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Am J Physiol Regul Integr Comp Physiol 284: R444-R454, 2003. First published October 10, 2002; doi:10.1152/ajpregu.00325.2002
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Vol. 284, Issue 2, R444-R454, February 2003

Lipolytic and metabolic response to glucagon in fasting king penguins: phase II vs. phase III

Servane F. Bernard, Marie-Anne Thil, and René Groscolas

Centre d'Ecologie et Physiologie Energétiques, Centre National de la Recherche Scientifique, 67087 Strasbourg, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · min-1 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 beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · kg-1 · min-1, 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 · kg-1 · min-1 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 beta -hydroxybutyrate (beta -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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  		Fig. 1.   Glycerol (A) and palmitate (B) specific activity before, during, and after infusion of glucagon (0.025 µg · kg-1 · min-1) in phase II () and phase III (triangle ) 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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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  		Table 1.   Glucagon and insulin levels before, during, and after infusion of glucagon in king penguins during phase II and phase III of fasting



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  		Fig. 2.   Plasma concentration of glucagon (A) and insulin (B) before, during, and after infusion of glucagon (0.025 µg · kg-1 · min-1) in phase III fasting king penguins. Values (means ± SE, n = 9) not sharing the same superscript letter are significantly different, P < 0.05.

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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  		Fig. 3.   Plasma concentration of glycerol (A) and nonesterified fatty acids (NEFA; B) before, during, and after infusion of glucagon (0.025 µg · kg-1 · min-1) in phase II () and phase III (triangle ) 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 in phase II, P < 0.05; b significantly different from basal values in phase III, P < 0.05.

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 BM-1 · min-1; 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 BM-1 · min-1, i.e., by 4 times) and in phase III birds (from 6.0 to 21.2 µmol · kg BM-1 · min-1, 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 BM-1 · min-1) and in phase III birds (from 15.5 to 47.3 µmol · kg BM-1 · min-1). 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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  		Fig. 4.   Rate of appearance (Ra) of glycerol (A) and NEFA (B) before, during, and after infusion of glucagon (0.025 µg · kg-1 · min-1) in phase II () and phase III (triangle ) 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 in phase II, P < 0.05; b significantly different from basal values in phase III, P < 0.05.

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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  		Fig. 5.   Regressions of glycerol (A) and NEFA (B) Ra expressed per unit of fat mass (FM) against plasma concentrations in phase II () and phase III (triangle ) fasting king penguins. Each point represents one observation; all data obtained during the infusion experiment (basal, glucagon, and postglucagon) were included in the calculations. Glycerol: phase II, Y = 2.567 + 0.821X, r2 = 0.81, n = 72, P < 0.001; phase III, Y = 3.030 + 0.919X, r2 = 0.78, n = 81, P < 0.001. NEFA: phase II, Y = 1.940 + 0.905X, r2 = 0.76, n = 72, P < 0.001; phase III, Y = 2.464 + 0.862X, r2 = 0.78, n = 81, P < 0.001.

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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  		Table 2.   Mean rates of TAG:FA cycling and percentage of reesterification during phases II and III in king penguins before and during glucagon infusion

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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  		Fig. 6.   Plasma concentration of uric acid (A) and glucose (B) before, during, and after infusion of glucagon (0.025 µg · kg-1 · min-1) in phase II () and phase III (triangle ) 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 in phase II, P < 0.05; b significantly different from basal values in phase III, P < 0.05.

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).

beta -OHBUT. Basal plasma beta -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 beta -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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  		Fig. 7.   Plasma concentration of beta -hydroxybutyrate (A) and triacylglycerols (B) before, during, and after infusion of glucagon (0.025 µg · kg-1 · min-1) in phase II () and phase III (triangle ) 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 in phase II, P < 0.05; b significantly different from basal values in phase III, P < 0.05.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 · kg-1 · min-1 (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 beta -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 beta -OHBUT levels can be suggested since it has been shown in vitro that beta -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 10-6 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 beta -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 beta -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R281-R283, 2002[Free Full Text].

2.   Barré, H, and Rouanet JL. Calorigenic effect of glucagon and catecholamines in king penguin chicks. Am J Physiol Regul Integr Comp Physiol 244: R758-R763, 1983[Abstract/Free Full Text].

3.   Bernard, SF, Fayolle C, Robin JP, and Groscolas R. Glycerol and NEFA kinetics in long-term fasting king penguins: phase II vs. phase III. J Exp Biol 275: 2745-2754, 2002.

4.   Bernard, SF, Mioskowski E, and Groscolas R. Blockade of fatty acid oxidation mimics the phase II-phase III transition in a fasting bird, the king penguin. Am J Physiol Regul Integr Comp Physiol 283: R144-R152, 2002[Abstract/Free Full Text].

5.   Bernard, SF, Reidy SP, Zwingelstein G, and Weber JM. Glycerol and fatty acid kinetics in rainbow trout: effects of endurance swimming. J Exp Biol 202: 279-288, 1999[Abstract].

6.   Beylot, M. Regulation of in vivo ketogenesis: role of free fatty acids and control by epinephrine, thyroid hormones, insulin and glucagon. Diabet Med 22: 299-304, 1996.

7.   Bleiberg-Daniel, F, Lamri Y, Feldmann G, and Lardeux B. Glucagon administration in vivo stimulates hepatic RNA and protein breakdown in fed and fasted rats. Biochem J 299: 645-649, 1994.

8.   Cahill, GF. Starvation in man. Clin Endocrinol Metab 5: 397-415, 1976[ISI][Medline].

9.   Campbell, RM, and Scanes CG. Growth hormone inhibition of glucagon- and cAMP-induced lipolysis by chicken adipose tissue in vitro. Proc Soc Exp Biol Med 184: 456-460, 1987[Abstract].

10.   Carlson, MG, Snead WL, and Campbell PJ. Regulation of free fatty acid metabolism by glucagon. J Clin Endocrinol Metab 77: 11-15, 1993[Abstract].

11.   Cherel, Y, Gilles J, Handrich Y, and Le Maho Y. Nutrient reserve dynamics and energetics during long-term fasting in the king penguin (Aptenodytes patagonicus). J Zool Lond 234: 1-12, 1994.

12.   Cherel, Y, and Groscolas R. Relationship between nutrient storage and nutrient utilization in long-term fasting birds and mammals. In: Proceedings of the 22nd International Ornithological Congress Durban, edited by Adams NJ, and Slotow RH.. Johannesburg, South Africa: BirdLife South Africa, 1999, p. 17-34.

13.   Cherel, Y, Leloup J, and Le Maho Y. Fasting in king penguin. II. Hormonal and metabolic changes during molt. Am J Physiol Regul Integr Comp Physiol 254: R178-R184, 1988[Abstract/Free Full Text].

14.   Cherel, Y, Robin JP, Walch O, Karmann H, Netchitailo P, and Le Maho Y. Fasting in king penguin. I. Hormonal and metabolic changes during breeding. Am J Physiol Regul Integr Comp Physiol 254: R170-R177, 1988[Abstract/Free Full Text].

15.   Cherrington, AD, Chiasson JL, Liljenquist JE, Jennings A, Keller U, and Lacy WW. The role of insulin and glucagon in the regulation of basal glucose production in the post-absorptive dog. J Clin Invest 58: 1407-1418, 1976[ISI][Medline].

16.   Chiasson, JL, Liljenquist JE, Sinclair-Smith BC, and Lacy WW. Gluconeogenesis from alanine in normal post-absorptive man---intrahepatic stimulatory effect of glucagon. Diabetes 24: 574-584, 1975[Abstract].

17.   Chu, CA, Sherck SM, Igawa K, Sindelar DK, Neal DW, Emshwiller M, and Cherrington AD. Effects of free fatty acids on hepatic glycogenolysis and gluconeogenesis in conscious dogs. Am J Physiol Endocrinol Metab 282: E402-E411, 2002[Abstract/Free Full Text].

18.   De Oya, M, Prigge WF, Swenson DE, and Grande F. Role of glucagon on fatty liver production in birds. Am J Physiol 221: 25-30, 1971[Free Full Text].

19.   Ferrannini, E, Barret EJ, Bevilacqua S, Jacob R, Walesky M, Sherwin RS, and DeFronzo RA. Effects of free fatty acids on blood amino acid levels in humans. Am J Physiol Endocrinol Metab 250: E686-E694, 1986[Abstract/Free Full Text].

20.   Freeman, BM, and Manning ACC The prandial state and the glycaemic and lipolytic responses of Gallus domesticus to catecholamines and glucagon. Comp Biochem Physiol A 47: 1145-1152, 1974[Medline].

21.   Goodman, MN, Larsen PR, Kaplan MM, Aoki TT, Young VR, and Ruderman NB. Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism. Am J Physiol Endocrinol Metab 239: E277-E286, 1980[Free Full Text].

22.   Grande, F, and Prigge WF. Glucagon infusion, plasma FFA and triglycerides, blood sugar, and liver lipids in birds. Am J Physiol 218: 1406-1411, 1970[Free Full Text].

23.   Groscolas, R. Metabolic adaptations to fasting in emperor and king penguins. In: Penguin Biology, edited by Davis LS, and Darby JT.. San Diego, CA: Academic, 1990, p. 269-296.

24.   Groscolas, R, and Bézard J. Effect of glucagon and insulin on plasma free fatty acids and glucose in the emperor penguin, Aptenodytes forsteri. Gen Comp Endocrinol 32: 230-235, 1977[ISI][Medline].

25.   Groscolas, R, Decrock F, Thil MA, Fayolle C, Boissery C, and Robin JP. Refeeding signal in fasting-incubating king penguins: changes in behavior and egg temperature. Am J Physiol Regul Integr Comp Physiol 279: R2104-R2112, 2000[Abstract/Free Full Text].

26.   Groscolas, R, and Rodriguez A. Glucose metabolism in fed and fasted emperor penguins (Aptenodytes forsteri). Comp Biochem Physiol A 70: 191-198, 1981.

27.   Hazelwood, RL. Pancreatic hormones, insulin/glucagon molar ratios, and somatostatin as determinants of avian carbohydrate metabolism. J Exp Zool 232: 647-652, 1984[ISI][Medline].

28.   Heimberg, M, Weinstein I, and Kohout M. The effects of glucagon, dibutyryl cyclic adenosine 3',5'-monophosphate and concentration of free fatty acid on hepatic metabolism. J Biol Chem 244: 5131-5139, 1969[Abstract/Free Full Text].

29.   Hendrick, GK, Wasserman DH, Frizzeli RT, Williams PE, Lacy DB, Jaspan JB, and Cherrington AD. Importance of basal glucagon in maintaining hepatic glucose production during a prolonged fast in conscious dogs. Am J Physiol Endocrinol Metab 263: E541-E549, 1992[Abstract/Free Full Text].

30.   Hoak, JC, Connor WE, and Warner ED. Toxic effect of glucagon-induced acute lipid mobilization in geese. J Clin Invest 47: 2701-2710, 1968[ISI][Medline].

31.   Issekutz, BJ, Shaw WAS, and Issekutz TB. Effect of lactate on FFA and glycerol turnover in resting and exercising dogs. J Appl Physiol 39: 349-353, 1975[Abstract/Free Full Text].

32.   Keller, U, and Shulman G. Effect of glucagon on hepatic fatty acid oxidation and ketogenesis in conscious dogs. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E121-E129, 1979[Free Full Text].

33.   Klasing, KC, and Jarrell VL. Regulation of protein degradation in chick muscle by several hormones and metabolites. Poult Sci 64: 694-699, 1985[ISI][Medline].

34.   Koubi, HE, Robin JP, Dewasmes G, Le Maho Y, Frutoso J, and Minaire Y. Fasting-induced rise in locomotor activity in rats coincides with increased protein utilization. Physiol Behav 50: 337-343, 1991[Medline].

35.   Kraus-Friedmann, N. Hormonal regulation of hepatic gluconeogenesis. Physiol Rev 64: 170-259, 1984[Free Full Text].

36.   Langslow, DR, Butler EJ, Hales CN, and Pearson AW. The response of plasma insulin, glucose and non-esterified fatty acids to various hormones, nutrients and drugs in the domestic fowl. J Endocrinol 46: 243-260, 1970[Abstract/Free Full Text].

37.   Le Maho, Y, Robin JP, and Cherel Y. Starvation as a treatment for obesity: the need to conserve body protein. News Physiol Sci 3: 21-24, 1988[Abstract/Free Full Text].

38.   Lefebvre, P. Glucagon and adipose tissue. Biochem Pharmacol 24: 1261-1266, 1975[ISI][Medline].

39.   Lowell, BB, and Goodman MN. Protein sparing in skeletal muscle during prolonged starvation. Dependence of lipid fuel availability. Diabetes 36: 14-19, 1987[Abstract].

40.   McClelland, GB, Hochachka PW, Reidy SP, and Weber JM. High-altitude acclimation increases the triacylglycerol/fatty acid cycle at rest and during exercise. Am J Physiol Endocrinol Metab 281: E537-E544, 2001[Abstract/Free Full Text].

41.   Miyoshi, H, Shulman GI, Peters EJ, Wolfe MH, Dariush E, and Wolfe RR. Hormonal control of substrate cycling in humans. J Clin Invest 81: 1545-1555, 1988[ISI][Medline].

42.   O'Neill, IE, and Langslow DR. The action of hydrocortisone, insulin, glucagon on chicken liver hexokinase and glucose-6-phosphatase and the plasma glucose and free fatty acid concentrations. Gen Comp Endocrinol 34: 428-437, 1978[ISI][Medline].

43.   Palokangas, R, Vihko V, and Nuuja I. The effects of cold and glucagon on lipolysis, glycogenolysis and oxygen consumption in young chicks. Comp Biochem Physiol A 45: 489-495, 1973[Medline].

44.   Robin, JP, Boucontet L, Chillet P, and Groscolas R. Behavioral changes in fasting emperor penguins : evidence for a "refeeding signal" linked to a metabolic shift. Am J Physiol Regul Integr Comp Physiol 274: R746-R753, 1998[Abstract/Free Full Text].

45.   Robin, JP, Fayolle C, Decrock F, Thil MA, Côté SD, Bernard S, and Groscolas R. Restoration of body mass in King Penguins after egg abandonment at a critical energy depletion stage: early vs. late breeders. J Avian Biol 32: 303-310, 2001.

46.   Robin, JP, Frain M, Sardet C, Groscolas R, and Le Maho Y. Protein and lipid utilization during long-term fasting in emperor penguins. Am J Physiol Regul Integr Comp Physiol 254: R61-R68, 1988[Abstract/Free Full Text].

47.   Steele, R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Med Sci 82: 420-430, 1959.

48.   Steiner, KE, Williams PE, Lacy WW, and Cherrington AD. Effect of insulin on glucagon-stimulated glucose production in the conscious dog. Metabolism 39: 1325-1333, 1990[ISI][Medline].

49.   Tomassone, R, Lesquoy E, and Miller C. La régression. Paris: Masson, 1983.

50.   Turcotte, LP, Richter EA, and Kiens B. Increased plasma FFA uptake and oxidation during prolonged exercise in trained vs. untrained humans. Am J Physiol Endocrinol Metab 262: E791-E799, 1992[Abstract/Free Full Text].

51.   Umpleby, AM, and Russel-Jones DL. The hormonal control of protein metabolism. Bailleres Clin Endocrinol Metab 10: 551-570, 1996[ISI][Medline].

52.   Vaughan, M, and Steinberg D. Effect of hormones on lipolysis and esterification of free fatty acids during incubation of adipose tissue in vitro. J Lipid Res 4: 193-199, 1963[Abstract].

53.   Wicklmayer, M, Rett K, Schwiegelshohn B, Wolfram G, Hailer S, and Dietze G. Inhibition of muscular amino acid release by lipid infusion in man. Eur J Clin Invest 17: 301-304, 1987[ISI][Medline].

54.   Witters, LA, and Trasko CS. Regulation of hepatic free fatty acid metabolism by glucagon and insulin. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E23-E29, 1979[Abstract/Free Full Text].

55.   Wolfe, RR. Radioactive and stable isotope tracers in biomedicine. In: Principles and Practice of Kinetic Analysis. New York: Wiley-Liss, 1992.

56.   Wolfe, RR, Peters EJ, Klein S, Holland OB, Rosenblatt J, and Gary HJ. Effect of short-term fasting on lipolytic responsiveness in normal and obese human subjects. Am J Physiol Endocrinol Metab 252: E189-E196, 1987[Abstract/Free Full Text].

57.   Wu, G, and Thompson JR. The effect of ketone bodies on protein turnover in isolated skeletal muscle from fed and fasted chicks. Int J Biochem 22: 263-268, 1991.

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