Am J Physiol Endocrinol Metab 286: E129–E135, 2004.
First published June 24, 2003; 10.1152/ajpendo.00486.2002.
Nonhepatic glucose production in humans
Alberto Battezzati,1,2 Andrea Caumo,2 Francesca Martino,2 Lucia Piceni Sereni,2
Jorgelina Coppa,3 Raffaele Romito,3 Mario Ammatuna,3 Enrico Regalia,3
Dwight E. Matthews,4 Vincenzo Mazzaferro,3 and Livio Luzi2
1
International Center for the Assessment of Nutritional Status, DiSTAM, Università degli Studi di Milano, 20133 Milano;
Department of Medicine, Istituto Scientifico H San Raffaele, 20132 Milano; and 3Liver Transplantation Unit, Istituto
Nazionale dei Tumori, 20133 Milano, Italy; and 4University of Vermont, Burlington, Vermont 05405
2
Submitted 7 November 2002; accepted in final form 30 May 2003
kidney; intestines; amino acids
IN PHYSIOLOGICAL CONDITIONS,
glucose production is assumed to
be mainly hepatic in humans, although it was shown that the
kidney and other organs can release newly formed glucose for
the use of other tissues (22). The quantification of the extent of
extrahepatic glucose production and the definition of its regulation are presently subject to debate. The kidney was studied
as a major nonhepatic gluconeogenic site, but uncertainities
related to methodological problems led to divergent measures
of renal glucose production, which accounted for 5% up to
30% of the whole body glucose production during the postabsorptive state (8, 11, 19, 25). Most of the experimental problems are related to the accuracy in measurement of the renal
blood flow (which is large), of the glycemic gradient across the
renal bed (which is small or even neutral, because the kidney
can simultaneously produce and take up glucose), and of the
Address for reprint requests and other correspondence: A. Battezzati, Amino
Acids and Stable Isotopes Laboratory, San Raffaele Scientific Institute, Via
Olgettina, 60, 20132 Milano, Italy (E-mail: [email protected]).
http://www.ajpendo.org
gradients in glucose tracer enrichments or specific activities
across the same districts (19). In the studies that found a
consistent contribution of the kidney to the postabsorptive
glucose production, lactate and glutamine were preferred to
alanine as gluconeogenic substrates (27). The kidney was also
sensitive to the insulin suppression of gluconeogenesis (18)
and to the gluconeogenic stimulation from epinephrine but not
to that of glucagon (26, 27).
In considering the difficulties of dissecting the hepatic from
the extrahepatic contributions to glucose production (including
these from the kidney and other potential gluconeogenic organs), it is imperative to find experimental models in humans
to unequivocally demonstrate the potential of gluconeogenic
organs to release glucose into the systemic circulation. The
anhepatic phase of liver transplantation is the lag of time in
which the recipient’s liver has been removed but the donor’s
liver has not yet been replaced. Although it is nonphysiological, this is a clear-cut model to demonstrate nonhepatic gluconeogenesis, because there is no liver to confound results with
its large output of glucose. A recent study used this model to
suggest that glucose is released at the rate of 1.3 mg䡠kg⫺1䡠min⫺1
in the absence of the liver (14). In the present study, we decided
to measure the glucose production during the anhepatic phase of
liver transplantation and to test whether the alanine and the
glutamine carbons could still be transferred to glucose in the
absence of the liver. We studied 14 patients who, in the absence
of the liver, did not receive exogenous glucose during this period.
We found that during the anhepatic phase 1) the glucose concentration slowly declined by 15%/h, 2) the glucose production was
65% that of healthy postabsorptive subjects, 3) carbons from
glutamine and to a lesser extent from alanine were incorporated in
glucose.
METHODS
Materials. D-[6,6-2H2]glucose, L-[3-13C]alanine, and L-[1,2C2]glutamine were purchased from MassTrace (Woburn, MA).
Chemical and isotopic purity of the tracers was determined by gas
chromatography-mass spectrometry (GC-MS). Before every infusion study, sterile solutions of the tracers were prepared with an
aseptic technique. Accurately weighed amounts of the labeled
compounds were dissolved in weighed volumes of sterile, pyrogenfree saline and filtered through a 0.22-␮m Millipore filter before
use. An aliquot of the sterile solution was initially verified to be
pyrogen free before administration to human subjects. Solutions
were prepared ⱕ24 h before use and were kept at 4°C before
administration.
13
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.
0193-1849/04 $5.00 Copyright © 2004 the American Physiological Society
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Battezzati, Alberto, Andrea Caumo, Francesca Martino, Lucia
Piceni Sereni, Jorgelina Coppa, Raffaele Romito, Mario Ammatuna, Enrico Regalia, Dwight E. Matthews, Vincenzo Mazzaferro,
and Livio Luzi. Nonhepatic glucose production in humans. Am J
Physiol Endocrinol Metab 286: E129–E135, 2004. First published
June 24, 2003; 10.1152/ajpendo.00486.2002.—Extrahepatic glucose
release was evaluated during the anhepatic phase of liver transplantation in 14 recipients for localized hepatocarcinoma with mild or
absent cirrhosis, who received a bolus of [6,6-2H2]glucose and L-[313
C]alanine or L-[1,2-13C2]glutamine to measure glucose kinetics and
to prove whether gluconeogenesis occurred from alanine and glutamine. Twelve were studied again 7 mo thereafter along with seven
healthy subjects. At the beginning of the anhepatic phase, plasma
glucose was increased and then declined by 15%/h. The right kidney
released glucose, with an arteriovenous gradient of ⫺3.7 mg/dl.
Arterial and portal glucose concentrations were similar. The glucose
clearance was 25% reduced, but glucose uptake was similar to that of
the control groups. Glucose production was 9.5 ⫾ 0.9 ␮mol䡠kg⫺1 䡠
min⫺1, 30% less than in controls. Glucose became enriched with 13C
from alanine and especially glutamine, proving the extrahepatic gluconeogenesis. The gluconeogenic precursors alanine, glutamine, lactate, pyruvate, and glycerol, insulin, and the counterregulatory hormones epinephrine, cortisol, growth hormone, and glucagon were
increased severalfold. Extrahepatic organs synthesize glucose at a rate
similar to that of postabsorptive healthy subjects when hepatic production is absent, and gluconeogenic precursors and counterregulatory
hormones are markedly increased. The kidney is the main, but
possibly not the unique, source of extrahepatic glucose production.
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GLUCOSE PRODUCTION DURING LIVER TRANSPLANTATION
Table 1. Characteristics of the subjects
ANHEP
Preintervention
n
Age, yr
Weight, kg
Height, cm
Platelets, 1,000/mm3
Albumin, g/l
Bilirubin total, mg/100 ml
Prothrombin time, %
CON
12
52⫾2
69.6⫾2.5
168⫾1
7
35⫾3
66.8⫾5.2
170⫾2
4.5⫾0.3
1.2⫾0.3
96⫾6
5.1⫾0.5
0.6⫾0.1
100⫾6
43%
43%
7%
7%
Values are means ⫾ SE; n, no./group. ANHEP, subjects during liver
transplant; POST, recipients 7 mo after transplant; CON, healthy control
subjects.
Subjects. The characteristics of the subjects under study are reported in Tables 1 and 2. Fourteen subjects were studied during the
anhepatic phase of liver transplantation (LTx). The subjects were
transplanted for localized hepatocarcinoma [single (⬍5) or multiple
(⬍2 cm diameter)] (17), with the exception of two subjects who
received the liver for carcinoid. It should be stressed that 86% of the
patients had mild cirrhosis or no cirrhosis at all. Accordingly, the
albumin and bilirubin concentrations, the prothrombin time, and the
number of platelets reflected only a minor impairment in liver function. All subjects were at stable weight before transplantation. For
most of them, a combination of the following medications was
prescribed according to clinical indications: diuretics (spironolactone
and/or furosemide), beta-blockers (propranolol or atenolol), lactulose,
or ursodeoxycholic acid. The patients were following an isocaloric
diet with ⱖ250 g of carbohydrate and 70–90 g of protein daily, with
salt restriction. The last meal before transplantation was consumed
6–10 h before the skin incision, from which time 5 h on average
elapsed for infusion of the tracer bolus during the anhepatic phase.
Thus the study was performed 11–15 h from the last meal. The bolus
of isotopes was repeated in 12 of the recipients 7 mo after transplantation (POST). Seven healthy control subjects were also studied with
the same protocols (CON). A number of the LTx subjects and the
POST and the CON subjects were simultaneously studied to trace the
glutamine, alanine, and leucine kinetics. The complete set of results
relative to the latter protocol has been reported (3). The protocol was
approved by the institutional ethics committees.
Surgical model of the anhepatic phase of LTx. For the purpose of
this work, the anhepatic phase began when the recipient’s liver was
removed and terminated when the circulation through the graft was
reestablished by unclamping the portal vein (17). The duration of this
phase was ⬃45–75 min. To use this model, the major difficulties are
caused by the short duration of the anhepatic phase (⬃60 min) and by
the changed metabolic environment abruptly induced by liver removal. For a number of reasons, we decided to measure the glucose
kinetics by administering tracers as a single injection during the
anhepatic phase and not by using a primed-continuous infusion
commencing before the anhepatic phase. First, with continuous infusions, the time required to reach a tracer steady state is several times
greater than the duration of the anhepatic phase itself; thus the
experiment should have begun hours before the anhepatic phase.
When we considered the possible displacement of body fluids resulting from the surgical procedure (blood losses and replacement with
hemoderivatives, and a fluid compartmentation due to portal and caval
veins clamping and unclamping) before the anhepatic phase, we
doubted that we could have excluded any carryover from the isotopic
AJP-Endocrinol Metab • VOL
Table 2. Arterial blood hematocrit, pH, hemogasanalysis,
and electrolyte concentrations in transplanted patients
Hematocrit, %
pH
PO2, Torr
PCO2, Torr
Sodium, mmol/l
Potassium, mmol/l
Calcium, mmol/l
Chloride, mmol/l
Gap anion, mmol/l
Preintervention
Anhepatic Phase
35.7⫾2.4
7.43⫾0.03
222.0⫾20.4
34.8⫾1.7
139.2⫾0.5
3.5⫾0.2
0.988⫾0.039
107.2⫾1.2
12.5⫾1.4
35.9⫾2.5
7.43⫾0.04
225.6⫾15.2
33.1⫾2.6
139.7⫾1.2
3.4⫾0.2
1.149⫾0.031*
104.3⫾1.0*
16.7⫾1.1*
Values are means ⫾ SE. *P ⬍ 0.05 vs. preintervention.
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CHILD-PUGH classification
No cirrhosis
Class A
Class B
Class C
14
48⫾3
69.0⫾2.1
168⫾1
115⫾28
3.6⫾0.1
1.9⫾1.3
75⫾9
POST
dilution before the anhepatic phase. For these reasons, the surgical and
medical teams agreed to limit the duration of the experiment to the
anhepatic phase and to warrant in that period a stable hemodynamic
condition without blood losses and administration of hemoderivatives.
During the anhepatic phase, the subjects received saline and other
electrolytes (Normosol R; Abbott Laboratories, Abbott Park, IL),
sevoflurane anesthesia with remifentanil and pancuronium bromide,
and, occasionally, calcium chloride, furosemide, and human albumin
solutions.
Tracer injection and sampling protocol. At the beginning of the
anhepatic phase, two basal samples of arterial blood were drawn with
spacing 5 min apart. Immediately thereafter, a bolus of [6,62
H2]glucose (69 mg/kg) was administered to quantitate the glucose
kinetics. A bolus of L-[3-13C]alanine (90 ␮mol/kg, n ⫽ 7) or L-[1,213
C2]glutamine (30 ␮mol/kg, n ⫽ 7) was simultaneously administered
to evaluate whether the labeled carbons of these tracers were incorporated into gluconeogenic glucose during the anhepatic phase. The
bolus was delivered in a central vein in 20 s, immediately followed by
a flush with 15 ml of saline. Arterial blood was drawn at the following
times after the bolus: 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, and every 15
min thereafter until the end of the anhepatic phase, and 15 min after
the portal unclamping, when the circulation through the grafted liver
was reestablished. In some subjects, 35–45 min after the beginning of
the anhepatic phase, blood samples were simultaneously drawn from
the arterial line and from the right renal vein (n ⫽ 6) or the portal vein
(n ⫽ 5).
The same protocol was followed in the POST (n ⫽ 6 with the
alanine bolus, n ⫽ 4 with the glutamine bolus) and CON (n ⫽ 4 with
the alanine bolus, n ⫽ 3 with the glutamine bolus) subjects, with the
only difference being that CON received one-half of the alanine dose
given the other groups (45 ␮mol/kg), and “arterialized” venous blood
was drawn from a dorsal vein of the subject’s hand that was cannulated in a retrograde way and heated in a warming box, as previously
described (1). In both CON and POST subjects, blood was drawn up
to 150 min after the bolus.
Aliquots of blood were placed in tubes containing EDTA and
stored on ice until the plasma was prepared by centrifugation at 4°C.
A 0.5-ml aliquot was withdrawn, defined amounts of [2H4]alanine and
[2H5]glutamine were added as an internal standard for quantitation of
alanine and glutamine plasma concentrations, and the plasma was
frozen at ⫺60°C. One-milliliter blood aliquots for the measurements
of glucagon and the catecholamines were placed in tubes containing
EDTA plus aprotinin and in tubes containing glutathione, respectively. Blood aliquots for insulin, cortisol, and growth hormone were
collected in tubes without additives for serum separation. Blood
aliquots for the determination of lactate, pyruvate, glycerol, and
␤-hydroxybutyrate were placed in tubes containing perchloric acid.
All blood samples were placed on ice until the plasma or serum was
prepared by centrifugation at 4°C (within 1.5 h from drawing). All
plasma and serum aliquots were frozen at ⫺60°C until later analysis.
GLUCOSE PRODUCTION DURING LIVER TRANSPLANTATION
AJP-Endocrinol Metab • VOL
the control groups we compared the clearances obtained by analyzing
the first 75 min of the tracer decay curve with those obtained by
analyzing the full set of data (150 min). We found that the clearances
were never overestimated ⬎10% when only the first 75-min data were
used. We reasoned that, for proving our hypothesis, a ⬍10% systematic error in glucose clearance (applied to both the study and control
groups) was not relevant compared with the advantage of performing
the study of the anhepatic metabolism with the tracer bolus technique.
Statistical analysis. We used t-tests for paired data to compare
patients during LTx (ANHEP) and POST, and t-tests for independent
data to compare ANHEP and CON, and we applied to both tests the
Bonferroni correction. During the anhepatic phase, the changes over
time in plasma substrate concentrations were defined as a significant
correlation (P ⬎ 0.05) with time by standard linear regression.
RESULTS
Glucose concentrations. The time course of the glucose
concentration in the plasma from arterial blood is reported in
Fig. 1. After laparotomy, glucose concentration increased to
reach a plateau of 174 ⫾ 11 mg/dl, which was maintained until
the portal vein was clamped, at which time the glucose concentration began to fall. During the anhepatic phase, glucose
concentration decreased at the rate of 18 ⫾ 3 mg䡠dl⫺1 䡠h⫺1.
Fifteen minutes after the liver replacement, the glucose concentration rose by 97 ⫾ 10 mg/dl. At all times, the glucose
concentration was significantly higher than in POST and in
CON, whereas no difference was apparent between the two
control groups.
In six subjects, the glucose concentration in the plasma from
the right renal vein was higher than in the artery (⫹3.7 ⫾ 0.9
mg/dl, P ⬍ 0.01, Fig. 2). This subset of subjects had a mean
glucose Ra of 7.5 ⫾ 1.4 ␮mol䡠kg⫺1 䡠min⫺1. In contrast, no
significant difference in glucose concentration was apparent
across the portal system by portal blood sampling.
Glucose kinetics. The time course of the concentration of
[6,6-2H2]glucose after the bolus is reported in Fig. 3. The
pattern of the bolus disappearance was similar among the
groups, even though the area under the curve during the
Fig. 1. Time course of glucose concentration. ANHEP, subjects during liver
transplant; POST, recipients 7 mo after transplant; CON, healthy control
subjects. Surgical stress increased glucose concentration until the portal vein
was clamped. After liver removal, glucose concentration slowly declined, but
it remained in the hyperglycemic range. Liver replacement immediately
increased the glucose concentration.
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Analytical methods. Plasma glucose concentration was measured
with the hexokinase method (2) (Boehringer Mannheim, Mannheim,
Germany).
The 2H2-glucose enrichments were measured by GC-MS after
preparation of the butyl-boronate derivative (1). Injections of the
samples were made into a GC-MS instrument (model 5970; HewlettPackard, Palo Alto, CA) operated by electron impact ionization as
previously described. Glucose 13C enrichment was measured by
GC-combustion associated to IRMS as described by us (13).
Plasma amino acid concentrations and enrichments were measured
by electron impact GC-MS. Before derivatization, amino acids were
isolated from plasma by use of cation-exchange columns as previously described (1). Amino acids eluted from the columns were
evaporated to dryness and derivatized to form the tert-butyldimethylsilyl (TBDMS) derivative. The [M-57]⫹ ions at m/z ⫽ 260, 261, and
264 were monitored for unlabeled alanine, [3-13C]alanine, and
[2H4]alanine, respectively. The [M-57]⫹ ions at m/z ⫽ 431, 433, and
436 were monitored for unlabeled glutamine, [1,2-13C2]glutamine,
and [2H5]glutamine, respectively. TBDMS-glutamine was chromatographically resolved from TBDMS-glutamate. For all measurements,
the background-corrected tracer enrichments in mole % excess were
calculated as previously defined.
Plasma hormone concentrations were measured by radioimmunoassay with commercial kits, as previously described (23). The catecholamine concentrations were measured by an HPLC method (12).
The concentrations of whole body lactate, pyruvate, glycerol, and
␤-hydroxybutyrate were measured as previously described (15).
Data analysis. Because the liver utilizes and produces glucose, it
stands to reason that its removal provokes a brisk change in both the
plasma clearance rate (PCR) and the rate of appearance (Ra). The
sudden change in PCR and Ra forces the glucose plasma concentration
to change during the anhepatic phase toward a new level (dictated by
the new ratio Ra/PCR); the rate of disappearance (Rd) changes
accordingly. To quantify PCR, Ra, and Rd after liver removal, we
analyzed the tracer disappearance curve, which, normalized to the
administered tracer dose, is the unitary impulse response of the
system. Characterization of the impulse response was performed using
standard noncompartmental techniques (7). Briefly, a two-exponential
function was found to be necessary and sufficient to describe satisfactorily the impulse response. The measurement error was assumed
white, gaussian, of zero mean, and with experimentally determined
standard deviation. Parameter estimation was performed by weighted
nonlinear least squares. Weights were chosen optimally, i.e., equal to
the inverse of the variance of the experimental error (6). The kinetic
parameters that were derived from the analysis of the impulse response were the initial distribution volume [V1 (ml/kg)], the plasma
clearance rate [PCR (ml䡠kg⫺1 䡠min⫺1)], the total distribution volume
[VT (ml䡠kg⫺1 䡠min⫺1)], and the total body mass [QT (mmol/kg)]. Ra
(mmol䡠kg⫺1 䡠h⫺1) was estimated from the impulse response and tracee
data by deconvolution (6), and Rd was then derived from the equation
of the accessible pool: Rd(t) ⫽ Ra ⫺ V1 䡠dC/dt, where C is the tracee
concentration, and dC/dt is its rate of change during the anhepatic
phase. The kinetic analysis of the impulse response measured in
normal subjects and posttransplant patients followed the same approach as that just outlined. Because in such groups the tracee was in
steady state, calculation of Ra and Rd from the impulse response was
straightforward: Ra ⫽ PCR䡠C, and Rd ⫽ Ra. The kinetic analysis in
the control groups (i.e., normal and posttransplant patients) was
performed by relying on the tracer data collected in the first 75 min of
the tracer decay curve. In this way, we ruled out the possibility that the
different observation periods for tracer decay (150 min in the control
groups and 60–75 min in patients during the anhepatic phase) could
differently affect the estimation of the kinetic parameters in the three
groups. It is worth pointing out that 75 min is a relatively short period
for the analysis of glucose kinetics, and thus we were exposed to the
risk of overestimating the slower component of glucose kinetics and,
in turn, overestimating PCR. To assess the magnitude of such error, in
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Table 3. Glucose concentration and kinetics
Concentration, mmol/l
Distribution volume, dl/kg
Total mass, mmol/kg
Clearance, ml䡠kg⫺1 䡠min⫺1
Ra, ␮mol䡠kg⫺1 䡠min⫺1
Rd, ␮mol䡠kg⫺1 䡠min⫺1
ANHEP
POST
CON
6.19⫾0.26
2.17⫾0.06
0.136⫾0.012
2.15⫾0.16
9.51⫾0.88
11.63⫾1.06
4.60⫾0.13‡
2.79⫾0.11*
0.129⫾0.005
2.72⫾0.19*
12.51⫾0.84*
12.51⫾0.84
4.84⫾0.17†
2.66⫾0.42
0.128⫾0.017
3.00⫾0.18*
14.65⫾1.06†
14.65⫾1.06
Values are means ⫾ SE. Ra, rate of appearance; Rd, rate of disappearance.
Total mass is the size of the tracer miscible pool. *P ⬍ 0.05, †P ⬍ 0.01, ‡P ⬍
0.001 vs. ANHEP.
Fig. 2. A: arteriovenous difference in glucose concentration across the right
(R) kidney. The renal venous sampling in 6 subjects demonstrated that the
kidney is a net releaser of glucose during the anhepatic phase. B: arteriovenous
difference in glucose concentration across the portal vein. Glucose concentration in the portal blood was not different from that in the arterial blood.
anhepatic phase was increased, indicating a 25% reduction in
both the glucose space and glucose clearance, as shown in
Table 3. The rate of glucose uptake was similar to that of
control groups. The rate of glucose production was 30%
decreased compared with the control groups.
The carbons from alanine and glutamine were incorporated
into glucose as shown in Fig. 4. The [13C]glucose concentration, normalized for the 13C dose infused, was markedly
reduced compared with the control groups when the precursor
was [13C]alanine, but it was similar to that of healthy subjects
when the precursor was [13C2]glutamine. The area under the
curve in the first 60 min after the bolus was only 10% of the
area of the control groups with alanine infused, and it was
similar to that of healthy control subjects when glutamine was
Fig. 3. Time course of labeled glucose after the bolus. The tracer disappearance profile was similar among the groups, even though the area under the
curve during the anhepatic phase was increased, indicating a 25% reduction in
glucose clearance.
AJP-Endocrinol Metab • VOL
Fig. 4. Incorporation in glucose of 13C from alanine (A) and glutamine (B).
After the bolus of labeled alanine and glutamine (solid lines), 13C was
incorporated in glucose (dashed lines). The [13C]glucose concentration (normalized to the bolus 13C dose) was markedly reduced compared with that of
control groups when the precursor was [13C]alanine, but it was similar to that
of healthy subjects when the precursor was [13C2]glutamine. The area under
the curve in the first 60 min after the bolus was only 10% that of control groups
with alanine infused, and it was comparable to that of healthy control subjects
when glutamine was infused. These data qualitatively show that liver removal
impaired alanine but not glutamine gluconeogenesis.
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infused. These data qualitatively show that the liver removal
impaired alanine but not glutamine gluconeogenesis.
Gluconeogenic precursors. The time course of glutamine
and alanine is reported in Fig. 5. In ANHEP, the alanine
concentration was three times greater and the glutamine concentration was 50% greater than in the control groups. The
GLUCOSE PRODUCTION DURING LIVER TRANSPLANTATION
liver replacement produced a marked reduction in circulating
alanine (⫺20 ⫾ 4%, P ⬍ 0.001) and glutamine (⫺22 ⫾ 4%,
P ⬍ 0.001) concentration within 15 min. The time course of
lactate, pyruvate, and glycerol is shown in Fig. 6. The three
metabolites were increased severalfold during the anhepatic
phase. The liver replacement further increased the concentration of lactate and pyruvate, whereas it decreased that of
glycerol.
Hormones. The hormone concentrations are reported in Fig.
7: insulin and all of the counterregulatory hormones were
markedly increased during the anhepatic phase. The hormone
concentrations were similar in CON and in POST, with the
exception of a modest increment in insulin and in C-peptide in
POST.
anhepatic phase might have occurred either because of a
substantially maintained glucose production or because of a
markedly decreased glucose uptake. The bolus of labeled
glucose allowed us to discriminate between the two possibilities on the basis of direct and model-independent considerations. The area under the tracer concentration curve
was 25% higher than in the control groups, indicating an
⬃25% reduction in glucose clearance. This finding was
expected, because deep anesthesia should have reduced the
brain glucose uptake. Because the circulating glucose concentration increased 30% on average, we deduced that the
glucose disappearance resulting from an increased glucose
concentration and an equally decreased glucose clearance
had to be grossly comparable to that in the control groups.
Glucose production had to be similar to the glucose disappearance minus the amount of glucose that disappeared from
the glucose space during the anhepatic phase. Assuming a
volume of 0.1 l/kg for the glucose-accessible pool, a glucose
decrement of 20 mg䡠dl⫺1 䡠h⫺1 would have indicated a glucose production ⬃0.3 mg䡠kg⫺1 䡠min⫺1 smaller than the
disappearance. On the basis of these qualitative observations, we could conclude that, during the anhepatic phase,
glucose is released at a rate close to that of postabsorptive
subjects. The calculations based on the two-exponential
model of the glucose system quantified the glucose fluxes
and confirmed the qualitatively drawn conclusions. We
excluded that glucose was administered from any exogenous
source during the anhepatic phase. Thus all of the released
glucose had to be of endogenous origin. To qualitatively
DISCUSSION
In this study we showed that, in certain conditions, the
extrahepatic tissues can synthesize an amount of glucose
that is close to the whole body glucose production in healthy
postabsorptive subjects. Our conclusion is based on several
observations. First, we found that, during the anhepatic
phase, the glucose concentration remained in the hyperglycemic range. The glucose concentration diminished during
that period of time, but it did so at a rate that was very slow
compared with what we would have expected if the glucose
production had been consistently reduced with the liver
removal. The decrement in circulating glucose was only
15%/h, whereas we previously found that it decreased by
30–40% within 30 min during hypoglycemic clamp studies
in which the glucose production was reduced by 50% and
the glucose disappearance was not appreciably changed (1,
4). This slow reduction in circulating glucose during the
AJP-Endocrinol Metab • VOL
Fig. 6. Time course of lactate, pyruvate, and glycerol concentrations. The 3
metabolites were increased severalfold during the anhepatic phase. Liver
replacement further increased the concentration of lactate and pyruvate,
whereas it decreased that of glycerol.
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Fig. 5. Time course of alanine and glutamine concentrations. In ANHEP, the
alanine concentration was 3 times greater and the glutamine concentration was
50% greater than in the control groups. Liver replacement produced a rapid
reduction in circulating alanine and glutamine.
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GLUCOSE PRODUCTION DURING LIVER TRANSPLANTATION
prove that it was at least in part of gluconeogenic origin, we
evaluated whether any carbon transfer occurred between
simultaneously infused 13C-labeled alanine or glutamine
tracers and glucose. Because we found detectable 13C enrichments into glucose (remarkably in the experiments in
which glutamine was infused), we concluded that extrahepatic organs have the potential to produce glucose from
amino acids and especially from glutamine.
We sampled the renal and the portal venous blood to locate
the possible sources of the glucose released during the anhepatic phase. We found an appreciable glycemic gradient across
the right kidney that was greater than previously found in
postabsorptive, starved, or hypoglycemic humans (8, 11, 18,
19, 25, 26). With the assumption of a conversion factor of 0.85
between plasma and whole blood glucose concentration and a
renal blood flow of 1.5 l/min during the anhepatic phase, a
plasma glucose gradient of 3.7 mg/dl in 70-kg individuals
would give a net glucose release of 3.7 ␮mol䡠kg⫺1 䡠min⫺1.
When we consider the fact that the kidney may utilize 0.5–1.5
␮mol䡠kg⫺1 䡠min⫺1 (8, 19), glucose production could have been
as large as 5.2 ␮mol䡠kg⫺1 䡠min⫺1, i.e., the majority (70%) of
the observed (7.5 ␮mol䡠kg⫺1 䡠min⫺1) endogenous glucose production. Given the analytical precision of the methods employed, it is possible that glucose release from the kidney could
have accounted for all of the endogenous glucose release,
although it is possible that minor contributions were provided
from other sources. The gradient across the portal vein-drained
viscera was not significantly different from zero. The absence
of a significant net extraction of glucose from the portal blood
does not exclude a gut glucose release, consistent with the
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Fig. 7. Hormone concentrations. Insulin and all of the counterregulatory
hormones were markedly increased during the anhepatic phase. Hormone
concentrations were similar in CON and in POST, with the exception of a
modest increment in insulin and in C-peptide in POST.
recent demonstration that the glucose-6-phosphatase gene is
expressed in the human intestine (24) and that the rat intestine
is a gluconeogenic organ (10).
Regarding the gluconeogenic precursors, lactate is one important candidate, because it is very abundant during the
anhepatic phase, and it was shown that it is a primary gluconeogenic precursor in the kidney of dogs (9) and humans
(27). We were interested in alanine and especially in glutamine, because the latter substrate is the most important
carrier of protein-derived carbons for gluconeogenesis (21).
Glutamine and alanine also shuttle the amino groups derived
from amino acid transamination to the organs capable of
disposing nitrogen. Once deaminated, the glutamine and the
alanine carbon skeletons are suitable gluconeogenic precursors, and gluconeogenesis may actually represent a means to
recycle these carbons without oxidizing them. We studied the
disposal of alanine and glutamine during the anhepatic phase,
and we found that their fluxes were actually increased in such
a condition (3). Glutamine and alanine disposal and cycling
through gluconeogenic precursors occur not only within the
liver with its periportal and perivenous hepatocytes, but also in
the kidney, in the gut, and in the brain (20, 28). These are the
candidate organs for nonhepatic glucose production, and all of
them express to various degrees the enzymes needed to handle
amino nitrogen and to release the newly synthesized glucose
(5, 24).
Despite the marked hyperinsulinemia, the counterregulatory hormones increased the glucose production before the
anhepatic phase. As expected, we found that surgery per se
increased the glucose concentration. This was probably due
to the combined effect of glucagon, epinephrine, and cortisol (16). When the liver was removed, part of this effect
could still be maintained through a possible effect of increased epinephrine on renal gluconeogenesis (26, 27). It
should be noted that, during the anhepatic phase, the glucose
production was close to that of postabsorptive subjects, but
this does not strictly mean that it was appropriate to the
degree of surgical stress and the consequent increment in the
counterregulatory hormones. The increment in glucose concentration after the skin incision until the removal of the
liver was probably sustained by an increment in glucose
production. Thus our experiments represented a sort of
challenge for the extrahepatic organs to produce the maximum possible amount of glucose, being strongly stimulated
by the counterregulatory hormones, surgical stress, and
abundance of gluconeogenic precursors.
In conclusion, we demonstrated that, in the absence of the
liver, extrahepatic organs can release glucose at a rate close to
that of healthy postabsorptive subjects. These results support
the concept of hepatorenal reciprocity, according to which the
renal glucose release compensates for the failure of hepatic
glucose release, and vice versa, to preserve normoglycemia
(29). The kidney accounts for most and possibly all of endogenous glucose release in anhepatic individuals due to an increase in the availability of gluconeogenic precursors and
catecholamines and cortisol, which would be expected to
increase renal gluconeogenesis. Part of the glucose was synthesized from circulating alanine, and a larger amount was
synthesized from glutamine.
GLUCOSE PRODUCTION DURING LIVER TRANSPLANTATION
GRANTS
This work was supported in part by grants from the Italian Ministero della
Sanità (RF 97.4–2126) and from Associazione Italiana Ricerca Cancro (98–01
no. 163).
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