretion from the endocrine pancreas has been proposed for many years. Early investigations demonstrated that hypothalamic polypeptides such as vasotocin and oxytocin are hyperglycemic in fowl (Langslow & Hates, 1971). More recent mammalian studies have shown that electrical stimulation of the ventro-medical hypothalamus (VMH) produces decreased insulin and increased giucagon levels in plasma (Frohman 8~ Bernardis, 1971). The poiypeptide somatostatin inhibits both insulin and giucagon secretion (Johnson et ai., 1975). Neurotensin and substance P (Brown & Vale, 1976) and other unidentified blood-borne substances which are contained and released from VMH inhibit insulin release coincident with augmented glucagon release (Moitz et al., 1977). Thus, the vertebrate hypothalamus may regulate insulin and giucagon secretion via direct neural innervation and the secretion of releasing or inhibiting factors which reach the pancreas via the peripheral circulation. Whether similar control mechanisms 01 pancreatic secretion exist in avian species remains to be determined.
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Acknowledyemenr-The author is indebted to Dr Herbert H. Caswell Jr. Head. Department of Biology. for his much appreciated and continuous encouragement and for providing an appropriate environment for raptor research and rehabilitation.
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