BACKGROUND
obesity, defined as abnormal or excessive fat accumulation, increases risks for multiple metabolic diseases, such as type  <dig> diabetes  and cardiovascular disease   <cit> .
adipose tissue is a highly specialized loose connective tissue able to store large quantities of triacylglycerol  and fat-soluble substances.
on a cytological level, adipose tissue is heterogeneous: the main parenchymal cells are the white adipocytes.
white adipocytes are characterized by a unilocular lipid droplet occupying 95% of the cell volume, and a ‘squeezed’ nucleus.
despite adipocytes represent ~90% of the tissue volume, other cells type are present in adipose tissue such as preadipocytes, endothelial cells, pericytes, multipotent stem cells and immune system cells .
it is generally regarded as a tissue without a specific anatomy.
however, there are increasing data supporting the idea that adipose depots are organized to form a large organ with discrete anatomy, specific vascular and nerve supplies, complex cytology, and high physiological plasticity  <cit> .
this organ is made up of several depots located in two main compartments of the body: subcutaneous adipose tissue  and visceral adipose tissues .
sat represents over 80% of total body fat and is most commonly distinguished in abdominal , gluteal-femoral depots .
vat, which is mostly associated with digestive organs, includes omental, mesenteric and epiploic adipose tissue depots.
the adipose organ contributes to many fundamental biological functions: thermogenesis, lactation, immune responses and obviously energy balance and energy substrates partitioning.
more than 50 years ago j. vague observed that not only fat mass but also adipose tissue distribution is clearly linked to cvd risk.
central obesity, characterized by an increase in vat and abdominal sat, confers increased risk of cvd, insulin resistance, t2dm and even all-cause mortality.
on the other hand peripheral obesity, characherized by preferential accumulation of gluteo-femoral fat is associated with lower risk and may be protective .
at an anatomical level, different depots differ in cellular composition, microvasculature, innervation, metabolic characteristics, extracellular matrix composition  <cit> .
moreover different studies showed a different pattern of adipokines secretion and endocrine function between abdominal and lower-body adipose .
however, the relative contribution of abdominal subcutaneous adipose depot to increased cardiovascular is still controversial  <cit> .
the heterogeneity between different subcutaneous depots has also been investigated: abdominal subcutaneous adypocytes differ from femoral ones in term of proliferation  <cit> , differentiation  <cit>  and fatty acid release and accumulation capacity  <cit> .
different adrenergic stimulation response  <cit>  and sexual hormones influence  <cit>  have also been reported, while data on possible differences in insulin effect among different subcutaneous depots are inconclusive  <cit> .
several studies suggest that different fat depots could arise from distinct precursors, derived from mesenchymal stem cells, with inherently different proliferative and adipogenic properties  <cit> .
gene expression profiling has identified different molecular signature of adipocyte lineage commitment.
these genes, such as nuclear transcriptional receptor protein coding gene nr2f <dig> , seem to play a major role in early cell-fate determination  <cit> .
similarly, another study underpinned that the profound functional differences between the upper-body and lower-body adipose tissues might be under control of site-specific sets of developmental genes, such as homebox  genes, a group of related genes that control the body plan of an embryo along the anterior-posterior axis  <cit> .
here we studied global mrna expression in gluteal and abdominal adipose tissues from eight healthy subjects, with a microarray approach, in order to detect regional differences in gene expression.
RESULTS
the anthropometric and metabolic characteristics of participants are shown in table  <dig> table  <dig> characteristics of all participants
in the microarray analysis,  <dig>  probes were detected in abdominal and gluteal adipose tissues.
considering  <dig>  fold change as lower limit, a total of  <dig> probes were differentially expressed between the abdominal and gluteal depot, corresponding for a total of  <dig> coding genes;  <dig> genes were up-regulated in abdominal versus gluteal adipose tissue .
most of the expression differences were modest, >80% in  <dig> – <dig> fold change range.
hierarchical clustering analysis of gene expression profiles in subcutaneous abdominal and gluteal adipose tissues of these  <dig> healthy subjects is represented in fig.
<dig> table  <dig> differential gene expression between abdominal and gluteal adipose tissue depots
hoxc12
<dig>
homo sapiens homeobox c <dig> , mrna
hoxb8
<dig>
homo sapiens homeobox b <dig> , mrna
tbx5
hoxa11
<dig>
homo sapiens homeobox a <dig> , mrna
hoxa13
<dig>
homo sapiens homeobox a <dig> , mrna
cyp19a1
nr2f1
<dig>
homo sapiens nuclear receptor subfamily  <dig>  group f, member  <dig> , mrna
hoxa5
<dig>
homo sapiens homeobox a <dig> , mrna
col4a5
igf2
pitx2
hoxa3
<dig>
homo sapiens homeobox a <dig> , transcript variant  <dig>  mrna
pck1
hoxb7
<dig>
homo sapiens homeobox b <dig> , mrna
foxp2
hoxc10
<dig>
homo sapiens homeobox c <dig> , mrna
skap2
adrb1
vegfc
pcdh9
tnnt3
tbc1d1
bdkrb2
tagln
des
tgfbi
nmt2
bold data was used to drive attention to the presence of many hox genes, in our gene list.
underlined data was used to identify genes involved in type  <dig> diabtes pathogenesis
fig.
<dig> gene clustering in abdominal and gluteal adipose tissue
we identified several hox genes expression differences between gluteal and abdominal depots .
mean fold change for hox gene group was  <dig>  , compared to  <dig>  mean fold change of entire group of differentially expressed genes.
hoxa <dig>  hoxa <dig>  hoxb <dig>  hoxb <dig>  were up-regulated in the abdominal depot, while hoxa <dig>  hoxa <dig>  hoxc <dig>  hoxc <dig> were up-regulated in gluteal adipose tissue, with hoxc <dig> expression being almost  <dig> times overexpressed in gluteal vs abdominal adipose tissue.
nr2f <dig> was up-regulated in abdominal vs gluteal adipose tissue .
in silico analysis with bioinformatics database support  showed that  <dig> out of  <dig> coding genes have been previously involved in type  <dig> diabetes pathogenesis : tbx <dig>  col4a <dig>  pitx <dig>  foxp <dig>  skap <dig>  adrb <dig>  vegfc, pcdh <dig> were up-regulated in the abdominal depot, while cyp19a <dig>  igf <dig>  pck <dig>  tnnt <dig>  tbc1d <dig>  bdkrb <dig>  tagln, des, tgfbi, nmt <dig> were up-regulated in gluteal adipose tissue.
discussion
adipose tissue is considered as the largest endocrine organ in humans, and includes numerous discrete anatomical depots.
there is evidence that different adipose tissue depots have different morphology, physiology and adipokine profiles.
although depot differences in adipocyte metabolism and endocrine function are clearly important in etiology of obesity related diseases, the relative contribution of vat compared to abdominal subcutaneous is still controversial.
moreover, there is little evidence of in vivo or in vitro differences between different subcutaneous adipose depot.
the advent of new technology that allows the characterization of entire transcriptomes has permitted to look from another perspective to adipose tissue depots heterogeneity and led to the hope that the properties of adipose tissue and differences between adipose tissue depots might be revealed to help discover new therapeutic avenues.
indeed, expression profiling has revealed significant differences in expression of hundreds of genes between different depots of adipose tissue in both rodents and humans , particularly developmental and patterning genes involved in cell differentiation, organogenesis, antero-posterior or dorso-ventral patterning.
these findings not only have contributed to help explain the distinct impact of these depots on the development of metabolic complications but also have suggested possible differences in developmental origin of these fat cells  <cit> .
adipose tissue has a mesodermal origin.
a layer of cells between the primitive endoderm and ectoderm migrates and spreads along the antero-posterior and dorso-ventral axes of the developing embryo generating the axial, intermediate, lateral plate, and paraxial mesoderm.
each of these embryologic tissues eventually gives rise to local adipose tissue.
vertebrate embryonic patterning and evolution of mesodermal tissues such as fat are controlled by several conserved developmental signaling systems.
the mesenchimal stem cell gives origin to an early precursor, the adipoblast, which develops into committed preadipocytes that under appropriate stimuli differentiate into mature adipocytes of different types.
in more recent studies, the differences in gene expression pattern have been shown to persist even after in vitro differentiation of preadipocytes, suggesting that the differences are independent of extrinsic factors and that different adipocyte progenitors are programmed through epigenetic modulation during early development, participating in determining functional differences observed between different adipose tissue depots  <cit> .
the most frequently observed differences in gene expression involve hox genes, a subset of homeobox genes.
a homeobox is a dna sequence, around  <dig> base pairs long, found within genes that are involved in the regulation of patterns of anatomical development  in animals, fungi and plants.
these genes encode homeodomain protein products that are transcription factors sharing a characteristic protein fold structure that binds dna.
through the dna-recognition properties of the homeodomain, homeoproteins are believed to regulate the expression of targeted genes and direct the formation of many body structures during early embryonic development.
many homeodomain proteins induce cellular differentiation by initiating the cascades of coregulated genes required to produce individual tissues and organs.
thus, homeobox genes are critical in the establishment of body axes during embryogenesis.
the hox genes in humans are organized in four chromosomal clusters: hoxa, hoxb, hoxc and hoxd.
in a  <dig> study from gesta et al., gene expression profiling has revealed that intraabdominal  adipocytes express higher levels of hoxa <dig>  hoxa <dig>  hoxc <dig> and nr2f <dig>  whereas subcutaneous fat has higher levels of hoxa <dig>  and hoxc <dig>  and in most cases, these differences are observed in both rodents and humans.
similar differences in development gene expression are observed in preadipocytes isolated from different adipose depots of rodents.
these differences in gene expression are large in magnitude , appear to be intrinsic, and persist during in vitro culture and differentiation, indicating that they are cell autonomous and independent of the tissue microenvironment.
in addition, the authors have shown that one of these developmental genes  exhibit changes in expression that closely correlate with the extent of obesity  and the pattern of fat distribution .
in recent study from karastergiou et al.
substantial differences in hox genes expression have been found between two different subcutaneous adipose tissue depots, gluteal and abdominal.
in our study, we confirm significant gene expression differences between abdominal and gluteal sat, as well seen in the hierarchical clustering analysis.
we underline differences in expression of developmental and patterning genes such as hox genes , as previously observed.
we have also noticed as some genes, that in previous studies have been proposed as moleculare signatures of vat, such as hoxa <dig> and nr2f <dig> appear to be up-regulated in abdominal compared to gluteal sat , suggesting a similarity between vat and abdominal sat .
furthermore, the finding that a consistent number of genes differentially expressed between gluteal and abdominal adipose depots have been previously correlated to pathogenesys of type  <dig> diabetes enforces our hypotesis that these depots may have a different impact in obesity associated metabolic complications.
limitations of the study
some limitations of the study must be underlined.
first, this research was conducted on a small sample of single sex subjects: males with an average age of 60 years old.
therefore our findings apply only to men in this specific age group.
secondly, we adopted > <dig>  fold change as significant threshold of differences between the two groups, as previously seen in similar researches, however this increases the possibility of false positive.
CONCLUSIONS
our study demonstrates that subcutaneous gluteal and abdominal adipose tissue depots have distinct expression profiles.
the finding of a different expression of hox genes, fundamental during the embryo development, suggests an early regional differentiation of sat, while the higher expression of hoxa <dig> and nr2f <dig>  two molecular signatures of visceral adipocytes, in a-sat suggests that this subcutaneous adipose depot could be more similar to vat compared to g-sat.
in conclusion we suggest that we should look at sat as composed of distinct depots with possibly different impact in obesity associated metabolic complications.

 obesity, defined as abnormal or excessive fat accumulation, increases risks for multiple metabolic diseases, such as type diabetes and cardiovascular disease. adipose tissue is a highly specialized loose connective tissue able to store large quantities of triacylglycerol and fat-soluble substances. on a cytological level, adipose tissue is heterogeneous: the main parenchymal cells are the white adipocytes. white adipocytes are characterized by a unilocular lipid droplet occupying 95% of the cell volume, and a ‘squeezed’ nucleus. despite adipocytes represent ~90% of the tissue volume, other cells type are present in adipose tissue such as preadipocytes, endothelial cells, pericytes, multipotent stem cells and immune system cells. it is generally regarded as a tissue without a specific anatomy. however, there are increasing data supporting the idea that adipose depots are organized to form a large organ with discrete anatomy, specific vascular and nerve supplies, complex cytology, and high physiological plasticity. this organ is made up of several depots located in two main compartments of the body: subcutaneous adipose tissue and visceral adipose tissues. sat represents over 80% of total body fat and is most commonly distinguished in abdominal, gluteal-femoral depots. vat, which is mostly associated with digestive organs, includes omental, mesenteric and epiploic adipose tissue depots. the adipose organ contributes to many fundamental biological functions: thermogenesis, lactation, immune responses and obviously energy balance and energy substrates partitioning. more than 50 years ago j. vague observed that not only fat mass but also adipose tissue distribution is clearly linked to cvd risk. central obesity, characterized by an increase in vat and abdominal sat, confers increased risk of cvd, insulin resistance, t2dm and even all-cause mortality. on the other hand peripheral obesity, characherized by preferential accumulation of gluteo-femoral fat is associated with lower risk and may be protective. at an anatomical level, different depots differ in cellular composition, microvasculature, innervation, metabolic characteristics, extracellular matrix composition. moreover different studies showed a different pattern of adipokines secretion and endocrine function between abdominal and lower-body adipose. however, the relative contribution of abdominal subcutaneous adipose depot to increased cardiovascular is still controversial. the heterogeneity between different subcutaneous depots has also been investigated: abdominal subcutaneous adypocytes differ from femoral ones in term of proliferation, differentiation and fatty acid release and accumulation capacity. different adrenergic stimulation response and sexual hormones influence have also been reported, while data on possible differences in insulin effect among different subcutaneous depots are inconclusive. several studies suggest that different fat depots could arise from distinct precursors, derived from mesenchymal stem cells, with inherently different proliferative and adipogenic properties. gene expression profiling has identified different molecular signature of adipocyte lineage commitment. these genes, such as nuclear transcriptional receptor protein coding gene nr2f, seem to play a major role in early cell-fate determination. similarly, another study underpinned that the profound functional differences between the upper-body and lower-body adipose tissues might be under control of site-specific sets of developmental genes, such as homebox genes, a group of related genes that control the body plan of an embryo along the anterior-posterior axis. here we studied global mrna expression in gluteal and abdominal adipose tissues from eight healthy subjects, with a microarray approach, in order to detect regional differences in gene expression.  the anthropometric and metabolic characteristics of participants are shown in table  table characteristics of all participants in the microarray analysis, probes were detected in abdominal and gluteal adipose tissues. considering fold change as lower limit, a total of probes were differentially expressed between the abdominal and gluteal depot, corresponding for a total of coding genes; genes were up-regulated in abdominal versus gluteal adipose tissue. most of the expression differences were modest, >80% in – fold change range. hierarchical clustering analysis of gene expression profiles in subcutaneous abdominal and gluteal adipose tissues of these healthy subjects is represented in fig.  table differential gene expression between abdominal and gluteal adipose tissue depots hoxc12 homo sapiens homeobox c, mrna hoxb8 homo sapiens homeobox b, mrna tbx5 hoxa11 homo sapiens homeobox a, mrna hoxa13 homo sapiens homeobox a, mrna cyp19a1 nr2f1 homo sapiens nuclear receptor subfamily group f, member, mrna hoxa5 homo sapiens homeobox a, mrna col4a5 igf2 pitx2 hoxa3 homo sapiens homeobox a, transcript variant mrna pck1 hoxb7 homo sapiens homeobox b, mrna foxp2 hoxc10 homo sapiens homeobox c, mrna skap2 adrb1 vegfc pcdh9 tnnt3 tbc1d1 bdkrb2 tagln des tgfbi nmt2 bold data was used to drive attention to the presence of many hox genes, in our gene list. underlined data was used to identify genes involved in type diabtes pathogenesis fig. gene clustering in abdominal and gluteal adipose tissue we identified several hox genes expression differences between gluteal and abdominal depots. mean fold change for hox gene group was, compared to mean fold change of entire group of differentially expressed genes. hoxa hoxa hoxb hoxb were up-regulated in the abdominal depot, while hoxa hoxa hoxc hoxc were up-regulated in gluteal adipose tissue, with hoxc expression being almost times overexpressed in gluteal vs abdominal adipose tissue. nr2f was up-regulated in abdominal vs gluteal adipose tissue. in silico analysis with bioinformatics database support showed that out of coding genes have been previously involved in type diabetes pathogenesis: tbx col4a pitx foxp skap adrb vegfc, pcdh were up-regulated in the abdominal depot, while cyp19a igf pck tnnt tbc1d bdkrb tagln, des, tgfbi, nmt were up-regulated in gluteal adipose tissue. discussion adipose tissue is considered as the largest endocrine organ in humans, and includes numerous discrete anatomical depots. there is evidence that different adipose tissue depots have different morphology, physiology and adipokine profiles. although depot differences in adipocyte metabolism and endocrine function are clearly important in etiology of obesity related diseases, the relative contribution of vat compared to abdominal subcutaneous is still controversial. moreover, there is little evidence of in vivo or in vitro differences between different subcutaneous adipose depot. the advent of new technology that allows the characterization of entire transcriptomes has permitted to look from another perspective to adipose tissue depots heterogeneity and led to the hope that the properties of adipose tissue and differences between adipose tissue depots might be revealed to help discover new therapeutic avenues. indeed, expression profiling has revealed significant differences in expression of hundreds of genes between different depots of adipose tissue in both rodents and humans, particularly developmental and patterning genes involved in cell differentiation, organogenesis, antero-posterior or dorso-ventral patterning. these findings not only have contributed to help explain the distinct impact of these depots on the development of metabolic complications but also have suggested possible differences in developmental origin of these fat cells. adipose tissue has a mesodermal origin. a layer of cells between the primitive endoderm and ectoderm migrates and spreads along the antero-posterior and dorso-ventral axes of the developing embryo generating the axial, intermediate, lateral plate, and paraxial mesoderm. each of these embryologic tissues eventually gives rise to local adipose tissue. vertebrate embryonic patterning and evolution of mesodermal tissues such as fat are controlled by several conserved developmental signaling systems. the mesenchimal stem cell gives origin to an early precursor, the adipoblast, which develops into committed preadipocytes that under appropriate stimuli differentiate into mature adipocytes of different types. in more recent studies, the differences in gene expression pattern have been shown to persist even after in vitro differentiation of preadipocytes, suggesting that the differences are independent of extrinsic factors and that different adipocyte progenitors are programmed through epigenetic modulation during early development, participating in determining functional differences observed between different adipose tissue depots. the most frequently observed differences in gene expression involve hox genes, a subset of homeobox genes. a homeobox is a dna sequence, around base pairs long, found within genes that are involved in the regulation of patterns of anatomical development in animals, fungi and plants. these genes encode homeodomain protein products that are transcription factors sharing a characteristic protein fold structure that binds dna. through the dna-recognition properties of the homeodomain, homeoproteins are believed to regulate the expression of targeted genes and direct the formation of many body structures during early embryonic development. many homeodomain proteins induce cellular differentiation by initiating the cascades of coregulated genes required to produce individual tissues and organs. thus, homeobox genes are critical in the establishment of body axes during embryogenesis. the hox genes in humans are organized in four chromosomal clusters: hoxa, hoxb, hoxc and hoxd. in a study from gesta et al., gene expression profiling has revealed that intraabdominal adipocytes express higher levels of hoxa hoxa hoxc and nr2f whereas subcutaneous fat has higher levels of hoxa and hoxc and in most cases, these differences are observed in both rodents and humans. similar differences in development gene expression are observed in preadipocytes isolated from different adipose depots of rodents. these differences in gene expression are large in magnitude, appear to be intrinsic, and persist during in vitro culture and differentiation, indicating that they are cell autonomous and independent of the tissue microenvironment. in addition, the authors have shown that one of these developmental genes exhibit changes in expression that closely correlate with the extent of obesity and the pattern of fat distribution. in recent study from karastergiou et al. substantial differences in hox genes expression have been found between two different subcutaneous adipose tissue depots, gluteal and abdominal. in our study, we confirm significant gene expression differences between abdominal and gluteal sat, as well seen in the hierarchical clustering analysis. we underline differences in expression of developmental and patterning genes such as hox genes, as previously observed. we have also noticed as some genes, that in previous studies have been proposed as moleculare signatures of vat, such as hoxa and nr2f appear to be up-regulated in abdominal compared to gluteal sat, suggesting a similarity between vat and abdominal sat. furthermore, the finding that a consistent number of genes differentially expressed between gluteal and abdominal adipose depots have been previously correlated to pathogenesys of type diabetes enforces our hypotesis that these depots may have a different impact in obesity associated metabolic complications. limitations of the study some limitations of the study must be underlined. first, this research was conducted on a small sample of single sex subjects: males with an average age of 60 years old. therefore our findings apply only to men in this specific age group. secondly, we adopted > fold change as significant threshold of differences between the two groups, as previously seen in similar researches, however this increases the possibility of false positive.  our study demonstrates that subcutaneous gluteal and abdominal adipose tissue depots have distinct expression profiles. the finding of a different expression of hox genes, fundamental during the embryo development, suggests an early regional differentiation of sat, while the higher expression of hoxa and nr2f two molecular signatures of visceral adipocytes, in a-sat suggests that this subcutaneous adipose depot could be more similar to vat compared to g-sat. in conclusion we suggest that we should look at sat as composed of distinct depots with possibly different impact in obesity associated metabolic complications. 
our data suggest that we should look at sat as composed of distinct depots with possibly different impact in obesity associated metabolic complications. expression profiling reveals significant differences in expression of several hox genes. the finding of a different expression of hox genes, fundamental during the embryo development, suggests an early regional differentiation of subcutaneous adipose depots. interestingly, two molecular signature of visceral adipocyte lineage, homebox genes hoxa and nr2f are up-regulated in a-sat versus g-sat by a fold change. moreover, the higher expression of hoxa and nr2f two molecular signatures of visceral adipocytes, in a-sat suggests that this subcutaneous adipose depot could be more similar to vat than g-sat. rna was isolated from g-sat and a-sat biopsy, from eight healthy subjects, and hybridized on rna microarray chips in order to detect regional differences in gene expression. there is recent evidence that differentiation between upper-body and lower-body adipose tissues might be under control of site-specific sets of developmental genes, such as homebox genes, a group of related genes that control the body plan of an embryo along the anterior-posterior axis. the relative contribution, in term of cardiovascular risk, of abdominal subcutaneous adipose tissue is still controversial with studies showing both a detrimental effect and a protective role. these regional differences have a key role in the pathogenesis of obesity-related diseases. our study shows that g-sat and a-sat have distinct expression profiles. here we studied global mrna expression in g-sat and a-sat with a microarray approach. accumulation of visceral adipose tissue is clearly associated with an increased risk of obesity-related diseases and all-cause mortality, whereas gluteal subcutaneous fat accumulation is associated with a lower risk.
 accumulation of visceral adipose tissue is clearly associated with an increased risk of obesity-related diseases and all-cause mortality, whereas gluteal subcutaneous fat accumulation is associated with a lower risk. the relative contribution, in term of cardiovascular risk, of abdominal subcutaneous adipose tissue is still controversial with studies showing both a detrimental effect and a protective role. animal and in vitro studies demonstrated that adipocytes from visceral and subcutaneous depots have distinct morphological, metabolic and functional characteristics. these regional differences have a key role in the pathogenesis of obesity-related diseases. there is recent evidence that differentiation between upper-body and lower-body adipose tissues might be under control of site-specific sets of developmental genes, such as homebox genes, a group of related genes that control the body plan of an embryo along the anterior-posterior axis. however, the possible heterogeneity between different subcutaneous regions has not been extensively investigated. here we studied global mrna expression in g-sat and a-sat with a microarray approach. rna was isolated from g-sat and a-sat biopsy, from eight healthy subjects, and hybridized on rna microarray chips in order to detect regional differences in gene expression.  a total of genes are significantly and differently expressed in a-sat and g-sat. expression profiling reveals significant differences in expression of several hox genes. interestingly, two molecular signature of visceral adipocyte lineage, homebox genes hoxa and nr2f are up-regulated in a-sat versus g-sat by a fold change.  our study shows that g-sat and a-sat have distinct expression profiles. the finding of a different expression of hox genes, fundamental during the embryo development, suggests an early regional differentiation of subcutaneous adipose depots. moreover, the higher expression of hoxa and nr2f two molecular signatures of visceral adipocytes, in a-sat suggests that this subcutaneous adipose depot could be more similar to vat than g-sat. our data suggest that we should look at sat as composed of distinct depots with possibly different impact in obesity associated metabolic complications. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords obesitymicroarrayadipose tissuesatvathomeobox genes042-2/2009issue-copyright-statement© the author 2017 