BACKGROUND
during the development of obesity, the many changes in adipose tissue eventually lead to the disruptions of its normal dynamic endocrine function.
besides its storage ability of excessive lipids, the adipose tissue produces and secretes many factors that are involved in the pathophysiology of obesity associated secondary diseases like diabetes, dyslipidemia, liver steatosis, hypertension and cardiovascular disease  <cit> .
obesity-associated organ-specific pathological states can be ensued from the dysregulation of circulating levels of adipokines and metabolites and ectopic fat storage within organs that normally do not accumulate fat, e.g.
liver and muscle.
excessive fat accumulation is accompanied by changes in tissue composition, architecture, and tissue remodeling  <cit> .
how these processes affect the function of tissues is not well understood.
it is known that individual differences in the development of adiposity are the result of genetic predisposition and environmental factors.
several studies in mice have identified quantitative trait loci  that influence various obesity related traits.
for example, qtls have been identified for regional adiposity  <cit> , diet induced obesity  <cit> , resistance to diet induced obesity  <cit> , juvenile obesity  <cit> , obesity associated diseases  <cit>  as well as lipid content in the liver  <cit> .
the dissection of fat depots  <cit> , dual energy x-ray absorptiometry   <cit> , or magnetic resonance imaging   <cit>  have been used to measure fat pad size and body fat distribution.
chromatographic techniques   <cit> , different biochemical reagent sets  <cit> , or enzymatic assays  <cit>  are used for metabolic profiling to obtain information about specific components such as cholesterol, triglycerides, glycerol and others.
most of these methods are time consuming and not suitable for liquid and solid samples without preparation.
furthermore, they cannot specify structural and compositional changes in the samples, simultaneously.
attenuated total reflectance  spectroscopy represents a well-established sampling method in infrared  spectroscopy  <cit> .
in atr-ftir spectroscopy an ir beam is typically guided through an ir transparent crystal  in a way that one  total internal reflections take place at the inner surface of the crystal.
this creates an evanescent or near-field standing wave at the boundary between the crystal and the surrounding media .
if the samples are prepared directly on the surface of the atr crystal their absorption properties can be observed as a result of attenuation of the evanescent wave.
the penetration depth of the radiation is typically at the order of the wavelength and depends on factors like the wavelength, the angle of incidence and of the refractive indices of the crystal material and the sample.
an atr spectrum thus carries only information of a thin sample layer close to the atr crystal.
the atr approach allows measurements of samples like blood, serum, or fully hydrated tissues samples, without too much interference from ir absorption of bulk water  <cit> .
drawbacks of the atr technique are possible molecular interactions between the crystal surface and the sample.
the ir method is based on the characteristic absorption of infrared radiation at specific wavelengths by functional groups like n–h, c=o, c–h and p=o  <cit> .
an ir spectrum carries specific information on the samples’ molecular composition and structure .
as each sample represents a highly characteristic combination of molecules, ir spectroscopy provides a phenotypic fingerprint that is complementary to genomic approaches to detect unique genetic variants between individuals.
its high sensitivity allows the detection of differences among subspecies or strains  <cit> .
one of the main advantages of the ir technique is speed.
results can be obtained within a few minutes.
furthermore, only a small amount of material with a minimal or no sample preparation is required to apply this method  <cit> .
therefore, atr-ftir spectroscopy can be considered as a high-throughput technique that is very practical for studies with high sample numbers as it is the case in genetic mapping experiments.
it is an automated, nondestructive, sensitive and reproducible technique, which is easy to perform  <cit> .
because of its many advantages, the ir spectroscopy has become established as a research tool in biomedical applications for body fluids  <cit> , pathological changes in tissues  <cit> , diagnosis of diseases  <cit> , and cell line classification and discrimination, in particular in cancerous tissue  <cit> .
in the present study, we applied the attenuated total reflectance fourier transform infrared spectroscopy  to obtain quantitative information about macromolecular composition in adipose tissue as major fat storage depot, and in liver and muscle, which may store ectopic fat.
the lipid to protein ratio and collagen integrity were also assessed to identify molecular and compositional changes within distinct tissues.
in the obese state, the expansion of adipose tissue is associated with an increased collagen content, which contributes to tissue stiffening and fibrosis  <cit> .
as remodeling of the tissue occurs, the alterations in the environment of the collagen amide bonds may cause changes in the collagen molecule structure.
unraveling of collagen triple helix can occur and be monitored from the spectral changes such as a shift in the absorbance frequency  <cit> .
the collagen band at  <dig> cm- <dig> is sensitive to the ordered structure of the triple helix.
reduction of the intensity of this band indicates collagen denaturation  <cit> .
therefore, changes in the area of this band account for changes in the ratio of areas of the amide ii band at  <dig> cm- <dig> to the collagen band at  <dig> cm- <dig>  which we refer to as collagen integrity  <cit> .
the goal of this study was to identify genes that regulate obesity in bxd recombinant inbred  strains of mice  <cit> .
the association of the genomic mosaic structure of the genomes of every ri strain with the phenotypic differences between strains allows the mapping of genomic regions accounting for the trait variance.
the bxd ri strains descend from crosses between c57bl/6j  and dba/2j  mice and subsequent inbreeding by repeated brother sister mating over more than  <dig> generations  <cit> .
they have been used to identify genetic factors contributing to health complications such as atherosclerosis  <cit> , blood pressure  <cit> , diabetes  <cit>  and obesity  <cit> .
the parental strains c57bl/6j and dba/2j, which were originally crossed to generate the set of bxd ri strains, also differ in their susceptibility to high fat diet induced obesity  <cit>  and regional fat storage patterns  <cit> .
combining the sensitive atr-ftir study with the well characterized genetic model, we found differences in the macromolecular composition of adipose and liver tissues among high fat diet-fed bxd ri strains, which led to the identification of new genomic regions that may control obesity-related diseases.
RESULTS
phenotypes of parental and bxd ri strains on high fat diet
after feeding a high fat diet over  <dig> weeks, b <dig> males gained more weight compared to d <dig> males .
they had more than two-fold higher relative contents of total fat, saturated fat, unsaturated fat, collagen and lipid to protein ratio  and  <dig>  fold lower relative collagen integrity in the adipose tissue.
similar results were found in the liver.
in contrast, muscles of b <dig> mice had a higher relative collagen integrity than d <dig> mice.
the value of each trait content was derived from spectral parameters for comparison and does not refer to the mass.
therefore, the unit is an arbitrary unit.
among the  <dig> bxd ri strains, the intra-strain variation was smaller than the inter-strain variation for all traits  indicating genetic heritability.
the  <dig> bxd ri strains also showed a large diversity for epididymal adipose tissue  and liver weights  and they differed widely in the macromolecular composition of epididymal adipose tissues and liver .
differences between strains were not significant in muscle.
many bxd ri strains exceeded the parental values for total, saturated and unsaturated fat contents, collagen content and lipid to protein ratio in adipose tissue and were closer to the f <dig> animals .
thus, the more extreme phenotypes of many bxd ri strains reflect novel combinations of additive and dominant allele effects in the bxd ri strains as well as novel epistatic interactions between the parental alleles  <cit> .
pearson’s correlation coefficients were estimated using strains means to evaluate the degree of relationship between traits in the population of all bxd ri strains .
high correlations  between traits imply that certain genes or genomic region could simultaneously influence these traits.
in our study, the weights of the epididymal adipose tissue and liver  as well as liver and muscle  were highly correlated.
for the macromolecular contents of all tissues, significant positive correlations were found between fat related traits.
negative correlations were observed between collagen and collagen integrity in all tissues examined.
in the epididymal adipose tissue, collagen integrity showed significant negative correlations with all traits except for the ratio of saturated to unsaturated fat .
collagen itself correlated positively with all other traits, except collagen integrity.
in the liver, no significant correlation was found between collagen and fat related traits.
in muscle, negative correlations of collagen with collagen integrity was found whereas glycogen was positively correlated with acyl chain length, and collagen integrity .
between tissues, significant correlations were found for collagen content and integrity between liver and muscle and between unsaturated fat content in epididymal adipose tissue and glycogen content in muscle.
the degree of significance was denoted as: p< <dig> *, p< <dig> **, p< <dig> ***, p values were adjusted for multiple testing.
genenetwork record id was given in parentheses for each trait.
regardless of the tissue, the estimated heritability was higher than  <dig>  for the content of fat, collagen and glycogen.
qtl analysis
linkage analyses provided evidence for two genomic regions contributing significantly to differences in the macromolecular composition of epididymal adipose tissue and liver.
a genomic region on chromosome   <dig> between  <dig> and  <dig> mb significantly affected the relative contents of total, saturated, and unsaturated fat, as well as the relative content of collagen, collagen integrity and lipid to protein ratio in adipose tissue .
the effect on the length of the acyl chain was suggestive.
the b <dig> allele of the chr  <dig> locus was the increasing allele for all traits, except for collagen integrity.
the pleiotropic effect of this locus is consistent with the high correlation between these traits .
bxd strains being homozygous for the b <dig> allele at the chr  <dig> qtl had about  <dig>  fold more relative contents of total, saturated and unsaturated fats, and collagen content and  <dig>  fold higher lipid to protein ratio than bxd strains being homozygous for the d <dig> allele.
the collagen integrity was  <dig>  fold lower in homozygous b <dig> than d <dig> carriers .
the degree of lrs significance was denoted as: suggestive*, significant**.
genome-wide significance  and suggestive  thresholds were calculated as likelihood ratio statistic  in linkage analyses computed for  <dig> permutations  <cit> .
the values of total, unsaturated and saturated fat, the ratio of saturated to unsaturated fat, collagen and collagen integrity, the ratio of lipid to protein and glycogen are relative values as described in experimental procedures.
genenetwork record id was given in parenthesis for each trait.
the  <dig> mb qtl region on chr  <dig> affecting the composition of adipose tissue is sparse of genes and harbours only  <dig> protein coding genes .
the genes encoding the radical s-adenosyl methionine domain containing  <dig>  and collectin sub-family member  <dig>  matched with the criteria for potential quantitative trait candidate genes, e.g.
location of the gene in the qtl region, expression in the target tissue, density of non-synonymous  snp and indels, cis-regulation of expression, and known or presumed gene function.
two nssnps between the parental strains b <dig> and d <dig> are located in exon  <dig> of rsad <dig>  the snp rs <dig> with the alternative alleles g and c in the strains b <dig> and d <dig>  respectively, leads to an amino acid exchange from aspartate  to glutamate  at the amino acid position  <dig> .
the snp rs <dig> with the alleles g and a in b <dig> and d <dig>  respectively, causes an amino acid exchange from proline  to leucine  at position  <dig> .
in the colec <dig> gene, the snp rs <dig> was found as non-synonymous polymorphism in exon  <dig> with the alleles c and g in b <dig> and d <dig>  respectively.
this snp causes an amino acid change from valine  to leucine  at the amino acid position  <dig> .
so far, no functional consequences were reported for those changes in the protein structures of rsad <dig> and colec <dig>
expression data of rsad <dig> and colec <dig> in adipose tissues of b <dig> and d <dig> on a standard diet are accessible at biogps  <cit> .
both genes are expressed in adipose tissue.
according to those data and normalisation of expression levels with housekeeping genes  rsad <dig> and colec <dig> transcript amounts are  <dig>  and  <dig>  times higher in b <dig> than in d <dig> mice, respectively, under standard diet conditions  <cit>  when d <dig> animals are heavier than b <dig> animals.
evidence for cis-regulation comes from genetic variation between b <dig> and d <dig> and expression qtls  for rsad <dig> and colec <dig> in segregating f2-populations.
for example, for both genes, eqtls were described for adipose tissues in the cross castxc57bl6/j , and in addition for colec <dig> in the cross c3h/jxc57bl6/j   mlratio; probeset  <dig> for colec <dig> in adipose tissue of cross castb6/b6cast f <dig>  database: ucla ctb6/b6ctf <dig> adipose  mlratio; probeset  <dig> for colec <dig> in adipose tissue of cross bh/hbf <dig>  database: ucla bhhbf <dig> adipose  mlratio).
because four out of  <dig> synonymous snps in the coding region of rsad <dig> that exist between b <dig> and d <dig> also occur between cast/j and b <dig> mice, it is very likely that the genetic variation in this gene could be responsible for expression or functional differences in both reference populations.
the measurement of transcript amounts of rsad <dig> and colec <dig> in our high fat diet fed bxd ri strains carrying the alternative b <dig> and d <dig> alleles of the respective genes revealed  <dig>  times higher mrna amounts of rsad <dig> of d <dig> carriers , while no expression difference was found for colec <dig>  .
these results suggest cis-acting genetic variation that interacts with environmental changes on the cellular level leading to differential gene activation of rsad <dig> under different diets.
a second significant qtl region affecting the lipid to protein ratio in the liver was found on chr  <dig> in an interval between  <dig> and  <dig> mb.
the same region influenced the relative content of total and saturated fat and acyl chain length.
for all traits, the b <dig> allele was the allele associated with an increase in phenotype value.
this gene-rich region harbours  <dig> protein coding genes , among them genes contributing to apolipoprotein production, mitochondrial function, and tissue structure.
because of the high gene number, it is presently not possible to perform a search for candidate quantitative trait genes.
in respect to glycogen content, no significant effect was found in adipose tissues.
suggestive genomic regions affecting glycogen content in the liver and in the muscle were detected on chromosomes  <dig> and  <dig>  respectively .
discussion
in the present study, a phenotypic characterization method, atr-ftir spectroscopy, was used to characterize structure and composition of adipose, muscle and liver tissue in  <dig> bxd ri mouse strains.
the main purpose of this study was to combine ir data with genetic information in order to identify specific gene loci responsible for the observed differences in the macromolecular tissue composition.
the sensitive tissue characterization and high quality of data obtained by atr-ftir technique allowed us to identify novel chromosomal regions contributing to distinct features of high fat diet induced obesity.
furthermore, the specific haplotype block structure of the set of bxd ri strains allows the mapping of trait associated effects to specific chromosomal regions: a  <dig> mb interval on chr  <dig> correlated with relative fat content in epididymal adipose tissue, and a  <dig> mb on chr  <dig> correlated with lipid to protein ratio in the liver.
we did not find significant qtls for the muscle phenotypes.
one reason for missing effects may be the lack of variance between the parental strains b <dig> and d <dig>  another reason may be the preparation method of the muscle tissue for atr-ftir measurements, because homogeneity is crucial in the ir spectroscopy to obtain accurate data from the tissue.
therefore, homogenous tissues like adipose and liver tissues give robust and reliable data.
in contrast, the quadriceps muscle is composed of four different muscle groups , which may differ in the macromolecular composition.
here, we used the whole sliver of the muscle.
this is likely the reason, why possible differences between mouse groups could not be detected.
the identified qtls on chr  <dig> and  <dig> in our study coincided with previously mapped qtls for adipose tissue, body and liver weights in the lgxsm advanced intercross line   <cit> .
but, the confidence intervals in our study are much smaller.
the small genomic qtl regions are the result of a high recombination rate during the generation of ri strains.
moreover, the whole genome sequence information of both parental strains b <dig> and d <dig> allows for the selection of candidate genes that could cause the phenotypic differences.
the mapping of highly correlated phenotypes such as the relative content of total, saturated and unsaturated fat and lipid to protein ratio to the same narrow chromosomal region on chr  <dig> suggests pleiotropic gene effects.
in other words, it is likely that a functional relationship exists between these correlated traits.
to nominate genes that might underlay the identified qtls, six filters were applied including location, expression in the target tissue, density of nssnp and indels, cis regulation of expression, and function of gene.
this approach and additional gene expression analyses which we performed with bxd ri strains on a hfd provided further data to suggest rsad <dig> on chr <dig> as a positional candidate gene affecting fat content variation in epididymal adipose tissue and the lipid to protein ratio in the liver, respectively.
higher transcript amounts of rsad <dig> transcripts were always found in the tissues with the lower fat deposition.
on sbd, d <dig> mice had leaner livers than b <dig> and on a hfd b <dig> was leaner than d <dig>  the observation of higher gene expression in the leaner tissue is in line with cis-acting genetic variation interacting with environmental factors that occur in different amounts in the cells, e.g.
fatty acids.
rsad <dig>  is an evolutionary conserved protein.
initially, it was identified as an antiviral protein of innate immunity to diverse pathogens  <cit> .
rsad <dig> is located on the cytosolic side of the endoplasmic reticulum  membrane through an n-terminal amphipathic α-helical domain.
this domain contains a leucine zipper motif, which is involved in protein-protein interactions and may affect the proper folding of the protein as well as anchoring the protein to the er  <cit> .
recently, it has also been shown that rsad <dig> co-localizes with the adipocyte differentiation-related protein , which is located in the outer phospholipid and protein layer of lipid droplets and interacts with farnesyl–diphosphate synthase, an enzyme, which is located on the er membrane and which is required for the generation of cholesterol  <cit> .
the overexpression of rsad <dig> inhibits the secretion of various soluble proteins, induces changes in the er morphology  <cit> .
the ability to change the er membrane could also affect the lipid droplet formation and morphology.
furthermore, rsad <dig> may also alter the lipid content and quantity in lipid droplets, which originate in the er, by binding farnesyl–diphosphate synthase  <cit> .
our gene expression data for rsad <dig> supports findings of other expression experiments showing that increased bodily fat content is associated with lower rsad <dig> expression  <cit> .
the co-localization of rsad <dig> on lipid droplets as lipid storage organelles, the association of lower gene expression with higher fat deposition and our genetic mapping results suggests that rsad <dig> controls the formation of lipid droplets while impairment of rsad <dig> likely enhances fat accumulation in lipid droplets.
it has been repeatedly demonstrated that obesity and obesity-induced inflammation can arise from er stress, by the accumulation of unfolded or misfolded proteins in the er lumen and by overburden the reserve capacity of the organelle to tolerate it  <cit> .
it has been shown that rsad <dig> is likely also required for the t-cell receptor mediated activation of nfkb and ap- <dig>  which are important regulators of inflammatory cytokine production in white adipose tissue .
given the functional properties of rsad <dig> for lipid biosynthesis and proper protein folding and er stress, rsad <dig> might affect the activation of inflammation in an obese status.
colec <dig> belongs to the c-type lectins of the collectin family that is composed of a collagen-like domain and a carbohydrate recognition domain  <cit> .
recently, colec <dig> has been identified as a member of the innate immune system.
the collectins have functions in inflammatory and allergic responses, apoptotic cell recognition and clearance, and in the adaptive immune system  <cit> .
in particular the function of apoptotic cell recognition and clearance of colec <dig> could contribute to the growth and maintenance of white adipose tissue by adipose tissue regeneration.
although we did not find gene expression differences of colec <dig>  mrna amounts, we cannot exclude protein quality changes that might affect the function of colec <dig> and thus the collagen content in epididymal adipose tissue.
the suggested candidate genes rsad <dig> and colec <dig> may also contribute to human diseases as mutations leading to deleterious functional changes in the proteins have been identified in humans   <cit> .
with regard to the qtl for lipid to protein ratio on chr <dig>  further fine mapping is necessary to reduce the number of positional candidate genes.
because this qtl resides in a gene-rich genomic region, many potential candidate genes were predicted.
among the  <dig> protein coding genes in the qtl interval there are several that might affect the lipid content and the lipid to protein ratio in the liver.
for fine mapping additional bxd strains could be phenotypically characterized, which harbor recombinations in the qtl confidence interval.
CONCLUSIONS
in the present study, we introduced the analytical technique of atr-ftir spectroscopy as a novel phenotyping method that allows to characterize the macromolecular composition of different tissues and that minimizes the measuring error arising from non-molecular methods.
using this sensitive method, we found differences among the bxd ri strains with respect to the trait of interest, which reflect genetic variation.
the analyses revealed two genomic regions that may have a function in obesity-induced tissue dsyfunction.
as candidate gene on chr  <dig> we suggest rsad <dig>  which may modulate lipid droplet contents and lipid biosynthesis, and colec <dig>  which may play a role in apoptotic cell clearance and maintenance of adipose tissue.
further functional studies, for example with transgenic or knockout models, are required to validate these candidate genes.
for the qtl on chr <dig>  fine mapping is necessary to reduce the number of candidate genes.
however, even if the qtl results are significant, they were identified in a small population of just  <dig> bxd strains.
therefore, a replication study with a bigger panel of bxd strains would be necessary to verify the findings.
our study represents a first and crucial step to identify genetic causes of alterations in the tissue specific macromolecular composition in the obese state.
these results will help to gain detailed insight into individual and tissue-specific responses to high fat diet as a function of macromolecular composition and genetic factors that are important in the pathogenicity of obesity and obesity-associated clinical complications.
furthermore, our results showed the suitability and efficiency of ir spectroscopy to detect component specific changes since modes of vibrations of each group are dependent on changes in chemical composition, structures, conformation and environment.
therefore, combination of analytical techniques such as mass and vibrational spectroscopy with genetic approaches are suggested to help in identifying tissues with altered physiology, increase the power of qtl analysis and support a better understanding of underlying mechanisms.

 during the development of obesity, the many changes in adipose tissue eventually lead to the disruptions of its normal dynamic endocrine function. besides its storage ability of excessive lipids, the adipose tissue produces and secretes many factors that are involved in the pathophysiology of obesity associated secondary diseases like diabetes, dyslipidemia, liver steatosis, hypertension and cardiovascular disease. obesity-associated organ-specific pathological states can be ensued from the dysregulation of circulating levels of adipokines and metabolites and ectopic fat storage within organs that normally do not accumulate fat, e.g. liver and muscle. excessive fat accumulation is accompanied by changes in tissue composition, architecture, and tissue remodeling. how these processes affect the function of tissues is not well understood. it is known that individual differences in the development of adiposity are the result of genetic predisposition and environmental factors. several studies in mice have identified quantitative trait loci that influence various obesity related traits. for example, qtls have been identified for regional adiposity, diet induced obesity, resistance to diet induced obesity, juvenile obesity, obesity associated diseases as well as lipid content in the liver. the dissection of fat depots, dual energy x-ray absorptiometry, or magnetic resonance imaging have been used to measure fat pad size and body fat distribution. chromatographic techniques, different biochemical reagent sets, or enzymatic assays are used for metabolic profiling to obtain information about specific components such as cholesterol, triglycerides, glycerol and others. most of these methods are time consuming and not suitable for liquid and solid samples without preparation. furthermore, they cannot specify structural and compositional changes in the samples, simultaneously. attenuated total reflectance spectroscopy represents a well-established sampling method in infrared spectroscopy. in atr-ftir spectroscopy an ir beam is typically guided through an ir transparent crystal in a way that one total internal reflections take place at the inner surface of the crystal. this creates an evanescent or near-field standing wave at the boundary between the crystal and the surrounding media. if the samples are prepared directly on the surface of the atr crystal their absorption properties can be observed as a result of attenuation of the evanescent wave. the penetration depth of the radiation is typically at the order of the wavelength and depends on factors like the wavelength, the angle of incidence and of the refractive indices of the crystal material and the sample. an atr spectrum thus carries only information of a thin sample layer close to the atr crystal. the atr approach allows measurements of samples like blood, serum, or fully hydrated tissues samples, without too much interference from ir absorption of bulk water. drawbacks of the atr technique are possible molecular interactions between the crystal surface and the sample. the ir method is based on the characteristic absorption of infrared radiation at specific wavelengths by functional groups like n–h, c=o, c–h and p=o. an ir spectrum carries specific information on the samples’ molecular composition and structure. as each sample represents a highly characteristic combination of molecules, ir spectroscopy provides a phenotypic fingerprint that is complementary to genomic approaches to detect unique genetic variants between individuals. its high sensitivity allows the detection of differences among subspecies or strains. one of the main advantages of the ir technique is speed. results can be obtained within a few minutes. furthermore, only a small amount of material with a minimal or no sample preparation is required to apply this method. therefore, atr-ftir spectroscopy can be considered as a high-throughput technique that is very practical for studies with high sample numbers as it is the case in genetic mapping experiments. it is an automated, nondestructive, sensitive and reproducible technique, which is easy to perform. because of its many advantages, the ir spectroscopy has become established as a research tool in biomedical applications for body fluids, pathological changes in tissues, diagnosis of diseases, and cell line classification and discrimination, in particular in cancerous tissue. in the present study, we applied the attenuated total reflectance fourier transform infrared spectroscopy to obtain quantitative information about macromolecular composition in adipose tissue as major fat storage depot, and in liver and muscle, which may store ectopic fat. the lipid to protein ratio and collagen integrity were also assessed to identify molecular and compositional changes within distinct tissues. in the obese state, the expansion of adipose tissue is associated with an increased collagen content, which contributes to tissue stiffening and fibrosis. as remodeling of the tissue occurs, the alterations in the environment of the collagen amide bonds may cause changes in the collagen molecule structure. unraveling of collagen triple helix can occur and be monitored from the spectral changes such as a shift in the absorbance frequency. the collagen band at cm- is sensitive to the ordered structure of the triple helix. reduction of the intensity of this band indicates collagen denaturation. therefore, changes in the area of this band account for changes in the ratio of areas of the amide ii band at cm- to the collagen band at cm- which we refer to as collagen integrity. the goal of this study was to identify genes that regulate obesity in bxd recombinant inbred strains of mice. the association of the genomic mosaic structure of the genomes of every ri strain with the phenotypic differences between strains allows the mapping of genomic regions accounting for the trait variance. the bxd ri strains descend from crosses between c57bl/6j and dba/2j mice and subsequent inbreeding by repeated brother sister mating over more than generations. they have been used to identify genetic factors contributing to health complications such as atherosclerosis, blood pressure, diabetes and obesity. the parental strains c57bl/6j and dba/2j, which were originally crossed to generate the set of bxd ri strains, also differ in their susceptibility to high fat diet induced obesity and regional fat storage patterns. combining the sensitive atr-ftir study with the well characterized genetic model, we found differences in the macromolecular composition of adipose and liver tissues among high fat diet-fed bxd ri strains, which led to the identification of new genomic regions that may control obesity-related diseases.  phenotypes of parental and bxd ri strains on high fat diet after feeding a high fat diet over weeks, b males gained more weight compared to d males. they had more than two-fold higher relative contents of total fat, saturated fat, unsaturated fat, collagen and lipid to protein ratio and fold lower relative collagen integrity in the adipose tissue. similar results were found in the liver. in contrast, muscles of b mice had a higher relative collagen integrity than d mice. the value of each trait content was derived from spectral parameters for comparison and does not refer to the mass. therefore, the unit is an arbitrary unit. among the bxd ri strains, the intra-strain variation was smaller than the inter-strain variation for all traits indicating genetic heritability. the bxd ri strains also showed a large diversity for epididymal adipose tissue and liver weights and they differed widely in the macromolecular composition of epididymal adipose tissues and liver. differences between strains were not significant in muscle. many bxd ri strains exceeded the parental values for total, saturated and unsaturated fat contents, collagen content and lipid to protein ratio in adipose tissue and were closer to the f animals. thus, the more extreme phenotypes of many bxd ri strains reflect novel combinations of additive and dominant allele effects in the bxd ri strains as well as novel epistatic interactions between the parental alleles. pearson’s correlation coefficients were estimated using strains means to evaluate the degree of relationship between traits in the population of all bxd ri strains. high correlations between traits imply that certain genes or genomic region could simultaneously influence these traits. in our study, the weights of the epididymal adipose tissue and liver as well as liver and muscle were highly correlated. for the macromolecular contents of all tissues, significant positive correlations were found between fat related traits. negative correlations were observed between collagen and collagen integrity in all tissues examined. in the epididymal adipose tissue, collagen integrity showed significant negative correlations with all traits except for the ratio of saturated to unsaturated fat. collagen itself correlated positively with all other traits, except collagen integrity. in the liver, no significant correlation was found between collagen and fat related traits. in muscle, negative correlations of collagen with collagen integrity was found whereas glycogen was positively correlated with acyl chain length, and collagen integrity. between tissues, significant correlations were found for collagen content and integrity between liver and muscle and between unsaturated fat content in epididymal adipose tissue and glycogen content in muscle. the degree of significance was denoted as: p< *, p< **, p< ***, p values were adjusted for multiple testing. genenetwork record id was given in parentheses for each trait. regardless of the tissue, the estimated heritability was higher than for the content of fat, collagen and glycogen. qtl analysis linkage analyses provided evidence for two genomic regions contributing significantly to differences in the macromolecular composition of epididymal adipose tissue and liver. a genomic region on chromosome between and mb significantly affected the relative contents of total, saturated, and unsaturated fat, as well as the relative content of collagen, collagen integrity and lipid to protein ratio in adipose tissue. the effect on the length of the acyl chain was suggestive. the b allele of the chr locus was the increasing allele for all traits, except for collagen integrity. the pleiotropic effect of this locus is consistent with the high correlation between these traits. bxd strains being homozygous for the b allele at the chr qtl had about fold more relative contents of total, saturated and unsaturated fats, and collagen content and fold higher lipid to protein ratio than bxd strains being homozygous for the d allele. the collagen integrity was fold lower in homozygous b than d carriers. the degree of lrs significance was denoted as: suggestive*, significant**. genome-wide significance and suggestive thresholds were calculated as likelihood ratio statistic in linkage analyses computed for permutations. the values of total, unsaturated and saturated fat, the ratio of saturated to unsaturated fat, collagen and collagen integrity, the ratio of lipid to protein and glycogen are relative values as described in experimental procedures. genenetwork record id was given in parenthesis for each trait. the mb qtl region on chr affecting the composition of adipose tissue is sparse of genes and harbours only protein coding genes. the genes encoding the radical s-adenosyl methionine domain containing and collectin sub-family member matched with the criteria for potential quantitative trait candidate genes, e.g. location of the gene in the qtl region, expression in the target tissue, density of non-synonymous snp and indels, cis-regulation of expression, and known or presumed gene function. two nssnps between the parental strains b and d are located in exon of rsad the snp rs with the alternative alleles g and c in the strains b and d respectively, leads to an amino acid exchange from aspartate to glutamate at the amino acid position. the snp rs with the alleles g and a in b and d respectively, causes an amino acid exchange from proline to leucine at position. in the colec gene, the snp rs was found as non-synonymous polymorphism in exon with the alleles c and g in b and d respectively. this snp causes an amino acid change from valine to leucine at the amino acid position. so far, no functional consequences were reported for those changes in the protein structures of rsad and colec expression data of rsad and colec in adipose tissues of b and d on a standard diet are accessible at biogps. both genes are expressed in adipose tissue. according to those data and normalisation of expression levels with housekeeping genes rsad and colec transcript amounts are and times higher in b than in d mice, respectively, under standard diet conditions when d animals are heavier than b animals. evidence for cis-regulation comes from genetic variation between b and d and expression qtls for rsad and colec in segregating f2-populations. for example, for both genes, eqtls were described for adipose tissues in the cross castxc57bl6/j, and in addition for colec in the cross c3h/jxc57bl6/j mlratio; probeset for colec in adipose tissue of cross castb6/b6cast f database: ucla ctb6/b6ctf adipose mlratio; probeset for colec in adipose tissue of cross bh/hbf database: ucla bhhbf adipose mlratio). because four out of synonymous snps in the coding region of rsad that exist between b and d also occur between cast/j and b mice, it is very likely that the genetic variation in this gene could be responsible for expression or functional differences in both reference populations. the measurement of transcript amounts of rsad and colec in our high fat diet fed bxd ri strains carrying the alternative b and d alleles of the respective genes revealed times higher mrna amounts of rsad of d carriers, while no expression difference was found for colec. these results suggest cis-acting genetic variation that interacts with environmental changes on the cellular level leading to differential gene activation of rsad under different diets. a second significant qtl region affecting the lipid to protein ratio in the liver was found on chr in an interval between and mb. the same region influenced the relative content of total and saturated fat and acyl chain length. for all traits, the b allele was the allele associated with an increase in phenotype value. this gene-rich region harbours protein coding genes, among them genes contributing to apolipoprotein production, mitochondrial function, and tissue structure. because of the high gene number, it is presently not possible to perform a search for candidate quantitative trait genes. in respect to glycogen content, no significant effect was found in adipose tissues. suggestive genomic regions affecting glycogen content in the liver and in the muscle were detected on chromosomes and respectively. discussion in the present study, a phenotypic characterization method, atr-ftir spectroscopy, was used to characterize structure and composition of adipose, muscle and liver tissue in bxd ri mouse strains. the main purpose of this study was to combine ir data with genetic information in order to identify specific gene loci responsible for the observed differences in the macromolecular tissue composition. the sensitive tissue characterization and high quality of data obtained by atr-ftir technique allowed us to identify novel chromosomal regions contributing to distinct features of high fat diet induced obesity. furthermore, the specific haplotype block structure of the set of bxd ri strains allows the mapping of trait associated effects to specific chromosomal regions: a mb interval on chr correlated with relative fat content in epididymal adipose tissue, and a mb on chr correlated with lipid to protein ratio in the liver. we did not find significant qtls for the muscle phenotypes. one reason for missing effects may be the lack of variance between the parental strains b and d another reason may be the preparation method of the muscle tissue for atr-ftir measurements, because homogeneity is crucial in the ir spectroscopy to obtain accurate data from the tissue. therefore, homogenous tissues like adipose and liver tissues give robust and reliable data. in contrast, the quadriceps muscle is composed of four different muscle groups, which may differ in the macromolecular composition. here, we used the whole sliver of the muscle. this is likely the reason, why possible differences between mouse groups could not be detected. the identified qtls on chr and in our study coincided with previously mapped qtls for adipose tissue, body and liver weights in the lgxsm advanced intercross line. but, the confidence intervals in our study are much smaller. the small genomic qtl regions are the result of a high recombination rate during the generation of ri strains. moreover, the whole genome sequence information of both parental strains b and d allows for the selection of candidate genes that could cause the phenotypic differences. the mapping of highly correlated phenotypes such as the relative content of total, saturated and unsaturated fat and lipid to protein ratio to the same narrow chromosomal region on chr suggests pleiotropic gene effects. in other words, it is likely that a functional relationship exists between these correlated traits. to nominate genes that might underlay the identified qtls, six filters were applied including location, expression in the target tissue, density of nssnp and indels, cis regulation of expression, and function of gene. this approach and additional gene expression analyses which we performed with bxd ri strains on a hfd provided further data to suggest rsad on chr as a positional candidate gene affecting fat content variation in epididymal adipose tissue and the lipid to protein ratio in the liver, respectively. higher transcript amounts of rsad transcripts were always found in the tissues with the lower fat deposition. on sbd, d mice had leaner livers than b and on a hfd b was leaner than d the observation of higher gene expression in the leaner tissue is in line with cis-acting genetic variation interacting with environmental factors that occur in different amounts in the cells, e.g. fatty acids. rsad is an evolutionary conserved protein. initially, it was identified as an antiviral protein of innate immunity to diverse pathogens. rsad is located on the cytosolic side of the endoplasmic reticulum membrane through an n-terminal amphipathic α-helical domain. this domain contains a leucine zipper motif, which is involved in protein-protein interactions and may affect the proper folding of the protein as well as anchoring the protein to the er. recently, it has also been shown that rsad co-localizes with the adipocyte differentiation-related protein, which is located in the outer phospholipid and protein layer of lipid droplets and interacts with farnesyl–diphosphate synthase, an enzyme, which is located on the er membrane and which is required for the generation of cholesterol. the overexpression of rsad inhibits the secretion of various soluble proteins, induces changes in the er morphology. the ability to change the er membrane could also affect the lipid droplet formation and morphology. furthermore, rsad may also alter the lipid content and quantity in lipid droplets, which originate in the er, by binding farnesyl–diphosphate synthase. our gene expression data for rsad supports findings of other expression experiments showing that increased bodily fat content is associated with lower rsad expression. the co-localization of rsad on lipid droplets as lipid storage organelles, the association of lower gene expression with higher fat deposition and our genetic mapping results suggests that rsad controls the formation of lipid droplets while impairment of rsad likely enhances fat accumulation in lipid droplets. it has been repeatedly demonstrated that obesity and obesity-induced inflammation can arise from er stress, by the accumulation of unfolded or misfolded proteins in the er lumen and by overburden the reserve capacity of the organelle to tolerate it. it has been shown that rsad is likely also required for the t-cell receptor mediated activation of nfkb and ap- which are important regulators of inflammatory cytokine production in white adipose tissue. given the functional properties of rsad for lipid biosynthesis and proper protein folding and er stress, rsad might affect the activation of inflammation in an obese status. colec belongs to the c-type lectins of the collectin family that is composed of a collagen-like domain and a carbohydrate recognition domain. recently, colec has been identified as a member of the innate immune system. the collectins have functions in inflammatory and allergic responses, apoptotic cell recognition and clearance, and in the adaptive immune system. in particular the function of apoptotic cell recognition and clearance of colec could contribute to the growth and maintenance of white adipose tissue by adipose tissue regeneration. although we did not find gene expression differences of colec mrna amounts, we cannot exclude protein quality changes that might affect the function of colec and thus the collagen content in epididymal adipose tissue. the suggested candidate genes rsad and colec may also contribute to human diseases as mutations leading to deleterious functional changes in the proteins have been identified in humans. with regard to the qtl for lipid to protein ratio on chr further fine mapping is necessary to reduce the number of positional candidate genes. because this qtl resides in a gene-rich genomic region, many potential candidate genes were predicted. among the protein coding genes in the qtl interval there are several that might affect the lipid content and the lipid to protein ratio in the liver. for fine mapping additional bxd strains could be phenotypically characterized, which harbor recombinations in the qtl confidence interval.  in the present study, we introduced the analytical technique of atr-ftir spectroscopy as a novel phenotyping method that allows to characterize the macromolecular composition of different tissues and that minimizes the measuring error arising from non-molecular methods. using this sensitive method, we found differences among the bxd ri strains with respect to the trait of interest, which reflect genetic variation. the analyses revealed two genomic regions that may have a function in obesity-induced tissue dsyfunction. as candidate gene on chr we suggest rsad which may modulate lipid droplet contents and lipid biosynthesis, and colec which may play a role in apoptotic cell clearance and maintenance of adipose tissue. further functional studies, for example with transgenic or knockout models, are required to validate these candidate genes. for the qtl on chr fine mapping is necessary to reduce the number of candidate genes. however, even if the qtl results are significant, they were identified in a small population of just bxd strains. therefore, a replication study with a bigger panel of bxd strains would be necessary to verify the findings. our study represents a first and crucial step to identify genetic causes of alterations in the tissue specific macromolecular composition in the obese state. these results will help to gain detailed insight into individual and tissue-specific responses to high fat diet as a function of macromolecular composition and genetic factors that are important in the pathogenicity of obesity and obesity-associated clinical complications. furthermore, our results showed the suitability and efficiency of ir spectroscopy to detect component specific changes since modes of vibrations of each group are dependent on changes in chemical composition, structures, conformation and environment. therefore, combination of analytical techniques such as mass and vibrational spectroscopy with genetic approaches are suggested to help in identifying tissues with altered physiology, increase the power of qtl analysis and support a better understanding of underlying mechanisms. 
the analysis of genotype-phenotype correlations revealed significant quantitative trait loci on chromosome for the content of fat and collagen, collagen integrity, and the lipid to protein ratio in adipose tissue and on chromosome for lipid to protein ratio in liver. in the present study, the analytical method of atr-ftir spectroscopy has been combined with a genetic approach to identify genetic differences responsible for phenotypic alterations in adipose, liver and muscle tissues. mice from bxd recombinant inbred mouse strains were put on high fat diet and gross-compositional changes in adipose, liver and muscle tissues were measured by atr-ftir spectroscopy. the results demonstrate that the analytical method of atr-ftir spectroscopy effectively contributed to decompose the macromolecular composition of tissues that accumulate fat and to link this information with genetic determinants. obesity-associated organ-specific pathological states can be ensued from the dysregulation of the functions of the adipose tissues, liver and muscle. using gene expression and sequence information, we suggest rsad and colec on chromosome as potential quantitative trait candidate genes. the candidate genes in the qtl regions may contribute to obesity-related diseases in humans, in particular if the results can be verified in a bigger bxd cohort. rsad may act as a modulator of lipid droplet contents and lipid biosynthesis; colec might play a role in apoptopic cell clearance and maintenance of adipose tissue. an increased level of rsad transcripts in adipose tissue of dba/2j compared to c57bl/6j mice suggests a cis-acting genetic variant leading to differential gene activation.
 obesity-associated organ-specific pathological states can be ensued from the dysregulation of the functions of the adipose tissues, liver and muscle. however, the influence of genetic differences underlying gross-compositional differences in these tissues is largely unknown. in the present study, the analytical method of atr-ftir spectroscopy has been combined with a genetic approach to identify genetic differences responsible for phenotypic alterations in adipose, liver and muscle tissues.  mice from bxd recombinant inbred mouse strains were put on high fat diet and gross-compositional changes in adipose, liver and muscle tissues were measured by atr-ftir spectroscopy. the analysis of genotype-phenotype correlations revealed significant quantitative trait loci on chromosome for the content of fat and collagen, collagen integrity, and the lipid to protein ratio in adipose tissue and on chromosome for lipid to protein ratio in liver. using gene expression and sequence information, we suggest rsad and colec on chromosome as potential quantitative trait candidate genes. rsad may act as a modulator of lipid droplet contents and lipid biosynthesis; colec might play a role in apoptopic cell clearance and maintenance of adipose tissue. an increased level of rsad transcripts in adipose tissue of dba/2j compared to c57bl/6j mice suggests a cis-acting genetic variant leading to differential gene activation.  the results demonstrate that the analytical method of atr-ftir spectroscopy effectively contributed to decompose the macromolecular composition of tissues that accumulate fat and to link this information with genetic determinants. the candidate genes in the qtl regions may contribute to obesity-related diseases in humans, in particular if the results can be verified in a bigger bxd cohort. collagenendoplasmic reticulumapoptosisremodelingliver steatosisviperincollectin-11 