Glycerol and NEFA kinetics in long-term fasting king penguins: phase II versus phase III
Centre d'Ecologie et Physiologie Energétiques, Centre National de la Recherche Scientifique, 67087 Strasbourg, France
* Author for correspondence (e-mail: rene.groscolas{at}c-strasbourg.fr)
Accepted 20 May 2002
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Summary |
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Key words: lipolysis, triacylglycerol:fatty acid cycling, fat stores, refeeding signal, bird, king penguin, Aptenodytes patagonicus
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Introduction |
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A large part of our knowledge on how animals adjust to prolonged fasting,
especially on the relationships between energy reserves, metabolic status and
behaviour, arises from studies on birds that spontaneously fast at certain
stages of their annual cycle. Among them are penguins (Spheniciforms), sea
birds living in the antarctic and subantarctic regions. Penguins feed
exclusively at sea and must fast on land for periods of up to four months
during breeding, especially during incubation
(Groscolas, 1990). Both mates
alternate at incubating (except in the emperor penguin, Aptenodytes
forsteri) and normally it is relief by the partner that terminates the
bird's fasting bouts and allows departure to sea for refeeding
(Groscolas, 1990
). At this
stage, most penguins are still in the phase II metabolic and endocrine status,
with only a small proportion of the birds at the onset of phase III
(Groscolas and Robin, 2001
).
However, the relieving partner can be delayed, forcing the incubating bird to
prolong its fast until eventually it abandons its egg and goes to sea for
feeding. This abandonment has also been observed in other sea birds such as
petrels (Chaurand and Weimerskirch,
1994
; Ancel et al.,
1998
). Recently, Groscolas et al.
(2000
) showed that in the king
penguin Aptenodytes patagonicus egg abandonment is preceded by a
progressive decrease in egg attendance and occurs when the birds had been
fasting into phase III for about one week. In fasting non-incubating emperor
penguins, and as observed in fasting rodents
(Koubi et al., 1991
), entrance
into phase III is associated with an increase in locomotor activity
(Robin et al., 1998
). These
observations have led to the hypothesis that a metabolic shift from
preferential use of body lipids to body proteins as energy sources would
trigger an endogenous refeeding signal
(Robin et al., 1998
;
Groscolas et al., 2000
). A
reduction of the contribution of fatty acid oxidation to energy production
could be a basic component of this signal. This suggestion is supported by the
finding that in rats fed high fat diets, and thus relying heavily on fat as
the main energy fuel (as do fasting penguins), a blockade of fatty acid
oxidation stimulates food intake (Langhans
and Scharrer, 1987
). Since in penguins the metabolic shift occurs
when fat stores are critically but not totally depleted, a decrease in the
rate of fatty acid oxidation could result from a reduction in the rate of
production of these substrates from adipose tissue (lipolysis).
Elucidating the mechanism of the refeeding signal that translates
alteration in energy metabolism into feeding behaviour is of interest in
understanding the long-term control of energy intake and body mass. The major
aim of the present study was therefore to determine whether a decrease in
lipid substrate release from adipose tissue is associated with entrance into
phase III in penguins. Most of our knowledge on fat mobilization and its
regulation in the fasting state derives from studies in short-term fasting
humans and laboratory animals, i.e. essentially during the fed state to phase
II transition (Belo et al.,
1976; Wolfe et al.,
1987
; Klein et al.,
1989
; Kalderon et al.,
2000
). Not only has lipolysis during long-term fasting rarely been
examined (Armstrong et al.,
1961
; Steele et al.,
1968
; Bortz et al.,
1972
), but no data is available for phase III fasted animals.
Since the fasting physiology of the king penguin is representative of that of
other birds (Cherel et al.,
1988a
) and to some extent of mammals
(Cherel and Groscolas, 1999
),
the present study may also provide useful information on whether and how the
lipolytic rate and NEFA availability are affected by this fasting
situation.
The rate of appearance (Ra) of non-esterified fatty acid (NEFA) and glycerol (an index of lipolysis) were measured using tracer methodology during phases II and III of fasting. Phase III birds were at the same stage of fat store depletion as incubating birds at the time of spontaneous egg abandonment. The large size and tameness of the king penguin allowed measurement of lipolytic fluxes in birds under field conditions for the first time.
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Materials and methods |
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Phase II of fasting
Eight animals were habituated to these conditions for 6 days before the
experiment. This time period is known to be sufficient to suppress the
confinement stress and for daily BM loss, body temperature and plasma fuel
level to reach a steady state in penguins
(Groscolas and Rodriguez,
1981). Thus, the fasting duration at Ra
measurement was about 8 days (6 days of captivity plus 1-3 days of fasting
ashore at capture), which is more than the 2-3 day duration of the transition
from the fed state to phase II reported for this species
(Cherel et al., 1988a
). BM at
Ra measurement (12 kg,
Table 1) was similar to that of
free-living male king penguins at the onset of the first incubation shift,
i.e. after about 8-10 days of fasting
(Robin et al., 2001
). It was
also about 2-2.5 kg higher than the 9.6-10 kg BM measured at entrance into
phase III in breeding, fasting male king penguins (Cherel et al.,
1988b
,
1994
).
|
Phase III of fasting
Nine animals were kept fasting and weighed every 3 days, then every day
until they reached a BM close to 9 kg. This mass corresponds to that measured
by Groscolas et al. (2000) in
king penguins abandoning their egg to refeed at sea, and is about 0.5-1 kg
lower than the BM at entrance into phase III. On average, 24±1 days of
fasting in the pen were necessary for the animals to reach the 9 kg BM. Based
on the previously reported rate of daily BM loss
(Cherel et al., 1988b
), it can
be estimated that birds had been in the phase III fasting situation for 5-7
days when Ra measurements were performed.
Catheterization and experimental setup
The day before the experiment, the nonanesthetized bird was cannulated with
a polyethylene catheter (length, 50 mm; external diameter, 1.1 mm) inserted
percutaneously into the marginal vein of each flipper and extended with a 2 m
long prolongator filled with saline. Catheterization of an artery for blood
sampling could not be securely performed in field conditions. We assumed that
any particular metabolism of the flipper (essentially feathers, bones and
tendons) is low and that flipper venous blood reflects whole body metabolism.
Until the experiment, catheters were kept patent by continuous infusion of
0.9% NaCl (12.5 ml day-1) using a small peristaltic pump. After
catheterization, the birds were allowed to habituate to the experimental setup
for 24 h. This setup was installed in the fenced area and consisted of a
wooden pen (70 cmx70 cm) with one wall high enough for us not to be seen
by the bird. Catheter prolongators were placed into a balance lever system to
avoid damaging the prolongators or tearing out catheters. It also allowed the
bird to move freely inside the pen (a few steps) and even to lie on its belly
or sleep with the bill under the shoulder, as was regularly observed during
tracer infusion. The free ends of catheter prolongators used for infusion and
blood sampling were brought outside the pen. From a distance these two
operations could then be performed without disturbing the animal. Once the
animal was in the experimental setup, particular care was taken to avoid any
intervention or noise. During isotope infusion, the air temperature ranged
from 10 to 15 °C, i.e. was within the thermoneutral range for the species
(Groscolas and Robin, 2001),
and the penguins were protected against wind but not against rain. Birds were
de-equipped the day after Ra measurement, marked on the
chest with Nyanzol dye to allow resighting and released in the colony next to
the beach. All the birds used in the study were resighted during the following
weeks, caught and weighed. All had restored their BM, which indicates that
they had been succesfully feeding at sea and that the experiments had no
impact on their health.
Continuous isotope infusions
Infusate preparation
The infusate was prepared daily according to Wolfe
(1992) and Turcotte et al.
(1992
) using
2-[3H]glycerol (Amersham, 40.7 Gbq mmol-1) and
1-[14C]palmitate (Amersham, 2.04 Gbq mmol-1) as tracers.
Palmitate is one of the most commonly used fatty acids for measuring NEFA
kinetics in mammals (Bonadonna et al.,
1990
). Besides its ready commercial availability, palmitate is the
second most abundant NEFA in mammals and its percentage contribution to NEFA
shows low interindividual variability. This was also observed in king penguins
in phase II and phase III (data not shown), suggesting that labeled palmitate
is as appropriate an indicator of NEFA kinetics in king penguins as in
mammals. 1-[14C]palmitate was supplied commercially in toluene. A
subsample in a sterile flask was evaporated to dryness and immediately
resuspended in ethanol to obtain a solution of 37 kBq µl-1. NaOH
(2 mmol l-1 in ethanol) was added to excess and the mixture was
evaporated to dryness. The water-soluble 1-[14C]palmitate sodium
salt was dissolved in heated sterile saline. The solution was cooled to about
30 °C and 3.5% delipidated bovine albumin solution was added so that the
palmitate/albumin molar ratio was 0.5. 2-[3H]glycerol was then
added and the infusate volume was adjusted with saline. Infusion of the
isotope mixture was performed using a calibrated syringe pump at 7 ml
h-1. 2-[3H]glycerol and 1-[14C]palmitate
infusion rates were 224.9±5.1x103 and
109.8±3.6x103 d.p.m. kg-1 min-1
(N=17), respectively, or less than 0.1 nmol glycerol kg-1
min-1 and less than 1.0 nmol palmitate kg-1
min-1. This corresponded to trace amounts of <0.002% of
Ra glycerol and <0.03% of Ra
palmitate.
Infusion protocol and blood sampling
To decrease the time necessary to reach the isotopic steady state, we
injected a priming dose. It was immediately followed by a 180-min continuous
infusion. The first blood sample was not taken until at least 120 min after
the beginning of the infusion, to ensure that the steady state was reached
(see Results). During the last hour of infusion, 3-4 blood samples of 5 ml
each were drawn. Immediately after sampling, the blood was centrifuged and the
plasma separated. Plasma was stored at -20 °C until analysis.
Analytical procedures
A portion of plasma (1 ml) was mixed with 25 ml chloroform:methanol (2:1,
v:v) according to Folch et al.
(1957). After extraction and
evaporation as described in Bernard et al.
(1999
), an aqueous and an
organic extract were obtained and resuspended in ethanol:water (1:1, v:v) and
hexane:isopropanol (3:2, v:v), respectively.
Glycerol
A volume of aqueous extract equivalent to 300 µl of plasma was used to
determine glycerol concentration. It was dried under nitrogen and resuspended
in hydrazine buffer. Glycerol concentration was measured enzymatically using
an Uvikon spectrophotometer at 340 nm. Total tritium activity was counted on
another sample of aqueous extract equivalent to 150 µl of plasma using
Ecoscint A scintillation fluid (National Diagnostics, Hessle Hull, England)
and a Wallac 1409 counter (Wallac, Turku, Finland). At this step of analysis,
tritium activity in the aqueous extract was found to be incorporated not only
into glycerol but also into other compounds, mostly glucose. Glycerol was
purified using thin layer chromatography and the percentage activity
determined according to Bernard et al.
(1999). The percentage of
tritium activity in glycerol was found to be similar in phase II
(28.9±3.7%) and phase III (32.3±4.5%). The specific activity of
glycerol was calculated as total tritium activity times percentage of activity
in glycerol divided by glycerol concentration.
Fatty acids
Total NEFA concentration was measured on 10 µl of plasma with an
analytical test-kit (NEFA C, Wako Chemicals, Osaka, Japan). The palmitate
concentration was obtained by multiplying NEFA concentration by the fractional
contribution of palmitate to total NEFA, determined by gasliquid
chromatography. Briefly, plasma lipids were extracted according to Dole and
Meinertz (1960) and separated
by thin layer chromatography (silica gel plate 60, Merck, Darmstadt, Germany)
using hexane:diethyl ether:acetic acid (70:30:1, v:v:v) as the developing
solvent. The NEFA fraction was isolated and converted to methyl esters using
14% boron trifluoride in methanol. Fatty acid methyl esters were separated and
quantified by gasliquid chromatography using a Chrompack CP 9001 gas
chromatograph equipped with an AT-WAX capillary column [0.25 mm (i.d.) x
30 m, 0.25 µm thickness, Alltech, Templeuve, France] and a flame ionization
detector. Helium was used as the carrier gas and the oven temperature was
maintained at 200 °C. Fatty acid peaks were identified by comparing their
retention times with authentic standards (Nu-Chek Prep, Elysian, MN, USA) and
quantified with an integrator (model SP 4290, Spectra-Physics, Les Ulis,
France).
Because phospholipids are not completely extracted by the method of Dole
and Meinertz (1960), the total
14C activity and its distribution in plasma lipids (TAG,
diacylglycerols, NEFA and phospholipids) was determined on the organic extract
obtained from extraction, according to Folch et al.
(1957
). Lipids were separated
by thin layer chromatography, as described above. Each fraction was scraped
into separate scintillation vials, resuspended in ethanol:water (1:1, v:v) and
counted in Ecoscint A scintillation fluid. Because no 14C is
incorporated into fatty acids other than palmitate, palmitate activity was
calculated by multiplying total 14C activity found in the organic
extract by the percentage activity found in the NEFA fraction. Palmitate
activity divided by palmitate concentration yielded palmitate specific
activity.
Other plasma metabolites and hormones
Plasma glucose and ß-hydroxybutyrate levels were determined on
deproteinized plasma by enzymatic methods (Test-Combination,
Boehringer-Mannheim GmbH, Germany). Uric acid and TAG levels were estimated by
enzymatic colorimetric methods using commercial kits (UA plus for uric acid
and Peridochrom triglycerides GPO-PAP for TAG; Boehringer-Mannheim GmbH,
Germany). Plasma glucagon and insulin levels were determined by
radioimmunoassay. Glucagon was estimated using a commercial kit (Linco, St
Charles, MS, USA); the intra- and inter-assay coefficients of variation were
6% and 7%, respectively. Insulin was estimated using the insulin-CT kit from
CIS bio international (Gif-sur-Yvette, France). The intra- and inter-assay
coefficients of variation were 5% and 6%, respectively. Plasma obtained after
reaching an isotopic steady state was used in all the measurements.
Calculations and statistics
Fat mass (FM) was calculated from BM (both in kg) as:
FM=0.552xBM-4.260 (r2=0.74, N=81,
P<0.0001). This equation was determined in a preliminary study for
king penguins with BM 8.5-14.7 kg (M.-A. Thil and R. Groscolas, unpublished
data).
Glycerol and palmitate Ra values were calculated using
the steady state equation of Steele
(1959):
Ra=tracer infusion rate (d.p.m. min-1)/specific
activity (d.p.m. mmol-1), and are expressed per unit of body mass
or fat mass. Ra NEFA was determined by dividing
Ra palmitate by the fractional contribution of palmitate
to total NEFA (on average 26% and 14% in phase II and III, respectively; see
Results).
TAG:FA cycling occurs both primarily (i.e. where fatty acids are
re-esterified in adipose tissue without entering the circulation) and
secondarily (i.e. where fatty acids arrive at their site of re-esterification
through the circulation) (Klein et al.,
1989; Wolfe et al.,
1990
). In the present study, only the absolute and relative rates
of primary cycling could be assessed. Upon hydrolysis, each TAG molecule
yields three NEFA and one glycerol molecules so that
3xRa glycerol represents total fatty acid release by
lipolysis. Glycerol cannot be metabolized directly because of the absence of
glycerokinase in adipocytes (Wolfe et al.,
1990
). Therefore, primary TAG:FA cycling was calculated according
to Wolfe et al. (1990
) as:
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Changes in glycerol and palmitate specific activities were assessed by two-way analysis of variance (ANOVA) or KruskalWallis ANOVA on ranks (when populations were not normal or homoscedastic). Relationships between plasma concentration and Ra glycerol and Ra NEFA were assessed by linear regression analysis. In all other cases, statistical differences were estimated using the Student's t-test or the MannWhitney Rank Sum test. Percentages were transformed to the arcsine of their square root before statistical analysis. Values are means ± S.E.M. The criterion of significance was P<0.05.
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Results |
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Isotopic steady state
In both fasting phases, the isotopic steady state existed during the last
60 min of infusion, as indicated by the non-significant changes in specific
activities of glycerol and palmitate (P>0.90;
Fig. 1A,B). Glycerol specific
activity was similar in phases II and III (P=0.84) and averaged
4.62±0.60x104 disints min-1
mol-1. Palmitate specific activity was significantly higher in
phase III (6.74±1.16x104 disints min-1
mol-1) than in phase II (4.09±0.45x104
disints min-1 mol-1) (P<0.05).
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Concentration and rate of appearance of lipolytic metabolites
Concentration and Ra glycerol, Ra
palmitate and Ra NEFA are shown in
Table 2. Glycerol and NEFA
concentrations were slightly but not significantly higher in phase III than in
phase II, the palmitate level in phase III being about two-thirds that of
phase II (P<0.01). The percentage contribution of palmitate to
total NEFA did not change throughout the infusion experiment. It was lower in
phase III (14±2%) than in phase II (26±3%; P<0.001).
When expressed relative to BM, Ra glycerol and
Ra palmitate were similar in phases II and III
(P>0.20) and averaged 5.85±0.51 and 2.43±0.22
µmol kg-1 min-1, respectively. Only
Ra NEFA was 1.5-fold higher in phase III than in phase II
(P<0.05). However, when expressed per unit of FM,
Ra glycerol, Ra palmitate and
Ra NEFA were 2.8-, 2.2- and 4.1-fold, respectively, higher
in phase III than in phase II (P<0.001).
|
As shown in Fig. 2A, there was a significant direct relationship between plasma concentration of glycerol (x) and Ra glycerol (y) in phases II and III. At any given plasma glycerol level, Ra glycerol was higher in phase III than in phase II when expressed per unit FM (P<0.05). When Ra glycerol was expressed relative to BM, the regression equations were not different between the two fasting situations (P>0.05; not shown). Fig. 2B illustrates that NEFA levels in plasma and Ra NEFA expressed per unit FM were directly related in phase III. This relationship was not significant in phase II but, at any given plasma NEFA level, Ra NEFA was lower than in phase III.
|
TAG:FA cycling
The rate of primary TAG:FA cycling expressed relative to BM and the
percentage of re-esterification were about 3.5-fold lower in phase III than in
phase II (P<0.01, Table
2). The rate of TAG:FA cycling expressed in relation to FM was not
different in the two fasting situations (P=0.53).
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Discussion |
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Here we report the first simultaneous in vivo measurements of
glycerol and NEFA kinetics in birds. How valid are the lipolytic fluxes
estimated by the tracer method we used in fasting penguins? In phase II
penguins, Ra glycerol and Ra NEFA
averaged 5.7 and 10.5 µmol kg-1 min-1, respectively.
Given the molecular weight of glycerol (92 g mol-1) and NEFA (on
average 280 g mol-1 in king penguins; R. Groscolas, unpublished
data), these rates are equivalent to a daily release of 9.1 and 50.8 g of
glycerol and NEFA, respectively. Assuming all glycerol and NEFA released into
the circulation are oxidized (no secondary recycling), these numbers
approximate daily loss. The daily loss of glycerol and NEFA can also be
estimated from daily BM loss, considering that (i) in the king penguin, lipids
(TAG) steadily contribute to 47% of BM loss during phase II
(Cherel et al., 1994) and (ii)
the glycerol and fatty acid moieties, respectively, contribute 10% and 90% of
TAG. To avoid repeated manipulations, daily BM loss was not measured in this
study. However, based on the 140 g day-1 BM loss observed in king
penguins under similar conditions (BM=12 kg, birds caught at pairing and
penned for 1 week; R. Groscolas, unpublished data), a daily loss of
approximately 7 g of glycerol and 59 g of fatty acids could be estimated. The
reasonable agreement between the two estimates supports the conclusion that
realistic lipolytic fluxes are obtained by continuous infusion of
2-[3H]glycerol and 1-[14C]palmitate in phase II fasting
king penguins. Estimates of the daily loss of glycerol and fatty acids from BM
loss cannot be made during phase III because the lipid contribution to daily
BM loss progressively decreases during this phase and is not known for animals
of 9 kg BM.
Lipolysis during prolonged fasting: phase II versus phase III
Lipolytic fluxes have rarely been measured during prolonged fasting in
mammals, and never during natural fasting or during phase III. The turnover of
two fatty acids has been measured in naturally fasting pups of elephant seal,
but no data on the Ra glycerol and Ra
NEFA were obtained (Castellini et al.,
1987). Table 3
summarizes data obtained in the only two mammal species, humans and dogs,
studied after fasting durations (1-4 weeks) comparable to that of the king
penguin during phase II. Ra glycerol in penguins is within
the range reported for humans, whereas Ra NEFA is at the
lower end of the range reported in dogs. More abundant data is available for
short-term fasting laboratory animals and humans. In chickens fasted for 2
days, Emmanuel et al. (1983
)
reported Ra glycerol (6.6 µmol kg-1
min-1) similar to that measured in phase II penguins. In resting
non-obese humans fasting for 60-96 h, Ra glycerol ranges
from 3.8 to 6.4 µmol kg-1 min-1
(Klein et al., 1986
;
Wolfe et al., 1987
;
Jensen et al., 2001
), and was
5.9 µmol kg-1 min-1 in dogs fasted for 18 h
(Nurjhan et al., 1988
).
Ra NEFA values of 16.5 and 10.0 µmol kg-1
min-1 have been reported in normal and obese humans, respectively,
fasted for 84 h, respectively (Wolfe et
al., 1987
). Thus, the lipolytic fluxes measured in the present
study of king penguins during phase II (Ra glycerol=5.7
µmol kg-1 min-1, Ra NEFA=10.5
µmol kg-1 min-1) are within the range reported for
fasting animals of comparable body size. The value of 41 % obtained for the
percentage of primary TAG:FA cycling in penguins during phase II is within the
range of 20-49 % reported for short-term fasting humans
(Wolfe et al., 1990
;
Campbell et al., 1994
) and
laboratory mammals (Commerford et al.,
2000
; Kalderon et al.,
2000
; McClelland et al.,
2001
). However, it must be mentioned that a percentage of
recycling that was lower than 13 % or even insignificant has been reported by
others (Coppack et al., 1994
).
It therefore seems that primary recycling is comparatively high in phase II
fasting king penguins.
|
In this study, the whole body lipolytic rate (Ra
glycerol in relation to BM; Klein et al.,
1986) remained unchanged during phase III in comparison to phase
II, despite a fourfold lower FM. It has been shown previously that in
penguins, entrance into phase III corresponds to a decrease in the
contribution of lipid to energy production, compensated by an increase in the
contribution of protein (Robin et al.,
1988
; Groscolas,
1990
). On the other hand, the resting metabolic rate expressed per
kg BM is not affected by entrance into phase III
(Dewasmes et al., 1980
;
Cherel et al., 1988a
). In our
experimental setup, animals in phase III did not show a higher level of
physical activity than animals in phase II. Thus it is likely that the
metabolic rate per kg BM was similar in the two fasting situations, which
suggests that in long-term fasting penguins, the whole body lipolytic rate is
related to energy needs rather than to FM. A similar suggestion has been made
for short-term fasting humans on the basis of the determination of the whole
body lipolytic rate at different levels of adiposity
(Klein et al., 1986
).
Lipolysis (Ra glycerol per FM unit;
Klein et al., 1986
) and NEFA
availability for oxidation (Ra NEFA per BM unit;
Klein et al., 1986
) were
respectively 2.8-fold and approx. 50 % higher in phase III than in phase II,
despite high depletion of fat stores in phase III. This observation indicates
that, in long-term fasting penguins, NEFA availability is not directly
proportional to the size of the TAG stores, i.e. that fat mass is not the only
regulator of fatty acid availability. A similar conclusion was drawn from the
comparison of Ra NEFA in obese and lean overnight-fasted
humans (Lillioja et al.,
1986
). Here we show that the same applies to prolonged fasted
animals with very small fat stores.
The increase in lipolysis observed in phase III could have been subject to
lipolytic hormone control. In birds, glucagon has a strong lipolytic effect
(Hazelwood, 1984) and its
intravenous injection markedly increases the concentration of circulating
plasma NEFA in the emperor penguin
(Groscolas and Bézard,
1977
). Here we found that the plasma level of glucagon was
3.5-fold higher in phase III than in phase II, in accordance with the previous
observation that in fasting non-incubating king penguins, glucagon increases
progressively during phase II and more sharply at entrance into phase III
(Cherel et al., 1988b
). This
increase in glucagonemia might have stimulated lipolysis. Unlike in mammals,
insulin has no antilipolytic effect in birds
(Hazelwood, 1984
), including
the emperor penguin (Groscolas and
Bézard, 1977
). Moreover, in the present study the plasma
concentration of this hormone was not different in birds fasting in phase II
and in phase III. It is therefore unlikely that insulin intervened in the
phase III increase of lipolysis.
The triacylglycerol:fatty acid substrate cycle and NEFA
availability
Compared to phase II, total fatty acid release via TAG hydrolysis
(3xRa glycerol) relative to total BM remained nearly
unchanged in phase III, whereas the percentage of fatty acids that were
primarily re-esterified decreased from 41 % to 12 %. Thus, the decrease in
primary TAG:FA cycling (-5.9 µmol kg-1 min-1)
accounts entirely for the increase in NEFA availability (+5.1 µmol
kg-1 min-1). Regulation of the primary TAG:FA cycle is
poorly understood (McClelland et al.,
2001). Potential factors include hormones, substrate
concentrations, adipose tissue blood flow and interactions among them
(Wolfe et al., 1990
;
McClelland et al., 2001
). It
is unlikely that the reduced percentage of primary TAG:FA cycling in phase III
was due to a direct inhibition of resterification within the adipocyte.
Indeed, the absolute primary TAG:FA cycling in relation to FM was not
significantly lower in phase III than in phase II. The availability of
glucose, which is a precursor of glycerol 3-phosphate
(Wolfe et al., 1990
;
McClelland et al., 2001
), was
probably not a limiting factor of primary re-esterification. Glucose
concentration was only slightly lower in phase III than in phase II, and in
penguins the glucose turnover rate did not change between phases II and III
(Groscolas and Rodriguez,
1981
). Blood flow per unit mass of adipose tissue is known to
increase as fat cell size decreases (Di
Girolamo et al., 1971
), and in the emperor penguin the average fat
cell size at entrance into phase III is fivefold smaller than at the onset of
the fast (Groscolas, 1990
).
Here, an increase in adipose tissue blood flow in the leaner birds is
supported by the finding that the regression lines between glycerol and NEFA
concentration and their respective Ra per kg FM are
shifted upwards during phase III (Fig.
2). Indeed, since the Ra of metabolites has
been shown to be strongly dependent on their convective transport through the
circulation, i.e. concentration x blood flow
(Weber et al., 1987
), a higher
Ra at a given concentration indicates a higher blood flow.
Thus, simultaneously with improving glucose supply to adipocytes, an increased
adipose tissue blood flow in phase III could have caused the reduced
percentage of primary TAG:FA cycling by providing adequate albumin binding
sites to carry away fatty acids released by lipolysis
(Leibel and Edens, 1990
;
Wolfe et al., 1990
).
Our method allowed us to measure only the primary TAG:FA cycling, so we do not know the fraction of Ra NEFA that is re-esterified secondarily after NEFA transit through the circulation. Such an estimation would have required determining fatty acid oxidation (from the measurement of energy expenditure, RQ and N2 excretion), which was not feasible under our field conditions. NEFA availability, i.e. NEFA available either for oxidation or secondary cycling after their release into the circulation, was increased in phase III. Because energy expenditure does not increase during phase III whereas the contribution of fatty acids to energy production decreases (see above), it is very likely that the rate of fatty oxidation was lower in phase III than in phase II. Consequently, it is conceivable that a larger part of the circulating NEFA was re-esterified back to TAG through secondary TAG:FA cycling in phase III than in phase II. A higher secondary cycling could counterbalance the decrease in primary cycling and prevent the remaining fatty acid stores from being oxidized, perhaps because they are needed for a more vital role than energy production.
Lipolysis and the refeeding signal
The major aim of this study was to examine whether a reduction in NEFA
availability through a decrease in the lipolytic rate is the first step of a
metabolic and endocrinal cascade that leads to the stimulation of feeding
behaviour in prolonged fasting penguins. Our results do not support this
possibility. Indeed, entrance into phase III, which is known to trigger the
refeeding signal, was not associated with a reduction but with an increase in
Ra NEFA, the whole body lipolytic rate remaining
unchanged. Thus, if entrance into phase III is due to the attainment of a
critical fat mass, the lipolytic rate does not appear to be the link between
reduced total body fat availability and increased protein catabolism. The
observation that NEFA availability may positively affect sparing of body
protein (Hasselblatt et al.,
1971) clearly does not apply in penguins during fasting phase III.
It cannot be ruled out that other information arising from adipose tissue,
including leptin secretion (Ahima et al.,
1996
), might inform the whole body or organs of fat store
availability. On the other hand, alteration of metabolic pathways other than
from adipose tissue can be suggested. Among them, a reduction of fatty acid
oxidation in tissues such as the liver should be considered first. In
prolonged fasting rats, entrance into phase III has been shown to be
associated with a rapid decrease in the total hepatic activity of carnitine
palmitoyltransferase and fatty acid oxidase, which are enzymes involved in
mitochondrial and peroxisomal fatty acid oxidation, respectively
(Andriamampandry et al., 1996
).
Such a decrease in hepatic fatty acid oxidation in the face of an increased
Ra NEFA could explain the transitory (several days)
increase in plasma NEFA concentration observed previously at entrance into
phase III in young (Cherel and Le Maho,
1985
) and adult (R. Groscolas, E. Mioskowski and J.-P. Robin,
unpublished data) king penguins. Here, plasma NEFA concentration was slightly
but not significantly higher in phase III than in phase II, perhaps because
the experiments were done a little before or after reaching the peak in plasma
NEFA concentration. A decrease in hepatic fatty acid oxidation in phase III
fasted king penguins is also suggested by the slight decrease in the plasma
concentration of ß-hydroxybutyrate, a product of ß-oxidation.
Although non-significant, this decrease at least indicates that the
progressive increase in plasma ß-hydroxybutyrate concentration that is
observed during phase II in penguins
(Groscolas, 1986
;
Cherel et al., 1988b
) has been
reversed. Altogether, these observations support the view of a reduced fatty
acid oxidation in penguins with the same energy status as those spontaneously
departing to refeed at sea. A reduction of hepatic fatty acid oxidation has
been shown to stimulate feeding behaviour in rats relying heavily on fat as
energy substrate (Langhans and Scharrer,
1987
). The same might apply in penguins, the reduction in fatty
acid oxidation apparently being independent of fatty acid production by
adipose tissue.
In conclusion, this study shows that at a stage of fasting (phase III) corresponding to spontaneous egg abandonment in fasting, incubating king penguins, lipolysis and NEFA availability are not depressed but are in fact increased. Thus, the phase III-associated refeeding signal that redirects behaviour from incubation towards the search for food does not appear to originate from a limited NEFA availability. The increased lipolysis seems related to a stimulated secretion of glucagon. On the other hand, the higher NEFA availability is attributable to a decrease in the primary TAG:FA cycling, suggesting that this cycling intervenes in the control of NEFA availability in prolonged fasting birds. The possibility that the metabolic and behavioural changes that accompany phase III are the result of a direct inhibition of fatty acid oxidation, and whether and how this inhibition is linked to the attainment of a critical depletion of fat stores, should be examined in further investigations.
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References |
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