Download Physiological responses to food deprivation in the house sparrow, a

Am J Physiol Regul Integr Comp Physiol 303: R551–R561, 2012.
First published July 11, 2012; doi:10.1152/ajpregu.00076.2012.
Physiological responses to food deprivation in the house sparrow, a species
not adapted to prolonged fasting
Anton Khalilieh,1 Marshall D. McCue,2 and Berry Pinshow1
1
Mitrani Department of Desert Ecology, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev,
Midreshet Ben-Gurion, Israel; and 2Department of Biological Sciences, St. Mary’s University, San Antonio, Texas
Submitted 21 February 2012; accepted in final form 5 July 2012
house sparrow; fasting; starvation; blood metabolites;
isotopes; breath testing
13
CO2; stable
endure long fasts during reproduction,
molting, hibernation, or migration (53), or simply because food
is scarce (7, 21, 41, 43). The physiological and behavioral
responses of species that are adapted to long fasts have been
the subjects of much attention; in brief, animals adapted to
fasting tend to preferentially mobilize carbohydrates and lipids,
while sparing precious body proteins (2, 14, 17, 18, 24). By
contrast, little attention has been paid to the responses to food
deprivation in species that are not considered well adapted to
fasting (27).
MANY WILD ANIMALS
Address for reprint requests and other correspondence: A. Khalilieh, Mitrani
Dept. of Desert Ecology, Jacob Blaustein Institutes for Desert Research,
Ben-Gurion Univ. of the Negev, 84990 Midreshet Ben-Gurion, Israel (e-mail:
[email protected]).
http://www.ajpregu.org
Fasting is a common occurrence in the lives of many avian
species. Most long distance migrants cannot feed in flight and
must exploit endogenous fuel deposits to meet energy demands
(5, 34, 57, 60, 67). Some of these birds fly nonstop for
thousands of kilometers, most notably shorebirds and waders
that cross oceans on their migration (26, 56, 57). Migrants are
not the only birds that experience long fasts. Many species of
marine birds (e.g., blue petrels, Halobaena caerulea, shorttailed shearwaters Puffinus tenuirostris, and sooty shearwaters,
P. griseus) forage in areas far from their nests, and some of
their trips can last for as long as 14 days (15, 25, 58). During
these periods, the chicks must fast until their parents return to
feed them (3, 9). King penguin (Aptenodytes patagonicus)
chicks fast for as long as 5 mo during the winter when the
parents leave them inland on land to forage in the ocean (16).
Small birds inhabiting temperate habitats often face severe
environmental conditions during winter, such as cold temperatures, snowfalls and storms, long nighttime roosting, reduced
foraging time, and unpredictable food resources that force
them to fast (7, 20, 43, 63). The adaptations of these birds to
fasting are crucial elements in their survival and involve a
variety of behavioral, physiological, and biochemical responses (34, 66).
The sequential physiological responses to fasting have been
categorized into discrete phases, delimited by the types of
metabolic fuel being oxidized, levels of blood metabolites, or
changes in rates of mass loss (2, 14, 16, 18, 39, 40, 46). After
the absorption of the last meal, Phase I begins wherein glycogen is apparently the predominant source of energy (2, 24, 66).
In some cases, stored glycogen may be completely exhausted
to maintain blood glucose levels (reviewed in Ref. 46). Phase
II is typically the longest phase in fasting-adapted species. In
this phase, lipid oxidation is ostensibly the major source of
energy and protein is spared from catabolism (13, 16). As a
consequence of increasing lipid catabolism, the levels of free
fatty acids, glycerol, and ␤-hydroxybutyrate in blood plasma
generally increase, and triacylglycerides decrease (14, 32). In
Phase III, the terminal phase of fasting, blood plasma concentrations of nitrogenous waste increase and lipid metabolites
decrease (13, 14, 18). From what is known about the metabolic
changes that take place during fasting in birds and mammals
adapted to long fasts, we hypothesized that bird species that do
not typically fast for long periods in nature also have three
distinct fasting phases; however, these are truncated in time. In
other words, such birds should undergo the same sequential
changes in fuel oxidation and blood metabolites during fasting
as those documented in fasting-adapted species, but each phase
is proportionally shorter. We examined this possibility using
both 13CO2 breath testing and the more the traditional approach
of blood metabolite chemical analyses, in fasting house spar-
0363-6119/12 Copyright © 2012 the American Physiological Society
R551
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Khalilieh A, McCue MD, Pinshow B. Physiological responses to food
deprivation in the house sparrow, a species not adapted to prolonged fasting.
Am J Physiol Regul Integr Comp Physiol 303: R551–R561, 2012. First
published July 11, 2012; doi:10.1152/ajpregu.00076.2012.—Many wild
birds fast during reproduction, molting, migration, or because of
limited food availability. Species that are adapted to fasting sequentially oxidize endogenous fuels in three discrete phases. We hypothesized that species not adapted to long fasts have truncated, but
otherwise similar, phases of fasting, sequential changes in fuel oxidization, and similar changes in blood metabolites to fasting-adapted
species. We tested salient predictions in house sparrows (Passer
domesticus biblicus), a subspecies that is unable to tolerate more than
⬃32 h of fasting. Our main hypothesis was that fasting sparrows
sequentially oxidize substrates in the order carbohydrates, lipids, and
protein. We dosed 24 house sparrows with [13C]glucose, palmitic
acid, or glycine and measured 13CO2 in their breath while they fasted
for 24 h. To ascertain whether blood metabolite levels reflect fastinginduced changes in metabolic fuels, we also measured glucose,
triacylglycerides, and ␤-hydroxybutyrate in the birds’ blood. The
results of both breath 13CO2 and plasma metabolite analyses did not
support our hypothesis; i.e., that sparrows have the same metabolic
responses characteristic of fasting-adapted species, but on a shorter
time scale. Contrary to our main prediction, we found that recently
assimilated 13C-tracers were oxidized continuously in different patterns with no definite peaks corresponding to the three phases of
fasting and also that changes in plasma metabolite levels accurately
tracked the changes found by breath analysis. Notably, the rate of
recently assimilated [13C]glycine oxidization was significantly higher
(P ⬍ 0.001) than that of the other metabolic tracers at all postdosing
intervals. We conclude that the inability of house sparrows to fast for
longer than 32 h is likely related to their inability to accrue large lipid
stores, separately oxidize different fuels, and/or spare protein during
fasting.
R552
FOOD DEPRIVATION IN THE HOUSE SPARROW
‘Glycogen’
13C-Glucose
13C-Palmitic
‘Lipids’
acid
Phase III
‘Protein’
Phase II
13C-Glycine
Phase I
Death
Relative rate of 13CO2
Production
A
Glucose
Death
Plasma metabolite
concentration
B
Fig. 1. A: sequential changes of oxidization rate of glucose (_ _ _) , palmitic acid
(_ • _) , and glycine (_ • • _), as they are expected to change in time in fasting
house sparrows during 24 h of fasting; B: blood concentrations of glucose (_ • _),
␤-hydroxybutyrate (_ _ _), and triglycerides (_ • • _) as they are expected to change
in time in fasting house sparrows during 24 h of fasting.
Phases I and II; however, it decreases in Phase III; 2) the
concentration of ␤-hydroxybutyrate increases throughout
Phase II but decreases during Phase III, while 3) the concentration of triacylglycerides decreases slowly and steadily during the three phases of fasting (Fig. 1B). Since changes in
blood metabolites are traditionally used as an indirect measure
for changes in metabolic fuels (30 –33, 35), we also predicted
that blood plasma metabolite concentrations correspond to the
sequential changes in recently assimilated substrates, as determined by breath testing.
MATERIALS AND METHODS
Permits. This research was done under permit no. 9017/2008 of the
Israel Nature and National Parks Protection Authority and under
permit BGU-R-06-2009 (to B. Pinshow) of the BGU Animal Care and
Ethics Committee.
Study organism. Sixty adult house sparrows were captured using
mist nets at Midreshet Ben-Gurion (30°52=N, 34°47=E), Israel, in July
2010. Each bird was banded with a uniquely numbered plastic leg
band (Red Bird, Mt. Aukum, CA). The birds were randomly divided
into groups of 10 and maintained in six outdoor aviaries (2.5 m ⫻ 1.5
m ⫻ 1.5 m), where they were provided with a diet of unhusked millet
seeds [⬃12% protein and 5% lipids by dry mass; (65)] and tap water
ad libitum. Once a week, the birds were provided also with crushed
chicken egg shells, vitamin-supplemented water, and fresh lettuce.
Thus the birds were acclimatized to seminatural conditions. One
month before experiments, that began in October 2010 and ended in
February 2011, the birds were gavaged twice with Baytril (Enrofloxacin, 0.1 ml), one week apart, to reduce the risk of infections that might
influence the overall health of birds and the oxidative dynamics of
tracers. All birds used were first fasted for 24 h to identify any weak
or sick individuals, and they were given a minimum of 12 days to
recover before subsequent fasting experiments. Perceptibly weak
individuals were released and replaced. The number of individuals
used differed according to the sample size requirements for each type
of measurement. None of the birds were used more than once in any
13
C tracer experiment; however, in cases where birds used for both
13
C tracer measurements and metabolite measurements, they were
first given a minimum of 19 days to recover. None of the birds were
reproductive or molting during experiments.
13
C isotope measurements and calculations. Breath tests were done
on a total of 24 adult house sparrows (12 males and 12 females, mean
mass (mb) ⫽ 27.4 ⫾ 1.84 g). In each sequence of measurements, four
birds were used that were allowed to feed from dawn (⬃0600 h) until
0800 h. They were then transferred to the laboratory and background
13
CO2 levels were measured. The birds were placed in 850-ml
metabolic chambers made from transparent, hermetically sealable
plastic containers (model HPL808, Lock&Lock, Hana Cobi, Korea)
maintained in a room where the air temperature was 25 ⫾ 0.5°C. We
chose this temperature because it is at the low end of the bird’s
thermal-neutral zone [22.5–35°C; (29), explained in Ref. 4]; the
temperature within the chambers never measurably exceeded 25.5°C.
Dry, CO2-free air was pumped through the chambers at 90 –140
ml/min, a rate high enough to prevent hypercapnic conditions (12, 50),
yet sufficient to ensure detectable levels of CO2 for isotope analysis.
The inlet and outlet ports were on opposite sides of the chamber, at
sparrow head height. After 30 min, a 50-ml sample of excurrent air
was collected from each chamber using a gas-tight, glass syringe
(Perfektum, Popper, New Hyde Park, NY) and was injected in to the
analyzer immediately (see below).
After collection of background 13CO2 samples, at 0900 h (experiment
hour 0), the birds were gavaged with 20 mg of one of three 13C-labeled
tracers, namely D-[1-13C]glucose, 98 –99%; [1-13C]palmitic acid, 99%; or
[1-13C]glycine, 99% (Cambridge Isotope Laboratories, Andover, MA),
suspended in 240 ␮l of sunflower seed oil, using a 1.0-ml syringe
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rows (Passer domesticus biblicus), a subspecies that is generally unable to survive ⬎32 h of fasting (Khalilieh and McCue,
unpublished observations).
Before the onset of fasting, carbon atoms in an animal’s diet
can be stored in its tissues, released in excreta, or oxidized and
exhaled in CO2. Assuming that the physiological fates of
13
C-labeled tracers are essentially the same as nonlabeled
ingested nutrients, by measuring 13C in exhaled breath, we
recently used organic compounds (glucose, fatty acids, and
amino acids) artificially enriched with 13C 1) to track recently
assimilated fuel oxidization in well-fed adult birds during the
postprandial phase (50) and 2) to examine how postprandial
fuel oxidization changed with the developmental and nutritional status of birds (49). The theoretical framework for using
enriched tracers to quantify directly nutrient oxidation during
prolonged fasting was recently summarized by McCue (47),
who also found that the three tracers we used were routed
preferentially into tissue within 2 h of administration in house
sparrows (51). The present study is the first to employ this
technique experimentally.
With the above assumption in mind, we tested three predictions
regarding fuel oxidation in fasting house sparrows following
meals labeled with artificially enriched 13C molecules. First,
fasted birds, dosed with [13C]glucose have exhaled breath 13CO2
enrichments during Phase I of fasting (Fig. 1A). Second, fasted
birds dosed with artificially enriched [13C]palmitic acid have
exhaled breath 13CO2 enrichments during fasting Phase II
reflecting fatty acid oxidation (Fig. 1A). Third, fasted birds
dosed with artificially enriched [13C]glycine, a glucogenic and
nonessential amino acid, have exhaled breath 13CO2 enrichments during fasting Phase III reflecting amino acid oxidation
(Fig. 1A). With respect to blood metabolites, we predicted that
1) fasting birds maintain constant blood glucose levels during
R553
FOOD DEPRIVATION IN THE HOUSE SPARROW
␦13C‰ PDB ⫽
冋共
13
C ⁄ 12C兲sample ⫺ 共 13C ⁄ 12C兲std
共 13C ⁄ 12C兲std
册
⫻ 103
(1)
where (13C/12C )sample is the ratio of 13C to 12C atoms in the sample
and (13C/12C )std is the ratio of the 13C to 12C atoms in the standard.
We used the international standard for (13C/12C)std, namely that of Pee
Dee Belemnite with a value of 0.01112329, in the calculations.
The instantaneous rates of the substrate oxidation (T; nmol/min)
was calculated using the following equation that combine equations 4
and 5 from (47):
T⫽
冋
·
APE ⫻ VCO2
k ⫻ m ⫻ ␪ ⫻ BRF
册
⫻ 104
(2)
where APE is the atom percent excess of 13CO2 above background
levels, V̇CO2 is the carbon dioxide production (ml CO2/min), k is the
volume of CO2 (ml) produced per milligram of tracer oxidized, m is
the molar mass of each tracer, ␪ is the number of isotopically enriched
atoms per tracer molecule, and BRF is the bicarbonate retention factor
[0.86; (62)]. The cumulative oxidation, f(T)dT (in ␮mol), was calculated using equation 5:
冋兰
1,440
f 共T兲dT ⫽
0
册
f 共T兲dT ⫻ 10⫺3
(3)
Blood metabolites. Fifty-six house sparrows (26 males and 30
females) were weighed to ⫾0.1 g, after being fed from dawn to
⬃0800 h. Fourteen birds were used in each of four sequences of blood
metabolite measurements. In each sequence, the birds were placed
individually in the 850-ml metabolic chambers, exposed to room air at
25 ⫾ 0.5°C, and allowed 15 min to habituate. Then blood samples
(⬇50 ␮l) were collected into heparinized capillary tubes from two
birds, by puncturing the brachial vein with a sterile, 26-g needle; the
process taking about a minute. The blood samples were analyzed for
plasma levels of triacylglycerides, glucose, and ␤-hydroxybutyrate.
The 12 remaining birds were gavaged with 20 mg of unlabeled
glucose suspended in 240 ␮l of sunflower seed oil using the method
describe above. After dosing, beginning at 0900 h, blood samples
were collected from pairs of birds at intervals during the 24-h fast: at
30 min, 1 h, 2 h, 3.5 h, 5.5 h, and 8 h. To help maintain plasma
volume, the birds were gavaged with 2 ml water 5 h after the first
blood sampling. Blood was sampled again from each pair of birds in
the same order at 10.5, 13.5, 15.5, 20, 22, and 24 h and was not
sampled more than twice from any individual. Plasma concentration
of triacylglycerides, glucose, and ␤-hydroxybutyrate were measured
using a blood chemistry analyzer (model ST, CardioCheck, Maria
Stein, OH) periodically calibrated with standard samples on strips
supplied by the manufacturer. The analyzer’s lower detection limit of
triacylglyceride concentration is 50 mg/dl.
Carbon dioxide production. Since V̇CO2 is a key input variable to
equation 2, we measured it in 21 sparrows (11 males and 10 females)
sparrows randomly chosen from our captive birds at 25 ⫾ 0.5°C, the
same ambient temperature at which all our measurements were made.
Individuals were weighed to ⫾ 0.1 g, after being fed for about 2.5 h
between dawn and 0830 h. V̇CO2 was measured starting at 0900 h (⫾
10 min) and continued for 24 h while fasting. V̇CO2 data were
collected from seven birds at a time and background, using an
open-flow, 8-channel, multiplexed, respirometry system (Qubit Systems, Kingston, Ontario, Canada). The measurement sequence was 5
min per bird and 5 min background air between birds, thus V̇CO2 was
measured for 6 birds each hour. To calculate V̇CO2 (as ml gas/min) we
used equation 4, appropriate for this system (42):
·
·
VCO2 ⫽ VI ⫻ 共FICO2 ⫺ FECO2兲
(4)
where V̇I is the flow rate of CO2-free dry air flowing into the metabolic
chamber (in ml/min), and FICO2 and FECO2 are, respectively, the
fractional concentrations of CO2 in incurrent and excurrent (dried) air
of the metabolic chambers. Flow rates were verified with a bubble
flow calibrator (Sensydyne, Gilian Gilibrator 2, Clearwater, FL), and
FCO2 was calibrated against dry nitrogen and O2, CO2, and N2
mixtures supplied by a three-way gas mixing pump (model M301/2-F,
H. Wösthoff, Bochum, Germany).
Data organization and statistical analysis. After careful visual inspection of the data, we divided the measurements for each tracer into 5
phases: 1) the postprandial phase (experiment hours 0 to 3, i.e., 0900 –
1200 h); 2) the postabsorptive phase, until the end of the first photophase
(hours 4 to 7); 3) fasting: the transition between the first photophase and
the scotophase (hours 8 to 11); 4) fasting: scotophase (hours 12 to 20);
and 5) fasting: the transition between the scotophase into the second
photophase (hours 21–24). Breath samples were taken from eight birds,
four times in stages 1, 2 and 3, and 5, and nine in stage 4.
Within each stage, we tested for normality of distribution using the
Shapiro-Wilk test and for homogeneity of variance using Bartlett’s
test. Wherever necessary, we transformed the data to comply with the
assumptions of the parametric tests used. To test for differences in
oxidation rates of tracers (13C breath test) we used repeated measures
(RM)-ANOVA. To test for differences in blood metabolite concentrations we used one-way ANOVA. Where ANOVA and RM-ANOVA
showed significant differences, we used the Tukey HSD post hoc test to
ascertain which means at which times were different. Data are
reported as means ⫾ SD, and null hypotheses were rejected at P ⬍
0.05. All statistical tests were done using Statistica 10 software
(StatSoft, Tulsa, OK), and all graphs were plotted using SigmaPlot
11.0 (Systat Software, San Jose, CA).
RESULTS
All the results that follow, based on equation 2 required V̇CO2
as an input variable. The average values for V̇CO2 during 24 h of
fasting, used in our calculations are shown in Fig. 2 that also
clearly illustrates the difference between awake (photophase)
and resting (scotophase) birds.
13
CO2 breath tests. The rates at which the different tracers
were oxidized was affected by fasting time. The instantaneous
rate of glucose tracer oxidization reached a peak of 132.3 ⫾
35.7 nmol/min during the postprandial phase, 30 min after
tracer administration (Fig. 3A). It then declined over the next
6.5 h that included the last 2.5 h of the postprandial phase and
4 h more, until hour 7 of the photophase (RM-ANOVA from
hour 0.5 until hour 3 of fasting, F3,21 ⫽ 147.90, P ⬍ 0.0001,
Tukey HSD, P ⬍ 0.0001; RM-ANOVA from hour 4 until hour
7 of fasting, F3,21 ⫽ 44.996, P ⬍ 0.0001, Tukey HSD, P ⬍
0.0001) (Fig. 3, A and B). However, glucose tracer oxidization
briefly stabilized between hours 8 and 9 (from 4.39 ⫾ 1.10 to
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attached to a 15-g silicon-tipped polyethylene feeding tube (FTP-15-78;
Instech Solomon, Plymouth Meeting, PA). Immediately after administration, the birds were returned to the metabolic chambers, and excurrent
gas samples were collected 30 min later and then at hourly intervals over
the next 24 h. Since birds were gavaged about 2 min apart, the timing for
each bird was standardized to the same 0 hour for the purposes of
calculations and analyses. This sequence was repeated so that the sample
size for each tracer was eight.
The 13CO2 content of collected breath samples (13CO2) was determined using an infrared 13CO2 analyzer (HeliFANplus, Fischer Analysen Instrumente, Leipzig, Germany). For quality control, some
randomly chosen breath samples were collected into 9-ml glass
Vacutainers (Greiner Bio-One, Kremsmunster, Austria) and analyzed
by conventional gas source isotopic ratio mass spectrometry (GSIRMS) through a Gas Bench II interface (Thermo-Fisher Scientific,
Waltham, MA). Delta (␦) 13CO2 of the samples were calculated using
Craig’s equation (19):
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FOOD DEPRIVATION IN THE HOUSE SPARROW
Postprandial Phase
3.2
Scotophase
Photophase
3.0
Photophase
2.8
2.6
VCO2 ml min -1
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
2
4
6
8
10
12
14
16
18
20
22
24
Time from begining of experiment (h)
Fig. 2. The rate of carbon dioxide (V̇CO2) production of 21 adult house sparrows
during 24 h of fasting. Measurements from hour 0 to hour 8 (0900 h-1700 h) are
during the photophase, when birds were awake. Between hours 9 and 21 is the
scotophase and birds were likely sleeping. From hour 22 to 24 is the photophase
of the second day and the birds were again awake. Values are means ⫾ SD for all
18 measurements for each hour, except for the first 30 min of the experiment, when
a total of 9 measurements were made. See text for details.
4.30 ⫾ 1.26 nmol/min, RM-ANOVA from hour 8 until hour
11 of fasting, F3,21 ⫽ 123.08, P ⬍ 0.0001, Tukey HSD, P ⫽
0.988) as the birds entered the transition between photophase
and scotophase (hours 8 –11 of fasting). During this phase, the
rate of glucose tracer oxidization decreased significantly between hours 9 and 11 (RM-ANOVA from hour 8 until hour 11
of fasting, F3,21 ⫽ 147.90, P ⬍ 0.0001, Tukey HSD, P ⬍
0.0002). In the scotophase, the rate of oxidization decreased
significantly between hours 12 and 17 (RM-ANOVA from
hour 12 until hour 20 of fasting, F8,56 ⫽ 12.337, P ⬍ 0.0001,
Tukey HSD, P ⬍ 0.0040) and stabilized thereafter until hour
20 of fasting (Tukey HSD, P ⫽ 0.9845). A significant increase
in rate of glucose tracer oxidization occurred during the photophase of the next day between hours 21 and 22 (RMANOVA from hour 21 until hour 24 of fasting, F3,21 ⫽ 8.589,
P ⬍ 0.0001, Tukey HSD, P ⬍ 0.0190) and remained relatively
constant for the remainder of the experiment (Tukey HSD, P ⬍
0.5036; Fig. 3B). Over the entire 24-hour experiment sparrows
oxidized only 10.8 ⫾ 1.3% of the tracer dose (Fig. 3C).
The mean rate of palmitic acid tracer oxidization peaked 1 h
after administration, 30 min longer into the experiment than the
appearance of the glucose peak, but still during the postprandial phase (Fig. 4A). At this point the mean instantaneous
oxidization rate was 5.8 ⫾ 1.2 nmol/min. The mean rate of
tracer oxidization decreased significantly between hours 1 and
3 (RM-ANOVA from hour 1 until hour 3 of fasting, F3,21 ⫽
21.385, P ⬍ 0.0001, Tukey HSD, P ⬍ 0.006), also still during
the postprandial phase. A second significant decrease in rate of
oxidation occurred between hours 4 and 7 during the photophase (RM-ANOVA from hour 4 until hour 7 of fasting,
F3,21 ⫽ 219.6, P ⬍ 0.0001, Tukey HSD, P ⬍ 0.0002), but it
stabilized as the birds entered the transition phase between
photophase and scotophase (hours 8 –11) at approximately
hour 8 (0.66 ⫾ 0.18 nmol/min) and remained relatively constant for the next 2 h, unlike the glucose tracer (Fig. 4, A and
B). However, during this phase, a significant decrease in the
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0
rate of palmitic acid tracer oxidization occurred between hours
10 and 11 (RM-ANOVA from hour 8 until hour 11 of fasting,
F3,21 ⫽ 36.04, P ⬍ 0.0001, Tukey HSD, P ⬍ 0.0002). During
the scotophase (hours 12–20) the rate of palmitic acid tracer
oxidization decreased between hours 12 and 14 (RM-ANOVA
from hour 12 until hour 20 of fasting, F8,56 ⫽ 8.083, P ⬍
0.0001, Tukey HSD, P ⬍ 0.0367), after which it stabilized for
the remainder of the scotophase (Tukey HSD, P ⫽ 0.9065) and
through the subsequent morning (RM-ANOVA from hour 21
until hour 24, F3,21 ⫽ 0.3685, P ⫽ 0.7765), again unlike the
glucose tracer. During the experiment, sparrows oxidized the
palmitic acid tracer less extensively than glucose, i.e., only
1.53 ⫾ 0.43% of the tracer was recovered in the bird’s breath
(Fig. 4C).
Glycine tracer oxidization peaked at 2705.3 ⫾ 687.4 nmol/
min during the postprandial phase, 30 min after its administration (Fig. 5A). A rapid decrease in oxidization rate occurred
during the first 3 h (RM-ANOVA from hour 0.5 until hour 3 of
fasting, F3,21 ⫽ 63.56, P ⬍ 0.0001, Tukey HSD, P ⬍ 0.0001),
yet this decrease significantly declined over the next 4 h of
fasting (RM-ANOVA from hour 4 until hour 7 of fasting,
F3,21 ⫽ 378.7, P ⬍ 0.0001, Tukey HSD, P ⬍ 0.0001) (Fig. 5,
A and B). Nevertheless, the rate of glycine tracer oxidization
stabilized as the birds entered the transition phase between
photophase and scotophase at approximately hour 8 (30.5 ⫾
3.1 nmol/min) and remained relatively constant for the next
hour (RM-ANOVA from hour 8 until hour 11 of fasting,
F3,21 ⫽ 58.23, P ⬍ 0.0001, Tukey HSD, P ⫽ 0.8950).
However, the rate of oxidization decreased significantly between hours 9 and 11 (Tukey HSD, P ⬍ 0.0002) during the
same phase. During the scotophase, the rate of glycine tracer
oxidization stabilized between hours 12 and 15 (RM-ANOVA
from hour 12 until hour 20 of fasting, F8,56 ⫽ 5.078, P ⬍
0.0001, Tukey HSD, P ⫽ 0.5242), yet decreased thereafter
until hour 19 (Tukey HSD, P ⬍ 0.002) and stabilized for the
next hour (Tukey HSD, P ⫽ 0.9214). During the photophase of
the next morning, the rate of glycine tracer oxidization increased significantly until the end of the experiment (RMANOVA from hour 21 until hour 24 of fasting, F3,21 ⫽ 5.078,
P ⬍ 0.0003, Tukey HSD, P ⬍ 0.002). Over the 24-h fast, the
sparrows oxidized the glycine tracer more extensively than
either glucose or palmitic acid tracers, and 61.8 ⫾ 7.6% of the
tracer was recovered in their breath (Fig. 5C).
Blood metabolites. Fasting time significantly affected plasma
concentrations of glucose, ␤-hydroxybutyrate, and triacylglycerides during the 24-h experiment (Fig. 6, A–C). Plasma
glucose concentration was relatively stable (Fig. 6A) during the
first 5.5 h; however, it decreased significantly between hours 7
and 8 (ANOVA, from hour 0 until the end of the experiment,
F12,91 ⫽ 7.9, P ⬍ 0.0001, Tukey HSD, P ⬍ 0.003). After this
point, the concentration of plasma glucose seemingly increased
until hour 13.5, although the trend was not significant (Tukey
HSD, P ⫽ 0.520). A second gradual decrease in plasma
glucose concentration occurred between hours 13.5 and 24,
where minimum mean concentration reached 229.8 ⫾ 43.4
mg/dl at hour 24 (Tukey HSD, P ⬍ 0.006).
The concentration of plasma ␤-hydroxybutyrate increased
continuously from the background level of 11.55 ⫾ 1.80 mg/dl
(n ⫽ 8) at 0 h, to 17.4 ⫾ 2.92 30 min after glucose administration, to 23.7 ⫾ 3.29 after 1 h, reaching 64.30 ⫾ 5.30 mg/dl
at hour 8 (Fig. 6B). Although the increase is unambiguously
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FOOD DEPRIVATION IN THE HOUSE SPARROW
clear due to the use of ANOVA on all the data at once,
statistical significance is apparent only at hour 3.5, just as the
birds became postabsorptive (ANOVA, from hour 0 until the
end of the experiment, F12,91 ⫽ 47.7, P ⬍ 0.0001, Tukey HSD,
P ⬍ 0.0005). Thereafter, the concentration of plasma ketone
bodies stabilized, with slight variations, from hour 8 until the
end of the 24 h of fasting (Tukey HSD, P ⬍ 0.4206).
Plasma triacylglyceride concentration declined rapidly from
30 min after glucose administration until hour 5.5 of fasting
(Fig. 6C); however, the decline only became statistically significant at hour 2 during the postprandial phase (ANOVA,
from hour 0 until the end of the experiment, F12,91 ⫽ 16.227,
P ⬍ 0.0001, Tukey HSD, P ⫽ 0.0102). Nevertheless, the
concentration of triacylglycerides remained relatively constant
after that, with slight variations, up to hour 20 of the experiment (Tukey HSD, P ⫽ 0.9997). By hour 22, some of the
triacylglyceride values fell below the range of detection (50
mg/dl), precluding statistical comparisons.
DISCUSSION
Since there are at least 12 subspecies of the house sparrow
(4), and some of them are resident in cold climates, we hesitate
to generalize about the responses we see in Passer domesticus
biblicus, although Blem (8) reports mean body fat between 5
and 12% in P. d. domesticus, increasing from low to high
latitudes in North America. With our initial assumptions and
the results of McCue et al. (51) in mind, the results of both
breath 13CO2 and plasma metabolite analysis in the present
study did not support our hypothesis that P. d. biblicus have the
same metabolic responses characteristic of fasting-adapted
species of similar body mass, but on a shorter time scale.
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Fig. 3. Instantaneous rates of oxidation of
glucose tracer by house sparrows. A: during
24 h of fasting; B: data are identical to A, but
the postprandial phase was excluded for rescaling; C: cumulative glucose tracer oxidation for the 24 h of fasting. Each point
represents the mean for 8 birds ⫾ SD. The
experimental period is divided into five phases:
postprandial, photophase, photophase-scotophase transition, scotophase, and scotophasephotophase transition (see MATERIALS AND
METHODS for justification). Different lower
case letters within each phase indicate significant differences according to post hoc Tukey
HSD comparisons. Values are means ⫾ SD.
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FOOD DEPRIVATION IN THE HOUSE SPARROW
Recently assimilated tracers were oxidized continuously in
different patterns and did not exhibit the sequential changes
corresponding to the three phases of fasting that we predicted.
In addition, changes in plasma metabolite levels did not follow
the typical pattern of fasting-adapted species.
The rates of tracer oxidization during the postprandial phase
(hours 0 –3) were highest during the 24 h of fasting. The
magnitude of oxidization rates differed greatly among the three
tracers, but the timing of peak glucose and glycine oxidization
were similar (⬃30 min after administration), occurring before
the peak oxidization of palmitic acid (⬃1 h after administration). Similar results for the oxidization of exogenous fuels in
house sparrows were found in an additional study, where
overall fuel catabolism during the postprandial phase in house
sparrows was examined (50).
After the postprandial phase, the glucose tracer was oxidized
with no clearly defined peaks during the first 6 h of the birds’
becoming postabsorptive (Fig. 3). The rate of glucose tracer
oxidization stabilized between hours 8 and 9 during the transition between photophase and scotophase, but because a
similar plateau in rate of oxidation of the other tracers, this is
likely related to the animal’s diel metabolic rate adjustment for
scotophase. Furthermore, contrary to our prediction, glucose
tracer oxidization decreased in rate and remained constant
between hours 9 and 21. In other words, we found no peak in
13
CO2 production in [13C]glucose-dosed birds during the period that would traditionally be considered Phase II of fasting
based on blood metabolites, suggesting the possibilities that
1) only a small amount of glucose was converted to lipids, or
2) carbohydrates were being oxidized along with fatty acids.
Furthermore, compound-specific analyses would be necessary
to quantify the amount of 13C from the glucose tracer was
incorporated into glycogen or recently synthesized lipid pools
(10, 45, 55).
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Fig. 4. Instantaneous rates of oxidation of
palmitic acid tracer by house sparrows.
A: during 24 h of fasting; B: data are identical to A, but the postprandial phase was
excluded for rescaling; C: cumulative palmitic
acid tracer oxidation for the 24 h of fasting.
Each point represents the mean for 8 birds ⫾
SD. The experimental period is divided into
five phases: postprandial, photophase, photophase-scotophase transition, scotophase,
and scotophase-photophase transition (see MATERIALS AND METHODS for justification). Different lower case letters within each phase
indicate significant differences according to
post hoc Tukey HSD comparisons. Values are
means ⫾ SD.
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FOOD DEPRIVATION IN THE HOUSE SPARROW
Unexpectedly, an increase in the rate of 13CO2 production,
following glucose tracer dosing, occurred between hours 21
and 24. This response was not observed for the fatty acid
tracer, suggesting that glucose-derived carbon stores were not
completely depleted during the first hours of fasting and were
conserved as a source of energy for the scotophase of the next
day. Glycogen sparing has been documented in ectothermic
animals (22, 28, 54), but we are not aware of any studies of
birds that indicate that glucose oxidation increases toward the
end of a bout of prolonged starvation. Nevertheless, transient
increases in glucose oxidation have been reported in flying
mammals arousing from torpor (11).
The palmitic acid tracer was continually oxidized after the
postprandial phase until hour 8 (Fig. 4). This result does not
support our prediction that lipid stores, in particular fatty acids,
provide the bulk of the energy needs during what would be
considered Phase II of fasting. Nevertheless, the rate of oxidization stabilized between hours 8 and 10 during the transition
from photo- to scotophase and into the first hour of the
scotophase, unlike the level of glucose tracer oxidation that
decreased in this period. The low cumulative oxidation of the
palmitic acid tracer may reflect extensive integration of the
tracer into a pool of lipids, e.g., structural membrane phospholipids, which is not readily available as an energy source (1, 48,
64). However, since tissue growth is minimal during starvation, this is an improbable explanation for our observations,
although tissue turnover might account for some of it. By
contrast, unlike the glucose tracer, the oxidation rates of the
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Fig. 5. Instantaneous rates of oxidation of
glycine tracer by house sparrows. A: during
24 h of fasting; B: data are identical to A, but
the postprandial phase was excluded for rescaling; C: cumulative glycine tracer oxidation for the 24 h of fasting. Each point
represents the mean for 8 birds ⫾ SD. The
experimental period is divided into five phases: postprandial, photophase, photophase-scotophase.......... transition, scotophase, and scotophase-photophase transition (see MATERIALS
AND METHODS for justification). Different lower
case letters within each phase indicate significant
differences according to post hoc Tukey HSD
comparisons. Values are means ⫾ SD.
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FOOD DEPRIVATION IN THE HOUSE SPARROW
palmitic acid tracer closely tracked the plasma triacylglyceride
values.
During fasting, the glycine tracer was continually oxidized
at rates far exceeding the other two tracers (Fig. 5). Moreover,
the data do not to support the prediction that the oxidation of
amino acids increases sharply toward the end of the fasting
period. Nevertheless, unlike the oxidation of the glucose and
palmitic acid tracers, the rate of glycine tracer oxidization did
not plateau during the transition from photophase to scotophase. The oxidation rate of glycine tracer was highest during
early scotophase, suggesting that house sparrows are limited in
their capacity to spare protein during fasting.
Our results indicate that fasting house sparrows do not
sequentially oxidize substrates in the order carbohydrates, then
lipids, and then protein as typically occur in fasting-adapted
species (14, 18, 40). Rather, these birds apparently oxidized
simultaneously a combination of recently assimilated sub-
strates, which we assume reflect the three endogenous fuel
types (51). Furthermore, the fact that the amino acid tracers
were oxidized more extensively than any of the other metabolic
tracers indicates that house sparrows do not spare body protein
during fasting which, in part, explains the inability of even
well-nourished house sparrows to tolerate more than 32 h of
fasting at rest. The phenomenon does not seem to be body
mass dependent, because many migratory species of similar
body mass to house sparrows accumulate large amounts of
lipid and can fast for far longer than can house sparrows (6,
30, 36 –38, 60).
In addition, our results suggest that house sparrows do not
increase their stores of lipids much to survive an overnight fast
but depend more on protein reserves. This conclusion is supported by the fact that sparrows are not particularly effective at
accumulating lipid stores as seen among fasting-adapted species (4, 8). Our data also indicate that our measures of the
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Fig. 6. Plasma concentrations of glucose (A),
␤-hydroxybutyrate (B), and triacylglycerides
(C) during 24 h of fasting in house sparrows.
Each point represents the mean for 8 birds ⫾
SD. Different lower case letters indicate significant differences according to post hoc Tukey
HSD comparisons. Values are means ⫾ SD.
FOOD DEPRIVATION IN THE HOUSE SPARROW
Perspectives and Significance
None of the results (13CO2 breath-testing or plasma metabolite analyses) supported our prediction that the subspecies of
house sparrow that we examined has a truncated, but otherwise
similar, blood metabolite pattern to that found in fastingadapted species. Rather, these results show that these birds,
unlike fasting-adapted species, were simultaneously oxidizing
recently assimilated carbohydrates, lipids, and amino acids
together, albeit at different rates, to meet their energy demands
during the first 8 h of fasting. To the best of our knowledge,
these are the first direct measurements of oxidation of multiple
metabolic fuels during fasting. In addition, we found that the
oxidation of the glucose tracer did not follow the same pattern
as plasma glucose concentration, rather it was inversely proportional to it, yet the rate of palmitic acid tracer oxidation was
reflective of triacylglyceride concentration during fasting. Furthermore, the results of the present study reveal that direct
measurements of substrate oxidation can complement more
traditional measurements of blood metabolites that, alone, offer
limited insight into the physiological transitions among different metabolic fuels.
ACKNOWLEDGMENTS
We especially thank Christian Voigt for lending us the 13CO2 analyzer and
Miri Ben-Hamo and Cynthia Downs for help with statistical analysis. We are
grateful for constructive comments and insights provided by three anonymous
reviewers, and by Dr. Leonard Z. Gannes for his helpful suggestions concerning the revised version. This is paper number 777 of the Mitrani Department
of Desert Ecology.
-1
hydroxybutyrate concnetration (mg dl )
until the end of the 24-h fast. This suggests that, in sparrows,
the capacity for lipid storage is meager and alone is not
sufficient to meet energy requirements during even a relatively
short fast. The breath-test results also failed to support the
prediction that house sparrows oxidize mainly lipids during
what would be expected to be Phase II of fasting. Moreover,
the results of the metabolite analysis suggest that the continued
elevation in blood ␤-hydroxybutyrate concentration is more
likely to result from protein catabolism than lipid oxidation
since the rate of glycine tracer oxidation was much higher than
that of glucose or palmitic acid during the period when ketone
levels were highest.
70
60
GRANTS
50
This work was supported was supported by US-Israel Binational Science
Foundation Grant 2005119 to B. Pinshow and Scott R. McWilliams and by
Sigma Xi Grant-in-Aid of Research number G200810150517 to A. Khalileh.
During the study M. D. McCue was supported by a Blaustein Post-Doctoral
Fellowship and a Fellowship from the Israel Committee for Higher Education.
40
30
20
y = 150.748 - 0.376x
DISCLOSURES
2
Plasma
P < 0.001; R = 0.889
No conflicts of interest, financial or otherwise, are declared by the authors.
10
200
220
240
260
280
300
320
340
360
380
Plasma glucose concentration (mg dl-1)
Fig. 7. Blood ␤-hydroxybutyrate concentration plotted against glucose concentration during 24 h of fasting of 56 house sparrows. Each point represents
the mean for 8 birds ⫾ SD.
AUTHOR CONTRIBUTIONS
Author contributions: A.K. and M.D.M. conception and design of research;
A.K. performed experiments; A.K. and M.D.M. analyzed data; A.K., M.D.M.,
and B.P. interpreted results of experiments; A.K. and B.P. prepared figures;
A.K. drafted manuscript; M.D.M. and B.P. edited and revised manuscript;
M.D.M. and B.P. approved final version of manuscript.
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changes in blood metabolites and in tracer oxidization, during
the relatively short period of fasting that house sparrows can
tolerate, were confounded by regular circadian adjustments in
metabolic rate, metabolites, and physiological fuel choice.
Consequently, it was difficult to identify clear fasting-phase
transitions in these animals.
In the present study of a species that is ostensibly not
adapted to prolonged fasting, blood glucose concentration
decreased significantly after the postprandial phase up to hour
8 of the experiment. Although blood glucose concentration
increased slightly after this point until hour 13.5, it decreased
significantly thereafter until the end of the 24-h fast. These
results also failed to support our prediction that glucose oxidation is similar to that found in fasting-adapted species but on
a shorter time scale.
Surprisingly, ␤-hydroxybutyrate increased from 30 min
after food removal (while the birds were still postprandial!),
until hour 8, failing to support our prediction that the
concentration of ␤-hydroxybutyrate does not increase at the
onset of fasting. Given the large differences in the extent to
which the glycine and the palmitic acid tracers were oxidized, at this time we attribute the source of the ketone
bodies to the oxidation of ketogenic amino acids. The level
then plateaued with slight fluctuations toward the end of the
fasting period. The increase was significant only after 3.5 h.
The increase in ␤-hydroxybutyrate concentration was inversely correlated with the decrease in glucose concentration (P ⬍ 0.001; Fig. 7). In humans and other mammals the
increase in plasma ␤-hydroxybutyrate initiates a drop in
plasma glucose concentration as the rate of glucose production decreases (52, 59), which might explain our results. The
high correlation between glucose and ␤-hydroxybutyrate
concentration suggests that ␤-hydroxybutyrate plays a key
role as an energy substitute for glucose, as has been documented in mammals (23, 44, 61).
The concentration of plasma triacylglycerides decreased
significantly only after hour 2 of the experiment, during the
postprandial phase, and remained low with slight variations,
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FOOD DEPRIVATION IN THE HOUSE SPARROW
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