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Original Research Communications
Diet-induced weight loss reduces colorectal inflammation: implications
for colorectal carcinogenesis
1–3
Swaroop Pendyala, Lisa M Neff, Mayte Sua
´rez-Farin
˜as, and Peter R Holt
ABSTRACT
Background: Epidemiologic data have shown that obesity indepen-
dently increases colorectal cancer (CRC) risk, but the mechanisms
are poorly understood. Obesity is an inflammatory state, and chronic
colonic inflammation induces CRC.
Objective: We conducted this proof-of-principle study to seek ev-
idence of obesity-associated colorectal inflammation and to evaluate
effects of diet-induced weight loss.
Design: We measured inflammatory cytokines, gene arrays, and mac-
rophage infiltration in rectosigmoid mucosal biopsies of 10 obese pre-
menopausal women [mean 6SD body mass index (in kg/m
2
): 35 6
3.5] before and after weight loss induced by a very-low-calorie diet.
Results: Subjects lost a mean (6SD) of 10.1 61% of their initial
weight. Weight loss significantly reduced fasting blood glucose,
total cholesterol, triglycerides, LDL, tumor necrosis factor-a
(TNF-a), and interleukin (IL)-8 concentrations (P,0.05). After
weight loss, rectosigmoid biopsies showed a 25–57% reduction in
TNF-a, IL-1b, IL-8, and monocyte chemotactic protein 1 concen-
trations (P,0.05). T cell and macrophage counts decreased by
28% and 42%, respectively (P,0.05). Gene arrays showed dra-
matic down-regulation of proinflammatory cytokine and chemokine
pathways, prostaglandin metabolism, and the transcription factors
STAT3 (signal transducer and activator of transcription 3) and nu-
clear transcription factor jB. Weight loss reduced expression of
FOS and JUN genes and down-regulated oxidative stress pathways
and the transcription factors ATF (activating transcription factor)
and CREB (cyclic AMP response element-binding).
Conclusions: Our data show that diet-induced weight loss in obese
individuals reduces colorectal inflammation and greatly modulates in-
flammatory and cancer-related gene pathways. These data imply that
obesity is accompanied by inflammation in the colorectal mucosa and
that diet-induced weight loss reduces this inflammatory state and may
thereby lower CRC risk. Am J Clin Nutr 2011;93:234–42.
INTRODUCTION
Epidemiologic evidence suggests obesity as an independent
risk factor for the development of several cancers, including
colorectal cancer (CRC) (1). Every 5-unit increase in body mass
index (BMI) increases risk of CRC by 30% in men and by 13% in
women (2). The prevalence of obesity has increased dramatically
in adult Americans from 14% in the 1970s to the current level of
34% (3–5). The increased prevalence of obesity indicates that the
incidence of CRC and other forms of cancer will become
a greater public health problem in the future.
In mouse models of genetic and chemically induced colitis,
obesity enhanced the development of colorectal neoplasia (6, 7).
Western-style diet consumption in mice caused obesity accom-
panied by increased oxidative stress, inflammation in the colon,
and development of colon tumors without genetic or chemical
manipulation (8, 9). Consistent are findings that caloric re-
striction protected mice from developing aberrant crypt foci and
colonic neoplasia (10) and decreased expression of colonic
cyclooxygenase-2 (COX-2) in the colon (11). In the Iowa
Women’s Health Study, an intentional weight loss of .20
pounds reduced colon cancer incidence by 9% and obesity-
related cancer mortality by 40% (12).
Several factors have been proposed to mediate the relation
between obesity and increased CRC risk. Obesity is associated
with elevated concentrations of insulin-like growth factor (IGF)-1,
which is a known stimulator of epithelial growth (13). Increased
plasma concentrations of inflammatory cytokines including tu-
mor necrosis factor-a(TNF-a) and interleukin (IL)-6 occur in
obesity and are elevated in subjects with colorectal adenomas
(14). Furthermore, antiinflammatory drugs, such as aspirin and
celecoxib, reduce colorectal adenoma incidence (15, 16). Sub-
group analyses in one study revealed aspirin to have a greater
protective effect in obese subjects than in lean subjects (17).
Obesity is associated with chronic low-grade inflammation in
the adipose tissue, liver, and coronary endothelium and is ac-
companied by increased oxidative stress (18), which through
intermediary molecules, increase the expression of the proto-
1
From the Clinical and Translational Science Center, The Rockefeller
University, New York, NY (SP, LMN, MS-F, and PRH), and St Luke’s
Roosevelt Hospital Center, New York, NY (SP).
2
Supported by a Clinical and Translational Science Award (grant
UL1RR024143) to the Rockefeller University from the National Center
for Research Resources, a component of the NIH, and an NIH Roadmap
for Medical Research.
3
Address correspondence to PR Holt, The Rockefeller University, Box
179, 1230 York Avenue, New York, NY 10065. E-mail: holtp@rockefeller.
edu.
Received August 23, 2010. Accepted for publication November 9, 2010.
First published online December 8, 2010; doi: 10.3945/ajcn.110.002683.
234 Am J Clin Nutr 2011;93:234–42. Printed in USA. Ó2011 American Society for Nutrition
oncogenes FOS and JUN that leads to increased transcription of
proinflammatory and cell-cycle regulatory genes that promote
carcinogenesis (19). Chronic inflammation can enhance the
initiation and progression of CRC (20).
Therefore, we hypothesized that obese humans would show
signs of chronic inflammation in their colorectal mucosa. To test
this hypothesis, obese women were studied before and after
a 10% weight loss induced by a very-low-calorie diet (VLCD),
which reduced rectosigmoid mucosal inflammatory cytokine
concentrations and inflammatory cell infiltration accompanied
by a decreased expression of gene pathways involved in in-
flammation and cancer, including gene sets regulated by TNF-a,
IL-6 and IL-17, prostaglandins, and oxidative stress. Weight loss
also decreased the expression of gene sets regulated by tran-
scription factors involved in colorectal inflammation and CRC,
such as signal transducer and activator of transcription 3
(STAT3), nuclear transcription factor jB(NF-jB), activating
transcription factor (ATF), and cyclic AMP response element-
binding (CREB).
SUBJECTS AND METHODS
Subjects
For this unblinded study, 10 healthy, obese [average (6SD)
BMI (in kg/m
2
): 34.8 63.5], premenopausal women (mean age:
43.2 68.3 y) were recruited from the community through ad-
vertisements. Study volunteers included 6 whites, 1 Hispanic, 2
African Americans, and 1 subject with a mixed racial back-
ground (Table 1). All subjects underwent a complete medical
examination, standard blood and urine tests, and an electrocar-
diogram. Subjects were weight stable (,2% change) for 6mo
before the study. Exclusion criteria included a history of cancer,
current treatment of weight loss, previous intestinal surgery,
a history suggestive of malabsorption, major medical problems,
consumption of medications with antiinflammatory properties
(eg, nonsteroidal antiinflammatory drugs, statins, glitazones, and
COX-2 inhibitors) or medications that could increase risk of
complications associated with consuming a VLCD (eg, lithium),
an allergy to soy products or aspartame, or contraindications to
undergoing a sigmoidoscopy and biopsy, including any history
of excessive bleeding. The study was approved by the In-
stitutional Review Board of The Rockefeller University (New
York, NY). Subjects were recruited between September 2007
and May 2008. Written informed consent was obtained from
each subject before participation in the study.
Experimental design
Seven subjects chose to complete the weight-loss phase of the
study in The Rockefeller University Hospital inpatient unit, and 3
subjects were closely monitored as outpatients. After screening,
subjects underwent baseline testing, which included fasting blood
determinations, urine analysis, and flexible sigmoidoscopy with
multiple mucosal biopsies. Subjects started a VLCD weight-loss
program and were monitored weekly by the study physicians. The
VLCD was continued until subjects had lost 8% of baseline
weight, at which time the initial measurements were repeated.
This degree of weight loss was chosen because a previous study
showed that this would lower rectosigmoid proliferation in
obese subjects by ’40% (21). Subjects were counseled that the
study required them to maintain their usual physical activity, and
this was emphasized throughout the study.
Subjects consumed a reduced-calorie, Western-style diet that
contained 50% of their calculated baseline caloric intake for 3 d
before initiation of the VLCD. Subjects were fed a commercially
available VLCD product (New Direction Program; Robard
Corporation, Mount Laurel, NJ) that provided ’782 kcal/d
with an energy distribution of carbohydrate, fat, and protein of
25%, 25%, and 50%, respectively, and daily requirements of
vitamins and minerals (Table 2). This VLCD provided a choice
of shakes, soups, bars, and pudding in different flavors; subjects
had 4 choices/d, consumed one item every 4 h, and drank 2L
water daily. Inpatients were monitored daily by the hospital
staff, and outpatients were monitored weekly during clinic vis-
its. Weekly blood tests for electrolytes and renal function tests
were performed to monitor for complications that can occur with
VLCD consumption.
Procedures and sample collection
Fasting electrolytes, liver function tests, renal function tests,
lipid profiles, and high-sensitivity C-reactive protein measure-
ments were performed by the clinical chemistry laboratory at
TABLE 1
Baseline characteristics of the 10 obese volunteers
1
Subject Age Weight Height BMI Race
y kg cm kg/m
2
1 49 74.8 158 30.2 H
2 43 104 159 41.3 W
3 39 92 171 31.4 W
4 49 95 158 38.3 M
5 52 97 172 32.8 AA
6 24 93 167.5 33.0 W
7 49 120 178 37.9 AA
8 37 98 170 34.0 W
9 47 83 158 33.4 W
10 43 103 170 35.5 W
Combined 43 682
2
96 612 166 67 34.8 64 6 W, 2 AA, 1 H, and 1 M
1
H, Hispanic; W, white; M, mixed; AA, African American.
2
Mean 6SD (all such values).
WEIGHT LOSS REDUCES COLORECTAL INFLAMMATION 235
Memorial Sloan-Kettering Cancer Center. Aliquots of serum
samples for cytokine measurements were stored at 280°C. Flexible
proctosigmoidoscopy was performed between 0800 and 0900 after
a 60-mL tap-water enema, and 12–14 rectosigmoid biopsies were
obtained from 4 quadrants between 10 and 20 cm from the anal
verge. Four biopsies for mucosal cytokine measurements were
immediately frozen in liquid nitrogen; 6 biopsies for gene ex-
pression analysis were immersed in RNAlater (catalog no. 7024;
Ambion) at 4°C for 24 h and frozen in liquid nitrogen. The re-
maining biopsies were frozen in optimum cutting temperature
compound (OTC; International Medical Equipment, Chico, CA)
and stored at 280°C for immunohistochemical analysis.
Sample analyses
Serum cytokines
Serum cytokines were measured in duplicate with the Human
Proinflammatory 9-Plex MULTI-SPOT 96-well plate kit (catalog
no. N05007A-1; Mesoscale Diagnostics, Gaithersburg, MD) (22)
for IL-2, IL-8, IL-12p70, IL-1b, IL-6, and IL-10, TNF-a, and
interferon-cwith a intraassay correlation 10% and interassay
variance 10%.With the use of this multiplex assay, only IL-6
and -8 and TNF-awere detectable with a CV 10% and re-
covery rate 80%. Monocyte chemotactic protein (MCP)-1 was
measured with a human Elisa kit (catalog no. DCP00; R&D
Systems, Minneapolis, MN) with an intraassay correlation 8%
and interassay variance 7%.
Mucosal cytokines
Two frozen mucosal biopsies were homogenized in RIPA
buffer [25 mmol tris HCl/L (pH 7.6), 150 mmol NaCl/L, 1% NP-
40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate;
catalog no. 89900; Pierce, Rockport, IL] with protease and
phosphatase inhibitors at 4°C (catalog nos. P8340 and P5726;
Sigma-Aldrich, Saint Louis, MO). The homogenate was
centrifuged at 15,000 rpm for 20 min at 4°C, and the supernatant
fluid was collected and stored at 280°C until further analysis.
Mucosal cytokines were measured in duplicate with the Proin-
flammatory 9-Plex MULTI-SPOT 96-well plate kit (catalog no.
N05007A-1; Mesoscale Diagnostics) as described for serum
cytokines. Only mucosal IL-6, IL-8, IL-1b, and TNF-awere
detectable with a CV 10% and recovery rate 80%. MCP-1
concentrations were measured as previously described. Mucosal
protein concentrations were measured with the Pierce BCA as-
say kit (catalog no. 23225; Thermo Fischer, Waltham, MA) with
Nanodrop technology (NanoDrop Technologies, Wilmington,
DE). Mucosal cytokine concentrations are presented as pico-
grams of each cytokine per milligram of mucosal protein.
Samples with cytokine concentrations 2.5 SD from the mean
were considered outliers and excluded from the final analysis.
Immunohistochemistry
Six-micron sections were cut at 220°C onto plus slides
(Fisher-Scientific, Waltham, MA) and stored at 280°C until as-
say. For immunohistochemical analyses, sections were quenched
in ice-cold acetone and rehydrated in phosphate buffered saline
(PBS). Sections were treated with purified mouse anti-human
antibodies to cluster of differentiation (CD) 3 (T cell marker) at
a 1:200 dilution (catalog no. 34740; BD Pharminogen, Franklin
Lakes, NJ) and to CD163 (macrophage marker) at a 1:500 di-
lution (catalog no. BM4041; Novus Biologics, Littleton, CO).
The secondary antibody, a biotin-labeled horse anti-mouse anti-
body at a 1:200 dilution was applied and followed by amplifi-
cation with an avidin-biotin complex (1:100 of avidin and 1:100
of biotin; catalog no. 6102; Vector Laboratories, Burlingame,
CA), and the signal was developed with chromogen-3-amino-9-
ethylcarbazole (Sigma-Aldrich). The area of the sections and
number of positive cells contained were counted manually with
the National Institutes of Health Image J software (NIH IMAGE J
1.42; National Institutes of Health, Bethesda, MD) and reported
as the number of positive cells per square millimeter. For each
subject before and after the weight-loss regimen, all cells were
counted in 3 separate sections .100 lm apart at a 10·magni-
fication. Each section was analyzed in a random fashion by
a single person blinded to their identity. A subset of sections were
analyzed 3 times in a similar fashion, and the CV was ,10%.
Mucosal morphology was assessed by hematoxylin (Fisher
Scientific, Waltham, MA) and eosin (Shandon, Pittsburg, PA)
staining of sections by using a standard protocol. Data from only
8 subjects are presented for CD163 staining because the staining
quality was inadequate for analysis in 2 subjects.
Gene expression
Total RNA was extracted from 2 rectosigmoid biopsy speci-
mens by using the Trizol method (Invitrogen, Carlsbad, CA).
RNA purification, quality assessment, and hybridization were
performed as previously described (23). Biotin-labeled com-
plementary RNA was hybridized onto Human HT-12v3 Ilumina
expression chips (Illumina Inc, San Diego, CA). Samples from
baseline and post–weight loss for each subject were analyzed on
the same chip. The arrays were scanned by the Illumina Bead-
Station laser scanning imaging system (Illumina).
For determinations of real-time polymerase chain reactions
(PCRs), 2 lg total RNA was used as a template for comple-
mentary DNA synthesis with a SuperScript-III First-Strand kit
(Invitrogen). Complementary DNA was diluted in water, and
amounts corresponding to 50 ng original RNA were used for
quantitative gene expression by real-time PCR. SYBR Green
PCR master mix (Applied Biosystems, Foster City, CA), primers
from Sigma-Aldrich, and the ABI Prism7900 real-time PCR
system (Applied Biosystems, Carlsbad, CA) were used for the
real-time PCR. Six down-regulated and 4 up-regulated genes
were assayed, which were normalized to the housekeeping gene
GAPDH to adjust for the sample loading and efficiency of the
reaction. Real-time PCR samples were assessed in duplicate,
and messenger RNA concentrations were expressed as the log
TABLE 2
Composition of the very-low-calorie diet
1
Nutrient Daily intake Percentage of energy
Energy (kcal) 782 100
Carbohydrates (g) 48 25
Fat (g) 23 26
Protein (g) 96 49
1
The diet was fortified with vitamins and minerals and provided
.100% of the Recommended Dietary Allowance on the basis of a
2000-kcal/d diet.
236 PENDYALA ET AL
ratio of relative expression concentrations before and after treat-
ment (see supplemental Table 1 under “Supplemental data” in the
online issue for a list of real-time PCRs).
Analysis of the gene-expression microarray data
Gene-expression data from the microarrays were analyzed
with the R software (version 2.1) and available packages from
Bioconductor (http://www.bioconductor.org). Data were nor-
malized by using robust spline normalization functions from the
lumi package (Bioconductor). A classical quality control was
performed. Expression concentrations with detection Pvalues
0.05 were considered detectable. Probes with detectable ex-
pression in at least one sample and with SD .0.1 were used in
further analysis. Expression values (log
2
transformed) were
modeled with the package limma (Bioconductor). Moderated
paired ttests were used to test the significance of the effect of
weight loss. Pvalues were adjusted for multiple comparisons by
using the Benjamini-Hochberg procedure. Genes with fold-
changes 1.2 and P0.05 were considered differentially ex-
pressed. Heat maps were generated by hierarchical clustering by
using one Pearson ras the distance measure and complete
linkage as the agglomeration procedure. The gene-expression
data were deposited in the public domain (transcription profiling
accession no. GSE20931).
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) was conducted with the
GSEA desktop application (http://www.broadinstitute.org/gsea)
(24). Our analysis of gene expression before and after weight loss
in each subject was performed in a paired fashion, and genes were
ranked by their effect size (ie, the mean fold change for each
subject). This rank-order list was used as the input to the GSEA.
For each gene set, the enrichment score (ES) was calculated by
using weighted Kolmogorov-Smirnov statistics to measure the
proximity of the gene set to the top of the weight-loss effect
ranked list. A high and positive ES indicated that the gene set or
pathway was collectively up-regulated by the weight-loss in-
tervention, whereas a negative ES indicated down-regulation.
The significance (Pvalue) of the observed ES was calculated by
using simulations. Normalized ESs were used to compare
analysis results across gene sets. The molecular signature da-
tabase available at the Broad Institute, MIT (http://www.
broadinstitute.org/gsea/msigdb), and gene sets created at the
Rockefeller University from the data generated by IL-17 stim-
ulation of keratinocytes (25, 26) were used to query the data. We
used sets from 3 different collections from the GSEA website
[ie, the curated canonical pathway database (C2), the Gene
Ontology database (C5), and the transcription factor database
(C3)]. Gene sets that were positively or negatively enriched with
P,0.05 and a false discovery rate (FDR) ,5% were consid-
ered significant, except for the transcription factor gene sets for
which an FDR ,10% was considered significant.
Statistical analyses
Two-tailed paired ttests were used to compare anthropo-
metric measurements, biochemical variables, serum and muco-
sal cytokine concentrations, and CD3 and CD163 cell counts at
baseline and after weight loss. P0.05 was considered sig-
nificant. S-Plus (S+) version 8.1 for Windows software package
(TIBCO Software Inc, Somerville, MA) was used for statistical
analyses.
RESULTS
Anthropometric and biochemical measurements
All 10 subjects successfully completed the study without
significant adverse events in a mean of 46.5 69.3 d. The VLCD
resulted in a mean (6SD) decrease in body weight of 10.1 61%
(P,0.01), decreased BMI of 10.2 61.4% (P,0.01), and
reduction in waist circumference of 8.3 64%, (P,0.01). The
weight-loss regimen also induced improvements in systolic and
diastolic blood pressures, fasting blood sugar concentrations,
total and LDL serum cholesterol and triglyceride concentrations,
and total white blood cell counts (Table 3). Serum electrolyte
concentrations, liver function tests, and renal function tests were
not significantly altered.
Serum and mucosal cytokines
Diet-induced weight loss resulted in mean reductions in serum
TNF-aconcentrations of 15% and in IL-8 concentrations of 30%
TABLE 3
Effect of diet-induced weight loss on anthropometric and biochemical measurements
1
Measurement Baseline After weight loss Percentage change P
Weight (kg) 96 612 86 611 10 61.4 ,0.01
BMI (kg/m
2
)3563.5 31 631061.4 ,0.01
Waist circumference (cm) 107 611 97 612 8.3 64.4 ,0.01
Systolic blood pressure (mm Hg) 121 68 107 612 11.6 67.7 ,0.01
Diastolic blood pressure (mm Hg) 79 6470661169.3 ,0.01
Fasting blood glucose (mg/dL) 95 610 85 66 9.7 613.2 0.03
Total cholesterol (mg/dL) 187 636 157 626 15 68.8 ,0.01
LDL (mg/dL) 110 628 94 619 13 611 0.01
Triglycerides (mg/dL) 122 645 93 636 22 618 ,0.01
HDL (mg/dL) 52 69456612614 0.02
hs-CRP (mg/dL) 0.8 60.5 0.9 60.7 1.4 60.5 0.85
WBC (1000/mm
3
) 6.7 61.3 5.6 61.4 15.2 618.1 0.02
1
All values are mean 6SDs; n= 10. hs-CRP, high-sensitivity C-reactive protein; WBC, peripheral white blood cell
count.
WEIGHT LOSS REDUCES COLORECTAL INFLAMMATION 237
(both P,0.05). There was also a trend toward decreased serum
contents by 9% (P= 0.097) (Figure 1) but no change in con-
centrations of IL-6 and high-sensitivity C-reactive protein.
Baseline serum MCP-1 concentrations were similar to those
previously reported in obese individuals (27). In the rec-
tosigmoid mucosa, concentrations of mucosal TNF-adecreased
by 57% (P,0.05), concentrations of IL-8 decreased by 44%
(P,0.05), concentrations of IL-1bdecreased by 38% (P,0.05),
and concentrations of MCP-1 decreased by 25% (P,0.05), and
there was a trend toward decreased IL-6 concentrations by 45%
(P= 0.06) (Figure 2). Data from one subject were excluded
from the analysis because concentrations of all cytokines from
her biopsies were .3 SDs from the mean of all subjects.
Immunohistochemistry
Standard hematoxylin and eosin staining of mucosal biopsies
at the onset and end of the study showed no identifiable changes
in total cellular content or mucosal morphology (data not shown).
However, immunohistochemical staining showed a 28% decrease
in CD3-positive T lymphocytes (P,0.01) (n= 10) and a 42%
decrease in CD163-positive macrophages (P,0.05) (n=8)
(Figure 3) at the end of the study.
Microarray analyses
Microarray analyses revealed that diet-induced weight loss
was accompanied by 1211 differentially expressed genes (with
P0.05) of which 70 genes (56 down-regulated and 14 up-
regulated genes) had a fold-change 1.2. This differential pattern
was strikingly shown in the heat map in Figure 4 (see supple-
mental Table 2 under “Supplemental data” in the online issue for
the differential pattern shown in each gene). Genes involved in
inflammation and cancer were down-regulated, including many
genes associated with cytokines and chemokine signaling such
as IL-8,CCL-21,CCL-19, and CCL4L1, as well as the proto-
oncogenes JUN and FOS. Genes coding for molecules for which
the circulating concentrations decreased with weight loss also
were down-regulated, including peptide YY (PYY), vasoactive
intestinal peptide (VIP), and somatostatin (SST).Genes that
coded for gastrointestinal substrate transport, including ATP-
binding cassette, subfamily A members 8 and 9 (ABCA8 and
ABCA9), and carnitine palmitoyltransferase-I (CPT-1A) were
up-regulated. Six down-regulated and 4 up-regulated genes were
identified from the differentially expressed gene list for valida-
tion by real-time PCR, and gene-fold changes confirmed the
microarray data findings (see supplemental Table 3 under
“Supplemental data” in the online issue).
FIGURE 1. Mean (6SD) changes (pg/mL) in serum cytokine and
chemokine concentrations in 10 subjects before and after diet-induced
weight loss. *P,0.05,
#
P,0.1. TNF, tumor necrosis factor; IL,
interleukin; MCP, monocyte chemotactic protein.
FIGURE 2. Mean (6SD) changes (pg/mg protein) in mucosal cytokines and chemokines in 9 subjects before and after diet-induced weight loss. *P,0.05,
#
P,0.1. TNF, tumor necrosis factor; IL, interleukin; MCP, monocyte chemotactic protein.
238 PENDYALA ET AL
Detecting changes in gene pathways by altered expression of
single genes relies on relatively large changes and may be in-
sensitive to many physiologically significant changes of lesser
magnitude. A more sensitive approach has been GSEA, which
examines many genes in a pathway for changes that may not, by
themselves, be significant but are significant in the aggregate
(28). In our analyses, we used gene sets curated in the canonical
pathway database, in the Gene Ontology database, IL-17 pathway
gene sets, and the transcription factor database from the GSEA
website. In the canonical pathway database, 74 gene sets were
down-regulated, and 24 gene sets were up-regulated (see sup-
plemental Tables 4 and 5 under “Supplemental data” in the
online issue); in the Gene Ontology database, 55 gene sets were
down-regulated, and none were up-regulated (see supplemental
Table 6 under “Supplemental data” in the online issue); and in
the IL-17 gene sets, 6 gene sets were down-regulated and 2 gene
sets were up-regulated (see supplemental Table 7 under “Sup-
plemental data” in the online issue). Gene sets related to in-
flammation and cancer that were down-regulated are highlighted
in Table 4 and include pathways activated by TNF-a, IL-6, IL-1,
IL-17, chemokine and cytokine signaling pathways, prostaglandin
synthesis pathways, and pathways involved in oxidative stress,
glutathione metabolism, and apoptosis. Up-regulated mucosal
gene pathways were involved in carbohydrate metabolism (see
supplemental Table 5 under “Supplemental data” in the online
issue). The reduction in oxidative stress pathways was supported
by down-regulation of downstream gene targets FOS and JUN.
Importantly, the analysis of changes in transcription factor
genes identified 38 down-regulated and 9 up-regulated tran-
scription factor gene sets (see supplemental Tables 8 and 9 under
“Supplemental data” in the online issue). These gene sets in-
cluded down-regulated transcription factor gene sets for STAT3,
FIGURE 3. Mean (6SD) changes (number of cells/mm
2
)inmucosalT
cells and macrophages before and after diet-induced weight loss. A: CD3-
positive T cells (n= 10). B: CD163-positive macrophages (n=8).*P,0.05.
FIGURE 4. Heat map of differentially expressed genes in rectosigmoid biopsies taken at baseline and after diet-induced weight loss in 10 individual
subjects. The red color indicates a higher expression, and the green color indicates a lower expression.
WEIGHT LOSS REDUCES COLORECTAL INFLAMMATION 239
STAT5A,STAT5B,NF-jB,ATF,CREB, and multiple serum re-
sponse factors, which are recognized as very important in co-
lorectal inflammation, proliferation, apoptosis, and oncogenesis,
which are shown in Table 5.
DISCUSSION
Epidemiologic studies indicated that obesity is accompanied
by an increased risk of CRC, and intentional weight loss reduced
this risk (2, 12). To explore potential mechanisms, we studied
effects of diet-induced weight loss on the colorectal mucosa in
obese women. A VLCD was used to induce a 10% weight loss,
which resulted in reduced rectosigmoid mucosal cytokine and
chemokine concentrations and fewer tissue macrophages and T
cells. Gene-expression analysis showed down-regulation of
multiple genes involved in inflammation and cancer, including
genes involved in cytokine and chemokine signaling and the
proto-oncogenes JUN and FOS. The gene-pathway analysis
showed down-regulation of pathways activated by TNF-a, IL-6,
IL-1, and IL-17 and pathways involved in chemokine and cy-
tokine signaling, prostaglandin synthesis, oxidative stress, and
glutathione metabolism. In addition, transcription factor path-
ways for STAT3,STAT5,NF-jB,ATF,CREB, and several serum
response factors were down-regulated. These results strongly
suggested that chronic inflammation was present in the colo-
rectal mucosa of obese individuals and that diet-induced weight
loss decreased inflammation and down-regulated inflammatory
and cancer gene pathways.
Obesity is accompanied by elevated circulating concentra-
tions of insulin and IGF-1, and changes in the insulin-IGF axis
TABLE 4
Negatively enriched gene sets related to inflammation and cancer in rectosigmoid biopsies before and after diet-induced weight loss in all 10 subjects
1
Name of gene set Size ES NES PFDR
CYTOKINE_ACTIVITY 72 20.51 22.17 ,0.001 ,0.001
CHEMOKINE_RECEPTOR_BINDING 34 20.65 22.30 ,0.001 ,0.001
CHEMOKINE_ACTIVITY 33 20.68 22.36 0.004 ,0.001
INFLAMMATORY_RESPONSE 108 20.46 22.02 ,0.001 ,0.001
TNFALPHA_ALL_UP 70 20.48 22.2 ,0.001 ,0.001
TNFALPHA_30MIN_UP 39 20.55 22.00 ,0.001 0.008
CROONQUIST_IL6_STROMA_UP 36 20.61 22.20 0.002 0.001
IL6_FIBRO_UP 42 20.50 21.82 ,0.001 0.009
IL1 KC UP 38 20.63 22.24 ,0.001 ,0.001
IL17ANDTNF KC UP 28 20.63 22.11 ,0.001 ,0.001
IL17 GAFFEN 30 20.56 21.95 ,0.001 ,0.001
IL17 KC UP 40 20.51 21.85 ,0.001 0.002
ADDITIVE IL17 AND TNFA KC 181 20.35 21.67 ,0.001 0.008
EICOSANOID_SYNTHESIS 15 20.67 21.88 0.004 0.027
PROSTAGLANDIN_AND_LEUKOTRIENE_METABOLISM 23 20.58 21.84 0.004 0.037
ICOSANOID_METABOLIC_PROCESS 15 20.7 21.99 ,0.001 0.042
HSA00590_ARACHIDONIC_ACID_METABOLISM 42 20.49 21.80 ,0.001 0.046
GLUTATHIONE_METABOLISM 30 20.65 22.24 ,0.001 0.001
GLUTATHIONE_TRANSFERASE_ACTIVITY 15 20.71 22.06 ,0.001 0.007
ELECTRON_TRANSPORT_CHAIN 87 20.66 22.86 ,0.001 ,0.001
HSA00190_OXIDATIVE_PHOSPHORYLATION 105 20.66 22.82 ,0.001 ,0.001
PROTEASOME_DEGRADATION 30 20.66 21.81 0.002 0.045
UBIQUINONE_BIOSYNTHESIS 14 20.66 22.02 ,0.001 0.001
PROTEASOME 17 20.66 22.04 ,0.001 ,0.001
1
Size, number of genes analyzed for each gene set; ES, enrichment score; NES, normalized ES; P,Pvalue of ES; FDR, false discovery rate. Gene sets
with P0.005 and an FDR 5% are shown.
TABLE 5
Negatively enriched transcription factor gene sets related to inflammation and cancer in rectosigmoid biopsies before
and after diet-induced weight loss in all 10 subjects
1
Name of gene set Size ES NES PFDR
CCAWWNAAGG_V$SRF_Q4 63 20.53 22.13 ,0.001 ,0.01
V$SRF_01 40 20.55 22.00 ,0.001 ,0.01
V$SRF_Q5_01 165 20.41 21.94 ,0.001 ,0.01
V$STAT3_01 16 20.62 21.82 0.005 0.01
V$STAT5A_01 169 20.37 21.77 ,0.001 0.01
V$STAT5B_01 171 20.37 21.74 ,0.001 0.02
V$ATF_B 133 20.35 21.63 0.001 0.06
V$CREB_Q4_01 150 20.34 21.58 0.001 0.07
V$NFKB_Q6_01 149 20.33 21.55 0.003 0.07
GGGNNTTTCC_V$NFKB_Q6_01 91 20.35 21.52 0.005 0.08
1
Size, number of genes analyzed for each gene set; ES, enrichment score; NES, normalized ES; P,Pvalue of ES;
FDR, false discovery rate. Gene sets with P0.005 and an FDR 10% are shown.
240 PENDYALA ET AL
are believed to be important in CRC pathogenesis in obesity
through the PIK3/Akt pathway (29) Insulin is mitogenic only at
supraphysiologic concentrations through the activation of the
IGF-1 pathway (30). Physiologic concentrations of IGF-1 are
growth promoting and mitogenic, and increased concentrations
of IGF-1, when adjusted for concentrations of its binding protein,
are associated with a modest increase in CRC risk (31). Leptin
concentrations also increase in obesity, and leptin has growth-
promoting, mitogenic, and antiapoptotic properties in colon
cancer cell lines but not in the normal epithelium (32) In the
Adenomatous polyposis coli (APC
min
) mouse, leptin did not
promote CRC or xenograft growth in mice (33). However,
obesity is recognized as a chronic inflammatory state with in-
creased serum cytokine concentrations, including those of TNF-a,
IL-6 and -8, and MCP-1 (27, 34), and evidence of inflammation
in adipose tissue, liver, skeletal muscle, and coronary arteries
(18). In our study, weight loss significantly reduced circulating
concentrations of TNF-a, IL-8, and MCP-1. Increased concen-
trations of obesity are accompanied by progressively higher
concentrations of serum cytokines and increased risk of co-
lorectal neoplasia (27, 35). This relation is supported by evi-
dence that cytokines have a procarcinogenic effect in colorectal
tissues (20). Reducing systemic TNF-aactivity (36) and knocking
out NF-jBin myeloid cells (37) decreased colonic tumors in
mouse colitis models. Thus, this diet regimen–induced reduction
in systemic cytokines might contribute to lowering colon neo-
plasia in obese individuals.
The current study showed that the diet-induced weight loss
reduced rectosigmoid mucosal concentrations of the inflam-
matory mediators TNF-a, IL-6, IL-8, IL-1b, and MCP-1. In
confirmation, the expression of gene pathways regulated by
TNF-aand IL-6 and -1balso decreased. In mouse models of
colitis-induced CRC, the inhibition of activated gene pathways
downstream of TNF-aand IL-6 decreased CRC. We also ob-
served a decreased expression of transcription factor pathways
implicated in colorectal inflammation, CRC, and other types of
cancer, including STAT3,NF-jB,ATF,CREB, and STAT5 (38).
STAT3 activation up-regulates inflammatory cytokines such as
IL-6, IL-1b, IL-8, MCP-1, and COX-2 (39). NF-jB induces the
expression of many inflammatory mediators and is a core tran-
scription factor in the immune response. Both STAT3 and
NF-jB signaling are recognized as pathways involved in in-
flammation-induced carcinogenesis (38). STAT3 and STAT5 can
expand regulatory T cells, which inhibit anti-tumor immunity
and promote tumor progression (40). An analogous pathway of
inflammation that leads to increased risk of liver cancer in
obesity was recently published (41). Thus, diet-induced weight
reduction down-regulates core transcription factors that are
crucial in inflammation and carcinogenesis and thereby may
reduce risk of CRC. The expression of gene pathways involved
in prostaglandin synthesis were also lower. In human obesity
and mouse obesity models, the increased expression of the
COX2 gene is a major regulator of colonic prostaglandin syn-
thesis. Caloric restriction has been shown to reduce colonic
COX2 expression in mouse obesity accompanied by decreased
numbers of aberrant crypt foci (11).
The current study showed that our experimental regimen
reduced rectosigmoid mucosal T cell and macrophage numbers
accompanied by decreased expression of IL-8,CCL19,CCL20,
CCL4L1, and gene pathways associated with chemokine ac-
tivity. These gene pathways are involved in mucosal leukocyte
accumulation, and their activity is increased in the colonic mu-
cosa of patients with inflammatory bowel disease during acute
exacerbations and down-regulated during remissions (42).
Oxygen free-radical formation and increased oxidative stress
link colonic inflammation and cancer formation (20). Obesity
enhances systemic oxidative stress, which, through intermediary
pathways, enhances the expression of proinflammatory and cell
cycle genes that can lead to tissue inflammation and DNA
damage and result in loss of tumor suppressor functions that are
characteristic of CRC (43). Western diet–consuming obese mice
have shown enhanced oxidative stress as well as inflammation in
the colorectal mucosa, (9) and calorie restriction has reduced
oxygen free-radical formation and DNA damage in rodent liver
and heart tissue (44, 45). Our study subjects showed down-
regulation of pathways involved with oxidative phosphorylation
and FOS and JUN genes that activate the transcription of
proinflammatory and cell proliferative mediators and can pro-
mote several human cancers (19). Thus, obesity creates an en-
vironment in the colon that promotes the initiation and
progression of CRC, and calorie restriction has an opposite
effect by reducing oxygen free-radical formation and decreasing
oxidative stress.
The current experimental model was chosen as a proof-of-
principle study for several reasons. Our experience with similar
translational studies has shown that endpoint data obtained in the
same individual obtained before and after an intervention pro-
vided optimum and reproducible information. A weight loss of
over 8% of initial weight was used because a previous study
showed that this would greatly lower proliferation rates in
humans (21). Previous experience permitted us to study only 10
subjects because the research staff of the Rockefeller University
Hospital has provided remarkable nutritional and environmental
control in intervention studies. One limitation is that the protocol
did not include control subjects such as obese individuals fed an
isoenergetic diet by using VLCD products. As a result, it is not
possible to determine whether the observed improvements in the
inflammatory and carcinogenesis pathways were changes in
energy balance, adiposity, or diet composition. However, un-
published data (P Holt, 2010) using dramatically differing diets
have not shown similar degrees of changes in mucosal cytokines
or gene array pathway analysis. Irrespective of the precise factors
responsible, our analysis showed a marked reduction in inflam-
matory and colon carcinogenesis endpoints with the intervention,
which indicated that obesity must be accompanied by increases in
these pathways. We recognize that the diet can alter luminal
microbiota diversity, which might influence epithelial cell sig-
naling (46) and, thus, contribute to some of the changes that we
observed.
In conclusion, our study shows that diet-induced weight loss in
obese women reduced colorectal mucosal inflammation as shown
by decreased inflammatory cytokines, inflammatory cells, down-
regulated inflammatory and cancer gene pathways, and tran-
scription factor gene sets that enhance inflammation and CRC risk.
Thus, obesity appeared to be accompanied by a low-grade in-
flammatory state in the colorectal mucosa, and diet-induced weight
loss decreased such inflammation. Because colorectal inflammation
is an important cofactor for colorectal carcinogenesis, such an
inflammatory state may contribute to enhanced CRC risk in obesity;
weight loss counteracts this effect and may thereby lower CRC risk.
WEIGHT LOSS REDUCES COLORECTAL INFLAMMATION 241
We thank Jan L Breslow for his advice and support, Jeanne Walker for her
close supervision of study volunteers, James G Krueger and Artemis Khatch-
erian for help with immunohistochemistry, and Juana Gonzalez for technical
help with the Mesoscale multiplex assay at the Clinical and Translational Sci-
ence Center immunology core facility.
The authors’ responsibilities were as follows—SP: study concept and de-
sign, data acquisition, data analysis and interpretation, and drafting of the
manuscript; LMN: study design and VLCD management; MS-F: statistical
analyses; and PRH: study concept and design, data interpretation, and drafting
of the manuscript. None of the authors had a conflict of interest.
REFERENCES
1. Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen M. Body-mass
index and incidence of cancer: a systematic review and meta-analysis of
prospective observational studies. Lancet 2008;371:569–78.
2. Larsson SC, Wolk A. Obesity and colon and rectal cancer risk: a meta-
analysis of prospective studies. Am J Clin Nutr 2007;86:556–65.
3. Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in
obesity among US adults, 1999-2008. JAMA 2010;303:235–41.
4. Mokdad AH, Serdula MK, Dietz WH, Bowman BA, Marks JS, Koplan
JP. The spread of the obesity epidemic in the United States, 1991-1998.
JAMA 1999;282:1519–22.
5. Kuczmarski RJ, Flegal KM, Campbell SM, Johnson CL. Increasing
prevalence of overweight among US adults. The National Health and
Nutrition Examination Surveys, 1960 to 1991. JAMA 1994;272:205–11.
6. Gravaghi C, Bo J, Laperle KM, et al. Obesity enhances gastrointestinal
tumorigenesis in Apc-mutant mice. Int J Obes (Lond) 2008;32:1716–9.
7. Weber RV, Stein DE, Scholes J, Kral JG. Obesity potentiates AOM-
induced colon cancer. Dig Dis Sci 2000;45:890–5.
8. Newmark HL, Yang K, Lipkin M, et al. A Western-style diet induces
benign and malignant neoplasms in the colon of normal C57Bl/6 mice.
Carcinogenesis 2001;22:1871–5.
9. Erdelyi I, Levenkova N, Lin EY, et al. Western-style diets induce oxi-
dative stress and dysregulate immune responses in the colon in a mouse
model of sporadic colon cancer. J Nutr 2009;139:2072–8.
10. Reddy BS, Wang CX, Maruyama H. Effect of restricted caloric intake
on azoxymethane-induced colon tumor incidence in male F344 rats.
Cancer Res 1987;47:1226–8.
11. Raju J, Bird RP. Energy restriction reduces the number of advanced
aberrant crypt foci and attenuates the expression of colonic transforming
growth factor beta and cyclooxygenase isoforms in Zucker obese (fa/fa)
rats. Cancer Res 2003;63:6595–601.
12. Parker ED, Folsom AR. Intentional weight loss and incidence of
obesity-related cancers: the Iowa Women’s Health Study. Int J Obes
Relat Metab Disord 2003;27:1447–52.
13. Sandhu MS, Dunger DB, Giovannucci EL. Insulin, insulin-like growth
factor-I (IGF-I), IGF binding proteins, their biologic interactions, and
colorectal cancer. J Natl Cancer Inst 2002;94:972–80.
14. Kim S, Keku TO, Martin C, et al. Circulating levels of inflammatory
cytokines and risk of colorectal adenomas. Cancer Res 2008;68:323–8.
15. Cole BF, Logan RF, Halabi S, et al. Aspirin for the chemoprevention of
colorectal adenomas: meta-analysis of the randomized trials. J Natl
Cancer Inst 2009;101:256–66.
16. Arber N, Eagle CJ, Spicak J, et al. Celecoxib for the prevention of
colorectal adenomatous polyps. N Engl J Med 2006;355:885–95.
17. Kim S, Baron JA, Mott LA, et al. Aspirin may be more effective in
preventing colorectal adenomas in patients with higher BMI (United
States). Cancer Causes Control 2006;17:1299–304.
18. Zeyda M, Stulnig TM. Obesity, inflammation, and insulin resistance–
a mini-review. Gerontology 2009;55:379–86.
19. Klaunig JE, Kamendulis LM, Hocevar BA. Oxidative stress and oxi-
dative damage in carcinogenesis. Toxicol Pathol 2010;38:96–109.
20. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:
860–7.
21. Steinbach G, Heymsfield S, Olansen NE, Tighe A, Holt PR. Effect of
caloric restriction on colonic proliferation in obese persons: implications
for colon cancer prevention. Cancer Res 1994;54:1194–7.
22. Chowdhury F, Williams A, Johnson P. Validation and comparison of two
multiplex technologies, Luminex and Mesoscale Discovery, for human
cytokine profiling. J Immunol Methods 2009;340:55–64.
23. Protiva P, Cross HS, Hopkins ME, et al. Chemoprevention of colorectal
neoplasia by estrogen: potential role of vitamin D activity. Cancer Prev
Res (Phila) 2009;2:43–51.
24. Subramanian A, Kuehn H, Gould J, Tamayo P, Mesirov JP. GSEA-P:
a desktop application for Gene Set Enrichment Analysis. Bioinformatics
2007;23:3251–3.
25. Nograles KE, Zaba LC, Guttman-Yassky E, et al. Th17 cytokines
interleukin (IL)-17 and IL-22 modulate distinct inflammatory and
keratinocyte-response pathways. Br J Dermatol 2008;159:1092–102.
26. Gaffen SL. Structure and signalling in the IL-17 receptor family. Nat
Rev Immunol 2009;9:556–67.
27. Kim CS, Park HS, Kawada T, et al. Circulating levels of MCP-1 and
IL-8 are elevated in human obese subjects and associated with obesity-
related parameters. Int J Obes (Lond) 2006;30:1347–55.
28. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment
analysis: a knowledge-based approach for interpreting genome-wide
expression profiles. Proc Natl Acad Sci USA 2005;102:15545–50.
29. Pollak M. Insulin and insulin-like growth factor signalling in neoplasia.
Nat Rev Cancer 2008;8:915–28.
30. Giovannucci E, Michaud D. The role of obesity and related metabolic
disturbances in cancers of the colon, prostate, and pancreas. Gastroen-
terology 2007;132:2208–25.
31. Wei EK, Ma J, Pollak MN, et al. A prospective study of C-peptide,
insulin-like growth factor-I, insulin-like growth factor binding protein-1,
and the risk of colorectal cancer in women. Cancer Epidemiol Bio-
markers Prev 2005;14:850–5.
32. Fenton JI, Hord NG, Lavigne JA, Perkins SN, Hursting SD. Leptin,
insulin-like growth factor-1, and insulin-like growth factor-2 are mi-
togens in ApcMin/+ but not Apc+/+ colonic epithelial cell lines. Cancer
Epidemiol Biomarkers Prev 2005;14:1646–52.
33. Aparicio T, Kotelevets L, Tsocas A, et al. Leptin stimulates the pro-
liferation of human colon cancer cells in vitro but does not promote the
growth of colon cancer xenografts in nude mice or intestinal tumori-
genesis in Apc(Min/+) mice. Gut 2005;54:1136–45.
34. Fain JN. Release of interleukins and other inflammatory cytokines by
human adipose tissue is enhanced in obesity and primarily due to the
nonfat cells. Vitam Horm 2006;74:443–77.
35. Adams KF, Leitzmann MF, Albanes D, et al. Body mass and co-
lorectal cancer risk in the NIH-AARP cohort. Am J Epidemiol 2007;
166:36–45.
36. Popivanova BK, Kitamura K, Wu Y, et al. Blocking TNF-alpha in mice
reduces colorectal carcinogenesis associated with chronic colitis. J Clin
Invest 2008;118:560–70.
37. Greten FR, Eckmann L, Greten TF, et al. IKKbeta links inflammation
and tumorigenesis in a mouse model of colitis-associated cancer. Cell
2004;118:285–96.
38. Grivennikov SI, Karin M. Dangerous liaisons: STAT3 and NF-kappaB
collaboration and crosstalk in cancer. Cytokine Growth Factor Rev,
2010;21:11–19.
39. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity:
a leading role for STAT3. Nat Rev Cancer 2009;9:798–809.
40. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune
cells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol
2007;7:41–51.
41. Park EJ, Lee JH, Yu GY, et al. Dietary and genetic obesity promote liver
inflammation and tumorigenesis by enhancing IL-6 and TNF expression.
Cell 2010;140:197–208.
42. Banks C, Bateman A, Payne R, Johnson P, Sheron N. Chemokine ex-
pression in IBD. Mucosal chemokine expression is unselectively increased
in both ulcerative colitis and Crohn’s disease. J Pathol 2003;199:28–35.
43. Klaunig JE, Kamendulis LM. The role of oxidative stress in carcino-
genesis. Annu Rev Pharmacol Toxicol 2004;44:239–67.
44. Gredilla R, Barja G, Lopez-Torres M. Effect of short-term caloric re-
striction on H2O2 production and oxidative DNA damage in rat liver
mitochondria and location of the free radical source. J Bioenerg Bio-
membr 2001;33:279–87.
45. Gredilla R, Sanz A, Lopez-Torres M, Barja G. Caloric restriction de-
creases mitochondrial free radical generation at complex I and lowers
oxidative damage to mitochondrial DNA in the rat heart. FASEB J 2001;
15:1589–91.
46. Neish AS. Microbes in gastrointestinal health and disease. Gastroen-
terology 2009;136:65–80.
242 PENDYALA ET AL
