Sleeve gastrectomy in rats improves postprandial lipid clearance by reducing intestinal triglyceride secretion.

Abstract

Postprandial hyperlipidemia is a risk factor for atherosclerotic heart disease and is associated with the consumption of high-fat diets and obesity. Bariatric surgeries result in superior and more durable weight loss than dieting. These surgeries are also associated with multiple metabolic improvements, including reduced plasma lipid levels. We investigated whether the beneficial effects of vertical sleeve gastrectomy (VSG) on plasma lipid levels are weight independent.
VSG was performed on Long-Evans rats with diet-induced obesity. Controls were sham-operated animals who were either pair-fed or ad libitum-fed. We measured fasting and postprandial levels of plasma lipid. To determine hepatic and intestinal triglyceride secretion, we injected the lipase inhibitor poloxamer 407 alone or before oral lipid gavage. (13)C-Triolein was used to estimate postprandial uptake of lipid in the intestine.
Rats that received VSG and high-fat diets had markedly lower fasting levels of plasma triglyceride, cholesterol, and phospholipid than obese and lean (pair-fed) controls that were fed high-fat diets. Rats that received VSG had a marked, weight-independent reduction in secretion of intestinal triglycerides. VSG did not alter total intestinal triglyceride levels or size of the cholesterol storage pool nor did it affect the expression of genes in the intestine that control triglyceride metabolism and synthesis. VSG did not affect fasting secretion of triglyceride, liver weight, hepatic lipid storage, or transcription of genes that regulate hepatic lipid processing.
VSG reduced postprandial levels of plasma lipid, independently of body weight. This resulted from reduced intestinal secretion of triglycerides following ingestion of a lipid meal and indicates that VSG has important effects on metabolism.

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Sleeve Gastrectomy in Rats Improves Postprandial Lipid Clearance by
Reducing Intestinal Triglyceride Secretion
M. A. STEFATER,* DARLEEN A. SANDOVAL,* A. P. CHAMBERS,* H. E. WILSON-PÉREZ,* S. M. HOFMANN,*
R. JANDACEK,‡P. TSO,‡S. C. WOODS,§and RANDY J. SEELEY*
Metabolic Disease Institute and *Department of Internal Medicine,‡Department of Pathology, and§Department of Psychiatry, University of Cincinnati College of
Medicine, Cincinnati, Ohio
BACKGROUND & AIMS: Postprandial hyperlipidemia
is a risk factor for atherosclerotic heart disease and is
associated with the consumption of high-fat diets and
obesity. Bariatric surgeries result in superior and more
durable weight loss than dieting. These surgeries are also
associated with multiple metabolic improvements, includ-
ing reduced plasma lipid levels. We investigated whether
the beneficial effects of vertical sleeve gastrectomy (VSG)
on plasma lipid levels are weight independent. METH-
ODS: VSG was performed on Long-Evans rats with diet-
induced obesity and pair-fed and ad libitum-fed rats that
received sham operations (controls). We measured fasting
and postprandial levels of plasma lipid. To determine
hepatic and intestinal triglyceride secretion, we injected
the lipase inhibitor poloxamer 407 alone or before oral
lipid gavage.13C-Triolein was used to estimate postpran-
dial uptake of lipid in the intestine. RESULTS: Rats that
received VSG and high-fat diets had markedly lower fast-
ing levels of plasma triglyceride, cholesterol, and phos-
pholipid than obese and lean (pair-fed) controls that were
fed high-fat diets. Rats that received VSG had a marked,
weight-independent reduction in secretion of intestinal
triglycerides. VSG did not alter total intestinal triglyceride
levels or size of the cholesterol storage pool nor did it
affect the expression of genes in the intestine that control
triglyceride metabolism and synthesis. VSG did not affect
fasting secretion of triglyceride, liver weight, hepatic lipid
storage, or transcription of genes that regulate hepatic
lipid processing. CONCLUSIONS: VSG reduced post-
prandial levels of plasma lipid, independently of body
weight. This resulted from reduced intestinal secre-
tion of triglycerides following ingestion of a lipid
meal and indicates that VSG has important effects on
metabolism.
Keywords: Bariatric Surgery; Very Low-Density Lipopro-
tein; VLDL; Bile Acid; Meal Pattern; Weight Reduction.
C
mia associated with consumption of high-fat diets. In
fact, even mild obesity has been reported to increase risk
of coronary heart disease by 50%, with more severe obesity
increasing risk as much as 3-fold.1Cardiovascular disease
is currently the leading cause of death in the United
States.2Obesity-related dyslipidemia commonly includes
ardiovascular disease in obese patients is a leading
cause of mortality, which can arise from dyslipide-
elevated total cholesterol, low-density lipoprotein, and
triglycerides and reduced levels of high-density lipopro-
tein cholesterol.3Dyslipidemia in addition to excess fat is
therefore an important goal of any ideal weight loss strat-
egy.
Bariatric surgery improves cardiovascular health by re-
ducing symptoms of and risk factors for cardiovascular
disease.4Dyslipidemia is a potent cardiovascular risk fac-
tor shown to be reduced in at least 70% of patients after
bariatric surgery.5Improvements have been documented
following RYGB,6–9gastric banding,10,11biliary-intestinal
bypass,11duodenal-jejunal bypass,12biliopancreatic diver-
sion,13ileal interposition,14and vertical sleeve gastrec-
tomy (VSG).15–17The degree of improvement to plasma
lipids appears to vary with each procedure, but it is yet
unknown which gastrointestinal manipulations are most
effective to reverse dyslipidemia. Importantly, for each of
these procedures, it is also unclear whether improvements
are due to weight loss or to an independent effect of the
surgery. In this study, we focused on VSG, where a reduc-
tion in gastric volume is created by the removal of gastric
mucosa along the greater curvature and fundus. We have
previously shown that this surgical model produces pro-
found and persistent weight loss in rats.18We hypothe-
sized that VSG would also reverse dyslipidemia secondary
to altered gastrointestinal function. Here, we demonstrate
that VSG improves dyslipidemia in a weight-independent
manner and that this reduction is due to attenuated
postprandial triglyceride production by the intestine.
Materials and Methods
Animals
Male Long-Evans rats (Harlan Laboratories, India-
napolis, IN; 250–300 g) were fed a high-fat butter, oil-
based diet (HFD; Research Diets, New Brunswick, NJ;
D12451; 45% fat; 4.73 kcal/g) or standard chow (Harlan-
Teklad, Indianapolis, IN) for 8 weeks prior to surgery and
maintained on this diet postsurgery. Rats were housed
Abbreviations used in this paper: ANOVA, analysis of variance; Apo,
apolipoprotein; CHOW, chow-fed animals; HFD, high-fat butter, oil-
based diet; NEFA, nonesterified fatty acids; PF, pair-fed; SHAM, sham-
operated animals; VLDL, very low-density lipoprotein; VSG, vertical
sleeve gastrectomy.
© 2011 by the AGA Institute
0016-5085/$36.00
doi:10.1053/j.gastro.2011.05.008
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under controlled conditions (12:12-hour light-dark cycle,
50%–60% humidity, 25°C, free access to water and food
except where noted). A subgroup of sham-operated rats
was pair-fed (PF) to the VSG group; amount of food eaten
by each VSG rat during the previous 24 hours was given to
each PF rat at a random time during the light/dark cycle.
For the tracer study, all animals were fed 15 g of HFD on
the day prior to the study, divided into 2 meals given at 0
and 6 hours after the onset of the dark cycle. Fat and lean
tissue masses were measured using nuclear magnetic res-
onance (Echo MRI: Echo Medical Systems, Houston, TX).
At the end of each study, animals were placed briefly in a
CO2chamber and then killed by decapitation. All pro-
cedures for animal use were approved by the University
of Cincinnati Institutional Animal Care and Use Com-
mittee. A summary of cohorts is provided in Supple-
mentary Table 1.
Surgical Procedures
For VSG, a laparotomy incision was made. The
stomach was isolated outside the abdominal cavity. Loose
gastric connections to the spleen and liver were released
along the greater curvature, and the suspensory ligament
supporting the upper fundus was severed. The lateral 80%
of the stomach was excised using an ETS 35-mm staple
gun (Ethicon Endo-Surgery, Cincinnati, OH), leaving a
tubular gastric remnant in continuity with the esophagus
and duodenum. Following reintegration of the stomach
into the abdominal cavity, the abdominal wall was closed
in layers. Sham surgery was laparotomy, isolation of the
stomach, and application of blunt pressure using forceps
along a line between the esophageal sphincter and the
pylorus. Postsurgery, rats received postoperative care for 3
days (Supplementary Figure 1), consisting of twice-daily
subcutaneous injections of 10 mL warm saline and 0.3 mL
buprenorphine. Daily subcutaneous meloxicam was ad-
ministered for 2 days postoperatively. Rats were given
access only to Osmolite OneCal liquid diet from 24 hours
preoperatively until solid food was returned 3 days after
surgery.
Measurement of Lipids
All blood was sampled from the tip of the tail
except when taken as trunk blood during death. Plasma
cholesterol, nonesterified fatty acids (NEFA), phospho-
lipid, and triglycerides were measured via colorimetric
assays using Infinity Reagents (Thermo Fisher Scientific,
Inc, Waltham, MA). For the study of triglyceride produc-
tion rate, rats were fasted for 24 hours, and baseline blood
samples were collected prior to intraperitoneal injection
of 1 g/kg poloxamer 407 (P-407; Sigma-Aldrich, St. Louis,
MO), and blood was taken at various time points there-
after. For the experiment described in Figure 3, an intra-
gastric gavage of 0.5 mL/kg olive oil was delivered 1 hour
prior to P-407 injection. For apolipoprotein (Apo) pro-
files, trunk blood was collected into EDTA after 24 hours
of fasting and stored at 4°C for fractionation via fast
protein liquid chromatography within 7 days.
For measurement of triglycerides and cholesterol in
liver and intestine, tissues from 24 hour-fasted rats were
flash frozen in isopentane, and lipid from 50 mg of tissue
was extracted in 2:1 chloroform/methanol. Triglyceride
and cholesterol content were measured as described
above, using colorimetric assays. Hepatic cholesterol es-
ters were measured using a Cholesterol/Cholesteryl Ester
Quantitation Kit (EMD Chemicas, Gibbstown, NJ) from
10-mg tissue homogenates. Plasma total sample bile acid
concentrations were determined using a kit from Bio-
Quant (San Diego, CA).
Lipid Absorption
Dietary lipid absorption of a 5-mL/kg intragastric
lipid emulsification (20% soybean oil, 1.2% egg phospho-
lipid, 2.5% glycerin, 2.5% sucrose polybehenate) was mea-
sured as previously described.19Briefly, 24 hour-fasted
rats received the emulsion via intragastric gavage. Ten-
milligram fecal samples were collected 24 hours later.
Fecal lipid content was assayed by gas chromatography of
fatty acid methyl esters. Dietary lipid absorption was
estimated using a ratio of total fecal fatty acids to sucrose
polybehenate.
Stable Isotope Study
On the day prior to the study, all rats were placed
in clean cages and given 10 g HFD at the onset of and 5 g
HFD 6 hours into the dark phase of the light-dark cycle.
All food was consumed prior to death. On the day of the
study, 1 g/kg P-407 was administered 1 hour prior to a
0.28 g intragastric lipid gavage containing 0.18 g olive oil
and 0.10 g 1,1,1,-13C3-Triolein. Rats were killed 4 hours
later. Intestines were removed immediately after death
and dissected into 4 equal length sections (M1, M2, M3,
and M4 from proximal to distal, respectively). After re-
moval of 1.5 cm from each segment for polymerase chain
reaction and histology, each gut segment was cleaned
thoroughly by removing mesenteric fat and rinsing inner
surface in 1% sodium dodecyl sulfate followed by phos-
phate-buffered saline. Each segment was placed in 10 mL
chilled phosphate-buffered saline for immediate homog-
enization on ice. Lipids were extracted from homogenate
aliquots using the method described by Folch et al.20
Ratio of13C to12C in the lipid fraction was measured
using gas chromatography combustion isotope ratio mass
spectroscopy and reported as excess enrichment above
baseline, as measured using samples from pluronic-
treated, ungavaged animals.
Intestinal and Liver Gene Expression
Tissue harvested 6 hours after a lipid gavage was
homogenized in RLT buffer using a tissue lyser (QIAGEN,
Inc, Valencia, CA). RNA was extracted using a QIAGEN
miniprep RNA extraction kit, and complementary DNA
was made using an iScript complementary DNA synthesis
kit (Bio-Rad Laboratories, Hercules, CA). Quantitative
polymerase chain reaction was performed using TaqMan
gene expression assays (Supplementary Table 2).
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Histology
To visualize intestinal morphology, tissue samples
were fixed in 4% paraformaldehyde and stored in 30%
sucrose. Seven-micrometer cryosections were stained with
Cresyl Violet. Digital images of sections were acquired
using a digital camera attached to a Zeiss microscope
(Zeiss, Thornwood, NY).
Biochemical Assays
Western blots were used to measure plasma
ApoB48 and ApoB100 content after a lipid gavage in the
presence of intraperitoneal P-407. Plasma samples were
denatured in one volume of radioimmunoprecipitation
assay buffer (Santa Cruz Biotechnologies, Santa Cruz, CA)
plus 3 volumes Laemmli buffer (Bio-Rad Laboratories)
and diluted with distilled water. Following 10 minutes at
95°C, samples were applied to a 4%–15% Tris-HCl poly-
acrylamide gel (Bio-Rad Laboratories) and run at 50 V for
4.5 hours. Proteins were transferred to nitrocellulose over-
night at 30 V. Blots were blocked at room temperature in
5% bovine serum albumin. Goat anti-human ApoB anti-
body (Millipore, Temecula, CA) was diluted 1:5000 in 5%
bovine serum albumin and incubated overnight at 4°C.
Blots were incubated in secondary antibody, 1:5000 horse-
radish peroxidase-conjugated rabbit anti-goat immuno-
globulin G (Millipore) in 5% milk for 1 hour at room
temperature. Immunoreactivity was quantified using Al-
pha Ease software (Alpha Innotech Corporation, San Le-
andro, CA).
Statistical Analysis
All data are expressed as mean ? standard error of
mean. Body weight, food intake, nuclear magnetic reso-
nance, and changes in plasma triglyceride or cholesterol
levels over time were analyzed via 2-way analysis of vari-
ance (ANOVA) (variables: treatment and time) with a
Bonferroni post hoc test where appropriate. Area-under-
the-curve comparisons, triglyceride production rates,
plasma lipid analytes at single time points, plasma biliru-
bin and bile acid measurements, single-gene expression
comparisons, comparison of gavage sizes, single-day body
weight and body composition measurements, stable iso-
tope enrichment in intestinal lipid samples, and total
tissue cholesterol and triglyceride content were analyzed
using 1-way ANOVA followed by a Tukey post hoc test
where appropriate. Two-tailed, unpaired t tests were used
where indicated.
Results
VSG Reduces Plasma Lipids
Four-hour fasting plasma lipids were measured 50
days postsurgery. When rats were weight stable and obese,
sham-operated (SHAM) animals were heavier compared
with VSG, sham-operated, chow-fed (CHOW), and sham-
operated, PF groups (Figure 1A). Weight loss after VSG is
a selective loss of fat mass (DePaula et al17and Figure 1B).
All animals in PF, SHAM, and VSG groups were main-
tained on HFD for the duration of the study. Plasma
cholesterol (Figure 1C, P ? .001), triglycerides (Figure 1D,
P ? .01), and phospholipids (Figure 1E, P ? .001) but not
NEFA (Figure 1F) concentrations were decreased after
VSG as compared with SHAM controls. Importantly, this
observed reduction after VSG is weight independent be-
cause cholesterol (Figure 1C, P ? .05), triglycerides (Figure
1B, P ? .01), and phospholipids (Figure 1E, P ? .001) were
all reduced after VSG vs PF animals, both groups exhib-
iting similar body weight (Supplementary Figure 2). Fur-
thermore, levels of all 4 lipids measured (cholesterol, trig-
lycerides, phospholipids, and NEFA) were similar in
CHOW and VSG groups (Figure 1A–D) despite lower
body weight and fat mass in the CHOW group (Figure 1A
and B), underscoring a weight-independent effect of VSG
on dyslipidemia.
To explore whether reductions in plasma triglycerides
were dependent on the duration of fasting, we measured
triglycerides in plasma sampled after 0, 4, 8, and 24 hours
of fasting. All animals consumed 15 g of HFD on the day
prior to the study. Body weight was significantly reduced
in VSG and PF animals on the day of the study (Body
weight, food intake, and body composition are presented
in Supplementary Figure 2). Consistent with the data
from Figure 1C, triglyceride levels were reduced in non-
fasting plasma from VSG animals as compared with
SHAM (P ? .001) and PF (P ? .01). This difference was
gradually diminished along the course of the fast (Figure
2A). At 4 and 8 hours of fasting, plasma triglycerides were
lower in VSG animals than SHAM (P ? .001 at both time
points) but no longer significantly different from PF. By
24 hours of fasting, no significant differences existed
between any of these groups. However, area under the
curve for triglycerides during the 24-hour experiment was
reduced for VSG compared with PF (P ? .05) or SHAM
(P ? .001, Figure 2B). At the same time, we did not detect
significant differences in plasma cholesterol levels among
the 3 groups (Figure 2C), and thus area under the curve
for 24-hour cholesterol was unaffected by treatment (Fig-
ure 2D). As reflected by total plasma cholesterol levels
after 24 hours of fasting (Supplementary Figure 3), VSG
improved fasting hypercholesterolemia by reducing both
very low-density lipoprotein (VLDL) and high-density li-
poprotein cholesterol (Figure 2E). Apo size did not appear
to be altered by VSG.
Triglyceride Production Rate and
Postprandial Lipid Clearance
To determine whether reduced plasma triglyceride
levels after VSG are due to attenuated VLDL production
and/or secretion, we administered an intraperitoneal in-
jection of the lipase inhibitor poloxamer 407 (P-407) (1
g/kg) to 24 hour-fasted animals. We found no changes in
rate of triglyceride appearance in the plasma across
groups following P-407 administration (Figure 3A and B).
These results indicate that VLDL production/secretion
rate is similar among the different experimental groups.
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Based on the observation that plasma triglycerides
were reduced most dramatically in VSG animals after
short periods of fasting, we hypothesized that VSG
might enhance postprandial lipid metabolism. We next
investigated whether intestinal chylomicron assembly
and secretion are affected by VSG. To answer this ques-
tion, we administered P-407 to 24 hour-fasted rats 1
hour prior to a 0.5-mL/kg olive oil gavage (average fat
content: 0.28 ? 0.004 g) and measured plasma triglyc-
erides for 6 hours after the gavage. We found a signif-
icantly reduced rate of triglyceride appearance in the
blood for VSG animals from 4 to 6 hours after the
gavage and that remained low throughout the 6-hour
duration of the study (treatment X time interaction,
P ? .0001; Figure 3C). Rate of triglyceride appearance
during the 6-hour experiment was lower in VSG ani-
mals than in either SHAM (P ? .01) or PF (P ? .001)
animals (Figure 3D). Postprandial plasma ApoB48 and
ApoB100 content was assayed to assess whether re-
duced postprandial plasma lipid concentration might
be due to the secretion of smaller or to fewer triglycer-
ide-rich particles. No significant changes to ApoB48 or
to ApoB100 content in the plasma were detected in
samples collected at the 6-hour time point (Figure 3E).
Triglyceride-to-total ApoB content also did not differ
among groups at this time point, although a trend
toward increased particle size was observed for PF an-
imals relative to SHAM and VSG animals (Figure 3E).
Figure 1. VSG reduces plasma lipids in a weight-independent manner. (A) Blood from 4-hour fasted animals was sampled on postoperative day 50.
At this time, VSG animals weighed less than SHAM (P ? .05) animals. CHOW rats were lighter than SHAM (P ? .001), VSG (P ? .05), and PF (P ?
.01) rats on the day of study. (B) Fat mass was reduced in VSG (P ? .001) and PF (P ? .05) animals as compared with SHAM. CHOW animals had
reduced fat mass as compared with SHAM (P ? .001), VSG (P ? .05), and PF (P ? .001) animals. Lean mass was unaffected by surgery, as
compared with SHAM and PF groups. CHOW animals had reduced lean mass as compared with SHAM (P ? .05) and VSG (P ? .05) animals. (C)
Plasma triglyceride levels were reduced in VSG and CHOW animals as compared with SHAM (P ? .01 vs VSG, P ? .001 vs CHOW) and PF (P ?
.01 vs VSG, P ? .001 vs CHOW) rats. (D) Plasma cholesterol levels were reduced by VSG as compared with SHAM (P ? .001) and PF (P ? .05). (E)
Phospholipid levels were reduced in plasma from VSG and CHOW animals as compared with either SHAM or PF rats (P ? .001 for all comparisons).
(F) Plasma NEFA levels were reduced in CHOW animals as compared with PF (P ? .05) but were not reduced significantly by VSG.
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VSG Does not Affect Hepatic Lipid Storage
Although fasting and thus hepatic triglyceride
secretion appears to be unchanged after VSG, other
metabolic changes might occur in the liver to affect
lipid homeostasis. For example, the liver secretes bile
acids that are critical for digestion and absorption of
fats. Recent evidence also suggest that bile acids serve
to activate nuclear transcription factors that can di-
rectly influence genes regulating lipid metabolism, glu-
cose metabolism, and gut peptide secretion.21Serum
bile acids have been reported to be increased following
bariatric surgery22,23but have not previously been mea-
sured following VSG. We found that plasma bile acids
were decreased in SHAM animals compared with
CHOW, VSG, and PF animals (Figure 4A). Plasma bil-
irubin levels were unaffected by weight or diet (Figure
4B). We did not detect any significant differences in
expression of hepatic CYP7A1, CYP27A1, HMG-CoA
reductase, or scavenger receptor class B, member 1
(SCARB1; Table 1), genes that regulate bile acid circu-
lation and metabolism. Neither hepatic triglyceride
(Figure 4D) nor cholesterol (Figure 4E) content was
Figure2. Reductionsinplasmalipidsaremostdramaticduringshortperiodsoffasting.Triglycerideandcholesterollevelsweremeasuredinplasma
sampled at 0, 4, 8, and 24 hours of fasting. (A) In unfasted blood, triglycerides were reduced by VSG as compared with SHAM (P ? .001) and PF
(P ? .01). Triglycerides were also reduced in VSG animals after 4 and 8 hours of fasting (P ? .001 vs SHAM at each time point), but this reduction
wasnotweightindependent(P?.05vsPF).(B)Areaunderthecurveforplasmatriglyceridesacross24hoursoffastingwasreducedforVSGanimals
as compared with either SHAM (P ? .001) or PF (P ? .05). (C) Plasma cholesterol did not differ significantly among groups across the 24-hour fast.
(D) Area under the curve for plasma cholesterol across the 24-hour study was unchanged by surgery or weight loss. (E) Cholesterol in fractionated
plasma from VSG rats did not reveal a shift in the size of plasma lipoproteins. However, VSG improved fasting hypercholesterolemia observed in
SHAM animals, as demonstrated by reduced cholesterol content in VLDL and HDL peaks. The reduction in HDL cholesterol content appears to be
unique to VSG vs PF or CHOW groups.
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affected by treatment in this study. Ratio of cholesterol
ester content to total cholesterol did not differ among
groups (Figure 4F), indicating that long-term hepatic
cholesterol storage was also unaffected by either VSG or
pair feeding. Consistent with this result, we did not
detect any differences in whole liver weight among
groups (Figure 4G). We next assayed the expression of
several genes (Table 1) related to hepatic lipid uptake
(CD36, FATP4, L-FABP) and esterification (ACAT2),
intracellular triglyceride
(MGAT2, DGAT1, MTP), lipoprotein composition
(ApoA2 and ApoB), and lipoprotein lipase (LPL) activ-
ity. VSG was not found to have any effect on hepatic
gene expression. ApoB expression was found to be re-
duced in PF vs SHAM with no effect of VSG (ANOVA,
P ? .0181; PF vs SHAM, P ? .05).
synthesisand packaging
Figure 3. VSG impairs dietary triglyceride secretion without affecting fasting triglyceride secretion. (A) Under fasted conditions, plasma triglycerides
were increased along a similar trajectory in all animals after a 1-g/kg intraperitoneal dose of P-407 (treatment X time, interaction P ? .6684; effect of
treatment, P ? .7272; effect of time, P ? .0001). (B) VSG did not affect the rate of appearance of triglycerides in the blood across the 24-hour
experiment (ANOVA: P ? .6053). (C) After an intraperitoneal injection of 1 g/kg of P-407, a 0.47-g/kg lipid gavage resulted in an attenuated
appearance of triglycerides in the plasma of VSG animals as compared with PF (P ? .001 at 4- and 6-hour time points) and SHAM (P ? .01 at 4-hour
time point and P ? .001 at 6-hour time point). Interaction of treatment and time, P ? .0001. (D) The rate of appearance of triglycerides in the plasma
during the 6-hour experiment was reduced by VSG as compared with SHAM (P ? .01) and PF (P ? .001) animals. (E) Plasma ApoB48 (P ? .1193),
ApoB100 (P ? .8792), and total ApoB (P ? .6486) content were unchanged 6 hours after the oral lipid load. VSG did not significantly alter the ratio
of triglyceride to total ApoB at this time point (P ? .3502).
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VSG Does not Affect the Size of Intestinal
Lipid Storage Pools
Enterocytes of the upper small intestine are known
to contain lipid storage pools.24Because VSG seemed to
have minimal effect on hepatic lipid processing, we hy-
pothesized that reduced postprandial intestinal triglycer-
ide secretion after VSG might be due to enhanced intes-
tinal lipid storage. Following P-407 administration and a
1,1,1,-13C3-Triolein-enriched olive oil gavage containing a
total of 0.28 g of lipid, uptake of lipids into the proximal
intestine was comparable among groups (Figure 5A). We
did not find any differences in intestinal weight (Figure
5B) or in triglyceride (Figure 5C) or cholesterol content
(Figure 5D) from whole intestinal segment homogenates.
Cresyl violet-stained sections from M2 were examined,
revealing no obvious differences in villus length or mor-
phology, which could affect lipid absorption (Figure 5E).
No differences in plasma triglycerides or cholesterol were
detected at this time point (Supplementary Figure 6).
Analysis of the expression of genes known to control intes-
tinal triglyceride metabolism and chylomicron synthesis did
not reveal any significant changes in VSG-operated animals.
Samples from M1 and M2 represent duodenum and jeju-
num, respectively, and so any changes to chylomicron for-
mation or secretion are expected to be effected in these
regions. Gene expression studies within these regions (Table
2) did not reveal any significant changes to transcripts
known to affect intestinal lipid uptake (CD36, FATP4,
Figure 4. VSG does not affect hepatic lipid storage. (A) Plasma bile acid content is increased by weight loss (vs SHAM: P ? .05 for VSG, P ? .05
for PF, and P ? .001 for CHOW; P ? .0006). (B) Plasma bilirubin levels did not differ among groups (P ? .0776). (C) Hepatic triglyceride content was
unaffected by VSG (P ? .3902). (D) Cholesterol content was unchanged in livers from VSG animals (P ? .3920). (E) No differences in the ratio of
hepaticcholesterolestertototalcholesterolcontentweredetected(P?.7771).(F)Wetliverweight(expressedasaratiotobodyweight)didnotdiffer
among groups (.2831).
Table 1. Expression of Genes Known to Affect Hepatic Lipid Uptake
GeneSHAM VSG PFP value
CD36
L-FABP (1)
FATP4
ACAT2
DGAT1
MGAT2
MTP
APOB
APOA2
APOC2
APOC3
CYP7A1
CYP27A1
HMGCR
SCARB1
100.0 ? 21.14
100.0 ? 14.82
100.0 ? 6.224
100.0 ? 16.91
100.0 ? 5.066
100.0 ? 13.39
100.0 ? 10.59
100.0 ? 7.986
100.0 ? 9.322
100.0 ? 8.895
100.0 ? 9.444
100.0 ? 27.04
100.0 ? 19.38
100.0 ? 15.36
100.0 ? 9.981
79.16 ? 10.78
71.11 ? 6.121
89.97 ? 5.556
165.8 ? 33.36
93.62 ? 4.983
66.71 ? 9.168
92.46 ? 4.408
79.74 ? 6.043
106.6 ? 6.865
85.2 ? 5.433
75.71 ? 5.019
46.71 ? 9.300
122.0 ? 7.334
129.3 ? 20.20
101.1 ?8.118
54.30 ? 10.40
79.15 ? 11.60
81.45 ? 5.817
78.03 ? 30.15
79.03 ? 7.389
67.41 ? 8.861
85.04 ? 10.40
67.55 ? 6.164
102.4 ? 4.575
94.64 ? 10.82
85.82 ? 8.247
85.96 ? 25.74
109.3 ? 13.47
68.08 ? 11.27
82.61 ? 9.082
.0975
.1598
.1329
.0981
.0656
.0507
.4932
.0107a
.7981
.4287
.0801
.1687
.4755
.0528
.2892
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Month 2011SLEEVE GASTRECTOMY IMPROVES POSTPRANDIAL LIPIDS7
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T2

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L-FABP, I-FABP, and ACAT2), triglyceride synthesis and
packaging (DGAT1, MGAT2, and MTP), chylomicron
composition and size (ApoAIV and ApoB), and lipopro-
tein lipase activity (ApoC2 and ApoC3).
Discussion
It has been speculated that plasma lipids are re-
duced in humans after bariatric surgery in part because of
conscious dietary changes. Here, we show that the im-
provement to lipid homeostasis after VSG is instead a
physiologic consequence of the surgery. We report lower
plasma lipid levels in VSG-operated rats than weight-
matched (PF) rats despite the fact that both groups were
maintained on the same amount of HFD.
Our data show that plasma triglyceride levels are mark-
edly reduced in VSG-operated rats, specifically after short
periods of fasting, suggesting that VSG primarily affects
postprandial lipid metabolism. Our findings indicate that
VSG improves postprandial triglyceride clearance through
an intestinal mechanism. Whereas endogenous triglycer-
Figure 5. VSG does not affect the size of intestinal lipid storage pools. (A)13C enrichment in M1 and M2 intestinal samples after13C-Triolein gavage
did not differ between groups (M1, P ? .1274; M2, P ? .9866). (B) Weight of neither M1 (P ? .5527), M2 (P ? .7453), M3 (P ? .9788), nor M4 (P ?
.8089) was affected by VSG surgery or by weight loss. (C) Triglyceride content did not differ among groups for any quartile of the small intestine (M1,
P?.6202;M2,P?.9445;M3,P?.2394;M4,P?.4164).(D)Differencesincholesterolcontentwerenotdetectedinanygutregion(M1,P?.5310;
M2, P ? .8361; M3, P ? .3177; M4, P ? .7376). (E) No obvious differences in the morphology of intestinal villi in cresyl violet-stained M2 sections
were observed.
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8 STEFATER ET AL GASTROENTEROLOGY Vol. xx, No. x
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ide secretion is not changed by VSG (Figure 3), the secre-
tion of dietary-derived triglyceride-rich particles is signif-
icantly reduced in VSG animals as compared either with
SHAM or PF controls. The ratios of triglyceride levels to
ApoB concentrations in plasma 6 hours after oral lipid
gavage indicate that VSG does not significantly alter the
size of TRLs secreted postprandially. However, the data do
trend toward the secretion of larger TRLs in PF animals
than in either SHAM or VSG animals and toward the
secretion of fewer TRLs in PF and VSG vs SHAM animals.
Intestinal enterocytes contain lipid storage pools that
may be acutely mobilized by the consumption of a meal.24
Thus, the degree of postprandial lipidemia depends not
only on intestinal lipid absorption, resynthesis, and secre-
tion but also on the extent of intestinal lipid storage. Here,
we demonstrate that VSG does not alter intestinal storage
capacity for a dose of oral fat known to produce reduced
postprandial lipidemia in VSG rats (Figure 5). We did not
detect any changes in the expression of a large panel of genes
known to regulate intestinal triglyceride metabolism and/or
chylomicron synthesis (Table 2). These data lead us to the
hypothesis that VSG does not induce long-term changes to
intestinal lipid metabolism. Importantly, gene expression
was assayed at a single postprandial time point. Whereas we
would expect that earlier transcriptional changes would be
maintained at the 6-hour time point, we cannot exclude the
possibility that key transcriptional changes occur at other
time points. However, the current data are consistent with
the hypothesis that smaller, more frequent meals in VSG
rats18might reduce postprandial plasma lipids by altering
the dynamics of lipid flux through the intestine.
In humans, it has been speculated that small, frequent
meal intake may have benefits for metabolic health. Al-
though the degree of lipidemia observed postprandially is
determined by the fat content of the meal,25it has recently
been shown that enterocytes abide by a “last in- last out”26
phenomenon whereby triacylglycerols consumed at break-
fast may not appear in the plasma until after lunch. Our
data raise the possibility that smaller meals consumed by
VSG animals18may reduce plasma lipid excursions by
reducing the size of the lipid pool, which is released from
the enterocyte postprandially. This would also explain
why VSG-operated rats exhibit lower plasma triglycerides
following a lipid gavage designed to eliminate intergroup
differences in meal size because the preceding meal was
smaller in the VSG animals. Thus, we hypothesize that
eating smaller, more frequent meals may alter the rate by
which stored intestinal lipids are mobilized, in effect “me-
tering out” triglycerides more gradually as they are re-
leased in smaller, more frequent boluses and leading to
weight-independent reductions in plasma lipid levels in
VSG rats during ad libitum feeding conditions. In hu-
mans, reduced postmeal lipid levels might translate into
reduced cardiovascular risk in patients receiving VSG be-
cause high postprandial lipid levels have been shown to
promote atherosclerosis.27
We were surprised to find malabsorption in VSG ani-
mals following a large intragastric lipid bolus (Supple-
mentary Figure 4). This malabsorption was despite a lack
of intestinal malabsorption during a 24-hour period of ad
libitum HFD consumption.18The fat content of the ga-
vaged bolus did not exceed the size of meals consumed ad
libitum: VSG animals freely consume small meals of
about 5.77 kcal (1.27 ? 0.10 g of HFD per meal, with a
caloric density of 4.54 kcal/g18), whereas the malabsorbed
gavage dose contained an average of 4.5 kcal (0.5 fat g).
However, the nonphysiologic nature of the gavaged meal
may be responsible for impaired lipid absorption in VSG
animals. Intragastric gavage bypasses the cephalic response
to food and is delivered more rapidly to the stomach than a
meal consumed ad libitum. Furthermore, meal size appears
to have a relationship to the degree of malabsorption be-
cause a smaller dose (0.28 g lipid; Figures 3 and 5) mini-
mized intergroup differences and increased absorption in all
groups. Equivalent postmeal levels of isotope-labeled lipid
across groups (Figure 5) eliminates the possibility that ma-
labsorption might account for intergroup differences. How-
ever, at larger doses of gavaged lipid, certain hypothesized
features of VSG, including accelerated gastric emptying,28–30
might promote malabsorption. Future exploration into the
effects of VSG on gastrointestinal physiology will help to
clarify these issues in the future.
Table 2. Expression of Genes Affecting Intestinal Lipid Metabolism Are Unaffected by VSG
M1M2
Gene SHAM VSG PFP valueSHAM VSG PFP value
CD36
FATP4
L-FABP (1)
I-FABP (2)
ACAT2
DGAT1
MGAT2
MTP
APOA4
APOB
APOC2
APOC3
100 ? 18.52
100 ? 13.16
100 ? 20.39
100 ? 20.46
100 ? 13.62
100 ? 17.05
100 ? 10.10
100 ? 15.62
100 ? 30.44
100 ? 22.71
100 ? 24.87
100 ? 17.77
69.96 ? 23.94
76.42 ? 17.40
61.03 ? 21.58
144.7 ? 63.31
89.71 ? 16.28
79.43 ? 15.15
75.01 ? 8.65
65.73 ? 14.41
97.55 ? 61.22
87.68 ? 30.61
74.03 ? 32.52
63.4 ? 17.27
128.5 ? 39.27
89.75 ? 23.22
96.29 ? 20.55
163.4 ? 60.26
93.44 ? 18.63
112.3 ? 24.94
93.52 ? 12.85
108.3 ? 25.57
162.6 ? 59.35
132.4 ? 41.95
137.4 ? 42.51
89.96 ? 24.15
.3469
.6533
.3477
.7320
.9084
.4614
.2234
.2356
.6469
.6143
.4229
.3952
100 ? 21.17
100 ? 15.05
100 ? 20.28
100 ? 21.08
100 ? 15.52
100 ? 19.05
100 ? 13.61
100 ? 18.59
100 ? 25.90
100 ? 24.86
100 ? 20.32
100 ? 19.68
57.96 ? 16.3
92.31 ? 12.17
62.23 ? 17.45
63.34 ? 16.14
133.9 ? 16.27
81.38 ? 14.06
86.7 ? 9.580
93.10 ? 16.06
81.49 ? 31.97
72.78 ? 19.96
91.04 ? 32.43
72.68 ? 23.36
81.32 ? 14.71
88.34 ? 10.62
76.87 ? 23.23
83.79 ? 18.52
105.1 ? 20.25
88.65 ? 14.14
98.8 ? 11.78
94.89 ? 12.97
113.7 ? 32.12
96.54 ? 17.76
104.1 ? 21.4
96.73 ? 20.79
.2392
.8235
.4131
.3653
.3200
.6997
.6455
.9531
.7584
.5927
.9388
.6156
BASIC AND
TRANSLATIONAL AT
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AQ: 28
AQ: 47
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Our data highlight several important points regarding
weight loss and lipid metabolism. First, weight loss either
via caloric restriction (PF) or VSG does not appear to alter
the expression of several target genes known to be affected
by HFD consumption. High-fat diets have been reported to
increase intestinal absorptive capacity by enhancing the ex-
pression of several genes including FATP4, I-FABP, L-FABP,
CD36, ApoC2, ApoAIV, and MTP.31We did not detect any
weight-dependent changes to the expression of these genes
in the proximal intestine, suggesting that weight loss alone
(without changes to dietary composition) cannot reverse the
hyperabsorptiveeffectofHFDontheintestine,atleastatthe
level of the transcriptional machinery.
Second, our data show, for the first time, that both VSG
and caloric restriction (PF) enhance plasma bile acid levels
(Figure 4) relative to diet-induced obese (SHAM) animals.
We have not defined a mechanism for weight-related alter-
ation to plasma bile acids, but we do report reduced expres-
sion of hepatic CYP7A1, the rate-limiting step in hepatic bile
acid synthesis, in VSG animals (Table 1). Plasma bile acid
levels are influenced by a number of variables, including
hepatic bile acid synthesis and ileal bile acid reabsorption
efficiency. Plasma bile acids might become elevated in VSG
vs CHOW or PF animals because of distinct mechanisms,
such as enhanced ileal reabsorption secondary to accelerated
delivery of bile acids to ileal transporters, but the current
data cannot distinguish among these explanations.
Collectively, these data provide critical insights into an
important secondary benefit to VSG as 1 model of bariatric
surgery. Atherosclerosis can be caused by high postprandial
lipid levels,27which are characteristic of obesity. As we de-
termined herein, the improvements to lipid homeostasis
observed in VSG-operated animals are most dramatic in the
postprandial state. Understanding mechanisms by which
VSG may elicit this improvement may thereby lead to the
developmentofnoveltherapiesforatheroscleroticcardiovas-
cular disease. (Supplementary Figure 5).
Supplementary Material
Note: To access the supplementary material
accompanying this article, visit the online version of
Gastroenterology at www.gastrojournal.org, and at doi:
10.1053/j.gastro.2011.05.008.
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25. Dubois C, Beaumier G, Juhel C, et al. Effects of graded amounts
(0–50 g) of dietary fat on postprandial lipemia and lipoproteins in
normolipidemic adults. Am J Clin Nutr 1998;67:31–38.
26. Jauregui RC, Mattes RD, Parks EJ. Dynamics of fat absorption and
effect of sham feeding on postprandial lipema. Gastroenterology
2010;139:1538–1548.
27. Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circu-
lation 1979;60:473–485.
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28. Melissas J, Koukouraki S, Askoxylakis J, et al. Sleeve gastrec-
tomy: a restrictive procedure? Obes Surg 2007;17:57–62.
29. Shah S, Shah P, Todkar J, et al. Prospective controlled study of
effect of laparoscopic sleeve gastrectomy on small bowel tran-
sit time and gastric emptying half-time in morbidly obese pa-
tients with type 2 diabetes mellitus. Surg Obes Relat Dis;6:
152–157.
30. Braghetto I, Davanzo C, Korn O, et al. Scintigraphic evaluation
of gastric emptying in obese patients submitted to sleeve
gastrectomy compared with normal subjects. Obes Surg 2009;
19:1515–1521.
31. Petit V, Arnould L, Martin P, et al. Chronic high-fat diet affects
intestinal fat absorption and postprandial triglyceride levels in the
mouse. J Lipid Res 2007;48:278–287.
Received February 16, 2011. Accepted May 10, 2011.
Reprint requests
Address requests for reprints to: Randy J. Seeley, PhD, 2180 E.
Galbraith Road, Cincinnati, Ohio 45237. e-mail: randy.seeley@uc.edu;
fax: (513) 297-0966.
Acknowledgments
The authors thank Elizabeth Parks and Rohit Kohli for providing
valuable advice in designing these studies and Jose Berger,
Mouhamadoul Toure, Ken Parks, and Kathi Smith for their surgical
expertise and Michelle Kirby and Therese Rider for technical
assistance with biochemical assays.
Author contributions: M.A.S.: study concept, design, execution,
analysis, and write-up. P.T., S.M.H., and R.J., D.A.S.: study design and
data interpretation. A.P.C., H.W.P., and D.A.S.: study execution,
interpretation, and discussion. S.C.W. and R.J.S.: study concept,
design, analysis, and writing.
Conflicts of interest
The authors disclose the following: Randy J. Seeley: Johnson &
Johnson (Ethicon Endo-Surgery), Zafgen, Merck, Pfizer, Mannkind,
Roche. Darleen A. Sandoval: Johnson & Johnson. The remaining
authors disclose no conflicts.
Funding
Supported by NIH grants DK54890 and DK083870 and Ethicon
Endo-Surgery.
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Page 12

Supplementary Table 1. Overview of Experimental Groups
CohortTreatment groups Experiments
B
● CHOW (n ? 18 or 19)
● SHAM (n ? 14)
● PF (n ? 15)
● VSG (n ? 18)
● CHOW (n ? 9)
● SHAM (n ? 8)
● PF (n ? 8)
● VSG (n ? 7)
● SHAM (n ? 8?12)
● PF (n ? 10?12)
● VSG (n ? 10?14)
● Plasma TG, cholesterol, phospholipid, and NEFA (Figure 1)
● Body weight and body composition data (Supplementary Figure 1)
C
● FPLC (Figure 3; pooled samples composed of plasma from 7 animals each)
● Plasma TG and cholesterol (Supplementary Figure 3)
● Plasma bile acids (Figure 4)
● Fasted intestinal triglyceride content (Supplementary Figure 6)
● Fasting plasma time course triglyceride and cholesterol (Figure 2)
● Hepatic and intestinal triglyceride secretion (Figure 3)
● ApoB Western blot (n ? 6/group; Figure 3E)
● Wet liver weight (Figure 4)
● Postprandial liver triglyceride, cholesterol, and cholesterol ester content (Figure 4)
● Liver gene expression studies (Figure 4)
● Stable isotope study (Figure 5)
● Wet intestinal weight (Figure 5)
● Postprandial intestinal triglyceride and cholesterol content (Figure 5)
● Postprandial intestinal cresyl violet staining (Figure 5)
● Hepatic (Table 1) and intestinal (Table 2) gene expression studies
● Body weight, food intake, and body composition (Supplementary Figure 2)
● Plasma triglyceride and cholesterol content (Supplementary Figure 5)
D
FPLC, fast protein liquid chromatography; TG, xxxx.
Supplementary Figure 1. Timeline of postsurgical care.
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AQ: 48
AQ: 49
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Supplementary Table 2. Taqman Gene Expression Assays
GeneTaqman gene expression assay
L32
ACAT2
MGAT2
DGAT1
MTP
CD36
ApoB
ApoAIV
ApoA2
FABP2
FABP1
FATP4
ApoC2
ApoC3
CYP7A1
CYP27A1
SCARB1
HMGCR
Rn00820748_g1
Rn01526240_gH
Rn00585985_s1
Rn00584870_m1
Rn01522970_m1
Rn00580728_m1
Rn01499054_m1
Rn00562482_m1
Rn00565403_m1
Rn00565061_m1
Rn00664587_m1
Rn01438951_m1
Rn01764530_g1
Rn00560743_g1
Rn00564065_m1
Rn00710297_m1
Rn00580588_m1
Rn00565598_m1
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Supplementary Figure 2. Body weight, food intake, and body composition following VSG. (A) VSG elicits body weight loss that is comparable with
that produced by PF. Body weight change for VSG-operated animals was significantly different from SHAM animals by postsurgical day 12 (P ?
.001), an effect that persisted for the duration of the study. Body weight change for PF animals was significantly different from SHAM by day 12 (P ?
.01) and for all days thereafter. Interaction of time and treatment, P ? .0001. (B) Presurgical body weight did not differ among groups. At the time of
death, VSG (P ? .01) and PF (P ? .05) animals had lost weight relative to SHAM controls. Interaction of treatment and time, P ? .0001. (C) Prior to
surgery, treatment groups were matched for fat mass. At the termination of the study, VSG (P ? .001) and PF (P ? .01) animals had significantly
reduced fat mass compared with SHAM controls. Interaction of treatment and time, P ? .001. (D) Lean mass did not differ among groups either
postsurgicallyorattheendofthestudy(interactionoftreatmentandtime,P?.1406).(E)VSGproducedtransientpostsurgicalfoodintakereduction,
but daily food intake thereafter was equivalent to SHAM.
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Supplementary Figure 3. VSG reduces diet-induced hypertriglyceridemia and hypercholesterolemia. (A) Twenty-four hour–fasting plasma cho-
lesterol is reduced in VSG animals as compared with CHOW animals (P ? .05). (B) Triglycerides are reduced in plasma from VSG animals as
compared with SHAM animals (P ? .05).
Supplementary Figure 4. 1 g/kg Lipid gavage induces fecal fat loss in VSG animals. (A) A similar rate of appearance of triglycerides in the plasma
is observed during the first 3 hours following a gavage containing 1 g/kg lipid. Plasma triglycerides reach an early, reduced peak in VSG animals, and
plasma triglycerides 3 (P ? .001) and 6 (P ? .05) hours after the gavage are lower for VSG than SHAM animals. (B) Enhanced clearance in VSG
animals is reflected by reduced area under the curve for plasma triglycerides during the 6-hour experiment (P ? .05 for SHAM vs VSG). (C) Fecal lipid
loss in VSG animals is nearly 3-fold higher than in SHAM animals (P ? .05; 92.1% absorption for SHAM vs 77.8% absorption for VSG). Absorption
was also lower in VSG animals as compared with pair-fed animals (87.7338% absorption), although not significantly.
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