Chronic Ethanol and Triglyceride Turnover in White Adipose
Tissue in Rats
Li Kang‡§, Xiaocong Chen§¶, Becky M. Sebastian§, Brian T. Pratt§, Ilya R. Bederman¶, James C. Alexander?,
Stephen F. Previs¶, and Laura E. Nagy**§¶1
andDepartmentsof**Gastroenterologyand§Pathobiology,ClevelandClinicFoundation,Cleveland, Ohio 44195
Chronic ethanol consumption disrupts whole-body lipid
metabolism. Here we tested the hypothesis that regulation of
triglyceride homeostasis in adipose tissue is vulnerable to long-
term ethanol exposure. After chronic ethanol feeding, total
body fat content as well as the quantity of epididymal adipose
controls. Integrated rates of in vivo triglyceride turnover in epi-
didymal adipose tissue were measured using2H2O as a tracer.
Triglyceride turnover in adipose tissue was increased due to a
2.3-fold increase in triglyceride degradation in ethanol-fed rats
compared with pair-fed controls with no effect of ethanol on
triglyceride synthesis. Because increased lipolysis accompanied
chronic ethanol feeding. Chronic ethanol feeding suppressed
anol feeding was not due to increased ?-adrenergic-mediated
lipolysis. Instead, chronic ethanol feeding markedly impaired
insulin-mediated suppression of lipolysis in conscious rats dur-
ing a hyperinsulinemic-euglycemic clamp as well as in adipo-
These data demonstrate for the first time that chronic ethanol
tissue. Furthermore, this enhanced rate of lipolysis was due to a
suppression of the anti-lipolytic effects of insulin in adipocytes
after chronic ethanol feeding.
ferent medical conditions, including hepatic diseases and car-
chronic ethanol exposure causes excessive lipid accumulation
in liver with the eventual development of hepatic steatosis (2).
These pathophysiological effects of ethanol can be modeled in
rats induces hepatic steatosis coupled with the development of
hyperlipidemia, characterized by elevated plasma cholesterol
alcohol-related disease progression. However, the effects of
chronic ethanol feeding on lipid metabolism in adipose tissue,
the biggest storage pool of lipids, are unknown.
Adipose tissue is a specialized connective tissue that func-
Serving as an energy reserve, adipose tissue synthesizes triglyc-
erides when energy intake exceeds energy output. During fast-
mobilizes free fatty acids and glycerol, providing other tissues
with metabolites and energy substrates (3). Mobilization of
termed lipolysis, is tightly regulated by a number of hormones.
which initiate lipolysis by the stimulation of ?-adrenergic
receptors, and insulin, which inhibits catecholamine-induced
and insulin-dependent signal transduction in a variety of cell
types, including adipocytes (5–8). For example, we have dem-
gic receptor-stimulated lipolysis (9) and suppresses insulin-
stimulated glucose uptake in isolated adipocytes (10, 11).
lytic response of adipocytes to insulin has not been examined.
In this study we investigated the effects of chronic ethanol
feeding over a 2-week period on the integrated rates of in vivo
of2H2O (12). After the administration of2H2O,2H in body
water equilibrates with the carbon-bound hydrogens of glyc-
erol 3-phosphate, and the rates of triglyceride synthesis and
degradation are determined by measuring the incorporation/
washout of2H to/from carbon 1 of triglyceride-bound glycerol
(13). The rate of triglyceride degradation during 2 weeks of
ethanol feeding was increased by 2.3-fold. Because increased
rates of lipolysis are associated with the development of insulin
resistance and fatty liver in other model systems, we therefore
investigated the mechanisms by which chronic ethanol
increased triglyceride degradation. We find that the ethanol-
* This work was supported by National Institutes of Health Grant AA 11876.
The costs of publication of this article were defrayed in part by the pay-
ment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-
cate this fact.
636-1493; E-mail: firstname.lastname@example.org.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 39, pp. 28465–28473, September 28, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
SEPTEMBER 28, 2007•VOLUME 282•NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 28465
by guest on July 20, 2015
induced increase in lipolysis in adipose was due to a loss of the
anti-lipolytic actions of insulin rather than an increase in stim-
ulation of lipolysis by ?-adrenergic receptor activation.
Materials—Male Wistar rats (150–160g) were purchased
from Harlan Sprague-Dawley (Indianapolis, IN). The Lieber-
DeCarli high fat ethanol diet was purchased from Dyets (Beth-
lehem, PA). Maltose dextrins were obtained from BioServ
(Frenchtown, NJ). The ethanol-L3K assay kit was purchased
percent excess) and [2H5]glycerol (98 atom percent excess)
were purchased from Isotec (Miamisburg, OH), ion-exchange
resins were from Bio-Rad, glycerokinase was from Roche
Applied Science, bis(trimethylsilyl)trifluoroacetamide with
10% trimethylchlorosilane was from Regis Technologies Inc.
(Morton Grove, IL), and gas chromatography-mass spectrom-
etry (GC-MS)2supplies were from Agilent Technologies
(Wilmington, DE). NEFA C kit for the measurement of plasma
icals USA, Inc. (Richmond, VA). Cilostamide, a phosphodies-
terase 3B (PDE3B)-selective inhibitor, was from BIOMOL
(Plymouth Meeting, PA). [3H]cAMP was from Amersham Bio-
sciences. Antibodies were from the following sources: anti-ex-
tracellular signal-regulated kinase, Upstate, Charlottesville,
blood glucose meter and blood glucose test strips were from
CVS (Woonsocket, RI), human insulin was from Eli Lilly (Indi-
anapolis, IN), rat insulin enzyme-linked immunosorbent assay
was from Mercodia Inc. (Winston Salem, NC), and all other
reagents were from Sigma.
Animal Protocol for Chronic Ethanol Feeding—Rats were
ethanol as 35% of total calories or pair-fed an isocaloric control
diet which substituted maltose dextrins for ethanol for 4 weeks
as previously described (9). Rats were housed in individual
with 12 h light-12 h dark (7:00 p.m.-7:00 a.m.) cycle. In studies
to determine2H2O-based triglyceride turnover rate, rats were
of2H) for 5 days; after that,2H2O was switched to tap water. A
total of 30 rats were euthanized over time after the intraperito-
neal injection of2H2O; 3 rats per group on day 0, and 2 rats per
group on days 3, 5, 7, 9, 11, and 14.
At the end of the feeding protocol, rats were anesthetized by
ml for pair-fed rats per 100 g of body weight of a mixture con-
taining 10 mg/ml acepromazine, 100 mg/ml ketamine, and 20
mg/ml xylazine. The lower dose of anesthetic for ethanol-fed
rats was used because of an increased sensitivity to the anes-
xylazine at these doses either have no effects or equivalent
pose tissue was frozen in liquid nitrogen and stored at ?80 °C.
Plasma samples were prepared by centrifugation at 16,100 ? g
for 2 min, and plasma ethanol concentration was measured
immediately by the ethanol-L3K kit. The rats used in these
studies were not fasted; all studies were carried out at 10:30
a.m., except the hyperinsulinemic-euglycemic clamps, which
were performed at 12:00 noon (as time 0 of the clamps). Proce-
dures involving animals were approved by the Institutional
versity or the Cleveland Clinic.
Body Composition Analysis—Percent body fat was deter-
mined in ethanol- and pair-fed rats by magnetic resonance
Bruker/Siemens Medspec 4T magnetic resonance imaging
scanner. Coronal, proton density weighted, spin echo images
(TR/TE ? 5910 ms/7 ms, resolution ? 860 ?m ? 860 ?m ? 2
mm, matrix ? 128 ? 256) were obtained for each animal.
Twenty images per rat were obtained with and without water
using the Amira image processing and visualization software
(Mercury Computer Systems, Inc.) to determine total body fat
volume to total body volume ratios.
The2H-Labeling of Body Water—The2H-labeling of body
et al. (16) and as modified previously (12, 13). Briefly, known2H
atom percent excess standards were prepared by mixing natu-
rally labeled water and 99.9%2H2O. Assays were performed
using 40 ?l of plasma or standard, 2 ?l of 10 N NaOH, and 4 ?l
incubation, the solution was extracted with 600 ?l of chloro-
form and dried with Na2SO4. The2H-labeling of acetone was
then determined by GC-MS. Ions of mass-to-charge ratios
(m/z) 58–60 were monitored.
ethanol at 70 °C for 2 h. After evaporation of ethanol, free glyc-
erol was recovered as previously described (12, 13). H2O (3 ml)
was added, and the solution was acidified to ?pH 1. After
extraction of fatty acids by diethyl ether (3? with 4 ml), the
glycerokinase at 37 °C for 2.5 h. The formed glycerol 3-phos-
phate was then purified by passing the solution over an AG
eluting the column with 4 N formic acid. The labeling of2H
bound to carbon 1 of glycerol 3-phosphate was determined by
reacting the glycerol 3-phosphate with 100 ?l of bis(trimethyl-
silyl)trifluoroacetamide plus 10% trimethylchlorosilane for 30
min at 75 °C. Isotope enrichment was determined by GC-MS.
Mathematical Modeling—The rates of triglyceride synthesis
2H-Labeling of Triglyceride-bound Glycerol—Frozen
2The abbreviations used are: GC-MS, gas chromatography-mass spectrome-
try; PDE, phosphodiesterase; Ra, the rate of appearance.
28466 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282•NUMBER 39•SEPTEMBER 28, 2007
by guest on July 20, 2015
Laura E. Nagy
J. Biol. Chem.
James C. Alexander, Stephen F. Previs and
Sebastian, Brian T. Pratt, Ilya R. Bederman,
Li Kang, Xiaocong Chen, Becky M.
TO INCREASED TRIGLYCERIDE
CHRONIC ETHANOL CONTRIBUTES
ACTION OF INSULIN AFTER
INHIBITION OF THE ANTI-LIPOLYTIC
Turnover in White Adipose Tissue in Rats:
Chronic Ethanol and Triglyceride
Metabolism and Bioenergetics:
doi: 10.1074/jbc.M705503200 originally published online August 7, 2007
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