Diet, gonadal sex, and sex chromosome complement influence white adipose tissue miRNA expression
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MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by targeting specific mRNA species for degradation or interfering with translation. Specific miRNAs are key regulators of adipogenesis, and are expressed at different levels in adipose tissue from lean and obese mice. The degree of lipid accumulation and distribution of white adipose tissue differs between males and females, and it is unknown whether sex differences in adipose tissue-specific miRNA expression may contribute to this dimorphism. Typically, sex differences are attributed to hormones secreted from ovaries or testes. However, the sex chromosome complement (XX versus XY) is also a determinant of sex differences and may regulate miRNA expression in adipocytes.
To identify sex differences in adipose tissue miRNA expression and to understand the underlying mechanisms, we performed high-throughput miRNA sequencing in gonadal fat depots of the Four Core Genotypes mouse model. This model, which consists of XX female, XX male, XY female, and XY male mice, allowed us to assess independent effects of gonadal type (male vs. female) and sex chromosome complement (XX vs. XY) on miRNA expression profiles. We have also assessed the effects of a high fat diet on sex differences in adipose tissue miRNA profiles. We identified a male–female effect on the overall miRNA expression profile in mice fed a chow diet, with a bias toward higher expression in male compared to female gonadal adipose tissue. This sex bias disappeared after gonadectomy, suggesting that circulating levels of gonadal secretions modulate the miRNA expression profile. After 16 weeks of high fat diet, the miRNA expression distribution was shifted toward higher expression in XY vs. XX adipose tissue. Principal component analysis revealed that high fat diet has a substantial effect on miRNA profile variance, while gonadal secretions and sex chromosome complement each have milder effects.
Our results demonstrate that the overall miRNA expression profile in adipose tissue is influenced by gonadal hormones and the sex chromosome complement, and that expression profiles change in response to gonadectomy and high fat diet. Differential miRNA expression profiles may contribute to sex differences in adipose tissue gene expression, adipose tissue development, and diet-induced obesity.
KeywordsmicroRNA Adipose tissue Sex differences Sex chromosome
Four core genotypes
- miR, miRNA
MicroRNAs (miRNAs) are small noncoding RNA molecules that modulate gene expression by targeting mRNA transcripts for degradation or interfering with mRNA translation [1, 2]. These 21- to 22-nucleotide molecules are derived from the processing of longer mRNAs transcribed from intergenic regions or introns of protein-coding genes [3, 4]. Approximately 2000 miRNA genes have been recognized in the human and mouse genomes, many of which are expressed in a tissue-dependent manner [5, 6]. Bioinformatic predictions estimate that 30–80% of mammalian mRNAs are targeted by miRNAs, and a given mRNA may be targeted by multiple miRNAs [7, 8]. In addition, a single miRNA can regulate multiple mRNA transcripts, potentially orchestrating coordinate regulation of several genes within a metabolic pathway [9, 10]. It is generally thought that miRNAs act to fine-tune mRNA levels, but there is also evidence of full repression of specific protein production in some cases [11, 12, 13].
miRNAs have important roles in the regulation of metabolic homeostasis. For example, miR-33a and miR-33b, which are embedded within genes for sterol regulatory element-binding proteins Srebf2 and Srebf1, respectively, have key roles in the modulation of cholesterol homeostasis [14, 15]. The discovery of the cellular roles of miR-33a/b in the repression of genes involved in cellular cholesterol export has revealed a new therapeutic target. Indeed, studies in mice and non-human primates have shown that antagonism of miR-33a/b reduces plasma lipid levels and atherosclerosis [16, 17]. Many other examples of important roles for miRNAs in metabolism have emerged recently (reviewed in 18). Among these are roles for miRNAs in the regulation of adipogenesis [18, 19, 20]. Specific miRNAs that enhance adipocyte differentiation (miR-30c, miR-143, miR-146b, and miR-378; [21, 22, 23, 24]) or inhibit adipocyte differentiation (miR-27, miR-130, and miR-138; [25, 26, 27]) have been identified. Some miRNAs that are induced during adipocyte development are dysregulated in obese mice [28, 29]. While a few specific miRNAs have been well characterized, the roles of the majority of miRNAs expressed in adipose tissue are unknown.
Many properties of mammalian adipose tissue accumulation, distribution, and metabolism differ between males and females . To understand the basis for sex differences in fat storage, previous studies have assessed differences in mRNA expression levels in male vs. females fat depots [31, 32]. It is likely that sex differences also occur in miRNA expression levels in adipose tissue, and that these differences may contribute to sex differences in mRNA levels. Consistent with this, sex differences in miRNA expression levels have been reported in brain , lung , and liver  and may influence sex-specific disease development and pathogenesis . However, the effect of sex on miRNA expression in adipose tissue has not been investigated.
The mechanisms underlying sex differences in miRNA levels have not been studied. Sex differences in metabolism can be attributed to both hormonal and genetic factors [37, 38]. Gonadal hormones have been considered to be the primary drivers of sex differences, and some miRNA levels in a variety of tissues are responsive to estradiols . Importantly, however, the sex chromosome complement also plays a major role in determining adiposity [40, 41], and it is possible that this is mediated in part by miRNAs. To dissect the contributions of gonadal secretions and sex chromosome complement to sex differences, we have used the Four Core Genotypes (FCG) mouse model. The FCG model generates mice with four combinations of gonads and sex chromosomes: XX female, XX male, XY female, and XY male mice . FCG mice have a Y chromosome that carries a mutation (denoted Y−) in the testis-determining gene, Sry, such that XY− mice develop ovaries rather than testes. In addition, FCG mice carry an autosomal Sry transgene that independently segregates from the Y− chromosome. Mice that inherit the Sry transgene develop testes. A cross between XX females and XY− Sry males produces four combinations of gonads and sex chromosomes (XX male and female, XY male and female). Using this model, we previously demonstrated that the presence of two X chromosomes leads to increased adiposity compared to XY mice, independently from effects of ovaries or testes .
Here, we performed RNA sequencing (RNA-seq) of small RNAs present in gonadal fat from FCG mice to identify sex differences in miRNA expression levels in adipose tissue. We determined that sex hormones and sex chromosomes each influence the miRNA expression profile. In addition, comparison of mice fed chow vs. high fat diets revealed sex-specific changes in miRNA profiles in response to diet-induced obesity. Our findings have implications for understanding sex differences in adipose tissue metabolism and the development of diet-induced obesity.
Four Core Genotypes (FCG) C57BL/6 mice were bred and genotyped as described previously [40, 43]. Briefly, XX female mice were mated with XY−(Sry+) male mice to generate XX, XX(Sry+), XY−, and XY−(Sry+) offspring, and genotyping was performed by PCR to detect the Sry transgene and a Y-chromosome–specific sequence. Where indicated, gonadectomy was performed at 75 days of age as described previously . During surgery, gonads were removed while leaving surrounding adipose tissue in place.
Gonadal males and females were housed in separate cages and maintained at 23 °C with a 12:12 h light:dark cycle. Gonadally intact females were analyzed without estrous cycle synchronization, such that gene expression values represent an average over the estrous phases. All mice were initially fed Purina mouse chow diet containing 5% fat (Purina 5001; PMI Nutrition International, St. Louis, MO). Where specified, mice were fed a high fat diet (60% calories from fat, Bio-Serv Diets #S3282, Flemington, NJ) for 16 weeks beginning at 3.5 months of age (4 weeks after gonadectomy). Adipose tissue was harvested from all mice at 7.5 months of age.
Mouse studies were conducted after approval by the Institutional Animal Research Committee of the University of California, Los Angeles.
RNA extraction and quality control
At the time of dissection, gonadal fat tissue was flash frozen in liquid nitrogen and stored at −80 °C. Small RNAs were isolated from 100 mg tissue samples using QIAzol and Qiagen’s miRNeasy Mini kit (Cat. 217004, Qiagen, Valencia, CA). After homogenization, samples were centrifuged at 12,000 × g for 10 min to separate the transparent lipid layer from the pink organic layer. Only the organic layer was used in chloroform extraction. All subsequent steps followed the Qiagen protocol. RNA samples were submitted to Agilent BioAnalyzer Eukaryote Total Nano-RNA chip analysis, yielding RNA integrity numbers of 7.5 or greater.
miRNA library preparation
The feasibility of using pooled sequencing libraries was assessed by sequencing miRNAs from individual samples separately and after pooling. Indexed libraries were generated from adipose tissue of three XX females fed a high fat diet. Average counts of mapped miRNAs from the individual libraries were compared to miRNA counts of the pooled library using Pearson’s product-moment correlation. For the remaining conditions, three samples of each genotype were pooled into equimolar amounts for library preparation. In total, twelve miRNA libraries were made: libraries for each of the four genotypes in chow-fed, gonadally intact mice, chow-fed, gonadectomized mice, and gonadectomized mice fed a high fat diet.
miRNA libraries were processed individually using a standard protocol from Illumina TruSeq Small RNA kit, with indices 1–12, and gel purified according to manufacturer’s instructions. Final sequencing library concentration (19.07 nM) was determined using KAPA library quantification qPCR kit (KK4854, Kapa Biosystems, Wilmington, MA). Sequencing was performed at the Broad Stem Cell Research Center core facility at UCLA, on Illumina HiSeq 2000.
Reference sequence determination
miRNA gene expression is typically quantified by counting reads that map to the miRNA genes. However, in some families of miRNAs, several genes give rise to identical mature sequences, thus it is impossible to distinguish which miRNA gene gave rise to the mature sequence based on sequencing alone. We performed reference preprocessing to compile a list of uniquely expressed mouse miRNA sequences, regardless of their gene of origin, so that quantification was done at the level of mature miRNA sequence, rather than at the gene level.
A Reference sequence was compiled based on all mature and precursor miRNA sequences available from the main repository of miRNA studies, miRBase version 18 (http://www.mirbase.org), and included all mature and precursor sequences. The respective mature sequences within precursor sequences were masked to “N”, to prevent mature sequences mapping both to mature and precursor. Thus, reads mapping to precursor sequences mapped to precursors exclusively.
In cases where several genes gave rise to the same mature miRNA sequence, that sequence was represented only once in the Reference under one name, with an additional column listing all matching genes as potential targets in the results. In total, 1007 mature miRNA sequences were unique (Additional file 1), while an additional 256 miRNAs grouped into 82 unique sequences (Additional file 2). In 7 cases, the miRNAs were not completely equivalent, with one miRNA being 1 base shorter than the other. In such cases, the shorter sequence would map to both mature sequences, while the longer one would map uniquely to the longer sequence. For these pairs, the counts of the shorter sequence were determined as the total counts mapping to longer sequence minus the unique mappings to longer sequence (Additional file 3).
Sequence read processing
Reads were demultiplexed in.qseq format based on perfect match to barcode sequence, and converted to.fastq format using in-house Perl scripts (available upon request). Read quality was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adaptor sequence was clipped using cutadapt tool (https://cutadapt.readthedocs.io). Clean reads 18–30 bp long were retained for subsequent mapping and analysis. Identical reads were collapsed using collapse.pl script from mirDeep package (https://www.mdc-berlin.de/8551903/en/). Reads were then mapped to the Reference with BWA aligner allowing for up to 1 mismatch and no gaps. Using these criteria, 80–90% of the reads mapped to the Reference sequence.
Read counting and result table
Reads that uniquely mapped to one target were counted towards that target. Reads that had both perfect and imperfect (1 mismatch) matches, were counted only towards the perfect match target. Results were summarized in a count table that listed the counts for each target in each sample (Additional file 4). Precursor miRNA counts are composed of reads mapping exclusively to precursor sequences, and not to any mature miRNA sequence in the Reference. For each Reference sequence, the total read count and the number of reads that originate from 1-mismatch reads were presented separately. The number of mismatched reads is a subset of total reads mapping to the Reference.
Percent abundance of miRNAs was calculated by normalizing the reads from one miRNA to the total number of reads mapped for all miRNAs. The mean of the four genotypes across all diet/gonadectomy conditions is represented in Fig. 2b.
Select miRNAs were validated with quantitative PCR, using individual samples that comprised the pooled samples that were sequenced. Taqman primers (Cat. 4427975, Thermo Fisher Scientific, Waltham, MA) were used according to manufacturer’s protocols. Briefly, 10 ng RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Cat. 4366596, Thermo Fisher Scientific). Quantitative real-time PCR was performed on Bio-Rad CFX Connect Real-Time PCR Detection System using SsoFast Probes Supermix (Cat. 172–5231, Bio-Rad, Hercules, CA). miRNAs of interest were normalized to two housekeeping genes: U6 small nuclear RNA and small nucleolar RNA (snoRNA) 251. Expression was quantified using ΔCt for miR-221 or standard curve for miR-133a, miR-192, and miR-205. Standard curves were generated using 4 serial dilution points of cDNA combined from all biological samples.
miRNA target prediction
miRNAs with exclusively male–female or XX–XY log2 fold changes greater than 0.5 were selected for target prediction. Targets for miRNAs were predicted by mirdb.org, version 5.0 . All targets with a prediction score of 95 or above were included in subsequent functional analysis using DAVID, version 6.7 [45, 46]. The functional annotation tool was used to identify enriched annotation terms in the KEGG Pathway. Unique KEGG pathways with a Bonferroni-corrected p-value < 0.05 were included in Additional file 5: Table S1.
Pearson’s product-moment correlation, exact binomial tests, Wilcoxon rank sum tests, and principal component analyses were performed using R and visualized with the R packages ggplot2 and ggbiplot [47, 48, 49]. Because each genotype and diet condition was measured in one pooled sample from each genotype/diet/sex condition, the present analysis did not have adequate power to assess the significance of treatment and genotypic effects on expression levels of individual miRNAs. Instead, we viewed each miRNA as an independent contributor to the overall miRNA expression profile of each genotype under each experimental condition, and we report patterns of expression among groups.
In qPCR experiments, groups were compared using two-way ANOVA (NCSS 2001; Number Cruncher Statistical Systems, Kaysville, UT), with gonadal sex and sex chromosome complement as independent factors. Values for miR-192 and miR-205 were log10-transformed before statistical analysis.
Study design to identify sex differences in adipose tissue miRNAs under three physiological conditions
Sex differences in adipose tissue miRNA profiles
To streamline our sequencing process for the twelve conditions shown in Fig. 1, we assessed the feasibility of pooling three biological replicates for miRNA sequencing for each condition. We sequenced samples from three individual mice (from XX female mice fed high fat diet) separately and after pooling together. We found that the average miRNA reads of the three individual samples were highly correlated with the values from the pooled sample (Fig. 2c, d; p < 2.2 × 10−16; r 2 = 0.9966 for the 183 most abundant miRNAs, r 2 = 0.9185 for the lower abundance miRNAs). Based on these findings, we concluded that pooled libraries were a satisfactory representation of individual biological samples. For analyses described below, we focused on the 183 most abundantly expressed adipose tissue miRNAs, as these are most likely to have physiological significance.
Adipose tissue miRNAs regulated by gonadal hormones
We hypothesized that male–female differences in miRNA expression profile were caused by differences in gonadal hormones. Gonadal hormones cause sex differences in two primary ways. First, they impose permanent, or “organizational,” effects during development, such as the effect of testosterone on male genital differentiation. Second, male and female gonadal hormones in the circulation have acute/reversible effects on processes such as gene expression. To investigate the effects of acute gonadal hormone action on miRNA expression, we gonadectomized adult FCG mice (at 10 weeks of age) and assessed miRNA profiles 5 months later, during which time circulating gonadal hormones were absent. With this experimental design, male–female differences that disappear after gonadectomy can be attributed to acute hormone effects.
Adipose tissue miRNA sex differences influenced by high fat diet or obesity
Several studies have shown that miRNA expression levels are regulated during adipogenesis or are altered in obesity [28, 51]. However, it is unknown whether sex influences miRNA expression levels in obese adipose tissue. To identify sex and/or sex chromosome factors that alter miRNA expression in obesity apart from the acute effect of gonadal hormones, we performed miRNA-seq on gonadal fat from FCG mice that were gonadectomized (10 weeks of age) and then fed a high fat diet (60% calories from fat) beginning 1 month later for 4 months.
We followed up on select miRNAs that had distinct sex-influenced expression patterns and/or were previously implicated in adipocyte biology. Quantitative real-time PCR (qPCR) of miR-133a-3p, previously shown to inhibit brown adipocyte differentiation , was upregulated in XX compared to XY mice in the chow-fed, gonadally intact group (Additional file 5: Figure S1A). This difference was abolished after gonadectomy, suggesting an interplay between gonadal hormones and sex chromosome complement. miR-192-5p levels were higher in males compared to females in gonad-intact mice, but this sex difference is lost after gonadectomy, and an XX > XY difference emerges (Additional file 5: Figure S1B). Conversely, miR-205-5p showed a sex-chromosome bias (XX > XY) in gonadally intact mice and a sex bias (M > F) in gonadectomized mice fed a chow diet (Additional file 5: Figure S1C). However, when mice were fed a high fat diet, miR-192-5p and miR-205-5p showed no sex or sex chromosome differences. miR-221-3p, which is located on the X chromosome and has been previously associated with obesity and adipogenesis , showed no sex or sex chromosome difference in mice fed a chow diet (Additional file 5: Figure S1D). However, mice fed high fat diet showed increased levels of miR-221-3p in XX male and female mice compared to XY mice. These miRNA species illustrate several distinct patterns of expression that are dependent on gonadal type, gonadal hormones, diet, and sex chromosome complement, and emphasize that sex differences exist in miRNAs that have been studied previously in the context of adipose tissue and obesity.
miRNAs are key regulators of gene expression through their effects on the degradation or translation of mRNA transcripts. In metabolic tissues, miRNAs influence gene expression related to adipogenesis, cholesterol homeostasis, and glucose homeostasis . These metabolic processes are also affected by sex. While there is evidence for sexual dimorphism in miRNA expression in brain, skeletal muscle, and immune cells , sex effects on miRNAs in adipose tissue have not been characterized. In this study, we used the FCG mouse model to identify sex chromosome complement and gonadal hormone effects on miRNA abundance in gonadal fat. We sequenced miRNAs in three distinct cohorts of FCG mice in different hormonal and dietary conditions to determine the effects of circulating gonadal hormones and high fat diet on sex differences in gonadal fat miRNAs. We selected the gonadal fat depot for our studies because it has been widely studied in the mouse obesity field. However, it is clear that differences exist between gonadal fat and other visceral fat depots, as well as subcutaneous fat depots, and it would ultimately be of interest to delineate sex differences across these distinct depots. Our analyses focused on the most abundant miRNAs, as we suspect that sex differences in their levels are the most likely to have significant physiological effects.
By comparing overall miRNA profiles across diet, gonadal state, gonadal type, and sex chromosome complement, we determined that each of these factors influence miRNA expression in white adipose tissue. In gonadally intact mice, we identified a male bias in miRNA expression levels and no significant bias between XX and XY mice. As expected, the distribution of male–female differences in the levels of miRNAs was altered by gonadectomy, suggesting that circulating gonadal hormones regulate miRNA levels and are responsible for some male–female sex differences. Gonadectomy also influences adiposity , which may impact miRNA expression. We have previously shown that gonadectomized XX mice have increased body weight and adiposity compared to gonadectomized XY mice . Because we studied mice 5 months after gonadectomy, we cannot distinguish whether changes in miRNA expression preceded increases in adiposity or resulted from them. However, male–female differences in miRNA levels do not correlate with XX–XY differences in body weight, suggesting that sex differences in adiposity do not determine sex differences in overall miRNA levels. Studies measuring miRNA levels after gonadectomy but before weight gain will be required to address these questions.
High fat diet feeding alters sex and sex chromosome differences in miRNA levels, suggesting a diet–sex interaction in miRNA expression. It is notable that the observed sex and sex chromosome biases result from changes in overall miRNA distributions and not from changes in a few specific miRNAs. High fat feeding revealed a sex chromosome bias in miRNA expression ratios that was not apparent in chow-fed animals. This raises the possibility that differential miRNA expression levels may contribute to the dramatic difference in adiposity of XX compared to XY mice in response to high fat diet . Thus, mice with two X chromosomes gain more fat than XY mice, and this difference is enhanced upon feeding a high fat diet . Further studies will be required to elucidate the physiological roles of individual sex-biased miRNAs in adipogenesis and diet-induced obesity.
Some miRNAs with distinct sex-biased patterns in expression, such as miR-133a and miR-221, have been implicated in adipogenesis [28, 52]. Other miRNAs may have yet unidentified roles in lipid metabolism. An initial analysis of potential target genes for sex-biased miRNAs that we identified revealed enrichment in specific metabolic pathways (Additional file 5: Table S1). These predictions suggest that pathways such as mTOR signaling and phosphatidylinositol signaling may be important in mediating sex differences in healthy and/or diseased adipocytes. Studies to determine the impact of these predicted target genes and corresponding pathways on sex differences in obesity provide a unique area for future research.
This study represents a first look at mechanisms underlying sex differences in miRNA profiles in adipose tissue. We demonstrate that miRNAs in adipose tissue are influenced by diet, gonadal state, gonadal type, and sex chromosome complement. Principal component analysis revealed that diet is the leading factor in miRNA expression covariance, while gonadal state, gonadal type, and sex chromosome complement were milder but important regulators. By recognizing the innate sex differences in miRNA levels, we can better understand sex differences in fat accumulation, distribution, and adipose pathophysiology.
We thank Rebecca McClusky for mouse colony management and surgeries.
This study was supported by the National Institutes of Health Ruth L. Kirschstein National Research Service Award GM007185 (JCL), NIDDK R01DK083561 (APA, KR, XC), R56DK083561 (KR, APA), and NHLBI P01HL28481 (AJL, YH-B, KR).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional files.
JCL, YH-B, and XC performed the experiments and analyzed the data. JCL, YH-B, and KR drafted the manuscript. KR, AJL, and APA conceived the experiments, organized the research team, and revised and edited the manuscript. RMC provided advice and statistical approaches for data analyses. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Mouse studies were conducted after approval by the Institutional Animal Research Committee of the University of California, Los Angeles.
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