Effects of fasting and glucocorticoids on hepatic
gluconeogenesis assessed using two independent methods in vivo
Richard E.
Goldstein1,
Luciano
Rossetti3,
Brett A. J.
Palmer1,
Rong
Liu3,
Duna
Massillon3,
Melanie
Scott2,
Doss
Neal2,
Phillip
Williams2,
Benjamin
Peeler1, and
Alan D.
Cherrington2
1 Department of Surgery, Vanderbilt University, and
the Nashville VA Medical Center; 2 Department of
Molecular Physiology and Biophysics, Vanderbilt University,
Nashville, Tennessee 37232; and 3 Division of
Endocrinology, Department of Medicine, Albert Einstein College of
Medicine, New York, New York 10461
 |
ABSTRACT |
The purpose of this study was to
compare the assessment of gluconeogenesis (GNG) in the overnight- and
prolonged-fasted states and during chronic hypercortisolemia using the
arteriovenous difference and
[14C]phosphoenolpyruvate-liver biopsy
techniques as well as a combination of the two. Two weeks before a
study, catheters and flow probes were implanted in the hepatic and
portal veins and femoral artery of dogs. Animals were studied after an
18-h fast (n = 8), a 42- or 66-h fast
(n = 7), and an 18-h fast plus a continuous infusion of
cortisol (3.0 µg · kg
1 · min
1) for
72 h (n = 7). Each experiment consisted of an
80-min tracer ([3-3H]glucose and
[U-14C]alanine) and dye equilibration period (
80 to 0 min) and a 45-min sampling period. In the cortisol-treated group,
plasma cortisol increased fivefold. In the overnight-fasted group,
total GNG flux rate (GNGflux), conversion of glucose
6-phosphate to glucose (GNGG-6-P
Glc), glucose
cycling, and maximal GNG flux rate (GNGmax) were 0.95 ± 0.14, 0.65 ± 0.06, 0.62 ± 0.06, and 0.70 ± 0.09 mg · kg
1 · min
1,
respectively. In the prolonged-fasted group, they were 1.50 ± 0.18, 1.18 ± 0.13, 0.40 ± 0.07, and 1.28 ± 0.10 mg · kg
1 · min
1, whereas in
the cortisol-treated group they were 1.64 ± 0.33, 0.99 ± 0.29, 1.32 ± 0.24, and 0.91 ± 0.13 mg · kg
1 · min
1. These
results demonstrate that GNGG-6-P
Glc and GNGmax were almost identical. However, these rates were
15-38% lower than GNGflux generated by a
combination of the two methods. This difference was most apparent in
the steroid-treated group, where the combination of the two methods
(GNGflux) detected a significant increase in gluconeogenic flux.
fasting; cortisol
 |
INTRODUCTION |
THE QUEST TO
DEVELOP accurate methods for determining the rate of
gluconeogenesis in vivo is ongoing. Many early studies used tracer
methods or arteriovenous (a-v) difference techniques to assess the
gluconeogenic rate in vivo (12, 23, 27, 28). In
1977, Chiasson et al. (5) combined these two approaches for the assessment of gluconeogenesis in the dog. The latter approach yielded two estimates of gluconeogenesis, the gluconeogenic efficiency (percent extracted gluconeogenic carbon converted to glucose) and the
gluconeogenic conversion rate (the rate of glucose production from the
gluconeogenic precursor given). Both represented minimal estimates of
gluconeogenesis due largely to dilution of the tracer in the
oxaloacetate (OAA) pool of the liver (25). This approach was further extended by calculating the minimal and maximal rates of
gluconeogenesis from circulating gluconeogenic precursors (3, 16,
51). The maximal rate was derived by measuring the net hepatic
uptakes of all gluconeogenic precursors using the a-v difference
technique and assuming that they were quantitatively converted to
glucose. The minimal rate was derived by multiplying the maximal rate
of gluconeogenesis by gluconeogenic efficiency (which is itself a
minimum). With both of these rates known, the true gluconeogenic rate
from circulating precursors could be bracketed. It should be noted that
this approach underestimates the actual maximal rate of hepatic
gluconeogenesis by an amount equal to the contribution of precursors
derived from within the liver, namely from intrahepatic proteolysis
and/or lipolysis.
Recently, Giaccari and Rossetti (13) and Rossetti et al.
(47) described a method which permits one to quantify
hepatic gluconeogenesis in a more direct manner. Like that of
Cherrington et al. (3), the method utilizes the infusion
of a labeled gluconeogenic precursor, such as
[14C]alanine, but also requires a biopsy of hepatic
tissue. HPLC analysis is used to determine the intrahepatic specific
activities of uridine diphosphoglucose (UDPglucose) and
phosphoenolpyruvate (PEP) (14). The direct
assessment of PEP specific activity overcomes the major limitation of
tracer methods previously used to estimate gluconeogenesis, the
dilution of tracer in the OAA pool. This information is also used to
calculate the rates of hepatic glucose cycling and the net
glycogenolytic contribution to hepatic glucose release. The requirement
for a liver biopsy limits the applicability of the method;
nevertheless, it represents a viable and independent alternative method
for determining the rate of hepatic gluconeogenesis in experimental
animals. It should be noted, however, that it measures hepatic
gluconeogenesis from lactate, pyruvate, and amino acids (both
circulating and intrahepatic) but does not account for gluconeogenesis
from glycerol. Thus it also underestimates the total hepatic
gluconeogenic rate to a certain extent.
The first aim of the present study, therefore, was to compare the
results obtained using the a-v difference technique with those obtained
simultaneously using the methodology of Rossetti and Giaccari
([14C]PEP technique) under conditions of overnight
fasting, which favors glycogenolysis, and more prolonged fasting, which
favors gluconeogenesis. The second aim was to combine information
gained from these two methodologies to develop a new, more powerful
approach to quantify hepatic gluconeogenesis in vivo.
Goldstein et al. (15) previously reported that chronic
hypercortisolemia caused only a marginal increase in gluconeogenesis as
assessed using the combined tracer a-v difference technique but that it
increased glucose cycling drastically. This finding was surprising in
view of in vitro observations (8, 11) and in vivo data
(11, 24) that characterize cortisol as a gluconeogenic hormone. Therefore, an additional aim of the present study was to
assess the gluconeogenic effects of hypercortisolemia in vivo using the
combined a-v difference-[14C]PEP approach and to
determine whether the combination of the methodologies would yield
further insight into the intrahepatic gluconeogenic fluxes associated
with hypercortisolemia.
 |
RESEARCH DESIGN AND METHODS |
Animals and surgical procedures.
Experiments were performed on a total of 22 mongrel dogs (16-27
kg) of either gender that had been fed a weight-maintaining diet of
meat and chow (Purina Mills, St. Louis, MO and KalKan, Vernon, CA; 31%
protein, 52% carbohydrate, 11% fat, and 6% fiber based on dry weight
once daily).
Animal preparation has been extensively described in previous articles
(1, 15). In brief, under general endotrachial anesthesia,
a sampling catheter was placed directly in the portal vein, and its tip
was positioned ~1 in. caudal to the portal vein's entry into the
liver. Sampling catheters were also placed in the left common hepatic
vein and the left femoral artery. Ultrasonic perivascular flow probes
(Transonic Systems, Ithaca, NY) were placed around the portal vein (7.0 mm in diameter) and the common hepatic artery (3.0 mm in diameter). An
infusion catheter was placed in the left jugular vein of all dogs that
were to receive chronic cortisol infusion.
Three days before an experiment in those animals designated to receive
cortisol, the jugular infusion catheter was exteriorized from its
subcutaneous pocket through a small incision made under local
anesthesia (2% xylocaine; Astra Pharmaceutical Products, Worcester,
MA). It was then connected to an infusion pump (Walkmed 2000; Infumed
Medex, Broomfield, CO), and the pump was placed in a jacket worn by the
dog while it moved freely about its cage.
On the day of the experiment, the ends of the catheters and flow probes
were exteriorized from the subcutaneous pocket under local anesthesia
(2% xylocaine, Astra Pharmaceutical Products). Angiocaths were
inserted into the cephalic veins for peripheral infusion of tracers.
The conscious dogs were then allowed to stand calmly in a Pavlov
harness for 30 min before the start of the experiment.
Animals in this study were maintained, and experiments performed, in
accordance with the guidelines of the Animal Care Committee of
Vanderbilt University in a facility accredited by the American Association for Accreditation of Laboratory Animal Care.
Experimental procedures.
Each experiment consisted of an 80-min tracer and dye equilibration
period (
80 to 0 min) followed by a 45-min sampling period. At time
80 min, a primed (63 µCi), constant infusion of
[3-3H]glucose (0.88 µCi/min) and a constant infusion of
[U-14C]alanine (6.5 µCi/min) were started in the
cephalic vein and continued throughout the experiment. In addition, at
80 min, a constant infusion of indocyanine green (0.1 mg · m
2 · min
1) was started
via the cephalic vein. Animals were studied after an overnight (18 h)
fast (Overnight Fast; n = 8), after a prolonged (42 or
66 h) fast (Prolonged Fast; n = 7), or after an
18-h fast plus a chronic cortisol infusion (18-h Fast + Cortisol;
n = 7). In the last protocol, a continuous intravenous
infusion of hydrocortisone (3.0 µg · kg
1 · min
1) was
begun 3 days before the experiment and continued throughout the
experiment. It should be noted that four dogs were fasted for 42 h
and three dogs were fasted for 66 h with no discernible differences; thus the data were combined. Blood from the femoral artery, hepatic vein, and portal vein was sampled six times during the
45-min sampling period. Immediately after the final sampling time, the
animals were anesthetized with pentobarbital sodium but not killed.
They were removed from the Pavlov harness while the tracers continued
to infuse. A midline laparotomy incision was made, the liver was
exposed, and two clamps precooled in liquid nitrogen were used to
simultaneously obtain biopsies in situ from the two largest hepatic
lobes (within 2 min of anesthesia). The hepatic tissue was immediately
removed, placed in liquid nitrogen, and stored at
70°C. The animals
were then euthanized using pentobarbitol sodium (390 mg/ml) and
necropsied to confirm proper positioning of the sampling catheters.
Collection and processing of blood samples.
Plasma glucose concentrations were determined using the glucose oxidase
method in a Beckman glucose analyzer (Beckman Instrument, Fullerton,
CA). Plasma glucose radioactivity (3H and 14C)
was determined by liquid scintillation counting after deproteinization with barium hydroxide and zinc sulfate (49), glucose
isolation with cation and anion exchange resins, and reconstitution in
1 ml of distilled water and 10 ml of Ecolite+ (ICN, Costa
Mesa, CA). Concentrations of indocyanine green were determined
spectrophotometrically (805 nm) in arterial and hepatic vein plasma
samples. Whole blood glucose values were assumed to equal 73% of
plasma values on the basis of extensive comparisons between whole blood
and plasma glucose in the dog (1). This yielded an
accurate estimate of net hepatic glucose output (NHGO) regardless of
glucose exchange between red blood cell and plasma water. Whole blood
lactate, alanine, glycerol, and
-hydroxybutyrate concentrations were
determined in samples deproteinized with 4% perchloric acid according
to the method developed by Lloyd et al. (26) for the
Technicon AutoAnalyzer. Whole blood glutamine and glutamate
concentrations were determined according to methods developed for the
AutoAnalyzer (54). Nonesterified fatty acid (NEFA)
concentrations were determined using the Wako NEFA C reagent kit (Wako
Chemicals, Richmond, VA). Immunoreactive insulin was measured using a
double-antibody RIA (39). Immunoreactive glucagon was
measured using a modification of the double-antibody insulin method
(39). Insulin and glucagon antibodies and 125I
tracers were obtained from Linco Research (St. Louis, MO). Cortisol was
determined by RIA (Clinical Assays Gamma Coat Radioimmunoassay Kit).
Plasma epinephrine and norepinephrine levels were determined by HPLC
(37). Labeled and unlabeled plasma alanine and lactate concentrations were determined with a short-column ion exchange chromatographic system that has been described previously
(4). Amino acid concentrations were measured by
reverse-phase HPLC using a modified version of the methods of
Bidlingmeyer et al. (2) and Heinrikson and Meredith
(18). UDPglucose and PEP concentrations and specific
activities in the liver were obtained in the Rossetti laboratory
through two sequential chromatographic separations by a modification of
a method previously described (13, 47). The major
modifications from the original method (14) were in the
HPLC columns, mobile phases, and run conditions. Two C18T
columns (Supelco) were used in series for all analyses. The mobile
phase (1 ml/min) was a 200 mM potassium phosphate buffer at pH 4.5 for
PEP and a 100 mM potassium phosphate buffer at pH 6.5 for UDPglucose.
The ion-pairing agent was tetrabutyl hydrogen sulfate at 5 mM.
Materials.
[3-3H]glucose (New England Nuclear, Boston, MA) was used
as the glucose tracer (500 µCi/0.005 mg), and
[U-14C]alanine (Amersham, Chicago, IL) was used as the
labeled gluconeogenic precursor (171 mCi/mmol). Indocyanine green was
purchased from Hynson, Westcott, and Dunning (Baltimore, MD) to measure
hepatic blood flow (HBF). The [3-3H]glucose infusate
contained cold glucose so that its final concentration was 1 mg/ml. The
indocyanine green infusate was prepared with sterile water, and the
[U-14C]alanine and [3-3H]glucose infusates
were prepared with 0.82% NaCl.
Calculations.
In these studies, ultrasonic flow probes were used to directly
determine hepatic artery and portal vein blood flow. When flow probes
were not functioning properly (i.e., n = 3),
indocyanine green dye was used to assess total hepatic plasma flow
(HPF). When indocyanine green dye was used, the proportion of HBF
provided by the hepatic artery was assumed to be 20% on the basis of
data compiled in our laboratory over many years with the use of Doppler flow probes in the dog. In the case of indocyanine green, HBF was
derived from the HPF: (HBF = HPF/1
hematocrit). Net
hepatic balances of glucose, alanine, lactate, glycerol, glutamine,
glutamate,
-hydroxybutyrate, and gluconeogenic amino acids were
determined by the formula [(Fa × Ca) + (Fp × Cp)
(Fh × Ch)], where Fa, Fp, and Fh represent blood flow
(ml · kg
1 · min
1) in the
hepatic artery, portal vein, and hepatic vein, respectively, and
Ca, Cp, and Ch represent the blood
metabolite concentrations in the three respective vessels. Net hepatic
glucose balance was determined as above after the plasma glucose levels
were converted to blood glucose levels (1). Net hepatic
fractional extraction of a metabolite was determined by the formula
[(Fra × Ca) + (Frp × Cp)
Ch]/[(Fra × Ca) + (Frp × Cp)], where Fra and Frp
represent the fraction of total HBF contributed by the hepatic artery
and portal vein, respectively. All net hepatic uptakes are depicted as
positive values. Glucose production (Ra) and glucose
utilization (Rd) were determined by the equations for
isotope dilution during a constant infusion of radioactive glucose
[3-3H]glucose, as modified by DeBodo et al.
(6). The [14C]glucose production rate was
determined using the tracer technique described by Chiasson et al.
(5). This production rate is assumed to represent hepatic
[14C]glucose production, although it technically reflects
whole body conversion of [14C]alanine to
[14C]glucose. Because [14C]glucose is
virtually all derived from the liver, one can neglect the small amount
of conversion of [14C]alanine to
[14C]glucose in the kidney (29)
[Ra actually reflects both hepatic glucose production
(HGP) and renal glucose production (RGP)]. Therefore, hepatic
glucose release (HGR) was defined as NHGO + hepatic glucose uptake
(HGU)
|
(1)
|
HGU was determined as described by Goldstein et al.
(15), where tracer-derived HGU was calculated by dividing
the net hepatic 3H balance by the inflowing
[3H]glucose specific activity. This represents the
[3H]glucose that is taken up by the liver and metabolized
(as opposed to being recycled to plasma glucose). Implicit in this
approach is the assumption that uptake of glucose occurs before the
addition of glucose to the blood. However, even if this assumption is
not true, the drop in [3H]glucose specific activity
across the liver is so small as to have little or no quantitative impact.
A maximal estimate of hepatic gluconeogenic flux
(GNGmax) was determined by calculating the net
hepatic uptake of the circulating gluconeogenic precursors (lactate,
glycerol, and the gluconeogenic amino acids), assuming that there was
100% conversion to glucose, and expressing the data as glucose
equivalents
(mg · kg
1 · min
1). Pyruvate
was not measured and was not entered into the calculation, as previous
data demonstrated that it represented only one-tenth the contribution
of lactate (46). The product of efficiency and
GNGmax yields a minimal rate of hepatic gluconeogenic flux (GNGmin).
The minimal and maximal rates of net hepatic glycogenolysis were
calculated as
|
(2)
|
|
(3)
|
where HGR is hepatic glucose release, NHLP is the net hepatic
lactate production, and GO is hepatic glucose oxidation. It should be
noted that NHLP = 0 when net hepatic lactate uptake occurs. Under
that circumstance, lactate uptake by the liver is considered a part of
gluconeogenesis. Hepatic GO was assumed to be equal to 0.3 mg · kg
1 · min
1 for all
studies. This rate is based on data from our previous studies and our
observation that it changes little under various physiological
conditions (17). NHLP, GO, and
GNGmax were expressed in glucose equivalents.
RGP was directly determined using renal vein sampling catheters
and flow probes in the last three animals in each protocol. To estimate
it in the other studies, RGP was calculated as the difference between
tracer-determined Ra and HGR.
The gluconeogenic rate was also determined by a modification of the
method of Rossetti et al. (47) and Giaccari and Rossetti (13). The method measures the relative contributions (%)
of plasma-derived glucosyl units [glucose cycling (GC)] and
PEP-derived glucosyl units (PEP-gluconeogenesis) to the hepatic glucose
6-phosphate (G-6-P) pool. Thus, as originally described,
total glucose production (TGO) = HGP + GC. When RGP is zero,
HGP equals the tracer-derived Ra rate determined using
[3-3H]glucose. Thus TGO = Ra + GC.
However, because RGP was expected to be nonzero under our experimental
conditions, HGR was substituted for Ra. The formula then
becomes TGO = HGR + GC. GC was calculated as TGO multiplied
by the percentage of the G-6-P pool derived from arterial
plasma glucose. Thus
|
(4)
|
|
(5)
|
The contribution of PEP-gluconeogenesis to hepatic
glucose-6-phosphatase flux (i.e., from G-6-P to glucose) was
determined by multiplying TGO by the fractional contribution of PEP to
the G-6-P pool. This is the gluconeogenic conversion of
G-6-P to glucose (GNGG-6-P
Glc),
and it was originally designated PEP-gluconeogenesis by Giaccari and
Rossetti (13) and Rosetti et al.
(47). Under the experimental conditions used,
G-6-P and UDPglucose are in equilibrium; thus
|
(6)
|
This approach assumes that the contribution of plasma
[14C]glucose to [14C]UDPglucose is
negligible. Indeed, when hepatic [14C]glucose uptake was
calculated (using [3H]glucose uptake by the liver), such
was shown to be the case. One can also calculate the glycogenolytic
contribution to TGO as
|
(7)
|
The method of Giaccari and Rossetti (13) can also
be used to provide an estimate of overall hepatic gluconeogenic flux from PEP to G-6-P by accounting for all carbons leaving the
G-6-P pool that were derived from PEP. This involves
combining their method and the a-v difference technique to yield the
overall hepatic gluconeogenic flux rate (GNGflux)
reflecting gluconeogenic flux from PEP to G-6-P
|
(8)
|
where NHLP is net hepatic lactate production, NHGlyU is net
hepatic glycerol uptake, and netGlyDep is the net deposition of glucose
carbons from G-6-P into glycogen. In the present experiment, all animals were studied in the postabsorptive period under euglycemic conditions, and netGlyDep was assumed to be negligible. This latter assumption is supported by recent data from this laboratory
(7), which showed that net hepatic
[14C]glucose uptake was negligible under euglycemic
hyperinsulinemic conditions. Another assumption in this calculation is
that all of the glycerol taken up by the liver is converted to
G-6-P. Whereas glycerol accounted for at most 0.21 mg · kg
1 · min
1 glucose
(see RESULTS), the amount of glycerol that was oxidized can
be considered to be negligible. In addition, by use of these methods,
the net rate of hepatic glycogenolysis can be estimated as
|
(9)
|
Data are reported as means ± SE. Statistical analyses were
performed using Student's t-test and one-way ANOVA to
compare values between groups. Data from the prolonged-fasted group and the cortisol group were compared with data obtained from the
overnight-fasted group. Statistical calculations were performed using
SPSS 6.1 for Macintosh. A P value <0.05 was used to define
statistical significance among the prolonged-fasted group, the cortisol
group, and the overnight-fasted group.
 |
RESULTS |
Arterial plasma hormone concentrations.
The plasma cortisol level was 1.3 ± 0.2 µg/dl after an
overnight fast (Fig. 1). There was no
significant change in this level after prolonged fasting.
However, the 3-day cortisol infusion increased the arterial plasma
cortisol level sixfold (P < 0.01). The plasma insulin
level was 10 ± 1 µU/ml in the overnight-fasted animals and
decreased slightly in response to prolonged fasting to 7 ± 1 µU/ml (P < 0.05). There was a significant increase
in the plasma insulin level to 16 ± 3 µU/ml in the
cortisol-treated animals (P < 0.05). The plasma
glucagon levels were not significantly different in any of the groups
(Fig. 1, A-C). The arterial plasma epinephrine and
norepinephrine levels were similar in the overnight- and
prolonged-fasted groups (60 ± 23 vs. 67 ± 24 and 119 ± 27 vs. 174 ± 49 pg/ml, respectively; Fig. 1, D and
E). On the other hand, the arterial plasma epinephrine level
was significantly decreased (26 ± 5 pg/ml, P < 0.05) by cortisol infusion, whereas norepinephrine was not changed
(107 ± 54 pg/ml).

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Fig. 1.
Arterial plasma cortisol (A), insulin (B),
and glucagon levels (C) in 3 groups of dogs. Also shown are
arterial plasma epinephrine (D) and norepinephrine levels
(E). In the Overnight Fast group (n = 8),
animals were studied after an 18-h overnight fast; in the Prolonged
Fast group (n = 7), animals were studied after
42-66 h of fasting; in the 18-h Fast + Cortisol group
(n = 7), animals were studied after an 18-h overnight
fast plus chronic infusion of cortisol (3.0 µg · kg 1 · min 1) for 3 days before the study. Values are means ± SE. Statistical
analysis was performed comparing data from Prolonged Fast and 18-h
Fast + Cortisol with those of Overnight Fast.
|
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Gluconeogenic amino acid precursors.
Although prolonged fasting tended to cause a slight decrease in the net
hepatic uptake of gluconeogenic amino acids, this decrease was not
statistically significant (P = 0.15). Cortisol treatment, however increased net hepatic uptake of the gluconeogenic amino acids (P < 0.001), primarily as a result of a
doubling of net hepatic alanine uptake (Table
1 and Fig.
2).
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Table 1.
Whole blood levels, net hepatic uptakes, and net hepatic fractional
extractions of gluconeogenic amino acids in Overnight Fast, Prolonged
Fast, and 18-h Fast + Cortisol groups of conscious dogs
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Fig. 2.
Net hepatic uptake of gluconeogenic amino acids
(A), net hepatic glycerol uptake (B), and net
hepatic lactate balance (C) in 3 groups of dogs. Values are
means ± SE.
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Lactate and glycerol metabolism.
The arterial blood lactate level was 700 ± 87 µM after
overnight fasting, 388 ± 29 µM with prolonged fasting
(P < 0.05), and 3,098 ± 361 µM in the 18-h
Fast + Cortisol group (P < 0.001). The liver
showed a tendency to produce lactate after an overnight fast (1.4 ± 1.1 µmol · kg
1 · min
1),
but it switched to net lactate consumption after a more prolonged fast
(7.7 ± 0.5 µmol · kg
1 · min
1;
Fig. 2). With chronic hypercortisolemia, the liver was a larger net
producer of lactate (20.0 ± 3.1 µmol · kg
1 · min
1,
P < 0.05). Thus only during prolonged fasting was
lactate a net contributor to hepatic gluconeogenesis.
The arterial blood glycerol level was 98 ± 17 µM after
overnight fasting and did not increase with prolonged fasting (97 ± 10 µM) or with steroid treatment (103 ± 21 µM). Net
hepatic glycerol uptake was 2.3 ± 0.5 µmol · kg
1 · min
1 after
overnight fasting and was not different (2.1 ± 0.2 µmol · kg
1 · min
1) after
prolonged fasting or after cortisol treatment (1.9 ± 0.6 µmol · kg
1 · min
1).
Glucose metabolism.
Arterial plasma glucose levels in the Overnight Fast, Prolonged Fast,
and 18-h Fast + Cortisol groups were 106 ± 2, 103 ± 3, and 110 ± 3 mg/dl, respectively. Glucose Ra decreased
slightly from 2.49 ± 0.12 to 2.30 ± 0.11 mg · kg
1 · min
1 as the fast
progressed, although this difference was not statistically significant
(Table 2). Chronic hypercortisolemia
markedly increased Ra to 3.72 ± 0.19 mg · kg
1 · min
1
(P < 0.001). Similarly, NHGO decreased from 1.99 ± 0.11 to 1.48 ± 0.18 mg · kg
1 · min
1 as the fast
progressed (P < 0.03). However, chronic
hypercortisolemia caused an even more marked decrease in NHGO to
0.79 ± 0.31 mg · kg
1 · min
1
(P < 0.001). HGU was only 0.31 ± 0.08 mg · kg
1 · min
1 after an
overnight fast but increased to 0.81 ± 0.08 mg · kg
1 · min
1 with
prolonged fasting (P < 0.001). In the 18-h Fast + Cortisol group, HGU was markedly elevated to 2.23 ± 0.21 mg · kg
1 · min
1
(P < 0.001). Interestingly, HGR was well preserved as
the fast progressed (2.29 ± 0.12 to 2.27 ± 0.13 mg · kg
1 · min
1), and even
in the steroid-treated animals (3.02 ± 0.44). Estimated RGP
(Table 2) was 0.22 ± 0.13 mg · kg
1 · min
1 after an
overnight fast, and prolonged fasting tended to decrease it, whereas
chronic hypercortisolemia tended to increase it, but these changes did
not reach statistical significance given the variances in the
measurement.
Gluconeogenic indexes using the a-v difference technique.
In the overnight-fasted group, GNGmax and
GNGmin were 0.70 ± 0.09 and 0.17 ± 0.08 mg · kg
1 · min
1,
respectively (Table 3). In the
prolonged-fasted group, these values increased significantly to
1.28 ± 0.10 and 0.62 ± 0.15 mg · kg
1 · min
1,
respectively (P < 0.001). This was primarily due to
increased net hepatic uptake of lactate. In the cortisol-treated group, the very slight increase (0.91 ± 0.13 mg · kg
1 · min
1) noted in
GNGmax compared with the overnight-fasted animals was not
statistically significant, nor was the small fall in
GNGmin.
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Table 3.
Gluconeogenic parameters of the [14C]PEP-liver biopsy
technique, the a-v difference technique, and the combined technique
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Calculations of minimal and maximal rates of net hepatic glycogenolysis
demonstrated significant decreases as fasting progressed but no
significant increase in response to steroid treatment.
Gluconeogenesis and GC determined using the
[14C]PEP-liver biopsy technique.
After an overnight fast, the hepatic
GNGG-6-P
Glc rate determined using the method
of Giaccari and Rossetti (13) was 0.65 ± 0.06 mg · kg
1 · min
1, and the GC
rate was 0.62 ± 0.07 mg · kg
1 · min
1 (Table 3).
Prolonged fasting caused a doubling in the hepatic GNGG-6-P
Glc rate (P < 0.02),
whereas GC decreased significantly (P < 0.05). Chronic
hypercortisolemia did not result in a significant increase in the
hepatic GNGG-6-P
Glc but did cause a doubling
in hepatic GC (P < 0.005).
Hepatic gluconeogenic flux to G-6-P and net hepatic glycogenolysis
determined using the [14C]PEP-liver biopsy technique
combined with the a-v difference technique.
The gluconeogenic flux rate derived by combining the
[14C]PEP technique and the a-v difference technique is a
more accurate reflection of new glucose carbons added to the
G-6-P pool. These rates are depicted in Table 3. After an
overnight fast, the gluconeogenic flux rate was 0.95 ± 0.14 mg · kg
1 · min
1. This rate
increased significantly to 1.50 ± 0.18 mg · kg
1 · min
1
(P < 0.05) with prolonged fasting and was also
increased to 1.64 ± 0.33 mg · kg
1 · min
1
(P < 0.05) by steroid treatment. Net glycogenolysis
was 1.46 ± 0.12 mg · kg
1 · min
1 after an
overnight fast and decreased significantly to 0.26 ± 0.08 mg · kg
1 · min
1 during
prolonged fasting. Net glycogenolysis was not significantly altered by
steroid treatment compared with overnight-fasted animals without
steroid treatment (1.25 ± 0.43 mg · kg
1 · min
1).
 |
DISCUSSION |
The primary purpose of this study was to compare determinations of
hepatic gluconeogenic rates obtained using the a-v difference technique
with those obtained using the [14C]PEP-liver biopsy
technique under conditions of modest or increased hepatic
gluconeogenesis in vivo. Hepatic gluconeogenic rates were determined
after an overnight 18-h fast and compared with those rates obtained
after a prolonged fast (42- to 66-h fast) and an overnight 18-h fast
following 3 days of chronic hypercortisolemia. With the recognition
that the [14C]PEP technique specifically reflects the
conversion of gluconeogenic G-6-P to glucose
(GNGG-6-P
Glc), which is a major component of
the overall gluconeogenic pathway, a second aim of this study was to
develop and evaluate a third methodology, in which components of the
a-v difference technique and the [14C]PEP-liver biopsy
technique were combined to yield insights into the gluconeogenic flux
from PEP to G-6-P under the three experimental conditions
cited. Although there exists no "gold standard" to which one can
compare the results of various methodologies, several major conclusions
can be drawn from the present study. 1) In the overnight-fasted animal and in the prolonged-fasted animal, there was a
close correlation between the maximal hepatic gluconeogenic flux
(GNGmax, determined using the a-v difference technique) and the directly determined GNGG-6-P
Glc
(determined using the [14C]PEP-liver biopsy technique);
2) although the techniques individually appeared to yield
similar results, they both underestimated (by 15-38%) the total
hepatic gluconeogenic flux rate (GNGflux) determined as a
result of combining the methodologies; and 3) chronic
hypercortisolemia resulted in a significant increase in
GNGflux, an effect that was clearly detected only by
combining the two methods and determining GNGflux.
In discussion of the methodology used in the present study, it is
important to consider the major assumptions and potential inaccuracies
of each of the methods. A limit to the a-v difference technique lies in
its inability to account for intrahepatic proteolysis (40,
44) and intrahepatic lipolysis (9). In addition,
GNGmax is, by definition, an estimate made with the
assumption that all gluconeogenic substrates taken up by the liver are
converted to glucose. The GNGmin requires the determination
of gluconeogenic efficiency, which is itself a minimal estimate due to
tracer dilution in the OAA pool of the liver (44). The
true gluconeogenic flux rate can be bracketed between the
GNGmin and GNGmax only with the assumption that
intrahepatic lipolysis and proteolysis contribute little to hepatic gluconeogenesis.
In their initial study describing the [14C]PEP-liver
biopsy technique, Giaccari and Rossetti (13) used
tracer-determined glucose production as the value for HGP.
Given that the experiments were performed in the rat and therefore a-v
sampling across liver could not be performed, Ra determined
with [3-3H]glucose was the only practical approximation
of HGP. However, RGP contributes to Ra, and thus
substituting Ra for HGP would lead to some overestimation
of hepatic gluconeogenesis. In the present study, HGR, which is a more
precise measurement of HGP, was calculated. Another limitation to the
method of Giaccari and Rossetti is that the technique cannot take into
account the contribution of glycerol to gluconeogenesis. As glycerol
enters the gluconeogenic pathway above PEP, its contribution to
gluconeogenesis is not reflected in the determination of PEP specific
activity, which will result in an underestimation of hepatic gluconeogenesis.
The validity of the calculation of gluconeogenesis using the method of
Rossetti et al. (47) has been examined under a number of
potential conditions related to cycling of 14C
gluconeogenic precursors. If there is gluconeogenic flux from PEP to
G-6-P but no flux of label back down to PEP, then
GNGG-6-P
Glc would closely approximate the
true gluconeogenic rate (given the caveats discussed). However, if
there is cycling of label from G-6-P to PEP and back to
G-6-P, the gluconeogenic rate would be overestimated by the calculation.
A more robust estimation of GNGflux was defined in
Eq. 8. Although the [14C]PEP-liver biopsy
technique, by itself, will account for the contribution of intrahepatic
proteolysis to gluconeogenesis, the error resulting from not accounting
for the contribution of glycerol can be minimized by adding net hepatic
glycerol uptake to the gluconeogenic estimate. Likewise, one can also
account for the G-6-P derived from PEP that goes down the
glycolytic pathway either to exit the liver as lactate or to be
oxidized in the tricarboxylic acid cycle. Equation 8 also
accounts for the G-6-P derived from PEP that contributes to
new glycogen formation.
One of the other modifications made to the original method of Giaccari
and Rossetti (13) and Rosetti et al. (47) was
to continuously infuse the [14C]alanine during the entire
course of the study rather than to bolus the tracer over the last 10 min of the study. This change raised the possibility that plasma
[14C]glucose could reenter the hepatic UDPglucose pool
and lead to an overestimation of the calculated gluconeogenic rate.
Calculations were made to determine the maximal overestimation in the
gluconeogenic rate that might result. This determination was based on
estimating the [14C]glucose counts that entered the liver
during the study and comparing those counts to the
[14C]UDPglucose content of the liver. In the
overnight-fasted animals, gluconeogenesis could have been overestimated
(data not shown) by a maximum of 4%, whereas in the prolonged-fasted
animals this overestimation could have been as much as 14%. The
largest possible error from this source could have occurred in the
steroid-treated animals, and even then the overestimate would have been
no more than 20%. Nevertheless, on the basis of these calculations, it is recommended that future applications of this technique employ an
infusion of [14C]alanine only during the last 20 min
before liver biopsy.
Previous data derived under similar conditions but analyzed using a
different approach failed to reveal significant enhancement in
gluconeogenesis by steroid treatment (15). This conclusion remained largely at odds with in vitro data demonstrating stimulatory effects of cortisol on phosphoenolpyruvate carboxykinase
(PEPCK) activity (44) and PEPCK mRNA transcription
(10). In the former study (15) and
in the present study, neither gluconeogenic efficiency nor the
GNGmax was significantly increased by steroid treatment. Furthermore, gluconeogenic conversion of alanine to glucose was only
modestly increased by the steroid (15). However, by use of
the combination of methodologies, a significant stimulatory effect of
glucocorticoids on gluconeogenic flux was detected in vivo, even in the
complex setting of enhanced GC. To the extent that
[14C]glucose derived from gluconeogenic flux accumulated
in hepatic glycogen (which was not measured), GNGflux would
have been underestimated.
Each of the two methods also yielded independent information on
different aspects of hepatic GC. Goldstein et al. (15)
previously described a significant difference between Ra
and NHGO associated with chronic hypercortisolemia. This difference
roughly equaled net HGU, which resulted from steroid treatment. The
[14C]PEP-liver biopsy technique detected enhanced
steroid-induced GC through the G-6-P pool. This accounted
for the additional glucose production that was undetected due to the
use of [3-3H]glucose rather than
[2-3H]glucose (51, 57). As previously shown,
if one used [2-3H]glucose as the tracer, Ra
determined using this tracer should equal TGO (47). Thus
the combination of the two methodologies detected a significant inward
movement of glucose carbons, even in the 18-h-fasted state.
In the present study, RGP was determined as the difference between
tracer-determined glucose production and HGR. This indirect assessment
of RGP is imprecise but nevertheless suggested that the kidneys
contributed at most ~0.3
mg · kg
1 · min
1 to TGO in
overnight- and prolonged-fasted dogs. In the 18-h-fasted steroid-treated group, the indirect measurement of RGP suggested that
the contribution of RGP to Ra might be increased two- to threefold. In the last few animals studied in each group and in additional animals studied under similar experimental conditions for
other purposes, RGP was directly assessed as described by McGuinness et
al. (36). In those animals, the directly determined RGP
was 0.1 ± 0.6 mg · kg
1 · min
1 after an
overnight fast (n = 6). This was not different from the
indirectly determined value and is not different from that reported by
McGuinness et al. (0.4 ± 0.4 mg · kg
1 · min
1). During
chronic hypercortisolemia (n = 5), directly assessed RGP was 0.2 ± 0.1 mg · kg
1 · min
1, whereas
after prolonged fasting (n = 6) it was 0.1 ± 0.1 mg · kg
1 · min
1. Thus, in
the animals in which RGP was directly assessed, it was 10% or less of TGO.
In the present study, hepatic GNGflux was equal to 37% of
TGO (Ra) after an 18-h fast, and this percentage increased
to 57% after more prolonged fasting. Although the steroid treatment
increased hepatic gluconeogenesis, the treatment also caused a
significant increase in Ra, thus accounting for the failure
of hepatic GNGflux (39%) to increase as a percentage of
total Ra. Hellerstein and colleagues (19, 20,
21) and Neese and colleagues (41, 42) have recently
assessed gluconeogenesis by mass isotopomer distribution
analysis by use of [2-13C]glycerol,
[3-13C]lactate, or [1-13C]lactate. In rats,
progressive fasting increased the ratio of gluconeogenesis as a
percentage of glucose production from ~50% at 5-6 h to 80% at
20-24 h (41). Hellerstein et al. (21)
further developed this methodology and have recently incorporated the glucuronate probe technique to measure the rate of glucose entry into
hepatic UDPglucose by the direct pathway. In a group of
overnight-fasted human subjects, gluconeogenesis accounted for 36% of
glucose production (19). After a 60-h fast, this
percentage increased to 78%. A similar rate was recently reported by
Tayek and Katz (52) for overnight-fasted human subjects.
In their study, gluconeogenesis accounted for 40% of glucose
production. In a separate group of overnight-fasted human subjects,
these same authors reported that gluconeogenesis accounted for 47% of
glucose production (54).
Recently Landau et al. (34) estimated the contribution of
gluconeogenesis to TGO in human subjects by use of
2H2O. The amount of 2H bound to
carbon-6 of glucose relative to that bound to carbon-2 provides a
measure of the fraction of glucose formed via
gluconeogenesis (30, 45). Using this
methodology, Landau et al. (32) reported that
gluconeogenesis represented 23-42% of total glucose production in
overnight-fasted human subjects and that this increased to 59-85%
after 42 h of fasting. Further refinement of the technique by Landau and colleagues (31, 33) utilized the ratio of
2H bound to carbon-5 of glucose relative to that bound to
carbon-2. By use of this technique, data indicated that gluconeogenesis accounted for 47% of glucose production at 14 h and increased to
67% at 22 h and to 93% after 42 h of fasting in human
subjects. These data of Landau et al. (32), Tayek and Katz
(52), and Hellerstein et al. (19) obtained
from human subjects are consistent with the overnight-fast data
obtained in the present study despite the species difference.
Because the assessment of gluconeogenesis in human subjects
necessitates a less invasive methodology than can be employed in animal
models, Rothman et al. (48) have pioneered 13C
nuclear magnetic resonance (NMR) spectroscopy to assess hepatic gluconeogenesis in humans. In their studies, gluconeogenesis accounted for 64% of TGO at 22 h of fasting and for 96% of glucose
production by 68 h of fasting. When the methodology was applied to
type 2 diabetic subjects, gluconeogenesis accounted for 88% of TGO
after 23 h of fasting (35). The gluconeogenic rates
obtained by Rothman et al. are higher than those obtained in the
present study and higher than those obtained in the human studies
discussed above. It is possible that the protocol design may have
accounted for the higher gluconeogenic rates that those authors
observed. For instance, the meal that subjects were fed before the
official start of the fast was relatively small. Thus the subjects may have entered the fast still relatively glycogen depleted and thus had
already progressed to a more gluconeogenic state. However, additional
data from the same group obtained using 13C NMR
spectroscopy to assess hepatic gluconeogenesis in humans indicated a
gluconeogenic rate of 60% in human subjects after an overnight fast
(49). It remains unclear why the gluconeogenic rates
obtained using NMR spectroscopy were slightly higher than those
obtained using other techniques. It should be remembered, however, that
the approach of Rothman et al. involves the measurement of the net
glycogenolytic rate, which is subtrated from tracer-determined glucose
production. Any RGP will thus manifest as hepatic gluconeogenesis. In
addition, it should be noted that, if glycogen synthesis were to be
augmented, the NMR technique would detect a decrease in gluconeogenesis that was not the result of a diminution in
the flux of pyruvate to G-6-P but was instead due to an
augmentation of glycogen synthesis. One must be careful when comparing
the results from the different gluconeogenic methods, because they measure slightly different things.
Figures 3,
4, and 5
summarize the glucose carbon fluxes within the liver. GNG represents
the gluconeogenic flux from PEP to G-6-P. After an overnight
fast (Fig. 3), net glycogenolysis was 1.46 mg · kg
1 · min
1. After a
prolonged fast (Fig. 4), net glycogenolysis decreased to 0.26 mg · kg
1 · min
1. With
steroid treatment (Fig. 5), net glycogenolysis was 1.25 mg · kg
1 · min
1. The
derived glycogenolytic rates after an overnight fast and during
prolonged fasting are consistent with rough approximations of the
glycogenolytic rates derived from published hepatic glycogen measurements compiled by Moore et al. (38) and Hendrick et
al. (22) at various fasted states. If one plots their data
and assumes that the decline in glycogen mass is linear over time, the
net glycogenolytic rate is
2.0
mg · kg
1 · min
1 at
20 h and
0.7
mg · kg
1 · min
1 at 40 h of fasting.

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|
Fig. 3.
Flux rates into and out of the hepatic glucose 6-phosphate
(G-6-P) pool after an 18-h overnight fast (Overnight Fast,
n = 8) are summarized in this figure. Total glucose
output (TGO) and glucose cycling (GC) were derived using the
[14C]phosphoenolpyruvate (PEP)-liver biopsy
technique. The gluconeogenic rate (GNG) is the gluconeogenic flux
(GNGflux) determined using the combined
[14C]PEP-liver biopsy technique plus the arteriovenous
(a-v) difference technique. All other rates into the liver from the
plasma compartment were determined solely by use of the combined-tracer
a-v difference technique. These include hepatic glucose uptake (HGU),
net hepatic glucose output (NHGO), hepatic glucose release (HGR), net
hepatic glycerol uptake (Glycerol), net hepatic lactate balance
(Lactate), and the net hepatic uptake of amino acid gluconeogenic
(amino acid precursors). Net hepatic glycogenolysis was calculated as
the difference between the known glucose carbons entering and leaving
the G-6-P pool. All rates are in
mg · kg 1 · min 1.
|
|

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|
Fig. 4.
Flux rates into and out of the hepatic G-6-P pool after
a 42- or 66-h fast (Prolonged Fast, n = 7) are
summarized in this figure. See legend of Fig. 3 for explanatory
details.
|
|

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|
Fig. 5.
Flux rates into and out of the hepatic G-6-P pool after
an 18-h fast during chronic hypercortisolemia (18-h Fast + Cortisol, n = 7) are summarized in this figure. See
legend of Fig. 3 for explanatory details.
|
|
Results from the two methods used in this study can also be combined to
yield estimates of the contribution of hepatic proteolysis to the PEP
pool under the three experimental conditions studied
|
(9)
|
where GNGflux is the gluconeogenic flux determined
using the combined methodology. With this equation, the rate of hepatic proteolysis after an overnight fast was calculated to be 0.24 ± 0.14 mg · kg
1 · min
1.
Prolonged fasting had little effect on this rate (0.22 ± 0.20 mg · kg
1 · min
1). However,
chronic steroid treatment significantly increased this rate to
0.76 ± 0.28 mg · kg
1 · min
1.
In summary, the results of the present study demonstrated that
combining the a-v difference technique with the
[14C]PEP-liver biopsy technique yielded a more complete
picture of hepatic glucose carbon flux than what one obtained using
either method alone. The combined technique did not merely decrease the limitations of the a-v difference and [14C]PEP-liver
biopsy techniques but, in some cases, significantly changed conclusions
such that the in vivo data reconciled with the in vitro data.
 |
ACKNOWLEDGEMENTS |
We thank Kareem Jabbour for technical help in the laboratory.
 |
FOOTNOTES |
Some of the data in these studies were presented at the American
Diabetes Association, Atlanta, GA, June 1995. These studies were
supported in part by a Veterans Affairs Merit grant, National Institute
of Diabetes and Digestive and Kidney Diseases Grant RO1 DK-18243, and
the Diabetes Research and Training Center (P60 DK-20593).
Address for reprint requests and other correspondence:
A. D. Cherrington, 710 RRB, Vanderbilt Univ. Medical Center,
Nashville, TN 37232-0615 (E-mail:
Alan.Cherrington{at}mcmail.vanderbilt.edu).
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.
10.1152/ajpendo.00320.2002
Received 16 July 2002; accepted in final form 17 July 2002.
 |
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