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Centre d'Ecologie et Physiologie Energétiques, Centre National de la Recherche Scientifique, 67087 Strasbourg, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study aims to determine how glucagon
intervenes in the regulation of fuel metabolism, especially lipolysis,
at two stages of a spontaneous long-term fast characterized by marked
differences in lipid and protein availability and/or utilization
(phases II and III). Changes in the plasma concentration of various
metabolites and hormones, and in lipolytic fluxes as determined by
continuous infusion of [2-3H]glycerol and
[1-14C]palmitate, were examined in vivo in a subantarctic
bird (king penguin) before, during, and after a 2-h glucagon infusion.
In the two fasting phases, glucagon infusion at a rate of 0.025 µg · kg
1 · min1
induced a three- to fourfold increase in the plasma concentration and
in the rate of appearance (Ra) of glycerol and
nonesterified fatty acids, the percentage of primary reesterification
remaining unchanged. Infusion of glucagon also resulted in a
progressive elevation of the plasma concentration of glucose and
-hydroxybutyrate and in a twofold higher insulinemia. These changes
were not significantly different between the two phases. The plasma
concentrations of triacylglycerols and uric acid were unaffected by
glucagon infusion, except for a 40% increase in plasma uric acid in
phase II birds. Altogether, these results indicate that glucagon in a
long-term fasting bird is highly lipolytic, hyperglycemic, ketogenic,
and insulinogenic, these effects, however, being similar in phases II
and III. The maintenance of the sensitivity of adipose tissue lipolysis
to glucagon could suggest that the major role of the increase in basal
glucagonemia observed in phase III is to stimulate gluconeogenesis
rather than fatty acid delivery.
lipolysis; ketone bodies; glucose; isotopic tracers; seabirds
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MAMMALS AND BIRDS adjust to long-term fasting by mobilizing their fat stores and sparing body proteins (8, 14, 21). However, the conservation of body protein that characterizes the so-called phase II of fasting is no longer maintained when a lower threshold of fat stores is reached. Then a metabolic shift occurs, and animals enter a new fasting state (phase III) corresponding to a simultaneous acceleration in the catabolism of protein and a decrease in the contribution of lipid to energy production (21, 46). This shift has been described in experimentally fasted laboratory mammals (21, 34) and in birds that spontaneously fast for prolonged periods at certain stages of their annual cycle, such as penguins (23, 37). Nevertheless, the way the metabolic shift is triggered and how the utilization of the metabolic fuels is regulated during phase II and phase III are poorly understood.
Among the various hormones that could intervene to regulate fuel utilization, glucagon is likely to play a major role. Although the evidence for a lipolytic action in humans is scarce (10), glucagon stimulates lipolysis in vitro in mammals (38, 52) and is the main lipolytic hormone in birds (9, 22, 36). It therefore plays a key role in the mobilization of fatty acids (FA) from adipose tissue. In mammals, glucagon also appears to influence the hepatic fate of released FA by diverting the circulating nonesterified FA (NEFA) into oxidation pathways and away from esterification products (28, 32), although this effect seems to depend on the nutritional state (54). Glucagon stimulates gluconeogenesis and glycogenolysis in mammals and birds (15, 16, 42), contributing to the regulation of glucose production and circulating level, including during a prolonged fast in the dog (29). While a direct role of glucagon in proteolysis has not been clearly established (51), this hormone at least indirectly influences protein metabolism by stimulating the hepatic uptake of released amino acids, and thus gluconeogenesis (48, 51). Finally, the action of glucagon on fuel metabolism may be through the secretion of other metabolic hormones. For example, it is known that glucagon is insulinogenic (38).
Most of the present knowledge on the metabolic role of glucagon arises from studies in mammals and birds in the fed state or during short-term fasting, but how glucagon intervenes in vivo in the regulation of lipolytic fluxes during prolonged periods of intense fat store mobilization such as during prolonged fasting is not understood. Studies on long-term fasting animals are limited to the determination of changes in plasma NEFA and glucose in response to glucagon injection in emperor penguins (24) and to the role of basal glucagon in hepatic glucose production in dogs (29). No study has considered the role of glucagon in fuel utilization, and notably in the regulation of lipolytic fluxes, in phase II and phase III. Using the king penguin as an animal model, we aimed to determine how glucagon intervenes in the control of birds' fuel metabolism in these two contrasted fasting situations. The king penguin is known to fast for ~1 mo during breeding, especially during incubation (23). Its fasting physiology is representative of that of other birds (14) and to some extent of mammals (12), and the phase II-phase III transition is well-characterized in this species. In particular, it has been shown that entrance into phase III is associated with an accelerated increase in the plasma concentration of glucagon (14). What is this increase in glucagonemia for? Does it allow compensating for a decreased lipolytic sensitivity of fat cells as their size becomes smaller, which would enable the maintenance of lipolytic fluxes, or is it rather related to other aspects of fuel metabolism such as protein catabolism or gluconeogenesis?
In the present study we investigate under field conditions the in vivo lipolytic, metabolic, and hormonal response to glucagon infusion in phases II and III fasting king penguins. This was done by measuring the plasma concentration of metabolites and hormones and by determining the lipolytic fluxes [rate of appearance (Ra) of NEFA and of glycerol] using tracer methodologies before, during, and after a 2-h glucagon infusion.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
This study was carried out at the breeding colony of the Baie du Marin (Possession Island, Crozet Archipelago; 46°25' S; 51°52' E) during December 1999-January 2000. Birds at this locality are accustomed to seeing people almost every day and are tame. The study was approved by the Ethical Committee of the Institut Français pour la Recherche et la Technologie Polaires and followed the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society (1). Seventeen male king penguins (Aptenodytes patagonicus) were identified from their song and caught while pairing, i.e., at the onset of the ~25-day breeding fast and after having been fasting ashore for 1-3 days. Their average body mass (BM) at capture was 13.15 ± 0.20 kg, and their size (beak and flipper length) was representative of the average size of males in this breeding colony. Birds were then kept in an outdoor fenced area (3 × 3 m) next to the colony under natural climatic conditions and within their thermoneutral range.Phase II of Fasting
Eight animals were habituated to these conditions for 6 days before the infusion 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 (26). Because at capture the birds had been fasting for 1-3 days, the length of the fast at the time of the infusion experiment was ~8 days, which is more than the 2- to 3-day duration of the transition from the fed state to phase II reported for this species (14). BM at the infusion experiment (~12 kg, see RESULTS) was similar to that of free-living male king penguins at the onset of the first incubation shift, i.e., after 8-10 days of fasting (45).Phase III of Fasting
Nine animals were kept fasting. They were weighed every 3 days and then every day until they reached a BM close to 9 kg. This BM was chosen for the phase III infusion experiment because it corresponds to the highest, but still reversible (45), state of energy depletion that is sustained on land by fasting-incubating king penguins. In this species, parents alternate at incubating, but when the relieving partner is delayed, the incubating bird prolongs its fast until it eventually abandons its egg and goes to sea for feeding. Egg abandonment occurs on average at a 9-kg BM (25), which is about 0.5-1 kg lower than the BM at entrance into phase III for breeding-fasting male king penguins (11, 14) and is reached after 5-7 days of phase III fasting. Here, 31 ± 1 days of fasting in the pen were necessary for the animals to reach this 9-kg BM.Catheterization and Experimental Setup
The infusion experiment lasted a total of 6 h and was separated into basal condition (3 h of tracer infusion without glucagon), glucagon infusion (2 h of tracer plus glucagon infusion), and postglucagon period (1 h of tracer infusion without glucagon). On the day before the infusion experiment, the unanesthetized bird was cannulated with a polyethylene catheter (50 mm long; 1.1-mm OD) inserted percutaneously into the large marginal vein of each flipper and extended with a 2-m-long section of tubing. Catheters were kept patent by infusion of saline (12 ml/day) using a small peristaltic pump. Catheterization of an artery for blood sampling for lipolytic flux measurements could not be adequately performed in field conditions. We assumed that any particular metabolism of the flipper (essentially feathers, bones, and tendons) is low and that the flipper venous blood reflects whole body metabolism. 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 small wooden pen (0.7 × 0.7 m) with one wall high enough to prevent us from being seen by the bird. Catheter extensions were placed into a balance lever system to avoid damage to the extensions or removal of catheters. It also allowed the bird to move freely (a few steps) inside the pen and even to lay on its belly or sleep with the bill under the shoulder, as was regularly observed during tracer infusion. The free ends of catheter extensions were brought outside the pen to allow intravenous infusion of isotopic tracers and glucagon into one flipper and blood sampling from the other one, from a distance, without disturbing the animal. Once the animal was in the experimental setup, care was taken to avoid any intervention or noise. On the day after the infusion experiment, the equipment was removed, and the penguins were weighed, 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 the following weeks, caught, and weighed. All had restored their BM, which indicates that they had been successfully feeding at sea and that the experiments had no impact on their health.Infusion Protocol and Preparation of the Infusates
At 0800, a primed constant-rate infusion of labeled glycerol and palmitate was started using a calibrated syringe pump and continued for 6 h (see above). The tracer infusate was prepared daily as described by Wolfe (55) and Turcotte et al. (50) using [2-3H]glycerol (Amersham, 40.7 GBq/mmol) and [1-14C]palmitate (Amersham, 2.04 GBq/mmol). Delipidated bovine serum albumin (catalog no. A-3803, Sigma) was used as a palmitate carrier. Maybe penguin plasma would have been a more suitable carrier, but under our field conditions we were not confident in preparing plasma with the required sterility. Bovine albumin has been commonly used to bind palmitate in numerous flux studies, and the time course of its administration in the present study was likely too short for a full immune response to take place. Palmitate is one of the most commonly used FA for measuring NEFA kinetics in mammals: it is the second most abundant NEFA and shows low interindividual variability in its percent contribution to NEFA. The same was observed here for king penguins, and we have determined previously that using palmitate to measure NEFA kinetics in penguins gives realistic estimates of Ra NEFA (3). Even if the absolute Ra values obtained with the methodology used cannot be fully ascertained, it is likely that the relative changes of Ra on which this study was based were correctly determined. Infusion rates of [2-3H]glycerol and [1-14C]palmitate were 221,700 ± 5,800 and 110,600 ± 3,800 dpm · kg1 · min1,
respectively (n = 17), which corresponded to trace
amounts of <0.002% of basal Ra glycerol and <0.03% of
basal Ra palmitate. Glucagon (bovine; catalog no. G-1774,
Sigma) dissolved in sterile saline was infused at 0.025 µg · kg1 · min1
using a calibrated syringe pump and the same catheter used for tracers.
This dose was chosen from similar studies in humans (10) and birds (2, 18, 22) after adjustment for BM. It was half the dose used to demonstrate a calorigenic effect of glucagon in 8- to
10-kg king penguin chicks (2) and 2.5 times the dose that
induced a barely detectable increase in the NEFA level in geese
(22). The maximum plasma glucagon level measured during glucagon infusion (1.5-2.0 ng/ml, see RESULTS) was
close to the higher basal glucagonemia reported in fasting king
penguins [1.7 ± 0.2 ng/ml toward the end of phase III (13,
14)] and may be therefore considered as physiological.
Based on data from a previous study (3), a delay of 120 min separated the beginning of the tracer infusion and the first blood
sampling (time 
60 min in the figures) to ensure that a steady state
had been reached for at least 1 h. Throughout the infusion
experiment, blood sampling was performed every 30 min. Five milliliters
of blood was collected at each sampling time, with EDTA used as an
anticoagulant. Immediately after blood was sampled, it was centrifuged
and the plasma was separated and stored at 20°C until analysis.
Determination of Glycerol and Palmitate Specific Activities
The specific activities of plasma glycerol and NEFA were determined as previously described (4, 5). A 1-ml aliquot of plasma was mixed with chloroform-methanol (2:1, vol/vol). After extraction and evaporation, an aqueous extract and an organic extract were obtained and resuspended in ethanol-water (1:1, vol/vol) and hexane-isopropanol (3:2, vol/vol), respectively. A volume of aqueous extract equivalent to 300 µl of plasma was used to determine the glycerol concentration. It was dried under nitrogen and resuspended in hydrazine buffer. Glycerol concentration was measured enzymatically. Total tritium activity was counted on another aliquot of aqueous extract equivalent to 150 µl of plasma using scintillation fluid (Ecoscint A, National Diagnostics) and a Wallac 1409 counter. At this step of analysis, tritium is found only in glycerol and glucose. The percent activity in glycerol was obtained by separating glycerol from glucose using thin-layer chromatography with chloroform-methanol (40:24, vol/vol) as the developing solvent. The glycerol and glucose fractions were resuspended in scintillation fluid for counting. The specific activity of glycerol was calculated as total tritium activity times the fraction of activity in glycerol divided by glycerol concentration.Total NEFA concentration was measured on 10 µl of plasma with an analytic test kit (NEFA C, Wako Chemicals). Palmitate concentration was obtained by multiplying NEFA concentration by the fractional contribution of palmitate to total NEFA, as determined by gas-liquid chromatography. Briefly, an aliquot of the lipid extract was separated by thin-layer chromatography using hexane-diethyl ether-acetic acid (70:30:1, vol/vol/vol) as the developing solvent. The NEFA fraction was isolated and converted to methyl esters using 14% boron trifluoride in methanol. FA methyl esters were separated and quantified using a gas chromatograph (Chrompack CP 9001) equipped with a capillary column (AT-WAX) and a flame ionization detector. The total 14C activity was counted on an aliquot of the organic extract, and its distribution in plasma lipids [triacylglycerols (TAGs), diacylglycerols, NEFA, and phospholipids] was determined after separation of lipids by thin-layer chromatography as described above. Each fraction was resuspended in ethanol-water (1:1, vol/vol) and counted in scintillation fluid (Ecoscint A). Because no 14C is incorporated in FA other than palmitate, palmitate activity was calculated by multiplying total 14C activity found in the lipid extract by the fraction of activity in NEFA. Palmitate activity divided by palmitate concentration yielded palmitate specific activity.
Other Metabolites and Hormones
Plasma glucose and-hydroxybutyrate (-OHBUT) were
determined on deproteinized plasma by enzymatic methods
(Test-Combination, Boehringer-Mannheim). Uric acid and TAG were
estimated by enzymatic colorimetric methods using commercial kits (UA
plus and Peridochrom triglycerides GPO-PAP, respectively,
Boehringer-Mannheim). Radioimmunoassay was used to measure plasma
glucagon (GL-32K kit, Linco) and insulin (insulin-CT kit from CIS bio
international). All measurements were made in the same run, and the
intra-assay coefficients of variation were 6 and 5% for glucagon and
insulin, respectively. Glucagon and insulin were measured every 30 min
during the whole infusion experiment in phase III birds and at the end
of each infusion period (basal, glucagon, postglucagon) in phase II birds.
Calculations and Statistics
Fat mass (FM) was calculated from BM as: FM (kg) = 0.552 × BM (kg) 4.260 (r2 = 0.74, n = 81, P < 0.0001). This
equation was determined in a preliminary study on king penguins with BM
ranging from 8.5 to 14.7 kg (M.-A. Thil and R. Groscolas, unpublished data).
During the basal period and the glucagon infusion, physiological and
isotopic steady states were maintained (P > 0.070;
Fig. 1), specific activities being
significantly lower during glucagon infusion than during the basal
period (P < 0.05 to 0.001). Glycerol and palmitate
Ra were therefore calculated with the steady-state equation
of Steele (47): Ra = tracer infusion rate
(dpm/min)/specific activity (dpm/mmol). In the postglucagon period, the
isotopic steady state was not significantly disrupted (Fig. 1), but
significant changes in plasma glycerol and palmitate concentrations
were observed. In this case, the Ra and the rate of
disappearance (Rd) of glycerol and palmitate were
calculated using the non-steady-state equations of Steele
(47). Because the distribution volume of glycerol and
palmitate is unknown in penguins, calculations were made using the
various values reported for mammals. Irrespective of the distribution volume [150-325 ml/kg for glycerol (31, 40);
40-50 ml/kg for palmitate (31, 56)], at all times of
the infusion experiment, Ra and Rd were not
significantly different from each other or from flux rates
(Rt) calculated using the steady-state equation (P > 0.05). Consequently, all fluxes are presented as
Ra calculated with the steady-state equation and expressed
per unit BM or FM. Ra NEFA was determined by dividing
Ra palmitate by the fractional contribution of palmitate to
total NEFA. This contribution did not change significantly during the
infusion experiment (P > 0.75) and averaged 24.5 ± 3.2 and 15.3 ± 2.4% in phases II and III, respectively. The
absolute and relative rates of primary TAG-FA cycling (i.e., where FA
are reesterified in adipose tissue without entering the circulation)
were calculated according to Wolfe et al. (56). It is
known that substantial and significant rates can be obtained only if
the ratio of Ra NEFA to Ra glycerol is substantially below 3 (55). The area enclosed by the
curves relating the concentration (see Fig. 3) or the Ra
(see Fig. 4) of glycerol and NEFA to time and the horizontal line
through the respective initial levels (time 0) were
calculated by integration from 0 to 180 min. These areas represent,
respectively, the net load in glycerol and NEFA per plasma liter and
the net glycerol and NEFA overproduction throughout the glucagon
infusion and postglucagon periods.
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Ra and Rd were compared with the Wilcoxon signed rank test. The identity of Ra and Rt was determined by verifying that the slope of the linear regression between them was not statistically different from unity, as according to Tomassone et al. (49). Statistical differences of means between phase II and phase III were assessed using the unpaired Student's t-test. Statistical significance of changes induced by glucagon infusion was assessed using two-way ANOVA with time and penguins as the main factors. When populations were not normal or homoscedastic, the Mann-Whitney Ranked-Sum test or Kruskal-Wallis ANOVA on ranks was used. When significant changes were detected with ANOVA, the Student-Newman-Keuls method was used to determine which means were different from basal values. Linear regression analysis with the F-test, after log-transformation to normalize data, was performed to examine the relationships between NEFA or glycerol plasma concentrations and Ra. Slopes and intercepts of the regressions were compared using the t-test. Values are means ± SE. The criterion of significance was P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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BM and FM
At the time of the infusion experiment, birds in phase II had a total BM of 12.17 ± 0.26 kg and a FM of 2.41 ± 0.14 kg. Compared with phase II, birds in phase III had a 25% lower BM (8.98 ± 0.05 kg) and a nearly four times lower FM (0.63 ± 0.02 kg) (P < 0.001).Hormones
Basal levels of plasma glucagon were 3.5-fold higher in phase III than in phase II (P < 0.001), whereas basal plasma insulin levels were similar in the two phases (P = 0.33; Table 1). As illustrated in Fig. 2A for phase III birds, glucagon infusion resulted in a severalfold increase in glucagonemia. High glucagon levels were maintained (P > 0.63) throughout infusion, a partial return toward basal levels being observed postglucagon. These high levels were not significantly different between the two phases, either at plateau (P = 0.54) or after 60 min into postglucagon (P = 0.60; Table 1). As illustrated in Fig. 2B for phase III birds, glucagon infusion induced about a twofold increase in plasma insulin levels (P < 0.001). These high insulin levels were maintained throughout and after glucagon infusion (P > 0.51) and were not significantly different between the two fasting phases (P = 0.54; Fig. 2 and Table 1).
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Plasma Glycerol and NEFA
Basal plasma glycerol levels were not significantly different between phase III and phase II (P = 0.06; Fig. 3A). In both phases, glucagon induced a rapid increase in plasma glycerol levels (P < 0.001) that were maintained steady (P > 0.14) and high throughout the infusion (on average 4.6 and 2.9 times the basal levels in phase II and phase III, respectively). Plateau plasma glycerol levels were the same in the two phases (0.23 ± 0.05 mmol/l; P = 0.40). During the postglucagon period, the plasma glycerol concentration decreased toward basal values in phase III birds (P < 0.05). Throughout glucagon infusion and postglucagon, integrals of plasma glycerol changes (i.e., the net load in glycerol per plasma liter; see Calculations and Statistics) did not differ significantly between the two phases (phase II = 22.1 ± 7.2 mmol/l, phase III = 18.3 ± 5.1 mmol/l; P = 0.58).
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Basal plasma NEFA levels were not significantly different between the two phases (P = 0.13; Fig. 3B). In both phases, glucagon infusion induced a significant increase in NEFA concentration (P < 0.001). In phase II birds, the maximum effect was observed at the end of infusion and postglucagon, NEFA concentration being on average 3.4-fold higher than in basal conditions (2.11 ± 0.18 vs. 0.61 ± 0.03 mmol/l). In phase III birds, NEFA concentration reached maximum levels after only 30 min of glucagon infusion and during postglucagon significantly decreased toward basal levels. The plateau that was maintained throughout infusion (P > 0.70) was 3.4 times the basal level (2.49 ± 0.36 vs. 0.74 ± 0.08 mmol/l) and nonsignificantly different from the plateau level observed in phase II birds (P = 0.15). Throughout glucagon infusion and postglucagon, integrals of plasma NEFA changes did not differ significantly between the two phases (phase II = 269 ± 44 mmol/l, phase III = 221 ± 36 mmol/l; P = 0.39).
Lipolytic Fluxes
Basal Ra (µmol · kg BM1 · min1; Fig.
4) was not significantly different
between the two phases (glycerol, P = 0.20) or was
slightly higher in phase III than in phase II (NEFA, P < 0.05). In both phases, glucagon infusion induced a significant
increase (P < 0.001) in Ra glycerol (Fig.
4A) and Ra NEFA (Fig. 4B), plateau values being maintained throughout infusion (P > 0.05). The increase in Ra glycerol was similar in phase II
(from 5.6 to 22.8 µmol · kg
BM1 · min1, i.e., by
4 times) and in phase III birds (from 6.0 to 21.2 µmol · kg
BM1 · min1, i.e., by
3.5 times). A similar 3-3.2-fold increase in Ra NEFA was also observed in phase II (from 10.8 to 35 µmol · kg
BM1 · min1) and in
phase III birds (from 15.5 to 47.3 µmol · kg
BM1 · min1). During
postglucagon, Ra glycerol and Ra NEFA declined
significantly in phase III (P < 0.05) but not in phase
II birds (P > 0.32). Throughout glucagon infusion and
postglucagon, integrals of Ra glycerol and NEFA changes
(i.e., the net overproduction of glycerol and NEFA per kilogram BM; see
Calculations and Statistics) did not differ significantly
between the two phases (P > 0.59).
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As can be seen in Figs. 3 and 4, the time courses and magnitude of the
changes in plasma concentrations and Ra were very similar. Actually, in both phases and throughout the whole infusion experiment, significant direct relationships (P < 0.001) were
found between plasma concentrations and Ra (log-transformed
data) whether Ra was expressed per kilogram BM or per
kilogram FM. When expressed relative to BM, the regression equations
were not different between the two fasting situations, either for
glycerol or for NEFA (P > 0.26, not shown). When
expressed relative to FM, the slopes of the relationships were not
significantly different between the two phases (P > 0.21), either for glycerol (Fig.
5A) or for NEFA (Fig.
5B), but at any given plasma concentration, Ra
was higher (P < 0.001) in phase III than in phase II.
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The infusion of glucagon resulted in about a fivefold increase in the
absolute rate of TAG-FA cycling in phases II and III respectively
(P < 0.005; Table 2),
whereas the percentage of intracellular cycling did not change
significantly in either phase (P > 0.25).
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Other Metabolites
Uric acid.
Birds in phase III had a 2.4-fold higher basal plasma uric level than
phase II birds (0.54 ± 0.11 vs. 0.22 ± 0.02 mmol/l; P < 0.001; Fig.
6A). Glucagon infusion had no
effect on plasma uric acid in phase III birds (P = 0.12), but it induced a significant 40% increase in phase II birds
(P < 0.05).
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Glucose. Basal plasma glucose levels were slightly higher in phase II than in phase III birds (P = 0.03; Fig. 6B). In both phases, glucagon induced a similar and progressive increase in glycemia (P < 0.001), by 32% and 45-70% at the end of infusion and postglucagon, respectively. The final glycemia was not different between the two phases (phase II = 19.49 ± 4.13 mmol/l, phase III = 15.67 ± 0.22 mmol/l; P = 0.12).
-OHBUT.
Basal plasma -OHBUT levels were similar in phases II and III
(P = 0.19; Fig.
7A). In both phases, glucagon
induced a similar and progressive increase in these levels
(P < 0.001), by 1.5 times at the end of infusion. The
final plasma level of -OHBUT was not different between the two
phases (phase II = 5.12 ± 0.53 mmol/l, phase III = 6.82 ± 0.41 mmol/l; P = 0.23).
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TAG. The plasma TAG concentration remained unchanged throughout the infusion experiment, both in phase II and in phase III (P > 0.16; Fig. 7B). On average, it was higher in phase II (0.88 ± 0.07 mmol/l) than in phase III (0.49 ± 0.01 mmol/l; P < 0.001).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Lipolytic and Metabolic Response to Glucagon in Long-Term Fasting Birds
The first objective of this study was to determine how glucagon intervenes in the in vivo regulation of energy substrate utilization in long-term fasting birds. Our results show that in such a fasting situation glucagon is highly lipolytic. This is demonstrated by the finding that during glucagon infusion there was a three- to fourfold increase in the Ra of glycerol and NEFA, which are the two lipid fuels derived from the hydrolysis of adipose tissue TAG. Glucagon infusion also induced an increase in the plasma concentration of NEFA and glycerol, and in phase II as in phase III, these increases were strongly parallel to that of Ra, as reflected by significant correlations between concentrations and Ra. To date, lipolytic fluxes have not been measured in birds during glucagon infusion, and our results therefore provide the first evidence that the changes in the plasma concentrations of NEFA and glycerol in this situation are mostly if not entirely due to changes in their production rates from adipose tissue.The major mechanism through which glucagon stimulates lipolysis in birds and mammals is by increasing the activity of hormone-sensitive lipase, the enzyme that hydrolyzes fat cell TAG (27). In mammals, insulin also intervenes in the control of lipolysis, being antilipolytic. This is not the case in birds (27), so that the increase in insulinemia observed after glucagon infusion most likely had no effect on NEFA and glycerol release. Another factor possibly regulating the release of NEFA from adipose tissue is primary recycling, i.e., the process where a part of NEFA released by TAG hydrolysis is reesterified back into TAG before entering the circulation. The finding that in our study glucagon infusion did not affect the percentage of primary reesterification argues against such a possibility for partly explaining the increase in Ra NEFA. A lack of effect of glucagon on primary recycling has not been observed previously in birds but has been reported for mammals (41).
The progressive increase in plasma -OHBUT after glucagon infusion
supports the view that in long-term fasting birds glucagon stimulates
ketogenesis. A direct effect of glucagon on ketogenesis has been
suggested in humans from the observation that glucagon increases the
percentage of NEFA used for ketogenesis (6), but whether
this occurs in birds is unknown. In the present work, it is likely that
the ketogenic effect of glucagon was mainly indirect, i.e., that it was
the consequence of an increased FA oxidation related to the
glucagon-induced increased availability of NEFA. An increased FA
oxidation in glucagon-infused fasting king penguins is supported by the
previous observation in this species of a mean 47% increase in
metabolic rate during the 2 h after a 15-min glucagon infusion at
a dose of 0.05 µg · kg1 · min1
(2). It is known that in fasting penguins 90-96% of
energy production is from lipid oxidation (23). The
preferential utilization of NEFA released through the lipolytic action
of glucagon for oxidation and ketogenesis in the liver of fasted king
penguins could be also suggested from the finding that the plasma
concentration of TAG did not change during glucagon infusion. An
increase in plasma TAG would have been expected if a substantial
proportion of the NEFA delivered to the liver was reesterified
(secondary recycling). However, the possibility that an increased
hepatic production of TAG is not reflected by an increase in their
circulating levels because of impaired hepatic TAG release cannot be
discarded. It has been suggested that glucagon might interfere with the
synthesis of apoprotein, thus impairing the hepatic release of TAG
(18). This could explain the development of fatty liver
and the moderate or insignificant changes in plasma TAG levels that
have been observed with glucagon treatment in fed and short-term fasted
geese (18, 22, 30).
Infusion of glucagon also resulted in a progressive increase in
glycemia that persisted after the infusion of glucagon was stopped. The
hyperglycemic effect of glucagon was less marked and lasted longer than
its lipolytic effect, as observed in other birds (20, 24,
43). Such a hyperglycemia was likely the result of an increased
glucose production, because glucagon is known to directly stimulate
glycogenolysis and gluconeogenesis (42). An indirect
stimulation of gluconeogenesis can also be postulated from the observed
increase in protein catabolism (see below), lipolysis, and NEFA
oxidation. Indeed, it has been shown in mammals that gluconeogenesis is
promoted by an increased supply of gluconeogenic precursors to the
liver, such as amino acids and glycerol (10, 35), and by
the increased NADH-to-NAD+ ratio and ATP and acetyl CoA
production resulting from enhanced hepatic NEFA oxidation (10,
17, 35). In the postglucagon period, the persistent increase in
glycemia that paralleled the decrease in plasma glucagon and the
maintenance of a high availability of NEFA and -OHBUT as metabolic
fuels might also be the consequence of a decreased rate of glucose
utilization. Such a glucose-sparing effect of these lipid fuels has
already been suggested in birds (20, 22, 24). The increase
in plasma glucose occurred despite the increase in plasma insulin, a
hormone known to counteract the hyperglycemic action of glucagon in
birds (27). This can be explained by the finding that
during and after glucagon infusion, the glucagon-insulin molar ratio,
to which glycemia is best correlated, remained higher than under basal
conditions. The increase in insulinemia that accompanied glucagon
infusion was probably related to the insulinogenic effect of glucagon
(27, 38), whereas postglucagon a high insulinemia was
possibly maintained in response to elevated blood glucose
(27).
Uric acid is the end product of protein degradation in birds, and its
plasma level is a good index of this degradation in penguins
(46). The 40% increase in plasma uric acid observed during glucagon infusion in phase II birds therefore suggests that in
this fasting stage glucagon stimulates protein catabolism. Studies in
chicks (33) and in mammals (51) have
demonstrated that glucagon has no direct role on muscle proteolysis or
peripheral release of amino acid. However, an indirect effect through
the glucagon-induced rise in -OHBUT levels can be suggested since it
has been shown in vitro that -OHBUT inhibits muscular protein synthesis in fasted chicks (57). A glucagon-induced
increase in hepatic proteolysis can be also postulated, as it has been observed in the fed and fasted rat (7). Whatever the
mechanism, the observation that increased protein catabolism was
concurrent with the glucagon-induced high NEFA availability argues
against the idea expressed for mammals that NEFA availability may be
directly responsible for protein conservation during prolonged
starvation (19, 39, 53). Such an accelerated protein
catabolism in the face of an increased NEFA availability has been
previously reported in fasting king penguins at the entrance into phase
III (3) or after pharmacological inhibition of FA
oxidation (4).
Response to Glucagon in Phase III vs. Phase II
The second objective of this study was to determine whether glucagon affects similarly the utilization of energy substrates in the two fasting situations characterized by large differences in fat stores, protein catabolism and hormonal status, and to gain insight on the role of the increase in glucagonemia in the phase III fasting status. In other words: is a change in the response to glucagon a component of the phase II-phase III transition?As stated in MATERIALS AND METHODS, our phase III birds were in the same state of fat store depletion as those that under field conditions spontaneously stop fasting and go to sea for refeeding. The lipolytic response to glucagon of these birds differed from that of phase II birds only in the time course of the changes in plasma concentration and Ra of NEFA, and to a lesser extent of glycerol. After the onset of glucagon infusion, plateau values of NEFA concentration and Ra were reached ~1 h sooner in phase III than in phase II birds. After glucagon, the basal values were rapidly overtaken in phase III but not in phase II penguins, the same tendency being observed for glycerol. These differences probably reflected a higher adipose tissue blood flow in phase III than in phase II, a higher blood flow carrying glucagon in and away faster.
On the other hand, and although having about a fourfold lower fat mass,
the phase III birds showed a lipolytic response to glucagon of a
magnitude comparable to that in phase II birds. Because glucagon
infusion resulted in similar plateau levels of plasma glucagon, these
results demonstrate that the lipolytic sensitivity to glucagon remains
unchanged in king penguins fasting into phase III for ~1 wk. It has
been shown in fasting emperor penguins that at a comparable stage of
phase III, the remaining adipose tissue is made only of small fat cells
(<50 µm in diameter), with no evidence for a decrease in fat cell
number (23). The same probably applies to the king
penguin, and it is therefore reasonable to conclude that on a per cell
basis, small fat cells in long-term fasting penguins are as sensitive
to the lipolytic action of glucagon as large fat cells. This conclusion
is reinforced by the finding that the amount of NEFA released in vitro
during incubation of pieces of adipose tissue with 106 M
glucagon was on a per cell basis similar in phase II (BM = 12 kg)
and phase III (BM = 9 kg) fasting king penguins (Groscolas, unpublished data). Finally, the maintenance of lipolytic capacity of
small fat cells in penguins is also supported by the finding that under
basal conditions lipolytic fluxes per kilogram BM were similar
(Ra glycerol) or slightly higher (Ra NEFA) in
phase III birds compared with those in phase II (Ref. 3;
this study). Thus entrance into phase III after a severe reduction of
FM in long-term fasting penguins does not seem related to a decreased basal lipolysis or to a reduced sensitivity to glucagon, the major lipolytic hormone in birds.
Not only the lipolytic response but also the responses of plasma
-OHBUT (increase) and TAG (maintenance) to glucagon were similar in
phases II and III. This observation suggests that the metabolic fate of
released NEFA was the same in the two fasting situations. More
specifically, in the two phases, a similar increase in plasma -OHBUT
resulted from a similar increase in NEFA availability (NEFA plasma
concentration and Ra). Consequently, it may be suggested that under conditions of high NEFA mobilization, the oxidative and
ketogenic capacities of the liver are maintained in fasting penguins at
a stage of energy depletion identical to that of birds spontaneously
departing to refeed at sea. Under free-living conditions, these birds
have to swim at a high rate of energy expenditure for hundreds of
kilometers before reaching their feeding grounds (44). It
is likely that by this time the remaining fat stores are mobilized at a
high rate. Thus the maintenance of the efficiency of the oxidative
machinery suggested by our data under such conditions would allow
penguins to cope with this high energy demand.
Compared with phase II birds, the response of phase III penguins to glucagon differs for plasma uric acid but not for plasma glucose and insulin. In accordance with previous reports, and reflecting accelerated protein catabolism (46), basal plasma uric acid was already increased in phase III birds. This might have impaired a further increase during glucagon infusion. The hyperglycemic and insulinogenic effect of glucagon was maintained in phase III, i.e., in a situation of stimulated protein catabolism. Because the lipolytic sensitivity of fat cells to glucagon is also maintained in this situation, it is unlikely that the role of the increase in basal plasma glucagon observed in phase III birds (Ref. 14; this study) is to stimulate lipolysis. The possibility remains that the major role of the increase in basal glucagon level would be to contribute to the maintenance of glycemia. This could occur by stimulating gluconeogenesis directly, by improving hepatic uptake of amino acids issued from the accelerated protein catabolism, or by stimulating hepatic oxidative and ketogenic capacities, hence ensuring ketone body production and an adequate supply of energy for gluconeogenesis.
Perspectives
In conclusion, this study has demonstrated that glucagon is highly lipolytic, hyperglycemic, and insulinogenic in long-term fasting king penguins. It also stimulates ketogenesis and protein catabolism (phase II) but has no effect on the percentage of primary TAG-FA cycling or on plasma TAG. The lipolytic response to glucagon was similar in phases II and III, indicating that entrance into phase III is not linked to changes in the lipolytic sensitivity of fat cells to glucagon. The role of the increase in basal plasma glucagon observed during phase III should thus be to maintain glycemia rather than to stimulate lipolysis. The maintenance of the glycemic, ketogenic, and insulinogenic effect of infused glucagon in phase III penguins also suggests that a modulation of the metabolic and hormonal response to glucagon is not a major component of the phase II-phase III transition in birds. However, the possibility that the effects of increased basal glucagonemia could be different in some aspects from those of artificially raised glucagon levels should be examined, including using somatostatin (if effective in penguins) and replacement infusion of insulin. From previous results, we have suggested that under conditions of basal NEFA availability, entrance into phase III could be related to a decreased capacity for FA oxidation (3, 4). In contrast, the present data suggest that oxidative capacity is maintained in phase III birds with highly increased NEFA availability. How NEFA availability affects the oxidative fate of these fuels during fasting should therefore be the subject of further studies.| |
ACKNOWLEDGEMENTS |
|---|
We thank E. Mioskowski for assistance in the sample analyses and C. Fayolle for field assistance.
| |
FOOTNOTES |
|---|
Financial support was provided by the Institut Français pour la Recherche et la Technologie Polaires (programme 119) and logistical support by the Terres Australes et Antarctiques Françaises. S. F. Bernard was the recipient of a grant from the Bettencourt Schueller Found.
Address for reprint requests and other correspondence: R. Groscolas, Centre d'Ecologie et Physiologie Energétiques, CNRS, 23 rue Becquerel, 67087 Strasbourg, France (E-mail: rene.groscolas{at}c-strasbourg.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 10, 2002;10.1152/ajpregu.00325.2002
Received 4 June 2002; accepted in final form 8 October 2002.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and phase III (
) fasting
king penguins. Values are means and T-bars show SE (phase II:
n = 8; phase III: n = 9).
a Significantly different from basal values
(P < 0.05); b nonsignificantly
different from basal and glucagon values.
