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Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and ...

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Preferential accumulation of fat in the upper body (apple shape) is associated with higher risk of developing metabolic syndrome relative to lower body fat (pear shape). We previously discovered that chromatin openness partially defined the transcriptome of preadipocytes isolated from abdominal and gluteofemoral fat. However, the molecular mechanisms underlying interindividual variation in body shape are unknown.

Fat Distribution in Women Is Associated With Depot-Specific Transcriptomic Signatures and Chromatin Structure.

Preferential accumulation of fat in the gluteo-femoral (GF) depot (pear shape) rather than in the abdominal (A) depot (apple shape), protects against the development of metabolic diseases but the underlying molecular mechanism is still unknown. Recent data, including our work, suggest that differential epigenetic marking is associated with regulation of genes attributed to distinct fat distribution. Here, we aimed to compare the genomic DNA methylation signatures between apple and pear-shaped premenopausal women. To investigate the contribution of upper and lower body fat, we used paired samples of A-FAT and GF-FAT, analyzed on the BeadChip Methylation Array and quantified the differentially methylated sites between the 2 groups of women. We found unique DNA methylation patterns within both fat depots that are significantly different depending on the body fat distribution. Around 60% of the body shape specific DNA methylation sites identified in adipose tissue are maintained  in cultured preadipocytes. As it has been reported before in other cell types, we found only a hand full of genes showing coordinated differential methylation and expression levels. Finally, we determined that more than 50% of the body shape specific DNA methylation sites could also be detected in whole blood derived DNA. These data reveal a strong DNA methylation program associated with adipose tissue distribution with the possibility that a simple blood test could be used as a predictive diagnostic indicator of young women who are at increased risk for progressing to the apple body shape with a higher risk of developing obesity related complications. https://clinicaltrials.gov/ct2/show/NCT02728635 and https://clinicaltrials.gov/ct2/show/NCT02226640, identifiers NCT02728635 and NCT02226640.

DNA Methylation as a Marker of Body Shape in Premenopausal Women.

Polycystic ovary syndrome (PCOS) is often associated with metabolic syndrome features, including central obesity, suggesting that adipose tissue (AT) is a key organ in PCOS pathobiology. In this study, we compared both abdominal (ABD) and gluteofemoral (GF) subcutaneous AT in women with and without PCOS. ABD and GF subcutaneous ATs from PCOS and BMI/WHR-matched control women were analyzed by RT-qPCR, FACS and histology. ABD and GF adipose-derived stem cell (ASC) transcriptome and methylome were analyzed by RNA-seq and DNA methylation array. Similar to the control group with abdominal obesity, the GF AT of PCOS women showed lower expression of genes involved in lipid accumulation and angiogenesis compared to ABD depot. FACS analysis revealed an increase in preadipocytes number in both AT depots from PCOS. Further pathway analysis of RNA-seq comparisons demonstrated that the ASCs derived from PCOS are pro-inflammatory and exhibit a hypoxic signature in the ABD depot and have lower expression of adipogenic genes in GF depot. We also found a higher CpG methylation level in PCOS compared to control exclusively in GF-ASCs. Our data suggest that ASCs play an important role in the etiology of PCOS, potentially by limiting expansion of the healthy lower-body AT.

Transcriptional and DNA Methylation Signatures of Subcutaneous Adipose Tissue and Adipose-Derived Stem Cells in PCOS Women.

Accumulation of fat above the waist is an important risk factor in developing obesity-related comorbidities independently of BMI or total fat mass. Deciphering the gene regulatory programs of the adipose tissue precursor cells within upper body or abdominal (ABD) and lower body or gluteofemoral (GF) depots is important to understand their differential capacity for lipid accumulation, maturation, and disease risk. Previous studies identified the HOX transcript antisense intergenic RNA (HOTAIR) as a GF-specific lncRNA; however, its role in adipose tissue biology is still unclear. Using three different approaches (silencing of HOTAIR in GF human adipose-derived stem cells [GF hASCs], overexpression of HOTAIR in ABD hASCs, and ChIRP-seq) to localize HOTAIR binding in GF hASC chromatin, we found that HOTAIR binds and modulates expression, both positively and negatively, of genes involved in adipose tissue-specific pathways, including adipogenesis. We further demonstrate a direct interaction between HOTAIR and genes with high RNAPII binding in their gene bodies, especially at their 3' ends or transcription end sites. Computational analysis suggests HOTAIR binds preferentially to the 3' ends of genes containing predicted strong RNA-RNA interactions with HOTAIR. Together, these results reveal a unique function for HOTAIR in hASC depot-specific regulation of gene expression.

Unique role for lncRNA HOTAIR in defining depot-specific gene expression patterns in human adipose-derived stem cells.

White adipose tissue (WAT) is a complex mixture of adipocytes and non-adipogenic cells. Characterizing the cellular composition of WAT is critical for identifying where potential alterations occur that impact metabolism. Most single-cell (sc) RNA-Seq studies focused on the stromal vascular fraction (SVF) which does not contain adipocytes and have used technology that has a 3' or 5' bias. Using full-length sc/single-nuclei (sn) RNA-Seq technology, we interrogated the transcriptional composition of WAT using: snRNA-Seq of whole tissue, snRNA-Seq of isolated adipocytes, and scRNA-Seq of SVF. Whole WAT snRNA-Seq provided coverage of major cell types, identified three distinct adipocyte clusters, and was capable of tracking adipocyte differentiation with pseudotime. Compared to WAT, adipocyte snRNA-Seq was unable to match adipocyte heterogeneity. SVF scRNA-Seq provided greater resolution of non-adipogenic cells. These findings provide critical evidence for the utility of sc full-length transcriptomics in WAT and SVF in humans.

Single cell full-length transcriptome of human subcutaneous adipose tissue reveals unique and heterogeneous cell populations.

Automated single-cell dispensing is incompatible with white adipose tissue (WAT) due to lipid-laden adipocytes. Single-nuclei RNA-Seq permits transcriptional profiling of all cells from WAT. Human WAT faces unique technical challenges in isolating nuclei compared to rodent tissue due to greater extra-cellular matrix content and larger lipid droplets. In this protocol, we detail how to isolate nuclei from frozen subcutaneous human WAT for single-nuclei RNA-Seq. For complete information on the generation and use of this protocol, please refer to Whytock et al. (2022)..

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

White adipose tissue (WAT) is a robust energy storage and endocrine organ critical for maintaining metabolic health as we age. Our aim was to identify cell-specific transcriptional aberrations that occur in WAT with aging. We leveraged full-length snRNA-Seq to characterize the cellular landscape of human subcutaneous WAT in a prospective cohort of 10 Younger (≤ 30 years) and 10 Older individuals (≥ 65 years) balanced for sex and body mass index (BMI). We highlight that aging WAT is associated with adipocyte hypertrophy, increased proportions of resident macrophages (M2), an upregulated innate immune response and senescence profiles in specific adipocyte populations, highlighting CXCL14 as a biomarker of this process. We also identify novel markers of pre-adipocytes and track their expression levels through pre-adipocyte differentiation. We propose that aging WAT is associated with low-grade inflammation that is managed by a foundation of innate immunity to preserve the metabolic health of the WAT.

A single nuclei atlas of aging human abdominal subcutaneous white adipose tissue.

Insulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous, however, this association remains controversial. The aim of this study was to perform an in-depth multifactorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active,  = 9), individuals with obesity (Obese,  = 9), and individuals with obesity, insulin resistance, and type 2 diabetes (T2D,  = 22). Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic-supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by Blue Native (BN)-PAGE and immunoblot. Tricarboxylic (TCA) cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. We reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity, supercomplex assembly, TCA cycle intermediates, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (body mass index, age, and aerobic capacity). We highlight that lean, active individuals have greater mitochondrial content, mitochondrial capacity, supercomplex assembly, and TCA cycle intermediates. These phenotypical changes are reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance. Clinical trial number: NCT01911104. Whether impaired mitochondrial capacity contributes to skeletal muscle insulin resistance is debated. Our multifactorial analysis shows no differences in skeletal muscle mitochondrial content, mitochondrial capacity, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity). We highlight that lean, active individuals have enhanced skeletal muscle mitochondrial capacity that is also reflected at the level of DNA methylation and gene transcription.

Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity.

Determining the mechanism driving body fat distribution will provide insights into obesity-related health risks. We used functional genomics tools to profile the epigenomic landscape to help infer the differential transcriptional potential of apple- and pear-shaped women's subcutaneous adipose-derived stem cells (ADSCs). We found that CCCTC-binding factor (CTCF) expression and its chromatin binding were increased in ADSCs from pear donors compared to those from apple donors. Interestingly, the pear enriched CTCF binding sites were located predominantly at the active transcription start sites (TSSs) of genes with active histone marks and YY1 motifs and were also associated with pear enriched RNAPII binding. In contrast, apple enriched CTCF binding sites were mainly found at intergenic regions and when identified at TSS, they were enriched with the bivalent chromatin signatures. Altogether, we provide evidence that CTCF plays an important role in differential regulation of subcutaneous ADSCs gene expression and may influence the development of apple vs. pear body shape.

Transcriptional Control of Subcutaneous Adipose Tissue by the Transcription Factor CTCF Modulates Heterogeneity in Fat Distribution in Women.

The genome of human adipose-derived stem cells (ADSCs) from abdominal and gluteofemoral adipose tissue depots are maintained in depot-specific stable epigenetic conformations that influence cell-autonomous gene expression patterns and drive unique depot-specific functions. The traditional approach to explore tissue-specific transcriptional regulation has been to correlate differential gene expression to the nearest-neighbor linear-distance regulatory region defined by associated chromatin features including open chromatin status, histone modifications, and DNA methylation. This has provided important information; nonetheless, the approach is limited because of the known organization of eukaryotic chromatin into a topologically constrained three-dimensional network. This network positions distal regulatory elements in spatial proximity with gene promoters which are not predictable based on linear genomic distance. In this work, we capture long-range chromatin interactions using HiChIP to identify remote genomic regions that influence the differential regulation of depot-specific genes in ADSCs isolated from different adipose depots. By integrating these data with RNA-seq results and histone modifications identified by ChIP-seq, we uncovered distal regulatory elements that influence depot-specific gene expression in ADSCs. Interestingly, a subset of the HiChIP-defined chromatin loops also provide previously unknown connections between waist-to-hip ratio GWAS variants with genes that are known to significantly influence ADSC differentiation and adipocyte function.

An Atlas of Promoter Chromatin Modifications and HiChIP Regulatory Interactions in Human Subcutaneous Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women. Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women.

Adeline Divoux

Translational Research Institute

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Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Adeline Divoux

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Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Translational Research Institute