Glucose production and substrate cycle activity in a fasting adapted animal, the northern elephant seal
Department of Biology, Sonoma State University, Rohnert Park, CA 94928, USA
* Author for correspondence (e-mail: champagn{at}sonoma.edu)
Accepted 22 December 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: glucose metabolism, glucose cycle, fasting, elephant seal
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Northern elephant seals fast for 23 months, during which time
metabolic requirements are met primarily through fatty acid oxidation
(Ortiz et al., 1978).
Postnatal development continues and the swimming and diving abilities
necessary for foraging at sea are acquired during the fast
(Reiter et al., 1978
). This
developmental period is evident in the increase in hematocrit, hemoglobin
concentration, mass specific blood volume, and myoglobin concentration that
occurs across the fast (Thorson and Le
Boeuf, 1994
). Based upon the observed developmental pattern, it
may be expected that glucose requirements would increase due to a persistent
requirement of some glucose-dependent tissues (e.g. CNS) and an increase in
the requirement of others (e.g. red blood cells). Estimates of the
contribution of glucose to metabolism during the fast are 15% of
average metabolic rate (AMR), but it is not known whether this increases with
time fasting (Keith and Ortiz,
1989
). Protein catabolism contributes less than 4% to the AMR
while fasting and declines with the progression of the fast
(Adams and Costa, 1993
;
Houser and Costa, 2001
;
Pernia et al., 1980
),
suggesting that contributions of protein to gluconeogenesis may decline across
the fast. However, blood glucose levels remain high throughout the fast
(135160 mg dl1;
Costa and Ortiz, 1982
;
Ortiz et al., 2003a
)
complicating the interpretation of the contribution of glucose to the
metabolism of the fasting and developing northern elephant seal weanling.
Monitoring glucose production across the postweaning fast can elucidate
whether decreased protein catabolism is inversely correlated with increased
glucose production, but accurate interpretation of how glucose is being
utilized cannot be addressed without giving consideration to pathways that
regulate glucose availability (e.g. substrate cycling;
Fig. 1) or provide substitutive
substrates for energy needs (e.g. ketone use by the CNS). Glucose cycling
provides a mechanism by which the total flux of glucose can be varied in
response to some regulating factor (Katz
and Rongstad, 1976; Newsholme, 1980) or substrate concentration
(Hue and Hers, 1974
). The
extent to which this variation can occur is related to the rate of cycling and
the endogenous glucose production (EGP, the combined output of gluconeogenesis
and glycogenolysis; Newsholme and
Crabtree, 1976
; Weber et al.,
1990
). More glucose can be made available for tissues if EGP
increases without a concomitant change in cycling, or rates of glucose cycling
decline without a concomitant reduction in endogenous glucose output (EGO;
equivalent to gluconeogenesis + glycogenolysis + glucose cycle activity).
Conversely, glucose availability can be reduced by increasing glucose cycle
activity without a change in EGO.
|
It is currently unknown how the demand for glucose varies with the progression of development and synthesis of glucose-demanding tissues in fasting elephant seal weanlings. Furthermore, it is unknown to what degree the glucose cycle plays a role in mediating glucose availability across the fast. The objectives of this study were to (1) assess glucose production and (2) investigate the glucose cycle as a possible regulatory mechanism for the availability of glucose in developing northern elephant seal pups during their postweaning fast. The study further investigated the relationship of glucose production and cycle activity to regulatory hormones and the relationship between glucose production and estimates of the contribution of glucose to the fasting metabolism of weanlings.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sample collection and processing
Using a bolus injection technique, a noncompartmental model was used to
describe the glucose kinetics of each seal
(Wolfe, 1992). Pups were
immobilized using an initial intramuscular injection of telazol
(teletamine/zolazepam HCl) at a dose of 1.0 mg kg1 and
administered intravenous doses of 50 mg ketamine and diazepam as needed to
maintain immobilization (all drugs from Fort Dodge Labs, Fort Dodge, IA, USA).
Mass was determined using a tripod and scale (MSI tension dynamometer,
±1.0 kg) in conjunction with a nylon restraint bag
(Ortiz et al., 1978
). Axillary
girth and curved length measurements were taken to provide an index of body
composition. Body composition index (BCI) was calculated as axillary girth
divided by the curved length. Blood samples were collected in chilled
heparinized vacutainers. Each animal was administered 0.1 mCi each of
[2-3H]glucose and [6-3H]glucose via the
extradural vein at the onset of the flux measurement. After injection, blood
samples were drawn at 5 min intervals for 30 min, then every 15 min thereafter
until 3 h post injection. Samples were stored on ice and transported to the
laboratory, centrifuged for 15 min at 800 g and 4°C, and
the plasma collected. Protein was precipitated from plasma by adding 1.5 ml of
barium hydroxide and zinc sulfate (0.3 N; Sigma-Aldrich, St Louis, MO, USA) to
1.0 ml plasma, vortexing, and chilling for 20 min in an icewater bath.
Samples were then centrifuged at 1800 g for 20 min and the
supernatant decanted and stored at 80°C until further analysis.
To distinguish 3H at the second carbon (C-2) from the sixth
carbon (C-6) of the glucose molecule, an enzymatic detritiation, developed by
Issekutz (1977) and modified
by Rooney et al. (1992
) was
utilized to selectively remove the 3H from C-2. Deproteinated
samples were thawed and passed through an ion exchange column containing
cation resin (AG 50W-X8 200-400 mesh hydrogen form) and anion resin (AG 1 X8
200-400 mesh formate form; both resins from Bio-Rad Laboratories, Hercules CA,
USA; Wolfe, 1992
). The eluate
was collected and lyophilized for 36 h to remove any 3H that had
exchanged with the plasma water. Dried samples were reconstituted in 1.0 ml of
133 mmol l1 phosphate buffer (pH 7.4) and divided into four
200 µl fractions. Two fractions were detritiated, two were non-detritiated,
and the remaining portion was used to determine glucose concentration of the
sample. To each fraction that was to be detritiated, 500 µl of a
detritiation solution was added. The detritiation solution consisted of 133
mmol l1 phosphate buffer, 8.4 mmol l1 ATP,
9.0 mmol l1 MgCl2, 2.4 units
ml1 hexokinase, and 10 units ml1
phosphoglucose isomerase (all reagents from Sigma, St Louis, MO, USA). The pH
of the final solution was adjusted to 7.4 with 1 mol l1
NaOH. To the non-detritiated aliquots, 500 µl of 133 mmol
l1 phosphate buffer was added. All samples were incubated in
a shaker water bath at 37°C for 2 h and were subsequently lyophilized
overnight. Samples were reconstituted in 500 µl of 1.0 mol
l1 H2SO4. Scintillation cocktail (6.5
ml; Econolite, Fisher, Pittsburg, PA, USA) was added to each sample and the
sample then agitated for 1 min. Sample activity was determined by liquid
scintillation counting on a Beckman LS 3801 scintillation spectrophotometer
(Beckman, Fullerton, CA, USA) using standard scintillation technique. A quench
correction factor was established for each sample based on a calculated H#
using a series of 3H standards with variable degrees of quench.
Glucose concentration of each sample was measured in duplicate on an YSI 2300
glucose autoanalyzer (YSI inc., Yellow Springs, Ohio, USA) and the specific
activity of counted samples determined. Since the non-detritiated fractions
contain [2-3H] and [6-3H]glucose, and the detritiated
fractions contain only [6-3H]glucose, the specific activity of
[2-3H]glucose (SA2) was determined by the equation:
![]() |
Single label tritiated glucose standards were run in parallel with samples to determine degree of detritiation of each isotope and correct for detritiation efficiency. Average detritiation of [2-3H]glucose was 96.3±2.9%. Within each assay detritiation efficiency was corrected for by multiplying sample SA6 by [2-3H]glucose standard detritiation efficiency. Average detritiation of [6-3H]glucose was less than 1.0%. Detritiation of [6-3H]glucose standards ranged from 3.0 to 7.0%; therefore, detritiation efficiency of less than |3|% was not corrected for, while efficiencies of 37% were adjusted for as above.
Hormone and metabolite analysis
Plasma samples drawn prior to tracer injection were thawed for use in
assays of insulin, glucagon, cortisol and glucose. Insulin was assayed using a
Sensitive Rat Insulin RIA kit (cat. no. SRI-13K, Linco Research Inc, St
Charles, MO, USA). This kit's stated specificity is 100% for rat, porcine,
sheep, hamster, and mouse. Glucagon was assayed with a Glucagon RIA kit (cat.
no. GL-32K, Linco Research Inc). Cortisol levels were measured using a
Cortisol RIA kit (cat. no. TKCO2, Diagnostic Products Corporation, Los
Angeles, CA, USA). All kits used for this study were validated by comparing
results from serially diluted samples of pooled elephant seal plasma to the
assay standard curve. Serially diluted pooled elephant seal plasma samples
displayed significant parallelism with the standard curves. The cortisol kit
has also been validated previously for this species
(Ortiz et al., 2001). Despite
the use of a sensitive insulin RIA kit, insulin values were frequently
measured near the lower detection limits of the assay and the mean intra-assay
coefficient of variation was 14.6%. Mean intra assay coefficient of variation
for glucagon was 5.9% and 5.5% for cortisol. Since insulin and glucagon are
antagonistic and it is believed that their molar ratios determine their
metabolic effect rather than absolute plasma concentrations (Kraus-Friedman,
1984), values of insulin and glucagon were used to calculate the
insulin:glucagon molar ratio.
Glucose concentration of plasma samples collected prior to isotope administration was processed in triplicate using a glucose autoanalyzer as above. ß-hydroxybutyrate (ßHBA) was assayed using a GM-7 Micro-Stat autoanalyzer (Analox Instruments Inc, Lunenburg, MA, USA).
Kinetic analysis
The rate of appearance of glucose was determined as:
![]() |
|
In vivo, hydrogen at C-2 of glucose is removed early in the conversion of glucose-6-phosphate to fructose-6-phosphate (Fig. 1). Therefore, endogenous glucose output (EGO= gluconeogenesis + glycogenolysis + glucose cycle activity) was measured as the rate of appearance of glucose with respect to [2-3H]glucose (Ra2). Hydrogen at C-6 is not removed until the phosphoenolpyruvate (PEP) and citric acid cycles, so endogenous glucose production (EGP=gluconeogenesis + glycogenolysis) was measured as the rate of appearance with respect to [6-3H]glucose (Ra6). Glucose cycle activity (GCA) was calculated as the difference between EGO and EGP (GCA=Ra2Ra6).
Statistical analysis
Early fasting and late fasting measurements were compared using paired
t-tests; only matched early and late fasting measurements were used
(seals 710 were removed for these comparisons) but reported means
(± S.E.M.) are calculated from all samples unless otherwise
stated. Paired longitudinal measurements of body condition and plasma hormone
concentration were used to examine relationships between changes in body
condition and regulatory hormones with changes in plasma glucose
concentration.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
EGO during early fasting was 3.16±0.87 mg glucose kg1 min1 and during late fasting was 2.43±0.24 mg glucose kg1 min1. There was no significant change in EGO over fasting duration (t=1.87, P=0.12). EGP during early fasting was 2.80±0.65 mg glucose kg1 min1 and during late fasting was 2.21±0.12 mg glucose kg1 min1. There was a strong trend towards decreased EGP with fasting duration (t=2.46, P=0.057).
There was a large degree of variation in GCA during both sampling periods. Early fasting GCA ranged from 0.045 to 0.764 mg glucose kg1 min1, mean 0.361 mg glucose kg1 min1. Late fasting GCA ranged from 0 to 0.475 mg glucose kg1 min1, mean 0.221 mg glucose kg1 min1. Mean GCA was 12.2±7.6% of EGP during early fasting and 10.1±10.4% during late fasting. Proportional GCA (pGCA=GCA/EGP) changed by less than 5% in four animals (seals 1, 3, 4 and 5), increased by 13.2% in seal 2, and decreased dramatically in seal 6, going from the highest proportional GCA, 21.4% to one of the lowest, 0.1%.
There was no significant change in plasma glucose over the study period (t=2.25, P=0.075); mean plasma glucose was 154.4±15.5 mg dl1 during early fasting and 143.5±8.5 mg dl1 during late fasting. ßHBA increased across the postweaning fast from 0.77±0.20 mmol l1 during early fasting to 1.65±0.39 mmol l1 during late fasting (t=5.54, P=0.004). Hormone levels measured in all study animals are presented in Table 2. Mean plasma insulin did not significantly change throughout the fast; mean concentration during early fasting was 67.6±10.5 pg ml1 while during late fasting it was 57.1±13.6 pg ml1. Mean plasma glucagon concentration significantly increased (t=2.96, P=0.031) between early fasting (51.6±14.0 pg ml1) and late fasting (69.4±17.0 pg ml1). Insulin:glucagon (I:G) molar ratio during early fasting was 0.83±0.22 and during late fasting was 0.49±0.16 and the ratio significantly decreased across the fast (t=5.27, P=0.003). Mean cortisol level during early fasting was 5.33±2.61 µg ml1 and 6.13±2.27 µg ml1 during late fasting. Cortisol levels showed a small but significant increase over the postweaning fast (t=2.84, P=0.037).
|
The magnitude of change in EGP decreased with the reductions in BCI
(Fig. 3,
EGP=1.257.13
BCI; r2=0.85,
P=0.01). The magnitude of change in plasma glucose (
PG) varied
inversely with that of glucagon (
PG= 2.100.85
glucagon;
r2=0.75, P=0.02) and directly with that of I:G
(Fig. 4,
PG=6.13+66.64
I:G; r2=0.78, P=0.02).
Decreased I:G ratio was associated with decreased plasma glucose. The change
in insulin was positively correlated with the rise in cortisol levels
(r=0.82, P=0.048). There was no relationship between the
changes in EGP and plasma glucose, insulin, glucagon, I:G, or cortisol
(P>0.5).
|
|
Considering only the early fasting samples, EGP decreased with plasma
glucose (EGP=7.960.0335PG, r2=0.63,
P=0.006) and with _HBA (EGP=4.582.31
_HBA,
r2=0.52, P=0.018). Proportional glucose cycle
activity decreased with days fasting (pGCA=0.4640.0203 days fasting,
r2=0.63, F=13.4, P=0.006). There was no
correlation between insulin, glucagon, I:G ratio or cortisol level with plasma
glucose or EGP (P>0.05). None of these relationships were
significant in the late fasting measurements (P>0.05), although,
glucagon concentration was negatively correlated with EGO
(r=0.84, P=0.036).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is unlikely that there is any significant change across the measurement
interval in the mass of the CNS or renal medulla that accompanies an increase
in mass-specific red blood cell volume
(Thorson and Le Boeuf, 1994),
suggesting that an overall increase in mass-specific glucose-dependent tissues
occurs with the progression of the fast. This is compounded by an apparent
increase in physical activity with time fasting that is related to the
development of diving. Despite this, gluconeogenesis decreased over the fast
while plasma glucose levels essentially remained the same. This suggests that
the mass-specific utilization of glucose becomes more efficient with the
progression of the fast, possibly due to a reduction in the utilization of
glucose by tissues that can exploit substrates other than glucose for energy.
This compensation would make more glucose available to glucose-dependent
tissues, and if it proportionally matched the consumption requirements of the
de novo synthesized glucose-dependent tissues, could result in the
stasis of plasma glucose levels observed across the fast.
Contrary to expectations in a fasting animal, glucose production values in
fasting northern elephant seal weanlings were higher than non-fasting adapted
species undertaking similar duration fasts
(Table 3). During the early
fasting measurement, average glucose production was 2.8 mg
kg1 min1 and mean mass was 104 kg,
resulting in an average of 2.3 moles of glucose produced per day. To explore
the energetic demands of glucose-dependent tissues, the glucose consumption of
the brain and red blood cells were estimated using measurements made in
elephant seals and closely related phocid species. Murphy et al.
(1980) and Hochachka
(1981
) estimated that, for a
500 kg Weddell seal Leptonychotes weddellii with 0.5 kg brain,
0.30.4 µmol g1 min1 of glucose
was required to support a CNS metabolic rate of 6 µmol ATP
g1 min1. Assuming a 250 g brain for a 100
kg elephant seal pup, and using the same mass-specific energy requirements as
estimated for the Weddell seal, glucose utilization for the brain amounts to
0.126 mol d1. Plasma ketone levels more than doubled over
the fasting period, and values were similar to those reported in Castellini
and Costa (1990
), but levels
remain much lower than required to induce CNS utilization of ketone as a fuel
source in non-fasting adapted mammals
(Felts et al., 1964
;
Nehlig and de Vasconcelos,
1993
). Since blood glucose is elevated in weanlings, it seems
likely that most of the energy for nervous system metabolism is supplied by
glucose. If the CNS of fasting elephant seals is capable of utilizing ketones
at low plasma concentrations (e.g. high flux but low hemococentration), the
demand for glucose would be further reduced. Nordøy and Blix
(1991
) measured ketone
turnover rates in fasting grey seals of 9.313.8 µmol
kg1 min1 and argued that ketone
utilization facilitates protein sparing in this species. For the calculations
made here, no ketone utilization by glucose-dependent tissues was assumed.
Thus our estimates are potentially biased upwards, and actual glucose
utilization may be less than estimated here if elephant seals are adapted to
ketone utilization at lower plasma concentrations.
|
At 2 weeks postweaning, elephant seal pups have approximately 6.05 l of red
blood cells (Thorson and Le Boeuf,
1994). Using rates of glucose consumption measured in red blood
cells by Castellini et al.
(1992
), and correcting for the
temperature dependence of enzymatic activity, it is estimated that red blood
cells require approximately 700 nmol glucose (ml RBC)1
h1. This comes to 0.102 moles glucose consumed by RBC per
day. Therefore, total glucose demand by CNS and erythrocytes is 0.228 moles
glucose per day; this represents only 10% of the glucose produced each day.
Renal glucose utilization accounts for 1025% of glucose turnover in
humans (Cersosimo et al., 1999
)
and as much as 30% in dogs (Cersosimo et
al., 1994
) in the post absorptive state. As reliable data on renal
glucose consumption during long duration fasting or in phocid seals are
unavailable at this time, we have not included estimates of glucose
utilization by the renal medulla in our calculations. Castellini et al.
(1987
) estimated that an
elephant seal pup of 98 kg had 686 µmoles glycerol min1
available for gluconeogenesis. If all available glycerol were converted to
glucose, it would account for 0.49 moles of glucose each day; leaving
1.8
moles of glucose production unaccounted for, but meeting the glucose
requirement of the glucose-dependent tissues. It is not practical for a
fasting adapted animal to make up the balance catabolizing lean tissue, and
previous research has demonstrated lean tissue loss to be low. Houser and
Costa (2001
) estimated that
over 6 weeks of fasting, pups lose only 1.8 kg of lean tissue. Assuming that
all of the lost lean mass produced amino acids in proportion to those observed
during the late fasting period in weanlings
(Houser and Crocker, 2004
),
and that the gluconeogenic precursors that make up the majority of the amino
acid pool (i.e., alanine, glutamine and glycine) are converted to glucose, it
is crudely estimated that
3.9 moles of glucose would be formed. This
value is certainly an overestimate but provides a theoretical ceiling to which
experimental results can be compared. Given this theoretical ceiling and
assuming a constant rate of production of 2.3 mol glucose
d1, the contribution of amino acids would only constitute
4% of the glucose produced over a 6-week period. Keith
(1984
), after injection of
U[14C]alanine, found only minor amounts of labeled carbon
incorporated into glucose, which agrees with estimates of a minimal
contribution of lean tissue to gluconeogenesis.
The apparent disparity between EGP and the estimated contribution of
glycerol and amino acids to gluconeogenesis suggests that other remaining
potential precursors (namely lactate and pyruvate) are utilized. Erythrocytes
represent a significant glucose-dependent tissue in this species. In adult
elephant seals 21% of their body mass is blood. In pups the value is closer to
11%, with hematocrit levels greater than 50%
(Thorson and Le Boeuf, 1994).
The primary mechanism by which erythrocytes meet metabolic costs is the
breakdown of glucose to lactate. Produced lactate may subsequently act as a
gluconeogenic precursor with resulting glucose released back into circulation.
This form of glucose cycling was first hypothesized by Cori
(1931
) as a process of
importance in the regulation of blood glucose. Owen et al.
(1969
) proposed that Cori
cycle activity may be important in fasting humans and glucose oxidation
findings of Keith and Ortiz
(1989
; <2.5% of EGP) in
elephant seals led them to suggest that the Cori cycle was the primary
mechanism of recycling of radioactive carbon in fasting elephant seal pups.
Tayek and Katz (1997
)
determined that Cori cycle activity accounted for 20% of gluconeogenesis after
an overnight fast in normal man and over 33% in some cancer patients.
In the fasting state, the energy to fuel gluconeogenesis is likely supplied
through fatty-acid oxidation. Glucose that is released following hepatic
gluconeogenesis could be utilized by erythrocytes and kidney medulla
via the Cori cycle. This would allow glucose to act as a shuttle for
ATP between fat oxidation in liver and glycolysis in glucose-dependent tissue.
By recycling glucose in this manner, elephant seals may provision erythrocytes
and the renal medulla using the energy from fat. However, liberally estimating
that erythrocyte and renal medulla glucose consumption are met via the Cori
cycle, the CNS meets its energetic needs through the complete oxidation of
glucose, and that the contributions to gluconeogenesis from glycerol and amino
acids liberated via lean mass catabolism are complete and irreversible, less
than 20% of EGP can be accounted for. This presents a conundrum if the rest of
the EGP is equated to Cori cycling since it is unknown which tissues would be
contributing to the remaining 80% of net glucose flux. Regardless, the rates
of gluconeogenesis as well as glucose recycling appear higher in elephant
seals than other fasting animals studied
(Katz et al., 1974). This
degree of futile cycling suggested by the glucose flux measurements made here
implies an energetically inefficient system and suggests that protection of
lean tissue is more important than energetic efficiency. Goodman et al.
(1980
) as well as Henry et al.
(1988
) suggested that
suppression of metabolic rate during long-term fasting is essential to
facilitate protein sparing. Sparing of amino acid precursors may take priority
over energy efficiency during extended fasts provided extensive lipid energy
reserves are available. This study found that weanlings maintaining body
composition over the fasting duration reduced glucose production to a greater
extent than weanlings greatly decreasing BCI
(Fig. 3). However, using our
methods we are unable to differentiate between the gluconeogenic substrates
that accounted for the decrease in glucose production. It remains unclear
whether the change in body composition impairs weanlings ability to spare
protein or if the loss in lipid reserves is associated with increased Cori
cycle activity.
The regulatory role of the glucose cycle is best viewed as a proportion of
EGP. Variation in proportional glucose cycle activity was high between
individuals, but taken as a whole, the proportion was nearly consistent
between early and late fasting periods. Values of pGCA found in this study are
similar to those reported in other species measured: 12.6% in post-absorptive
dog (Issekutz, 1977) and from
1125% in post-absorptive humans
(Shulman et al., 1985
;
Neely et al., 1992
;
Rooney et al., 1992
;
Heaney et al., 1997
). There is
no evidence to suggest that the glucose cycle varies the regulation of glucose
availability across the fast; rather, it changes in parallel with EGP. It is
worth noting that the high plasma glucose concentration after extended fasting
observed in elephant seals is near that of post-absorptive carnivores
(Opazo et al., 2004
). A change
in GCA may be expected if the demand for glucose changes over the fast,
provided there was a premium on glucose availability. In contrast, the
northern elephant seal weanling may stabilize glucose utilization by making
glucose available for consumption in excess, but by reducing the sensitivity
of non-target tissues to glucose uptake. It has been suggested that elephant
seals are insensitive to insulin and there was no relationship between GCA,
hormone levels or I:G, suggesting an insensitivity of regulatory processes to
hormonal variation. This contrasts with previous studies in non-fasting
adapted mammals which demonstrated that both insulin
(Rooney et al., 1992
) and
glucagon increase GCA (Issekutz,
1977
; Miyoshi et al.,
1988
). However, Wolfe
(1992
) has argued that
alterations in GCA in response to hormonal changes are not due to a direct
affect on GCA, but that GCA changes in proportion to total flux; i.e. glucose
cycling is a passive consequence of total glucose production and not under
hormonal control. The results of this study agree with this assertion.
A small but significant increase in cortisol was measured across the study
period. Increased cortisol with fasting is common in seals
(Guinet et al., 2004;
Ortiz et al., 2003b
). Cortisol
has been shown to increase gluconeogenesis
(Friedmann et al., 1967
;
Issekutz and Allen, 1972
) as
well as increasing glucose recycling
(Goldstein et al., 1993
).
However, we found no relationship between cortisol levels and glucose
production or cycle activity. Ortiz et al.
(2003b
) measured a much larger
increase in cortisol, 5.8 µg dl1 early in the postweaning
fast and 14.1 µg dl1 at the end of the fast.
Previous studies have shown a reduction in plasma insulin concentration
with the progression of fasting, but the effect of fasting on glucagon is less
clear. Glucagon did not change over the fasting duration in rat
(Goodman et al., 1980), while
in man glucagon levels rose early in the fast and then returned to baseline
values (Marliss et al., 1970
).
In contrast, glucagon increased across the fast in penguin
(Cherel et al., 1988
). Previous
research in elephant seals has found decreases in insulin and increases in
glucagon over the postweaning fast (Ortiz
et al., 2003a
). Ortiz et al.
(2003a
) measured insulin
levels of 35 µU ml1 and glucagon levels of
80
pg ml1 in pups fasting less than 1 week; and 23 µU
ml1 insulin and
190 pg ml1 glucagon
in pups fasting 68 weeks. The study by Ortiz et al. involved a much
greater sample size (N=40) and values measured were higher than those
of this study, especially glucagon during late fasting. Although the pattern
of variation in hormone levels is similar between the two studies, the large
disparities in glucagon and cortisol values can only be speculated upon until
more data are collected on the levels of these hormones at similar intervals.
Some possible explanations for these discrepancies include the smaller sample
size of the current study and the possibility that there were sampling
differences within the 68 week late fasting sampling range.
In this study, as expected, the insulin-glucagon ratio decreased over the
fast. A similar trend is found in fasting penguins
(Cherel et al., 1988) and
humans undergoing a long duration fast
(Streja et al., 1977
),
although absolute hormone levels were much higher in both penguins and humans.
Decreasing I:G ratio is indicative of an upregulation of catabolic processes
and stimulates glucose release by the liver and renal cortex. Contrary to
expectations, decreased I:G ratio was accompanied by decreased levels of
plasma glucose (Fig. 4).
Increased levels of glucagon should lead to phosphorylation and inactivation
of pyruvate kinase and stimulation of phosphoenolpuyruvate carboxykinase
transcription and activity, allowing for an increase in gluconeogenesis
(Jiang and Zhang, 2003
).
Previous studies in this species have not detected an insulin response to
glucose injection (Kirby and Ortiz,
1994
) and it has been proposed that elephant seals do not closely
regulate blood glucose by the conventional insulin-glucagon push pull model.
In this study we found no relationship between insulin, glucagon, or I:G ratio
to EGP or GCA. This lack of correlation between hormone levels and glucose
production support the conclusions of Kirby and Ortiz
(1994
). However, this may also
be due to the high degree of individual variation in EGP and GCA. More
controlled longitudinal studies involving hormonal manipulations may be needed
to reveal regulatory effects of hormones that are masked by individual
variability.
Placing the results of the glucose flux experiment and hormonal
measurements into proper context cannot be done without some consideration
being given to potential confounding factors. One such factor is the use of
immobilizing drugs during the measurement period. The impact of ketamine and
tiletamine/zolazepam on glucose kinetics is unknown and there was a large
degree of variation in the total amount of drugs required to maintain
immobilization during the measurement period, 2.07.5 ml of ketamine;
initial immobilization with tiletamine/zolazepam was standardized to animal
mass. Despite this variation, there was no relationship between the amount of
drugs administered with EGO, EGP or GCA (F=0.6-1.8,
r2=0.040.10, P=0.230.45) suggesting
that there was no effect of the maintenance schedule on glucose production or
cycle activity. Another potential source of variation may be the incomplete
removal of tritium from the administered tracers via the predicted pathways,
or loss via unaccounted for pathways. Errors due to these processes may cause
EGP to be underestimated by 3% and EGO by as much as 20%
(Landau, 1993). There are no
data on in vivo loss of label in seals and we report uncorrected data
in this study with the understanding that these values represent conservative
estimates of both GCA and EGP. This underestimation is probably the cause of
zero and slightly negative GCA values that were occasionally observed.
In summary, we measured unexpectedly high levels of EGP in weaned elephant seals that decreased across the duration of the fast. GCA did not appear to play an important role in regulating EGP across the fast. Proportional glucose cycle activity was highly variable between individuals, but, overall, was nearly consistent between early and late fasting periods and was similar to those reported in other species in which measurements have been made. High levels of EGP relative to estimated glucose utilization and gluconeogenic substrate availability suggest extensive recycling of glucose and a potential increased importance of Cori cycle activity in fasting elephant seals.
List of abbreviations
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, S. H. and Costa, D. P. (1993). Water conservation and protein metabolism in northern elephant seal pups during the postweaning fast. J. Comp. Physiol. B 163,367 -373.[Medline]
Castellini, M. A., Costa, D. P. and Huntley, A. C. (1987). Fatty acid metabolism in fasting elephant seal pups. J. Comp. Physiol. B 157,445 -449.[Medline]
Castellini, M. J. and Costa, D. P. (1990). Relationships between plasma ketones and fasting duration in neonatal elephant seals. Am. J. Physiol. 259,R1086 -R1089.[Medline]
Castellini, M. A., Castellini, M. and Kirby, V. L. (1992). Effects of standard anticoagulants and storage procedures on plasma glucose values in seals. J. Am. Vet. Med. Assn. 201,145 -148.[Medline]
Cersosimo, E., Judd, R. L. and Miles, J. M. (1994). Insulin regulation of renal glucose metabolism in conscious dogs. J. Clin. Invest. 93,2584 -2589.[Medline]
Cersosimo, E., Garlick, P. and Ferretti, J. (1999). Insulin regulation of renal glucose metabolism in humans. Am. J. Physiol. 276,E78 -E84.[Medline]
Cherel, Y., Leloup, J. and Le Maho, Y. (1988). Fasting in king penguin. II. Hormonal and metabolic changes during molt. Am. J. Physiol. 254,R178 -R184.[Medline]
Cori, C. F. (1931). Mammalian carbohydrate
metabolism. Physiol. Rev.
11,143
-275.
Costa, D. P. (1991). Reproductive and foraging energetics of pinnipeds: implications for life history paterns. In The Behavior of Pinnipeds (ed. D. Renouf). London: Chapman and Hall.
Costa, D. P. and Ortiz, C. L. (1982). Blood chemistry homeostasis during prolonged fasting in the northern elephant seal. Am. J. Physiol. 242,R591 -R595.[Medline]
Cowan, J. S., Vranic, M. and Wrenshall, G. A. (1969). Effects of preceding diet and fasting on glucose turnover in normal dogs. Metabolism 18,319 -330.[CrossRef][Medline]
Dunn, A., Katz, J., Golden, S. and Chenoweth, M.
(1976). Estimation of glucose turnover and recycling in rabbits
using various [3H, 14C]glucose labels. Am. J. Physiol.
230,1159
-1162.
Felts, P. W., Crofford, O. B. and Park, C. R. (1964). Effect of infused ketone bodies on glucose utilization in the dog. J. Clin. Invest. 43,638 -646.[Medline]
Friedmann, N., Exton, J. and Park, C. (1967). Interaction of adrenal steroids and glucagon on gluconeogenesis in perfused rat liver. Bioch. Biophys. Res. Commun. 29,113 -119.[CrossRef][Medline]
Goldstein, R. E., Wasserman, D. H., McGuinness, O. P., Lacy, D. B., Cherrington, A. D. and Abumrad, N. N. (1993). Effects of chronic elevation in plasma cortisol on hepatic carbohydrate metabolism. Am. J. Physiol. 264,E119 -E127.[Medline]
Goodman, M. N., Larsen, P. R., Kaplan, M. M., Aoki, T. T., Young, V. R. and Ruderman, N. B. (1980). Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism. Am. J. Physiol. 239,E277 -E286.[Medline]
Guinet, C., Servera, N., Mangin, S., Georges, J. Y. and Lacroix, A. (2004). Change in plasma cortisol and metabolites during the attendance period ashore in fasting lactating subantarctic fur seals. Comp. Biochem. Physiol. 137A,523 -531.
Heaney, A. P., Harper, R., Ennis, C., Rooney, D. P., Sheridan, B., Atkinson, A. B. and Bell, P. M. (1997). Insulin action and hepatic glucose cycling in Cushing's syndrome. Clin. Endocrinol. 46,735 -743.[CrossRef][Medline]
Henry, C. J., Rivers, J. P. and Payne, P. R. (1988). Protein and energy metabolism in starvation reconsidered. Eur. J. Clin. Nutr. 42,543 -549.[Medline]
Hochachka, P. W. (1981). Brain, lung, and heart functions during diving and recovery. Science 212,509 -514.[Medline]
Houser, D. S. and Costa, D. P. (2001). Protein catabolism in suckling and fasting northern elephant seal pups (Mirounga angustirostris). J. Comp. Physiol. B 171,635 -642.[Medline]
Houser, D. S. and Crocker, D. E. (2004). Age, sex, and reproductive state influence free amino acid concentrations in the fasting elephant seal. Physiol. Biochem. Zool. 77,838 -846.[CrossRef][Medline]
Hue, L. and Hers, H. G. (1974). Utile and futile cycles in the liver. Biochem. Biophys. Res. Commun. 58,540 -548.[Medline]
Issekutz, B. (1977). Studies on hepatic glucose cycles in normal and methylprednisolone-treated dogs. Metabolism 26,157 -169.[CrossRef][Medline]
Issekutz, B. and Allen, M. (1972). Effect of catecholamines and methylprednisolone on carbohydrate metabolism of dogs. Metabolism 21,48 -59.[CrossRef][Medline]
Jiang, G. and Zhang, B. B. (2003). Glucagon and regulation of glucose metabolism. Am. J. Physiol. 284,E671 -E678.
Katz, J. and Rognstad, R. (1976). Futile cycles in the metabolism of glucose. In Current Topics in cellular Regulation, vol. 10 (ed. B. L. Horecker and E. R. Stadtman), pp. 237-289. New York: Academic Press.[Medline]
Katz, J. and Tayek, J. A. (1998). Gluconeogenesis and the Cori cycle in 12-, 20-, and 40-h-fasted humans. Am. J. Physiol. 275,E537 -E542.[Medline]
Katz, J., Dunn, A., Chenoweth, M. and Golden, S. (1974). Determination of synthesis, recycling and body mass of glucose in rats and rabbits in vivo 3H- and 14C-labelled glucose. Biochem. J. 142,171 -183.[Medline]
Keith, E. O. (1984). Glucose metabolism in fasting northern elephant seal pups. In Biology, pp.101 . Santa Cruz: University of California.
Keith, E. O. and Ortiz, C. L. (1989). Glucose kinetics in neonatal elephant seals during postweaning aphagia. Mar. Mamm. Sci. 5,99 -115.
Kirby, V. L. and Ortiz, C. L. (1994). Hormones and fuel regulation in fasting elephant seals. In Elephant Seals: Population Ecology, Behavior, and Physiology (ed. B. J. Le Boeuf and L. R. M.), pp. 374-386. Berkeley: University of California Press.
Kraus-Friedmann, N. (1984). Hormonal regulation
of hepatic gluconeogenesis. Physiol. Rev.
64,170
-259.
Landau, B. R. (1993). Measuring glucose and fructose-6-phosphate cycling in liver in vivo. Metabolism 42,457 -462.[CrossRef][Medline]
Marliss, E. B., Aoki, T. T., Unger, R. H., Soeldner, J. S. and Cahill, G. F., Jr (1970). Glucagon levels and metabolic effects in fasting man. J. Clin. Invest. 49,2256 -2270.[Medline]
Miyoshi, H., Shulman, G. I., Peters, E. J., Wolfe, M. H., Elahi, D. and Wolfe, R. R. (1988). Hormonal control of substrate cycling in humans. J. Clin. Invest. 81,1545 -1555.[Medline]
Murphy, B., Zapol, W. M. and Hochachka, P. W.
(1980). Metabolic activities of heart, lung, and brain during
diving and recovery in the Weddell seal. J. Appl.
Physiol. 48,596
-605.
Neely, R. D., Rooney, D. P., Bell, P. M., Bell, N. P., Sheridan, B., Atkinson, A. B. and Trimble, E. R. (1992). Influence of growth hormone on glucose-glucose 6-phosphate cycle and insulin action in normal humans. Am. J. Physiol. 263,E980 -E987.[Medline]
Nehlig, A. and de Vasconcelos, A. P. (1993). Glucose and ketone body utilization by the brain of neonatal rats. Prog. Neurobiol. 40,163 -221.[CrossRef][Medline]
Newsholme, E. and Crabtree, B. (1976). Substrate cycles in metabolic regulation and in heat generation. Biochem. Soc. Symp. 41,61 -109.[Medline]
Nordøy, E. S. and Blix, A. S. (1991). Glucose and ketone body turnover in fasting grey seal pups. Acta Physiol. Scand. 141,563 -571.
Opazo, J. C., Soto-Gamboa, M. and Bozinovic, F. (2004). Blood glucose concentration in caviomorph rodents. Comp. Biochem. Physiol. A 137, 57-64.
Ortiz, C. L., Costa, D. P. and Le Boeuf, B. J. (1978). Water and energy flux in elephant seal pups fasting under natural conditions. Physiol. Zool. 51,166 -178.
Ortiz, R. M., Wade, C. E. and Ortiz, C. L. (2001). Effects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups. Am. J. Physiol. 280,R790 -R795.
Ortiz, R. M., Noren, D. P., Ortiz, C. L. and Talamantes, F.
(2003a). GH and ghrelin increase with fasting in a naturally
adapted species, the northern elephant seal (Mirounga
angustirostris). J. Endocrinol.
178,533
-539.
Ortiz, R. M., Houser, D. S., Wade, C. E. and Ortiz, C. L. (2003b). Hormonal changes associated with the transition between nursing and natural fasting in northern elephant seals (Mirounga angustirostris). Gen. Comp. Endocrinol. 130, 78-83.[CrossRef][Medline]
Owen, O. E., Felig, P., Morgan, A. P., Wahren, J. and Cahill, G. F., Jr (1969). Liver and kidney metabolism during prolonged starvation. J. Clin. Invest. 48,574 -583.[Medline]
Pernia, S. D., Hill, A. and Ortiz, C. L. (1980). Urea turnover during prolonged fasting in the northern elephant seal. Comp. Biochem. Physiol. 65B,731 -734.
Reiter, J., Stinson, N. L. and Le Boeuf, B. J. (1978). Northern Elephant Seal Development: The Transition from Weaning to Nutritional Independence. Behav. Ecol. Sociobiol. 3,337 -367.[CrossRef]
Rooney, D. P., Neely, R. D., Ennis, C. N., Bell, N. P., Sheridan, B., Atkinson, A. B., Trimble, E. R. and Bell, P. M. (1992). Insulin action and hepatic glucose cycling in essential hypertension. Metabolism 41,317 -324.[Medline]
Shulman, G. I., Ladenson, P. W., Wolfe, M. H., Ridgway, E. C. and Wolfe, R. R. (1985). Substrate cycling between gluconeogenesis and glycolysis in euthyroid, hypothyroid, and hyperthyroid man. J. Clin. Invest. 76,757 -764.[Medline]
Streja, D. A., Steiner, G., Marliss, E. B. and Vranic, M. (1977). Turnover and recycling of glucose in man during prolonged fasting. Metabolism 26,1089 -1098.[CrossRef][Medline]
Tayek, J. A. and Katz, J. (1997). Glucose production, recycling, Cori cycle, and gluconeogenesis in humans: relationship to serum cortisol. Am. J. Physiol. 272,E476 -E484.[Medline]
Thorson, P. H. and Le Boeuf, B. J. (1994). Developmental aspects of diving in northern elephant seal pups. In Elephant Seals: Population Ecology, Behavior, and Physiology (ed. B. J. Le Boeuf and R. M. Laws), pp.271 -289. Berkeley: University of California Press.
Weber, J.-M., Klein, S. and Wolfe, R. R.
(1990). Role of the glucose cycle in control of net glucose flux
in exercising humans. J. Appl. Physiol.
68,1815
-1819.
Wolfe, R. R. (1992). Radioactive and stable isotope tracers in biomedicine: principles and practice of kinetic analysis. New York: Wiley-Liss Inc.