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.

1

12

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

3

Gao

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: j.liu@mail.xjtu.edu.cn

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： h.r.griffiths@surrey.ac.uk

Word count: 6045

Reference numbers: 31

2

45

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

6

Gao

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.

3

78

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

9

Gao

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.

4

1011

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

12

Gao

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.

5

1314

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

15

Gao

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

6

1617

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

18

Gao

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

7

1920

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

21

Gao

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

8

2223

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

24

Gao

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

9

2526

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

27

Gao

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

10

2829

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

30

Gao

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

11

3132

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

33

Gao

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

12

3435

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

36

Gao

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

13

3738

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

39

Gao

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

14

4041

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

42

Gao

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

15

4344

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

45

Gao

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.

16

4647

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

48

Gao

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

17

4950

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

51

Gao

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.

18

5253

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

54

Gao

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

19

5556

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

57

Gao

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

20

5859

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

60

Gao

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

21

6162

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

63

Gao

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

22

6465

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

66

Gao

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

23

6768

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

69

Gao

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

24

7071

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

72

Gao

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

25

7374

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

75

Gao

(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].

26

7677

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

78

Gao

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.

27

7980

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

81

Gao

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

28

8283

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

84

Gao

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

29

8586

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

87

Gao

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

References

1. Adachi T, Inoue M, Hara H, Maehata E, Suzuki S. Relationship of plasma

extracellular-superoxide dismutase level with insulin resistance in type 2 diabetic

patients. J Endocrinol 181: 413–417, 2004.

2. Adachi T, Toishi T, Wu H, Kamiya T, Hara H. Expression of extracellular

superoxide dismutase during adipose differentiation in 3T3-L1 cells. Redox Rep 14:

34-40, 2009.

3. Arner E, Westermark PO, Spalding KL, Britton T, Rydén M, Frisén J, Bernard S,

30

8889

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678

90

Gao

Arner P. Adipocyte turnover: relevance to human adipose tissue morphology.

Diabetes 59: 105-109, 2010.

4. Bowler RP, Nicks M, Olsen DA, Thøgersen IB, Valnickova Z, Højrup P, Franzusoff

A, Enghild JJ, Crapo JD. Furin proteolytically processes the heparin-binding region

of extracellular superoxide dismutase. J Biol Chem 277: 16505-16511, 2002.

5. Constantino L, Gonçalves RC, Giombelli VR, Tomasi CD, Vuolo F, Kist LW, de

Oliveira GM, Pasquali MA, Bogo MR, Mauad T, Horn A Jr, Melo KV, Fernandes C,

Moreira JC, Ritter C, Dal-Pizzol F. Regulation of lung oxidative damage by

endogenous superoxide dismutase in sepsis. Intensive Care Med Exp 2: 17-28.

2014

6. Cui R, Gao M, Qu S, Liu D. Overexpression of superoxide dismutase 3 gene blocks

high-fat diet-induced obesity, fatty liver and insulin resistance. Gene Ther 21: 840-

848, 2014.

7. Deng J, Sitou K, Zhang Y, Ru Y, Hu Y. Analyzing the Chinese landscape in anti-

diabetic drug research: leading knowledge production institutions and thematic

communities. Chin Med 11: 13-23, 2016.

8. Flegal KM, Kit BK, Orpana H, Graubard BI. Association of all-cause mortality with

overweight and obesity using standard body mass index categories: a systematic

review and meta-analysis. JAMA 309: 71-82, 2013.

9. Furukawa S1, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y,

Nakayama O, Makishima M, Matsuda M, Shimomura I. Increased oxidative stress

in obesity and its impact on metabolic syndrome. J Clin Invest 114: 1752-1761,

2004.

31

9192

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

93

Gao

10. González-Muniesa P, Mártinez-González MA, Hu FB, Després JP, Matsuzawa Y,

Loos RJF, Moreno LA, Bray GA, Martinez JA. Obesity. Nat Rev Dis Primers. 3:

17034-17052, 2017. Review.

11. Griess B, Tom E, Domann F, Teoh-Fitzgerald M. Extracellular superoxide

dismutase and its role in cancer. Free Radic Biol Med 112: 464-479, 2017. Review.

12. Hicks CL. Occurrence and consequence of superoxide dismutase in milk products:

a review. J Dairy Sci 637: 1199-1204,1980.

13. Isogawa A, Yamakado M, Yano M, Shiba T. Serum superoxide dismutase activity

correlates with the components of metabolic syndrome or carotid artery intima-

media thickness. Diabetes Res Clin Pract 86: 213–218, 2009.

14. Kwon MJ, Kim B, Lee YS, Kim TY. Role of superoxide dismutase 3 in skin

inflammation. J Dermatol Sci 67: 81-87, 2012. Review

15. Laurila JP, Laatikainen LE, Castellone MD, Laukkanen MO. SOD3 reduces

inflammatory cell migration by regulating adhesion molecule and cytokine

expression. PLoS One 4: e5786, 2009.

16. Li B and Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data

with or without a reference genome. BMC Bioinformatics 12: 323-339, 2011.

17. Marklund SL, Holme E, Hellner L. Superoxide dismutase in extracellular fluids.

Clin Chim Acta 126: 41–51, 1982.

18. Marklund SL. Extracellular superoxide dismutase and other superoxide dismutase

isoenzymes in tissues from nine mammalian species. Biochem J 222: 649–655,

1984.

32

9495

702

703

704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722

723

96

Gao

19. McGown C, Birerdinc A, Younossi ZM. Adipose tissue as an endocrine organ. Clin

Liver Dis 18: 41-58, 2014. Review.

20. Moller DE, Kaufman KD. Metabolic syndrome: a clinical and molecular perspective.

Annu Rev Med 56: 45-62, 2005.

21. Nguyen CT, Sah SK, Kim TY. Inhibitory effects of superoxide dismutase 3 on

Propionibacterium acnes-induced skin inflammation. Sci Rep 8: 4024-4036, 2018.

22. Nightingale H, Kemp K, Gray E, Hares K, Mallam E, Scolding N, Wilkins A.

Changes in expression of the antioxidant enzyme SOD3 occur upon differentiation

of human bone marrow-derived mesenchymal stem cells in vitro. Stem Cells Dev

21: 2026-2035, 2012.

23. Ninomiya Y, Sugahara-Yamashita Y, Nakachi Y, Tokuzawa Y, Okazaki Y,

Nishiyama .Development of rapid culture method to induce adipocyte differentiation

of human bonemarrow-derived mesenchymal stem cells. Biochem Biophys Res

Commun 394: 303-308, 2010.

24. Olsen DA, Petersen SV, Oury TD, et al. The intracellular proteolytic processing of

extracellular superoxide dismutase (EC-SOD) is a two-step event. J Biol Chem 279:

22152-22157, 2004.

25. Ookawara T, Imazeki N, Matsubara O, Kizaki T, Oh-Ishi S, Nakao C, et al. Tissue

distribution of immunoreactive mouse extracellular superoxide dismutase. Am J

Physiol 275: C840–847, 1998.

26. Orr JS, Kennedy AJ, Hasty AH. Isolation of adipose tissue immune cells. J Vis Exp

75: e50707, 2013.

27. Pinto A, Immohr MB, Jahn A, Jenke A, Boeken U, Lichtenberg A, Akhyari P. The

33

9798

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

99

Gao

extracellular isoform of superoxide dismutase has a significant impact on

cardiovascular ischaemia and reperfusion injury during cardiopulmonary bypass.

Eur J Cardiothorac Surg 50:1035-1044, 2016.

28. Sandstrom, J.; Karlsson, K.; Edlund, T.; Marklund, S. L. Heparin-affinity patterns and

composition of extracellular superoxide dismutase in human plasma and tissues.

Biochem J 294: 853– 857, 1993.

29. Schmittgen TD and Livak KJ. Analyzing real-time PCR data by the comparative

C(T) method. Nat. Protoc 3: 1101-1108, 2008.

30. Smitka K, Marešová D. Adipose Tissue as an Endocrine Organ: An Update on Pro-

inflammatory and Anti-inflammatory Microenvironment. Prague Med Rep 116: 87-

111, 2015.

31. Griess B, Tom E, Domann F, Teoh-Fitzgerald M. Extracellular superoxide dismutase

and its role in cancer. Free Radic Biol Med. 2017 Nov;112:464-479.

34

100101

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

102