The inhibition of gluconeogenesis
following alcohol in humans
SCOTT Q. SILER,1 RICHARD A. NEESE,1,2 MARK P. CHRISTIANSEN,2
AND MARC K. HELLERSTEIN1,2
1Department of Nutritional Sciences, University of California, Berkeley 94720-3104;
and 2Division of Endocrinology and Metabolism, Department of Medicine,
University of California, San Francisco, California 94110
Siler, Scott Q., Richard A. Neese, Mark P. Christiansen, and Marc K. Hellerstein. The inhibition of gluconeogenesis following alcohol in humans. Am. J. Physiol. 275
(Endocrinol. Metab. 38): E897–E907, 1998.—Accurate quantification of gluconeogenic flux following alcohol ingestion in
overnight-fasted humans has yet to be reported.
[2-13C1]glycerol, [U-13C6]glucose, [1-2H1]galactose, and acetaminophen were infused in normal men before and after the
consumption of 48 g alcohol or a placebo to quantify gluconeogenesis, glycogenolysis, hepatic glucose production, and intrahepatic gluconeogenic precursor availability. Gluconeogenesis decreased 45% vs. the placebo (0.56 6 0.05 to 0.44 6 0.04
mg·kg21 ·min21 vs. 0.44 6 0.05 to 0.63 6 0.09 mg·kg21 ·min21 ,
respectively, P , 0.05) in the 5 h after alcohol ingestion, and
total gluconeogenic flux was lower after alcohol compared
with placebo. Glycogenolysis fell over time after both the
alcohol and placebo cocktails, from 1.46–1.47 mg · kg21 · min21
to 1.35 6 0.17 mg · kg21 · min21 (alcohol) and 1.26 6 0.20
mg · kg21 · min21, respectively (placebo, P , 0.05 vs. baseline).
Hepatic glucose output decreased 12% after alcohol consumption, from 2.03 6 0.21 to 1.79 6 0.21 mg · kg21 · min21 (P , 0.05
vs. baseline), but did not change following the placebo.
Estimated intrahepatic gluconeogenic precursor availability
decreased 61% following alcohol consumption (P , 0.05 vs.
baseline) but was unchanged after the placebo (P , 0.05
between treatments). We conclude from these results that
gluconeogenesis is inhibited after alcohol consumption in
overnight-fasted men, with a somewhat larger decrease in
availability of gluconeogenic precursors but a smaller effect
on glucose production and no effect on plasma glucose concentrations. Thus inhibition of flux into the gluconeogenic precursor pool is compensated by changes in glycogenolysis, the fate
of triose-phosphates, and peripheral tissue utilization of
plasma glucose.
mass isotopomer distribution analysis; glucose production;
stable isotopes
ALCOHOL, OR ETHANOL
(EtOH), has long been thought to
be a potent inhibitor of gluconeogenesis. Krebs et al.
(21) were among the first to demonstrate this by
perfusing rat livers with gluconeogenic precursors in
the presence and the absence of EtOH. They concluded
from this model that the change in hepatic redox state
was responsible for inhibition of gluconeogenesis by
EtOH. Several investigators have studied the incorporation of labeled lactate (22) or alanine (5, 23) into
glucose before and during infusion of EtOH in normal,
healthy humans. EtOH-induced reductions of 65–75%
in the incorporation of lactate and/or alanine into
glucose were reported in these studies. Similar reductions have been observed in non-insulin-dependent
diabetics as well (5, 40).
Inhibition of gluconeogenesis has also been implicated in the etiology of alcoholic hypoglycemia in the
clinical setting. Freinkel et al. (9) presented indirect
evidence for the inhibition of gluconeogenesis by EtOH
when they were unable to counteract EtOH-induced
hypoglycemia with infusion of glucagon in normal
subjects who had fasted 24–48 h. Other groups have
measured a decrease in hepatic glucose production as
hypoglycemia developed during the infusion of EtOH
after a 2- to 3-day fast (43, 53, 54). Because hepatic
glycogen levels after a long fast are already substantially depleted (35), these findings suggest that the
decrease in hepatic glucose production after EtOH
infusion likely reflects a reduction in gluconeogenesis.
Direct quantitative evidence for the inhibition of
gluconeogenesis in humans by EtOH has yet to be
presented, however, with reliable techniques. New techniques for the measurement of total gluconeogenesis
(i.e., from all precursors) with stable isotope tracers
have recently been developed (15, 24, 25, 33, 48, 49),
including mass isotopomer distribution analysis (MIDA;
Refs. 15 and 33). Our goal in this study was to directly
measure the effects of EtOH on gluconeogenesis, glycogenolysis, and other hepatic metabolic fluxes in normal,
overnight-fasted humans by combining MIDA with
probes of intrahepatic metabolism (11, 12, 29) and
other isotopic techniques (13, 16, 15). Portions of the
material presented here have been previously reported
in abstract form (45).
METHODS
Materials. [2-13C1]glycerol, [U-13C6]glucose, and [1-2H1]galactose were purchased from CIL (Andover, MA) and/or Isotec
(Miamisburg, OH). Isotopic purity was .98% for all tracers
used. Acetaminophen was purchased from Mallinkrodt
(Phillipsburg, NJ).
Subject characteristics. Volunteers were recruited by advertisement and gave written informed consent before enrolling
in the study. All protocols were approved by the University of
California at San Francisco Committee on Human Research
and the University of California at Berkeley Committee for
the Protection of Human Subjects. All five subjects were
males and moderate consumers of EtOH (,120 g/wk). Subject
characteristics are listed in Table 1. None had any history of
alcoholism, and all the subjects had normal liver enzyme
levels in blood. All subjects but one had normal serum lipid
levels; fasting serum triglyceride levels were elevated in one
individual (409 mg/dl). The data generated from this subject
were similar to the data from the other subjects and, thus, are
included here. None of the subjects had history of any medical
diseases, metabolic disorders, diabetes mellitus, or a family
history of diabetes mellitus, and none were using medications
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
E897
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GLUCONEOGENESIS AFTER ETHANOL
Table 1. Subject characteristics
Age, yr
Weight, kg
BMI, kg/m2
Body Fat, %
Total Body Water, liters
Triglycerides, mg/dl
Total Cholesterol, mg/dl
28.2 6 1.3
78.4 6 6.2
25.5 6 1.9
14.2 6 6.6
56.3 6 13.6
179 6 134
177 6 21
Values are means 6 SD of 5 subjects. One subject had triglycerides .400 mg/dl but was included in the data set. BMI, body mass index. See
for details.
METHODS
with known metabolic effects. Body composition was measured with bioelectrical impedance analysis (model no. 1990B,
Valhalla Scientific, San Diego, CA). Body fat and total body
water were calculated according to the equations of the
manufacturer.
Study design. Subjects were admitted twice to the General
Clinical Research Center at San Francisco General Hospital
for separate studies. During one admission, four alcoholic
beverages containing 12 g EtOH (40% vodka, Absolut, Ahus,
Sweden) mixed with sugar-free lemonade (Kraft General
Foods, White Plains, NY) were administered, whereas during
the other admission the sugar-free lemonade was given as a
placebo. Each EtOH cocktail contained 84 kcal from EtOH
and 4 kcal from the sugar-free lemonade, whereas the placebo
cocktails contained 4 total kcal (as measured by bomb calorimetry). The order of the treatment was randomized, with the
second admission following 1 wk after the first.
The infusion protocol is shown in Fig. 1. After an evening
meal (40% total daily caloric requirement, 55:30:15, carbohydrate:fat:protein) at 1700, intravenous infusion of isotopes
was begun at 0400. [U-13C6]glucose (0.02 mg · kg21 · min21 )
and [1-2H1]galactose (0.05 mg · kg21 · min21 ) were infused at a
constant rate after a 1-h priming bolus at 0400. Acetaminophen was also infused at a constant rate (3.7 mg/min),
starting at 0400. Acetaminophen was prepared for infusion by
Dr. Lou Tomimatsu (University of California San Francisco
School of Pharmacy). Acetaminophen (dense powder; 5 g;
USP) was mixed into 100 ml final volume containing propylene glycol (40 ml), ethanol (10 ml), and 5% dextrose (as 100
ml). An aliquot of this solution was added to 0.45% saline and
infused to deliver 4 ml/h (containing 200 mg acetaminophen,
0.4 g ethanol, 1.6 g proplyene glycol, and 200 mg glucose per
h). A constant infusion of [2-13C1]glycerol (0.25 mg · kg lean
body mass21 · min21 ) was begun at 0500. All infusions were
terminated at 1300. Cocktails (EtOH or placebo) were administered at 0800, 0830, 0900, and 0930. Blood was drawn
before isotope administration (baseline), in the hour before
the cocktails (pre-EtOH), and half-hourly thereafter (postEtOH). Urine samples were collected at 0800 and at 1300,
representing pre-EtOH and post-EtOH time points, respectively. No food was consumed until the termination of the
isotope infusions.
Fig. 1. Study design. Arrows indicate time
points (400–1300). See METHODS for details. EtOH, ethanol; BSL, baseline.
Metabolite and hormone isolation and measurement.
Plasma glucose concentrations were determined with a glucose analyzer (YSI, Yellow Springs, OH). Blood alcohol concentrations were measured with a standard kit (Sigma, St. Louis,
MO). Insulin (Diagnostic Products, Los Angeles, CA) and
glucagon (ICN Biochemicals, Costa Mesa, CA) concentrations
were measured by radioimmunoassay.
Glucose and glycerol were isolated from plasma by ionexchange chromatography as described previously (16, 15,
33). Plasma was deproteinized with perchloric acid (1:2),
desalted with 6 N KOH, and loaded with a water wash onto
gravity-flow columns. One set of columns contained an anionexchange resin (AG 1-X8, Bio-Rad, Hercules, CA) and the
other contained a cation-exchange resin (AG 50W-X8, BioRad). The two columns were used in sequence, with the
eluent collected completely and lyophilized. The samples
were then divided and derivatized three ways: glucose pentaacetate, aldonitrile pentaacetate, and glycerol triacetate.
Glucose pentaacetate and glycerol triacetate were prepared
by combining 100 µl of 2:1 acetic anhydride:pyridine with the
lyophilized sample at room temperature for 15 min. After
drying under nitrogen gas, the samples were reconstituted in
ethyl acetate for gas chromatography-mass spectrometry
(GC-MS) analysis. The aldonitrile derivative was formed by
combining the lyophilized sample with hyroxylamine in pyridine (2%) for 30 min at 100°C. After cooling, 100 µl of 2:1
acetic anhydride:pyridine was added to the solution and kept
at room temperature for 15 min. The solution was then
reconstituted in ethyl acetate after drying under nitrogen
gas.
Acetaminophen-glucuronide (GlcUA) was isolated from
urine and derivatized to either the saccharic acid or the
methyl-tetraacetate derivative, as previously described (13,
15, 29, 31). Briefly, the urine was acidified to pH 1.0 with HCl
and neutralized with NaOH. After centrifugation, the supernatant was injected onto a Waters 0.2 3 10 cm reverse-phase
C18 resolve high-performance liquid chromatography column
in a radial compression module system (Waters, Milford, MA)
with a C18 precolumn. The mobile phase was 2% acetonitrile
in water with 1 ml glacial acetic acid per liter. Absorbance was
measured at 254 nm with a variable wavelength ultraviolet
detector (160 Absorbance Detector, Beckman, San Ramon,
GLUCONEOGENESIS AFTER ETHANOL
CA). The flow rate was as follows: 6.0 ml/min initially,
decreasing to 1.0 ml/min from 0.8 to 1.7 min, and then
increasing to 6.0 min. The cycle was terminated at 6 min.
Acetaminophen-GlcUA eluted between 1.0 and 1.2 min with
this chromatographic profile. The peak was collected manually from the column effluent and lyophilized. The lyophilized
sample was then converted to one of two derivatives, saccharic acid or methyl-tetraacetate. The method for the conversion to the dimethyl-tetraacetate saccharic acid derivative
was a modification of the method reported by Mehltretter
(31). Concentrated nitric (35 µl) acid and sodium nitrite (25
µl; 0.5 g/ml) were added to the lyophilized sample and the
mixture was heated at 60°C for 1 h. The sample was then
lyophilized and methylated by the addition of 0.5 N methanolic HCl for 8 h of heat at 80°C. Acetylation was achieved as
described above for plasma glucose, with an acetic anhydridepyridine mixture. The methyl-tetraacetate derivative was
formed by following the saccharic acid protocol, with the
exclusion of the nitric acid-sodium nitrite reaction.
MS. GC-MS was performed with an HP 5971 instrument
(Hewlett-Packard, Palo Alto, CA). The measurement of the
M6 enrichments of glucose pentaacetate and saccharic acid
were determined by comparison with a standard curve. The
M1 enrichment of glycerol triacetate was also determined by
comparison with a standard curve.
The M1 and M2 isotopomers of the aldonitrile and saccharic
acid derivatives were used for the measurement of gluconeogenesis. We employed analytical guidelines for optimizing MS
accuracy and reliability of gluconeogenesis estimates that
have been outlined previously (7, 33). Briefly, these guidelines
include 1) frequent testing of natural abundance (baseline)
samples to confirm instrument accuracy for each mass isotopomer (33); 2) meeting requirements that baseline fractional
abundances for all mass isotopomers be within 2% (0.0030 for
M1, 0.0005 for M2 ) of theoretical values for instrument
performance to be considered acceptable; 3) rejection of data
as below the detection limit for reliable quantitation if
enrichments for any mass isotopomer were less than 0.0050
[0.50 mole percent excess (MPE)]; 4) preinjection of samples
to establish concentrations present and reinjection to maintain ion abundances within a constant range for the baseline
and all samples analyzed to avoid concentration effects on
isotope ratios (36); and 5) administration of [2-13C1]glycerol at
doses that maintain the triose-phosphate pool enrichments in
the range (33) between 0.10 and 0.20.
The M1 enrichments from [1-2H1]galactose in glucose pentaacetate and methyl-GlcUA were determined by correcting
for underlying 13C distribution with the aldonitrile and
saccharic acid derivatives (7). Because some M11 isotopic
species in glucose and methyl-GlcUA are produced from
13C-glycerol, the M
11 in the glucose pentaacetate and methylGlcUA derivatives (which contain label from both
[1-2H1]galactose and [13C]glycerol) must be corrected to account for the M11 in the aldonitrile and saccharic acid
derivatives (which do not contain 1-2H1 label) to determine
true [1-2H1]glucose and GlcUA enrichments. For this correction, at first the contribution to the pentaacetate and methylGlcUA isotopomeric pattern from [13C]glycerol incorporation
was determined, on the basis of aldonitrile and saccharic acid
analyses of the same samples. A theoretical standard curve
was then generated in which various combinations of the two
unknowns (natural abundance and 1-2H1-labeled molecules)
were superimposed on the known isotopomeric distribution
from the gluconeogenesis-derived molecules. The slope and
intercept for M11-glucose and GlcUA as a function of the
proportion of [1-2H1]glucose was then calculated from this
standard curve, which was applied to the measured M11-
E899
glucose pentaacetate and methyl-GlcUA derivatives, to establish [1-2H1]glucose and GlcUA enrichments. A unique standard curve was generated for each time point on the basis of
the gluconeogenic contribution observed in the concurrent
aldonitrile and saccharic acid derivatives, because a unique
contribution from gluconeogenic 13C needed to be accounted
for in each sample (7, 33).
All GC-MS analyses of glucose were performed with a 60 m
DB-17 column (J & W Scientific, Folsom, CA), and glycerol
analyses were performed with a 10 m DB-225 column (J & W
Scientific). Chemical ionization with methane and selected
ion monitoring were used for all analyses (13, 15, 33).
The GC-MS temperature profiles for each derivative were
as follows: the initial temperature for the glucose pentaacetate derivative was 150°C and rose to 270°C at a rate of
40°C/min. The column was held at 270°C for 9.5 min at the
end of the run. The 331, 332, 333, and 337 ions were analyzed,
representing the M0, M1, M2, and M6 mass isotopomers,
respectively. The temperature for the aldonitrile derivative
rose from a starting value of 120°C at a rate of 40°C/min to a
final temperature of 270°C. The ions monitored were the 328
(M0-aldonitrile tetraacetate), 329 (M1 ), and 330 (M2 ). The
methyl-GlcUA derivative was analyzed using an initial temperature of 100°C and rising to 180°C at a rate of 40°C/min
then rising 4°C/min until 252°C, and rising to 280°C at
45°C/min. The M0 of 317, M1 of 318, and M2 of 319 were
monitored for the measurement of methyl-GlcUA enrichments. The saccharic acid derivative was separated by using
a starting temperature of 150°C and rising at a rate of
40°C/min to a final temperature of 270°C, which was held for
9.75 min. The ions monitored for the saccharic acid derivative
are the 347 (M0 ), 348 (M1 ), 349 (M2 ), and 353 (M6 ) ions. The
glycerol triacetate derivative was analyzed using an initial
temperature of 75°C and rising 45°C/min to 160°C, 5°C/min
to 171°C, and 40°C/min to a final temperature of 220°C. The
M0 and M1 of 159 and 160, respectively, were collected.
Calculations. The rates of appearance (Ra ) of plasma
glucose and intrahepatic UDP-glucose (via acetaminophenGlcUA) were calculated by the dilution technique (13, Table
2). Ra UDP-glucose was calculated based on the assumption of
a constant and complete entry of galactose into the UDPglucose pool in the liver (13).
Fractional gluconeogenesis in both plasma glucose and
hepatic UDP-glucose (as measured in secreted acetaminophenGlcUA) was calculated by MIDA (Table 2) as described in
detail elsewhere (14, 33). With the use of combinatorial
probabilities, the enrichment of the true gluconeogenic precursor (triose-phosphate) can be inferred from the ratio of the
excess double-labeled to single-labeled species (EM2/EM1 ) of
glucose. The precursor-product relationship can then be used
to calculate the fractional contribution of gluconeogenesis to
plasma glucose or hepatic UDP-glucose production.
The fractional contribution of plasma glycerol to plasma
gluconeogenesis was calculated as the ratio of the calculated
(by MIDA) triose-phosphate enrichment to the measured
plasma glycerol enrichment. The absolute rate of contribution
of plasma glycerol to plasma gluconeogenesis was calculated
by multiplying the fractional contribution of plasma glycerol
to plasma gluconeogenesis by the absolute rate of plasma
gluconeogenesis (Table 2).
The fractional release of labeled hepatic UDP-glucose in
plasma glucose (R) was calculated from the recovery of
infused [1-2H1]galactose in plasma glucose (15), on the assumption that all galactose passes through hepatic UDP-glucose
(Fig. 2, Ref. 13). The M1 enrichment of glucose was multiplied
by Ra glucose and divided by the galactose infusion rate to
calculate R (Table 2). The deuterium-influenced M1 enrich-
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GLUCONEOGENESIS AFTER ETHANOL
Table 2. Equations for calculated parameters
1) Ra glucose (mg · kg21 · min21 ) 5 (Iglc /M6 glucose enrich) 2 Iglc
2) Ra UDP-glucose (mg · kg21 · min21 ) 5 (Igal/[1 2 2H1 ]GUA
enrich) 2 Igal
3) Fractional GNG 5 EM1 glucose enrich/A*1 glucose enrich
4) Fractional UDP-GNG 5 EM1 GUA enrich/A*1 GUA enrich
5) Fractional contribution of plasma glycerol to GNG 5 trioseP enrich/plasma glycerol enrich
6) Fraction of plasma glucose from UDP-glucose 5 R 5 [1 2 2H1]
glucose enrich/[1 2 2H1 ]GUA enrich
7) Fractional direct pathway 5 D 5 M6 GUA enrich/M6 glucose
enrich
8) Absolute GNG (mg · kg21 · min21 ) 5 Ra glucose 3 fractional
GNG
9) Absolute glycogen to glucose flux (mg · kg21 · min21
) 5 (1 2 fractional GNG) 3 Ra glucose
10) Absolute UDP-GNG (mg · kg21 · min21 ) 5 Ra UDPglucose 3 fractional UDP-GNG
11) Corrected Rd(H) 5 D 3 Ra UDP-glucose 3 (1 2 R)
12) Total GNG pathway flux (mg · kg21 · min21 ) 5 (absolute
GNG) 1 [absolute UDP-GNG 3 (1 2 R)] 2 [fractional
GNG 3 corrected Rd(H)]
13) GNG partitioning coefficient into plasma glucose 5 absolute
GNG/(absolute GNG 1 absolute UDP-GNG)
14) Absolute contribution from plasma glycerol to GNG
(mg · kg21 · min21 ) 5 absolute GNG 3 fractional contribution
of plasma glycerol to GNG
Ra , rate of appearance or turnover/flux; Rd(H), rate of direct
pathway hepatic glucose disposal; Iglc and Igal , infusion rates of
glucose and galactose, respectively; M6 , M16 isotopomer of glucose;
GUA, glucuronate; GNG, gluconeogenesis; EM1 , excess M11 isotopomer; A*1 , asymptotic value for EM1 as calculated by mass isotopomer distribution analysis (MIDA); enrich, enrichment; triose-P,
triose-phosphates.
Fig. 2. Key pathways of gluconeogenesis after EtOH. glc, glucose; gal, galactose; glc-6P, glucose 6-phosphate; glc-1P,
glucose 1-phosphate; GUA, glucuronate; aceta, acetaminophen; AcHO, acetaldehyde; OAA, oxaloacetate; AcCoA,
acetyl-coenzyme A; pyr, pyruvate; PEP,
phosphoenolpyruvate; triose-P, triosephosphates; glyc-3P, glycerol 3-phosphate; glyc, glycerol. * Metabolic reaction that includes interconversion
between NAD and NADH.
ment of plasma glucose was corrected for the underlying 13C
labeling pattern determined by MIDA (15). The direct pathway contribution from blood glucose to hepatic UDP-glucose
was calculated as the ratio of the M6 enrichment in acetaminophen-GlcUA to the M6 enrichment in plasma glucose (Ref. 15,
Table 2).
Absolute gluconeogenesis was calculated as the product of
Ra glucose and fractional gluconeogenesis (Table 2). The
glycogenolytic contribution to plasma glucose was calculated
as the difference between Ra glucose and absolute gluconeogenesis (Table 2). Absolute UDP-gluconeogenesis was calculated
as the product of fractional UDP-gluconeogenesis and Ra
UDP-glucose. Total gluconeogenesis was calculated as the
sum of absolute plasma gluconeogenesis and UDP-gluconeogenesis, after correcting for duplicate fluxes (i.e., gluconeogenic flux that passed through UDP-glucose, then cycled into
plasma glucose or entered plasma glucose then UDP-glucose
by the direct pathway; Table 2, Ref. 15). The partitioning
coefficient for glucose 6-phosphate was calculated as the ratio
of absolute plasma gluconeogenesis to the sum of plasma
gluconeogenesis and UDP-gluconeogenesis (Table 2). A more
detailed explanation of the above calculations is presented in
Ref. 15.
Statistics. Multifactorial ANOVA with time and treatment
as variables was performed for all parameters except blood
alcohol concentration, for which time was the only variable.
Tukey’s post hoc test was used to determine statistical
significance (P , 0.05).
RESULTS
Plasma metabolite and hormone concentrations. Blood
alcohol concentrations rose significantly from undetectable levels to peak values of 14.7 6 3.2 mM and decayed
linearly thereafter (Fig. 3) in the EtOH protocol. Plasma
glucose concentrations did not change significantly
throughout either the EtOH or placebo studies. At
0800, 1000, 1200, and 1300, plasma glucose concentrations were 83.6 6 7.6, 80.7 6 4.9, 79.7 6 6.6, and 83.0 6
GLUCONEOGENESIS AFTER ETHANOL
Fig. 3. Blood alcohol concentration after EtOH. * Statistically significant differences vs. before EtOH (8:00) values.
3.9 mg/dl, respectively, in the placebo phase and 84.2 6
7.2, 82.3 6 10.0, 78.7 6 6.3, and 76.0 6 5.5 mg/dl,
respectively, in the EtOH phase of the study. Plasma
glucagon and insulin values over time were similar
(and not statistically different) in both the EtOH and
placebo studies (Fig. 4).
Plasma glucose flux, gluconeogenesis, and glycogenolysis. In the EtOH treatment protocol (Fig. 5, Table 3),
the Ra of plasma glucose (Ra glucose) fell over time from
a value of 2.03 6 0.21 mg · kg21 · min21 to 1.79 6 0.21
mg · kg21 · min21 (statistically significant vs. pre-EtOH,
Fig. 4. Insulin (A) and glucagon (B) concentrations after EtOH or
placebo. Statistically significant differences were not observed within
or between treatments.
E901
Fig. 5. Rate of appearance of plasma glucose (Ra glucose) after EtOH
or placebo. Calculated by isotope dilution. # Statistically significant
differences vs. pretreatment values.
P , 0.001). Ra glucose did not change significantlyover
time in the placebo treatment protocol (1.91 6 0.20
mg · kg21 · min21 initially to 1.92 6 0.22 mg · kg21 · min21
at the end of the infusion). There were no statistically
significant differences between the EtOH and placebo
treatments for Ra glucose.
The fractional gluconeogenic contribution to plasma
glucose (Fig. 6A, Table 4) dropped significantly (P ,
0.001) from an initial value of 28.0 6 4.7% to 23.5 6
1.7% after EtOH, and it rose from 23.2 6 1.3% to 32.3 6
4.3% after consumption of placebo. Significant differences were observed between treatments for several
time points. Fractional gluconeogenesis reached relatively stable values after both EtOH and placebo treatments. Absolute plasma gluconeogenic fluxes changed
in the same manner as fractional gluconeogenesis (Fig.
6B, Table 3). Initial rates of absolute gluconeogenesis
were 0.56 6 0.08 mg · kg21 · min21 and 0.44 6 0.05
mg · kg21 · min21 in the EtOH and placebo treatments,
respectively. After consumption of EtOH, the steadystate value was 0.44 6 0.04 mg · kg21 · min21 , representing a statistically significant decrease from baseline of
21% (P , 0.05). In contrast, the absolute rate of plasma
gluconeogenesis rose 42% (P , 0.05 vs. pre-EtOH) to
0.63 6 0.09 mg · kg21 · min21 in the placebo treatment.
The response of absolute gluconeogenesis after the
placebo and EtOH treatments was significantly different (P , 0.001).
The contribution from glycogen to plasma glucose
flux dropped in both treatments over time from similar
initial values of 1.47 6 0.22 (EtOH) and 1.46 6 0.16
(placebo) mg · kg21 · min21 (Fig. 6C, Table 3). Glycogenolysis fell 8% after EtOH and 14% after placebo, to 1.35 6
0.17 mg · kg21 · min21 and 1.26 6 0.20 at the end of each
infusion, respectively. There were no statistically significant differences between treatments, although the placebo 1230 and 1300 time points were significantly lower
(P , 0.05) than the pretreatment values.
Plasma glycerol enrichments. Plasma glycerol enrichments rose after EtOH and decreased after consumption of the placebo, reflecting a change in adipose
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GLUCONEOGENESIS AFTER ETHANOL
Table 3. Metabolic fluxes: comparison of EtOH vs. placebo
Measurement
Pre-EtOH
Post-EtOH
Preplacebo
Postplacebo
Fractional GNG, %
Ra glucose, mg · kg21 · min21
Absolute plasma GNG, mg · kg21 · min21
Glycogenolysis, mg · kg21 · min21
Ra UDP-glucose, mg · kg21 · min21
Fractional UDP-GNG, %
Absolute UDP-GNG, mg · kg21 · min21
Total gluconeogenic flux, mg · kg21 · min21
27.9 6 4.8*†
2.03 6 0.21*
0.56 6 0.08*†
1.47 6 0.22
0.99 6 0.04
23.5 6 1.8†
1.79 6 0.21
0.44 6 0.04†
1.35 6 0.17
1.09 6 0.16
16.1 6 1.6
0.17 6 0.03
0.52 6 0.04†
23.2 6 1.7*†
1.91 6 0.20
0.44 6 0.05*†
1.46 6 0.16*
0.95 6 0.15
32.2 6 4.4†
1.92 6 0.22
0.63 6 0.09†
1.26 6 0.20
1.04 6 0.13
24.2 6 7.1
0.25 6 0.09
0.75 6 0.10†
Values are means 6 SD. Values for posttreatment glycogenolysis are final values measured just before termination of infusion. Values for
rate of appearance (Ra ) of UDP-glucose and total gluconeogenic fluxes are based on time averages from analysis of urine samples pre- and
posttreatment. Pretreatment values for fractional UDP gluconeogenesis (GNG), absolute UDP GNG, and total gluconeogenic flux are not
shown because isotopic steady state was not achieved at time of measurement. Conditions and calculations are as described in text.
* Significant difference from posttreatment value; † significant difference between treatments.
lipolysis (17). The pre-EtOH plasma glycerol enrichment was 49.4 6 7.2% and rose to a steady-state
value of 58.6 6 10.1% after EtOH; these values corresponded to endogenous Ra glycerol values of 2.53 6
0.73 µmol · kg21 · min21 before EtOH and 1.85 6 0.77
µmol · kg21 · min21 after EtOH. The preplacebo plasma
glycerol enrichment was 47.0 6 8.3% and decreased to
38.2 6 7.0% at the end of the infusion (endogenous Ra
glycerol there was equal to 2.38 6 1.27 µmol·kg21 ·min21
before placebo and 3.78 6 1.95 µmol · kg21 · min21 after
placebo). These changes were not statistically significant. The contribution of plasma glycerol to gluconeogenesis was calculated to be 24 6 2% before EtOH, 35 6
5% after EtOH, 28 6 5% before placebo, and 32 6 7%
after placebo. The absolute contribution of glycerol to
gluconeogenesis was 0.14 6 0.01 mg · kg21 · min21 before
EtOH, 0.15 6 0.02 mg · kg21 · min21 after EtOH, 0.12 6
0.02 mg · kg21 · min21 before placebo, and 0.20 6 0.04
mg · kg21 · min21 after placebo. These differences were
not statistically significant.
Intrahepatic metabolite enrichments and fluxes. Intrahepatic triose-phosphate enrichments ( p) responded
differently to the two treatments. Values of p increased
by 61% after EtOH, from a before EtOH enrichment of
0.121 6 0.012 to 0.195 6 0.012 at the end of the infusion
(Fig. 6D, Table 3). In contrast, p did not change in the
placebo treatment, maintaining a steady value of
0.127 6 0.011. The differences between the two treatments were statistically significant (P , 0.001) for the
time points from 900 through 1300, and the post-EtOH
values were significantly different from the pre-EtOH
values.
The fractional gluconeogenic contribution to UDPglucose was significantly lower after EtOH (16.1 6
1.6%) compared with after placebo (24.2 6 7.1%). Ra
UDP-glucose, representing the intrahepatic turnover of
UDP-glucose, did not change significantly in either the
EtOH or placebo studies (Table 3). Ra UDP-glucose was
0.99 6 0.04 mg · kg21 · min21 before and 1.09 6 0.16
mg · kg21 · min21 after EtOH and 0.95 6 0.15
mg · kg21 · min21 before and 1.04 6 0.14 mg · kg21 · min21
after placebo. The absolute rate of UDP gluconeogenesis (calculated as the fractional contribution from
gluconeogenesis to UDP-glucose multiplied by Ra UDPglucose) after EtOH was 0.17 6 0.03 mg · kg21 · min21
and after placebo was 0.25 6 0.09 mg · kg21 · min21
(Table 3).
The fractional recovery of labeled hepatic UDPglucose in plasma glucose (R) did not change after
EtOH (from 25 6 3% to 27 6 4%) or placebo (from 27 6
1% to 25 6 6%). Incorporation of [1-2H1]galactose into
plasma glucose was stable over time and reproducibly
measured (Fig. 7). The direct plasma glucose contribution to UDP-glucose decreased significantly (P , 0.05)
after EtOH but not after the placebo; the pre-EtOH
value was 29 6 5%, and the post-EtOH value was 18 6
6%, whereas the pre- and postplacebo values were 22 6
8% and 20 6 9%, respectively. Total gluconeogenic flux
(plasma gluconeogenesis 1 UDP-gluconeogenesis) was
significantly higher after the placebo (0.75 6 0.10
mg · kg21 · min21 ) compared with after EtOH (0.45 6
0.05 mg · kg21 · min21, Table 4). The glucose 6-phosphate
partitioning coefficient for gluconeogenic flux (10) was
not different (75–80% of flux into plasma glucose) after
either EtOH or placebo ingestion.
DISCUSSION
The consumption of EtOH (48 g) inhibited gluconeogenesis in normal, healthy, overnight-fasted men.
Plasma gluconeogenic flux fell by 21% after EtOH
instead of rising by 43% after placebo (a significantly
different response between the two treatments); total
gluconeogenic flux (plasma gluconeogenesis 1 UDPgluconeogenesis) was also lower. The effect of EtOH on
plasma gluconeogenesis (Fig. 6B, Table 3) can therefore
be calculated to be a 45% inhibition (79%/143% of
baseline values for EtOH/placebo treatments). The
increase in triose-phosphate enrichment (61%, Fig. 6D
and Table 4) was of a similar magnitude as that in the
inhibition of gluconeogenesis. The borderline significant increase in glycogenolysis compared with placebo
prevented a greater reduction in hepatic glucose output
after EtOH.
In previous studies (5, 22, 23), gluconeogenesis was
assessed by the infusion of radioactive precursors
([14C]lactate or [14C]alanine) with and without EtOH.
In each of these studies, the specific activity of plasma
lactate or alanine was compared with the specific
activity of plasma glucose to estimate gluconeogenic
E903
GLUCONEOGENESIS AFTER ETHANOL
tion in gluconeogenesis after EtOH observed in our
study and the 65–75% changes reported in several of
these previous studies.
Measurement of label incorporation from a plasma
precursor into plasma glucose is not a quantitative
index of gluconeogenesis, however. Dilution occurs as
the labeled metabolite moves from the plasma into the
hepatocyte, particularly as the carbon skeleton enters
the TCA cycle (Fig. 2; Refs. 18 and 21). The extent of
this dilution is not constant, however, and changes with
different physiological conditions (18, 21). Use of MIDA
avoids this problem by measuring the enrichment of
the intrahepatic triose-phosphates. Quantification of
the enrichment of the triose-phosphate pool with MIDA
not only enabled us to calculate fractional gluconeogenesis but also allowed us to observe changes in the
availability of the intracellular precursors for gluconeogenesis (i.e., the metabolic flux into the precursor
triose-phosphates). Whereas there was no change in
triose-phosphate enrichment over time after the placebo, triose-phosphate enrichments increased considerTable 4. Calculation of fractional GNG by MIDA:
comparison of EtOH vs. placebo
Subject
M1
M2
EM1
1
2
3
4
5
Mean
SD
0.1798
0.1759
0.1654
0.1767
0.1710
0.0355
0.0338
0.0310
0.0343
0.0316
0.0430
0.0376
0.0302
0.0416
0.0313
1
2
3
4
5
Mean
SD
0.1902
0.1922
0.1765
0.1981
0.1828
0.0378
0.0378
0.0337
0.0392
0.0344
0.0534
0.0548
0.0409
0.0622
0.0431
1
2
3
4
5
Mean
SD
0.1696
0.1747
0.1760
0.1868
0.1899
0.0325
0.0335
0.0325
0.0366
0.0359
0.0335
0.0383
0.0388
0.0489
0.0508
EM2
P, MPE
Fractional
GNG, %
Preplacebo
0.0108
0.0090
0.0068
0.0102
0.0071
0.145
0.131
0.113
0.139
0.115
0.129
60.014
24.5
23.2
21.7
24.6
22.1
23.2
61.30
0.145
0.131
0.119
0.133
0.118
0.129
60.011
31.8
35.4
27.6
37.9
29.0
32.3*
64.3
0.124
0.119
0.105
0.139
0.118
0.121
60.012
21.7
25.7
29.1
28.9
34.4
28.0
64.7
0.199
0.199
0.184
0.21
0.0181
0.195*†
60.012
22.9
22.4
21.8
25.8
24.8
23.5*†
61.7
Postplacebo
Fig. 6. A: fractional gluconeogenesis calculated by mass isotopomer
distribution analysis (MIDA) as described in METHODS. B: absolute
gluconeogenesis calculated as the product of fractional gluconeogenesis and Ra glucose. C: glycogenolysis calculated as difference between Ra glucose and absolute gluconeogenesis. D: gluconeogenic
precursor pool enrichment calculated by MIDA as described in
METHODS. q, Measurements made during EtOH treatment; j, measurements made during the placebo treatment. * Statistically significant differences between treatments. # Statistically significant differences vs. pretreatment values.
0.0131
0.0130
0.0093
0.0150
0.0099
Pre-EtOH
0.0078
0.0088
0.0085
0.012
0.0116
Post-EtOH
flux. Kreisberg et al. (22) observed a 66% reduction in
the incorporation of labeled lactate into glucose. Similarly, the incorporation of [14C]alanine into glucose was
inhibited 75% after EtOH (23). Clore and Blackard (5)
also measured a 50% reduction in the incorporation of
labeled alanine into glucose with administration of
EtOH. Labeled lactate and glycerol have been similarly
infused into non-insulin-dependent diabetics with and
without EtOH (5, 40); a 71% and a 65% reduction in the
incorporation of each into glucose was observed. There
appears to be a disparity between the 45–50% reduc-
1
2
3
4
5
Mean
SD
0.1915
0.1887
0.1731
0.1979
0.1914
0.0412
0.0404
0.0338
0.0433
0.0393
0.0550
0.0526
0.0360
0.0609
0.0523
0.0164
0.0158
0.0095
0.0191
0.0148
Values for posttreatment glycogenolysis are final values measured
just before termination of infusion. Conditions and calculations are
as described in text. P, precursor pool enrichment for GNG; MPE,
molar percent excess. * Significant difference vs. pretreatment value;
† significant difference between treatments.
E904
GLUCONEOGENESIS AFTER ETHANOL
Fig. 7. Incorporation of [1-2H1]galactose into plasma glucose in
individual subjects. Stability of calculated plasma M1 enrichments
during placebo (A) and EtOH (B).
ably after EtOH. The 61% increase in precursor enrichment likely represents at least a comparable decrease
in triose-phosphate flux, that is, dilution by input from
unlabeled precursors. There is some evidence to suggest that the liver is not exclusively responsible for
glycerol uptake from the blood (26, 39), particularly in
the presence of EtOH (27). We cannot therefore be
certain about the input rate of label ([2-13C1]glycerol),
and, thus, the dilution rate of unlabeled metabolites
into the triose-phosphate pool. Even so, if ethanol
inhibits hepatic uptake of glycerol (27), the effect
should be to reduce triose-phosphate enrichment; instead, we observed higher triose-phosphate enrichments (Fig. 6D). It is therefore possible that the actual
inhibition in intracellular dilution (i.e., triose-phosphate flux) and gluconeogenic precursor availability is
even greater than the 61% we have calculated. This
reduction in intracellular gluconeogenic precursor availability is similar to the reduction in the incorporation of
the individual gluconeogenic precursors into plasma
glucose discussed above (i.e., 60–75% inhibition, Refs.
5, 22, 23, and 40). Previous observations may therefore
reflect inhibition of labeled metabolite entry into the
gluconeogenic precursor pool.
It has previously been shown that when normal,
healthy volunteers were starved for 2–3 days, infusion
of EtOH elicited a <30% reduction in Ra glucose (43,
53). As hepatic glycogen levels are substantially reduced after a 2- to 3-day fast (35), rates of glucose
production are presumed to reflect exclusively the rates
of gluconeogenesis. The reduction in gluconeogenesis
that we observed with EtOH after an overnight fast (ca.
45%) was a little higher than that observed in Ra
glucose after a 2- to 3-day fast in these previous studies
(43, 53).
One potential concern is whether the administration
of acetaminophen, with propylene glycol-ethanol for
solubilization, might have affected the baseline results.
The infusion rate of the propylene glycol-ethanol solution was 4 ml/h or 2 g/h. We have previously compared
fractional gluconeogenesis in overnight-fasted normal
humans, measured by MIDA with [2-13C1]glycerol, in 21
subjects with and 17 subjects without intravenous
acetaminophen-propylene glycol-ethanol (Neese, Siler,
and Hellerstein, unpublished observations). The gluconeogenic values were 31 6 4% with acetaminophen
infusion and 33 6 6% without it. The presence of
acetaminophen-propylene glycol-ethanol clearly does
not affect baseline measurements of gluconeogenesis.
It should be noted that the dilution of glucose tracer
in the plasma glucose pool reflects whole body glucose
production. Recent work (46) has supported the view
that the kidney contributes to glucose production.
Direct measurement of renal glucose output and gluconeogenesis after EtOH has not been reported. Because
our measurement of gluconeogenesis, in principle, includes renal gluconeogenesis that may be present (33),
it is therefore likely that both renal gluconeogenesis
and glucose production are included in our results. The
stability of the plasma glucose concentrations despite
significantly lower Ra glucose after EtOH consumption
signifies a reduced metabolic clearance rate for blood
glucose. This observation is consistent with previous
reports (2, 44, 56) of impaired peripheral glucose removal during euglycemic hyperinsulinemic clamp conditions.
The fraction of plasma glucose from gluconeogenesis
in these subjects as measured by MIDA was lower than
some previous estimates with different techniques (25,
42), although consistent with some others. Previs et al.
(38) reported that measurement of gluconeogenesis
with [13C]glycerol and MIDA systematically underestimated fractional gluconeogenesis. These authors did
not isolate or measure triose-phosphate pools in the
liver but inferred that their lower-than-expected values
of gluconeogenesis would be compatible with an isotopic gradient of labeled glycerol across the hepatic
lobule. It is unlikely that our values of gluconeogenesis
are systematically underestimated, however, for several reasons.
First, several laboratories (not only our own) have
reported appropriately high values of gluconeogenesis
when MIDA with [2-13C1]glycerol is used in the manner
and at the infusion rates that we initially described (15,
33) and use here. Fractional gluconeogenesis reaches
90–95% in 48-h-fasted rats (16, 33, 37) and 85–90% in
mice (51). In perfused livers from fasted rats, it is .90%
(37); in Kenyan children with malaria it is up to 96%
with a mean value of 75% (7); in neonatal humans, it is
up to 88–100% of glucose production, with a mean
value of 72% (47); in fasted adults it reaches 92% (15);
GLUCONEOGENESIS AFTER ETHANOL
and in overnight-fasted humans (50) or fasted rats (32)
it is not affected by adding [13C1]alanine to [13C1]glycerol,
contrary to the prediction of the labeled-glycerol gradient model (38).
Second, the reported values of gluconeogenesis in the
literature are extremely variable among individuals.
Landau et al (25) reported values ranging between 33
and 69% based on 2H2O incorporation; Rothman et al.
(42) reported a range between 46 and 81% with NMR
spectroscopy. Prolongation of fasting from 14 to 22 h
increased the gluconeogenic contribution from 47 to
67% (25) by the 2H2O method. Thus the composition or
energy sufficiency of recent or chronic dietary intake,
duration of fasting, and exercise habits could clearly
have a substantial impact on gluconeogenesis. Comparison between studies should therefore be made with
caution, and it is premature to assume what the
fractional gluconeogenic values should have been.
Finally, some variations in the estimates of fractional gluconeogenesis were obtained with the use of
[U-13C3]- instead of [2-13C1]glycerol (38). It is possible
that very low enrichments of (M16) species of glucose
could lead to some technical difficulties in their accurate measurement. Indeed it appears that the lowest
estimates of fractional gluconeogenesis were obtained
with uniformly labeled substrates. In perfused livers,
however, estimates with either [U-13C3]glycerol (38) or
[2-13C1]glycerol (37, 38) yielded similar estimates for
gluconeogenesis.
Accordingly, it seems unlikely that gluconeogenesis
is systematically underestimated by MIDA from
[2-13C1]glycerol. In any event, our directional comparisons within subjects (before vs. after interventions)
would not be affected even if systematic underestimation were present here.
It appears from the results of this study that the
partial preservation of gluconeogenesis is achieved at
the expense of other fates of triose-phosphate flux.
Alternative metabolic fates of intrahepatic triosephosphates include entry into the pentose-phosphate
shunt, passing down the glycolytic pathway back to
pyruvate, conversion to glycerol 3-phosphate, and UDPgluconeogenesis. There is some evidence from work in
hepatocytes for a decrease in pentose-phosphate shunt
flux in the presence of EtOH (41). A decrease in the flux
from triose-phosphate to pyruvate has not been directly
measured by us or others. The pathway does contain a
redox-sensitive metabolic reaction (conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate at
glyceraldehye-3-phosphate dehydrogenase) (Fig. 2). The
decreased availability of NAD due to the oxidation of
EtOH by alcohol dehydrogenase could reduce metabolite flux through this reaction as well as the rest of the
pathway. There is also evidence for a directional flux
from triose-phosphate to glycerol 3-phosphate under
the influence of EtOH. Yu and Cronholm (57) have
provided evidence for a conversion from the triosephosphates to glycerol 3-phosphate in studies with
rats, and elevated hepatic glycerol 3-phosphate concentrations with EtOH have been observed in other studies (4, 30, 34). Quantification of the flux from the
E905
triose-phosphates to glycerol 3-phosphate under the
influence of EtOH has not yet been determined, however.
One of the possible fates of triose-phosphate flux is
glycogen synthesis (the indirect pathway, Ref. 19). We
were able to quantify the partitioning of the flow of
newly synthesized glucose 6-phosphate (via gluconeogenesis) into either plasma glucose (plasma gluconeogenesis) or into glycogen (UDP-gluconeogenesis). The
glucose 6-phosphate partitioning coefficient did not
change over time in either the EtOH or placebo treatments in this study, and neither did the UDP-glucose
flux; if there were a diversion of glucose 6-phosphate
flow away from glycogen synthesis and toward plasma
glucose, UDP-glucose flux would be expected to decrease with EtOH. These results suggest that there is
no decrease in the flux of triose-phosphates toward
UDP-gluconeogenesis.
It is unlikely that changes in catecholamines could
explain our observation of stable values for Ra glucose
in these subjects after EtOH ingestion. When given in
larger doses than were administered in this study,
EtOH has been shown to initiate a response from the
sympathetic nervous system (3). The decrease in lipolysis that we observe after EtOH consumption (opposite
to the effect catecholamines would have) suggests that
the sympathetic nervous system was active minimally,
if at all.
In conclusion, EtOH elicits an inhibition of gluconeogenesis in normal, overnight-fasted men and a somewhat larger decrease in intracellular gluconeogenic
precursor availability but a smaller effect on plasma Ra
glucose and no effect on plasma glucose concentrations.
These results suggest that inhibition of flux into the
gluconeogenic precursor pool (and subsequently into
plasma glucose) is compensated by changes in glycogenolysis, by diversion of triose-phosphates from other
kinetic fates and into gluconeogenic pathways, and by
reduced peripheral tissue clearance of plasma glucose.
We gratefully acknowledge the nurses at the San Francisco
General Hospital GCRC for their help. We also acknowledge Mark
Hudes for assistance with statistical analyses.
This work was supported in part by National Institute on Alcohol
Abuse and Alcoholism Grant 1 RO3 AA10693–01 and National
Center for Research Resources Grant RR-00083 from the Division of
Research Resources for the General Clinical Research Center.
Address for reprint requests: M. K. Hellerstein, Dept. of Nutritional Sciences, Univ. of California, Berkeley, CA 94720-3104 or
Division of Endocrinology and Metabolism, SF General Hospital,
U.C. San Francisco, CA 94110.
Received 30 September 1997; accepted in final form 17 July 1998.
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