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Gao
Original Research Communication
SOD3 is secreted by adipocytes and mitigates high-fat diet-induced obesity,
inflammation and insulin resistance
Authors: Dan Gao1a, Sijun Hu2, Xuewei Zheng3, Wenjuan Lin3, Jing Gao1, Kewei
Chang4, Daina Zhao1, Xueqiang Wang1, Jinsong Zhou4, Shemin Lu5, Helen R Griffiths6*,
Jiankang Liu1*
Affiliations:
1. Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical
Information Engineering of the Ministry of Education, School of Life Science and
Technology and Frontier Institute of Science and Technology, Xi'an Jiaotong University,
Xi'an, Shaanxi 710049, China.
2. Department of Gastroenterology, Xijing Hospital of Digestive Diseases, State Key
Laboratory of Cancer Biology and Institute of Digestive Diseases, Xi'an, Shaanxi
710000, China.
3. The Key Laboratory of Biomedical Information Engineering of the Ministry of
Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an,
Shaanxi, China.
4. Department of Human Anatomy, Histology and Embryology, School of Basic Medical
Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061,
China.
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5. Department of Biochemistry and Molecular Biology, School of Basic Medical
Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061,
China.
6. Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH,
U.K.
a: Present address
Department of Human Anatomy, Histology and Embryology, School of Basic Medical
Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061,
China.
Running title: SOD3 and obesity
* Corresponding authors :
Jiankang Liu, Ph.D.
Center for Mitochondrial Biology and Medicine, School of Life Science and Technology,
Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China.
Telephone: +86 (0) 29 8266 5849
Email: [email protected]
Helen R Griffiths, Ph.D.
Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, U.K.
Telephone: +44 (0) 1483 689586
Email: [email protected]
Word count: 6045
Reference numbers: 31
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Grayscale illustrations: 2
Color illustrations: 9 (online 11 and hardcopy 0)
Key words: SOD3; adipose tissue; lipid metabolism, metabolic pathways; obesity
Abstract
Aims: To study the expression and regulatory role of SOD3 in adipocytes and adipose
tissue.
Results: SOD3 expression was determined in various tissues of adult C57BL/6J mice,
human adipose tissue and epididymal (eWAT), subcutaneous (sWAT) and brown (BAT)
adipose tissue of high-fat diet (HFD)-induced obese mice. SOD3 expression and
release were evaluated in adipocytes differentiated from primary human preadipocytes
and murine bone marrow-derived mesenchymal stem cells. The regulatory role for
SOD3 was determined by SOD3 lentivirus knockdown in human adipocytes and global
SOD3 KO mice. SOD3 was expressed at high levels in white adipose tissue and
adipocytes were the main cells expressing SOD3 in adipose tissue. SOD3 expression
was significantly elevated in adipose tissue of HFD-fed mice. Moreover, SOD3
expression and release were markedly increased in differentiated human adipocytes
and adipocytes differentiated from mouse bone marrow-derived mesenchymal stem
cells compared to undifferentiated cells. In addition, SOD3 silencing in human
adipocytes increased expression of genes involved in metabolic pathways such as
PPARγ and SEEBP1c and promoted the accumulation of triglyceride. Finally, global
SOD3 KO mice were more obese and insulin resistant with enlarged adipose tissue and
increased triglyceride accumulation.
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Innovation: Our data showed that SOD3 is secreted from adipocytes and regulates
lipid metabolism in adipose tissue. This important discovery may open up new avenues
of research for the cytoprotective role of SOD3 in obesity and its associated metabolic
disorders.
Conclusion: SOD3 is a protective factor secreted by adipocytes in response to
HFD-induced obesity and regulates adipose tissue lipid metabolism.
1. Introduction
Obesity is one of the major health problems worldwide and the number of
overweight or obese people has increased rapidly over the last three decades [10]. A
primary characteristic of obesity is the accumulation of adipose tissue in subcutaneous
and visceral compartments, which is attributed to enlargement of adipocytes and
proliferation of adipose progenitors [3]. As a consequence of obesity, an increase of
adipose tissue has been observed in several major diseases, such as type 2 diabetes,
dyslipidemia, hypertension, cardiovascular disease and fatty liver disease [8].
Adipose tissue expresses and secretes a variety of adipokines, i.e., enzymes, pro-
inflammatory and anti-inflammatory cytokines, hormones, peptides and other
biologically active molecules, which actively regulate whole body metabolism, energy
homeostasis and inflammatory processes [19, 30]. Adipose tissue dysfunction is
reflected by an altered adipokine secretion pattern, such as reduced circulating
concentrations of adiponectin and increased pro-inflammatory cytokines, which
subsequently contribute to insulin resistance and a pro-inflammatory state.
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SOD3 is one of three superoxide dismutase (SOD) family members and is primarily
localized within ECM and at the cell surface [31] and catalyzes the dismutation of
superoxide anion radical to hydrogen peroxide. In contrast, SOD1 and SOD2 are
located intracellularly in the cytoplasm and mitochondria respectively. SOD3 was
previously reported to protect against adipose tissue inflammation and insulin resistance
[6]. Furthermore, serum SOD3 levels were shown to be negatively correlated to insulin
resistance in type 2 diabetes) [1] and metabolic syndrome [13]. In our previous study,
we also detected SOD3 in the culture media of epidydimal adipose tissue from adult
C57BL6/J mice. Together these data suggest that SOD3 may be secreted from adipose
tissue and may have a close link to obesity and obesity-associated metabolic disorders.
In the present study, we investigated the hypothesis that adipocytes secrete SOD3
which functions as a protective factor for adipose tissue. We first studied the tissue
expression pattern of SOD3 in adult C57BL/6J mice and its cellular location within
adipose tissue. Then, we determined SOD3 expression in different adipose tissues from
normal diet (ND) and 4 weeks and 12 weeks high fat diet (HFD)-fed obese mice.
Furthermore, we examined SOD3 expression and secretion in differentiated adipocytes
from human primary preadipocytes and bone marrow-derived mesenchymal stem cells
in vitro and examined the regulatory role of SOD3 by siRNA SOD3 knockdown in
human adipocytes and in SOD3 KO mice fed with ND and HFD.
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Results
SOD3 is expressed at high levels in adipose tissue and adipocytes
We initially examined SOD3 mRNA expression in various tissues of adult C57BL/6
mice. As shown in Fig. 1A, SOD3 mRNA was highly expressed in kidney, eWAT, BAT,
aorta and lung tissues. Consistent with the mRNA expression pattern, we detected both
intact and cleaved form of SOD3 and found high levels in lung, kidney, eWAT, BAT,
aorta and serum (Fig. 1B-C). Moreover, we isolated adipocytes and stromal-vascular
(SV) fractions from adipose tissue of adult C57BL/6 mice and found that adipocytes
predominantly expressed SOD3 in adipose tissue compared to the low expression in SV
fractions (Fig. 1D-E). This result was further confirmed by the SOD3
immunohistochemistry staining showing adipocytes were SOD3 positive in human
subcutaneous adipose tissue (Fig. 1F).
SOD3 expression is increased in adipose tissue of HFD-fed obese mice
To investigate the relationship between SOD3 and obesity, we examined SOD3
expression in adipose tissue from adult C57BL/6 mice fed with a ND or a HFD for 4
weeks and 12 weeks. There was a significant increase of body weight and adipose
tissue in mice fed with HFD for 4 weeks and 12 weeks (Fig. 2A and 2B). SOD3
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expression was increased in eWAT, sWAT and BAT of HFD-fed mice for 4 weeks (Fig.
2C-D) and remained elevated for 12 weeks (Fig 2E-J) while SOD3 expression in serum
and lung were not different between ND and HFD groups (Supplementary Fig. S1). In
contrast to SOD3 expression, SOD1 expression was reduced in three adipose depots
from HFD-fed mice while SOD2 expression remained unchanged in eWAT and BAT but
was reduced in sWAT of HFD-fed mice (Fig. 2E-J).
SOD3 expression is increased in adipocytes differentiated from murine bone
marrow-derived mesenchymal stem cells
To study the relationship between lipid storage and SOD3 expression in
adipocytes, we examined SOD3 expression in adipocytes differentiated from murine
BM-MSCs. As shown in Fig. 3A, we observed the presence of lipid droplet-containing
cells at differentiation day 6, 9 and 12 (Fig. 3A). Also, the mRNA of adiponectin and
adipocyte differentiation marker PPARγ expression were markedly induced in these
cells at differentiation day 6 (Fig. 3C and 3D). Furthermore, there was a significant
increase of SOD3 mRNA (Fig. 3C, right panel), protein expression (Fig. 3D-E) and
release determined by Western blotting (Fig. 3F-G) in differentiated adipocytes
compared to undifferentiated BM-MSCs.
SOD3 expression is increased in differentiated human adipocytes
Next, we studied the expression and secretion of SOD3 in human primary
preadipocytes and after differentiation to adipocytes. Differentiation of pre-adipocytes
resulted in a phenotype of mature adipose tissue indicated by the storage of lipid
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droplets and triacylglycerol (Fig. 4A-B). We compared SOD1, SOD2 and SOD3 mRNA
expression during preadipocyte differentiation. As shown in Fig. 4C, preadipocyte (D0
cells) expressed high levels of SOD1 and SOD2 mRNA but lower levels of SOD3
mRNA. In contrast, differentiated adipocytes (D9-D12 cells) expressed all three SOD
isoforms at similar levels. Furthermore, while SOD2 mRNA levels were more stable in
preadipocytes and adipocytes, SOD1 and SOD3 mRNA levels were significantly
increased by differentiation (Fig. 4C). Consistent with the mRNA data, we found an
increase of SOD3 (both intact and cleaved) in differentiated adipocytes from day 6 to
day 9 (Fig. 4C). Consistent with mRNA data, SOD3 protein levels in lysates and release
in cell culture media determined by Western blotting were increased in differentiated
adipocytes compared to undifferentiated preadipocytes (D0 cells) (Fig. 4D, 4F, 4I-J). In
addition, both SOD1 and SOD2 protein levels were significantly increased in
differentiated adipocytes (Fig. 4E, 4G-H).
SOD3 silence affects metabolic pathways and increased lipid accumulation in
adipocytes
To investigate the regulatory role of SOD3 in human adipocytes, we transfected
adipocytes with human SOD siRNAs (Supplementary Fig. S5) to knockdown SOD3
expression in adipocytes and followed with a RNA-Seq analysis (Supplementary Fig.
S5A-B). In total, 253 differentially expressed genes were identified with 93 genes
upregulated and 160 genes downregulated by SOD3 knockdown (Fig. 5C). Next, we did
pathway enrichment analysis for total 253 differentially expressed genes and found 60
pathways with statistically significant differences (Supplementary Fig. S5C). The most
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enriched pathway was metabolic pathways (Supplementary Fig. S5D). We further
analyzed the individual genes in metabolic pathways. In total, there were 20
differentially expressed genes enriched in metabolic pathways, which fall into glycan,
lipid and amino acid metabolism (Supplementary Fig. S5E). We found that SOD3
silencing affected genes involved in TAG synthesis (GPAT3 and DGAT2) and long
chain polyunsaturated fatty acid metabolism (CYP2C8). In contrast, a reduction of
genes involved in sphingolipid ceramide degradation (ASAH1) and very long chain fatty
acid oxidation (ACADVL) were also observed. We also validated the above gene
expression differences by real-time PCR and confirmed the effects of SOD3 knockdown
on these metabolic pathways (Supplementary Fig. S5F).
To investigate whether SOD3 knockdown affects lipogenesis, we treated human
differentiating adipocytes (D3) with SOD3 shRNA lentivirus until day 12. Consistent with
the siRNA treatment, lentivirus treatment led to a marked reduction of SOD3 mRNA
(Fig. 5C) and protein levels (Fig. 5D) in human adipocytes. The lipid content was
analyzed by Oil Red O and quantified at 510nm. As shown in Fig. 5A and 5B, we found
a significant increase in lipid content of SOD3 knockdown cells. Moreover, we found an
increase of genes involved in adipogenesis (PPARγ and FABP4), lipogenesis
(SREBP1c, ACC1, SCD1, CD36, GPAT3 and DGAT2), fatty acids β-oxidation (CPT1α,
FATP1, PPARα and Acox), and lipolysis (perillipin 1) in SOD3 knockdown adipocytes.
Consistent with the mRNA data, we observed an increase of protein expression of
PPARγ, SREBP1c, GPAT3 and DGAT2, p-HSL and ATGL in SOD3 knockdown
adipocytes (Fig. 5D and 5E). In addition, we also observed an increase in lipid content
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and similar mRNA pattern in preadipocytes treated with SOD3 shRNA lentivirus
(Supplementary Fig. 6S).
SOD3 knockout (KO) mice are more obese and insulin resistant than WT mice
To investigate the role of SOD3 in obesity in vivo, we generated global SOD3 KO
mice (Fig. 7B, supplementary Fig. S4). SOD3 KO and WT control mice were fed with a
normal diet (ND, 10% kcal fat content) and a high-fat diet (HFD, 60% kcal fat content)
for 12 weeks. Overall, mice fed with HFD showed higher calorie intake than mice fed
with ND (Supplementary Fig. S7B). Moreover, we found a lower energy expenditure and
activity in SOD3 KO mice under HFD compared to WT while there was no difference
between KO and WT on ND (Supplementary Fig. S7C-H). Consistent with the higher
calorie intake and lower activity in KO mice, we observed a marked increase of body
weight in SOD3 KO mice starting from HFD feeding for 5 weeks. By the end of 12
weeks, SOD3 KO mice were significantly heavier than WT mice on HFD (50.93±2.87
vs 42.27±3.17, P<0.05) (Fig. 6C). We also found that SOD3 KO mice had higher body
weight than WT mice irrespective of diet (Fig. 6C). The increase of body weight was
mainly due to increased white adipose tissue such as eWAT and sWAT (Fig. 6D).
Interestingly, we found that both eWAT and sWAT weight was increased in SOD3 KO
mice on ND while only sWAT was increased in SOD3 KO mice compared to WT mice
on the HFD. Consistent with the obesity phenotype, the SOD3 KO mice had higher
circulating blood glucose, insulin and leptin levels that associated with reduced
circulating adiponectin (Fig. 6 E-H). Moreover, SOD3 KO also had impaired glucose
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tolerance (Fig. 6I-J) and insulin resistance (Fig. 6K-L) compared to WT mice under both
ND and HFD.
SOD3 KO mice show higher level of adipose tissue hypertrophy than WT mice
To further understand the role of SOD3 in HFD-induced adipose tissue expansion, we
analyzed the adipose tissue morphology by H&E staining. We found a marked increase
of adipocyte size in both eWAT and sWAT from SOD3 KO mice on ND (Fig. 7A-C).
Consistent with the tissue weight data, we found that sWAT adipocyte size was also
increased in SOD3 KO mice while eWAT adipocyte size was increased to a similar
extent as WT mice on HFD (Fig. 7C). In agreement with the adipocyte size, triglyceride
content was increased (Fig. 7D-E). To understand the cause of increased adipocyte
size and triglyceride levels in adipose tissue from SOD3 KO mice, we analyzed a series
of genes involved in adipogenesis, lipogenesis, lipolysis and fatty acid β-oxidation in
both eWAT and sWAT by real-time PCR and Western blotting. In eWAT, we observed
an overall reduction in lipid metabolism pathways such as adipogenesis (PPARγ and
adiponectin), lipogenesis (ACC1), lipolysis (p-HSL) and fatty acid β-oxidation (PPARα)
in KO mice compared to WT mice (Fig. 7F and 7H) while there was less extent of
reduction of these markers in sWAT of KO mice (Fig. 7G and 7I).
SOD3 KO mice show enhanced adipose tissue inflammation than WT mice
Obesity-induced adipose tissue expansion is associated with increased adipose
tissue inflammation, we therefore examined whether SOD3 knockout increases adipose
tissue macrophage infiltration, a hallmark for adipose tissue inflammation. As shown in
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Fig 9A, eWAT from SOD KO mice showed an increase in F4/80 or CD11c positive cells
under HFD compared to WT mice while there were no obvious F4/80 or CD11c positive
cells in eWAT from SOD3 KO and WT mice under ND condition. Next, we quantified the
macrophages in eWAT and sWAT from SOD KO and WT mice by flow cytometry. In
eWAT, SOD3 KO mice showed a mild but significant increase of total macrophages
(F4/80+CD11b+) under both ND and HFD compared to WT mice (Fig. 9C), which was
mainly contributed to by the increase of M1 macrophages (F4/80+CD11b+CD11c+) (Fig.
9D). Although the increase of total macrophages in sWAT was less dramatic and only
increased in SOD3 KO mice under HFD condition (Fig. 9F), there was significant
increase of M1 macrophages in sWAT from SOD3 KO mice compared to WT (Fig. 9G).
Consistent with the flow cytometry data, we found an increase of macrophage marker
expression, such as F4/80, CD68, CD206 and CD11c, and the inflammatory cytokine,
TNFα, in eWAT from SOD3 KO mice compared to WT mice under ND (Fig. 9I). Feeding
with HFD did not further increase these markers in SOD3 KO mice.
SOD3 KO mice show higher level of serum lipids and inflammatory cytokines
than WT mice
To determine the systemic metabolic and inflammatory status of SOD3 KO mice, we
measured circulating levels of lipids such as FFA, total cholesterol and triglycerides and
two inflammatory cytokines TNFα and IL-6. We found that SOD3 KO mice had higher
levels of FFA, total cholesterol and triglyceride levels under both ND and HFD
conditions compared to WT mice (Fig. 10A-C). Similarly, serum TNFα was also higher
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in SOD3 KO mice under both conditions than WT mice (Fig. 10D) while IL-6 levels was
only elevated in SOD3 KO mice under HFD condition (Fig. 10E).
Discussion
The main findings of the present work are that adipose tissue and adipocytes
express high levels of SOD3 and its expression is increased in adipose tissue in
response to HFD-induced obesity. Using SOD3 KO mice and SOD3-silenced human
adipocytes, we found a potential role for SOD3 in regulating adipose tissue lipid
homeostasis. Our study has identified SOD3 as a protective factor secreted by
adipocytes in response to HFD-induced obesity and revealed its possible role in
regulating adipose tissue lipid metabolism.
It has been widely acknowledged that adipose tissue is an active endocrine organ,
which can secrete a variety of molecules including proteins, lipids and other bioactive
molecules. Our proteomic work has identified SOD3 as a secreted protein from
epidydimal adipose tissue (unpublished data). To verify and extend this finding, we
compared the SOD3 mRNA and protein expression in various tissues of adult mice and
found that SOD3 expression is tissue-specific. It is highly expressed in both white and
brown adipose tissue compared to other high expressed tissues such as lung, kidney
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and aorta. This result was consistent with a previous study that showed a higher SOD3
activity and expression in lung, kidney, aorta and adipose tissue [18,25].
SOD3 was initially identified as a protein predominantly found in various
extracellular fluids such as milk, lymph, synovial fluid and plasma [12, 17]. Further
studies showed that SOD3 is present as an intact and a cleaved form once secreted.
The intact form of SOD3 has a high affinity to cell surface proteoglycans through its
positively charged heparin-binding domain and therefore is anchored to the cell surface
[35]. In contrast, the cleaved forms of SOD3 lose their affinity for proteoglycans due to
an intracellular proteolytic process mediated by furin endopeptidase and are released
into the extracellular milieu and circulation [4, 24]. The majority (>90%) of SOD3 is
secreted into the extracellular space and binds to cell surface proteoglycans such as
heparin sulphate [28]. Consistent with the previous work, we detected both intact and
cleaved forms of SOD3 in adipose tissue and serum. There was an equivalent amount
of intact and cleaved forms of SOD3 in tissues including adipose tissue but the cleaved
form of SOD3 was more prevalent in serum, consistent with its lack of heparin binding
site. To further investigate the cell types that express SOD3 in adipose tissue, we
separated adipocytes and SV fractions from adult C57BL/6J mouse epidydimal adipose
tissue and found that adipocyte fractions were the main cells expressing SOD3 in
adipose tissue. This result was further supported by immunohistochemical staining of
SOD3 in human subcutaneous adipose tissue. In addition, we also observed a marked
increase SOD3 expression and release in adipocytes differentiated from human
preadipocytes and in BM-MSCs differentiated adipocytes. Together these data suggest
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that SOD3 is expressed highly in adipose tissue and secreted mainly by adipocytes
within adipose tissue.
The SOD family has three members including the cytosolic SOD1, the
mitochondrial SOD2 and the extracellular SOD3. SOD3 and SOD1 share some
similarity because they require Cu and Zn for enzyme activity. In our study, we
examined the SOD3 expression in adipocytes generated from murine BM-MSCs and
primary human preadipocytes. We observed an increase of SOD3 mRNA, protein
expression and release in differentiated adipocytes compared to BM-MSCs. Moreover,
we observed a marked increase (~80 fold) in SOD3 mRNA levels in differentiated
human adipocytes compared to preadipocytes. This result is consistent with previous
reports that SOD3 expression was elevated in adipocytes derived from 3T3-L1 and
human mesenchymal stem cells and compared to undifferentiated cells [2, 22].
Obesity alters the function of adipose cells, causing changes in the expression and
secretion of adipokines into the blood [20]. Since we have identified SOD3 as an
adipokine, we studied its association with obesity. Feeding mice with HFD for 4 weeks
induced a mild increase of body weight and adipose tissue weight. SOD3 expression
was also significantly elevated in three adipose depots (eWAT, sWAT and BAT),
suggesting that SOD3 is positively associated with adipose tissue expansion during
obesity. Long-term (12 weeks) HFD feeding induced more obvious increase of body
weight and adipose tissue mass and associated with higher SOD3 levels in adipose
tissue, suggesting that SOD3 is closely linked to adipose tissue expansion and obesity.
In contrast to SOD3, SOD1 expression was significantly reduced in three adipose
depots and SOD2 expression was only reduced in sWAT. Whether the upregulation of
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SOD3 in white and brown adipose tissues in obese mice exerts a compensatory effect
for the reduction of other SOD1 or SOD2 remains to be determined. Downregulation of
SOD3 in human adipocytes did not affect SOD1 and SOD2 mRNA expression
(Supplementary Fig. S3), suggesting that SOD1 and SOD2 expression is regulated
independently of SOD3.
The role of SOD3 as an antioxidant enzyme in ROS-mediated tissue inflammation
and damage have been extensively studied in lung, skin and vascular disease such as
ischemia and atherosclerosis [5,14,27]. However, its role in obesity and adipose tissue
is not clear. An increase in ROS production has been reported during obesity [9] and
the corresponding increase in expression of SOD3 may be an adaptive response to
oxidative stress. To further understand its role, using RNA-Seq technology, we analyzed
the overall transcriptome of human adipocytes after SOD3 silencing. We identified 60
pathways that were significantly affected by SOD3 silencing. Metabolic pathways were
the top hits. Further analysis revealed that the pathways of glycan biosynthesis and
metabolism, lipid metabolism and amino acid metabolism were the major metabolic
pathways affected. Specifically, we found that SOD3 silencing significantly affected
several genes involved in glycan biosynthesis (B3GALT4 and ST6GALT1 upregulated;
B4GALT3 and DPM2 down regulated) and degradation (IDS downregulated). Since
SOD3 binds to the cell surface proteoglycan such as heparin sulfate, we speculated that
the reduction of IDS, an enzyme responsible for heparin sulfate lysosomal degradation,
could be the consequence of lower SOD3 production and secretion, leading to a
negative feedback to the glycan metabolic pathways.
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To clarify its role in obesity in vivo, we generated the global SOD3 KO mice and fed
them with normal diet and high-fat diet (HFD) for 12 weeks. Obesity was greater in
SOD3 KO mice under HFD feeding for 12 weeks compared to WT mice. The increase
of body weight in SOD3 KO mice under HFD was primarily contributed by the
enlargement of white adipose tissue especially the sWAT. The increased adipocyte size
was associated with increased accumulation of lipids in both eWAT and sWAT. To
understand the role of SOD3 in lipid accumulation in adipocytes, we treated
differentiating adipocytes with SOD3 lentivirus and observed an up-regulation of PPARγ
and SREBP1c, two important transcription factors involved in lipogenesis. Also, we
found an increase of two triglyceride synthetic genes GPAT3 and DGAT2 in SOD3
silenced adipocytes, suggesting a potential role of SOD3 in regulation of triglyceride
synthesis. In addition, we found an increase of p-HSL and ATGL expressions in SOD3
silenced adipocytes. The increased expression of p-HSL and ATGL may be a
compensatory effect to the increased lipogenesis and lipid storage in adipocytes.
In addition to the development of obesity, SOD3 KO mice were more insulin
resistant compared to WT mice, possibly due to obesity-induced adipose tissue and
systemic inflammation. In support of this, eWAT from SOD KO mice showed more
F4/80 or CD11c positive cell accumulation than WT mice under HFD condition,
suggesting an increase of macrophage infiltration into adipose tissue. Furthermore,
SOD3 KO mice showed an increase of inflammatory M1 macrophages
(F4/80+CD11b+CD11c+) in both eWAT and sWAT under ND condition, suggesting that
the increase of M1 macrophages could contribute to the adipose tissue inflammation in
SOD3 KO mice. Consistent with the increase of local adipose tissue inflammation, there
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was also a systemic increase of circulating pro-inflammatory cytokines such as TNFα
and IL-6 in SOD3 KO mice, which may contribute to the development of insulin
resistance. Consistent with our findings, several studies have reported an anti-
inflammatory role of SOD3 in inflammatory diseases. For example, SOD3
overexpression significantly reduces inflammatory cell migration in ischemic conditions
by regulating adhesion molecule and cytokine expression [15]. Furthermore, a recent
study demonstrated an inhibitory effect of SOD3 on Propionibacterium acnes-induced
skin inflammation [21]. Mechanistically, SOD3 suppresses Propionibacterium acnes-
induced skin inflammation through inhibition of TLR2/p38/NF-κB and NLRP3
inflammasome activation. Moreover, a previous study increasing SOD3 levels in liver
and blood by gene transfer protected HFD-fed obese mice from developing obesity,
obesity-induced adipose tissue inflammation, insulin resistance and liver steatosis [6],
suggesting a beneficial role of SOD3 in regulation of obesity-induced inflammation and
associated metabolic disorders.
In summary, it is well established that the expansion of adipose tissue during
obesity is associated with an increase of lipotoxicity, which could lead to the dysfunction
of adipocytes and contribute to obesity-associated metabolic disorders. Here, we have
demonstrated that SOD3 is a potential adipokine that is secreted to elevated levels in
adipose tissue during early (4 weeks feeding) and later stages (12 weeks feeding) of
HFD-diet-induced obesity. In addition, silencing SOD3 in human adipocytes significantly
affected genes involved in metabolic pathways especially lipid metabolism, suggesting a
potential role of SOD3 in maintaining lipid homeostasis in adipocyte.
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Innovation
SOD3 has been shown to protect various tissue including lung, kidney and skin
from oxidative stress. In our study, we found high SOD3 expression in adipose tissue
and adipocytes that was further increased during obesity. Therefore, we explored
whether SOD3 could be a protective factor in adipose tissue in obesity. We
demonstrated that SOD3 is secreted by adipocyte at elevated levels in adipose tissue of
HFD-diet-induced obesity and plays a novel protective role through regulating lipid
metabolism in adipocytes. These findings may open new avenues of research for the
cytoprotective role of SOD3 in obesity and its associated metabolic disorders.
Materials & Methods
Animals
SOD3 knockout mice
SOD3 KO mice were generated and provided by CRISPR/Cas 9 (Cyagen,
Biosciences, Suzhou, China). SOD3 KO and C57BL/6J WT mice (8 weeks old, male)
were fed with a normal diet (ND, 10% kcal fat content) and a high-fat diet (HFD, 60%
kcal fat content) for 12 weeks (n=6-8 per group).
C57BL/6J male mice (3 weeks old) were purchased from Xi’an Jiaotong University
Health Science Centre Animal Facility (Xi’an, China). After 1 week of acclimatization,
mice were divided into two groups: mice fed with a ND (control group, 10% kcal fat
content) and with a HFD (HFD group, 60% kcal fat content) (Research Diets Inc., New
Brunswick, NJ, USA) for 4 weeks and 12 weeks. All animals were housed in a
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temperature (25±2 C) and humidity (40%-60%) controlled animal room and maintained
on a 12 h light/12 h dark cycle with free access to food and water. All the procedures
were approved by the Animal Use and Care Committee of the School of Life Science
and Technology, Xi’an Jiaotong University and performed in accordance with the U.S.
Public Health Services Guide for the Care and Use of Laboratory Animals.
Glucose tolerance and insulin tolerance tests
For the glucose tolerance test (GTT), mice were fasted for 15 hours and challenged with
D-glucose (1 g/kg body weight) by i.p. injection. Glucose levels in blood samples from
the tail vein were monitored at various time points (0, 15, 30, 60 and 120 minutes) after
glucose infusion with a OneTouch Glucose Meter (LifeScan). For the insulin tolerance
test (ITT), mice were fasted for 3 hours and were intraperitoneally injected with insulin
(0.75 IU/kg body weight for ND- or HFD-fed mice, respectively), followed by
measurement of blood glucose as above.
Energy expenditure measurement
Food intake was measured on weekly base from WT and SOD3 KO mice under ND and
HFD. Energy expenditure was determined using FMS Field Metabolic System (Sable
Systems International, USA).
Locomotion activity measurement
To assess locomotor activity, an open field test was performed in an open field chamber
composed of a square arena (40X40cm) with opaque gray walls. Mice were placed
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individually in the center of the open field box and was allowed to move freely for 10
min. The chamber was wiped with 70% ethanol between tests. The distance moved
were recorded using SMART subject automated tracking system (San Diego
Instruments, San Diego, USA).
Serum biochemical analysis for lipids and inflammatory cytokines
Serum levels of glucose and lipids were analyzed with commercial biochemical assay
kits (Nanjing Jiancheng, Nanjing, China) by an automatic biochemical analyzer
(BECKMAN AU2700). Circulating levels of insulin, adiponectin, leptin, TNFα and IL-6
were quantified by ELISA kits (R&D Systems).
Sample preparation
Adult male C57BL/6J mice (n=3) were sacrificed by cervical dislocation and various
tissues including heart, lung, kidney, spleen, pancreas, liver, adipose tissue,
gastrocnemius muscle, aorta, brain and stomach were dissected and snap frozen in
liquid nitrogen and stored at -80 °C for later use.
Similar to the adult C57BL/6J mice, four weeks and twelve weeks ND and HFD-fed
mice (n=8-10 per group) were fasted overnight before being sacrificed. Blood samples
were collected by cardiac puncture and left at room temperature for at least 30 min. The
samples were homogenized, then centrifuged at 1000 g for 10 min and the
supernatants were collected and stored at -80C before analysis. Adipose tissues such
as epidydimal white adipose tissue (eWAT), subcutaneous white adipose tissue (sWAT),
brown adipose tissue (BAT) and lung tissue were dissected and snap frozen in liquid
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nitrogen and stored at -80 C for later use. For each group, four to five mice were used.
Adipose tissue homogenization procedure
Adipose tissues of eWAT, sWAT and BAT were homogenized by a tissue
homogenizer at 70 Hz for 90 sec (Tissuelyser-48, Shanghai Jingxin Co. Ltd, Shanghai,
China) in ice-cold (4°C) lysis buffer (pH 7.4) containing the following: (50 mM Tris·HCl
pH 6.7, 10% glycerol, 4% SDS, 2% 2-mercaptoethanol) with freshly added protease
inhibitor cocktail (Sigma, MO, USA). Homogenates were then centrifuged (4°C) for 10
min at 10,000 x g and the supernatant were collected. Total protein was determined
using a BCA assay kit (Pierce Biotechnology, Rockford, USA).
Isolation of adipocytes and stromal vascular fraction (SV) from mouse adipose issue
Adipocytes and SVF were prepared from adult C57BL/6 mice as previously
described [26]. In brief, eWAT was isolated in ice-cold PBS containing 1 mg/) ml
collagenase Ⅱ(Sigma, Product No. C6885) and minced into small segments. eWAT as
digested at 37 °C for 1 h with intermittent mixing. After digestion, the solution was
centrifuged, buoyant adipocytes were removed, and the cell pellet was retrieved as
SVF.
Human primary preadipocyte culture and differentiation
Human white preadipocytes derived from subcutaneous adipose tissue of a female
Caucasian subject (BMI 21 kg/m2, age 44 yr) were obtained from PromoCell
(Heidelberg, Germany, Product No. C-12730). Cells were cultured in preadipocyte
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growth medium supplemented with preadipocyte growth factors and 100 U/ml penicillin
and 100 µg/ml streptomycin (ScienCell, USA, Product No. 7211 and 7252) at 37°C in a
humidified atmosphere of 5% CO2 / 95% air. Passages less than 10 were used in all
experiment. Preadipocytes were seeded at 40,000/cm2 and grown in 24-well plates until
confluence. At confluence, cells were induced to differentiate (day 0) by incubation for 3
days in Dulbecco’s modified Eagle’s medium (DMEM)-Ham’s F-12 (1:1) medium (Gibco
BRL, Grand Island, NY, Product No. 11330-032) containing 32 µM biotin (Sigma,
Product No. B4639), 1 µM dexamethasone (Sigma, Product No. D4902), 200 µM 3-
isobutyl-1-methylxanthine (Sigma, Product No. I7018), 100 nM insulin (Sigma, Product
No. I9278), 11 nM L-thyroxine 8 µM rosiglitazone (Sigma, Product No. R2408) and 100
U/ml penicillin, 100 µg/ml streptomycin. After induction, cells were cultured in
maintenance medium containing 3% fetal bovine serum (FBS, Biological Industries,
Israel), 100 nM insulin, 32 µM biotin, and 1 µM dexamethasone until full differentiation
into adipocytes. Cell lysates and culture media at D0, D3, D6, D9 and D12 were
collected.
Murine bone marrow-derived mesenchymal stem cells culture and differentiation to
adipocytes
Murine bone marrow-derived mesenchymal stem cells (BM-MSCs) were purchased
from ScienCell (Sandiago, California, USA, Product No. ZQ0465). BM-MSCs were
grown in MSC growth medium (ScienCell, Product No. 7501) at 37 °C in a humidified
atmosphere of 5% CO2/95% air. Cell passages less than 10 were used in all
experiment. BM-MSCs were differentiated to adipocytes according to a method reported
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previously with slight modifications [28]. At confluence, the cells were incubated with
250 nM dexamethasone, 500 nM 3-isobutyl-1-methylxanthine, 5 μg/ml insulin, 1 μM
rosiglitazone (all from Sigma) for up to 12 days with fresh above media change every
three days. Cell lysates and culture media at D0, D3, D6, D9 and D12 were collected.
siRNA transfection
Human adipocytes at day 9 were transfected with 30 nM siRNA using
Lipofectamine RNAiMAX (Thermo Fisher Scientific, Rockford, IL, USA) for 72 h. Non-
targeting siRNA control (5′-UUCUCCGAACGUGUCACGU-3′) and siRNAs for human
SOD3 (#1 5′-AGUGGAUCCGAGACAUGUA-3′; #2 5 ′-GCCUCCAUUUGUACCGAAA-3′;
#3 5′-CCUUUGACCUGACGAUCUU-3′) were purchased from Shanghai GenePharma
Co., Ltd. (Shanghai, China).
Lentivirus treatment
Human preadipocytes (D0) or differentiating adipocytes (D3) grown in 24-well plate
were infected with SOD3 shRNA lentivirus at 107 Tu/ml (Shanghai Genechem Co., Ltd;Shanghai, China) for 4 hours and then incubated in adipocyte maturation medium with
fresh media change every three days until day 11 or day 12 respectively.
Western blotting
Total cellular protein from tissue and cells was prepared with lysis buffer (50 mM
Tris-HCl pH 6.7, 10% glycerol, 4% SDS, 2% 2-mercaptoethanol) with freshly added
protease inhibitor cocktail (Sigma-Aldrich, MO, USA). For determination of the levels of
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SOD3 and adiponectin in cell culture media, we firstly concentrated the cell culture
media (e.g. 1 ml) through a centrifuge filter (Amicon Ultra-0.5, Millipore) with 3 kD cutoff
at 12000 rpm for 20 min at 4°C. Then the concentrated media was mixed with 10X SLB
and incubated at 95°C for 10 min. Protein concentrations were measured by BCA
protein assay kit (Thermo Scientific, Rockford, IL, USA). Protein samples (20 µg/lane or
10 -30 μl concentrated media/lane) were resolved by 10% SDS-PAGE gels, transferred
onto a nitrocellulose membrane (Millipore, Bedford, MA, USA) by wet transfer (Trans
Blot, Bio-Rad) at 300 mA for 90 min. For immunodetection, the membrane was blocked
for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.1% Tween-20
and 5% BSA and incubated overnight at 4 °C with the antibody for mouse SOD3
(1:1000 dilution) (R&D systems, Minneapolis, MN, USA, Product No. AF4817),
mouse/human SOD1 (1:1000 dilutions, R&D systems. Product No. A0215),
mouse/human SOD2 (1:1000 dilution, Cell Signaling Technology, Beverley, MA, USA,
Product No. 13194S), mouse adiponectin (1:1000 dilution, Cell Signaling Technology ,
Product No. 27898S), mouse PPARγ (1:1000 dilution, Proteintec, Product No. 16643-1-
AP), mouse GPAT3 (1:1000 dilution, Proteintec, Product No. 20603-1-AP), human
DGAT2 (1:1000 dilution, Abcam, Product No.237613), mouse SREBP1c (1:1000
dilution, Arigo Biolaboratories, Product No. ARG62627), CPT1a (1:1000 dilution,
Proteintec, Product No. 15184-1-AP), PPARα (1:1000 dilution, Proteintec, Product No.
15540-1-AP), phospho-HSL (Ser563) (1:1000 dilution, Cell Signaling Technology,
Product No.4139S), HSL (1:1000 dilution, Cell Signaling Technology, Product No.
4107S), ATGL (1:1000 dilution, Cell Signaling Technology, Product No.2138S), human
adiponectin (1:1000 dilution, R&D systems, Product No. AF1065), human SOD3
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(1:1000 dilution) (R&D systems, Product No. AF3420), in 5% BSA TBS and 0.1%
Tween-20 followed by an anti-goat secondary antibody (Jackson Immunoresearch,
West Grove, PA, USA) at 1:2000 dilution. Signals were detected by chemiluminescence
(Bio-Rad, Hercules, CA, USA) and scanned using a ChemiScope 3300 Mini System
(Clinx Science Instruments, Shanghai, China). The membrane was further probed with
α-tubulin (1:1000 dilutions, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Product
No. sc-58666) or GAPDH (1:2000 dilution, Proteintec, Product No. 60004-1-Ig) or
HSP90 (1:1000 dilution Cell Signaling Technology, Product No. 4874S) as a loading
control.
Real-time PCR
Total RNA was extracted from tissue (~50-100 mg) or cells using Trizol (Roche,
Basel, Switzerland) and the RNA concentration determined from the absorbance at 260
nm. First strand cDNA was reverse transcribed from 1 g of total RNA using the
PrimeScript RT reagent kit (TaKaRa Biotechnology, Dalian, China) followed by real-time
PCR using specific primers. The primer sequences were listed in supplementary table
1.
Real-time PCR applications were performed with SYBR-Green Master Mix
(TaKaRa Biotechnology, Dalian, China) using a real-time PCR system (MX3006P,
Agilent Technologies, California, USA) and PCR cycling conditions were as follows: 95
°C for 5 min followed by 40 cycles (95 °C for 15 sec, 60 °C for 30 sec and 72°C for 20
sec). All samples were normalized to the β-actin values and the results expressed as
fold changes of Ct value relative to controls using the 2−ΔΔct formula [29].
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Flow cytometry
Samples of SVF cells from eWAT and sWAT were first incubated with anti-mouse
CD16/CD32 Fc Block (clone 2.4G2) (1:200) in 100 μl of PBS at 4 °C for 10 min. Cells
were then incubated with primary antibodies (Alexa488 anti-F4/80, PE anti-CD11b, APC
anti-CD11c and PE-Cy7 anti-CD206; all 1:200) (all from BioLegend) at 4°C for 1h. After
washing, cells were analyzed by flow cytometry (ACEA NovoCyte, USA).
SOD3 immunohistochemistry
Human subcutaneous adipose tissues were obtained from a cadaver at the
Department of Human Anatomy, Histology and Embryology at Xi’an Jiaotong University
Health Centre and was approved by the Ethical Committee of the Medical Health
Centre, Xi’an Jiaotong University. Adipose tissue was fixed in 10% (v/v) formalin
solution for 72 h, followed by incubation in 70% ethanol for 24 h and then embedded in
paraffin and cut into 5 μm sections and mounted on a charged glass slides. The slides
were deparaffinized by xylene, followed by dehydration. Antigen retrieval was carried
out by heating the sections in citric buffer (pH=6.0) in a microwave at maximum heating
for 20 min. The sections were then rinsed and blocked using 5% BSA, followed by
incubation overnight at 4°C with anti-human SOD3 (1:50) (Abcam, Cambridge, MA,
USA, Product No. ab80946) or moue IgG (control). The sections were then incubated
goat anti-mouse secondary antibody, SP kit (Zhongshan Gold Bridge, Beijing, China)
and DAB substrate (Zhongshan Gold Bridge, Beijing, China). After SOD3 staining, the
slides were co-stained with hematoxylin.
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Oil Red O staining
Lipid accumulation was determined by Oil Red O staining. At day 3, 6, 9 and 12
after the initiation of differentiation, cells were washed with PBS and fixed with 10%
formalin at room temperature for 30 min. The cells were then stained with 0.3% Oil Red
O solution (Sigma) at room temperature for 1 h. After three washes with PBS, the red-
stained lipid droplets were visualized under a light microscope and photographed. To
quantify the lipid content, Oil Red O-stained lipid droplets were extracted with 100%
isopropanol and its absorbance was measured at 510 nm.
RNA-Seq analysis
Triplicate RNA samples from NC and siRNA (#2) treated adipocytes were prepared
and sequenced on BGISEQ-500 platform (Beijing Genomics Institution, Shenzhen,
China). Gene expression levels were obtained as the FPKM (fragment per kilobase of
exons per million reads). quantified by a software package called RSEM [21]. NOISeq
method was used to screen differentially expressed genes (DEGs) between groups.
Statistical analysis was performed and DEGs were selected with the criteria of fold
change ≥ 1.40, P ≤ 0.05.
Pathway enrichment analysis
Pathway enrichment analysis was based on the kobas 3.0
(http://kobas.cbi.pku.edu.cn/index.php) and ggplot2 package of R (version 3.5.0) was
also used for visualization, respectively. Statistical analysis was performed with P < 0.05
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considered as significant.
Statistical analysis
Data were expressed as mean ± SEM. Differences between two groups were
analyzed by Student’s unpaired t-test; one-way ANOVA coupled with Bonferroni’s t-test
was employed for comparison of multi-groups. Differences were considered as
statistically significant when P<0.05.
Acknowledgements
This work is supported in part by the National Basic Research Programs (973 Program
No. 2015CB553602), the National Natural Science Foundation of China (NFSC) (Grant
No. 31770917; 31570777) to JL; the National Natural Science Foundation of China
(NFSC) (Grant No. 81600686, 81873665), Shaanxi Provincial Postdoctoral Science
Foundation (Grant No. 2017BSHYDZZ45) and China Postdoctoral Science Foundation
(Grant No. 2018T111074) to DG; the BBSRC China Partnering Award (grant no.
BB/M028100/2) to HRG. In addition, we thank Mr. Shengfeng Ji from the Department of
Human Anatomy, Histology and Embryology in Xi’an Jiaotong University Health Science
Center for providing the human subcutaneous adipose tissue. We thank the Beijing
EcoTech Ltd. Co providing the FMS Field Metabolic System (Sable Systems
International, USA). We also thank Prof. Dongmin Li for scientific discussion of the
manuscript. Thanks also to Mr. Weixiao Yang from Xi’an Academy of Fine Arts for the
advice on the artwork and schematic graph.
Author disclosure Statement
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No competing financial interests exist for any author. All authors declare that there
is no conflict of interest associated with this article.
Abbreviations:
BAT= brown adipose tissue
BM-MSCs=bone marrow-derived mesenchymal stem cells
DEGs=differentially expressed genes
eWAT= epididymal adipose tissue
HFD= high-fat diet
ND=normal diet
SOD=superoxide dismutase
sWAT= subcutaneous adipose tissue
TBS=Tris-buffered saline
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