BACKGROUND
serum testosterone levels decline gradually and progressively with aging in men.
low levels of testosterone are associated with metabolic disorders, including obesity, dyslipidemia, hypertension, and insulin resistance  <cit> , all of which contribute to the development of nonalcoholic fatty liver disease   <cit> .
recent studies have shown that men with low serum testosterone levels have a higher risk of developing hepatic steatosis  <cit> .
moreover, animal studies have demonstrated that the incidence of hepatic steatosis is increased in testosterone-deficient male mice  <cit> .
these findings indicate the important role of testosterone in the pathophysiology of nafld.
however, nutritional parameters, especially a high-cholesterol diet, have been also associated with the development of nafld  <cit> .
hypercholesterolemia induced by dietary cholesterol has recently been indicated as a major risk factor for hepatic steatosis  <cit> .
in the absence of testosterone, mice develop hypercholesterolemia when fed a diet with no added cholesterol  <cit> .
hypercholesterolemia and elevated serum cholesterol levels are also observed in hypogonadal men  <cit> .
therefore, these previous findings suggest that testosterone deficiency and testosterone deficiency-induced hypercholesterolemia may aggravate the severity of nafld induced by a high-cholesterol diet.
however, the mechanisms underlying the effects of testosterone deficiency on the promotion of diet-induced hepatic steatosis remain unclear.
previous studies have used rodent models to investigate the influence of testosterone on the development of hepatic steatosis.
for example, hepatic androgen receptor-knockout male mice receiving a high-fat diet develop insulin resistance and hepatic steatosis  <cit> .
moreover, dihydrotestosterone suppresses diet-induced hepatic steatosis in castrated male rats  <cit> , and hepatic lipid deposition is elevated in testicular feminized mice  compared with intact wild-type littermate controls  <cit> .
although rodents are commonly used to study hepatic steatosis and have provided valuable insights into the pathogenesis of this disease, the relevance of these models to human disease is limited  <cit> .
as an alternative to rodent models, large animal models, such as pigs, may be more relevant to the study of nafld because these models more closely mimic human physiology and anatomy  <cit> .
importantly, castration-induced sex hormone deficiency in male pigs results in obesity, elevated serum triglycerides , and increased cholesterol levels  <cit> , some of the typical causes of nafld, suggesting that pigs are suitable models for studying the effects of sex hormones on nafld development.
despite the importance of testosterone in the regulation of hepatic steatosis, the genomic mechanisms through which testosterone deficiency aggravates diet-induced hepatic steatosis are still unclear.
transcriptome profiling is an effective and widely used tool to identify critical genes and pathways involved in pathological processes.
sequencing-based and hybridization-based approaches represent the two major approaches used in transcriptomics studies  <cit> .
high-throughput rna sequencing  has recently become an attractive method of choice in the study of transcriptomes, providing several advantages over microarrays.
in this study, we sought to gain insights into the roles of testosterone in modulating hepatic steatosis using a porcine model of testosterone deficiency and diet-induced hepatic steatosis.
additionally, we examined the contribution of liver genes to the development of hepatic steatosis induced by testosterone deficiency and a high-cholesterol diet using rna-seq.
to the best of our knowledge, this is the first description of differences in whole-transcriptome patterns occurring in the liver following castration in pigs fed a high-cholesterol diet.
RESULTS
body weights and serum testosterone levels
pigs were separated into three groups: intact male pigs fed a high-fat and high-cholesterol  diet ; castrated male pigs fed an hfc diet ; and castrated male pigs fed an hfc diet and given testosterone replacement therapy .
the body weights of pigs in each group were calculated and found to be linearly elevated over time.
initial body weights were similar in pigs from the im, cm, and cmt groups .
castrated pigs fed an hfc diet gained less weight than did pigs in the other groups.
with testosterone replacement, the body weights of cmt pigs increased and were similar to the body weights of pigs in the im group .figure  <dig>
effects of castration and testosterone treatment on body weights and serum testosterone concentrations.
body weights.
serum testosterone concentrations.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
data are expressed as means ± sems, n =  <dig> per group.
**p <  <dig> .
serum testosterone concentrations were not significantly different between groups before the study began .
as expected, castration caused a significant decrease in serum testosterone levels.
the concentration of serum testosterone at 12 weeks was significantly lower in cm pigs than in im pigs .
testosterone treatment increased serum testosterone levels to  <dig>  ±  <dig>  ng/ml .
liver weight, body fat content, and serum biochemical parameters
liver weight, body fat content, and serum biochemical parameters after 12 weeks of hfc feeding are presented in table  <dig>  the liver weights in cm pigs were higher than those observed in im pigs, but lower than those in cmt pigs .
liver weight indexes in cm pigs were higher than those in im and cmt pigs, but these differences were not significant .
cm pigs had higher body fat percentages than im  and cmt pigs .
serum alanine aminotransferase  levels were significantly increased in cm pigs compared with im + hfc pigs  and significantly reduced in cmt pigs by testosterone replacement .
aspartate aminotransferase  levels were highest in cm pigs, but the differences were not significant among groups .
compared with im and cmt pigs, cm pigs showed obviously lower serum levels of superoxide dismutase  but higher levels of malondialdehyde .
serum free fatty acid  levels tended to be higher in cm pigs than in im and cmt pigs.
fasting serum glucose and insulin levels and homeostatic model assessment of insulin resistance  were not significantly different between cm and im pigs .
however, testosterone replacement significant reduced serum insulin levels and homa-ir in cm pigs .table  <dig>
liver weight, body fat content and serum biochemical parameters in miniature pigs fed a high-fat and high-cholesterol diet
im
cm
cmt
*p <  <dig>  and **p <  <dig> , im vs. cm; #
p <  <dig>  and ##
p <  <dig> , cmt vs. cm.
serum lipids
to analyze the effects of castration and testosterone treatment on lipid metabolism in hfc-fed pigs, serum lipids were measured at  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and 12 weeks postcastration.
serum total cholesterol , high-density lipoprotein cholesterol , low-density lipoprotein cholesterol , and tgs were significantly increased in miniature pigs fed an hfc diet .
cm pigs had higher tc levels than did im and cmt pigs from the fourth week after hfc supplementation .
there were no significant differences in serum tc levels between im and cmt pigs .
serum ldl-c followed a pattern similar to that of tc in all groups of pigs .
however, testosterone deficiency caused by castration had no significant influence on serum hdl-c levels in hfc-fed pigs .
serum tg levels in cm pigs were higher than those in im pigs at weeks  <dig> and  <dig>  and those in cmt pigs beginning at week  <dig> .figure  <dig>
effects of castration and testosterone treatment on serum lipid levels.
serum total cholesterol  levels.
serum low-density lipoprotein cholesterol  levels.
serum high-density lipoprotein cholesterol  levels.
serum triglyceride  levels.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
data are expressed as means ± sems, n =  <dig> per group.
*p <  <dig>  and **p <  <dig> , im vs. cm; #
p <  <dig>  and ##
p <  <dig> , cmt vs. cm.
liver histopathology and hepatic lipids
liver histology results showed that testosterone deficiency induced by castration significantly increased hepatic fat accumulation in pigs fed an hfc diet .
cm pigs developed massive micro- or macrovesicular steatosis, while the hepatocytes of im pigs showed only mild microvesicular steatosis .
moreover, castration significantly aggravated hepatic inflammation in pigs fed an hfc diet .
testosterone treatment clearly improved hepatic steatosis and reduced hepatic necroinflammation in cm pigs .
oil red o staining confirmed the occurrence of increased lipid deposition in the livers of cm pigs .figure  <dig>
effects of testosterone on liver histology and hepatic lipids.
liver sections stained with hematoxylin and eosin.
micro- and macrovesicular steatosis are indicated by arrow heads, and inflammation is indicated by arrows.
oil red o staining of fat in hepatocytes.
increased lipid deposition is indicated by arrows.
hepatic triglyceride  content in im, cm, and cmt pigs.
hepatic cholesterol  content in im, cm, and cmt pigs.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
data are expressed as means ± sems, n =  <dig> per group.
*p <  <dig> ; **p <  <dig> .
consistent with the observed increase in lipid deposition, we also detected marked increases in tg contents in the livers of cm pigs .
moreover, testosterone treatment reduced hepatic tg contents in cm pigs .
likewise, hepatic cholesterol contents in cm pigs were also significantly higher than those in im and cmt pigs .
gene expression analysis by rna-seq
to obtain a global view of the hepatic transcriptome responses to testosterone deficiency and testosterone treatment in pigs fed an hfc diet, we performed comparative rna-seq analyses of the liver transcriptomes.
cdna libraries were constructed using total rna isolated from the livers of im, cm, and cmt pigs.
using an illumina hiseq  <dig> sequencer, we obtained approximately  <dig> ,  <dig> , and  <dig>  million high quality clean reads from im, cm, and cmt pigs, respectively.
for each sample, ~87% of reads could be mapped to the pig reference genome; of these mapped reads, ~79% aligned with unique genes unambiguously .table  <dig>
summary of the sequencing reads alignment to the reference genome
statistics term
im
cm
cmt
in total,  <dig>   <dig>  and  <dig> expressed genes were detected in the livers of im, cm, and cmt pigs, respectively.
of these genes, a total of  <dig> genes were expressed in all three groups;  <dig>   <dig>  and  <dig> genes were identified commonly between each pair of groups , while  <dig>   <dig>  and  <dig> genes were discovered exclusively for im, cm, and cmt, respectively .
to identify differentially expressed genes  in the livers of im, cm, and cmt pigs, gene expression data from each group were compared using degseq software  <cit> .
significance scores were corrected for multiple testing using the benjamini-hochberg correction  <cit> .
we used the following criteria to identify degs:  gene expression level greater than or equal to  <dig> fragment per kilobases of exon per million fragments mapped  in all samples;  change in expression level greater than or equal to  <dig>  fold; and  a false discovery rate  of less than  <dig>   <cit> .
thus, a total of  <dig> ,  <dig> , and  <dig>  degs were detected in the livers of pigs for cm versus im, cmt versus cm, and cmt versus im groups, respectively .
in total,  <dig> different genes were differentially expressed between any two groups .figure  <dig>
rna-seq analyses of differentially expressed genes  in livers of im, cm, and cmt pigs.
the numbers of upregulated and downregulated degs between groups.
venn diagram showing the overlap of degs between groups.
heat map for hierarchical cluster analysis of degs between samples.
red: upregulated genes; yellow: downregulated genes.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
gene ontology  enrichment analysis of degs
to gain further insights into the biological functions of degs, we performed go analyses by querying each deg identified in the livers of im, cm, and cmt pigs against the go database.
go analysis of significantly upregulated degs between cm and im pigs revealed genes that were mainly enriched in immune-related processes, such as the type i interferon signaling pathway, the cytokine-mediated signaling pathway, immune responses, innate immune responses, antigen processing and presentation, and apoptosis .
downregulated degs were mainly involved in metabolism-related processes, such as lipid metabolism, fatty acid metabolism, fatty acid beta-oxidation, bile acid metabolic processes, steroid metabolic processes, ketone body biosynthetic processes, cholesterol metabolic processes, and the tricarboxylic acid and gluconeogenesis pathways .
upregulated degs between cmt and cm pigs revealed genes that were mainly involved in metabolism-related processes, such as lipid metabolism, fatty acid metabolism, fatty acid beta-oxidation, steroid metabolism, bile acid metabolism, cholesterol metabolism, ketone body biosynthesis, and the tricarboxylic acid and gluconeogenesis cycles, while downregulated degs were mainly enriched in immune-related processes, such as the type i interferon signaling pathway, the cytokine-mediated signaling pathway, innate immune responses, immune responses, apoptosis, antigen processing and presentation, inflammatory responses, and responses to oxidative stress .
the enriched go analyses of all significant degs between any two groups were also investigated and were found to be mainly involved in small molecule metabolism, metabolic processes, lipid metabolism, fatty acid metabolism, steroid metabolism, cholesterol metabolism, fatty acid beta-oxidation, immune responses, inflammatory responses, apoptosis, and responses to oxidative stress .figure  <dig>
gene ontology  and pathway analysis of differentially expressed genes .
top  <dig> significant go terms  associated with the identified degs.
the vertical axis represents the go category, and the horizontal axis represents the -log <dig> of the significant go terms.
top  <dig> significant pathways involving degs.
the vertical axis represents the pathway category, and the horizontal axis represents the -log <dig> of the significant pathways.
greater -log <dig> scores correlated with increased statistical significance.
pathway analysis of degs
pathway analysis using the kegg database was performed to determine significant pathways involving the degs identified in this study.
our results showed that  <dig> pathways were significantly enriched for the identified degs .
the enriched pathways are listed in additional file  <dig>  moreover, pathway analysis showed that these genes were mainly involved in metabolic pathways, steroid hormone biosynthesis, the ppar signaling pathway, peroxisomes, fatty acid degradation, pyruvate metabolism, steroid biosynthesis, the nuclear factor-kappab  signaling pathway, primary bile acid biosynthesis, antigen processing and presentation, the tricarboxylic acid cycle, synthesis and degradation of ketone bodies, and glycerolipid metabolism .
series-cluster analysis of degs
to refine the set of genes that were differentially expressed between any group, we categorized the  <dig> degs into seven possible model profiles  to enrich for gene expression tendencies using the short time-series expression miner  program  <cit> .
we identified two patterns of gene expression  with significance .
profile  <dig> contained  <dig> genes that were characterized by decreased expression in cm pigs but increased expression in the cmt group.
in contrast, profile  <dig> contained  <dig> genes that were characterized by increased expression in cm pigs but exhibited decreased expression in the cmt group.
significantly enriched go terms from profiles  <dig> and  <dig> are illustrated in figure 6b and additional files  <dig> and  <dig>  cluster analysis showed that the significantly enriched go terms from profile  <dig> were closely correlated with immune and inflammatory responses.
the included genes were enriched in the type i interferon signaling pathway, cytokine-mediated signaling pathways, regulation of immune responses, innate immune responses, immune responses, and antigen processing and presentation .
profile  <dig> genes were also enriched in apoptotic processes and positive regulation of chemokine secretion .figure  <dig>
significant gene expression tendencies and gene ontology  analysis.
differentially expressed genes between the groups described in additional file  <dig>  were separated into seven possible model profiles, including two significant gene expression tendencies .
profile  <dig> included  <dig> genes that were decreased in cm pigs, but increased in cmt pigs.
profile  <dig> included  <dig> genes that were increased in cm pigs, but decreased in cmt pigs.
significantly enriched go terms in profiles  <dig> and  <dig>  the top  <dig> biological functions and the case genes in each cluster are listed.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
the genes involved in immune and inflammatory responses are displayed and clustered in figure 7a.
to verify the observed differential mrna expression of immune and inflammatory response genes, we performed quantitative real-time rt-pcr  using rna samples extracted from the livers of im, cm, and cmt pigs.
the hepatic expression of six immune and inflammatory response-related genes  was analyzed in the three groups.
consistent with rna-seq results, the expression of these six genes was upregulated in cm pigs and downregulated in cmt pigs after testosterone treatment .
in addition to qrt-pcr validation experiments, we also employed immunohistochemistry to examine the protein expression levels of ccl <dig>  cxcl <dig>  and irf <dig> , which are involved in the development of nafld.
based on immunostaining results, we observed similar differences in expression at the protein level.
staining levels tended to be increased in the livers of cm pigs compared to those observed in im pigs .
however, this positive staining was reduced after testosterone treatment .figure  <dig>
expression of genes involved in immune and inflammatory responses.
heat map for genes involved in immune and inflammatory responses.
expression levels of six genes detected by quantitative real-time rt-pcr  were in agreement with the rna-seq results.
data are expressed as means ± sems, n =  <dig> per group.
immunohistochemical assessment of chemokine  ligand  <dig> , chemokine  ligand  <dig> , and interferon-regulatory factor  <dig> , showing expression changes that were in accordance with the rna-seq and qrt-pcr results.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
scale bars = 100 μm.
profile  <dig> genes were mainly enriched in various metabolic subprocesses, including fatty acid metabolism, fatty acid beta-oxidation, steroid biosynthesis, cholesterol and bile acid metabolism, and glucose metabolism .
we found that the most genes enriched in fatty acid metabolic processes were involved in fatty acid oxidation .
it is well known that impaired fatty acid oxidation contributes to nafld; thus, we analyzed the genes participating in fatty acid oxidation further.
carnitine palmitoyltransferase 1a  encodes the rate-limiting enzyme in fatty acid oxidation, which was downregulated  <dig> -fold in the livers of cm pigs and upregulated  <dig> -fold in the livers of cmt pigs.
peroxisome proliferator-activated receptor delta  belongs to a class of ligand-dependent transcription factors involved in hepatic lipid metabolism and was downregulated  <dig> -fold in the livers of cm pigs and upregulated  <dig> -fold in the livers of cmt pigs.
moreover, the acadl, acox <dig>  aco, and ecil genes, encoding long-chain acyl-coa dehydrogenase, palmitoyl acyl-coa oxidase  <dig>  acyl-coa oxidase, and mitochondrial enoyl-coa isomerase, respectively, were  <dig> -,  <dig> -,  <dig> -, and  <dig> -fold downregulated in the livers of cm pigs and  <dig> -,  <dig> -,  <dig> -, and  <dig> -fold upregulated in the livers of cmt pigs, respectively .figure  <dig>
expression of genes involved in fatty acid oxidation and cholesterol, bile acid, and glucose metabolism.
heat map for genes involved in fatty acid oxidation.
heat map for genes involved in cholesterol and bile acid metabolism.
heat map for genes involved in glucose metabolism.
expression levels of six genes detected by quantitative real-time rt-pcr  that were in agreement with the rna-seq results.
data are expressed as means ± sems, n =  <dig> per group.
western blot analysis of peroxisome proliferator-activated receptor delta  and carnitine palmitoyltransferase 1a  showing expression changes that were in accordance with rna-seq and qrt-pcr results.
im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy.
data are expressed as means ± sems, n =  <dig> per group.
*p <  <dig> .
enriched genes mediating cholesterol, bile acid, and glucose metabolism are also displayed in figure  <dig>  genes involved in cholesterol biosynthesis , cholesterol esterification , cholesterol transport and absorption , and bile acid metabolic processes  were downregulated in the livers of cm pigs, but restored by testosterone treatment .
several genes encoding enzymes involved in glucose metabolism were also downregulated in cm pigs.
for example, pck <dig>  encoding the rate-limiting gluconeogenesis enzyme phosphoenolpyruvate carboxykinase , and pfkfb <dig>  encoding the glycolysis enzyme 6-phosphofructo-2-kinase, were decreased in the livers of cm pigs, but increased in the livers of cmt pigs after testosterone treatment .
we performed qrt-pcr to verify the mrna expression profiles of the fatty acid oxidation-related genes cpt1a, ppard, acadl, aco, acox <dig>  and 3-hydroxyacyl-coa dehydrogenase .
consistent with rna-seq analysis, the expression of these six genes was downregulated in cm pigs and upregulated in cmt pigs after testosterone treatment .
we then measured the protein expression levels of cpt1a and ppard by western blot analysis.
the protein levels of cpt1a and ppard were decreased in the livers of cm pigs as compared to im pigs and were increased in cmt pigs after testosterone treatment .
pathway-act-network analysis
to further understand the importance of pathway interactions and to screen key pathways for significant roles in nafld induced by testosterone deficiency and an hfc diet, we built a pathway-act-network according to the direct or systemic interactions assigned between pathways in the kegg database .
as shown in figure  <dig>  some degs involved in key pathways during nafld were identified, including metabolic pathways, fatty acid degradation, pyruvate metabolism, and the citrate cycle.
moreover, glycerolipid metabolism and the nf-κb signaling pathway in the interaction network were also predicted to play important roles.figure  <dig>
pathway-act-network analysis.
the pathway-act-network was built according to the interactions with pathways identified in the kegg database.
cycle nodes represent pathways, and the arrow between two nodes represents an interaction target between pathways.
the size of nodes represents the power of the interaction among the pathways .
red nodes represent significant pathways and green nodes represent nonsignificant pathways.
to display a detailed pathway of fatty acid degradation and the relative differences in genes influenced by testosterone, a putative pathway of fatty acid degradation was constructed based on kegg mapping.
enzymes and proteins involved in regulating fatty acid degradation  were decreased in cm pigs, but recovered in cmt pigs .
we also constructed a putative pathway of peroxisome-related processes according to kegg mapping .
similarly, several genes involved in regulating peroxisome processes, such as pmp <dig> and pex <dig>  were decreased in cm pigs, but increased in cmt pigs.
discussion
in this work, we studied the effects of testosterone on the development of hepatic steatosis in pigs fed an hfc diet.
our data indicated that serum cholesterol  and tg levels were significantly higher in cm pigs than in im pigs.
moreover, testosterone deficiency exacerbated liver injury and increased hepatic tg contents  in cm pigs.
however, testosterone replacement attenuated these effects in cm pigs, suggesting that testosterone may play a protective role in diet-induced hepatic steatosis.
in previous studies, researchers have used rodent models to evaluate the effects of testosterone on nafld; however, several of these were genetic animal models, i.e., androgen receptor -knockout mice  <cit>  and tfm mutant mice  <cit> .
these models may have some utility for specific research aims, but most humans that develop nafld have few genetic defects.
other studies have also used orchidectomized mouse models; however, these animals do not have increased adiposity  <cit>  or have reduced serum tg levels  <cit> , which are inconsistent with findings in humans.
we showed here that a porcine model of nafld induced by testosterone deficiency and hfc diet mimicked human fatty liver disease in terms of histological analyses and metabolic profiles.
to the best of our knowledge, the present study is the first to demonstrate that testosterone deficiency aggravates diet-induced hepatic steatosis in a porcine model.
testosterone deficiency also caused significant changes in body fat percentages and serum biochemical parameters.
previous studies have shown that enzymes involved in fatty acid synthesis are increased in adipose tissues of castrated male pigs  <cit> .
thus, the higher body fat content observed in cm pigs may be due to the increased fatty acid synthesis in adipose tissue  <cit> .
estradiol has been reported to decrease fatty acid synthesis by increasing the phosphorylation of acetyl coenzyme-a carboxylase in rats fed a high-fat diet  <cit> .
moreover, previous studies have reported that serum alt levels are significantly elevated in ovariectomized mice fed an hfc diet  <cit> , and estradiol administration reduces hepatic mda content but increases sod activity in rats with hepatic fibrosis  <cit> .
thus, the significant changes in body fat and serum parameters induced by castration may be partly attributed to a reduction in estradiol  <cit> .
however, we did not measure hepatic estradiol levels, and elucidation of the role of estrogens in the regulation of fat metabolism and serum parameters was not the primary goal of our study; these will be investigated in future studies.
in this study, we found that testosterone deficiency did not affect serum fasting glucose and insulin levels or homa-ir in cm pigs compared to im pigs.
however, testosterone replacement significantly reduced serum insulin levels and homa-ir in cm pigs.
while previous studies have shown that androgen deficiency increases fasting glucose levels and reduces insulin sensitivity   <cit> , the effects of testosterone on glucose metabolism and ir in clinical observations and experimental studies have not been consistent.
for example, several studies have shown that low serum testosterone levels are associated with ir in men and that androgen therapy improves ir and fasting glucose levels in men  <cit> .
however, other studies have found that reduction of testosterone levels in men did not significantly affect fasting glucose levels or ir  <cit> .
moreover, castration results in slightly increased fasting glucose levels, but has no effects on fasting insulin concentrations and ir in rats  <cit> .
however, nikoleanko et al.
<cit>  showed that fasting glucose, fasting insulin, and homa-ir levels were not elevated in castrated male rats compared to intact animals and animals receiving testosterone replacement therapy.
using different mouse strains and diets, inoue et al.
<cit>  also indicated that castration does not induce serum glucose and insulin levels or related ir markers in any of the mouse models studied.
to date, few reports have described the effects of testosterone on glucose metabolism and ir in pigs.
our previous study showed that there were no differences in serum levels of glucose and insulin between intact and castrated male pigs fed a normal diet  <cit> .
in the present study, we still found that testosterone deficiency did not change fasting serum glucose and insulin levels in pigs fed an hfc diet.
our results were not consistent with those obtained by christofferson et al.
<cit> , who found that castration results in disturbed glucose metabolism.
these discrepancies may be explained by breed differences and the different experimental model of testosterone deficiency.
little is known regarding the molecular mechanisms underlying the testosterone deficiency-mediated promotion of fat deposition in the liver.
hepatic steatosis is regulated by multiple metabolic pathways.
previous studies have shown that disturbances in fatty acid oxidation account for excess lipid storage in the liver  <cit> .
we found that many genes involved in fatty acid oxidation were downregulated in the livers of cm pigs, and these effects were prevented by testosterone replacement.
ppard regulates lipid oxidation processes  <cit> , but its role in hepatic lipid metabolism remains unclear.
we found that the levels of ppard mrna and protein were significantly reduced in the livers of cm pigs, and testosterone treatment restored the expression of ppard in cmt pigs.
these results are consistent with those obtained by barroso et al.
<cit>  and bolic et al.
<cit> , who showed that ppard activation stimulates hepatic fatty acid oxidation and alleviates diet-induced hepatic steatosis.
moreover, this observation suggests that ppard may be regulated by sex hormones and plays key roles in the regulation of hepatic steatosis; this provides a novel view of ppard function in the modulation of hepatic lipid metabolism.
in addition to its effects on lipid metabolism, ppard has been reported to play roles in the regulation of glucose metabolism and ir  <cit> .
studies in genetic and diet-induced ir or diabetic mice have shown that ppard exerts beneficial effects to ameliorate ir  <cit> .
because of our data regarding ppard, and since the involvement of ppard in ir is well established, we would expect that markers of ir in cm pigs should be elevated compared with those in im and cmt pigs.
however, there was no difference in fasting glucose, insulin, or homa-ir levels in cm pigs compared to im pigs.
previous studies have indicated that ppard regulates glucose flux and ir through the pentose phosphate pathway  <cit> .
however, our transcriptomics data did not reveal any changes in the expression levels of glucose-6-phosphate dehydrogenase  and phosphogluconate dehydrogenase   in the livers of cm pigs compared to those of im pigs.
moreover, ppard has been reported to reduce glucose production indirectly via activation of ampk rather than through a direct mechanism  <cit> .
in our study, expression levels of ampk and adiponectin signaling genes  were not changed in cm pigs compared to im pigs.
activation of ppard has failed to enhance severe ir  <cit> .
moreover, in recent studies in rats and mice fed a high-fat diet, heterogeneous effects of ppard on ir have been observed  <cit> .
thus, the reasons for the discrepancy between ppard expression and ir markers in this study may be due to the different effects of ppard on ir in different species  <cit> .
however, testosterone replacement significantly improved fasting insulin and homa-ir levels and restored ppard expression in cm pigs, despite the observation that gonadectomy apparently did not change fasting insulin, glucose, or homa-ir levels compared to those in intact pigs.
cpt-1a is the rate-limiting enzyme of mitochondrial β-oxidation  <cit> .
we found that hepatic cpt1a mrna and protein levels were significantly reduced in the livers of cm pigs and subsequently restored in cmt pigs receiving testosterone supplementation.
other fatty acid oxidation-related genes, such as acadl, acox <dig>  aco, hadh, and short chain enoyl-coa hydratase  were also downregulated in the livers of cm pigs.
taken together, our results provided strong evidence that testosterone deficiency aggravated hepatic fat accumulation in cm pigs partly due to decreased hepatic fatty acid oxidation.
our results are consistent with a human study in which men with short-term hypogonadism showed decreased whole-body lipid oxidation  <cit> .
moreover, lin et al.
<cit>  observed reduced fatty acid oxidation in isolated primary hepatocytes from hepatic ar-knockout  mice, consistent with our results.
peroxisomes have many important functions in lipid metabolism, including fatty acid β-oxidation.
a previous study reported that impaired peroxisome function contributes to nafld in mice  <cit> .
in this study, we also found that the pmp <dig>  pex <dig>  and pem <dig> genes, which are involved in peroxisome-related processes, were downregulated in the livers of cm pigs.
these results suggested that testosterone deficiency led to increased hepatic steatosis in cm pigs by affecting peroxisome function.
in addition to the decrease in liver fat oxidation, increased fat synthesis may also contribute to elevated liver fat accumulation  <cit> .
tgs are the main lipids that accumulate during hepatic steatosis  <cit> .
glycerol 3-phosphate acyltransferase  and diacylglycerol acyltransferase  catalyze the initial and final steps in tg synthesis, respectively  <cit> .
increased levels of dgat <dig> mrna occur in the livers of humans with nafld  <cit> , indicating the importance of dgat <dig> in fatty liver development.
mice lacking dgat <dig> have reduced tissue tg levels and are protected against hepatic steatosis  <cit> .
studies using gpat-knockout mice  <cit>  and mice overexpressing gpat in the liver  <cit>  have implicated this enzyme in hepatic steatosis.
stearoyl-coa desaturase  catalyzes the de novo biosynthesis of monounsaturated fatty acids , which are critical substrates for the synthesis of tgs by gpat  <cit> .
to date, few studies have been conducted to explore the effects of testosterone on hepatic tg synthesis enzymes.
senmaru et al.
<cit>  reported that orchidectomized mice fed hfd showed significantly increased dgat <dig> expression.
a recent study revealed that scd <dig> expression was significantly elevated in tfm mice than in wild-type controls  <cit> .
these observations are consistent with our findings that the expression of the tg synthesis genes gpat, dagt, and scd were significantly upregulated in the livers of cm pigs and that testosterone replacement reduced their expression in cm pigs.
our results suggested that testosterone deficiency might increase liver fat accumulation by inducing hepatic tg synthesis.
acetyl-coa carboxylase alpha  and fatty acid synthase  are also key lipogenic enzymes involved in hepatic lipid deposition.
a recent study showed that hepatic acaca and fasn expression levels were elevated in tfm mice compared with wide-type littermates  <cit> .
however, nikolaenko et al.
<cit>  found that there were no significant differences in liver fasn protein levels in c + hfd mice after castration.
these results are inconsistent with our findings, which showed decreased acaca and fasn expression in the livers of cm pigs.
these differences might be species-specific or result from different experimental models of hepatic steatosis.
in addition, hepatic fatty acid synthesis requires acetyl-coa generated from multiple metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and fatty acid β-oxidation  <cit> .
thus, the observed decrease in acaca and fasn expression may be due to impaired glucose metabolism or fatty acid oxidation in the present study.
the spectrum of nafld ranges from simple fatty liver  to nonalcoholic steatohepatitis .
the “two-hit” theory of nafld progression proposes that inflammation, oxidative stress, and apoptosis play critical roles in the pathological progression of nafld  <cit> .
gene expression profiling indicated that several immune and inflammatory response genes were activated in the livers of cm pigs.
chemokines direct the trafficking of immune cells to sites of inflammation  <cit> .
chemokine  ligand  <dig>  is one of the most important chemokines involved in inflammation, and elevated levels of ccl <dig> have been confirmed in the livers of patients with nafld  <cit> .
moreover, increased hepatic and serum mcp- <dig> expression levels have been described in diet-induced nafld  <cit> .
expression levels of c-c chemokine receptor  <dig>  and chemokine  ligands  <dig>   <dig>   <dig>  are increased in livers during hfd-induced hepatic steatosis  <cit> .
we found that testosterone deficiency significantly increased mrna and protein levels of the ccl <dig>  cxcl <dig>  ccr <dig>  cxcl <dig>  and cxcl <dig> chemokines in the livers of cm pigs, and testosterone replacement abolished these effects.
our data suggested that the induction of pro-inflammatory chemokines contributes to the mechanism of inflammatory recruitment in hepatic steatosis induced by testosterone deficiency and an hfc diet.
moreover, clinical and experimental observations have shown that tumor necrosis factor  may have a pathogenic role during nafld development, specifically by modulating chronic lobular inflammation with hepatocellular injury  <cit> .
we found that hepatic tnf expression was reduced in cm pigs.
this is inconsistent with previous observations  <cit> .
dela pena et al.
<cit>  demonstrated that knocking out the tnf gene does not prevent diet-induced nafld in mice.
similar studies by deng et al.
<cit>  showed that tnfr1-knockout mice still develop hepatic steatosis.
taken together, these studies suggest that tnf may not be a critical mediator of inflammation in this experimental form of hepatic steatosis.
in the present study, numerous immunity and inflammation-related processes, including antigen processing and presentation, cytokine-mediated signaling pathways, and chronic inflammatory responses were induced in the livers of cm pigs.
interestingly, previous studies have shown marked induction of numerous immunity- and inflammation-related pathways in ppard−/− mice  <cit> .
moreover, activation of ppard may improve hepatic steatosis via directly suppressing the expression of cytokines or transcription factors associated with inflammation  <cit> .
these observations suggest that testosterone deficiency may induce immune and inflammatory responses through modulation of hepatic ppard expression.
moreover, testosterone has been suggested to act directly on immune cells by repressing transcription factors   <cit> .
in our study, cm pigs had higher fos expression than im pigs.
moreover, testosterone replacement significantly reduced the increased fos expression in cmt pigs.
these results suggested that testosterone may inhibit liver fos expression in cm pigs, thereby regulating the expression of genes involved in the immune response  <cit> .
additionally, previous studies have shown that testosterone can bind to intracellular receptors located in immune cells and activate hormone-responsive genes  <cit> .
in our study, androgen receptor expression was reduced by about  <dig>  fold in the livers of cm pigs compared to those in im and cmt pigs, suggesting that testosterone may interact with immune cells through its receptor.
therefore, further studies are needed to elucidate the precise mechanisms mediating the observed responses.
oxidative stress plays critical roles during the development of nafld  <cit> .
sod <dig> is an important antioxidant enzyme that can reduce reactive oxygen species and protect hepatocytes.
sod <dig> deficiency caused high levels of oxidative stress in the liver, resulting in hepatic lipid accumulation in mice  <cit> .
in the present study, hepatic sod <dig> gene expression was significantly decreased in the livers of cm pigs compared to im pigs, suggestive of a reduced defense mechanism against oxidative stress.
in contrast to sod <dig>  cytochrome p <dig> 2e <dig>  exerts pro-oxidant activity and may enhance oxidative stress.
cyp2e <dig> expression in the liver is increased in humans and animal models of nafld  <cit> .
moreover, cyp2e1-null mice are protected against hfd-induced obesity and hepatic steatosis  <cit> .
we showed that testosterone deficiency significantly induced hepatic expression of cyp2e <dig> and that testosterone replacement abolished these effects.
our data are in accordance with a previous study showing higher levels of cyp enzymes  in hepatic tissues of castrated ethanol-fed micropigs than in noncastrated counterparts  <cit> .
increased oxidative stress may induce hepatocyte apoptosis, resulting in more severe liver injury  <cit> .
although hepatic apoptosis was not detected histologically, it is important to note that several genes associated with apoptosis, including b-cell leukemia/lymphoma- <dig> , caspase- <dig> , and bcl- <dig> associated-x , were upregulated in the livers of cm + hfc pigs in response to testosterone deficiency in the present study.
similar to our study, nikolaenko et al.
<cit>  demonstrated that testosterone treatment improved diet-induced hepatic apoptosis in mice fed a high-fat diet.
the limitations of the current study include the small number of animals studied and the lack of confirmation of the results using human samples.
despite the fact that pigs have served as a suitable animal model for studying nafld, few animal models can entirely reflect the natural course and causative background of human nafld.
thus, it would be best to validate the identified degs in patients with hepatic steatosis.
furthermore, the dietary animal model used here did not manifest high serum tg levels, although pigs developed severe hypercholesterolemia and hepatic steatosis.
the amount and type of dietary fat and diet duration may have influenced these effects.
these issues should be addressed in future studies.
CONCLUSIONS
in summary, we demonstrated here that testosterone deficiency may aggravate hepatic steatosis and hypercholesterolemia in pigs fed an hfc diet, and these effects were improved by testosterone treatment.
importantly, hepatic transcriptomic analysis revealed that increased hepatic steatosis induced by testosterone deficiency and an hfc diet was mediated by altered expression of genes involved in multiple metabolic processes.
we observed abnormal regulation of fatty acid metabolism and found that the expression of fatty acid oxidation-related genes was reduced in the livers of cm pigs.
moreover, genes associated with immune and inflammatory responses, oxidative stress, and apoptosis also contributed to the increased hepatic steatosis.
these observations provide a molecular basis for understanding the mechanisms through which testosterone deficiency aggravates the progression of diet-induced hepatic steatosis.

 serum testosterone levels decline gradually and progressively with aging in men. low levels of testosterone are associated with metabolic disorders, including obesity, dyslipidemia, hypertension, and insulin resistance, all of which contribute to the development of nonalcoholic fatty liver disease. recent studies have shown that men with low serum testosterone levels have a higher risk of developing hepatic steatosis. moreover, animal studies have demonstrated that the incidence of hepatic steatosis is increased in testosterone-deficient male mice. these findings indicate the important role of testosterone in the pathophysiology of nafld. however, nutritional parameters, especially a high-cholesterol diet, have been also associated with the development of nafld. hypercholesterolemia induced by dietary cholesterol has recently been indicated as a major risk factor for hepatic steatosis. in the absence of testosterone, mice develop hypercholesterolemia when fed a diet with no added cholesterol. hypercholesterolemia and elevated serum cholesterol levels are also observed in hypogonadal men. therefore, these previous findings suggest that testosterone deficiency and testosterone deficiency-induced hypercholesterolemia may aggravate the severity of nafld induced by a high-cholesterol diet. however, the mechanisms underlying the effects of testosterone deficiency on the promotion of diet-induced hepatic steatosis remain unclear. previous studies have used rodent models to investigate the influence of testosterone on the development of hepatic steatosis. for example, hepatic androgen receptor-knockout male mice receiving a high-fat diet develop insulin resistance and hepatic steatosis. moreover, dihydrotestosterone suppresses diet-induced hepatic steatosis in castrated male rats, and hepatic lipid deposition is elevated in testicular feminized mice compared with intact wild-type littermate controls. although rodents are commonly used to study hepatic steatosis and have provided valuable insights into the pathogenesis of this disease, the relevance of these models to human disease is limited. as an alternative to rodent models, large animal models, such as pigs, may be more relevant to the study of nafld because these models more closely mimic human physiology and anatomy. importantly, castration-induced sex hormone deficiency in male pigs results in obesity, elevated serum triglycerides, and increased cholesterol levels, some of the typical causes of nafld, suggesting that pigs are suitable models for studying the effects of sex hormones on nafld development. despite the importance of testosterone in the regulation of hepatic steatosis, the genomic mechanisms through which testosterone deficiency aggravates diet-induced hepatic steatosis are still unclear. transcriptome profiling is an effective and widely used tool to identify critical genes and pathways involved in pathological processes. sequencing-based and hybridization-based approaches represent the two major approaches used in transcriptomics studies. high-throughput rna sequencing has recently become an attractive method of choice in the study of transcriptomes, providing several advantages over microarrays. in this study, we sought to gain insights into the roles of testosterone in modulating hepatic steatosis using a porcine model of testosterone deficiency and diet-induced hepatic steatosis. additionally, we examined the contribution of liver genes to the development of hepatic steatosis induced by testosterone deficiency and a high-cholesterol diet using rna-seq. to the best of our knowledge, this is the first description of differences in whole-transcriptome patterns occurring in the liver following castration in pigs fed a high-cholesterol diet.  body weights and serum testosterone levels pigs were separated into three groups: intact male pigs fed a high-fat and high-cholesterol diet; castrated male pigs fed an hfc diet; and castrated male pigs fed an hfc diet and given testosterone replacement therapy. the body weights of pigs in each group were calculated and found to be linearly elevated over time. initial body weights were similar in pigs from the im, cm, and cmt groups. castrated pigs fed an hfc diet gained less weight than did pigs in the other groups. with testosterone replacement, the body weights of cmt pigs increased and were similar to the body weights of pigs in the im group.figure effects of castration and testosterone treatment on body weights and serum testosterone concentrations. body weights. serum testosterone concentrations. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. data are expressed as means ± sems, n =  per group. **p < . serum testosterone concentrations were not significantly different between groups before the study began. as expected, castration caused a significant decrease in serum testosterone levels. the concentration of serum testosterone at 12 weeks was significantly lower in cm pigs than in im pigs. testosterone treatment increased serum testosterone levels to  ±   ng/ml. liver weight, body fat content, and serum biochemical parameters liver weight, body fat content, and serum biochemical parameters after 12 weeks of hfc feeding are presented in table  the liver weights in cm pigs were higher than those observed in im pigs, but lower than those in cmt pigs. liver weight indexes in cm pigs were higher than those in im and cmt pigs, but these differences were not significant. cm pigs had higher body fat percentages than im and cmt pigs. serum alanine aminotransferase levels were significantly increased in cm pigs compared with im + hfc pigs and significantly reduced in cmt pigs by testosterone replacement. aspartate aminotransferase levels were highest in cm pigs, but the differences were not significant among groups. compared with im and cmt pigs, cm pigs showed obviously lower serum levels of superoxide dismutase but higher levels of malondialdehyde. serum free fatty acid levels tended to be higher in cm pigs than in im and cmt pigs. fasting serum glucose and insulin levels and homeostatic model assessment of insulin resistance were not significantly different between cm and im pigs. however, testosterone replacement significant reduced serum insulin levels and homa-ir in cm pigs.table liver weight, body fat content and serum biochemical parameters in miniature pigs fed a high-fat and high-cholesterol diet im cm cmt *p <  and **p < , im vs. cm; # p <  and ## p < , cmt vs. cm. serum lipids to analyze the effects of castration and testosterone treatment on lipid metabolism in hfc-fed pigs, serum lipids were measured at and 12 weeks postcastration. serum total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and tgs were significantly increased in miniature pigs fed an hfc diet. cm pigs had higher tc levels than did im and cmt pigs from the fourth week after hfc supplementation. there were no significant differences in serum tc levels between im and cmt pigs. serum ldl-c followed a pattern similar to that of tc in all groups of pigs. however, testosterone deficiency caused by castration had no significant influence on serum hdl-c levels in hfc-fed pigs. serum tg levels in cm pigs were higher than those in im pigs at weeks and and those in cmt pigs beginning at week.figure effects of castration and testosterone treatment on serum lipid levels. serum total cholesterol levels. serum low-density lipoprotein cholesterol levels. serum high-density lipoprotein cholesterol levels. serum triglyceride levels. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. data are expressed as means ± sems, n =  per group. *p <  and **p < , im vs. cm; # p <  and ## p < , cmt vs. cm. liver histopathology and hepatic lipids liver histology results showed that testosterone deficiency induced by castration significantly increased hepatic fat accumulation in pigs fed an hfc diet. cm pigs developed massive micro- or macrovesicular steatosis, while the hepatocytes of im pigs showed only mild microvesicular steatosis. moreover, castration significantly aggravated hepatic inflammation in pigs fed an hfc diet. testosterone treatment clearly improved hepatic steatosis and reduced hepatic necroinflammation in cm pigs. oil red o staining confirmed the occurrence of increased lipid deposition in the livers of cm pigs.figure effects of testosterone on liver histology and hepatic lipids. liver sections stained with hematoxylin and eosin. micro- and macrovesicular steatosis are indicated by arrow heads, and inflammation is indicated by arrows. oil red o staining of fat in hepatocytes. increased lipid deposition is indicated by arrows. hepatic triglyceride content in im, cm, and cmt pigs. hepatic cholesterol content in im, cm, and cmt pigs. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. data are expressed as means ± sems, n =  per group. *p < ; **p < . consistent with the observed increase in lipid deposition, we also detected marked increases in tg contents in the livers of cm pigs. moreover, testosterone treatment reduced hepatic tg contents in cm pigs. likewise, hepatic cholesterol contents in cm pigs were also significantly higher than those in im and cmt pigs. gene expression analysis by rna-seq to obtain a global view of the hepatic transcriptome responses to testosterone deficiency and testosterone treatment in pigs fed an hfc diet, we performed comparative rna-seq analyses of the liver transcriptomes. cdna libraries were constructed using total rna isolated from the livers of im, cm, and cmt pigs. using an illumina hiseq sequencer, we obtained approximately,, and million high quality clean reads from im, cm, and cmt pigs, respectively. for each sample, ~87% of reads could be mapped to the pig reference genome; of these mapped reads, ~79% aligned with unique genes unambiguously.table summary of the sequencing reads alignment to the reference genome statistics term im cm cmt in total, and expressed genes were detected in the livers of im, cm, and cmt pigs, respectively. of these genes, a total of genes were expressed in all three groups; and genes were identified commonly between each pair of groups, while and genes were discovered exclusively for im, cm, and cmt, respectively. to identify differentially expressed genes in the livers of im, cm, and cmt pigs, gene expression data from each group were compared using degseq software. significance scores were corrected for multiple testing using the benjamini-hochberg correction. we used the following criteria to identify degs: gene expression level greater than or equal to fragment per kilobases of exon per million fragments mapped in all samples; change in expression level greater than or equal to fold; and a false discovery rate of less than. thus, a total of,, and degs were detected in the livers of pigs for cm versus im, cmt versus cm, and cmt versus im groups, respectively. in total, different genes were differentially expressed between any two groups.figure rna-seq analyses of differentially expressed genes in livers of im, cm, and cmt pigs. the numbers of upregulated and downregulated degs between groups. venn diagram showing the overlap of degs between groups. heat map for hierarchical cluster analysis of degs between samples. red: upregulated genes; yellow: downregulated genes. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. gene ontology enrichment analysis of degs to gain further insights into the biological functions of degs, we performed go analyses by querying each deg identified in the livers of im, cm, and cmt pigs against the go database. go analysis of significantly upregulated degs between cm and im pigs revealed genes that were mainly enriched in immune-related processes, such as the type i interferon signaling pathway, the cytokine-mediated signaling pathway, immune responses, innate immune responses, antigen processing and presentation, and apoptosis. downregulated degs were mainly involved in metabolism-related processes, such as lipid metabolism, fatty acid metabolism, fatty acid beta-oxidation, bile acid metabolic processes, steroid metabolic processes, ketone body biosynthetic processes, cholesterol metabolic processes, and the tricarboxylic acid and gluconeogenesis pathways. upregulated degs between cmt and cm pigs revealed genes that were mainly involved in metabolism-related processes, such as lipid metabolism, fatty acid metabolism, fatty acid beta-oxidation, steroid metabolism, bile acid metabolism, cholesterol metabolism, ketone body biosynthesis, and the tricarboxylic acid and gluconeogenesis cycles, while downregulated degs were mainly enriched in immune-related processes, such as the type i interferon signaling pathway, the cytokine-mediated signaling pathway, innate immune responses, immune responses, apoptosis, antigen processing and presentation, inflammatory responses, and responses to oxidative stress. the enriched go analyses of all significant degs between any two groups were also investigated and were found to be mainly involved in small molecule metabolism, metabolic processes, lipid metabolism, fatty acid metabolism, steroid metabolism, cholesterol metabolism, fatty acid beta-oxidation, immune responses, inflammatory responses, apoptosis, and responses to oxidative stress.figure gene ontology and pathway analysis of differentially expressed genes. top significant go terms associated with the identified degs. the vertical axis represents the go category, and the horizontal axis represents the -log of the significant go terms. top significant pathways involving degs. the vertical axis represents the pathway category, and the horizontal axis represents the -log of the significant pathways. greater -log scores correlated with increased statistical significance. pathway analysis of degs pathway analysis using the kegg database was performed to determine significant pathways involving the degs identified in this study. our results showed that pathways were significantly enriched for the identified degs. the enriched pathways are listed in additional file moreover, pathway analysis showed that these genes were mainly involved in metabolic pathways, steroid hormone biosynthesis, the ppar signaling pathway, peroxisomes, fatty acid degradation, pyruvate metabolism, steroid biosynthesis, the nuclear factor-kappab signaling pathway, primary bile acid biosynthesis, antigen processing and presentation, the tricarboxylic acid cycle, synthesis and degradation of ketone bodies, and glycerolipid metabolism. series-cluster analysis of degs to refine the set of genes that were differentially expressed between any group, we categorized the degs into seven possible model profiles to enrich for gene expression tendencies using the short time-series expression miner program. we identified two patterns of gene expression with significance. profile contained genes that were characterized by decreased expression in cm pigs but increased expression in the cmt group. in contrast, profile contained genes that were characterized by increased expression in cm pigs but exhibited decreased expression in the cmt group. significantly enriched go terms from profiles and are illustrated in figure 6b and additional files and cluster analysis showed that the significantly enriched go terms from profile were closely correlated with immune and inflammatory responses. the included genes were enriched in the type i interferon signaling pathway, cytokine-mediated signaling pathways, regulation of immune responses, innate immune responses, immune responses, and antigen processing and presentation. profile genes were also enriched in apoptotic processes and positive regulation of chemokine secretion.figure significant gene expression tendencies and gene ontology analysis. differentially expressed genes between the groups described in additional file were separated into seven possible model profiles, including two significant gene expression tendencies. profile included genes that were decreased in cm pigs, but increased in cmt pigs. profile included genes that were increased in cm pigs, but decreased in cmt pigs. significantly enriched go terms in profiles and the top biological functions and the case genes in each cluster are listed. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. the genes involved in immune and inflammatory responses are displayed and clustered in figure 7a. to verify the observed differential mrna expression of immune and inflammatory response genes, we performed quantitative real-time rt-pcr using rna samples extracted from the livers of im, cm, and cmt pigs. the hepatic expression of six immune and inflammatory response-related genes was analyzed in the three groups. consistent with rna-seq results, the expression of these six genes was upregulated in cm pigs and downregulated in cmt pigs after testosterone treatment. in addition to qrt-pcr validation experiments, we also employed immunohistochemistry to examine the protein expression levels of ccl cxcl and irf, which are involved in the development of nafld. based on immunostaining results, we observed similar differences in expression at the protein level. staining levels tended to be increased in the livers of cm pigs compared to those observed in im pigs. however, this positive staining was reduced after testosterone treatment.figure expression of genes involved in immune and inflammatory responses. heat map for genes involved in immune and inflammatory responses. expression levels of six genes detected by quantitative real-time rt-pcr were in agreement with the rna-seq results. data are expressed as means ± sems, n =  per group. immunohistochemical assessment of chemokine ligand, chemokine ligand, and interferon-regulatory factor, showing expression changes that were in accordance with the rna-seq and qrt-pcr results. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. scale bars = 100 μm. profile genes were mainly enriched in various metabolic subprocesses, including fatty acid metabolism, fatty acid beta-oxidation, steroid biosynthesis, cholesterol and bile acid metabolism, and glucose metabolism. we found that the most genes enriched in fatty acid metabolic processes were involved in fatty acid oxidation. it is well known that impaired fatty acid oxidation contributes to nafld; thus, we analyzed the genes participating in fatty acid oxidation further. carnitine palmitoyltransferase 1a encodes the rate-limiting enzyme in fatty acid oxidation, which was downregulated -fold in the livers of cm pigs and upregulated -fold in the livers of cmt pigs. peroxisome proliferator-activated receptor delta belongs to a class of ligand-dependent transcription factors involved in hepatic lipid metabolism and was downregulated -fold in the livers of cm pigs and upregulated -fold in the livers of cmt pigs. moreover, the acadl, acox aco, and ecil genes, encoding long-chain acyl-coa dehydrogenase, palmitoyl acyl-coa oxidase acyl-coa oxidase, and mitochondrial enoyl-coa isomerase, respectively, were -, -, -, and -fold downregulated in the livers of cm pigs and -, -, -, and -fold upregulated in the livers of cmt pigs, respectively.figure expression of genes involved in fatty acid oxidation and cholesterol, bile acid, and glucose metabolism. heat map for genes involved in fatty acid oxidation. heat map for genes involved in cholesterol and bile acid metabolism. heat map for genes involved in glucose metabolism. expression levels of six genes detected by quantitative real-time rt-pcr that were in agreement with the rna-seq results. data are expressed as means ± sems, n =  per group. western blot analysis of peroxisome proliferator-activated receptor delta and carnitine palmitoyltransferase 1a showing expression changes that were in accordance with rna-seq and qrt-pcr results. im: intact male pigs fed an hfc diet; cm: castrated male pigs fed an hfc diet; cmt: castrated male pigs fed an hfc diet and given testosterone replacement therapy. data are expressed as means ± sems, n =  per group. *p < . enriched genes mediating cholesterol, bile acid, and glucose metabolism are also displayed in figure  genes involved in cholesterol biosynthesis, cholesterol esterification, cholesterol transport and absorption, and bile acid metabolic processes were downregulated in the livers of cm pigs, but restored by testosterone treatment. several genes encoding enzymes involved in glucose metabolism were also downregulated in cm pigs. for example, pck encoding the rate-limiting gluconeogenesis enzyme phosphoenolpyruvate carboxykinase, and pfkfb encoding the glycolysis enzyme 6-phosphofructo-2-kinase, were decreased in the livers of cm pigs, but increased in the livers of cmt pigs after testosterone treatment. we performed qrt-pcr to verify the mrna expression profiles of the fatty acid oxidation-related genes cpt1a, ppard, acadl, aco, acox and 3-hydroxyacyl-coa dehydrogenase. consistent with rna-seq analysis, the expression of these six genes was downregulated in cm pigs and upregulated in cmt pigs after testosterone treatment. we then measured the protein expression levels of cpt1a and ppard by western blot analysis. the protein levels of cpt1a and ppard were decreased in the livers of cm pigs as compared to im pigs and were increased in cmt pigs after testosterone treatment. pathway-act-network analysis to further understand the importance of pathway interactions and to screen key pathways for significant roles in nafld induced by testosterone deficiency and an hfc diet, we built a pathway-act-network according to the direct or systemic interactions assigned between pathways in the kegg database. as shown in figure  some degs involved in key pathways during nafld were identified, including metabolic pathways, fatty acid degradation, pyruvate metabolism, and the citrate cycle. moreover, glycerolipid metabolism and the nf-κb signaling pathway in the interaction network were also predicted to play important roles.figure pathway-act-network analysis. the pathway-act-network was built according to the interactions with pathways identified in the kegg database. cycle nodes represent pathways, and the arrow between two nodes represents an interaction target between pathways. the size of nodes represents the power of the interaction among the pathways. red nodes represent significant pathways and green nodes represent nonsignificant pathways. to display a detailed pathway of fatty acid degradation and the relative differences in genes influenced by testosterone, a putative pathway of fatty acid degradation was constructed based on kegg mapping. enzymes and proteins involved in regulating fatty acid degradation were decreased in cm pigs, but recovered in cmt pigs. we also constructed a putative pathway of peroxisome-related processes according to kegg mapping. similarly, several genes involved in regulating peroxisome processes, such as pmp and pex were decreased in cm pigs, but increased in cmt pigs. discussion in this work, we studied the effects of testosterone on the development of hepatic steatosis in pigs fed an hfc diet. our data indicated that serum cholesterol and tg levels were significantly higher in cm pigs than in im pigs. moreover, testosterone deficiency exacerbated liver injury and increased hepatic tg contents in cm pigs. however, testosterone replacement attenuated these effects in cm pigs, suggesting that testosterone may play a protective role in diet-induced hepatic steatosis. in previous studies, researchers have used rodent models to evaluate the effects of testosterone on nafld; however, several of these were genetic animal models, i.e., androgen receptor -knockout mice and tfm mutant mice. these models may have some utility for specific research aims, but most humans that develop nafld have few genetic defects. other studies have also used orchidectomized mouse models; however, these animals do not have increased adiposity or have reduced serum tg levels, which are inconsistent with findings in humans. we showed here that a porcine model of nafld induced by testosterone deficiency and hfc diet mimicked human fatty liver disease in terms of histological analyses and metabolic profiles. to the best of our knowledge, the present study is the first to demonstrate that testosterone deficiency aggravates diet-induced hepatic steatosis in a porcine model. testosterone deficiency also caused significant changes in body fat percentages and serum biochemical parameters. previous studies have shown that enzymes involved in fatty acid synthesis are increased in adipose tissues of castrated male pigs. thus, the higher body fat content observed in cm pigs may be due to the increased fatty acid synthesis in adipose tissue. estradiol has been reported to decrease fatty acid synthesis by increasing the phosphorylation of acetyl coenzyme-a carboxylase in rats fed a high-fat diet. moreover, previous studies have reported that serum alt levels are significantly elevated in ovariectomized mice fed an hfc diet, and estradiol administration reduces hepatic mda content but increases sod activity in rats with hepatic fibrosis. thus, the significant changes in body fat and serum parameters induced by castration may be partly attributed to a reduction in estradiol. however, we did not measure hepatic estradiol levels, and elucidation of the role of estrogens in the regulation of fat metabolism and serum parameters was not the primary goal of our study; these will be investigated in future studies. in this study, we found that testosterone deficiency did not affect serum fasting glucose and insulin levels or homa-ir in cm pigs compared to im pigs. however, testosterone replacement significantly reduced serum insulin levels and homa-ir in cm pigs. while previous studies have shown that androgen deficiency increases fasting glucose levels and reduces insulin sensitivity, the effects of testosterone on glucose metabolism and ir in clinical observations and experimental studies have not been consistent. for example, several studies have shown that low serum testosterone levels are associated with ir in men and that androgen therapy improves ir and fasting glucose levels in men. however, other studies have found that reduction of testosterone levels in men did not significantly affect fasting glucose levels or ir. moreover, castration results in slightly increased fasting glucose levels, but has no effects on fasting insulin concentrations and ir in rats. however, nikoleanko et al. showed that fasting glucose, fasting insulin, and homa-ir levels were not elevated in castrated male rats compared to intact animals and animals receiving testosterone replacement therapy. using different mouse strains and diets, inoue et al. also indicated that castration does not induce serum glucose and insulin levels or related ir markers in any of the mouse models studied. to date, few reports have described the effects of testosterone on glucose metabolism and ir in pigs. our previous study showed that there were no differences in serum levels of glucose and insulin between intact and castrated male pigs fed a normal diet. in the present study, we still found that testosterone deficiency did not change fasting serum glucose and insulin levels in pigs fed an hfc diet. our results were not consistent with those obtained by christofferson et al., who found that castration results in disturbed glucose metabolism. these discrepancies may be explained by breed differences and the different experimental model of testosterone deficiency. little is known regarding the molecular mechanisms underlying the testosterone deficiency-mediated promotion of fat deposition in the liver. hepatic steatosis is regulated by multiple metabolic pathways. previous studies have shown that disturbances in fatty acid oxidation account for excess lipid storage in the liver. we found that many genes involved in fatty acid oxidation were downregulated in the livers of cm pigs, and these effects were prevented by testosterone replacement. ppard regulates lipid oxidation processes, but its role in hepatic lipid metabolism remains unclear. we found that the levels of ppard mrna and protein were significantly reduced in the livers of cm pigs, and testosterone treatment restored the expression of ppard in cmt pigs. these results are consistent with those obtained by barroso et al. and bolic et al., who showed that ppard activation stimulates hepatic fatty acid oxidation and alleviates diet-induced hepatic steatosis. moreover, this observation suggests that ppard may be regulated by sex hormones and plays key roles in the regulation of hepatic steatosis; this provides a novel view of ppard function in the modulation of hepatic lipid metabolism. in addition to its effects on lipid metabolism, ppard has been reported to play roles in the regulation of glucose metabolism and ir. studies in genetic and diet-induced ir or diabetic mice have shown that ppard exerts beneficial effects to ameliorate ir. because of our data regarding ppard, and since the involvement of ppard in ir is well established, we would expect that markers of ir in cm pigs should be elevated compared with those in im and cmt pigs. however, there was no difference in fasting glucose, insulin, or homa-ir levels in cm pigs compared to im pigs. previous studies have indicated that ppard regulates glucose flux and ir through the pentose phosphate pathway. however, our transcriptomics data did not reveal any changes in the expression levels of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in the livers of cm pigs compared to those of im pigs. moreover, ppard has been reported to reduce glucose production indirectly via activation of ampk rather than through a direct mechanism. in our study, expression levels of ampk and adiponectin signaling genes were not changed in cm pigs compared to im pigs. activation of ppard has failed to enhance severe ir. moreover, in recent studies in rats and mice fed a high-fat diet, heterogeneous effects of ppard on ir have been observed. thus, the reasons for the discrepancy between ppard expression and ir markers in this study may be due to the different effects of ppard on ir in different species. however, testosterone replacement significantly improved fasting insulin and homa-ir levels and restored ppard expression in cm pigs, despite the observation that gonadectomy apparently did not change fasting insulin, glucose, or homa-ir levels compared to those in intact pigs. cpt-1a is the rate-limiting enzyme of mitochondrial β-oxidation. we found that hepatic cpt1a mrna and protein levels were significantly reduced in the livers of cm pigs and subsequently restored in cmt pigs receiving testosterone supplementation. other fatty acid oxidation-related genes, such as acadl, acox aco, hadh, and short chain enoyl-coa hydratase were also downregulated in the livers of cm pigs. taken together, our results provided strong evidence that testosterone deficiency aggravated hepatic fat accumulation in cm pigs partly due to decreased hepatic fatty acid oxidation. our results are consistent with a human study in which men with short-term hypogonadism showed decreased whole-body lipid oxidation. moreover, lin et al. observed reduced fatty acid oxidation in isolated primary hepatocytes from hepatic ar-knockout mice, consistent with our results. peroxisomes have many important functions in lipid metabolism, including fatty acid β-oxidation. a previous study reported that impaired peroxisome function contributes to nafld in mice. in this study, we also found that the pmp pex and pem genes, which are involved in peroxisome-related processes, were downregulated in the livers of cm pigs. these results suggested that testosterone deficiency led to increased hepatic steatosis in cm pigs by affecting peroxisome function. in addition to the decrease in liver fat oxidation, increased fat synthesis may also contribute to elevated liver fat accumulation. tgs are the main lipids that accumulate during hepatic steatosis. glycerol 3-phosphate acyltransferase and diacylglycerol acyltransferase catalyze the initial and final steps in tg synthesis, respectively. increased levels of dgat mrna occur in the livers of humans with nafld, indicating the importance of dgat in fatty liver development. mice lacking dgat have reduced tissue tg levels and are protected against hepatic steatosis. studies using gpat-knockout mice and mice overexpressing gpat in the liver have implicated this enzyme in hepatic steatosis. stearoyl-coa desaturase catalyzes the de novo biosynthesis of monounsaturated fatty acids, which are critical substrates for the synthesis of tgs by gpat. to date, few studies have been conducted to explore the effects of testosterone on hepatic tg synthesis enzymes. senmaru et al. reported that orchidectomized mice fed hfd showed significantly increased dgat expression. a recent study revealed that scd expression was significantly elevated in tfm mice than in wild-type controls. these observations are consistent with our findings that the expression of the tg synthesis genes gpat, dagt, and scd were significantly upregulated in the livers of cm pigs and that testosterone replacement reduced their expression in cm pigs. our results suggested that testosterone deficiency might increase liver fat accumulation by inducing hepatic tg synthesis. acetyl-coa carboxylase alpha and fatty acid synthase are also key lipogenic enzymes involved in hepatic lipid deposition. a recent study showed that hepatic acaca and fasn expression levels were elevated in tfm mice compared with wide-type littermates. however, nikolaenko et al. found that there were no significant differences in liver fasn protein levels in c + hfd mice after castration. these results are inconsistent with our findings, which showed decreased acaca and fasn expression in the livers of cm pigs. these differences might be species-specific or result from different experimental models of hepatic steatosis. in addition, hepatic fatty acid synthesis requires acetyl-coa generated from multiple metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and fatty acid β-oxidation. thus, the observed decrease in acaca and fasn expression may be due to impaired glucose metabolism or fatty acid oxidation in the present study. the spectrum of nafld ranges from simple fatty liver to nonalcoholic steatohepatitis. the “two-hit” theory of nafld progression proposes that inflammation, oxidative stress, and apoptosis play critical roles in the pathological progression of nafld. gene expression profiling indicated that several immune and inflammatory response genes were activated in the livers of cm pigs. chemokines direct the trafficking of immune cells to sites of inflammation. chemokine ligand is one of the most important chemokines involved in inflammation, and elevated levels of ccl have been confirmed in the livers of patients with nafld. moreover, increased hepatic and serum mcp- expression levels have been described in diet-induced nafld. expression levels of c-c chemokine receptor and chemokine ligands are increased in livers during hfd-induced hepatic steatosis. we found that testosterone deficiency significantly increased mrna and protein levels of the ccl cxcl ccr cxcl and cxcl chemokines in the livers of cm pigs, and testosterone replacement abolished these effects. our data suggested that the induction of pro-inflammatory chemokines contributes to the mechanism of inflammatory recruitment in hepatic steatosis induced by testosterone deficiency and an hfc diet. moreover, clinical and experimental observations have shown that tumor necrosis factor may have a pathogenic role during nafld development, specifically by modulating chronic lobular inflammation with hepatocellular injury. we found that hepatic tnf expression was reduced in cm pigs. this is inconsistent with previous observations. dela pena et al. demonstrated that knocking out the tnf gene does not prevent diet-induced nafld in mice. similar studies by deng et al. showed that tnfr1-knockout mice still develop hepatic steatosis. taken together, these studies suggest that tnf may not be a critical mediator of inflammation in this experimental form of hepatic steatosis. in the present study, numerous immunity and inflammation-related processes, including antigen processing and presentation, cytokine-mediated signaling pathways, and chronic inflammatory responses were induced in the livers of cm pigs. interestingly, previous studies have shown marked induction of numerous immunity- and inflammation-related pathways in ppard−/− mice. moreover, activation of ppard may improve hepatic steatosis via directly suppressing the expression of cytokines or transcription factors associated with inflammation. these observations suggest that testosterone deficiency may induce immune and inflammatory responses through modulation of hepatic ppard expression. moreover, testosterone has been suggested to act directly on immune cells by repressing transcription factors. in our study, cm pigs had higher fos expression than im pigs. moreover, testosterone replacement significantly reduced the increased fos expression in cmt pigs. these results suggested that testosterone may inhibit liver fos expression in cm pigs, thereby regulating the expression of genes involved in the immune response. additionally, previous studies have shown that testosterone can bind to intracellular receptors located in immune cells and activate hormone-responsive genes. in our study, androgen receptor expression was reduced by about fold in the livers of cm pigs compared to those in im and cmt pigs, suggesting that testosterone may interact with immune cells through its receptor. therefore, further studies are needed to elucidate the precise mechanisms mediating the observed responses. oxidative stress plays critical roles during the development of nafld. sod is an important antioxidant enzyme that can reduce reactive oxygen species and protect hepatocytes. sod deficiency caused high levels of oxidative stress in the liver, resulting in hepatic lipid accumulation in mice. in the present study, hepatic sod gene expression was significantly decreased in the livers of cm pigs compared to im pigs, suggestive of a reduced defense mechanism against oxidative stress. in contrast to sod cytochrome p 2e exerts pro-oxidant activity and may enhance oxidative stress. cyp2e expression in the liver is increased in humans and animal models of nafld. moreover, cyp2e1-null mice are protected against hfd-induced obesity and hepatic steatosis. we showed that testosterone deficiency significantly induced hepatic expression of cyp2e and that testosterone replacement abolished these effects. our data are in accordance with a previous study showing higher levels of cyp enzymes in hepatic tissues of castrated ethanol-fed micropigs than in noncastrated counterparts. increased oxidative stress may induce hepatocyte apoptosis, resulting in more severe liver injury. although hepatic apoptosis was not detected histologically, it is important to note that several genes associated with apoptosis, including b-cell leukemia/lymphoma-, caspase-, and bcl- associated-x, were upregulated in the livers of cm + hfc pigs in response to testosterone deficiency in the present study. similar to our study, nikolaenko et al. demonstrated that testosterone treatment improved diet-induced hepatic apoptosis in mice fed a high-fat diet. the limitations of the current study include the small number of animals studied and the lack of confirmation of the results using human samples. despite the fact that pigs have served as a suitable animal model for studying nafld, few animal models can entirely reflect the natural course and causative background of human nafld. thus, it would be best to validate the identified degs in patients with hepatic steatosis. furthermore, the dietary animal model used here did not manifest high serum tg levels, although pigs developed severe hypercholesterolemia and hepatic steatosis. the amount and type of dietary fat and diet duration may have influenced these effects. these issues should be addressed in future studies.  in summary, we demonstrated here that testosterone deficiency may aggravate hepatic steatosis and hypercholesterolemia in pigs fed an hfc diet, and these effects were improved by testosterone treatment. importantly, hepatic transcriptomic analysis revealed that increased hepatic steatosis induced by testosterone deficiency and an hfc diet was mediated by altered expression of genes involved in multiple metabolic processes. we observed abnormal regulation of fatty acid metabolism and found that the expression of fatty acid oxidation-related genes was reduced in the livers of cm pigs. moreover, genes associated with immune and inflammatory responses, oxidative stress, and apoptosis also contributed to the increased hepatic steatosis. these observations provide a molecular basis for understanding the mechanisms through which testosterone deficiency aggravates the progression of diet-induced hepatic steatosis. 
moreover, serum levels of total cholesterol, low-density lipoprotein cholesterol, and triglycerides were markedly increased in cm pigs. compared to im and cmt pigs, cm pigs had lower serum levels of superoxide dismutase but higher levels of malondialdehyde. these results deepened our understanding of the molecular mechanisms of testosterone deficiency-induced hepatic steatosis and provided a foundation for future investigations. gene expression analysis revealed that upregulated genes in the livers of cm pigs were mainly enriched for genes mediating immune and inflammatory responses, oxidative stress, and apoptosis. impaired metabolic processes, enhanced immune and inflammatory responses, oxidative stress, and apoptosis may contribute to the increased hepatic steatosis induced by testosterone deficiency and an hfc diet. kegg analysis showed that metabolic pathways, fatty acid degradation, pyruvate metabolism, the tricarboxylic acid cycle, and the nuclear factor-kappab signaling pathway were the major pathways altered in cm pigs. to gain insight into the role of testosterone in modulating hepatic steatosis, we investigated the effects of testosterone on the development of hepatic steatosis in pigs fed a high-fat and high-cholesterol diet and profiled hepatic gene expression by rna-seq in hfc-fed intact male pigs, castrated male pigs, and castrated male pigs with testosterone replacement. surprisingly, the downregulated genes mainly included those that regulate metabolism-related processes, including fatty acid oxidation, steroid biosynthesis, cholesterol and bile acid metabolism, and glucose metabolism. recent studies have indicated that low serum testosterone levels are associated with increased risk of developing hepatic steatosis; however, the mechanisms mediating this phenomenon have not been fully elucidated. this study demonstrated that testosterone deficiency aggravated hypercholesterolemia and hepatic steatosis in pigs fed an hfc diet and that these effects could be reversed by testosterone replacement therapy. testosteronenonalcoholic fatty liver diseasehepatic steatosisminiature pigsrna-seqissue-copyright-statement© the author 2015 serum testosterone levels were significantly decreased in cm pigs, and testosterone replacement attenuated castration-induced testosterone deficiency. cm pigs showed increased liver injury accompanied by increased hepatocellular steatosis, inflammation, and elevated serum alanine aminotransferase levels compared with im pigs. testosterone replacement decreased serum and hepatic lipid levels and improved liver injury in cm pigs.
 recent studies have indicated that low serum testosterone levels are associated with increased risk of developing hepatic steatosis; however, the mechanisms mediating this phenomenon have not been fully elucidated. to gain insight into the role of testosterone in modulating hepatic steatosis, we investigated the effects of testosterone on the development of hepatic steatosis in pigs fed a high-fat and high-cholesterol diet and profiled hepatic gene expression by rna-seq in hfc-fed intact male pigs, castrated male pigs, and castrated male pigs with testosterone replacement.  serum testosterone levels were significantly decreased in cm pigs, and testosterone replacement attenuated castration-induced testosterone deficiency. cm pigs showed increased liver injury accompanied by increased hepatocellular steatosis, inflammation, and elevated serum alanine aminotransferase levels compared with im pigs. moreover, serum levels of total cholesterol, low-density lipoprotein cholesterol, and triglycerides were markedly increased in cm pigs. testosterone replacement decreased serum and hepatic lipid levels and improved liver injury in cm pigs. compared to im and cmt pigs, cm pigs had lower serum levels of superoxide dismutase but higher levels of malondialdehyde. gene expression analysis revealed that upregulated genes in the livers of cm pigs were mainly enriched for genes mediating immune and inflammatory responses, oxidative stress, and apoptosis. surprisingly, the downregulated genes mainly included those that regulate metabolism-related processes, including fatty acid oxidation, steroid biosynthesis, cholesterol and bile acid metabolism, and glucose metabolism. kegg analysis showed that metabolic pathways, fatty acid degradation, pyruvate metabolism, the tricarboxylic acid cycle, and the nuclear factor-kappab signaling pathway were the major pathways altered in cm pigs.  this study demonstrated that testosterone deficiency aggravated hypercholesterolemia and hepatic steatosis in pigs fed an hfc diet and that these effects could be reversed by testosterone replacement therapy. impaired metabolic processes, enhanced immune and inflammatory responses, oxidative stress, and apoptosis may contribute to the increased hepatic steatosis induced by testosterone deficiency and an hfc diet. these results deepened our understanding of the molecular mechanisms of testosterone deficiency-induced hepatic steatosis and provided a foundation for future investigations. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords testosteronenonalcoholic fatty liver diseasehepatic steatosisminiature pigsrna-seqissue-copyright-statement© the author 2015 