Differential Gene Expression in Adipose Tissue From Obese Human Subjects During Weight Loss and Weight Maintenance

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Differential gene expression in adipose tissue from obese human

subjects during weight loss and weight maintenance1–5


Lovisa E Johansson, Anders PH Danielsson, Hemang Parikh, Maria Klintenberg, Fredrik Norström, Leif Groop,
and Martin Ridderstråle

ABSTRACT substantial weight loss seen after bariatric surgery, are powerful in
Background: Differential gene expression in adipose tissue during protecting against at least some of these risk factors, particularly
diet-induced weight loss followed by a weight stability period is against diabetes (6–8).
poorly characterized. Markers of these processes may provide a deep- Obesity is a complex trait to which both genes and envi-
er understanding of underlying mechanisms. ronment provide important contributions. Thus far, very few
Objective: We aimed to identify differentially expressed genes in genetic variants have been convincingly shown to be associated
human adipose tissue during weight loss and weight maintenance with obesity (9). Among the more investigated genes, for which

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after weight loss. function also has been replicated, are the genes FTO and MC4R
Design: RNA from subcutaneous abdominal adipose tissue from 9 (10, 11). The majority of these genes seem to regulate events
obese subjects was analyzed by using a complementary DNA mi- in the central nervous system (ie, appetite and satiety). In ad-
croarray at baseline after weight loss on a low-calorie diet and after dition to genetic-association studies, gene expression arrays
weight maintenance. in different tissues may help to identify genes that predispose
Results: Subjects lost 18.8 6 1.8% of weight and maintained this individuals to obesity. Several microarray studies have in-
loss during weight maintenance (1.1 6 2.1%; range: 29.3 to 10.6%). vestigated differentially expressed genes in normal-weight and
Most differentially expressed genes exhibited a reciprocal regulation obese subjects and in obese subjects before and after weight
and returned to baseline after weight loss (2163 genes) and weight loss (2, 9–13). In this study, we aimed to investigate the global
maintenance (3175 genes). CETP and ABCG1, both of which partic- adipose tissue gene expression profile before and after weight
ipate in the HDL-mediated reverse cholesterol transport (RCT), were loss induced by a low-calorie diet (LCD)6 and then after a pe-
among the most upregulated of the 750 genes that were differentially
riod of maintained reduced weight after weight loss supported
expressed after both processes. Several genes involved in inflamma-
by group therapy in the same individuals. With consideration
tion were downregulated. The use of real-time polymerase chain re-
of changes that occur both during acute weight loss and in the
action confirmed or partially confirmed the previously implicated
weight-maintenance phase, we hoped to detect genes that are
genes TNMD and MMP9 (both downregulated), PNPLA3 (upregu-
regulated during these events and to identify genes that show
lated), and CIDEA and SCD (both reciprocally regulated).
sustained regulation and, therefore, may be involved in the
Conclusions: The beneficial effects of weight loss should be inves-
tigated after long-term weight maintenance. The processes of weight
loss and weight maintenance should be viewed as biologically dis- 1
From the Department of Clinical Sciences Malmö, Clinical Obesity
tinct. CETP and ABCG1 may be important mediators of these effects (LEJ, APHD, MK, and MR) and the Department of Clinical Sciences Mal-
through HDL-mediated RCT. Am J Clin Nutr 2012;96:196–207. mö, Unit of Diabetes and Endocrinology (HP and LG), Lund University
Diabetes Centre, Lund University, and the Department of Endocrinology
(FN), Skåne University Hospital, Malmö, Sweden.
2
None of the supporting sources had any influence on the study.
3
INTRODUCTION Supported by the Swedish Research Council, the Novo Nordisk Foun-
dation, the Skåne Regional Council, ALF, the Påhlsson Foundation, and the
Obesity is a serious threat to health through different mech- Craaford Foundation.
anisms. Because of a sedentary lifestyle including a high-caloric 4
Present address for HP: Laboratory of Translational Genomics, Division
diet, obesity is increasing rapidly all over the world, particularly in of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Be-
subjects who are genetically susceptible to develop the disease (1, thesda, MD.
5
2). Obesity increases risk of several cardiovascular risk factors, not Address correspondence to APH Danielsson, Department of Clinical
least through increased risk of developing type 2 diabetes (3, 4). Sciences Malmö, Clinical Obesity, Lund University, Clinical Research Cen-
There is also increased risk of different forms of gastrointestinal ter, Skåne University Hospital, Entrance 72, Building 91, Level 12, SE-205
02 Malmö, Sweden. E-mail: anders.danielsson@med.lu.se.
cancer, breast cancer in women, and prostate cancer in men. Life 6
Abbreviations used: FDR, false discovery rate; KEGG, Kyoto Encyclopedia
expectancy is reduced by about 14 y for an obese 40-y-old subject of Genes and Genomes; LCD, low-calorie diet; MMP, matrix metalloproteinase;
who also smokes, with an equal risk contribution from obesity and OGTT, oral-glucose-tolerance test; PCR, polymerase chain reaction.
smoking (5). Meanwhile, lifestyle changes, including weight loss, Received June 29, 2011. Accepted for publication April 24, 2012.
dietary recommendations, and physical activity as well as the more First published online May 30, 2012; doi: 10.3945/ajcn.111.020578.

196 Am J Clin Nutr 2012;96:196–207. Printed in USA. Ó 2012 American Society for Nutrition

Supplemental Material can be found at:


http://ajcn.nutrition.org/content/suppl/2012/06/20/ajcn.111.0
20578.DC1.html
WEIGHT LOSS AND ADIPOSE TISSUE GENE EXPRESSION 197
duction phase, the participants attended a 6-mo follow-up pe-
riod (167 6 37 d) with group sessions led by a dietitian to
maintain their reduced weights. The variation was explained
by the design of the study by which assessments were driven
by goal attainment and program length rather than a fixed time
FIGURE 1. Study design. Subcutaneous abdominal adipose tissue schedule. Per protocol, the second assessment and biopsy were
biopsies were obtained after an oral-glucose-tolerance test at the following to take place after the LCD period or when subjects had lost
3 occasions: baseline (before weight loss), after 10% body-weight 10% of their initial body weight, and the third assessment
reduction by an LCD, and after group therapy to achieve weight stability
(65%). LCD, low-calorie diet. and biopsy were to take place at the completion of the pro-
grams, which were running around the year. Study participants
beneficial effects of weight loss rather than in the process of attended different groups, each of which included ;10 par-
losing weight itself. ticipants with an identical program for weight loss and weight
maintenance but who did not partake in the current study.
SUBJECTS AND METHODS During twelve 1-h sessions, groups were given counseling
concerning diet and exercise by a dietitian and a physical
Study design and subjects therapist, respectively. The behavioral counseling was based on
The study design addressed the following 2 concerns: whether cognitive therapy and was patient oriented with a focus on
weight reduction with an LCD was associated with changes in solutions in daily life. By protocol design, only samples from
gene expression (the difference between baseline and after weight subjects who had lost 10% of their initial body weight during
loss) and whether this effect was sustained after persons had the LCD period and maintained this weight (65%) after group

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returned to a normal caloric intake (ie, the similarity between therapy were eligible for the study. All participants fulfilled
weight loss and weight maintenance). Thus, we tested 2 hy- these criteria. Fasting blood samples and anthropometric data
potheses rather than a time series and did not adjust for repeated (body weight with subjects in light clothing) were obtained,
measures that would have been appropriate if a control group had and a standard oral-glucose-tolerance test (OGTT; 75g) was
been included. We used the term “reciprocal regulation” when performed at the following 3 time points: baseline, after weight
a transcript was shown to be either upregulated or downregulated reduction when subjects no longer lost weight, and after the
during weight loss and then oppositely regulated during weight group-therapy weight-maintenance phase. Subcutaneous ab-
maintenance. Twelve consecutive obese subjects who were re- dominal adipose tissue biopsies were obtained by using needle
ferred to the obesity outpatient unit at the Department of En- aspiration under local anesthesia (2 mL 10% lidocain) imme-
docrinology, Skåne University Hospital, were asked to participate diately after the OGTT (+120 min) at the 3 occasions. This
in the current study on the basis of a structured weight-reduction approach was chosen to achieve conditions equal to the post-
program (Figure 1). Participants were asked to refrain from prandial situation and to evaluate glucose tolerance. The local
activities that could influence their weight before the start of the ethics committee approved the study, and all participants gave
program, and as far as could be determined, participants were their written consent. In total, 12 obese Swedish subjects were
weight stable the preceding 6 mo [mean (6SEM) weight was included [age: 38 6 11 y; BMI (in kg/m2): 43.4 6 5.5; 4 men
132.6 6 6.8 kg (range: 101–179 kg) 181 6 21 d before the first and 11 women]. Sufficient amounts of abdominal subcutaneous
assessment point, when it was 132.8 6 6.7 kg (range: 103–176 adipose tissue–biopsy RNA were obtained from 9 of 12 sub-
kg); P = 0.8)]. Participants (n = 12) were prescribed an LCD jects, and these amounts were included in the microarray
(1200 kcal/d) for ;3 mo (101 6 26 d). After the weight-re- analysis (Table 1). One of the 27 biopsies (obtained at the

TABLE 1
Clinical characteristics of study subjects at baseline (before weight loss), after 10% body-weight reduction by
consumption of an LCD, and after group therapy to achieve weight stability (65%)1
Baseline Weight loss Maintenance of reduced weight
2
Men/women (n) 3/6 3/6 3/6
Age (y) 40 6 3.73 40 6 4.2 40 6 3.7
Weight (kg) 129 6 7.3 105 6 7.8 106 6 6.7
Weight loss (%)4 — 218.8 6 1.9 217.7 6 1.8
BMI (kg/m2) 42.7 6 1.4 34.9 6 1.95 35.2 6 1.56
fP glucose (mmol/L) 5.0 6 0.2 4.6 6 0.2 4.7 6 0.1
fS insulin (pmol/L) 10.9 6 2.0 3.9 6 0.85 6.2 6 0.96,7
HOMA-IR 2.4 6 0.4 0.8 6 0.25 1.3 6 0.26,7
fP HDL (mmol/L) 1.1 6 0.1 1.0 6 0.1 1.3 6 0.16,7
fP triglycerides (mmol/L) 1.5 6 0.3 0.8 6 0.15 0.8 6 0.26
1
fP, fasting plasma; fS, fasting serum; LCD, low-calorie diet.
2
Biopsy from one woman after weight loss was not included in the microarray analysis.
3
Mean 6 SEM (all such values).
4
Comparison of weight change in percentage from baseline.
5
P , 0.05 between baseline and weight loss (Wilcoxon’s rank-sum test).
6
P , 0.05 between baseline and weight stability (Wilcoxon’s rank-sum test).
7
P , 0.05 between weight loss and weight stability (Wilcoxon’s rank-sum test).
198 JOHANSSON ET AL

second assessment) was excluded from the analysis because of (Affymetrix), and detection of hybridized complementary RNA
improper sampling (Figure 1). was performed according to manufacturer’s recommendation at
the SCIBLU Microarray Resource Centre, Lund University. The
Adipose tissue biopsies and RNA extraction HG-U133 Plus 2.0 GeneChip DNA microarray contains 54,675
probe sets for interrogation, including known genes and ex-
Biopsies were immediately put in RNAlater solution (Ambion),
pressed sequenced tags. For each array, the percentage present
and after overnight incubation at 4°C, biopsies were snap frozen
call was .50%, and the 3# to 5# ratio of housekeeping genes
at 280°C until additional processing. RNA was extracted by using
GAPDH and HSAC07 was ,3, which showed a good and uni-
Tri reagent (Sigma-Aldrich) and an RNeasy Midi kit (Qiagen).
form in vitro transcription. All gathered array data were uploaded
The RNA was further concentrated by using RNeasy MiniElute
to the Gene Expression Omnibus database at the National Center
kit for purifying and concentrating RNA (Qiagen) and a SpeedVac
for Biotechnology (http://www.ncbi.nlm.nih.gov/geo/). The ac-
centrifugal evaporator (DNA 120 SpeedVac; Thermo Savant).
cession number to the dataset is GSE35411.
RNA quality was assessed by using an Agilent 2100 Bioanalyzer
platform for sizing, quantification, and quality control for RNA
(Agilent). RNA was stored at 280°C until additional analysis. Microarray data extraction and analysis
We used the AffyProbeMiner web resource (14) to regroup
Microarray individual probes into consistent probe sets and remap probe
The synthesis of biotin-labeled complementary RNA, hy- sets to the correct sets of messenger RNA transcripts for the HG-
bridization to DNA microarray HG-U133 Plus 2.0 GeneChip U133 Plus 2.0 array, which resulted in 23,798 probe sets. The

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TABLE 2
Top 15 downregulated and upregulated genes during weight loss1
Acute weight Fold
Gene Probe set2 Baseline loss change FDR3

kg kg
Top 15 upregulated (fold)
RARRES1 221872_at 76.45 6 134 222.51 6 52.57 2.91 0.018
RARRES1 206391_at, 206392_s_at 62.33 6 7.51 151.54 6 31.92 2.43 0.036
IGSF6 206420_at 25.73 6 1.92 58.31 6 11.38 2.27 0.045
ATP1B1 227556_at, 201242_s_at 418.5 6 41.75 837.56 6 153.5 2.00 0.047
C7 202992_at 240.19 6 48.21 477.1 6 111.33 1.99 0.024
EXPH5 214734_at 28.07 6 2.15 54.14 6 4.59 1.93 0.014
DTNA 205741_s_at 31.75 6 1.66 60.65 6 7.15 1.91 0.025
MT1M 217546_at 134.28 6 7.65 249.72 6 55.84 1.86 0.050
SRPX 204955_at 1851.62 6 174.61 3382.64 6 419.41 1.83 0.020
RGS17 220334_at 37.8 6 4.58 67.36 6 9.45 1.78 0.045
EGFLAM 226911_at 199.98 6 17.49 353.47 6 67.09 1.77 0.040
CIDEA 221295_at 830.75 6 99.15 1437.16 6 132.03 1.73 0.013
CHKB and CPT1B 210069_at 62.39 6 5.03 107.71 6 16.67 1.73 0.028
CA12 214164_x_at, 215867_x_at 127.55 6 10.12 215.25 6 34.04 1.69 0.026
C1orf51 227285_at 45.82 6 8.08 77.11 6 13.26 1.68 0.028
Top 15 downregulated (fold)
NQO1 201467_s_at 650.52 6 68.38 334.76 6 28.57 0.51 0.011
NQO1 201468_s_at, 243763_x_at 4175.27 6 308.98 2134.49 6 205.06 0.51 0.013
CLCA2 206164_at, 206165_s_at 42.87 6 5.57 20.56 6 2.91 0.48 0.017
THBS4 204776_at 802.79 6 168.59 354.06 6 82.08 0.44 0.014
LOC55908 220437_at 508.72 6 156.46 222.57 6 75.11 0.44 0.021
SCD 200832_s_at, 217200_x_at, 207986_x_at, 200831_s_at, 4544.31 6 475.67 1965.77 6 559.11 0.43 0.017
223839_s_at, 211162_x_at,
ALDOC 202022_at 1562.86 6 213.02 674.17 6 143.27 0.43 0.017
MXRA5 209596_at 942.3 6 108.5 393.21 6 67.38 0.42 0.011
FADS1 208963_x_at, 208964_s_at 313.81 6 64.66 125.82 6 28.78 0.40 0.031
FADS1 208962_s_at, 208963_x_at 292.92 6 68.01 105.45 6 25.28 0.36 0.023
FADS2 202218_s_at 257.67 6 42.25 91.05 6 19.22 0.35 0.025
B4GALT6 206233_at 161.22 6 32.05 52.71 6 9.09 0.33 0.021
LTF 202018_s_at 286.95 6 126.6 93.25 6 23.94 0.32 0.017
SCD 211708_s_at, 211162_x_at 1835.13 6 310.64 532.18 6 238.55 0.29 0.019
SFRP2 223121_s_at, 223122_s_at 959.02 6 210.7 251.85 6 54.29 0.26 0.014
1
Data were obtained by using the robust multiarray average method for normalization. Data from baseline measurements were based on 9 subjects. Data
from weight-loss measurements were based on 8 subjects. The gene list was sorted on the fold change for the comparison between baseline and weight loss.
2
Affymetrix.
3
FDR, false discovery rate defined by using the OCplus R package (Bioconductor; http://www.bioconductor.org) (18).
4
Mean 6 SEM (all such values).
WEIGHT LOSS AND ADIPOSE TISSUE GENE EXPRESSION 199
TABLE 3
Top 15 downregulated and upregulated genes during weight maintenance1
Acute weight Weight Fold
Gene Probe set2 loss maintenance change FDR3

kg kg
Top 15 upregulated (fold)
FADS1 208962_s_at, 208963_x_at 105.45 6 25.284 492.01 6 137.87 4.67 0.022
FADS1 208963_x_at, 208964_s_at 125.82 6 28.78 561.97 6 159.21 4.47 0.023
SCD 211708_s_at, 211162_x_at 532.18 6 238.55 2198.82 6 313 4.13 0.020
LTF 202018_s_at 93.25 6 23.94 352.9 6 172.45 3.78 0.021
ELOVL6 204256_at, 210868_s_at 88.67 6 57.11 318.21 6 92.56 3.59 0.023
FADS2 202218_s_at 91.05 6 19.22 318.14 6 35.67 3.49 0.018
THBS4 204776_at 354.06 6 82.08 1135.03 6 177.47 3.21 0.023
LOC55908 220437_at 222.57 6 75.11 700.24 6 206.13 3.15 0.025
B4GALT6 206233_at 52.71 6 9.09 160.59 6 32.5 3.05 0.020
FASN 212218_s_at 633.08 6 142.53 1848.95 6 108.19 2.92 0.019
SFRP2 223121_s_at, 223122_s_at 251.85 6 54.29 669.38 6 110 2.66 0.020
ALDOC 202022_at 674.17 6 143.27 1768.51 6 174.07 2.62 0.024
SCD 200832_s_at, 217200_x_at, 207986_x_at, 200831_s_at, 1965.77 6 559.11 5128.9 6 357.66 2.61 0.021
223839_s_at, 211162_x_at
KRT14 209351_at 27.61 6 4.48 70.44 6 15.01 2.55 0.028
SLC2A5 204430_s_at, 204429_s_at 77.82 6 18.83 196.24 6 40.76 2.52 0.037

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Top 15 downregulated (fold)
IGSF6 206420_at 58.31 6 11.38 18.5 6 2.13 0.32 0.026
CCL18 209924_at, 32128_at 767.48 6 237.48 228.39 6 74.31 0.30 0.029
IFI30 201422_at 2201.56 6 372.93 651.02 6 113.04 0.30 0.025
CSN1S1 208350_at 136.41 6 45.71 39.89 6 6.51 0.29 0.043
CHI3L1 209396_s_at, 209395_at 479.39 6 99.72 140.09 6 45.72 0.29 0.024
DHRS9 223952_x_at, 224009_x_at 352.05 6 70.16 101.05 6 29.26 0.29 0.020
AADACL1 225847_at 34.23 6 8.58 9.77 6 0.54 0.29 0.033
ATF3 202672_s_at 139.17 6 41.21 39.29 6 4.04 0.28 0.029
RARRES1 206391_at, 206392_s_at 151.54 6 31.92 42.63 6 1.85 0.28 0.024
BCL2A1 205681_at 112.45 6 25.78 29.87 6 8.03 0.27 0.034
ALCAM 201952_at, 201951_at 302.4 6 74.46 77.23 6 7.93 0.26 0.027
MMP9 203936_s_at 523.08 6 152.63 128.81 6 20.71 0.25 0.048
ACP5 204638_at 570.75 6 103.55 131.65 6 19.79 0.23 0.019
RARRES1 221872_at 222.51 6 52.57 48.39 6 4.92 0.22 0.024
PLA2G7 206214_at 280.99 6 82.01 44.6 6 7.63 0.16 0.023
1
Data were obtained by using the robust multiarray average method for normalization. Data from baseline measurements were based on 9 subjects. Data
from weight-loss measurements were based on 8 subjects. The gene list was sorted on the fold change for the comparison between baseline and weight loss.
2
Affymetrix.
3
FDR, false discovery rate defined by using the OCplus R package (Bioconductor; http://www.bioconductor.org) (18).
4
Mean 6 SEM (all such values).

normalization of the intensity of the microarray data and removal use of modified t statistics. We considered only those probe sets
of technical variation can have a major impact on results of as differentially expressed between groups that fulfilled each of
differential expression analyses, and many methods are available the following 2 requisites: had a global FDR ,0.05 by using the
(15). We applied the robust multiarray average method, which empirical operating characteristics function and a confidence
implements model-based background adjustment, quantile-based level 0.95 [which is equivalent to 5% of the FDR (19)] by using
normalization, and summarization on the basis of a multiarray the patterns from gene expression method. Both of these methods
model fit by using a median polish algorithm (16). We conducted provided correction for multiple comparisons. Gene lists were an-
filtering on the basis of the Microarray Suite 5.0 present/absent alyzed by using the WebGestalt system (http://bioinfo.vanderbilt.
calls, which classified each probe set as expressed above the edu/webgestalt/option.php) for the enrichment analysis of the Kyoto
background (present call) or not (absent or marginal call). We Encyclopedia of Genes and Genomes (KEGG) pathways by using
included probe sets, which have a detection call as present call in the hypergeometric test (20).
50% of arrays in one group (17), which left 13,463 of 23,798
probe sets for additional analysis. To identify differentially ex-
pressed genes for interclass paired comparisons, we used a permu- Quantitative real-time polymerase chain reaction analysis
tation that was based on the false discovery rate (FDR) approach of gene expression
calculated by using the empirical operating characteristics RNA from a total of 26 samples was transcribed by using the
function in the OCplus R package (Bioconductor; http://www. QuantiTect Whole Transcriptome kit (Qiagen) according to man-
bioconductor.org) (18) as well as the patterns from gene ex- ufacturer’s recommendations. Gene expression was analyzed by
pression method (19) for log (base 2)–transformed data with the using a TaqMan assay on demand on the 7900HT Fast Real-Time
200 JOHANSSON ET AL
TABLE 4
Top 15 downregulated and upregulated genes during the complete weight-loss program1
Weight Fold
Gene Probe set2 Baseline maintenance change FDR3

kg kg
Top 15 upregulated (fold)
CETP 206210_s_at 77.91 6 14.314 286.98 6 88.46 3.68 0.039
CA3 204865_at 26.64 6 6.62 80.95 6 19.19 3.04 0.041
PNPLA3 233030_at, 220675_s_at 86.48 6 13.63 250.29 6 61.68 2.89 0.050
SLC2A5 204430_s_at, 204429_s_at 91.98 6 15.04 202.68 6 36.52 2.20 0.041
AZGP1 209309_at, 217014_s_at 131.24 6 21.76 282.26 6 39.64 2.15 0.048
ADSSL1 226325_at 117.86 6 14.13 241.79 6 21.78 2.05 0.040
GLUL 200648_s_at, 217202_s_at 573.21 6 31.05 1052.17 6 114.84 1.84 0.037
STOX1 1553202_at 15.6 6 1.22 28.61 6 4.47 1.83 0.040
CDKN2C 211792_s_at, 204159_at 1403.41 6 149.11 2434.63 6 190.72 1.73 0.048
ENPEP 204845_s_at, 204844_at 72.72 6 8.41 126.12 6 15.68 1.73 0.048
AGTR1 205357_s_at, 208016_s_at 457.28 6 38.48 784.75 6 86.18 1.72 0.042
APOB 223579_s_at, 205108_s_at 46.12 6 5.9 77.6 6 11.4 1.68 0.048
NID2 204114_at 630.94 6 61.29 1061.2 6 95.44 1.68 0.045
ABCG1 211113_s_at, 204567_s_at 40.99 6 4.52 68.66 6 9.27 1.68 0.041
DBP 209782_s_at 120.78 6 4.33 193.84 6 22.02 1.60 0.044
Top 15 downregulated (fold)

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TNMD 220065_at 3172.74 6 314.96 1621.38 6 288.98 0.51 0.037
TNFRSF11B 204932_at 34.04 6 5.76 17.14 6 1.53 0.50 0.050
UCHL1 201387_s_at 1049.94 6 95.38 514.56 6 64.45 0.49 0.037
SCIN 1552365_at 118.49 6 25.05 56.91 6 9.46 0.48 0.041
CD9 201005_at 598.17 6 113 275.18 6 26.64 0.46 0.042
DHRS9 223952_x_at, 224009_x_at 242.74 6 55.18 99.74 6 25.84 0.41 0.038
PENK 213791_at 97.97 6 15.45 38.85 6 4.81 0.40 0.043
SFRP4 204051_s_at 617.83 6 54.66 242.97 6 40.68 0.39 0.037
EGFL6 219454_at 5080.15 6 540.97 1934.63 6 473.62 0.38 0.043
SFRP4 204052_s_at 318.66 6 37.03 112.69 6 25.33 0.35 0.037
ACP5 204638_at 425.13 6 115.02 126.87 6 18.09 0.30 0.041
ALCAM 201952_at, 201951_at 284.63 6 102.17 74.97 6 7.35 0.26 0.048
CSN1S1 208350_at 173.85 6 49.19 43.14 6 6.59 0.25 0.038
MMP9 203936_s_at 544.32 6 105.04 119.19 6 20.64 0.22 0.038
CHI3L1 209396_s_at, 209395_at 589.97 6 160.77 128.72 6 41.89 0.22 0.043
1
Data were obtained by using the robust multiarray average method for normalization. Data from baseline measure-
ments were based on 9 subjects. Data from weight-loss measurements were based on 8 subjects. The gene list was sorted on
the fold change for the comparison between baseline and weight loss.
2
Affymetrix.
3
FDR, false discovery rate defined by using the OCplus R package (Bioconductor; http://www.bioconductor.org) (18).
4
Mean 6 SEM (all such values).

PCR system (Applied Biosystems) according to the manufactu- were performed with Number Cruncher Statistical Systems 2004
rer’s recommendation. Five genes (CIDEA: Hs00154455_m1; software (NCSS). The correlation between CETP/ABCG1 gene
MMP9: Hs00234579_m1; PNPLA3: Hs00228747_m1; SCD: expression and blood HDL concentrations was tested by using
Hs01682761_m1; and TNMD: Hs00943212_m1) were selected for Spearman’s rank correlation.
confirmation of the microarray data by using real-time polymerase
chain reaction (PCR) on the basis of a significant fold change and
biological significance. Data were analyzed with the use of the DCt RESULTS
(comparative cycle time) method by using cyclophilin A as an
endogenous control (4326316E; Applied Biosystems). Cyclophilin Clinical characteristics
A was selected as an appropriate and stable control after testing
Clinical characteristics of study subjects at the 3 OGTT and
a subset of complementary DNA samples on a Human Endoge-
biopsy occasions (at baseline, after weight loss, and after group
nous Control Array (4367563; Applied Biosystems).
therapy to maintain the reduced weight) are shown in Table 1.
The LCD treatment resulted in an 18.8 6 5.4% (from 129 6 22
to 106 6 20 kg) loss of initial body weight (P = 0.0094) during
Statistical analysis of clinical data an average time of 101 6 26 d in 9 subjects from whom biopsies
The significance of differences in variables between different were analyzed by using a microarray. The achieved weight loss
time points was calculated by using the following robust tests: the was maintained throughout the group-therapy period (167 6 37
Kruskal-Wallis 1-factor ANOVA on ranks and Wilcoxon’s rank- d; P = 0.70; from 105 6 22 to 106 6 20 kg after group therapy;
sum test for difference in medians. All statistical calculations 1.1 6 2.1%; range: 29.3 to 10.6%). Insulin sensitivity improved
WEIGHT LOSS AND ADIPOSE TISSUE GENE EXPRESSION 201
KEGG analysis was performed on all significantly differen-
tially expressed genes shown during weight loss, during weight
maintenance, and for the whole process (ie, from baseline to
follow-up after weight maintenance). Results are shown sepa-
rately for pathways that were significantly upregulated or
downregulated (Table 5). During weight loss, genes involved in
metabolism, including fatty acid metabolism, were down-
regulated, whereas genes that are important for biogenesis and
cell proliferation were upregulated. On the contrary, during the
weight-maintenance phase, upregulated genes seemed to belong
to pathways involved in metabolism, whereas downregulated
genes belonged to pathways involved in immune regulatory
function. As for the individual transcripts, the results of the
pathway analysis allowed for groupings on the basis of regu-
lation (Table 6). After conservative Benjamini-Hochberger
adjustment, only changes for ribosomes, citric acid cycle, lyso-
some, and glycosylphosphatidylinositol-anchor biosynthesis were
considered significant, and all other pathways were considered to
be reciprocally regulated (ie, returned toward baseline at follow-
FIGURE 2. Venn diagram. Several regulated genes showed overlap up after weight maintenance regardless of whether they were
between comparisons.

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upregulated or downregulated during weight loss).

significantly as a result of weight loss (P = 0.0033) and de-


Real-time PCR confirmation of candidate genes
creased slightly during the weight-maintenance phase (P =
0.038; Table 1). Triglyceride concentrations decreased during Five genes were selected for confirmation of the microarray
weight loss, and this decrease was maintained during weight results by using real-time PCR on the basis of being present in
maintenance, whereas HDL-cholesterol concentrations showed the top 15 genes (Tables 2–4), which exemplified different
improvement during the weight-maintenance phase (Table 1). patterns of regulation (sustained and reciprocal), and previ-
ous identification and implication in obesity in the literature
Microarray gene expression (Figure 4). Confirmation was partial; although not all tran-
script changes were significant at all time points, the data
Weight loss resulted in the differential expression of 2163
pointed in the same direction as the microarray data. The gene
genes, 1104 of which were upregulated and 1059 of which were
encoding CIDEA was reciprocally upregulated and downregulated;
downregulated, with fold changes that ranged from 0.26 to 2.91
the MMP9 was downregulated during weight maintenance,
(Table 2). During weight maintenance, 1877 genes were
PNPLA3 was consistently upregulated, SCD was reciprocally
downregulated and 1298 genes were upregulated, with a fold-
downregulated and upregulated; and TNMD was consistently
change range of 0.16–4.67 (Table 3). As a result of the whole
downregulated (Figure 4). For the reciprocally regulated genes
process, from baseline to weight maintenance, 750 genes were
CIDEA and SCD, there was no significant alteration as a result of
differentially expressed, 483 of which were downregulated and
the entire process.
267 of which were upregulated, with fold changes that ranged
from 0.22 to 3.68 (Table 4). There were overlaps between all
comparisons (Figure 2). Because CETP and ABCG1 are in- DISCUSSION
volved in HDL-mediated reverse cholesterol transport, and the For most people, maintaining a reduced weight is a difficult but
change in HDL was one of the most prominent changes after important task to fully obtain the beneficial effects of weight loss.
weight loss (Table 1), we tested for a correlation. CETP (r = Genes that show sustained regulation during weight loss and
0.42, P = 0.034) and ABCG1 (r = 0.55, P = 0.0004) expression weight maintenance may be involved in the mechanisms for such
amounts were correlated to the prevailing HDL concentrations favorable effects. The results presented in this article indicate that
at the 3 assessments (Figure 3). weight loss and weight maintenance should be viewed as 2

FIGURE 3. Scatter plots. The prevailing HDL concentration correlated to the relative expression of CETP (r = 0.42, P = 0.034) (A) and the relative
expression of ABCG1 (r = 0.55, P = 0.0004) at 3 assessments (B).
TABLE 5 202
KEGG pathway analysis of genes significantly regulated in relation to weight loss and weight maintenance1
Reference Observed Expected Ratio of P (hypergeometric P (Benjamini-Hochberger
Regulation KEGG pathway KEGG ID genes genes genes enrichment test) adjusted)

Baseline to weight loss


Up Ribosome 3010 85 73 4.66 15.66 5.56 · 10280 6.39 · 10278
Up Spliceosome 3040 123 31 6.75 4.6 4.85 · 10213 2.79 · 10211
Up RNA degradation 3018 57 9 3.13 2.88 0.0036 0.138
Up Adherens junction 4520 75 10 4.11 2.43 0.0076 0.1748
Up Lysine degradation 310 42 7 2.3 3.04 0.0073 0.1748
Up Wnt signaling pathway 4310 146 15 8.01 1.87 0.0143 0.2382
Up RNA polymerase 3020 27 5 1.48 3.38 0.0145 0.2382
Up Cell cycle 4110 123 13 6.75 1.93 0.0175 0.2516
Up Dorso-ventral axis formation 4320 23 4 1.26 3.17 0.0346 0.4421
Up Notch signaling pathway 4330 46 6 2.52 2.38 0.0386 0.4439
Down Metabolic pathways 1100 1039 133 53.76 2.47 7.09 · 10223 1.04 · 10220
Down Oxidative phosphorylation 190 114 29 5.9 4.92 5.00 · 10213 3.65 · 10211
Down Parkinson disease 5012 111 27 5.74 4.7 9.9510 · 10–12 4.84 · 10210
Down Alzheimer disease 5010 155 32 8.02 3.99 1.40 · 10211 5.11 · 10210
Down Huntington disease 5016 167 26 8.64 3.01 4.71 · 1027 1.38 · 1025
Down Valine, leucine, and isoleucine 280 43 11 2.22 4.94 8.40 · 1026 0.0002
degradation
Down Ubiquitin-mediated proteolysis 4120 131 19 6.78 2.8 4.43 · 1025 0.0009
Down Cardiac muscle contraction 4260 70 12 3.62 3.31 0.0002 0.0024
Down GPI-anchor biosynthesis 563 24 7 1.24 5.64 0.0002 0.0024
Down Fatty acid metabolism 71 41 9 2.12 4.24 0.0002 0.0024
Weight loss to weight maintenance
Up Metabolic pathways 1100 1039 183 66.13 2.77 3.18 · 10238 4.83 · 10236
JOHANSSON ET AL

Up Oxidative phosphorylation 190 114 46 7.26 6.34 1.15 · 10225 8.74 · 10224
Up Parkinson disease 5012 111 43 7.06 6.09 3.11 · 10223 1.58 · 10221
Up Alzheimer disease 5010 155 47 9.87 4.76 4.43 · 10220 1.68 · 10218
Up Huntington disease 5016 167 45 10.63 4.23 4.47 · 10217 1.36 · 10215
Up Citrate cycle (TCA cycle) 20 30 17 1.91 8.9 2.25 · 10213 5.70 · 10212
Up Pyruvate metabolism 620 40 19 2.55 7.46 5.92 · 10213 1.29 · 10211
Up Glycolysis/gluconeogenesis 10 60 22 3.82 5.76 5.48 · 10212 1.04 · 10210
Up Propanoate metabolism 640 32 15 2.04 7.36 2.12 · 10210 3.58 · 1029
Up Valine, leucine, and isoleucine 280 43 17 2.74 6.21 3.58 · 10210 5.44 · 1029
degradation
Down Ribosome 3010 85 50 7.61 6.57 8.20 · 10231 1.30 · 10228
Down Lysosome 4142 116 45 10.39 4.33 2.41 · 10218 1.90 · 10216
Down Viral myocarditis 5416 67 22 6 3.67 4.01 · 1028 2.11 · 1026
Down Toll-like receptor 4620 99 26 8.87 2.93 3.86 · 1027 1.52 · 1025
signaling pathway
Down Leukocyte transendothelial 4670 114 27 10.21 2.64 2.11 · 1026 4.17 · 1025
migration
Down MAPK signaling pathway 4010 265 48 23.74 2.02 1.87 · 1026 4.17 · 1025
(Continued)

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TABLE 5 (Continued )

Reference Observed Expected Ratio of P (hypergeometric P (Benjamini-Hochberger


Regulation KEGG pathway KEGG ID genes genes genes enrichment test) adjusted)

Down Antigen processing and 4612 81 22 7.26 3.03 1.61 · 1026 4.17 · 1025
presentation
Down Fc c R-mediated phagocytosis 4666 93 24 8.33 2.88 1.51 · 1026 4.17 · 1025
Down Regulation of actin cytoskeleton 4810 208 39 18.63 2.09 7.21 · 1026 0.0001
Down Intestinal immune network for 4672 48 15 4.3 3.49 1.14 · 1025 0.0002
IgA production
Baseline to weight maintenance
Up Ribosome 3010 85 13 1.21 10.75 2.17 · 10210 1.50 · 1028
Up Citrate cycle (TCA cycle) 20 30 4 0.43 9.37 0.0008 0.0276
Up Tyrosine metabolism 350 44 4 0.63 6.39 0.0035 0.0604
Up Fatty acid metabolism 71 41 4 0.58 6.86 0.0027 0.0604
Up Glycerolipid metabolism 561 44 3 0.63 4.79 0.0246 0.0998
Up Glycerophospholipid metabolism 564 67 4 0.95 4.2 0.0152 0.0998
Up Lysine degradation 310 42 3 0.6 5.02 0.0217 0.0998
Up Oxidative phosphorylation 190 114 5 1.62 3.08 0.0235 0.0998
Up Pyruvate metabolism 620 40 3 0.57 5.27 0.0191 0.0998
Up Glycolysis/gluconeogenesis 10 60 4 0.85 4.69 0.0105 0.0998
Down Lysosome 4142 116 20 2.82 7.08 5.79 · 10212 5.56 · 10210
Down Proteasome 3050 42 7 1.02 6.85 6.24 · 1025 0.003
Down GPI-anchor biosynthesis 563 24 5 0.58 8.56 0.0002 0.0064
Down Other glycan degradation 511 15 4 0.37 10.96 0.0004 0.0096
Down Porphyrin and chlorophyll 860 33 5 0.8 6.22 0.0011 0.0211
metabolism
Down Nicotinate and nicotinamide 760 24 4 0.58 6.85 0.0025 0.04
metabolism
Down Glycosphingolipid 604 15 3 0.37 8.22 0.0052 0.0555
biosynthesis – ganglio series
WEIGHT LOSS AND ADIPOSE TISSUE GENE EXPRESSION

Down Metabolic pathways 1100 1039 39 25.29 1.54 0.0049 0.0555


Down Regulation of actin cytoskeleton 4810 208 12 5.06 2.37 0.0051 0.0555
Down Sphingolipid metabolism 600 37 4 0.9 4.44 0.0121 0.1162
1
Analyses were performed by using the WebGestalt system (http://bioinfo.vanderbilt.edu/webgestalt/option.php) (20). The reference gene set used was affy_hg_u133_plus_2. Only the 10 most significant
regulations are included. GPI, glycosylphosphatidylinositol; ID, identification number; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; TCA, tricarboxylic acid.
203

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204 JOHANSSON ET AL
TABLE 6
Summary of regulated pathways1
Weight maintenance

Up-regulation Down-regulation No regulation

Weight loss
Up-regulation No pathways Ribosomes [2 No pathways
No pathways Y
Down-regulation Oxidative phosphorylation [ No pathways Glycosylphosphatidylinositol-
anchor biosynthesis pathway2
Metabolic pathways Y
No regulation Pathways for the citric acid cycle2 Pathways for lysosome*
Pyruvate metabolism Pathways for actin cytoskeleton
Glycolysis/gluconeogenesis
1
[ indicates that the end level was higher than the baseline level; Y indicates that the end level was lower than the baseline level.
2
Significant using the Hypergeometric test with the adjusted P value using Benjamini-Hochberger.

biologically distinct events. Most genes and pathways in the mediate the release and activation of growth factors as well as
adipose tissue identified by the complementary DNA microarray cleave cell-surface receptors (25). TNMD constitutes another
appeared to be reciprocally regulated during weight loss and interesting example. Kolehmainen et al (13) investigated the

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weight maintenance and returned to or toward baseline when effect of weight loss and weight maintenance on adipose tis-
subjects were followed up as they remained weight stable after sue gene expression by using a long-term moderate weight-
weight loss. These genes and pathways likely orchestrate and/or reduction program. They showed that TNMD was highly
reflect catabolic events in the tissue, which is exemplified by the regulated and associated with features of metabolic syndrome
downregulation and upregulation of pathways for metabolism (13). In a study by Saiki et al (27), it was shown that TNMD was
identified in the KEGG analysis. It is more likely that genes that highly expressed in obese subjects compared with in lean con-
are important for the long-term benefits of weight loss show late trols (27). In addition, it was shown that TNMD expression was
changes or sustained regulation throughout the process of weight downregulated during diet-induced weight loss. We showed this
loss and maintenance. Very few genes or pathways showed such gene to be downregulated by weight loss and the entire process on
sustained regulation, which illustrated the separation of phases. the array but only significantly so during weight maintenance
Inflammatory-related genes previously shown to be downregulated when real-time PCR was used to replicate findings. However, all
during weight loss were confirmed only in the later maintenance conformation data trended in the same direction as the array data,
phase (10, 11, 21). and the differences may have simply been a result of statistical
In a recent study with a design similar to ours, Mutch et al (22) power or isoform-specific differences. The biological relevance of
compared gene expression in subcutaneous fat from subjects who TNMD in obesity and weight loss has been confirmed repeatedly
maintained or regained weight after an LCD. Our study con- (28). Tenomodulin is believed to mediate antiangiogenic activity
firmed some of the findings of Mutch et al (22) concerning single and can modulate endothelial cell proliferation (29, 30). Variants
genes, such as SCD and CIDEA, and KEGG pathways (eg, fatty in TNMD are associated with adiposity and glucose metabolism,
acid metabolism, citric acid cycle, oxidative phosphorylation, which support a functional role in obesity (31). Taken together,
and apoptosis). The LCD-induced upregulation of CIDEA was low amounts of tenomodulin and matrix metallopeptidase 9 or
seen only in weight maintainers. The 2 studies were different in related proteins may be important for the beneficial effects of
that all subjects in the current study met the criteria for weight weight loss. PNPLA3, which encodes the adipocyte transacylase
maintenance, and gene expression was studied at this phase in adiponutrin, constitutes an inverse example to TNMD, which is
addition to before and after the LCD. Therefore, we were unable upregulated as a result of the whole process. This effect is likely
to confirm differences in post-LCD gene expression between because of the increase in insulin sensitivity and biopsies that are
weight maintainers and weight regainers. From our data, it was obtained after a glucose load because PNPLA3 expression has
also apparent that some of these signature changes shown in previously been shown to be regulated by insulin in a glucose-
subjects who were able to maintain weight returned to baseline dependent fashion (32). Adiponutrin has also been implicated in
over time as illustrated by the analysis of the follow-up biopsy obesity, on both transcriptional and genetic grounds and, more
here. recently, has emerged as a major candidate gene for nonalcoholic
We were able to verify the regulation of several previously fatty liver disease (33–35).
known genes involved in metabolism in relation to weight loss. The majority of genes, including some candidate genes that have
MMP9 and TNMD were downregulated as a result of both attracted previous attention, were differentially expressed and re-
weight loss and maintenance. Matrix metalloproteinases (MMPs) ciprocally reregulated during weight loss and weight maintenance.
are important for extracellular matrix remodeling, and they have Our data confirmed previous studies that indicated that the ex-
been implicated in the development of obesity and obesity- pression of a large number of genes involved in lipid-handling
related heart failure (23–25). MMPs are believed to have an change as a result of weight loss (36, 37). The CIDEA gene
effect on adipocyte biology because the inhibition of MMPs constitutes one such example (12, 36). CIDEA has been shown to
resulted in more adipocytes and reduced adipocyte cell size (26). be important for the regulation of lipolysis and metabolic com-
MMPs also have the ability to generate bioactive molecules and plications of obesity. Cidea knockout mice have a lean phenotype
WEIGHT LOSS AND ADIPOSE TISSUE GENE EXPRESSION 205
and show resistance to diet-induced obesity and diabetes (36, 38, 39). Here, CIDEA was highly up-regulated after weight loss,
confirming previous studies (12, 36, 38), but returned to baseline
during weight maintenance. This suggests that CIDEA may have
been induced to protect against weight loss, more precisely against
energy depletion by lipolysis. Meanwhile, it is important to keep in
mind that there may be species differences because cidea in mice
is exclusively expressed in brown adipose tissue (12). The gene
coding for stearoyl–coenzyme A desaturase, which is an enzyme
involved in the lipogenesis of unsaturated fatty acids and associ-
ated with insulin resistance (40, 41), constitutes another example.
SCD was downregulated during weight loss but upregulated during
the weight-maintenance phase. Our data supported previous studies
that showed the downregulation of SCD in adipose tissue during
weight reduction, although this did not seem to be a result of
improved insulin sensitivity, as has been suggested, because insulin
sensitivity was more or less conserved during weight maintenance
when SCD expression returned to baseline (12, 42, 43).
The notion that inflammatory genes play a role in the path-
ogenesis of obesity or complications of obesity has been sug-
gested several times (44–49). As a result of weight loss by either

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diet or surgery, the number of infiltrating macrophages in adipose
tissue decreases (11, 46, 50). It is likely that some of the genes
related to inflammation that we and others have picked up in
microarrays come from macrophages rather than from adipocytes
(11, 48, 50). We chose to analyze the effect of weight loss on
adipose tissue as a whole and considered it to be a functional
interdependent unit. We showed that the expression profile of
genes related to inflammation improved as a result of weight loss.
This improvement was seen during weight maintenance rather
than during the weight-reduction phase. These results contrast
with those of Clément et al (11), who investigated differentially
expressed genes in the adipose tissue of female subjects before
and after weight loss by using a diet that was very low in calories
for 28 d. A main finding was the improvement of the in-
flammatory gene expression profile, but this improvement was
already seen as a result of weight loss. The studies are not entirely
comparable, and differences in the results may be explained by
differences in designs. The achieved weight reduction was
smaller than that in our study, and the duration of the hypocaloric
phase was shorter (7% compared with 18%, respectively, and 28
compared with 101 d, respectively).
In any microarray, there are numerous candidates for future
research, and confirmation and array investigations are, in es-
sence, hypothesis-generating studies (51). One of the most im-
portant areas should now be to identify genes or pathways that are
responsible for the long-term beneficial effects of weight loss
rather than reciprocally regulated genes that orchestrate the
process. Energy-related pathways, such as the citric acid cycle,
FIGURE 4. Comparison of the mean 6 SEM expression of 5 genes by lipid metabolism, oxidative phosphorylation, pyruvate metabo-
using a microarray and real-time PCR. Open bars represent fold changes in
expression from the microarray data, and filled bars represent fold changes in
lism, glycolysis, and gluconeogenesis, were shown to be upre-
expression after confirmation by using real-time PCR. *P , 0.05 (calculated gulated in this category. Also, inflammatory genes, such as
by using the OCplus R package (Bioconductor; http://www.bioconductor. CHI3L1, MMP9, and ALCAM, were downregulated, although
org) (18) for robust multiarray average normalized expression data from their exact roles in the pathogenesis of obesity or complications
the microarray and for confirmation data by using real-time PCR and
Wilcoxon’s rank-sum test. Acute weight loss was determined as the
of obesity remain to be established. From a clinical perspective,
difference in expression between baseline and after weight loss, weight the presence of ABCG1 and CETP in the list of the top 15 up-
stability was determined as the difference in expression between after regulated transcripts may be of particular interest because they
weight loss and after weight maintenance, and overall weight loss was participate in HDL metabolism. ABCG1 encodes ATP-binding
determined as the expression difference between baseline and after weight
maintenance. The confirmatory analysis included 9 individuals who were
cassette subfamily G member 1, which is a member of the ATP-
analyzed by using a microarray. mRNA, messenger RNA; PCR, polymerase binding cassette transporter family involved in the transfer of
chain reaction. cholesterol esters from macrophage foam cells to HDLs in the
206 JOHANSSON ET AL

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