Characterization of weight loss and weight regain mechanisms after Roux-en-Y gastric
bypass in rats
Ana Guijarro1, Susumu Suzuki1, Chung Chen2, Henriette Kirchner1, Frank A. Middleton3, Sergiy
Nadtochiy4, Paul S. Brookes4, Akira Niijima5, Akio Inui6and Michael M. Meguid1
1Surgical Metabolism and Nutrition Laboratory, Neuroscience Program, Department of Surgery;
University Hospital, SUNY Upstate Medical University, Syracuse, NY 13210.
2Department of Statistics, Management Information and Decision, Whitman School of
Management, Syracuse University, Syracuse, NY 13244.
3Microarray Core Facility, Neuroscience Program, Physiology Department, SUNY Upstate
Medical University, Syracuse, NY 13210.
4Department of Anesthesiology & Mitochondrial Research Interest Group, University of
Rochester Medical Center, Rochester NY 14642.
5Department of Physiology, Niigata University, Niigata, Japan,
6Department of Behavioral Medicine, Kagoshima University Graduate School of Medical and
Dental Sciences, Sakuragtaoka, Kagoshima, Japan
Correspondence author: Michael M. Meguid, MD, PhD, Department of Surgery, University
Hospital, 750 East Adams Street, Syracuse, NY 13210.
Tel: 315-491 2863
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Articles in PresS. Am J Physiol Regul Integr Comp Physiol (July 11, 2007). doi:10.1152/ajpregu.00171.2007
Copyright © 2007 by the American Physiological Society.
Roux-en-Y gastric bypass (RYGB) is the most effective therapy for morbid obesity, but it
has a ~20% failure rate. To test our hypothesis that outcome depends on differential
modifications of several energy-related systems, we used our established RYGB model in
Sprague-Dawley diet-induced obese (DIO) rats to determine mechanisms contributing to
success (RGYB-S) or failed (RYGB-F) RYGB. DIO rats were randomized to RYGB, sham
operated Obese, and sham operated obese pair fed-linked to RYGB (PF) groups. BW, caloric
intake (CI) and fecal output (FO) were recorded daily for 90 days, food efficiency (FE) was
calculated, and morphologic changes were determined. D-xylose and fat absorption were
studied. Glucose-stimulated vagal efferent nerve firing rates of stomach were recorded. Gut,
adipose and thyroid hormones were measured in plasma. Mitochondrial respiratory complexes
in skeletal muscle, expression of energy-related hypothalamic and fat peptides, receptors and
enzymes were quantified. A 25% failure rate occurred. RYGB-S, RYGB-F and PF rats vs.
Obese showed rapid BW decrease, followed by sustained BW loss in RYGB-S. RYGB-F and PF
gradually increased BW. BW loss in RYGB-S rats is achieved not only by a RYGB-induced
decreased CI and increased FO, but also via SNS activation, driven by increased PYY, CRF
and orexin signaling, decreasing FE and energy storage, demonstrated by reduced fat mass
associated with the up-regulation of mitochondrial UCP-2 in fat. These events override the
compensatory response to the drop in leptin levels aimed at conserving energy.
Key words: Food efficiency; gastric bypass; gut and adipose hormones; mitochondria; obesity
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Bariatric operations are currently the only treatment for morbid obesity (defined as a
body mass index of 39 or greater, or weight that is at least 50% above normal). Of the 180,000-
200,000 bariatric operations performed in 2006, ~80% were Roux-en-Y gastric bypass (RYGB).
RYGB also reverses and ameliorates the major cardiovascular and metabolic risk factors,
including type 2 diabetes mellitus and hyper- and dys-lipidemia (12; 72), reduces the long-term
mortality and morbidity associated with obesity and decreases health care costs (15).
Despite the success of the RYGB operation, 20% of patients “ fail to maintain long term
weight loss” . Non surgically-related failure usually occurs within the first three postoperative
years (12), suggesting a metabolic-endocrine compensatory etiological mechanisms.
Weight loss induced by RYGB occurs bi-phasically. Initially there is rapid weight loss
lasting 1-3 years, followed by prolonged weight stabilization (39). Both occur despite a gradual
increase in caloric intake during the same period (72). Our RYGB model in diet induced obese
Sprague-Dawley rats (52) also shows a post-operative bi-phasic weight change pattern. In this
model, ~10-13 rat days is equivalent to 1 human year (62) thus allowing us to follow rats
postoperatively for a considerably long time compared with human subjects.
Conventional surgical belief is that weight loss following RYGB occurs by a) the small
gastric pouch that limits caloric intake; and b) the Roux-en-Y loop of hindgut which short-circuits
the remaining gastrointestinal tract, decreasing nutrient absorption. However, a large body of
evidence suggests that after RYGB, hormonal components play a major role in reducing food
intake and decreasing weight (6; 17).
Other mechanisms are initiated while creating the gastric pouch because it divides vagal
parasympathetic and sympathetic fibers (60), therefore influencing the afferent signals to the
brain, while efferent signals can also be disrupted, which may play a role in the outcome of this
operation. However, these mechanisms do not sufficiently account for the maintenance of body
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weight loss or regain after RYGB, suggesting that nutrient-hormone changes could define a
successful or failed operative outcome.
Mitochondria play a fundamental role in thermogenesis and energy regulation. Major
modifications in oxidative phosphorylation and mitochondrial metabolism occur in obesity (53).
Proton leak across the mitochondrial inner membrane, which is catalyzed by uncoupling
proteins (26) and that bypasses ATP production, may account for 25% of the standard
metabolic rate in the rat (66). Hepatic mitochondrial proton leak is positively correlated with
standard metabolic rate (10), and is affected by the thyroid status, being 4-fold greater in
hyperthyroid rats (33). Skeletal muscle mitochondria are also relevant in the context of energy
homeostasis. Thus, a decrease in skeletal muscle mitochondrial mass and function is
associated with accelerated rate of fat recovery (catch-up fat) and insulin resistance which are
characteristic features of weight recovery after caloric restriction (16).
We postulate that the outcome after RYGB depends on differential modifications of
several energy-related systems. To test our hypothesis we studied the changes in body weight,
food intake, nutrient absorption, fecal output, food efficiency, body composition and
metabolically-relevant nutrient and hormones in diet-induced obese Sprague-Dawley male rats
and examined the changes in electrical activity of the efferent vagal fibers innervating the gastric
pouch. In addition, we measured numerous transcripts in the hypothalamus and subcutaneous
fat involved in the modulation of food intake, metabolism and energy homeostasis as well as
mitochondrial function in skeletal muscle.
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MATERIALS AND METHODS
The Committee for the Humane Use of Animals at SUNY Upstate Medical University
approved the experiments, and animal care was in accordance with guidelines established by
the National Institute of Health.
The pre-operative protocol to induce obesity in the Sprague-Dawley male pups, our
operative procedures in which the subdiaphragmatic vagal trunks are preserved and the post-
operative management of the rats were previously described (52). To induce obesity, pups were
fed a high energy diet (HED, D12266, Research Diets®, New Brunswick, NJ) for 16 weeks (74).
After inducing obesity, obese rats were stratified according to body weight to ensure similar
average starting body weight before the following surgical procedures: 1.Sham operated obese
controls (Obese), which continued to eat ad libitum; 2. Roux-en-Y gastric bypass rats (RYGB)
and 3. Sham operated obese rats that were pair fed (PF) by being given the same amount of rat
chow to eat, as consumed the previous day by its paired RYGB rat. After surgery, rats were fed
with rat chow (Rat Chow Diet # 5008; Ralston Purina, St Louis, MO).
After the procedure, rats were followed for 90 post-operative days and then euthanized,
in a non-fasting state, starting at 9am. Because eight rats were euthanized in one day and the
harvesting of blood and tissue took approximately 60 minutes, this would result in fasting rats for
different periods that would introduce the variable of ‘ fast-duration’ , which has metabolic
consequences of relevance to our investigation. Therefore to avoid this variable the rats were
not fasted. The groups consisted of Obese, n = 10; RYGB, n = 12, [consisting of RYGB rats that
successfully lost weight: RYGB-S, n = 8; and RYGB rats that failed to sustain weight loss by
regaining weight: RYGB-F, n = 4], and PF, n = 8 (see General Statistical Analysis).
Based on the observed weight loss pattern derived from the above study, only Obese (n
= 10) and RYGB (n = 12) rats had absorption studies done on POD 30, 60 and 90.
Body weight, caloric intake, fecal output, food efficiency and body composition
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Postoperatively, daily body weight, food intake and fecal output were measured
gravimetrically for 90 days. Food intake was computed as caloric intake, because between post-
operative days 1-8 the rats had a liquid diet. They also had liquid stool and thus fecal weight
was measured only after solid food was given. Fecal output is expressed as fecal weight per
one g food intake x 100. Food efficiency, an index of nutrient assimilation into body mass, was
calculated as a ratio of change in body weight to cumulative caloric intake and expressed as
At 90 days the rats were anesthetized using isoflurane, decapitated and muscle
(representing the combined weight of left gastrocnemius plus soleus), fat depots (abdominal
subcutaneous, mesenteric, epididymal and retroperitoneal) and visceral organs (liver, heart and
pancreas) were dissected out and weighed, reflecting morphological changes and body
composition. Fat mass was the sum of the weights of the fat depots, while fat free mass was
calculated by subtracting fat mass from body weight. The above parameters were expressed as
a percent of body weight.
D-xylose and fat absorption
Before testing, rats were food deprived for 16h. D-xylose solution (0.5 g/kg; 20% w/v)
was gavaged via a trans-oral tube into the gastric pouch of RYGB rats or the stomach of Obese
without anesthesia. Urine was collected for 5h to measure the amount of excreted D-Xylose (23).
D-xylose absorption was calculated as (%) = urine D-Xylose (g)/administered D-Xylose (g) x
100. To measure fat absorption stool was collected for 72 h before POD 30, 60 and 90 and its
fat content was extracted and measured (76). Fat absorption (%) per day = Fat intake (g) - Stool
fat (g) (44). A PF group of rats was not included at the time of the study, because their
gastrointestinal tract was not surgically altered and was thus anatomically similar to the Obese.
We made the assumption that the absorption would thus be the same, based on supporting
data from our lab, in a different experiment, showing no differences in the disaccharidase
activity in proximal, distal jejunum and ileum in PF rats compared to Obese.
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Metabolically-relevant nutrients and hormones
After decapitation, mixed arterial and venous blood, in none fasted rats, was collected
into EDTA-rinsed tubes. It was centrifuged at 3,000 rpm for 10 min at 4°C for plasma. Samples
were stored for a limited time at –80°C before analysis in duplicate. Plasma glucose, total
cholesterol, triglycerides, and free fatty acids (FFA) were measured by enzymatic colorimetric
kits (WAKO®, Richmond, VA). Plasma polypeptide YY (PYY) (DSL®, Webster, TX), CCK,
ghrelin (Phoenix®, Belmont, CA), insulin (ALPCO®, Windham, NH), leptin (R&D Systems®,
Minneapolis, MN), free triiodotyronine 3 (T3) (Alpha Diagnostic, TX) were measured using
Efferent vagal nerve firing rates of gastric pouch
In a smaller separate set of rats (Obese, n=3 and RYGB, n=4) repeated measurements
of vagal efferent nerve firing rates of the gastric pouch were calculated before and after a
glucose challenge at 90 days. The methodological details of similar studies were previously
described in detail (54). Rats were food deprived for 6 h and anesthetized using intra-peritoneal
injection of urethane (1g/kg BW). The abdomen was opened and using a dissection microscope
the vagal gastric efferent nerve was isolated and divided. The central cut end was placed on a
silver wire recording electrodes to record efferent discharges. 5 % glucose in 5 ml water was
gavaged into the gastric pouch of RYGB rats or the stomach of Obese. The mean number of
vagal efferent firing impulses per 5 seconds was determined in 10 successive 50 second
periods. This measurement was repeated every 30 minutes for 180 to 210 minutes in response
to the 5% glucose solution. These results were compared between the groups after conversion
of raw data to standard pulses by a window discriminator, which separated study generated
discharges from background noise.
Blue native gels electrophoresis of skeletal muscle mitochondrial protein complexes
To determine the relative amounts of mitochondrial respiratory complexes contained
within skeletal muscle (gastrocnemius and soleus) samples at 90 days, frozen tissue (75 mg)
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was homogenized (glass Dounce homogenizer) in 2 ml of extraction buffer (440 mM sucrose,
20 mM MOPS, 1 mM Di-Na+ EDTA, 0.2 mM PMSF, pH7.2 at +40C), and centrifuged at 500 x g
for 2 min. The pellet was discarded, and 1ml of supernatant was centrifuged at 14,000 x g for 10
min. The pellet from this spin (nominal mitochondrial fraction) was resuspended in 200 µl of
buffer (0.75 mM Aminocaproic acid, 50 mM BisTris, 1% (w/v) dodecyl-β-D-maltoside, pH7 at
40C), and was kept on ice for 10min. with occasional mixing. The mixture was then centrifuged
at 14,000 x g for 10 min, and to the supernatant (100 µl) was added 9 µl of Coomassie Blue
suspension (0.5 M Aminocaproic acid, 5% Coomassie Blue) before loading directly onto gel.
Blue-native gel electrophoresis was performed as previously described with minor modifications
(11). Gels were run at a constant 40 V for 1h with Hi-Blue cathode buffer (50 mM Tricine, 15
mM BisTris, 0.02% Coomassie Blue, pH7 at +40C), and then at 20 V overnight with Lo-Blue
cathode buffer (50 mm Tricine, 15 mM BisTris, 0.002% Coomassie Blue, pH7 at + 40C). Gels
were stained in a solution of Coomassie Blue R-250 and G-250 (0.05 w/v each in 10 % acetic
acid and 25% isopropanol, pH 7 at +40C) for 3 hours, and then destained in 10 % acetic acid
+25% isopropanol. The relative amount of mitochondrial respiratory complexes contain within
skeletal muscle samples was assessed by optical densitometry with Scion Image software
(Scion Corp, Frederick, MD).
Short tandem repeats (STRs)
We genotyped 8 short tandem repeats (STRs) that are highly polymorphic between
Sprague-Dawley and other closely-related strains (Wistar, Wistar-Kyoto, Spontaneously
Hypertensive). The STRs were amplified using hepatic DNA from one RYGB-S and three
RYGB-F rats. The hepatic DNA was purified using the MasterPure kit (Epicentre
Biotechnologies, Madison, WI) and PCR was performed to amplify the STR sequences using
primers based on the Rat Genome Database records for specific STR sequences (and provided
by Drs. R. Hoopes and S. Scheinman at SUNY Upstate, NY). The STRs were chosen to
interrogate three different chromosomes (1-3) at approximately every 60 megabases. The 8
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STRs included were: D1RAT196, D1RAT193, D1MIT32, D2RAT6, D2RAT88, D2RAT171,
D3MGH16, and D3MIT13. The PCR products of these reactions were resolved on a 4%
agarose/ethidium bromide stained gel by electrophoresis (80V, 1 hour) and compared to the
SSLP expected size database for different rat strains available on the Rat Genome Database.
Microarray of whole hypothalamus and peripheral fat
To examine the possibility that changes in expression of specific genes and gene groups
might underlie the failure of certain rats to sustain weight loss following RYGB, we performed
gene expression analysis with oligonucleotide microarrays (RAE230 2.0, Affymetrix) on three
groups of male rats: RYGB-S (n=8), RYGB-F (n=3) and PF controls (PF, n=3). For each animal,
total RNA was isolated from homogenates of an entire hypothalamus from one hemisphere and
a sampling of subcutaneous abdominal fat, using the RNeasy kit (Qiagen). We had a large
number of successful animals (RYGB-S rats) with tissue available. Thus, we chose to use a
partial pooling design for establishing the baseline expression values for the RYGB-S rats which
showed the expected outcomes. For RYGB-F and PF groups, the RNA from three individual
animals was used for microarray analysis. For the RYGB-S group, the eight total RNA samples
for each tissue type were divided into three separate pools (each of which contained RNA from
2 or 3 rats) for microarray analysis using an equal quantity of RNA from each animal. In total, 18
GeneChip hybridizations were performed comparing the three treatment groups (RYGB-S,
RYGB-F, PF) and the two tissue types (hypothalamus and fat) using three independent
replicates for each. Amplification and labeling of RNA was performed using the WT-Ovation™
RNA Amplification System (NuGen), and hybridization, washing, staining and scanning of the
GeneChips performed according to standard protocol (GeneChip Expression Analysis Technical
Manual 701021 rev 5, Affymetrix). After scanning, the microarray images were processed using
GeneChip Operating System (GCOS) software to obtain performance metrics, and prepared for
data analysis using the RMA normalization method (36). After obtaining RMA-based estimates
of gene expression levels, individual transcripts in each of the three experimental groups and
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two tissue types were compared using a two-way analysis of variance (2 x 3 ANOVA)
comparing tissue type (hypothalamus or fat) and treatment group (RYGB-S, RYGB-F, PF).
Because our primary aim was on determining possible causes of the failure to sustain weight
loss in the RYGB-F animals, we focused the present analysis on a subset of 667 probes that
were determined to be involved in feeding, digestion, or general carbohydrate, lipid and protein
metabolism using the online NetAffx tool (Affymetrix). A list of these probes is posted at
http://www.upstate.edu/sunymac/Meguid.xls. A table of the genes showing a significant main
effect of treatment in either tissue type (uncorrected p < 0.05) was generated, and post-hoc
analyses (Fisher’ s Protected LSD) were performed to determine the specific comparisons
showing significant effects.
After the operation, time series plots of the daily body weight, food intake and fecal
output was plotted for each individual rat to show changing patterns. To determine the change
or inflection point (change point day) of each daily characteristic during the rapid and gradual
weight loss periods after operation for 90-days, a switching quadratic trend analysis was
performed, to identify the change point day of mean body weight, caloric intake and fecal output
in Obese, RYGB and PF rats. The exact time point was estimated based on the smallest
average error(s) and the highest adjusted R2for each possible time period between t=0 and
t=90. When the switching quadratic trend analysis was applied to the body weight series of each
individual rat and the mean body weight series of each of the study groups, it revealed two
separated RYGB cohorts: RYGB-weight loss (RYGB-S) and RYGB-weight regain (RYGB-F).
Thus further comparisons and analyses were conduct separately with respect to the four
experimental groups: Obese, RYGB-S, RYGB-F and PF. In the RYGB-S, RYGB-F and PF rats
a switching quadratic trend model was used to optimally analyze the data, while a switching
linear trend model fitted the Obese rat data. A linear trend model for change point analysis as
used for mean daily caloric intake and fecal output fitted the postoperative Obese, RYGB-S,
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RYGB-F and PF rat data more optimally. Comparisons of daily body weight, caloric intake, fecal
output, feeding efficiency and organ weight in the four study groups were analyzed using one-
way ANOVA and Tukey’ s pair-wise multiple-comparison test with the join error rate equal to
0.05. To assess the differences in D-xylose and fat absorption between Obese and RYGB rats
and with time one-way ANOVA and Tukey’ s pair-wise multiple-comparison test with the join
error rate equal to 0.05 was done and data are reported as Mean ± S.E.M. Vagal nerve firing
rates of gastric pouch. Differences in efferent firing rates were evaluated using one-way ANOVA
and t-test. Data are reported as Mean ± S.E.M. Microarray data were analyzed by two-way
ANOVA and post-hoc analyses (Fisher’ s Protected LSD) were performed to determine the
specific comparisons showing significant effects. Densitometric data from blue native gels were
analyzed by two-way ANOVA, with p < 0.05 being considered significant.
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Body weight and caloric intake
These inter-related indices are plotted together in Figure 1A and B. The preoperative
body weight of all RYGB rats (n = 12) was 765.76 ± 8.10 g. The switching quadratic trend
analysis of daily body weight showed that after RYGB operation, 4 of the 12 rats, after an initial
period of body weight loss, began to regain weight i.e.: the RYGB operation failed (RYGB-F,
n=4), so that by postoperative day 90 they weighed more (p < 0.001) than the remaining RYGB
rats that successfully continued to lose weight (RYGB-S, n = 8), as shown in Figure 1A.
The change point day, as well as the quantitative changes in mean body weight and
mean daily change in body weight before and after change point day is shown in Table 1. In the
immediate postoperative period the Obese lost 7.0 ± 0.7 % of their initial body weight until
change point day on day 9 (Table 1). With the switch to solid chow, the Obese gained weight
until the end of the study, when they weighed 4.3 ± 3.5 % more than the initial body weight.
After the operation RYGB-S rats also lost weight rapidly (15.4 ± 0.6 % of their initial body
weight) but, despite the switch to chow, continued to lose weight until change point day at day
22 (Table 1) after which body weight loss stabilized, decreasing by 33.5 ± 2.6%, compared to
their starting body weight, on day 90. Initially, the RYGB-F and the PF rats rapidly lost body
weight until change point day at days 24 and 23 , respectively (at which point RYGB-F rats had
lost 10.8 ±1.4 %, while PF rats had lost 7.7 ± 1.5 %). This was followed by a 10.9 ± 2.3 % regain
in body weight for RYGB-F rats and an 8.8 ± 5.0 % in PF rats. By day 90, the RYGB-S rats had
lost a greater percentage of body weight (p < 0.001) vs.RYGB-F rats (14.2 ± 1.1%) and PF rats
(13.4 ± 3.5%). The body weight pattern of RYGB-F and PF rats was similar with no difference in
the percentage of body weight loss (p = 0.997), which however was lower than their initial body
weight by day 90.
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The preoperative caloric intake was 101.4 ± 6.1 Kcal/day. After operation, when rats
were started on a liquid diet, the Obese ate 46.1 ± 4.7 Kcal/d, while RYGB-S rats ate 16.9 ± 2.0
Kcal/d and RYGB-F rats ate 15.9 ± 1.9 (Figure 1B). On day 9 chow was provided and caloric
intake increased in all groups, as shown in Figure 1B. This occurred during the same time that
body weight was decreasing until their respective change point day. As shown in Table 2, the
change point day was on day 25 in Obese; day 34 in RYGB-S, day 30 in RYGB-F and day 34 in
PF rats. After each change point day, caloric intake remained relatively stable until the end of
The quantitative changes in mean caloric intake and in mean daily change in caloric
intake before and after change point day are shown in Table 2. Although caloric intake
increased between day 9 and change point day in all groups, it remained significantly lower in
RYGB-S and in PF rats vs. Obese (p = 0.005, and p = 0.006, respectively). Caloric intake in
RYGB-F rats vs. Obese was lower, but it was not statistically different (p = 0.105, Table2).
Between change point day and day 90 mean caloric intake in RYGB-S rats was lower vs. the
remaining groups (p < 0.001), while in RYGB-F rats it was higher vs. both RYGB-S and PF rats
(p < 0.001), although it was not different vs. Obese (p = 0.920). Caloric intake in the PF rats vs.
Obese was lower (p < 0.001). Throughout the study there were no differences (p > 0.05, Table
2) in the daily mean change in caloric intake among the groups.
Because immediately after operation rats were on a liquid diet, their liquid stools were
not quantified. After the introduction of solid chow, feces became formed and were quantitated
and as shown in Table 3, from day 9 to change point day, there were no differences in the ratio
of fecal weight per one g food intake among groups.
But from change point day to the end of the study, RYGB-S and RYGB-F rats put out
significantly greater amounts of feces vs. both the Obese and PF cohorts in whom
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gastrointestinal tract was intact (p < 0.001). RYGB-S rats had also a greater fecal output than
RYGB-F rats (p < 0.001). No differences were found between PF group and Obese (p = 0.752).
D-xylose and fat absorption
At day 30, D-xylose absorption (Figure 2A) decreased in RYGB-S rats vs. Obese (p =
0.001), while in RYGB-F rats it was similar to Obese (p = 0.725). D-xylose absorption at day 60
and 90 was not significantly different in both RYGB groups vs. Obese (p > 0.05), while there
was no differences between RYGB-S and RYGB-F rats at any time point (p > 0.05).
In contrast, fat absorption (Figure 2B) was significantly lower in both RYGB-S and
RYGB-F rats vs. Obese at POD 30, 60 and 90 (p < 0.001) but no differences were found
between RYGB-S and RYGB-F rats (p > 0.05) at any time point, nor within each group with time
(p > 0.05).
Between days 0 to 8 the food efficiency was negative in all groups (Figure 3A) and in
RYGB-S rats was significantly lower than in Obese (p < 0.001), RYGB-F (p = 0.046) and PF (p
= 0.019) rats. Both RYGB-F (p = 0.022) and PF (p = 0.002) rats had a lower food efficiency than
Obese rats but there were not significant differences between them (p = 0.999). From day 9 to
change point day (Figure 3B) no differences were found among groups (p > 0.05) although
RYGB-S rats had a lower food efficiency compared to the remaining groups, but which was not
statistically different (p > 0.05). From change point day to day 90 (Figure 3C) food efficiency
improved, becoming positive in Obese, RYGB-F and PF rats, while remaining negative and
significantly lower in the RYGB-S rats compared to Obese (p = 0.002), RYGB-F (p = 0.029) and
PF (p = 0.011) rats.
At day 90, in RYGB-S (p < 0.001), RYGB-F (p < 0.001) and PF (p = 0.007) rats total fat
mass (expressed as a percentage of body weight) was lower than in Obese (Table 4). In RYGB-
S rats, total fat weight was also lower than in PF rats (p < 0.001), while there were no
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differences between RYGB-F rats and PF group (p = 0.543). The percentage of fat mass in
RYGB-S was lower than in RYGB-F rats but the difference was not statistically significant (p =
0.110). The reduction in the percentage of fat mass relative to Obese was 71.5 ± 3.4% in
RYGB-S rats, 47.1 ± 11.1% in RYGB-F rats and 27.4 ± 5.5% in PF group.
The percentage of fat free mass (Table 4) was higher in RYGB-S (p < 0.001), RYGB-F
(p < 0.001) and PF (p = 0.007) compared to Obese.. In RYGB-S rats, the percentage of fat free
mass was higher than in PF rats (p < 0.001), while there was no statistical differences between
RYGB-F and PF (p = 0,263) or RYGB-S and RYGB-F (p = 0.110).
At day 90, liver weight, as a percentage of body weight (Table 4), was lower PF rats
compared to Obese (p = 0.016), RYGB-S (p < 0.001) and RYGB-F (p = 0.002). There was no
difference RYGB-S vs. Obese rats (p = 0,423), RYGB-F rats and Obese (p = 0,277) or RYGB-S
vs. RYGB-F (p = 0,877). The pancreas weight, expressed as a percentage of body weight, did
not change with RYGB or caloric restriction (p > 0.05, Table 4). As shown in Table 4, the
percentage of heart weight was higher in RYGB-S rats compared to both Obese (p < 0.001) and
PF rats (p = 0.023). No differences were found among the remaining groups (p < 0.05). The
percentage of the skeletal muscle weight was not different among groups (p < 0.05; Table 4).
Metabolically-relevant nutrients and hormones
Relative to Obese plasma glucose was decreased in RYGB-S and RYGB-F rats (p <
0.001) and PF group (p = 0.010), as shown in Table 4. Plasma glucose was also decreased in
RYGB-S rats vs. PF group (p = 0.042), but was not different between both RYGB groups (p =
0.999) or between RGYB-F and PF rats (p = 0.204).
Total cholesterol was lower in RYGB-S rats vs. Obese (Table 4, p < 0.001) and it was
lower in RYGB-F rats and PF group vs. Obese, but not statistically different. No differences
were found among the remaining groups (p > 0.05). A decrease in plasma triglycerides occurred
in RYGB-S (p = 0.001), RYGB-F rats (p = 0.018) and PF group (p = 0.005) compared to Obese.
Free fatty acid (FFA) concentrations were similar in all groups (p > 0.05).
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No differences were detected among groups in total and acylated ghrelin, and
cholecystokinin (CCK) although acylated ghrelin in both RYGB groups was slightly lower than in
Obese and PF rats (p > 0.05). Peptide YY (PYY) concentrations in RYGB-S rats were more
than 3.7-fold higher than in Obese (p < 0.001) and 2.7-fold higher than in PF group (p = 0.012).
There were no differences between RYGB-F rats and PF group (p = 0.977).
A lower plasma insulin in RYGB-S rats (Table 4, p = 0.001) and PF group (p = 0.002) vs.
Obese was measured. There were no differences between PF group and RYGB-F rats or
between RYGB-S and RYGB-F rats (p = 0.354 and p = 0.993, respectively).
Relative to Obese, plasma leptin was lower in RYGB-S rats (p < 0.001), RYGB-F rats (p
= 0.001) and PF group (p = 0.020; Table4). Plasma leptin levels in RYGB-S rats were 14.8-fold
lower than in Obese and 8.7-fold lower compared to PF group (p = 0.004). In the RYGB-F and
PF rats leptin concentration was 2.8-fold and 1.7-fold lower than Obese, respectively. No
differences were found between RYGB-F rats and PF group (p = 0.504) or both RYGB-S and
RYGB-F rats (p = 0.209).
Plasma T3 was significantly lower in RYGB-S rats compared to Obese (p = 0.003),
RYGB-F rats and PF group (p < 0.001), while no differences were found among Obese, RYGB-
F rats and PF group (p > 0.05).
Efferent vagal nerve firing rates of gastric pouch
Figure 4A (Top of Panels A and B) shows a representative tracing upper gastrointestinal
vagal efferent firing rate before and after a 5% glucose gavage in one Obese rat and one RYGB
rat. These data are shown quantitatively in the lower graph, while the mean values of Obese
and RYGB rats are shown in Figure 4C. A significant increase in vagal celiac efferent firing rate
occurred 60 min after glucose gavage; and peaked at 120 min, remaining elevated for the
duration of the study in Obese (Figure 4A and C). In contrast (Figure 4B and C), in RYGB rats a
5% glucose gavage into the gastric pouch did not change the firing rate.
Blue-Native gel electrophoresis of mitochondrial protein complexes
Page 16 of 53
Figure 5A shows a representative blue native gel of mitochondrial complexes from
skeletal muscle of RYGB-S and RYGB-F rats studied at day 90, while Figure 5B reveals a
significant increase in the levels of complexes I, II and IV, of mitochondrial oxidative
phosphorylation in RYGB-F rats. Because samples were prepared from identical quantities of
tissue these data are indicative of greater mitochondrial mass per unit tissue in the RYGB-F
Short tandem repeats (STRs)
The lengths of the STRs from the RYGB-S and RYGB-F rats were all in the expected
ranges for Sprague-Dawley-derived rat strains except for the D1RAT193 STR of one RYGB-F
rat. Notably, however, evidence for heterogeneity was found (at least two different STRs) at five
of the eight loci (D1RAT193, D1MIT32, D2RAT88, D3MGH16 and D3MIT13). These data
suggested that the success or failure of RYGB was not simply related to genetic background
differences that might have been present between the animals in the random bred groups.
Microarray of whole hypothalamus and peripheral fat.
Quality. We performed a total of 18 GeneChip hybridizations comparing two tissue types
(hypothalamus and subcutaneous abdominal fat) and three treatment groups (RYGB-S, RYGB-
F, and PF) using 3 independent replicate samples for each comparison group. Overall, the 18
microarray hybridizations performed equally well, with % Present Call rates, Scale Factors,
RawQ, 3’ /5’ ratios for housekeeping genes, and Average Signals of Present and Absent genes
highly comparable within each tissue type. Thus, all arrays were included in the subsequent
RMA-based data analysis.
Single Gene Results. We initially used a 2 x 3 ANOVA to test a specific list of 667 probes
involved in feeding or metabolic function for significant effects of treatment group in either tissue
type and subsequently performed post-hoc analyses (Fisher’ s Protected LSD) to determine the
source of the main effect in RYGB-F and PF animals compared with RYGB-S animals. A total of
60 out of 667 tested transcripts demonstrated a significant main effect of treatment for either
Page 17 of 53
tissue type (Table 5). Of these, 44 transcripts showed significant main effects in the
hypothalamus only, 14 transcripts showed significant main effects in the fat tissue, and two
transcripts showed significant main effects in both tissues in this paradigm. The entire set of
data for the 667 genes of interest is available at the following link
http://www.upstate.edu/sunymac/Meguid.xls. All of the microarray data generated in this study
have been published at the NCBI Gene Expression Omnibus (Accession# GSE8314).
Examination of the specific transcripts showing significant changes in expression
indicated several involved in aerobic or anaerobic glycolysis (e.g., aldolase, enolase, lactate
dehydrogenase, glyceraldehyde-3 phosphate dehydrogenase), as well as fat metabolism, fat
cell differentiation, or fatty acid transport (e.g., multiple isoforms of fatty acid desaturases and
fatty acid transporters, hydroxysteroid (17-beta) dehydrogenase, adipose differentiation related
protein). In addition, numerous transcripts involved in signaling mechanisms that regulate FI or
metabolism were also observed as significantly changed (e.g., β-1 adrenergic receptor,
cannabinoid receptor-1, cholecystokinin, corticotrophin releasing hormone receptor-2, orexin,
orexin receptor-2 (OX2R), leptin receptor, PYY, ionotropic glutamate receptor and thyroid
hormone receptor-α). Some examples are shown in Figure 6. Notably, many of these genes are
the same as those (or directly related to those) showing significant changes in the plasma of
RYGB-F or PF animals (e.g., glucose, triglycerides, peptide YY, leptin, and T3 hormone; see
Table 4) and can thus the two data sets can be considered to provide independent validation of
these effects at the transcriptional and protein levels.
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The successful and progressive decline in body weight and thus the weight loss pattern
after RYGB in diet-induced obese rats is remarkably similar to the two-stage, weight loss pattern
defined in humans after successful RYGB for the morbidly obese (39), as well as that achieved
in response to behavior and/or life style modification (24), treatment with anti-obesity drugs (8),
or being on a very low-calorie diet (34), and to that previously observed in our 60 day RYGB
study (63). In our model, as well as in patients undergoing successful RYGB, a greater
magnitude of weight loss is achieved than with non-pharmacological or currently available
pharmacological treatments, including endocannabinoid receptor blockade therapy (34; 57).
This rapid weight loss is followed by a weight-loss plateau, which after RYGB in successful
patients, is maintained for life (72). Since 10-13 rat days are approximately equivalent to one
human year (62) weight loss is maintained for the equivalent of approximately 7 to 9 years in
our model and represents a successful loss of primarily fat mass (Table 4), indicative of lipid
oxidation (Table 4: total cholesterol and triglycerides).
In our study, a biphasic weight change was observed in all groups. The initial decrease
in body weight is attributed to the combined effects of operative stress and suboptimal caloric
intake due to the postoperative liquid diet. However, this is a common factor in all groups. The
provision of solid chow on day 9, after operation, led to an increase in caloric intake in all groups
despite which RYGB and PF rats continued to lose weight during this time, suggesting that both
the effects of the operation, as well as the caloric restriction by itself (PF group) triggered
peripheral metabolic signals, which continued to induce weight loss even during a simultaneous
increase in caloric intake. Because in this group of rats, switching quadratic trend analysis
revealing the differences between those rats that successfully lost weight and those which
started to regain weight was performed at day 90, we did not have a PF cohort for each RYGB
group. Thus, the information gained from these PF rats poses some limitations on the
interpretation of their data.
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In comparison to other therapies such as very low calorie diet (34), the RYGB-induced
rapid weight loss phase is even more striking, because it occurred when i) a concurrent and
continuous increase in caloric intake occurred and ii) during a similar time frame i.e. 10-35 post
RYGB days which is equivalent to the initial 2-3 years after RYGB in humans (Figure 1A and B).
In both RYGB-S rats and human RYGB outcome, weight loss and caloric intake stabilized and
plateaued at approximately the same time scale, suggesting that this stabilization in energy
intake may also account in part for the plateau in body weight. In our model, the lower mean
caloric intake in RYGB-S rats compared to Obese, RYGB-F and PF rats (Table 2) partly
explains the greater degree of weight loss. But caloric restriction may not be the sole cause of
this difference. PF rats had a significantly lower caloric intake than RYGB-F rats but had a
similar degree of weight reduction during the body weight plateau. Furthermore, in RYGB-F rats
caloric intake was the same as in Obese but their body weight was lower suggesting that other
factors contribute to explain the differences. The effect of caloric restriction on body weight
observed in PF rats is similar to that described in mice, where the initial period of weight loss is
followed by a period of weight regain (25).
Another factor contributing to the greater degree of weight loss in RYGB-S vs. both
RYGB-F and PF rats is their higher fecal output (Table 3) despite the fewer calories ingested by
RYGB-S rats (Table 2). The greater fecal loss found in both RYGB groups vs. Obese and PF
rats maybe explained by the rearranged gastrointestinal tract in RYGB, which reduces the
length of the gastrointestinal tract in which nutrient absorption occurs, as shown by a decrease
of both D-xylose and fat absorption (Figure 2). The greater fecal output measured in RYGB-S
rats might reflect their lower food efficiency. While RYGB is thought to cause weight loss, in part
via malabsorption, and although transiently true (9), once the gut has adapted, malabsorption is
not observed. When the temporal changes in small bowel absorption after RYGB was
compared, D-xylose absorption during the rapid weight loss period was less than during the
gradual weight loss period, suggesting that small bowel adaptation with time increased the
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efficiency of absorption. Could this contribute to the slowing of weight loss manifested by the
plateau? An increase in PYY was associated with intestinal cell proliferation after intestinal
resection and during adaptation to short bowel syndrome (2) leading to enhanced absorption
and delayed intestinal transit time (4; 74). Together the above suggests that the rise in PYY as
previously reported by us (74) contribute to the temporal adaptation in D-xylose absorption.
The weight loss plateau in the RYGB-S rats reflects compensatory processes that
opposes weight loss to the ~80% fat mass loss, as shown in Figure 3 (40). A similar response
also occurred in RYGB-F (~44%) and PF (~41%) rats although to a lesser degree. In RYGB-F
and PF rats, which started to regain weight, the compensatory processes opposing weight loss
are more robust than in RYGB-S rats leading to a lower body weight loss. In obese subjects the
degree of fat mass loss induced by the RYGB operation exceeds the 10% reduction in body
weight (46) which was accompanied by a reduction in energy expenditure beyond that predicted
by loss of body mass (21). In these obese study subjects SNS tone and concentrations of leptin
and thyroid hormones are decreased, while PNS tone is increased.
The plateau in weight loss seen in behavior life style modification and with anti-obesity
drugs is due to a reduction in resting energy expenditure, reflecting the compensatory
processes that oppose weight loss. These processes, also referred to as the “ starvation
response” , are the result of a decline in leptin levels, which counterbalance the decreased
caloric intake (50). This response is aimed at conserving energy through an increase in food
efficiency (68). In our model we found that the decrease in plasma leptin is accompanied by
enhanced food efficiency during the plateau in body weight loss in both RYGB-F and PF rats,
but this response was less robust in RYGB-S rats, whose food efficiency remained negative
despite the even greater reduction in plasma leptin. This lower food efficiency can explain the
lower weight of RYGB-S rats compared with the remaining groups and the reason why mean
daily body weight change remains negative in this group of rats.
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Does successful RYGB reset the leptin “ set point” (concentration of plasma leptin
perceived by the hypothalamus as a state of energy sufficiency (27)) by lowering it? If that is the
case, in RYGB-S rats the decrease in leptin is not perceived as a state of energy insufficiency,
and consequently food efficiency remains negative, while in RYGB-F and PF rats the reduced
plasma leptin triggers an increase in food efficiency that promotes an increase in weight. At
such lower leptin concentrations it could also activate the SNS by acting on the hypothalamus
(70). How the leptin “ set point” is reset by successful RYGB is a crucial question that remains to
be clarified. The increased leptin receptor expression in the hypothalamus of RYGB-S rats
(Figure 6) may account, in part, for this hypothetical lower leptin “ set point” , although it does not
rule out other changes including a higher affinity of these receptors. However, taking into
account that RYGB-S rats had a decrease in T3 levels and in thyroid hormone receptor-α in
hypothalamus, we would expect a decrease in resting energy expenditure and therefore an
increase in food efficiency. However, food efficiency remains negative in these rats.
In finding a decrease T3 in RYGB-S rats, we expect a lower metabolic rate, because T3
is involved in the regulation of energy expenditure, modulating both obligatory and adaptive
thermogenesis (71). T3 increases metabolic rate by up regulating uncoupling proteins (UCPs)
expression in skeletal muscle, heart and white adipose tissue and modulating UCP-1 in BAT
(45) leading to an increase in heat production and a decrease in the thermodynamic efficiency
of ATP synthesis (71). In caloric restriction a decrease in T3, T4 and TSH plasma levels occurs,
while ICV leptin increase T3 production in normal rats (19). Therefore, the lower RYGB-S leptin
levels along with their lower caloric intake may contribute to the reduction in T3 plasma levels.
On the other hand, T3 induces a rise in the expression of genes coding for enzymes of
lipogenesis (45; 55) and stimulates food intake by acting directly on the VMN (41). This
orexigenic effect is also achieved by the inhibition of leptin production (51). Taking the sum of
these effects into account, the lower T3 levels in RYGB-S rats would explain the reduction on fat
pad mass, caloric intake and body weight. Furthermore, the reduction in T3 could be part of the
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compensatory processes dampening the excessive increase in energy expenditure during the
negative energy state induced by successful RYGB (32).
A more plausible interpretation of our data is that in RYGB-S rats leptin “ set point” does
not change, but the compensatory processes that oppose weight loss triggered by the
decreased leptin levels is offset by the increase in PYY levels in these rats (Table 4), because in
high-fat diet-induced obese mice the administration of PYY3–36 reduces food intake and body
weight, decreases food efficiency and adiposity, increases fatty acid oxidation and increases
fecal energy loss (1). In contrast, in both RYGB-F and PF rats, PYY was not different from
Obese and consequently there is no compensation for the reduction in plasma leptin,
contributing to a lower weight loss than in RYGB-S rats. Moreover, we have found a higher
hypothalamic PYY expression in RYGB-S rats compared to RYGB-F rats (Figure 6), suggesting
an inhibition of the NPY/AgRP neurons in the arcuate nucleus (ARC) and an activation of the
POMC/CART neurons leading to an increase in α-MSH. Confirmation of this suggestion is
provided by our previous report of a significant decrease of NPY in ARC and in both
parvocellular and magnocellular-paraventricular (PVN) nuclei with a simultaneous increase in α-
MSH and 5-HT-1β receptors 10 days after RYGB (67).
The sum of these changes decreases food intake and increases SNS activity causing an
increase in energy expenditure in peripheral tissues (3). In addition, the higher hypothalamic
expression of CRF receptor-2 in the RYGB-S rats (Figure 6), counterbalances this
compensatory response to decreased leptin levels in RYGB-S rats, since CRF, when
administered ICV or microinjected into pre-optic area or dorsomedial hypothalamus (DMH),
elicits a sustained increase in sympathetic nerve activity to interscapular BAT and its
temperature, and also increases expired CO2and heart rate (13) and may also account for the
lower food intake found in RYGB-S rats. Similarly, ICV administration of orexin-A induces an
increase in body temperature (79) and enhances metabolic rate and alters respiratory quotient
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in mice (49). In our study, both orexin mRNA and orexin receptor-2 (OX2R) mRNA were
elevated in the hypothalamus of RYGB-S. Therefore, in RYGB-S rats we would expect an
increase in metabolic rate and in both physical activity and body temperature, leading to greater
energy expenditure and to their lower body weight. However, the reduction found in ionotropic
glutamate receptor expression in the hypothalamus of RYGB-S rats would argue against a role
of increased CRF and orexin activity inducing an increase in energy expenditure in these rats,
because the effects of both neuropeptides on thermogenesis are mediated by ionotropic
glutamate receptors (13). Such a discrepancy could be due to measuring the expression of
these receptors in the whole hypothalamus, whereas the glutamate receptors that mediate CRF
and orexin effects on thermogenesis are localized in the DMH. Furthermore, L-glutamate has
either stimulatory or inhibitory effects on sympathetic innervation to BAT depending on the
hypothalamic nucleus into which it is injected (78).
Both central and peripheral administration of anandamide increased food intake and
body weight in rodents (37) by acting on CB1 R (14). Could the RYGB procedure, therefore,
also affect the endocannabinoid system? Although we found higher hypothalamic expression of
cannabinoid receptor 1 (CB1 R) in RYGB-S vs. RYGB-F rats, they had a marked elevation in
hypothalamic expression of arachydonate 15-lipoxygenase, an enzyme involved in the
degradation of both anandamide and 2-arachydonylglycerol (42). The CB1 R antagonist
SR141716 induces long-lasting weight loss, independent in part of food intake, by deceasing
fatty acid synthesis (56), increasing metabolic activities (38; 47) and/or energy expenditure as
suggested by the increase in basal oxygen consumption in ob/ob mice after chronic
intraperitoneal administration (47), while CB1 R activation increases the hepatic gene
expression of the lipogenic enzymes in mice (56) and induces long-lasting hyperthermia (64).
By 90 days after operation, in RYGB-S rats, we documented significant loss of fat mass,
(Table 4). This reduction was associated with a decrease in insulin levels leading to the
mobilization of muscle and liver substrates such as glycogen or triglycerides. In fact, we found a
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reduction in hepatic (77) and intramyocellular triglycerides (unpublished data) in RYGB rats,
which is in accordance with an increase in fat catabolism, documented by the decrease in
plasma triglycerides and cholesterol found in the present study. The reduction in caloric intake
did not affect the weight of liver, pancreas and skeletal muscle in RYGB rats but decreased the
weight of the liver in PF rats in accordance with what was reported by other investigators who
found that a 40% caloric restriction in rats diminished the weight of liver, kidneys and heart (75).
Liver and heart, along with brain and kidney, are the most metabolically active organs (31) and,
although these organs make up less than 10% of the total body weight, they account for the
majority of the basal energy expenditure (28). Therefore, drastic reduction in the weight of the
liver in PF rats could be interpreted as an attempt to reduce energy expenditure aimed at
conserving energy and avoiding a further reduction in body weight. On the contrary, RYGB
operation seems to overcome this effect of caloric restriction given that the weight of the liver in
RYGB rats showed a slight, although not statistical, increase compared to Obese and was
markedly higher than in PF rats. Furthermore, in RYGB-S rats, the weight of the heart was
higher than in Obese and PF rats. This could contribute to higher energy expenditure in RYGB
rats, particularly in RYGB-S rats, leading to a greater weight loss.
In Obese, PF and RYGB-F rats the increase in caloric intake that followed the
diminished food intake (secondary to the stress of the operation and the lower caloric intake
during liquid diet intake) was accompanied by an increased weight leading to a higher fat mass
and compared to RYGB-S rats. The process of accelerated rate of fat recovery (catch-up fat) is
a recognized risk factor for the development of adult obesity following dieting. After
semistarvation, rats refed the same amount of a low-fat diet as controls have lower energy
expenditure due to reduced thermogenesis favoring accelerated fat deposition and show normal
glucose tolerance but higher plasma insulin after a glucose load at a time when their body fat
and plasma FFA had not exceeded those of controls. Isocaloric refeeding on a high-fat diet
results in even lower energy expenditure and thermogenesis and greater increased fat
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deposition and led to even higher plasma insulin and elevated plasma glucose after a glucose
load (22). Because UCP-2 and UCP-3 increase lipid utilization (69), these proteins may confer
resistance to weight gain. Although not statistically significant in our microarray comparisons
(which used a small number of samples), we found a biological increase in UCP-2 expression in
both hypothalamus (1.5 fold) and abdominal subcutaneous fat (2.8 fold) in RYGB-S rats (data
not shown), which would explain the absence of weight regain in these rats. The higher
hypothalamic expression of UCP-2 in RYGB-S vs. RYGB-F and PF rats is relevant in the
regulation of energy homeostasis. UCP-2 is coexpressed with CRF in PVN, with vasoactive
peptide and oxytocin in the supraoptic nucleus, and with NPY in the ARC (35) neurons
expressing these proteins. Moreover, all these neurons and those expressing MCH and orexin
are targeted by axon terminals containing UCP-2, while UCP-2-producing neuronal perikarya
express receptors for leptin in the ARC (35). The mitochondria of these UCP-2-producing
neurons have increased proton leak generating heat that controls the neuronal activity in the
hypothalamic peptidergic systems involved in energy homeostasis.
An increase in SNS activity, via activation of α-2 adrenergic receptors on β-cells, may
account for the reduction in insulin levels in RYGB-S rats (48). Also, the stimulation of SNS
driven by the increased PYY contributes to their greater decrease in fat mass by promoting
lipolysis in adipocytes via activation of β3-adrenergic receptor that causes an increase in both
the expression and the activity of UCP1 and UCP2 (48). The higher UCP-2 expression in
subcutaneous fat, along with the elevated plasma PYY and hypothalamic PYY mRNA, found in
RYGB-S rats is in accordance with the enhanced activation of SNS in RYGB-S rats.
The decrease in plasma glucose levels is due to the lower caloric intake, because it also
occurs in PF rats. But the fact that the glucose levels in RYGB-S rats were lower than in PF
rats, suggests another mechanism, such as the activation of β2-adrenergic receptor in skeletal
muscle by SNS stimulation leading to an increase in glucose oxidation (5), concurring with the
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higher plasma PYY levels and the simultaneous higher hypothalamic PYY expression in RYGB-
We did not find changes in FFA probably because of the dual effect of SNS activation.
On one hand, the stimulation of the hormone-sensitive lipase leads to the breakdown of the
triglycerides (48) decreasing plasma triglyceride concentration and thus increasing FFA levels,
while on the other hand, the SNS activation also stimulates fatty acid oxidation in skeletal
muscle (5) leading to the absence of net changes in plasma FFA. Similar changes in peripheral
nutrients and hormonal have been reported by other investigators in humans and in biliary
pancreatic models of weight loss (6; 29). However, the data concerning the changes in plasma
ghrelin remain inconsistent because it may be related to the size of the gastric pouch, afferent
limb length, current BMI and the degree of weight loss and the division of some vs. all
autonomic fibers innervating the stomach and the foregut (18).
The vagus is considered a sensory rather than an effector nerve since it contains
approximately 95% afferent and 5% efferent fibers (61). Although we try to preserve the vagus
nerve in our model the possibility exists that in RYGB-F rats this nerve and its gastric fibers
were unintentionally divided or injured. Therefore, the satiety signals borne by the afferent
branch, such as CCK, which enhances the process of satiation by activating the abdominal
vagal afferent neurons that innervate the stomach and the duodenum (73) and acting
synergistically with leptin (58), would not reach the nucleus of the solitary tract and the area
postrema in the brainstem. Moreover, some of the vagal afferent fibers innervating the stomach
are also distension responsive (65). So that, if the vagal afferent pathway was divided or
seriously interrupted in our RYGB-F rats, these vagally dependent satiety signals would not
reach the brain, thus leading to the greater caloric intake observed in RYGB-F rats. The putative
loss of these vagal signals would contribute to the RYGB operation failure inducing a
comparable degree of weight reduction in RYGB-F rats as that achieved in RYGB-S rats (Figure
1A and B). If that was the case, the weight reduction in RYGB-F rats could be attributed to just a
Page 27 of 53
reduction of caloric intake. Supporting this explanation is the fact that the degree and pattern of
weight loss was the same in RYGB-F and in PF rats.
On the other hand, the stimulation of the efferent vagus promotes energy storage by
opposing the effects of SNS activity (50). In our study, vagal celiac efferent firing rate in RYGB
rats did not change after glucose gavage into the gastric pouch (Figure 4) suggesting that
RYGB operation leads to the loss of the vagal efferent signal from the brain resulting in
decreased peristalsis, nutrient absorption and partitioning of nutrients into adipose tissue, and
thus lower energy storage (43). This interpretation concurs with our previous finding showing
that RYGB delays gastric emptying and increases intestinal transit time (74). Therefore, we
postulate that in RYGB-S rats the nerve pathway coming from the dorsal motor nucleus of the
vagus nerve was divided or injured, eliminating or attenuating these energy preservative effects
of efferent vagus, while in RYGB-F rats it was preserved, explaining why they had a greater
body weight compared to RYGB-S. However, if this was the case, we should also have
observed higher insulin levels in RYGB-F rats compared to RYGB-S rats, because vagal
stimulation activate β-cells to produce more insulin.
A decrease in skeletal muscle mitochondrial mass and function is associated with the
accelerated rate of fat recovery (catch-up fat) and insulin resistance that are characteristic
features of weight recovery after caloric restriction (16). Our findings are consistent with this
fact, because in RYGB-S rats, lower caloric intake was associated with lower mitochondrial
mass than RYGB-F rats (Figure 5). The lower number of mitochondria in RYGB-S rats did not
lead to lower energy expenditure and thus to a greater fat mass. Instead, a lower fat mass was
found in RYGB-S rats, suggesting that a disruption in the fat storage process or alterations in
adipogenesis in RYGB-S rats had occurred, as supported by our finding of the higher
expression of adipose differentiation related protein in subcutaneous fat in these rats (Figure 6).
The impaired adipogenesis in RYGB-F and PF rats potentially leads to a progressive adipocyte
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hypertrophy, and an increased deposition of fat in skeletal muscle, liver, myocardium and
Could it be that successful RYGB induces changes in mitochondria morphology and
function (smaller mitochondria have lower oxidative capacity and lower ATP production (30))
and thus induces lower energy storage and anabolic capacity? Fiber composition in skeletal
muscle depends on both developmental factors and physiological cues such as patterns of
innervation, functional demands or hormonal signals (59). RYGB could induced muscle
plasticity aimed at increasing the proportion of glycolytic fibers (fast-twitch fibers), which have
lower dependency on lipids and higher capacity to shift between glucose and lipids as fuel
substrates than slow-twitch glycolytic fibers. This postulate is supported by the fact that while
fast-twitch fibers lack rich mitochondrial networks, slow-twitch fibers have the highest
mitochondrial content of any muscle type fibers and could be adapted to the depleted fat stores
in RYGB-S rats which become relatively more dependent on glucose utilization, as suggested
by our finding of significantly higher levels of mRNA 1-alpha enolase in RYGB-S, an enzyme
involved in glycolysis (Figure 6).
In skeletal muscle, UCP-2 and UCP-3 expression are up-regulated during starvation and
down-regulated during refeeding (69), emphasizing UCPs role in the regulation of lipids as fuel,
rather than just as mediators of thermogenesis (69). Thus, their suppression would account for
the increase in metabolic efficiency and for the subsequent energy conservation to overcome
the energy deficit resulting from starvation. The decrease in mitochondrial mass in RYGB-S
rats, which have a lower caloric intake and greater loss of fat mass vs. RYGB-F, which have a
higher caloric intake and body weight and therefore a lower energy deficit, occurs as a natural
response to the state of energy deficiency sensed by the RYGB-S rats. Supporting this idea is
the fact that during the body weight plateau, food efficiency is higher than during the previous
period (Figure 2). However, we can also invoke other mechanisms in RYGB-S rats overriding
the enhanced efficiency of body fat deposition resulting from the decrease in UCP-2 and UCP-3
Page 29 of 53
expression. One of these mechanisms would be an increase in the expression and/or activity of
UCP-1 in BAT dissipating energy as heat (7) and resulting from enhanced sympathetic neural
modulation, associated with the higher plasma PYY, and the increased hypothalamic
expression of PYY, CRF receptor-2, OX2R and orexin. The reduction in fat mass in RYGB-S
results in a higher ratio of surface area to volume and reduced insulation leading to higher
Although we expected an increase in mitochondrial mass in RYGB-S vs. RYGB-F rats,
the discrepancy may be due to an increase in the expression and/or activity of UCP-2 and UCP-
3 in skeletal muscle, whose magnitude was sufficiently large to overcome the decrease in
mitochondrial mass. The increase in the expression of UCP-2 in subcutaneous fat (2.8-fold) and
hypothalamus (1.5-fold) in RYGB-S vs. RYGB-F rats (data not shown) would support this
argument and would result in increased mobilization of lipids and their utilization (69) accounting
for the lower fat mass in the RYGB-S rats.
Finally, to determine if the success or failure of the RYGB operation was not due to
different genetic back ground in the Sprague-Dawley rats, we screened for the possibility of
genetic admixture effects in these random bred Sprague-Dawley rats. Because the lengths of
the STRs from the RYGB-S and RYGB-F rats were all in the expected ranges for Sprague-
Dawley-derived rat strains (except for the D1RAT193 STR of one RYGB-F rat but not the other
rats; data not shown), these results indicate that it is unlikely that failure of RYGB was due to
genetic background differences. However, evidence for heterogeneity was found at least two
different STRs at five of the eight loci (D1RAT193, D1MIT32, D2RAT88, D3MGH16 and
D3MIT13). The sum of these data indicate that there was considerable variation between rats of
even the same treatment group, and strongly suggest that the demonstrated failure of RYGB in
some rats was due to common physiological or epigenetic adaptations associated with the
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The present findings provide evidence that success of RYGB inducing a sustained
weight loss engages many different systems and physiological processes that effectively
oppose the compensatory processes to weight loss (starvation response). Specifically, this
success is achieved not only by a RYGB-induced decreased caloric intake and increased fecal
output, but also via an activation of the SNS, driven by increased PYY, CRF and orexin
signaling, decreasing food efficiency and energy storage, demonstrated by reduced fat mass
associated with the up-regulation of mitochondrial UCP-2 in fat. These events override the
compensatory response to the drop in leptin levels aimed at conserving energy, which is more
robust in those rats that failed RYGB. Also our data suggest an important role of the vagus in
the outcome of RYGB surgery, whose contribution to the weight loss in this procedure has not
been adequately explored.
Page 31 of 53
We thank Andrzej Krol PhD, Robert Ploutz-Snyder PhD for their scientific advice and Karen
Hughes AAS and Karen Gentile M.P.S. for their technical assistance.
Michael M. Meguid is supported, in part, by the American Society for Bariatric Surgery,
American Diabetes Association and a SUNY Upstate Intramural Fund grant. Paul S Brookes is
funded by NIH-HL071158.
Page 32 of 53
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Figure 1. Body weight (BW) pattern (Panel A) and caloric intake (Panel B) in Obese, RYGB-S,
RYGB-F and PF rats from operation day (Day 0) to 90 days after surgery. Data are represented
as mean ± S.E.M.
Figure 2. Food efficiency from Days 0 to 8 (Panel A), from day 9 to change point day (Panel B)
and from change point day to postoperative day 90 (Panel C). Data are expressed as the mean
± S.E.M. * p < 0.05 and *** p < 0.005 compared to Obese; # p < 0.05 compared to RYGB rats.
Figure 3. D-xylose (Panel A) and fat (Panel B) absorption in Obese, RYGB-S and RYGB-F rats
at postoperative day 30, 60 and 90. Data are expressed as mean ± S.E.M. *** p < 0.005
compared to Obese.
Figure 4. Vagal nerve firing rates of gastric pouch. Representative tracings of UGI vagal
efferent firing rate before and after a 5% glucose gavage in one Obese rat (Panel A, top) and
one RYGB rat (Panel B, top) and their corresponding quantitative representation (lower graphs
on Panels A and B). Panel C shows the mean values of Obese and RYGB rats. Data are
represented as mean ± S.E.M. * p < 0.05 compared to Obese.
Figure 5. Mitochondrial respiratory complexes content in skeletal muscle. Representative
picture of mitochondrial respiratory complexes separated by blue-native gel electrophoresis
(Panel A) and quantitative analysis of mitochondrial respiratory complexes content (Panel B).
Data are represented as mean ± S.E.M. # p < 0.05 compared to RYGB-S rats.
Figure 6. Expression changes of selected feeding/metabolic-related genes in the hypothalamus
and subcutaneous abdominal fat of RYGB-S, RYGB-F and PF rats. Data are represented as
mean ± S.E.M. # p < 0.05 compared to RYGB-S rats.
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Table 1. Mean body weight (BW, g) and mean change in daily BW (Mean ∆ daily BW; g/day)
during 90 postoperative days in Obese, RYGB-S, RYGB-F and PF rats.
Data are mean ± S.E.M. *** p < 0.005 compared to Obese; # p < 0.05, ### p < 0.005 compared
to RYGB-S rats. CPD: Change point day.
Group CPD Parameter Day 0 to 8 Day 9 to CPD CPD to Day 90
Obese 9 Mean BW
Mean daily BW ∆
+1.1 ± 0.2
22 Mean BW
Mean daily BW ∆
RYGB-F 24 Mean BW
Mean daily BW ∆
PF 23 Mean BW
Mean daily BW ∆
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Table 2. Mean caloric intake (CI; Kcal/day) and mean change in daily caloric intake (Mean ∆
daily CI; Kcal/day) during 90 postoperative days in Obese, RYGB-S, RYGB-F and PF rats.
Group CPD Parameter Day 9 to CPD CPD to Day 90
Obese 25 Mean CI
Mean daily CI ∆
RYGB-S 34 Mean CI
Mean daily CI ∆
RYGB-F 30 Mean CI
Mean daily CI ∆
PF 34 Mean CI
Mean daily CI ∆
Data are mean ± S.E.M. ** p < 0.01, *** p < 0.005 compared to Obese; ### p < 0.005,
compared to RYGB-S rats; $$$ p < 0.005 compared to RYGB-F rats. CPD: Change point day.
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Table 3. Percent of Fecal weight/1g food intake during 90 postoperative days in Obese, RYGB-
S, RYGB-F and PF rats.
Group CPD Day 9 to CPD CPD to day 90
Data are mean ± S.E.M. *** p < 0.005 compared to Obese ; ### p < 0.005, compared to RYGB-
S rats; $$$ p < 0.005 compared to RYGB-F rats. CPD: Change point day.
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Table 4. Body composition (fat mass, fat free mass and organ weights expressed as a percentage of
body weight) and plasma nutrient and hormone concentrations at day 90 after either sham operation
(Obese and PF groups) or RYGB-S and RYGB-F rats.
Parameter Obese RYGB-S RYGB-F PF
Fat mass (%) 12.5 ± 0.8 3.6 ± 0.4*** 6.6 ± 1.4*** 9.1 ± 0.7**###
Fat free mass (%) 87.5 ± 0.8 96.4 ± 0.4*** 93.4 ± 1.4*** 90.9 ± 0.7**###
Liver weight (%) 2.71 ± 0.06 2.96 ± 0.13 3.13 ± 0.23 2.18 ± 0.13***###$$$
Pancreas weight (%) 0.30 ± 0.01 0.40 ± 0.01 0.32 ± 0.07 0.35 ± 0.03#
Heart weight (%) 0.21 ± 0.00 0.31 ± 0.03 0.26 ± 0.02 0.24 ± 0.01
Muscle weight (%) 0.49 ± 0.03 0.55 ± 0.04 0.54 ± 0.04 0.53 ± 0.03
Glucose (mg/dl) 176.7 ± 7.6 124.9 ± 5.2*** 126.5 ± 7.0*** 149.0 ± 3.2*#
Total Cholesterol (mg/dl) 103.7 ± 8.9 35.87 ± 8.3*** 51.5 ± 24.3 64.9 ± 11.8
Triglycerides (mg/dl) 160.0 ± 21.8 60.3 ± 11.2*** 61.8 ± 20.3* 68.5 ± 9.2**
FFA (mg/dl) 0.5 ± 0.1 0.3 ± 0.1 0.3 ± 0.0 0.4 ± 0.1
Total Ghrelin (ng/ml) 1.7 ± 0.3 3.0 ± 0.5 2.8 ± 0.4 3.1 ± 0.5
Acylated Ghrelin (fmol/ml) 12.3 ± 1.9 9.5 ± 2.5 6.8 ± 1.7 12.8 ± 3.4
PYY (pg/ml) 25.9 ± 3.1 95.1 ± 29.3*** 40.5 ± 0.0 35.2 ± 4.4#
CCK (ng/ml) 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.2 ± 0.0
Insulin (µg/L) 1.8 ± 0.2 0.5 ± 0.1*** 1.3 ± 0.7 0.5 ± 0.1***
Leptin (µg/ml) 11.0 ± 0.9 0.7 ± 0.1*** 4.0 ± 1.8*** 6.5 ± 1.6*###
Free T3 (pg/ml) 4.6 ± 0.2 3.3 ± 0.2*** 5.9 ± 0.7### 4.9 ± 0.3###
Data are expressed as mean ± S.E.M. * p < 0.05,** p < 0.01 and *** p < 0.005 compared to
Obese; # p < 0.05, ### p < 0.005 compared to RYGB-S rats, $$$ p < 0.005 compared to
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Table 5. Feeding/metabolic-related genes showing differential expression in RYGB-S, RYGB-F,
or PF rats.
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Figure 1. Body weight (BW) pattern (Panel A) and caloric intake (Panel B) in Obese
controls, RYGB-S, RYGB-F and PF rats from operation day (Day 0) to 90 days after
surgery. Data are represented as mean
160x192mm (300 x 300 DPI)
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Figure 2. D-xylose (Panel A) and fat (Panel B) absorption in Obese, RYGB-S and RYGB-F
rats at postoperative day 30, 60 and 90. Data are expressed as mean ÷ S.E.M. *** p <
0.005 compared to Obese.
160x193mm (300 x 300 DPI)
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Figure 3. Food efficiency from Days 0 to 8 (Panel A), from day 9 to change point day Download full-text
(Panel B) and from change point day to postoperative day 90 (Panel C). Data are
expressed as the mean ± S.E.M. * p < 0.05 and *** p < 0.005 compared to Obese; # p <
0.05 compared to RYGB rats.
141x199mm (300 x 300 DPI)
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