SSRN Id3666869 PDF

New insights into aging and obesity-associated aging characteristics of female
subcutaneous adipose tissue revealed by integrative analysis of multi-omics data
Zichao Lia,1, Shun Wangb,1, Shaojie Liuc,1, Ziwen Xud, Xiaowei Yie, Yunchuan Wang a, Jiaqi Liu a, Liang
Luo a, Xiaozhi Bai a, Xujie Wang a, Peng Ji a, Hongtao Wang a,*, Dahai Hua,*
a
Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University,
710032 Xi’an, Shaanxi, China
b
Department of Computer Science, Xiamen University, 316005 Xiamen, Fujian, China
c
Department of Urology, Xijing Hospital, Fourth Military Medical University, 710032 Xi’an, Shaanxi,
China
d
Fourth Military Medical University, 710032 Xi’an, Shaanxi, China
e
Department of Oto-Rhino-Laryngology, West China Hospital, West China Medical School, Sichuan
University, 610041Chengdu, Sichuan, China.
*Corresponding authors: wanght@fmmu.edu.cn (Hongtao Wang); hudhai@fmmu.edu.cn (Dahai.Hu)
1
These authors contributed equally to this work.
Abstract
Background: The subcutaneous adipose tissue (SAT) plays a role in the redistribution of adipose tissue
and systemic functions during aging. However, the changes in SAT during aging are not well
understood.
Methods: We integrated the multi-omics expression profiling data of 37 female individuals (age 23-72)
retrieved from the GEO database to construct the aging profiles of SAT. Weighted gene correlation
network analysis (WGCNA) was employed to determine the co-expression patterns of differential
genes, aging-specific biological processes, and regulatory networks at different aging stages. The
differential co-expression (DiffCoEx) analysis was further performed to explore the effects of obesity
on the SAT during aging.
Findings: A landscape of the aging profiles for differential genes, miRNAs, and gene methylation data
This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3666869
was first provided. Moreover, we provided an improved understanding of the age-dependent variations
in the biological states of the SAT. The integrative analysis indicated that only the regulatory
relationship of differentially expressed genes and miRNAs (DEGs-DEMs)was significant. While there
was no evidence confirming that the differential expression of senescence-related genes could be
regulated by methylation in SAT aging. Our research mainly elucidated that in normal aging, SAT
primarily experiences the immunosenescence, alterations of metabolism, and defective angiogenesis in
the different aging stages. Moreover, a higher risk of metabolic disorders, developing chronic
nonhealing wounds, and accelerated senescence in aging of obese females was revealed by the
DiffCoEx analysis of SAT.
Interpretation: Aging and obesity mutually influence the pathological states and decreased clinical
applications of the SAT. We additionally show that SAT is a potential novel model for studying aging
and aging-related metabolic disorders in the future.
Funding: This work was supported by the National Natural Science Foundation of China (No.
81971835; No. 81772071; No. 81772072).
Keywords: female subcutaneous adipose tissue, aging, obesity, multi-omics profiling, Weighted gene
correlation network analysis (WGCNA), differential co-expression (DiffCoEx) analysis.
Research in context
Evidence in this study
In the aging process, the systemic adipose tissue experienced the redistribution and aging related
function alterations. Previous studies on the aging related changes in the biological status of the
adipose tissue mainly focused on the visceral and ectopic fat. However, there are no systematic studies
on the changes of the biological states of SAT in aging. The development of obesity is partly attributed
to the excessive accumulation of SAT and leads to many disorders. Since the excessive deposited SAT
constitutes a dysfunctional state for the obese population, whether and how obesity affects the aging
process of SAT is important and an interesting direction to study.
Added value of this study
SAT was first utilized as the biological model to explore the aging process and the interaction between
aging and obesity. Our research originally integrated multi-omics microarray data to provide the
landscape of the aging spectrum. In the normal aging process, SAT experienced the specific aging
related biological processes, which led to many pathological states and decreased clinical application of
SAT. Further analysis identified the characteristic the biological processes of SAT aging for the obese
population compared to the non-obese population.
Implications of all available evidence
SAT is an ideal model to study human aging, obesity, and the interaction relationships between them.
SAT aging and obesity are two mutually reinforcing process and both of them could cause certain
metabolic disorders and the limitations of clinical application. Our results provide a potential novel
approach for the frontier of targeted therapy of aging and obesity.
1. Introduction
Aging is characterized by progressive decline of systemic physiological functions and represents a
major risk factor for chronic illnesses, including cardiovascular disease, metabolic disorders, and
cancers1. As age advances, systemic adipose tissues also undergo aging-related changes, especially fat
redistribution, which results in diminished subcutaneous adipose tissue (SAT) in the lower-body and
increased visceral and ectopic fat2. Adipose tissue dysfunction during aging is associated with
adipocyte senescence, which leads to disrupted cellular structures, as well as impaired insulin response
and secretory profiles3-4. Previous studies have reported age dependent functional variations in visceral
adipose tissue (VAT) and ectopic fat5. Chronic low-grade inflammation, metabolic disorders like insulin
resistance, and interference with homeostasis are aging-related features associated with VAT in human
and animals5-6. Despite storing about two-thirds of systemic fat and having multiple physiological
functions, few studies have investigated the widely distributed SAT during aging.
Obesity is often indicated by a body mass index (BMI) ≥30 and is associated with excessive
accumulation of white adipose tissue viscerally and subcutaneously. Due to its increasing incidence,
obesity is a major public health concern. In the US it is estimated that by 2030, one in two adults will
have obesity and one in four will have severe obesity7. Multiple studies have reported that during
obesity, dysfunctional excessive preadipocytes, VAT adipogenesis, and ectopic fat cause metabolic and
inflammation disorders8. Obesity is also associated with increased risk of type 2 diabetes,
cardiovascular disease, and cancer3, 8-9. Here, we hypothesize that obesity influences all aspects of fat in
physiology, including aging. However, there is little evidence on the effects of obesity on the aging
process of adipose tissue.
Here, we evaluated differential gene expression, miRNA and methylation data to profile female SAT
aging in Youth to Middle age. Analysis of aging-related genes revealed that female SAT aging is
characterized by immunosenescence, metabolic changes and defective angiogenesis. Our analysis also
identified the core molecular regulatory networks associated with female SAT aging and the
characteristics of SAT aging under the influence of obesity. Our data provide a better understanding of
the biological changes in SAT aging in non-obese and obese females. SAT is widely used in plastic and
reconstructive surgery 10-11

and there is evidence that many systemic metabolic diseases are associated
with SAT disorders 12-13. Therefore, our findings may guide targeted treatment of SAT aging associated
metabolic disorders, and improve outcomes for fat grafting in elderly and obese patients. Due to its
accessibility, SAT may be an ideal model for human aging studies.
2. Materials and methods
2.1. Microarray data
mRNA (GSE25401), miRNA (GSE25470) and methylation (GSE24884) datasets were downloaded
from GEO (Sheet S1 and Table S1). These datasets contained 56 female obesity samples14-15, of which
37 were selected and split into 6 groups according to age (youth: 20-30 years old, middle age: 40-50
years old, Elder: ≥60 years old) and obesity status (non-obese and obese).
2.2. Aging spectrums construction based on DEGs, DEMs and DMGs
Aging spectrums were constructed on the basis of differential gene expression profiles, miRNAs and
methylation, and visualized by heatmaps. Differentially expressed genes (DEGs) and differentially
expressed miRNAs (DEMs) between groups were identified using RankProd package16 in R. P ≤0·05
was set as cutoff threshold for DEGs and DEMs. Differential gene methylation (DMGs) was identified
using limma R package17 based on p ≤0·05 and differential mean methylation levels of ≥5% between
groups.
2.3. Integrative analysis of multi-omics interaction patterns
Molecular regulatory networks were constructed to uncover underlying intermolecular interactions
between the identified core aging related biological processes of SAT. Protein-protein interaction (PPI)
analysis for DEGs was done using STRING18 to construct differential co-expression network and
regulatory networks. Network node colors correspond to co-expression modules. Target interaction
relationships between DEMs and 3’UTRs of DEGs were downloaded from mirWalk219. Spearman
correlation coefficient between the expression patterns of DEMs and DEGs <-0·5 indicated existence of
regulatory relationships in networks. The same threshold of Spearman correlation coefficient was set to
identify DEGs-DMGs regulatory relationships. Interaction and regulatory networks between
DEGs-DEGs and DEGs-DEMs were visualized using CytoScape20.
2.4. Weighted gene co-expression network analysis (WGCNA)
We obtained the DEGs in different age groups of obese and non-obese females, and required their
union set which can be considered as aging-related DEGs (4560 DEGs) for further constructing the
scale-free gene co-expression network using “WGCNA” package 21

(Figure S3A). In this scale-free
network, only a few nodes were highly connected to others. Here, we used the signed correlation
method to transform the correlation coefficient of co-expression between nodes as follows: adjacency
(between nodes) = (one+correlation)/two, which weakened negative correlations and enhanced positive
ones. Next, the soft thresholding power, β, was set to 14 based on the ‘pick soft threshold’ function of
WGCNA. This soft thresholding power enabled the generation of an appropriate scale-free
co-expression network. The dynamic tree cut method was used to hierarchically cluster genes using the
dissimilarity matrix (1-TOM)22. The minimum cluster size required 50 genes, and the clustered
modules with high similarity were merged with a height cut-off of 0·4.
2.5. Differential gene co-expression analysis using DiffCoEx
To quantify differences in overall aging-related co-expression patterns in SAT between non-obese and
obese females, we performed differential co-expression (DiffCoEx) analysis based on WGCNA23.
Adjacency matrices for non-obese and obese cases microarray datasets were calculated separately and
adjacency values between nodes defined as follows: if correlations coefficient was >0, adjacent values
equaled the correlation coefficient; if correlations coefficient was ≤0, adjacent values were set as 0.
Topological overlap matrix was derived from the matrix of differences in powered adjacencies between
non-obese and obese cases and used as a distance metric. Genes were clustered by average hierarchical
clustering, so gene co-expression modules were partitioned with unique and similar variation patterns
between the 2 populations. DiffCoEx modules were further identified using the hybrid method of
dynamic tree cutting. Hierarchical clustering tree was cut at a height of 0·94. The minimum cluster size
of each module was 20 and merged cut height ≤0.2.
2.6. GSEA and GOBP enrichment analysis
The genes of co-expression modules from WGCNA and DiffCoEX analyses were intersected with
DEGs in different age groups for further enrichment analyses (Figure S4A-B. Next, Gene Set
Enrichment Analysis (GSEA)24 and Gene Ontology Biological Process (GOBP) enrichment analyses of
the selected DEGs in different groups were performed using Cluster Profiler R package25. Gene
expression profiling matrix and DEGs sets were divided according to age groups and obesity status.
Aging related biological-processes pathways with p ≤0 · 05 were identified as biological processes
involved in SAT aging.
3. Results
3.1. Multi-omics aging profiles of subcutaneous adipose tissue
The multi-omics expression profiling data obtained from 37 females were grouped as shown in Table1.
Our analyses offered an improved understanding of age-dependent variations in gene, miRNA and gene
methylation expression profiles during SAT aging. The SAT aging profiles for non-obese cases
uncovered 1860 DEGs (1148 (Youth vs Middle Age), 833 (Middle Age vs Elder)), 56 differentially
expressed miRNA (DEMs) (34 (Youth vs Middle Age), 25 (Middle Age vs Elder)) and 332 differential
gene methylation (DMGs) (204 (Youth vs Middle Age), 164(Middle Age vs Elder)) between the age
groups (p ≤0 · 05). Additionally, SAT aging-related expression profiles were evaluated in obese cases.
During aging, 1623 DEGs (711 (Youth vs Middle Age), 1019 (Middle Age vs Elder)), 95 DEMs (40
(Youth vs Middle Age), 60 (Middle Age vs Elder)) and 348 DMGSs (249 (Youth vs Middle Age), 205
(Middle Age vs Elder)) in all age groups (p ≤0 · 05; Table 2, Figure 1). A panorama of SAT aging
miRNAs and gene methylation spectrums for non-obese cases is shown in Figure S1A and Figure S2A
based on DEMs and DMGs. Because the DEGs aging spectrum is too broad, representative DEGs that
may be involved in the aging process are shown (Figure 3B)
3.2. Integrative analysis of multi-omics aging profiles
To elucidate the regulatory relationships between DMGs-DEGs-DEMs, we performed an integrative
analysis of multi-omics expression profiles for each case based on aging profiles. Remarkably, rich
regulatory relationships emerged between DEGs and DEMs. 500 regulatory relationships with
moderate correlation intensity ((r<-0·5, p≤0·05) between DEGs and DEMs in non-obese cases were
subjected to further study (Figure S1B). Based on differential gene methylation profiles and gene
expression profiles, none of the DEGs were considered to be regulated by methylation during SAT
aging. (Figure S2B). Although 111 DMGs were identified between different age groups, there was no
powerful evidence confirming the existence of regulatory patterns between aging associated DEGs and
DMGs in non-obese cases, so as to obese female (r<-0·5, p ≤0·05; Figure S2B, Table S2, S3).
Moreover, 119 and 308 methylated genes were identified in non-obese and obese cases (Sheet S2，S3).
In non-obese cases, these aging-irrelevant methylated genes were mainly involved in molecular
functions, cell signals and communication, cellular response, and metabolism (Figure S2C-D)
3.3. Identification of SAT aging related DEGs expression patterns
To further investigate the expression pattern of aging-related genes, we used WGCNA to identify
significant aging-related gene modules among 4560 genes (Figure S3A). Similar expression patterns
were grouped into modules via hierarchical average linkage clustering (Figure S3B). To ensure
relatively balanced scale independence and mean connectivity, a soft-thresholding power of 14 was set
for network construction (Figure S3C-D). From the gene expression profiles of non-obese cases, this
analysis identified 11 gene modules involving 4138 genes (Table S4, Figure 2A) and 10 modules,
closely associated with the aging process but with different patterns in SAT selected (Figure 2B). Thus,
together with the DEGs uncovered in the above analysis, 1754 DEGs remained for analysis at different
aging stages.
3.4. Aging-related specific biological processes of SAT
Next, we analyzed the selected enrichment DEGs using gene ontology biological processes (GOBP) to
identify aging-related pathways. Interestingly, comparison of the aging process between Youth-Middle
age and Middle Age-Elder, revealed shared specific BP terms. Significantly enriched GOBP pathways
in Youth or Middle age groups showed that advancing age was associated with an upregulation of
pathways related to inflammatory states (GO 0006954, GO 0001816, GO 0032943, GO 0042116),
tumor necrosis factor (GO 0032640, GO 0071706), lipid deposition and metabolism (GO 0008610, GO
0006629, GO 0044255, GO 0006633, GO 0032365). The Middle age group was also associated with
reduced immune response (GO 0070227, GO 0071887, GO 0002700, GO 0002274), energy
metabolism (GO 0046034, GO 0140053, GO 0006091) and angiogenesis (GO 1905288, GO 1905459)
(Figure 2C). In the aging process between Middle age and Elder, GOBP pathways associated with
carbohydrate metabolism (GO 0044262, GO 0006006, GO 0010675, GO 0006109, GO 0016052)
emerged as the main processes in the Elder group. Additionally, lower enrichment of functional
pathways implied a weakening of immune responses (GO 0045321, GO 0046649, GO 0002520, GO
0006955) and energy metabolism (GO 0046034) during aging (Figure 2C) Gene set enrichment
analysis (GSEA) was further used to characterize aging-related biological processes. During the SAT
aging process, GOBP enrichment pathways were associated with inflammatory states, immune
response, lipid metabolism, carbohydrate and energy metabolism. Additionally, the aging process was
accompanied by specific fat cell differentiation (GO 0045444) from Youth to Middle age, and B cell
mediated immunity (GO 0019724) from Middle age to Elder (Figure 2D)
3.5. SAT aging related interaction network and core nodes
Based on the aging related molecules and identified GOBP pathways (Sheet S4), we constructed an
interaction network to show the regulatory relationship between selected DEGs and DEMs. This
analysis highlighted 7 subnetworks that were then used to explore the regulatory patterns of our
focused aging related biological processes (immune response, inflammatory states, chronic
inflammatory cells, TNF, energy and carbohydrate metabolism and lipid metabolism) more explicitly
(Figure 3A). This process identified 70 significant core genes. A heatmap was then drawn from the
interaction network and associated gene modules based on WGCNA. Similarly, 28 miRNAs presented
in heatmap were considered core nodes, as they could modulate expression of core genes, altering the
aging related biological processes in the interaction network (Figure 3B-C)
3.6. SAT aging related module differences in obese and non-obese females
To explore SAT aging pattern differences in obese and non-obese females, differential co-expression
(DiffCoEx) analysis was done for aging-related genes. Of the 4560 genes analyzed, 539 were assigned
arbitrarily to one of 7 modules with various colors denoting various correlations representing different
expression patterns in obese and non-obese females (Figure 4A). The green, black, blue, brown, and
yellow modules contained 240 genes that were significantly co-expressed during the SAT aging process
in non-obese females. The red and turquoise modules, containing 299 genes, were notably co-expressed
in obese females during aging (Table S5). Bubble plot analysis showed that different aging stages
between obese and non-obese females were accompanied by different co-expression module patterns
(Figure 4B). The differential co-expression network was visualized using Cytoscape (Figure 4C).
3.8. Aging related functional enrichments and regulatory patterns in obese and non-obese females
Next, GOBP enrichment analysis for specific co-expression modules in each group revealed that SATs
of obese ad non-obese females experienced different biological processes during aging (Sheet S5).
During aging, non-obese female SAT experienced improved production of monocyte chemotactic
protein-1 (MCP-1, GO 0010243) while age progression from Youth to Middle Age encountered
increasing chronic inflammatory responses. Meanwhile, up-regulated lipid metabolism related
pathways (GO 1900076, GO 0031670) and down-regulated DNA duplication (GO 0044271) were also
significant. Additionally, highly enriched glycolytic fermentation (GO 0048523) was upregulated
during progression from Middle Age to Elder group (Figure 5A).
Our analysis found that during age progression from Middle Age to Elder groups, obese females SATs
experienced increased inflammatory mediators (GO 0009888, GO 0048869), immune response
suppression (GO 0099111, GO 0032984), wound healing (GO 0002697), mitochondrial gene
expression (GO 0030705), angiogenesis (GO 0035509, GO 0031622) and superoxide dismutase
activity (GO 0062012) (Figure 5A) Interestingly, based on GOBP analysis of pathways involved in
specific co-expression modules during aging, most aging related alterations of GOBP pathways in
non-obese females were identified in progression from Youth to Middle Age. For obese females, they
present in progression from Middle Age to Elder.
Based on DiffCoEx analysis, we constructed the molecular regulatory networks on our focused
aging-related biological processes under the influence of obesity. Each part in the networks revealed
the interaction patterns of genes and miRNAs involved in aging-related core biological alterations
(immune response, inflammatory state, mitochondria-related proteins, oxidative stress and lipid
metabolism) (Figure 5B). Finally, 8 genes and 2 miRNAs involved in various aging related biological
functions or at the center of the networks, were chosen for display (Figure 5C-D). The selected
molecules were regarded as key nodes with significantly different co-expression patterns and might be
the decisive factors in respective SAT aging processes in obese and non-obese females.
4. Discussion
SAT, along with visceral and yellow fat, constitute the systemic adipose tissue. Previous studies have
shown that during visceral fat aging, dysfunctional adipogenesis alters normal physiology, promoting
various pathological conditions, including metabolic disorders . Dynamic redistribution between

26-27
SAT and VAT during aging implies that as with VAT, SAT-associated physiological changes also
occur28. However, few studies have investigated SAT aging profiles. Here, we first profiled the SAT
aging landscape using multi-omics analysis, and identified specific aging-related biological processes,
interaction networks and associated core molecules that may be considered factors in SAT aging.
Aging is associated with immunosenescence, which weakens immunity and enhances susceptibility to
infections and cancer29. Immunosenescence in SAT is linked to altered immune and inflammatory
processes in aging. GOBP analysis of immune related pathways and regulatory patterns of highlighted
core molecules (Figure 2C, 3A) found that increased apoptosis and decreased lymphocytes and
leukocytes activation contribute suppressed immune response during aging. Additionally, development
of chronic low-grade SAT inflammatory state also characterized progression from Youth to Middle Age,
accompanied by elevated cytokine production as well as mononuclear and macrophage infiltration
(Figure 2C). We find that highly expressed proinflammatory factors, including IL6 and cytokines, are
closely linked to lipotoxicity and metabolic disorders30-31. This inflammatory SAT state may cause
regional chronic pain and dysfunction of parenchymal cells adjacent to inflammatory adipose tissue32-33.
Immunosenescence was associated with immune suppression, particularly humoral immunity, during
progression of Middle Age to Elder (Figure 2C-D).
Insulin resistance, an important feature of type 2 diabetes34-35, is associated with SAT. During
progression from Youth to Middle Age, SAT TNF production and mitochondrial gene expression may
promote insulin resistance (Figure 2C)36-37. We find that increased TNF superfamily cytokines
expression correlates with aging and may promote insulin resistance by interfering with response to
insulin. Besides, redundant TNFs and cytokines may accelerate fat catabolic processes and suppress
preadipocytes differentiation38, causing release of more fatty acids, lipid metabolites, and
proinflammatory factors into surrounding tissues and blood, promoting insulin resistance39-40. Previous
studies have shown that insulin resistance and diabetes are closely associated with impaired
mitochondrial function in humans and rodents41-42. Remarkably, we observed significantly reduced
mitochondrial gene expression in SAT during aging (Figure 2C), which may impair mitochondrial fat
oxidation and cooperate with TNF to promote insulin resistance.
In this study, we characterized altered metabolic patterns and decreased ATP production in SAT during
aging and observed that metabolism transitioned from enhanced lipid metabolism to enhanced
carbohydrate metabolism (Figure 2C, 3A). Various studies have shown that body fat redistribution
during aging increases trunk fat (represented by abdominal fat), and reduces peripheral SAT43-44.
During progression from Youth to Middle Age, increased lipid and fatty acid biosynthesis, and reduced
lipid transport may synergistically contribute to excessive deposition of abdominal SAT (Figure 2C),
which may affect energy metabolism and lead to abdominal obesity associated pathologies in Middle
Age. In later aging processes, significantly upregulated carbohydrate metabolism may be a
compensatory mechanism for ATP supply changes following decreased fat mass in the Elder group
(Figure 2C).
Previous studies show that defective angiogenesis is common in the Elder group45. Consistently, SAT
angiogenesis is suppressed during aging, enhancing vascular smooth muscle cell apoptosis in
progression from Youth to Middle Age (Figure 2C). Excessive deposition of abdominal SAT and
reduced angiogenesis in Middle Age contribute to regional hypoxia and chronic inflammation9. Thus,
aging related defective angiogenesis is unfavorable for wound healing and fat graft survival after
autologous fat transplantation46-47.
We find that excessive VAT and SAT accumulation contributes to obesity, which characterizes SAT
aging. Our data implies that SAT between obese and non-obese females exhibits different aging
features at specific stages. DiffCoEx analysis revealed that changes in aging-related biological
processes in non-obese females mainly ocurred during progression from Youth to Middle Age. In obese
females the changes were more significant during pregression from Middle Age to Elder (Figure
5A-B).
DiffCoEx analysis showed that SAT immunosenescence and inflammatory state presented different
aging-associated variation patterns in obese and non-obese females. In non-obese females, the
characteristically SAT upregulated monocyte chemotactic protein-1 (MCP-1) may mediate chronic
inflammation during progression from Youth to Middle Age (Figure 5A)48. In obese females, during
progression from Middle age to Elder, suppression of natural killer cell differentiation and the
biological activity of immunoglobulin contribute to nonspecific immunity immunosenescence. Our
data showed that obesity aggravates SAT aging-related inflammation, mainly by enhancing infiltration
by inflammatory cells and cytokines (Figure 5A). Furthermore, reduction in HDAC4, VAV1 and TNF
expression in obese people suppressed development and activation of T and B cells, and promoted SAT
immunosenescence and inflammation49-50 (Figure 5C). Besides, inflammation, along with impaired
wound healing-related inflammatory responses and suppression of SAT angiogenesis, may cooperate to
promote chronic wounds and lower fat graft survival in obese females51-52 (Figure 5A)
DiffCoEx analysis shows that dysfunctional SAT metabolism and accelerated aging are remarkable
aging characteristics in obese females. Since compensatory lipid catabolism and metabolism, and
glycolytic fermentation were impaired in obese females, regional females SAT may have experiencd
excessive lipid deposition and insufficient energy supply. Additionally, increased lipid production and
deposition was associated with enhanced compensatory LPL expression in Middle Age for non-obese
female but not in obese female. Decreased FGF19 expression in obese people suppressed lipid
metabolism53 (Figure 5C). The 2 factors contribute excessive regional lipid accumulation and lipid
metabolism dysfunction in obese females54. Moreover, elderly obese females are more likely to develop
type II diabetes. Our data suggest that decreased SAT mitochondrial gene expression during aging may
reduce fatty acid beta-oxidation levels and elevate the risk of metabolic disorders for obese females55.
Additionally, during progression from Middle Age to Elder, obese female SAT was in an accelerated
aging status attributable to increased oxidative stress due to reduced superoxide dismutase activity56
(Figure 5A).
SAT is widely distributed in the human body and exhibits aging-related features that are desirable in
aging models57-58. Systemic aging is accompanied by immunosenescence, inflammation and impaired
angiogenesis as demonstrated in numerous studies5, 59, including ours. SAT metabolic change is unique,
and may improve our understanding of metabolic variation patterns associated with aging and
aging-related metabolic disorders. Integrative analysis of SAT multi-omics aging profiles implied that
there was little regulatory relationship between aging associated DEGs and DMGs. Therefore, we
believe that methylation has little influence on the expression of aging related genes during SAT aging
in obese and non-obese females. Relative to non-obese females, methylation is more likely to modulate
gene expression in obese individuals (Sheet S3). These findings suggest that future studies on aging
related regulatory relationships should focus on DEGs-DEMs interaction networks and the differential
gene methylation characteristics during SAT aging in obese and non-obese females.
Our study has the following limitations. First, the multi-omics expression profiling data used were all
obtained from GEO. Since no project fully matched our research direction, only relevant sections of the
dataset were selected for further exploration. Second, all data analyzed in this study were collected
from females and therefore may not reflect SAT in males. Third, sampling was limited to abdominal
SAT and therefore is not representative of peripheral SAT. Fourth, we did not uncover strong evidence
that genes in our focused biological processes were regulated by methylation, and only the interaction
network between genes and miRNAs was demonstrated. Fifth, we didn’t perform experimental
validation of key molecules and pathways. Despite these limitations, the robustness of the selected
datasets and appropriate statistical models, insured reliable conclusions. We will next perform single
cell sequencing using a similar experimental design and experimental verification to validate our
observations.
In conclusion, herein, for the first time, we systematically describe the entire landscape of aging-related
molecules, biological processes, and core regulatory networks for the SAT (Fig.6). In the aging process,
gene expression and methylation profiles exhibited variation patterns, while there was no distinct
regulatory relationship between them. SAT showed specific aging-related characteristics in the different
aging stages. We established that immunosenescence, chronic inflammatory state, impaired
mitochondria, alterations of the metabolic patterns, and defective angiogenesis are typical physiological
characteristics of the female SAT associated with aging. These aging-related physiological
characteristics of the SAT were shown to contribute to numerous pathological states (such as insulin
resistance, regional excessive lipid deposition, and chronic pain) and decrease the fat graft survival,
further affecting the clinical application of SAT. Moreover, comparing the aging patterns of SAT
between obese and non-obese females, we found that the characteristics of SAT aging in obese females
were more remarkable from the middle age to the elderly age. The aging process of SAT for obese
females was more likely characterized by nonspecific immune impairment, reduced metabolic
compensatory, exacerbated impaired mitochondria and angiogenesis, and increased oxidative stress.
Therefore, the SAT of obese females showed an accelerated aging process with higher risks of type 2
diabetes and developing chronic nonhealing wounds compared with non-obese females during aging.
Since the aging SAT has biological processes similar to the aging systemic tissue and has its specific
metabolic characteristics, we speculate that SAT is a potential novel biological model to explore aging
and aging-related disorders. This is the first systematic study exploring the aging process of the human
SAT and the relationship between the aging process and obesity. Our findings provide novel insights
into the physiological changes of SAT during aging, which will guide future anti-aging and obesity
research.
Acknowledgement
We thank the following institution for giving us powerful support for raw data collection and
multi-omics data analysis: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth
Military Medical University. The authors thank Freescience Academic Services
(freescience@zju.edu.cn) for English language editing and review services.
Funding source
This study was funded by the National Natural Science Foundation of China (No. 81772071; No.
81971835; No. 81772072).
Declaration of Competing Interests
All authors declare no competing interests.
Author contributions
DHH, HTW and ZCL contributed to the study and experimental design. ZCL, SW and SJL contributed
to raw data collection. ZCL, SW, and XWY contributed to the multi-omics data analysis. ZCL,YCW,
JQL, LL and XJB contributed to the quality control and verification of data. SJL, XJW and PJ
contributed to the additional data analysis. ZWX and SW contributed to the quality control of the figure
and table. ZCL, SJL, and SW wrote the manuscript. All authors read and approved the final manuscript.
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Figure legends
Fig.1 Identification of differentially expressed mRNAs, miRNAs and methylation in aging processes
for both obese and non-obese female. Volcano Plots showed the up-regulated (log2(fold change)>0) and
down-regulated (log2(fold change)<0) differential molecules respectively in each group. Venn diagram
represented unique differential molecules in each aging stage and the shared differential molecules that
are up-regulated or down-regulated at the same time in two aging stages, i.e. YG(Youth Group) vs
MG(Middle Age Group) and MG(Middle Age Group) vs EG(Elder Group) .
Fig.2 The co-expression patterns of genes and visualization of core GOBP pathways of SAT in aging
process. a. Visualization of gene cluster tree. Based on the adjacency-based dissimilarity of the
hierarchical clustering genes, the tree diagram corresponding to color was established by the dynamic
tree cutting method recognition module. 11 modules were identified with specific co-expression
patterns and notated with different colors. b. 10 aging-related gene co-expression modules with
different enrichment status of SAT in each group. Enrichment Score indicted the ratio of DEGs in a
specific module to all genes involved in the WGCNA network. Fisher P Value also presented the
enrichment level of DEGs in each gene module with a significant level< 0.05. c. Functional enrichment
analysis for selected DEGs. 30 GOBP pathways for up- or down-regulated DEGs were used to
demonstrated with bubble plot. Gene counts(n) showed the overlap between DEGs and the significant
genes from a pathway. P value represented the enrichment status of the selected DEGs, with a
significant level< 0.05. d. Gene set enrichment analysis of genes based on age groups. GOBP items,
normalized enrichment score (NES) and P value were also presented in chart.
Fig.3 The regulatory networks and heatmaps of core molecules. a. The regulatory networks of
DEGs-DEmiRNAs involved in the core biological processes in SAT aging. The color of each genes in
the network corresponds to its positioned co-expression modules based on WGCNA. b. Heat map of
DEGs involved in core biological processes in different age groups. DEGs were distributed in different
color groups, in consistence with their co-expression modules. c. Heat map of DEmiRNAs involved in
core biological processes in different age groups. DEmiRNAs were assigned in different color groups
based on the positioned co-expression modules of their regulated DEGs.
Fig.4 DiffCoEx analysis based on aging related genes between obese and non-obese female. a. The
comparative correlation heatmap showed the differentially co-expressed modules between obese and
non-obese female based on aging related genes. The upper diagonal of the main matrix shows a
correlation between pairs of genes among the non-obese female (dark color corresponds to higher
correlations; light, lower correlations). The lower diagonal of the heatmap shows a correlation between
the same gene pairs among the obese female. Color bars at the edge of the square showed modules in
which genes co-expressed differently between obese and non-obese female (second column). b. 7
aging-related differential co-expression modules with different enrichment status of SAT in each group.
Enrichment Score indicted the ratio of DEGs in a specific module to all genes involved in the
DiffCoEx network. The significant level of Fisher P Value is < 0.05. c. Differential co-expression
network of SAT between obese and non-obese female. Nodes in different colors represented genes in
different modules. Red lines showed stronger correlation in obese female than in non-obese female;
blue lines show stronger correlation for non-obese female. The thickness of the connections between
two nodes represented the correlation strength.
Fig.5 Specific aging related alteration of biological processes and core genes in obese or non-obese
female based on DiffCoEx analysis. a. On basis of DiffCoEx analysis, specific aging related GOBP
enrichment pathways for obese or non-obese female were demonstrated with bubble plots. Gene
counts(n) showed the overlap between DEGs and the significant genes from a pathway. P value
represented the enrichment status of the selected DEGs, with a significant level< 0.05. b. The
regulatory networks of DEGs-DEmiRNAs involved in the core biological processes based on DiffCoEx
analysis of aging process between obese and non-obese female. The color of each gene in the network
corresponds to its positioned modules based on DiffCoEx analysis. “*” represented the core molecules
potentially performing the important functions in SAT aging. c. Box plots of expression of selected
core genes of SAT for both obese and non-obese female in different age groups. d. Box plots of
expression of selected core miRNAs of SAT for both obese and non-obese female in different age
groups.
Fig.6 The redistribution and alterations of biological processes of SAT, and high-risk diseases related to
adipose tissue status. First, we showed the redistribution of SAT in aging. More SAT was accumulated
in Middle Age among non-obese female. Hypertrophy and a disorganized array of adipocytes were
exhibited among obese female. Second, we showed the changes in biological status of SAT in different
age groups and listed the high-risk diseases, which was increased caused by the variations of biological
status of SAT in aging process. Third, we showed the specific aging-related variation patterns of SAT
affected by obesity and more significantly increased risks of presented diseases were tagged for obese
female.
DEmRNA DEmiRNA DMR
Obese Nonobese Obese Nonobese Obese Nonobese
Youth 3 6 3 6 3 6
Middle Age 11 9 11 9 11 9
Elder 3 5 3 5 3 5
Table1 The expression profiling data were obtained from 37 female, which were divided into groups
according to ages and obese status.
Non-obese population Obese population
YG vs.MG MG vs. EG YG vs.MG MG vs. EG
Up-regulated 557 436 388 451

DEmRNAs
Down-regulated 591 397 323 568
Up-regulated 12 14 6 35
DEmiRNAs
Up-regulated 117 100 150 135

DMRs
Table 2 Description of DEGs, DEMs and DMGs for SAT in different age groups among non-obese and
obese female.
Supplementary tables, figures and legends
Annotated
Data set Platform Probe
molecules
GPL6244
mRNA Profiling GSE25401 32331 16180
(Affymetrix Human Gene 1.0 ST)
GPL8786
miRNA Profiling GSE25470 (Affymetrix Multispecies 847 805
miRNA-1 Array)
GPL8490
Methylation Profiling GSE24884 (Illumina HumanMethylation27 27578 12701
BeadChip)
Table S1 The data sets platforms probes and annotated molecules of the multi-omics chip data
including gene profiling, miRNA profiling and gene methylation profiling.
Spearman Corr.(r) P value
AKAP6 -0.308270677 0.185681755
TOR2A -0.248120301 0.290194328
AMELX -0.239097744 0.308552435
MIA2 -0.060150376 0.801607069
DHX33 0.012030075 0.961971391
ZNF512 0.012431351 0.962231851
ZNF408 0.081203008 0.73347559
MYEOV 0.159458454 0.501889551
ASB9 0.181954887 0.440913257
BRSK1 0.184962406 0.433273062
AADACL2 0.210526316 0.371305188
CD1C 0.231578947 0.324387987
RNF14 0.326315789 0.160224304
ERRFI1 0.333834586 0.150382557
PRPH2 0.357894737 0.121794087
BTBD2 0.393984962 0.086660675
Table S2. Spearman correlation coefficient (r) and P value of the correlation between the expression
of 16 selected DEGs and methylation level among non-obese female. “r < −0.5, P ≤ 0.05” denoted
statistical significance.
Spearman Corr.(r) P value
IL17A -0.279411765 0.276432988
TRIM55 -0.237745098 0.356747173
MUC1 -0.205103015 0.429704159
IL23R -0.031862745 0.905925224
NFKBIB -0.019607843 0.94347712
ALG13 0.056372549 0.831480414
COMT 0.115196078 0.659518278
NR5A2 0.139705882 0.592010128
ADD3 0.151960784 0.559403278
SMCO4 0.178921569 0.490666292
ABCC9 0.18872549 0.466762825
COL3A1 0.208333333 0.420812044
TLR3 0.367647059 0.14710262
ITLN2 0.424019608 0.091305667
UNC5D 0.705882353 0.002128774
Table S3. Spearman correlation coefficient (r) and P value of the correlation between the expression
of 15 selected DEGs and methylation level among obese female. “r < −0.5, P ≤ 0.05” denoted statistical
significance.
WGCNA Module turquoise blue brown yellow green red
Gene Numbers 526 509 505 411 407 405
WGCNA Module black pink magenta purple greenyellow out of modules
Gene Numbers 387 294 257 245 192 422
Table S4 The numbers of co-expression DEGs in each gene module based on WGCNA for non-obese
female.
WGCNA Module black blue brown green
Gene Numbers 38 80 43 39
WGCNA Module yellow red turquoise out of modules
Gene Numbers 40 38 261 4021
Table S5 The numbers of differential co-expression genes in each gene module based on WGCNA
between obese and non-obese female.
Fig. S1 Aging spectrum of miRNA and the DEMs-DEGs interaction relationships. a. Heat map of the
expression of 56 DEMs in different age groups among non-obese female. b. Flowchart for exploring
the DEMs-DEGs interaction relationships among non-obese female.
Fig. S2 Aging spectrum of gene methylation, flowchart and functional enrichments. a. Heat map of the
expression of 111 DMGs in different age groups among non-obese female. b. Flowchart for exploring
the regulatory relationships between aging related gene expression and methylation of SAT. “r < -0.5，P
≤0.05” in Spearman correlation analysis denoted statistical significance. c and d. Top 20 GOBP
pathways of the aging-irrelevant methylated genes among non-obese(c) and obese female (d),
respectively.
Fig. S3 Analysis of the network topology for adjacency matrix weighting parameters (power). a. 4560
genes united by the 10 parts were considered as the potential aging-related DEGs for WGCNA.
b.Hierarchical average linkage clustering for the above screened DEGs. Branches of the dendrogram
represent DEGs with similar expression patterns. c and d. The x-axis represents soft threshold (power),
and the y-axis represents the scale free fitting index and connectivity for each power. The
soft-thresholding power for network construction was set at 14.
Fig.S4 Flowchart for target co-expression DEGs and DiffCoEx DEGs. a. Flowchart for the target
co-expression DEGs in non-obese female. The Up- and Down-regulated co-expression modules of
WGCNA were separately intersected with the corresponding co-expression DEGs in different age
groups for further enrichment analysis. b. Flowchart for the target DiffCoEx DEGs between non-obese
and obese female. The DiffCoEx modules of WGCNA were intersected with the corresponding DEGs
profiling in different groups divided by age and obese status.
Sheet S1 Detailed information of the 37 samples and their sample ID (GSM) corresponding to
multi-omics data.
Sheet S2 Spearman correlation coefficient (r) and P value of the correlation between gene methylation
profiling and gene expression profiling among non-obese female.
Sheet S3 Spearman correlation coefficient (r) and P value of the correlation between gene methylation
profiling and gene expression profiling among obese female.
Sheet S4 The description of the aging-related biological processes among non-obese female based on
GOBP enrichment analysis.
Sheet S5 The description of specific aging-related biological processes based on DiffCoEx analysis
between non-obese and obese female.
er has not been peer reviewed. Electronic copy available at: https://ss
　 DEmRNA DEmiRNA DMR
Obese Nonobese Obese Nonobese Obese Nonobese
Youth 3 6 3 6 3 6
Middle Age 11 9 11 9 11 9
Elder 3 5 3 5 3 5
Table1 The expression profiling data were obtained from 37 female, which were divided into groups
according to ages and obese status.
　　 Non-obese population Obese population
　　 YG vs.MG MG vs. EG YG vs.MG MG vs. EG
Up-regulated 557 436 388 451

DEmRNAs
Up-regulated 12 14 6 35
DEmiRNAs
Up-regulated 117 100 150 135

DMRs
Table 2 Description of DEGs, DEMs and DMGs for SAT in different age groups among non-obese
and obese female.

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New insights into aging and obesity-associated aging characteristics of female

subcutaneous adipose tissue revealed by integrative analysis of multi-omics data

710032 Xi’an, Shaanxi, China

University, 610041Chengdu, Sichuan, China.

*Corresponding authors: wanght@fmmu.edu.cn (Hongtao Wang); hudhai@fmmu.edu.cn (Dahai.Hu)

on the SAT during aging.

primarily experiences the immunosenescence, alterations of metabolism, and defective angiogenesis in

DiffCoEx analysis of SAT.

and aging-related metabolic disorders in the future.

81971835; No. 81772071; No. 81772072).

correlation network analysis (WGCNA), differential co-expression (DiffCoEx) analysis.

Evidence in this study

process of SAT is important and an interesting direction to study.

Added value of this study

population compared to the non-obese population.

Implications of all available evidence

approach for the frontier of targeted therapy of aging and obesity.

Aging is characterized by progressive decline of systemic physiological functions and represents a

process of adipose tissue.

reconstructive surgery 10-11

accessibility, SAT may be an ideal model for human aging studies.

2. Materials and methods

2.1. Microarray data

2.2. Aging spectrums construction based on DEGs, DEMs and DMGs

2.3. Integrative analysis of multi-omics interaction patterns

Molecular regulatory networks were constructed to uncover underlying intermolecular interactions

identify DEGs-DMGs regulatory relationships. Interaction and regulatory networks between

DEGs-DEGs and DEGs-DEMs were visualized using CytoScape20.

2.4. Weighted gene co-expression network analysis (WGCNA)

scale-free gene co-expression network using “WGCNA” package 21

2.5. Differential gene co-expression analysis using DiffCoEx

obese females, we performed differential co-expression (DiffCoEx) analysis based on WGCNA23.

of each module was 20 and merged cut height ≤0.2.

2.6. GSEA and GOBP enrichment analysis

Aging related biological-processes pathways with p ≤0 · 05 were identified as biological processes

involved in SAT aging.

3.1. Multi-omics aging profiles of subcutaneous adipose tissue

may be involved in the aging process are shown (Figure 3B)

3.2. Integrative analysis of multi-omics aging profiles

To elucidate the regulatory relationships between DMGs-DEGs-DEMs, we performed an integrative

3.3. Identification of SAT aging related DEGs expression patterns

3.4. Aging-related specific biological processes of SAT

pathways related to inflammatory states (GO 0006954, GO 0001816, GO 0032943, GO 0042116),

carbohydrate metabolism (GO 0044262, GO 0006006, GO 0010675, GO 0006109, GO 0016052)

pathways implied a weakening of immune responses (GO 0045321, GO 0046649, GO 0002520, GO

3.5. SAT aging related interaction network and core nodes

aging related biological processes in the interaction network (Figure 3B-C)

increasing chronic inflammatory responses. Meanwhile, up-regulated lipid metabolism related

during progression from Middle Age to Elder group (Figure 5A).

experienced increased inflammatory mediators (GO 0009888, GO 0048869), immune response

present in progression from Middle Age to Elder.

various pathological conditions, including metabolic disorders . Dynamic redistribution between

accompanied by elevated cytokine production as well as mononuclear and macrophage infiltration

progression of Middle Age to Elder (Figure 2C-D).

mitochondrial function in humans and rodents41-42. Remarkably, we observed significantly reduced

oxidation and cooperate with TNF to promote insulin resistance.

Age. In later aging processes, significantly upregulated carbohydrate metabolism may be a

autologous fat transplantation46-47.

biological activity of immunoglobulin contribute to nonspecific immunity immunosenescence. Our

aging models57-58. Systemic aging is accompanied by immunosenescence, inflammation and impaired

aging stages. We established that immunosenescence, chronic inflammatory state, impaired

Military Medical University. The authors thank Freescience Academic Services

(freescience@zju.edu.cn) for English language editing and review services.