Interaction of free fatty acids and epinephrine in regulating
hepatic glucose production in conscious dogs
Chang An
Chu,
Pietro
Galassetti,
Kayano
Igawa,
Dana K.
Sindelar,
Doss W.
Neal,
Mark
Burish, and
Alan D.
Cherrington
Department of Molecular Physiology and Biophysics,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232
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ABSTRACT |
To determine the effects of an increase in
lipolysis on the glycogenolytic effect of epinephrine (EPI), the
catecholamine was infused portally into 18-h-fasted conscious dogs
maintained on a pancreatic clamp in the presence [portal (Po)-EPI+FFA,
n = 6] and absence (Po-EPI+SAL, n = 6)
of peripheral Intralipid infusion. Control groups with high glucose
(70% increase) and free fatty acid (FFA; 200% increase; HG+FFA,
n = 6) and high glucose alone (HG+SAL,
n = 6) were also included. Hepatic sinusoidal EPI levels were elevated (
568 ± 77 and
527 ± 37 pg/ml,
respectively) in Po-EPI+SAL and EPI+FFA but remained basal in HG+FFA
and HG+SAL. Arterial plasma FFA increased from 613 ± 73 to
1,633 ± 101 and 746 ± 112 to 1,898 ± 237 µmol/l in
Po-EPI+FFA and HG+FFA but did not change in EPI+SAL or HG+SAL. Net
hepatic glycogenolysis increased from 1.5 ± 0.3 to 3.1 ± 0.4 mg · kg
1 · min
1
(P < 0.05) by 30 min in response to portal EPI but did
not rise (1.8 ± 0.2 to 2.1 ± 0.3 mg · kg
1 · min
1)
in response to Po-EPI+FFA. Net hepatic glycogenolysis decreased from
1.7 ± 0.2 to 0.9 ± 0.2 and 1.6 ± 0.2 to 0.7 ± 0.2 mg · kg
1 · min
1
by 30 min in HG+FFA and HG+SAL. Hepatic gluconeogenic flux to glucose
6-phosphate increased from 0.6 ± 0.1 to 1.2 ± 0.1 mg · kg
1 · min
1
(P < 0.05; by 3 h) and 0.7 ± 0.1 to
1.6 ± 0.1 mg · kg
1 · min
1
(P < 0.05; at 90 min) in HG+FFA and Po-EPI+FFA. The
gluconeogenic parameters remained unchanged in the Po-EPI+SAL and
HG+SAL groups. In conclusion, increased FFA markedly changed the
mechanism by which EPI stimulated hepatic glucose production,
suggesting that its overall lipolytic effect may be important in
determining its effect on the liver.
gluconeogenesis; glycogenolysis
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INTRODUCTION |
A RISE IN PLASMA
EPINEPHRINE stimulates adipose tissue lipolysis, muscle
glycogenolysis, and hepatic glucose production (9, 11, 20, 25,
29). The stimulatory effect of epinephrine on hepatic glucose
production is thought to arise both from its direct action on the liver
and from its indirect actions on adipose tissue and muscle. The
latter results in increased availability of gluconeogenic precursors
and free fatty acids (FFA) to the liver (13, 28, 29).
Our recent studies (4, 5) have shown that the direct
effect of epinephrine on hepatic glucose production arises from a
selective stimulation of glycogenolysis. When epinephrine was infused
intraportally so that portal vein epinephrine increased in the absence
of a rise in arterial epinephrine, hepatic glycogenolysis rose
markedly, whereas gluconeogenesis failed to change. In contrast, when
epinephrine was infused peripherally, both arterial and portal vein
epinephrine increased, and as a result the catecholamine simultaneously
stimulated the liver, muscle, and adipose tissue. The effect of
epinephrine on adipose tissue and muscle in turn increased the delivery
of FFA and gluconeogenic precursors to the liver. As a result, even
though the hepatic sinusoidal epinephrine concentration was the same as
when the catecholamine was given portally, it was then able to increase
gluconeogenic flux, albeit at the cost of a reduced glycogenolytic effect.
Earlier in vitro studies (32, 33) showed that FFA can
increase glucose production by stimulating hepatic gluconeogenesis. Studies of the relationship between FFA elevation and glucose production have also been carried out in humans (2, 3, 17, 24,
26, 30). Saloranta et al. (26) showed that
an increase in FFA availability resulting from an infusion of
Intralipid increased gluconeogenesis and glucose production in type 2 diabetic patients. In agreement with this, Boden and colleagues
(2, 3) showed that, in both normal and type 2 diabetic
subjects, increasing and decreasing plasma FFA stimulated and inhibited
gluconeogenesis, respectively. Recently Stingl et al. (30)
showed that an increase in plasma FFA decreased glycogenolysis in
normal men, although plasma insulin rose, making interpretation of the
data difficult. The aim of the present study, therefore, was to
determine the effect of elevated FFA and glycerol on the glycogenolytic
and gluconeogenic actions of portally infused epinephrine.
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METHODS AND MATERIALS |
Experiments.
Experiments were carried out on 24 18-h-fasted conscious mongrel dogs
(20-30 kg) of either sex that had been fed a standard diet of meat
and chow, as described elsewhere (5). The animals were
housed in a facility that met American Association for the Accreditation of Laboratory Animal Care guidelines, and the protocols were approved by the Vanderbilt University Medical Center Animal Care Committee.
A laparotomy was performed 16-18 days before each experiment to
implant catheters and Doppler flow probes into or around appropriate blood vessels, as described elsewhere (5). Each dog was
used for only one experiment. All dogs studied had 1) a
leukocyte count <18,000/mm3, 2) a
hematocrit >35%, 3) a good appetite, and 4)
normal stools.
Each experiment consisted of a 100-min tracer equilibration and hormone
adjustment period (
140 to
40 min), a 40-min basal period (
40 to 0 min), and a 180-min test period (0 to 180 min). In all studies, a
priming dose of purified [3-3H]glucose (42 µCi) was
given at
140 min, followed by a constant infusion of
[3-3H]glucose (0.35 µCi/min),
[U-14C]alanine (0.35 µCi/min), and indocyanine green
(ICG; 0.1 mg · m
2 · min
1).
An infusion of somatostatin (0.8 µg · kg
1 · min
1)
was started at
130 min to inhibit endogenous insulin and glucagon secretion. Concurrently, intraportal replacement infusions of insulin
(300 µU · kg
1 · min
1)
and glucagon (0.5 ng · kg
1 · min
1)
were started. The plasma glucose level was monitored every 5 min, and
euglycemia was maintained by adjusting the rate of insulin infusion.
The final alteration in the insulin infusion rate was made
30 min
before the start of the basal period, and the rate of insulin infusion
(mean of 245 µU · kg
1 · min
1)
remained unchanged thereafter. The study included four groups: those
maintained on a pancreatic clamp in the presence of portal epinephrine
infusion (8 ng · kg
1 · min
1) and the
presence [portal (Po)-EPI+FFA, n = 6] or absence
(Po-EPI+SAL, n = 6) of peripheral Intralipid infusion
and control groups with high glucose (70% increase) and FFA
(HG+FFA; 200% increase, n = 6) and high
glucose alone (HG+SAL, n = 6) (Fig.
1). Epinephrine (8 ng · kg
1 · min
1),
in a solution of 0.07% ascorbic acid, was infused during the test
period via the splenic and jejunal vein catheters in the Po-EPI+SAL and
Po-EPI+FFA groups. Data from some of the dogs (5 of 6) in the
Po-EPI+SAL and HG+SAL groups were published previously (4). In the Po-EPI+FFA and HG+FFA groups, Intralipid
(20%, 0.02 ml · kg
1 · min
1)
and heparin (0.5 unit · kg
1 · min
1)
were infused via a saphenous vein during the epinephrine and glucose
infusions, respectively. Arterial glucose levels in the Po-EPI+FFA,
HG+FFA, and HG+SAL groups were clamped to the level in the Po-EPI+SAL
group by an infusion of exogenous glucose (20% dextrose) given through
the right cephalic vein. Blood pressure and heart rate were measured
using methods described elsewhere (5).

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Fig. 1.
Protocol. Po, portal; EPI, epinephrine; SAL, saline; FFA,
free fatty acid; Po-EPI+SAL and Po-EPI+FFA, 18-h-fasted conscious dogs
maintained on a pancreatic clamp in the presence and absence,
respectively, of peripheral Intralipid infusion; HG+FFA and HG+SAL,
control groups with high glucose (HG) plus FFA and high glucose alone,
respectively.
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Plasma [3H]- and [14C]-, and
[12C]glucose, blood lactate, glycerol,
-hydroxybutyrate (BOHB), alanine, glutamine, glutamate, glycine, serine, threonine, and plasma FFA were determined using previously described methods (5). Plasma glucose was converted to
blood glucose, as previously described, before calculation of net
hepatic glucose balance (NHGB) (5). The levels of insulin,
glucagon, cortisol, epinephrine, and norepinephrine were also
determined as described elsewhere (5).
Doppler flow probes and ICG were used to estimate total hepatic blood
flow (5). Because the infusion of Intralipid impaired the
ICG absorbance, the data in the figures and tables were those calculated with Doppler-measured flows. The NHGB and fractional extraction of blood glucose, lactate, glycerol, BOHB, alanine, other
gluconeogenic amino acids, and plasma FFA in the present study were
calculated using arteriovenous difference (a-v) methods described
elsewhere (5). The average inflowing hepatic sinusoidal plasma epinephrine levels were calculated using Doppler-determined plasma flow, as previously described (5).
Total glucose production (Ra) and utilization
(Rd) were determined using both one- and two-compartment
models, as previously described (4, 6). The results were
similar regardless of which approach was employed because the
deviations from steady state were minimal. The Ra and
Rd data shown in the figures and tables are those
calculated using the two-compartment method. Endogenous glucose
production was calculated by subtracting the glucose infusion rate from
Ra. It should also be noted, because the kidneys produce a
small amount of glucose, that the rate of endogenous glucose production
determined by the tracer method slightly (0.2 mg · kg
1 · min
1)
overestimates total hepatic glucose release (18). Because epinephrine was administrated intraportally in the Po-EPI+SAL and
Po-EPI+FFA groups and the rise in arterial plasma epinephrine was not
significant, epinephrine infusion can be concluded to have had no
effect on renal glucose production. Therefore, renal glucose production
should have been minimal and equal in the four groups.
Gluconeogenesis, as classically defined, is the synthesis and
subsequent release of glucose from noncarbohydrate precursors. Carbon
produced from flux through the gluconeogenic pathway does not
necessarily have to be released as glucose; it can also be stored in
glycogen. Therefore, we make a distinction between gluoneogenic flux to
glucose 6-phosphate (G-6-P; conversion of precursors to G-6-P, G-6-P-neogenesis) and gluconeogenesis
(release of glucose derived from gluconeogenic flux). In the present
studies, we estimated hepatic gluconeogenic flux to G-6-P
(in glucose equivalents), net hepatic gluconeogenic flux (in glucose
equivalents), and net hepatic glycogenolytic flux.
Gluconeogenic flux to G-6-P (4, 6) was
determined by summing the net hepatic uptake rates of the gluconeogenic
precursors (alanine, glycine, serine, threonine, glutamine, glutamate,
glycerol, lactate, and pyruvate) and dividing by two to account for the incorporation of three carbon precursors into the six-carbon glucose molecule. The validation of this approach has been described elsewhere (14).
Net hepatic gluconeogenic flux was determined by subtracting the summed
net hepatic output rates (when such occurred) of the substrates noted
above (in glucose equivalents) and hepatic glucose oxidation (HGO) from
gluconeogenic flux to G-6-P. HGO was assumed to be 0.2 mg · kg
1 · min
1
in each experiment on the basis of data from our earlier studies in the
overnight-fasted dog (15). This parameter was not directly measured, because it is difficult to differentiate between the small
signal and the high inherent noise in the measurement and because it
changes minimally in response to a variety of signals.
Net hepatic glycogenolytic flux (6, 7) was estimated by
subtracting net hepatic gluconeogenic flux from NHGB. A positive number
therefore represents net glycogen breakdown, whereas a negative number
indicates net glycogen synthesis.
Ideally, the gluconeogenic flux rate would be calculated using
unidirectional hepatic uptake and output rates for each substrate, but
this would be difficult, as it would require the simultaneous use of
multiple stable isotopes, which could themselves induce a mild
perturbation of the metabolic state. Therefore, NHGB was used instead,
necessitating consideration of the limits of this approach. There is
little or no production of gluconeogenic amino acids or glycerol by the
liver, so in this case the compromise is of little consequence. Such
is, however, not the case for lactate. Our estimate of the rate of
gluconeogenic flux to G-6-P will be quantitatively accurate
only if we assume that lactate flux is unidirectional at a given moment
(i.e., either in or out of the liver). In a given cell, this does not
seem like an unreasonable assumption in light of the reciprocal control
of gluconeogenesis or glycogenolysis. It has been suggested, however,
that there is spatial separation of metabolic pathways so that
gluconeogenic periportal hepatocytes synthesize glucose and glycogen
primarily from lactate and other noncarbohydrate precursors, whereas
glycolytic perivenous hepatocytes generally consume plasma glucose,
which is predominantly oxidized or released as lactate. Therefore, it is possible that, under normal nutritional conditions, in a net sense,
hepatic gluconeogenic flux and glycolytic flux occur simultaneously, with lactate output or uptake occurring in different cells. To the
extent that flux occurs in both directions simultaneously, the
arteriovenous (a-v) difference method will underestimate the absolute
gluconeogenic flux to G-6-P. It should be noted, however, that net hepatic gluconeogenic flux and net hepatic glycogenolytic flux
can be calculated accurately regardless of whether the assumption related to whether or not simultaneous gluconeogenic and glycolytic substrate fluxes occur is valid or not. To the extent that intrahepatic proteolysis or lipolysis contributes to gluconeogenic flux to G-6-P, our approach will underestimate the process and thus
overestimate glycogenolysis. Our previous work (15) has
shown that the contribution of these two processes in the
overnight-fasted dog is minimal.
Gluconeogenic efficiency was assessed using a double-isotope technique
described elsewhere (5). Because the conversion of
[14C]alanine to [14C]glucose by the kidney
is minimal (19), [14C]glucose production was
almost exclusively attributable to the liver.
Statistical analysis.
All statistical comparisons were made using repeated-measures ANOVA
with post hoc analysis by univariate F-tests or the paired Student's t-test where appropriate. Statistical
significance was accepted at P < 0.05. Data are
expressed as means ± SE.
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RESULTS |
Hormone levels.
The arterial plasma levels of insulin, glucagon, norepinephrine, and
cortisol remained at basal values in all groups throughout the study (Table 1). The arterial
plasma levels of epinephrine did not change significantly during the
portal infusion of epinephrine or saline (Fig.
2). The increment in portal vein plasma
levels of epinephrine was indistinguishable in the Po-EPI+SAL (32 ± 10 to 687 ± 89 pg/ml; P < 0.05) and the
Po-EPI+FFA (50 ± 14 to 659 ± 50 pg/ml; P < 0.05) groups. Similarly, the increments in the hepatic sinusoidal
levels of epinephrine were identical in the Po-EPI+SAL group (42 ± 14 to 568 ± 77 pg/ml; P < 0.05) and the Po-EPI+FFA group (57 ± 14 to 527 ± 37 pg/ml;
P < 0.05).
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Table 1.
Arterial plasma insulin and glucagon during basal and test periods for
Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in the presence
of a pancreatic clamp in conscious 18-h-fasted dogs
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Fig. 2.
Arterial (A), portal (B), and
hepatic sinusoidal plasma (C) levels of epinephrine
during the basal and test periods in experiments conducted using a
pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.
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Hepatic blood flow, arterial blood pressure, and heart rate.
Hepatic blood flow remained basal and stable in all groups (Table 2).
Likewise, the mean arterial blood pressure and heart rate did not
change significantly in any group (Table
2).
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Table 2.
Hepatic blood flow, mean arterial blood pressure, and heart rate during
basal and test periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and
HG+FFA groups in the presence of a pancreatic clamp in conscious
18-h-fasted dogs
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Arterial plasma level, net hepatic uptake, and hepatic fractional
extraction of FFA.
The arterial plasma FFA level remained unchanged in the Po-EPI+SAL and
HG+SAL groups (Fig. 3). Peripheral
infusion of Intralipid and heparin increased the arterial plasma FFA
level from 613 ± 73 to 1,633 ± 101 µmol/l and from
746 ± 112 to 1,898 ± 237 µmol/l (both P < 0.05) by the end of the study in the Po-EPI+FFA and the HG+FFA
groups, respectively (Fig. 3).

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Fig. 3.
Arterial plasma level (A) as well as net
hepatic uptake (B) and fractional extraction (C)
of FFA during the basal and test periods in experiments conducted using
a pancreatic clamp in conscious 18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.
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Net hepatic uptake of FFA remained unchanged in the Po-EPI+SAL and
HG+SAL groups but increased from 2.5 ± 0.4 to 5.3 ± 0.7 µmol · kg
1 · min
1
and from 2.5 ± 0.4 to 4.4 ± 0.8 µmol · kg
1 · min
1
(both P < 0.05) in the Po-EPI+FFA and HG+FFA groups,
respectively (Fig. 3). Net hepatic fractional extraction of FFA did not
change significantly in any group (Fig. 3).
Arterial blood level and net hepatic balance of glycerol and BOHB.
The arterial blood glycerol level remained unchanged in the Po-EPI+SAL
and HG+SAL groups (Fig. 4). Peripheral
infusion of Intralipid and heparin increased arterial blood level of
glycerol from 67 ± 5 to 202 ± 14 µmol/l and 76 ± 8 to 182 ± 10 µmol/l (both P < 0.05) in the
Po-EPI+FFA and HG+FFA groups, respectively. Net hepatic glycerol uptake
remained unchanged in the Po-EPI+SAL and HG+SAL groups but increased
from 1.5 ± 0.2 to 3.7 ± 0.6 µmol · kg
1 · min
1
and from 1.5 ± 0.3 to 3.2 ± 0.3 µmol · kg
1 · min
1
(both P < 0.05) in the Po-EPI+FFA and HG+FFA groups,
respectively (Fig. 4).

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Fig. 4.
Arterial blood levels and net hepatic balances of
glycerol and -hydroxybutyrate (BOHB) during the basal and test
periods in experiments conducted using a pancreatic clamp in conscious
18-h-fasted dogs. Values are means ± SE. *P < 0.05 vs. its own basal.
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The arterial blood level of BOHB remained unchanged in the Po-EPI+SAL
and HG+SAL groups throughout (Fig. 4). Peripheral infusion of
Intralipid increased arterial blood BOHB from 15 ± 3 to 33 ± 6 (120 min) µmol/l and from 28 ± 6 to 46 ± 13 (90 min)
µmol/l (both P < 0.05) in the Po-EPI+FFA and HG+FFA
groups, respectively, after which it remained stable. Net hepatic
output of BOHB remained unchanged in the Po-EPI+SAL and HG+SAL groups
(Fig. 4). On the other hand, it increased from 0.5 ± 0.1 to
1.2 ± 0.4 µmol · kg
1 · min
1
(P < 0.05) in the Po-EPI+FFA group but did not change
significantly (0.9 ± 0.3 to 1.2 ± 0.4 µmol · kg
1 · min
1)
in the HG+FFA group (Fig. 4).
Arterial blood levels and net hepatic balances of lactate and
gluconeogenic amino acids.
The arterial blood lactate level did not change significantly in any
protocol. In response to glucose infusion alone, the liver gradually
switched to net lactate production. In response to portal epinephrine
infusion alone, the liver immediately switched to net lactate output
and remained producing lactate throughout the experiment. In the
presence of glucose infusion, Intralipid infusion increased net hepatic
lactate uptake to 6.1 ± 1.6 µmol · kg
1 · min
1
(Fig. 5; P < 0.05). In
the presence of portal epinephrine infusion, Intralipid infusion caused
an even greater increase in net hepatic lactate uptake (8.6 ± 1.5 µmol · kg
1 · min
1,
P < 0.05; Fig. 5).

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Fig. 5.
Arterial blood levels (A) and net hepatic
balances (B) of lactate during the basal and test periods in
experiments conducted using a pancreatic clamp in conscious 18-h-fasted
dogs. Values are means ± SE. *P < 0.05 vs. its
own basal.
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The fractional extraction of alanine increased 41 and 61% (both
P < 0.05) in the Po-EPI+SAL and Po-EPI+FFA groups,
respectively, during the 3-h test period (Table
3). Neither the arterial blood alanine
level nor the net hepatic alanine balance changed significantly in any
group (Table 3). Similarly, the arterial blood levels, the net hepatic
balances, and the fractional extractions of glutamate, glutamine,
glycine, serine, and threonine were not significantly changed at any
point in any group (Table 3).
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Table 3.
Arterial blood levels and net hepatic balances of alanine, glutamate,
glutamine, glycine, serine, and threonine during basal and test
periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in
the presence of a pancreatic clamp in conscious 18-h-fasted dogs
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Glucose kinetics.
The increment in the arterial blood glucose level was similar in all
four groups (Fig. 6). Hyperglycemia per
se decreased net hepatic glucose output from 2.1 ± 0.2 to
0.7 ± 0.4 mg · kg
1 · min
1
within 60 min (P < 0.05), where it remained. Net
hepatic glucose output decreased to a similar extent (2.1 ± 0.2 to 0.8 ± 0.3 mg · kg
1 · min
1;
P < 0.05) in the HG+FFA group. Portal infusion of
epinephrine increased net hepatic glucose output from 2.1 ± 0.3 to 3.1 ± 0.6 mg · kg
1 · min
1
within 30 min (P < 0.05), after which it fell back to
the basal rate by 60 min. In the presence of Intralipid infusion, the
increment in portal epinephrine increased net hepatic glucose output
only from 1.9 ± 0.2 to 2.5 ± 0.3 mg · kg
1 · min
1
within 30 min, after which it did not change. The changes in tracer-determined endogenous glucose production mirrored those observed
in net hepatic glucose output (Fig. 6).

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Fig. 6.
Arterial blood level (A) and net hepatic
output of glucose (B) as well as tracer-determined glucose
production (C) during the basal and test periods in
experiments conducted using a pancreatic clamp in conscious 18-h-fasted
dogs. Values are means ± SE. *P < 0.05 vs. its
own basal.
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Tracer-determined glucose utilization increased from 2.6 ± 0.3 to
3.8 ± 0.4 (P < 0.05), from 2.5 ± 0.3 to
3.1 ± 0.2 [not significant (NS)], from 2.4 ± 0.2 to
3.5 ± 0.3 (P < 0.05), and from 2.8 ± 0.3 to 3.8 ± 0.4 mg · kg
1 · min
1
(P < 0.05) in the Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and
HG+FFA groups, respectively (Table 4).
The whole body glucose clearance tended to decrease in the presence of
an elevated FFA level (
0.5 ml · kg
1 · min
1
in the Po-EPI+FFA and HG+FFA groups) but remained unchanged in the absence of FFA infusion (Table 4).
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Table 4.
Tracer-determined Rd and clearance during basal and test
periods for Po-EPI+SAL, Po-EPI+FFA, HG+SAL, and HG+FFA groups in
the presence of a pancreatic clamp in conscious 18-h-fasted dogs
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Gluconeogenesis and glycogenolysis.
Hepatic gluconeogenic flux to G-6-P did not change
significantly at any time in either the Po-EPI+SAL or the HG+SAL group (Fig. 7). Peripheral infusion of
Intralipid and heparin increased hepatic gluconeogenic flux to
G-6-P from 0.7 ± 0.2 to 1.6 ± 0.3 mg · kg
1 · min
1
by 90 min and from 0.6 ± 0.2 to 1.2 ± 0.2 mg · kg
1 · min
1
by 150 min (Fig. 7, both P < 0.05) in the Po-EPI+FFA
and HG+FFA groups, respectively. By the end of the study, net hepatic
gluconeogenic flux was 0.2 ± 0.1 and 0.2 ± 0.2 mg · kg
1 · min
1
in the HG+SAL and Po-EPI+Sal groups, respectively, indicating that flux
up the gluconeogenic pathway and down the glycolytic pathway were
essentially in balance. FFA increased net hepatic gluconeogenic flux to
1.0 ± 0.2 and 1.2 ± 0.3 mg · kg
1 · min
1
in the HG+FFA and EPI+FFA groups, respectively, indicating a significant shift in favor of net gluconeogenic flux. Hepatic gluconeogenic efficiency did not change significantly in either the
Po-EPI+SAL or the HG+SAL group (Fig. 7). On the other hand, peripheral
infusion of Intralipid increased hepatic gluconeogenic efficiency by
160 ± 10 and 75 ± 6% (both P < 0.05; Fig.
7) in the Po-EPI+FFA and HG+FFA groups, respectively. Overall hepatic gluconeogenic flux to G-6-P contributed 116 ± 14 mg/kg
during the 3-h test period in the HG+FFA group. Portal infusion of
epinephrine failed to have an impact on the rate (127 ± 21 mg/kg
for 3 h). Peripheral infusion of Intralipid, on the other hand,
increased overall hepatic gluconeogenic flux to G-6-P by
119 ± 18 mg/kg (103%; P < 0.05) and 67 ± 15 mg/kg (58%; P < 0.05) in the Po-EPI+FFA and HG+FFA
groups, respectively (Fig. 8).

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Fig. 7.
Hepatic gluconeogenic flux rate (A), change in
gluconeogenic efficiency (B), and net hepatic glycogenolysis
(C) during the basal and test periods in experiments
conducted using a pancreatic clamp in conscious 18-h-fasted dogs.
Values are means ± SE. *P < 0.05 vs. its own
basal.
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Fig. 8.
Increase in overall gluconeogenic and glycogenolytic
rates as well as the change in net hepatic glycogenolytic rate (at 30 min) during the test period in experiments conducted using a pancreatic
clamp in conscious 18-h-fasted dogs. Values are means ± SE.
*P < 0.05 vs. HG group. #P < 0.05 vs.
other groups in the same panel. G-6-P, glucose
6-phosphate.
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Hyperglycemia per se decreased the net hepatic glycogenolytic rate from
1.6 ± 0.2 to 0.7 ± 0.2 mg · kg
1 · min
1
within 30 min (Fig. 7, P < 0.05), after which it
slowly drifted back to 0.9 ± 0.2 mg · kg
1 · min
1
by the end of the study. Peripheral infusion of Intralipid in the
presence of hyperglycemia abolished net hepatic glycogenolysis (from
1.7 ± 0.2 to
0.1 ± 0.1 mg · kg
1 · min
1,
P < 0.05) by the end of the study. Portal infusion of
epinephrine increased the net hepatic glycogenolytic rate from 1.5 ± 0.3 to 3.1 ± 0.4 mg · kg
1 · min
1
within 30 min (P < 0.05), after which it slowly
drifted back to 1.9 ± 0.3 mg · kg
1 · min
1
(Fig. 7). When Intralipid infusion was added to portal epinephrine infusion, the net glycogenolytic rate rose from 1.8 ± 0.2 to
2.1 ± 0.3 mg · kg
1 · min
1
and then fell to 1.2 ± 0.2 mg · kg
1 · min
1
by 180 min (Fig. 7). Overall net hepatic glycogenolysis in the HG+SAL
group contributed 121 ± 15 mg/kg over the 3-h test period. Relative to this, it increased by 253 ± 31 mg/kg (209%;
P < 0.05) and by 97 ± 11 mg/kg (80%;
P < 0.05), respectively, in the Po-EPI+SAL and
Po-EPI+FFA groups (Fig. 8). It decreased by 38 ± 6 mg/kg (31%; P < 0.05) in the HG+FFA group (Fig. 8).
 |
DISCUSSION |
Previous studies (9, 25, 29) have shown that
epinephrine simultaneously increases lipolysis and hepatic glucose
production. Normally, a rise in the hepatic sinusoidal epinephrine
level is accompanied by a concomitant rise in arterial epinephrine and, as a result, by an increase in the plasma FFA level. In recent studies
(4, 5), we showed that a selective increase in the hepatic
sinusoidal epinephrine level (occurring without an increase in arterial
epinephrine) increased net hepatic glycogenolysis but had no effect on
hepatic gluconeogenesis. The aim of the present study was to assess the
effects of a simulated rise in lipolysis on epinephrine's ability to
increase net hepatic glycogenolysis and/or gluconeogenesis in vivo. The
arterial levels of insulin, glucagon, epinephrine, norepinephrine, and
cortisol remained at basal values in all four groups. Additionally, the
arterial blood glucose concentrations were kept at a similar
hyperglycemic level in each group, thereby simplifying data
interpretation. Finally, the increments in the hepatic portal and
sinusoidal epinephrine levels were indistinguishable in the Po-EPI+SAL
and Po-EPI+FFA groups. As a result, we were able to clearly examine the
effects of a rise in FFA and glycerol on epinephrine's hepatic
glycogenolytic and gluconeogenic actions.
Intralipid and heparin infusion increased the arterial level and net
hepatic uptake of FFA ~2.5- and 1.8-fold, respectively, in the
presence of hyperglycemia (HG+FFA). Hepatic gluconeogenic flux to
G-6-P and gluconeogenic efficiency were increased by
~100% (0.6 ± 0.2 to 1.2 ± 0.2 mg · kg
1 · min
1,
P < 0.05) and 75% (33 ± 5% to 58 ± 10%,
P < 0.05), respectively. Neither hepatic gluconeogenic
flux to G-6-P nor gluconeogenic efficiency was significantly
changed in the presence of hyperglycemia alone. It is clear, therefore,
that Intralipid infusion had a stimulatory effect on hepatic
gluconeogenesis in the presence of hyperglycemia in vivo. This is in
line with previous in vitro findings (32, 33), which
indicated that a rise in the availability of FFA in liver increased
gluconeogenesis. It should be noted that, in the present study, the
arterial blood level and net hepatic uptake of glycerol increased from
76 ± 8 to 182 ± 10 µmol/l (P < 0.05) and
from 1.5 ± 0.3 to 3.2 ± 0.3 µmol · kg
1 · min
1
(P < 0.05), respectively, during the infusion of
Intralipid. However, even if all of the extra glycerol taken up by the
liver were entirely converted to glucose (
1.7
µmol · kg
1 · min
1
glycerol
0.15
mg · kg
1 · min
1
glucose), it could account for only 25% of the observed increase in
net gluconeogenic flux (0.6 ± 0.2 to 1.2 ± 0.2 mg · kg
1 · min
1).
Therefore, the data clearly indicate that the increase in net hepatic
uptake of FFA results in a stimulation of hepatic gluconeogenic flux to
G-6-P even in the presence of hyperglycemia. This
enhancement of gluconeogenic flux to G-6-P is also evident
from the observation that the increase in net hepatic uptake of FFA was
associated with a significant increase in net hepatic lactate uptake.
Hyperglycemia per se increased liver glycolysis and thus increased net
hepatic lactate output. Addition of Intralipid switched net hepatic
lactate balance from output to uptake. Because both the arterial levels
and net hepatic output of BOHB increased during Intralipid infusion, it
is apparent that
-oxidation of FFA in the liver increased. Increased
-oxidation could stimulate gluconeogenic flux by producing more ATP
for the support of gluconeogenesis, by increasing the availability of
the NADH for the glyceraldehyde-3-phosphate dehydrogenase reaction, and
by activating pyruvate carboxylase via an increase in acetyl-CoA and
other thioesters (1, 22, 23). In addition, the increase in
citrate that occurs in response to increased FFA could inhibit
phosphofructokinase (PFK), thus at the same time limiting
glycolysis (22). It should be noted that an inhibition of
glycolysis would increase the net flux of carbon to G-6-P
even if hepatic lactate production and uptake do occur simultaneously,
as suggested by Radziuk and Pye (21). It has also been
suggested that increased intracellular fatty acid metabolites (fatty
acyl-CoAs, diacylglycerol, ceramides) can cause defects in insulin
signaling in the liver (27).
Because neither hepatic gluconeogenic flux to G-6-P, net
hepatic gluconeogenic flux, nor overall hepatic gluconeogenic
efficiency was significantly changed in the Po-EPI+SAL group, the
present data are consistent with our earlier finding (4)
that a selective increase in hepatic sinusoidal epinephrine alone has
no effect on hepatic gluconeogenesis. The addition of Intralipid
infusion to portal epinephrine infusion, however, resulted in an
increase in hepatic gluconeogenic flux to G-6-P from
0.7 ± 0.2 to 1.6 ± 0.3 mg · kg
1 · min
1
(230%, P < 0.05). Because the increment in overall (3 h) gluconeogenic flux to G-6-P was greater (119 ± 18 mg/kg) in the Po-EPI+FFA group than in the HG+FFA group (67 ± 15 mg/kg), even though both groups had similar increases in net hepatic
FFA uptake, it is clear that the increase in FFA caused a gluconeogenic
effect of epinephrine to be manifested. It is possible that epinephrine
stimulated
-oxidation (26, 30) and thereby further
increased gluconeogenic flux. Indeed, the change in ketogenesis was
greater in the Po-EPI+FFA group than in the HG+FFA group. In addition,
both net hepatic lactate uptake and the hepatic fractional extraction
of alanine increased to the greatest extent in response to Intralipid
infusion in the presence of portal epinephrine infusion. Once again, an increase in net hepatic glycerol uptake can explain only part of the
increase in gluconeogenic flux. Even if all of the extra glycerol taken
up by the liver were converted to glucose (
2.2 µmol · kg
1 · min
1
glycerol
0.2
mg · kg
1 · min
1
glucose), it could account for only 22% of the increase in net gluconeogenic flux (0.7 ± 0.2 to 1.6 ± 0.3 mg · kg
1 · min
1).
Therefore, the present data strongly suggest that the ability of
increased lipolysis to allow expression of epinephrine's gluconeogenic action at the liver results from the stimulatory effect of FFA.
The increment in hepatic sinusoidal epinephrine increased net hepatic
glycogenolysis by 1.5 ± 0.2 mg · kg
1 · min
1
by 30 min. Because hyperglycemia per se decreased net hepatic glycogenolysis by 0.9 ± 0.2 mg · kg
1 · min
1
at that time, the effect of the selective increase in hepatic sinusoidal epinephrine on net hepatic glycogenolysis was 2.4 ± 0.2 mg · kg
1 · min
1
(30 min; Figs. 7 and 8). In the presence of Intralipid
infusion, the same increment in hepatic sinusoidal epinephrine raised
net hepatic glycogenolysis above baseline by only 0.3 ± 0.1 mg · kg
1 · min
1
at 30 min. Therefore, Intralipid infusion diminished the effect of the
increment in hepatic sinusoidal epinephrine on net hepatic glycogenolysis to 1.2 ± 0.2 mg · kg
1 · min
1
(50% inhibition). The overall (3-h) net glycogenolytic effect attributable to the rise in portal epinephrine was 253 ± 31 mg/kg. However, this was diminished to 97 ± 11 mg/kg when
Intralipid infusion was added to the rise in portal epinephrine.
Therefore, a 62% inhibition was observed when the overall (3-h) impact
was examined. One can thus conclude that a rise in the circulating FFA
level can inhibit the glycogenolytic effect of epinephrine on hepatic
glucose production by more than one-half. This suggests that, when
plasma epinephrine increases due to adrenal secretion, its effect on
the adipose tissue significantly reduces the glycogenolytic effect that
it would otherwise have on the liver. Because glycogen and fat are the
two major fuel stores used by the body for energy, the physiological
significance of this inhibition might be to preserve the liver glycogen
content to allow a timely response to an unexpected need by the central
nervous system when glucose is scarce, such as during fasting,
hypoglycemia, or prolonged exercise.
The next question is how a high level of FFA can bring about an
inhibitory effect on glycogen breakdown. Previous in vivo studies in
the dog (10, 12) and human (16, 34) showed that infusing gluconeogenic precursors, i.e., alanine, lactate, and
glycerol, increased hepatic gluconeogenesis but did not change hepatic
glucose production. They therefore suggested that a rise in
gluconeogenesis itself may trigger an inhibition of hepatic glycogenolysis (8). Because the initial change (i.e., 30 min) in hepatic gluconeogenic flux was minimal, regardless of the
protocol (the time at which the glycogenolytic effect was fully
manifested), it seems likely that the inhibitory effect of high FFA
levels on epinephrine's glycogenolytic action resulted from a direct inhibition associated with the rapid increase in fat oxidation by the
liver. Indeed, an increase in FFA oxidation would increase citrate
levels, raising the possibility that it might inhibit PFK and blunt
glycogen breakdown.
In conclusion, the data from the present studies indicate that
epinephrine's lipolytic action limits its glycogenolytic effect and
augments its gluconeogenic/glycolytic effects on the liver in vivo.
Clearly, the impact of epinephrine on the liver is a function of both
its direct actions on the liver and the metabolite changes that
accompany the catecholamine to the liver.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Mary Courtney Moore, David Wasserman, Dale
Edgerton, and Owen P. McGuinness for valuable comments and careful
review of this manuscript. We specially appreciate assistance from Jon
Hastings, Melanie Scott, Maya Emshwiller, Pam Venson, Wanda Snead, Paul
Flakoll, and Yang Ying.
 |
FOOTNOTES |
This research was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grants 2RO1 DK-18243 and 5P60
DK-20593 (Diabetes Research and Training Center), and National
Institutes of Health Grant 2P30 DK-26657 (Nutrition Center).
Address for reprint requests and other correspondence:
A. D. Cherrington, Dept. of Molecular Physiology and
Biophysics, 702 Light Hall, Vanderbilt University School of Medicine,
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.00565.2001
Received 28 December 2001; accepted in final form 19 August 2002.
 |
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