many diseases of high public health importance , such as type 2 diabetes and metabolic syndrome , are profoundly influenced by obesity status ( 1 ) .
however , individuals with a similar body mass index ( bmi ) can exhibit substantially different metabolic profiles for glucose tolerance , lipids , and blood pressure . in the nhanes study ,
adult population ) exhibit a better metabolic profile than age - adjusted bmi expectations and are categorized as  metabolically healthy obese 
an increase in white adipose tissue ( wat ) mass in response to chronic positive energy imbalance is a defining feature of obesity .
however , several structural / histological characteristics and depot - specific ( subcutaneous and visceral ) distributions of adipose tissue differ between metabolically healthy and unhealthy obese individuals ( 4 ) . the location and cellular mechanisms of tissue expansion ( hyperplasia and hypertrophy ) greatly affect the normal physiological function of adipose tissue ( 5 ) .
this process may be mechanistically linked to the development of obesity - associated metabolic disorders ( 6;7 ) .
we and others have shown significant dysregulation in the adipose tissue transcriptome with obesity ( 811 ) .
many genes are correlated with obesity ( bmi ) and adiposity ( % fat mass ) . however , pinpointing which defining genes underlie the heterogeneity of metabolic features among obese individuals and determine metabolically healthy and unhealthy phenotypes remains challenging .
the purpose of this study is to identify an adipose tissue transcriptomic signature that may determine metabolic health in obese subjects .
defining metabolically healthy and unhealthy obese subjects based on clinical and biochemical characteristics is difficult ( 2;3 ) .
thus , we performed unsupervised hierarchical clustering analysis using data from obesity - associated adipose tissue transcripts to first identify comparatively homogenous sub - groups among obese subjects .
we hypothesized that these transcriptionally homogenous sub - groups could differ in metabolic characteristics , and that a subset of obesity - associated transcripts in subcutaneous adipose tissue are involved in defining metabolic health in obese subjects .
we then performed additional statistical and bioinformatics analyses to compare sub - groups of obese subjects .
the sample included a subset ( 30 obese men and women ) from the 170 subjects recruited for the university of arkansas for medical sciences ( uams ) gene expression study ( 8;12 ) .
these 30 subjects were all obese ( bmi  30 kg / m ) , non - diabetic , and  40 years old .
they had been characterized by frequently sampled intravenous glucose tolerance tests ( fsivgt ) , and genome - wide gene expression data were available from subcutaneous adipose tissue biopsy samples .
three additional subjects met the selection criteria mentioned above , but genome wide expression data of acceptable quality were unobtainable for these subjects .
methods for recruitment , physical examination , physiological experiments , and obtaining biopsies have been published . in brief , european - american or african - american men and women in general good health , between 19 and 60 years of age , and had a bmi between 19 and 45 kg / m were recruited .
all participants had a screening visit , at which time height , weight , and waist and hip circumference were measured ; body fat was determined by dual x - ray absorptiometry ( dxa ) scan ( hologic qdr-4500 ) ; fasting blood samples for lipid measurements were taken ; and a standard 75-g oral glucose tolerance test ( ogtt ) was done . at the second visit , adipose biopsies were obtained from fasting subjects .
insulin modified ( 0.03 u / kg ) frequently sampled intravenous glucose tolerance tests ( fsigt ) were performed to determine insulin sensitivity ( si ) and acute insulin response ( airg ) .
biopsies were obtained from abdominal subcutaneous fat near the umbilicus under local anesthesia ( lidocaine ) using a bergstrom needle .
each biopsy is made by opening the bergstrom needle and applying suction using a 120 ml syringe .
samples were rinsed immediately in sterile normal saline , quick - frozen in liquid nitrogen , and stored at 80c for further use .
our current study was approved by the institutional review board at wake forest school of medicine .
insulin was measured by the uams clinical research center core laboratory using an immuno - chemiluminometric assay ( invitron limited , monmouth , uk ) .
plasma triglyceride , total cholesterol , and hdl cholesterol concentrations were measured directly ( at labcorp ) by enzymatic colorimetric methods .
total rna was isolated from whole adipose tissue using the rnaeasy lipid tissue mini kit ( qiagen inc - usa , valencia , ca ) .
the quantity and quality of the isolated total rna samples were determined by ultraviolet spectrophotometry ( nanodrop , thermo scientific , pittsburgh , pa ) and electrophoresis ( experion nucleic acid analyzer , biorad laboratories , inc . , hercules , ca ) , respectively . high - quality rna with rin ( rna integrity number )
> 8 was used for genome - wide transcriptome analysis . genome - wide transcriptome analysis and initial array processing
were done at the center for human genomics core laboratory ( wake forest school of medicine ) using humanht-12 v4 expression beadchip ( illumina , san diego , ca ) whole genome gene expression arrays according to the vendor - recommended standard protocol ( 8) . raw expression intensity was background subtracted and normalized by the average normalization algorithm as implemented in genomestudio gene expression module v1.0 application software ( illumina ) .
we performed unsupervised hierarchical clustering to identify homogeneous groups among obese subjects based on adipose tissue transcript expression . a dissimilarity matrix based on pearson s correlation coefficient between each individual
was generated using z - score - normalized values for each probe and seriation under multiple fragment heuristics .
the hierarchical clustering - based tree and heat map was generated using mcquitty s criteria implemented in permutmatrix version 1.9.3en software ( 13 ) . for the current analysis , we chose a set of 1595 obesity - associated probes ( associated with % fat mass and bmi independent of age , gender and race ) that may define the heterogeneity of metabolic features among obese individuals , based on our previous study ( 8) . to estimate the differences in gluco- and cardio - metabolic phenotypes between the two groups ( two major groups / clusters assigned by unsupervised hierarchical clustering analysis ) , we used nonparametric mann - whitney u - test and univariate analysis of variance , with age , gender , and race as covariates . to identify the transcripts with significantly different expression between the two groups , we utilized nonparametric wilcoxon statistics on normalized data as implemented in statistical analysis for microarray ( sam ) software ( 14 ) .
we considered results significant for a false discovery rate ( q value )  5% , and fold difference between groups of > 1.5 , based on 1000 permutations .
additional validation of sam analysis was performed by t - test ( p 0.05 ) on log2 transformed expression data .
bioinformatic analysis was performed to investigate the enrichment of differentially expressed genes in known biological pathways or gene ontology ( go ) categories .
we performed canonical pathway analysis and interaction network analysis of differentially expressed genes using ingenuity pathway analysis ( ipa ; https://analysis.ingenuity.com , build version : 313398 m ; content version : 18841524 )
. pathways with a corrected p - value ( benjamini and hochberg p - value ) 0.05 were considered significantly enriched in our gene lists .
additional annotations of differentially expressed genes were performed by singular enrichment analysis ( sea ) using the david v6.7 functional annotation tool ( http://david.abcc.ncifcrf.gov/)(15 ) .
detailed analysis parameters for sea using the functional annotation chart module of david are described elsewhere ( 9 ) . in the sea analysis , a category with a gene count > 5 , an ease score 0.05 ( modified fisher s exact p - value ) , and fdr 5% was considered as a significant threshold .
we performed unsupervised hierarchical clustering to identify homogeneous groups among obese subjects based on adipose tissue transcript expression . a dissimilarity matrix based on pearson s correlation coefficient between each individual
was generated using z - score - normalized values for each probe and seriation under multiple fragment heuristics .
the hierarchical clustering - based tree and heat map was generated using mcquitty s criteria implemented in permutmatrix version 1.9.3en software ( 13 ) .
for the current analysis , we chose a set of 1595 obesity - associated probes ( associated with % fat mass and bmi independent of age , gender and race ) that may define the heterogeneity of metabolic features among obese individuals , based on our previous study ( 8) . to estimate the differences in gluco- and cardio - metabolic phenotypes between the two groups ( two major groups / clusters assigned by unsupervised hierarchical clustering analysis ) , we used nonparametric mann - whitney u - test and univariate analysis of variance , with age , gender , and race as covariates . to identify the transcripts with significantly different expression between the two groups
, we utilized nonparametric wilcoxon statistics on normalized data as implemented in statistical analysis for microarray ( sam ) software ( 14 ) .
we considered results significant for a false discovery rate ( q value )  5% , and fold difference between groups of > 1.5 , based on 1000 permutations .
additional validation of sam analysis was performed by t - test ( p 0.05 ) on log2 transformed expression data .
bioinformatic analysis was performed to investigate the enrichment of differentially expressed genes in known biological pathways or gene ontology ( go ) categories .
we performed canonical pathway analysis and interaction network analysis of differentially expressed genes using ingenuity pathway analysis ( ipa ; https://analysis.ingenuity.com , build version : 313398 m ; content version : 18841524 ) .
pathways with a corrected p - value ( benjamini and hochberg p - value ) 0.05 were considered significantly enriched in our gene lists .
additional annotations of differentially expressed genes were performed by singular enrichment analysis ( sea ) using the david v6.7 functional annotation tool ( http://david.abcc.ncifcrf.gov/)(15 ) .
detailed analysis parameters for sea using the functional annotation chart module of david are described elsewhere ( 9 ) . in the sea analysis , a category with a gene count > 5 , an ease score 0.05 ( modified fisher s exact p - value ) , and fdr 5% was considered as a significant threshold .
white adipose tissue modulates both glucose and lipid homeostasis in humans via its master regulatory role in controlling whole - body lipid flux ( 16 ) . under conditions of chronic energy overload ,
however , decompensation of this adaptive homeostatic mechanism ( largely regulated by the interaction of genetic and epigenetic factors with dietary components ) may lead to adipose tissue dysfunction and is likely to be the causal link for metabolic abnormalities in a subset of obese subjects .
after unsupervised hierarchical clustering of 1595 obesity - associated ( independent of age , gender and race ) transcripts , we classified the 30 obese subjects into two groups of 16 and 14 subjects , respectively ( figure 1a ) . these two groups were not significantly different in bmi . however , group 2 subjects had significantly higher total cholesterol ( p= 0.005 ) , ldl - cholesterol ( p= 0.006 ) , 2h - insulin during ogtt ( p=0.015 ) and lower insulin sensitivity ( si , p= 0.029 ) compared to group 1 ( figure 1b ) . despite similar bmi , obese subjects in group 2 showed poor gluco - and cardio - metabolic phenotype .
thus , our study revealed a distinct signature of adipose tissue transcripts that can differentiate obese subjects with comparatively better metabolic health ( group 1 ) from obese subjects with poor metabolic heath ( group 2 ) .
group 2 subjects showed higher fat mass ( 43.6  6.7% ) compared to group 1 subjects ( 35.5  6.8% ) , but this difference was not statistically significant after adjustment for age , gender , and race .
thus , the observed difference in metabolic characteristics between the two groups can not be attributed to the differences in total adiposity .
however , we have not analyzed depot - specific distribution or histological characteristics of white adipose tissue in these subjects .
we identified 158 transcripts with 1.5 fold difference in expression between groups 1 and 2 ( supplementary table 1 ) .
group 2 had significant up - regulation of 141 genes and down - regulation of 17 genes .
highly up - regulated genes included mmp9 ( matrix metallopeptidase 9 ; 4.1 fold , p= 5.6810 ) and spp1 ( secreted phosphoprotein 1 or osteopontin ; 3.2 fold , p= 3.5410 ) ; while the top down - regulated genes included ndrg4 ( n - myc downstream - regulated gene family member 4 , 2.1 fold , p= 7.0110 ) and gins3 ( gins complex subunit 3 ; 1.8 fold , p= 1.5810 ) ( figure 1c ) . two large genome - wide association studies ( gwas ) identified significant association of ecg qt interval ( a quantitative measure of cardiac repolarization ) in human chromosome 16q21 , a genomic interval which includes several candidate genes including ndrg4 and gins3 ( 17;18 ) . ndrg4 knockdown in zebra fish embryos is associated with defective cardiac morphogenesis and function , including weak contractility due to marked reduction in proliferative myocytes ( 19 ) .
similarly gins3 / zebra fish embryos show a significant defect in cardiac repolarization ( 20 ) . in our study , the distribution of age and race was not significantly different between groups , but distribution of gender was significant .
thus , we compared only women from group 2 ( n= 12 ) and group 1 ( n=7 ) to identify differentially expressed genes . despite the lower statistical power , results remain consistent . of 158 differently expressed genes ,
126 were significantly differentially expressed ( p 0.05 ) in the women - only analysis .
the fold changes of these genes were strongly correlated between the overall cohort and the women only ( overall vs women - only analysis , r=0.83 , p= 8.7610 ) .
thus , the differential expression of genes between groups as identified by unsupervised hierarchical clustering analysis can mainly be attributed to differences in metabolic characteristics of the subjects .
transcripts differentially expressed between the two groups were significantly enriched for the functional categories of genes ( table 1 ) .
these genes were primarily associated with cardiovascular disease , including peripheral arterial occlusive disease due to atherosclerotic lesions ( enrichment p = 2.8110 ) .
the ingenuity knowledge base shows an interaction network of 31 differentially expressed genes involved in different cardiovascular disease - related processes ( figure 1d ) .
these differentially expressed genes were also enriched in several canonical pathways involved in immune and inflammatory response , including the complement system ( p= 8.3210 ) , trem1 signaling ( p= 3.0910 ) and il-8 signaling ( p= 3.0910 ) ( table 1 ) .
similarly david analysis identified signaling in the immune system ( p= 3.9410 , 21 genes , reactome pathway database ) as the most strongly enriched pathway .
stronger upregulation of genes in pathways involved in immune and inflammatory responses , in metabolically unhealthy obese subjects , may be the link between obesity , cardiovascular disease , and type 2 diabetes ( 7 ) .
genes in immune and inflammatory response pathway in adipose tissue were associated with insulin resistance ( 9 ) , which could explain the significantly lower insulin sensitivity among group 2 subjects in our study .
genes in these pathways may become novel therapeutic targets in preventing obesity - associated diseases .
in conclusion , our study provides evidence that the perturbation in the adipose tissue gene expression network may be critical in defining metabolic health , including cardiometabolic phenotypes in obese subjects .
further functional studies will be required to define the causal relationship of these genes with cardio- and gluco - metabolic phenotypes in obesity .
 obese subjects with a similar body mass index ( bmi ) exhibit substantial heterogeneity in gluco- and cardio - metabolic heath phenotypes . 
 however , defining genes that underlie the heterogeneity of metabolic features among obese individuals and determining metabolically healthy and unhealthy phenotypes remain challenging . 
 we conducted unsupervised hierarchical clustering analysis of subcutaneous adipose tissue transcripts from 30 obese men and women 40 years old . despite similar bmis in all subjects , we found two distinct subgroups , one metabolically healthy ( group 1 ) and one metabolically unhealthy ( group 2 ) . 
 subjects in group 2 showed significantly higher total cholesterol ( p=0.005 ) , ldl cholesterol ( p=0.006 ) , 2h - insulin during ogtt ( p=0.015 ) and lower insulin sensitivity ( si , p=0.029 ) compared to group 1 . 
 we identified significant up - regulation of 141 genes ( e.g. mmp9 and spp1 ) and down - regulation of 17 genes ( e.g. ndrg4 and gins3 ) in group 2 subjects . 
 intriguingly , these differentially expressed transcripts were enriched for genes involved in cardiovascular disease - related processes ( p=2.8110113.741002 ) and pathways involved in immune and inflammatory response ( p=8.321050.04 ) . 
 two down - regulated genes , ndrg4 and gins3 , have been located in a genomic interval associated with cardiac repolarization in published gwass and zebra fish knockout models . 
 our study provides evidence that perturbations in the adipose tissue gene expression network are important in defining metabolic health in obese subjects . 
Introduction
Subjects and Methods
Data Analysis
Results and Discussion
Supplementary Material