streptozotocin-induced type 2 diabetic mice

Journal Pre-proof Protective role of adiponectin against testicular impairment in high-fat diet/ streptozotocin-induced type 2 diabetic mice Mayank Choubey, Ashutosh Ranjan, Puran S. Bora, Amitabh Krishna PII:

S0300-9084(19)30309-8

DOI:

https://doi.org/10.1016/j.biochi.2019.10.014

Reference:

BIOCHI 5777

To appear in:

Biochimie

Received Date: 15 June 2019 Accepted Date: 25 October 2019

Please cite this article as: M. Choubey, A. Ranjan, P.S Bora, A. Krishna, Protective role of adiponectin against testicular impairment in high-fat diet/streptozotocin-induced type 2 diabetic mice, Biochimie, https://doi.org/10.1016/j.biochi.2019.10.014. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Abstract Type 2 diabetes (T2D) is the most common endocrine and metabolic disorder, leading to reproductive impairments and infertility in male. Our recent study showed crucial role of adiponectin in the regulation of testicular functions, and the circulating level of adiponectin declines in diabetes. The current study thus aimed to examine the efficacy of adiponectin in improving testicular dysfunction in high fat diet/streptozotocin-induced T2D mice. T2D was induced in pre-pubertal mice fed with high fat diet for ~10 weeks followed by single treatment of streptozotocin. T2D mice showed presence of increased body mass, hyperglycemia, hyperinsulinemia, insulin resistance, increased oxidative stress, and declined serum testosterone compared to vehicle-treated control mice. The spermatogenic, steroidogenic, metabolic, and antioxidative parameters were evaluated in T2D mice treated with adiponectin for both two and four weeks. The exogenous administration of adiponectin to T2D mice showed enhanced testicular expression of steroidogenic markers, insulin receptor and GLUT8, increase in intra-testicular concentrations of glucose and lactate and activity of antioxidant enzymes compared to the levels in untreated T2D mice. This suggests that treatment of adiponectin effectively improves testicular functions by increasing expression of insulin receptor-mediated increased transport of energy substrate (glucose and lactate) and a marked reduction in oxidative stress are the possible mechanism by which adiponectin effectively improves testicular function in T2D mice.

1

Protective role of adiponectin against testicular impairment in high-fat diet/streptozotocin-

2

induced type 2 diabetic mice

3

4

Mayank Choubey1, Ashutosh Ranjan1, Puran S Bora2, and Amitabh Krishna*,1

5

6

1

7

Pradesh, India. First author. E-mail: [email protected]

Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi-221005, Uttar

8

9 10

2

Department of Ophthalmology, Jones Eye Institute, Pat & Willard Walker Eye Research Center,

4301 West Markham, University of Arkansas for Medical Sciences, Arkansas. 72205, USA.

11

12

*

13

Varanasi-221 005, Uttar Pradesh, India. E-mail: [email protected]

Corresponding author: Department of Zoology, Institute of Science, Banaras Hindu University,

14

15

16

17

18

19

20 1|Page

21

Abstract

22

Type 2 diabetes (T2D) is the most common endocrine and metabolic disorder, leading to

23

reproductive impairments and infertility in male. Our recent study showed crucial role of

24

adiponectin in the regulation of testicular functions, and the circulating level of adiponectin

25

declines in diabetes. The current study thus aimed to examine the efficacy of adiponectin in

26

improving testicular dysfunction in high-fat diet/streptozotocin-induced T2D mice. T2D was

27

induced in pre-pubertal mice fed with high-fat diet for ~10 weeks followed by single treatment

28

of streptozotocin. T2D mice showed presence of increased body mass, hyperglycemia,

29

hyperinsulinemia, insulin resistance, increased oxidative stress, and declined serum testosterone

30

compared to vehicle-treated control mice. The spermatogenic, steroidogenic, metabolic, and

31

antioxidative parameters were evaluated in T2D mice treated with adiponectin for both two and

32

four weeks. The exogenous administration of adiponectin to T2D mice showed enhanced serum

33

testosterone and expression of testicular steroidogenic markers proteins, insulin receptor and

34

GLUT8 proteins, increase in intra-testicular concentrations of glucose and lactate and activity of

35

LDH and antioxidant enzymes compared to the levels in untreated T2D mice. This suggests that

36

treatment of adiponectin effectively improves testicular functions by increasing expression of

37

insulin receptor-mediated increased transport of energy substrate (glucose and lactate) and a

38

marked reduction in oxidative stress are the possible mechanism by which adiponectin

39

effectively improves testicular function in T2D mice.

40

41

Keywords: Adiponectin; Type 2 Diabetes; Insulin resistance; Testicular impairment;

42

Antioxidants;

43

44

2|Page

45

1. Introduction

46

Type 2 diabetes (T2D) is a metabolic disorder affecting approximately 9% of the population

47

worldwide [1]. The prevalence of T2D is increasing among children and adolescents at an

48

alarming rate [2]. Major contributory factor for developing T2D in youth include lifestyle

49

factors, increased consumption of fast food, obesity, stress etc. [3]. T2D is characterized by

50

chronic hyperglycemia, hyperinsulinemia, insulin resistance, and increased oxidative stress [4],

51

as well as a disturbance in glucose homeostasis and defects in insulin signaling [5]. Patients with

52

T2D experience a number of dysfunctions, including prominent effects on brain, kidney, retina,

53

heart, and the male reproductive system [6, 7]. The majority of diabetic patients have

54

disturbances in reproductive functions, such as impaired spermatogenesis, increased apoptosis of

55

germ cells, and reduced steroidogenesis, leading to decreased libido, increased impotency and

56

infertility [8]. These dysfunctions are attributable to both testicular metabolic (nutritional) and

57

endocrine activities together with changes in the hypothalamic-pituitary-testicular (HPT) axis

58

[9].

59

Several recent studies suggest important role of metabolic (or nutritional) factors in the

60

regulation of normal spermatogenesis [10; 11]. The testis is one of the most dynamic endocrine

61

organ requires sufficient input of nutrients such as carbohydrates, lipids, amino acids, vitamins,

62

and metal ions etc. for the normal development of germ cells, differentiation of spermatozoa, and

63

synthesis of testosterone. Over consumption of high fat resulted in increased circulating level of

64

cholesterol, fatty acid, and fat-soluble toxicants which have been shown to cause impaired

65

spermatogenesis and reduced synthesis of testosterone [12; 13]. Altered metabolic activities as

66

found in individuals with obesity or T2D was shown to have impaired testicular activity

67

including spermatogenesis [5], However, the molecular and cellular basis of testicular

68

abnormalities occurring with diabetes remains to be fully elucidated. Glucose is the major

69

mediator of nutritional effect on spermatogenesis [14]. Glucose uptake in the testis is carried out

3|Page

70

mainly by the family of glucose transporters (GLUT 1, 3, 5, and 8) [5, 15, 16]. Our previous

71

findings along with other studies clearly showed involvement of GLUT8, regulated by both LH

72

and insulin in increased transport of glucose to the testis of mice [14, 17, 18]. In a previous

73

study, treatment of diabetic rats with insulin restored the normal sperm count either indirectly by

74

acting on hypothalamic-pituitary-gonadal axis [19], or directly by reducing testicular oxidative

75

stress [20]. Although multiple mechanisms have been proposed in the past by which T2D affects

76

male reproductive function, effective treatments that can restore T2D-induced testicular

77

impairment remain elusive.

78

Adiponectin is a most abundant adipokine, produced and secreted by the adipocytes that plays an

79

important metabolic role both centrally and peripherally. Adiponectin acts through its receptors,

80

AdipoR1 and AdipoR2, and is localized and expressed in various tissues including testis, where

81

it modulates male reproductive functions [21]. Our recent study identified AdipoR1 in Leydig

82

cells, and AdipoR2 in the seminiferous tubules of the mice [11]. These findings suggest an

83

important contribution of adiponectin for male fertility [22]. In another recent study, our group

84

showed that a marked decline in adiponectin may be responsible for decreased testosterone

85

synthesis and reduced spermatogenesis as found in the testis of aged mice [23]. Additionally, an

86

earlier study showed the presence of insulin resistance and diabetic-like features in the testis of

87

mice deficient in adiponectin [24]. The aim of the present study is therefore to elucidate the

88

significance of adiponectin in ameliorating T2D-mediated testicular dysfunctions in the mice.

89

Mice fed with a high-fat diet (HFD) and treated with a single high dose of streptozotocin (STZ)

90

were used as the experimental animal model.

91

2. Material and Methods

92

2.1. Animal Handling and Ethical issues

93

All the procedures involving animals were conducted in accordance with the ethical principles

94

adopted by the CPCSEA, GOI, India (No. 1802/G0/Re/S/15/CPCSEA) and were approved by the 4|Page

95

Institutional Animal Ethical Committee (No. F.Sc/88/IAEC/2017-18/1423-3), Institute of

96

Science, Banaras Hindu University, Varanasi, India. Male Parkes strain mice (~ 4-5 weeks) used

97

for the present study were maintained under standard laboratory conditions i.e. hygienic

98

conditions in a photo-periodically controlled (L:D 12:12) animal house with controlled

99

temperature (24 ± 2 ºC) and humidity and free access to food and water.

100

2.2. Adiponectin peptide

101

The 18 amino acid adiponectin peptide [NH2-LQVYGDGDHNGLYADNVN-COOH] dissolved

102

in phosphate buffer saline (PBS, 100 mM) was used in this study. This structure of the peptide

103

used in our study resembles mouse adiponectin peptide, having the globular domain (amino acid

104

residues 216-233) at C-terminus of adiponectin protein (247 amino acid), which is highly

105

conserved among mammalian species. This adiponectin globular domain showed a stronger

106

affinity with AdipoR1 receptor and has a moderate affinity with AdipoR2 receptor [25].

107

2.3. Animal model and experimental approach

108

Firstly, fourty male Parkes strain male were randomly distributed as a control (n=10) group, fed

109

with regular chow and a T2D (n=30) group, fed with high fat diet (HFD) for 8-12 weeks. This

110

duration of high fat diet was selected and validated in our laboratory that was able to induce

111

insulin resistance condition with increased serum insulin level. Further to achieve the induction

112

of true T2D model, resembling some of the key features of the human metabolic syndrome, the

113

animals from T2D group were injected intraperitoneally with freshly prepared streptozotocin

114

(STZ) (180 mg/kg of body weight) diluted in 100 µL sodium citrate buffer (0.1 M, pH 4.5) in

115

accordance with the previous study [26]. The HFD/STZ-induced diabetic mice model developed

116

characteristics of true T2D as previously described by our team [27]. Mice from the control

117

group only received the vehicle citrate solution in equivalent volume. Blood glucose level were

118

monitored after one week of STZ injection, and the presence of features viz. increased body

119

weight, hyperglycemia (fasting serum glucose concentration) (Accu-Check Active digital

5|Page

120

glucometer, Roche Diabetes Care, India), hyperinsulinemia (fasting serum insulin levels)

121

(Rat/Mouse Insulin ELISA kit (Millipore, United States), insulin sensitivity, and homeostatic

122

model assessment value of insulin resistance (HOMA-IR) were the basis for selection of T2D

123

mice for further experiment. Out of thirty animals in HFD/STZ group, twenty-one animals

124

having fasting serum glucose concentration >250 mg/dL were selected and considered T2D and

125

rest nine animals were discarded from the experiment. Finally, mice were divided into total four

126

groups; group 1 is vehicle control (n=10); group 2 is diabetic mice (n=7); group 3 is diabetic-

127

treated with adiponectin for 2 weeks short term (DT_APN_ST) (n=7) and group 4 is diabetic-

128

treated with adiponectin for 4 weeks long term (DT_APN_LT) (n=7), respectively. Adiponectin

129

dosage (50 µg/day/animal) and duration (~max 4 weeks) was selected in accordance with the

130

earlier reports [28, 29]. The maximum duration of adiponectin-treatment (4 weeks) was selected

131

in our experiment, as in mouse, one full spermatogenic cycle (twelve stages) completes in over

132

five weeks consisting of four cycles of 8.4 days each, to release mature spermatozoa from

133

spermatogonial stem cell. At the end of the experiment, animals were killed by decapitation

134

under a mild dose of anesthetic ether and blood was collected. Serum was collected from blood

135

and kept at -20⁰C for further assay. One testis of each animal was kept at –80⁰C for enzyme

136

assay and

137

immunohistochemistry.

138

2.4. Insulin Sensitivity and HOMA-IR Value

139

In

140

[(14.1/(glucose)(insulin))*100]

141

[(insulin)(glucose)/14.1], in accordance to the criteria described previously [30].

142

2.5. Histological analysis

143

The histological studies were performed as described previously [23]. In brief, Bouin’s fixed,

144

paraffin-embedded testes were sectioned at 6 µm. One set of slides were used for hematoxylin

brief,

the

other

insulin

was

fixed

sensitivity and

in

was

Bouin’s

fluid

calculated

HOMA-IR

value

for

histological

according according

to to

analysis

and

the

formula

the

formula

6|Page

145

and eosin staining whereas an alternate set was used for IHC. Stage VII-VIII spermatogenesis in

146

the testicular sections were identified according to the criteria described previously [31]. This is

147

the most commonly observed stage of spermatogenesis, highly dependent on androgen, and

148

seminiferous tubules have a maximal mean diameter in this stage of the spermatogenic cycle.

149

The epithelial height and diameter of seminiferous tubules of control, diabetic, and adiponectin-

150

treated diabetic testis were measured with motic image software using Nikon-E200.

151

2.6. Immunohistochemistry

152

IHC was performed according to the method described earlier [23]. Testis of control and diabetic

153

mice were analyzed by IHC, for adiponectin, AdipoR1, and AdipoR2. The testicular sections

154

were deparaffinized in xylene followed by hydration through graded alcohol. The sections were

155

then treated with 3% H2O2 in methanol for blocking endogenous peroxidase activity. The

156

sections were incubated with blocking serum for 1 hr, followed by incubation with the primary

157

antibody (dilution given in Table 1) overnight at 4 ºC. The sections were washed in PBS and

158

incubated with the horseradish peroxidase tagged secondary antibody (dilution given in Table 1)

159

for 2.5 hr at room temperature. After incubation with secondary antibody, sections were washed

160

and incubated with the chromogen substrate (0.1%; 3,3 diaminobenzidine tetrahydrochloride in

161

0.5 M Tris pH -7.6 and 0.01% H2O2) in dark for 1-2 min. Slides were dehydrated and mounted

162

with DPX and analyzed under a light microscope (Nikon, Tokyo, Japan) and shot.

163

2.7. Western blotting

164

The testes were pooled and homogenized in suspension buffer (0.01 M Tris pH 7.6, 0.001 M

165

EDTA pH = 8.0, 0.1 M NaCl, 1 µg/ml aprotinin, 100 µg/ml PMSF) to produce 20% (w/v)

166

homogenate. The extraction of protein and immunoblotting was performed as described

167

previously [23]. An equal amount of protein (60 µg) per lane, as estimated by the method of

168

Bradford [32], was loaded on to 10% SDS–PAGE for electrophoresis. Thereafter, proteins were

169

transferred electrophoretically to PVDF membrane (Millipore India Pvt. Ltd.) overnight at 50V, 7|Page

170

4ºC. The membranes were blocked for 1 hr with PBS (0.1 M, pH 7.4; NaH2PO4 -16 mM;

171

Na2HPO4 64 mM; NaCl 154 mM; 0.02% Tween 20) containing 5% dry fat-free milk and

172

incubated with primary antibodies (dilution given in Table 1) for 3 hr at room temperature.

173

Membranes were then washed with three changes of PBST over 10 min. Immunoreactive bands

174

were detected by incubating the membranes with horseradish peroxidase tagged secondary

175

antibody (at a dilution of 1:4000); for 1.5 hr. Finally, the blot was washed three times with PBST

176

and developed with enhanced chemiluminescence (ECL) detection system (BioRad, United

177

States). Blots for each protein were repeated three times. The densitometric analysis of the blots

178

was performed by scanning and quantifying the bands for density using computer-assisted image

179

analysis (Image J 1.38x, NIH, USA). The densitometric data were presented as the mean of the

180

raw integrated density value ± SEM. The bands obtained were normalized to β-actin (Sigma

181

Aldrich, St. Louis, MO, United States).

182

2.8. Testosterone assay

183

Intra-testicular testosterone levels were estimated using the ELISA kit (Diametra, USA)

184

according to the procedures as described in the manufacturer manual. In brief, 25 µl of the

185

standards, control or samples (testicular homogenate or media) were added to each well of the

186

ELISA plate. Subsequently, the enzyme conjugate solution was added to each of these wells. The

187

ELISA plate was then incubated at 37 ºC for 1 h. The wells were then aspirated and washed 3

188

times with wash solution. Then, 100 µl of the tetramethylbenzidine chromogen (TMB) solution

189

was added to each well and the plate was incubated at room temperature for 15 min. Finally, 100

190

µl of stop solution (0.2 M sulfuric acid) was added and the optical density (OD) was noted at 450

191

nm using a microplate reader (BioRad, United States).

192

2.9. Intra-testicular glucose concentration, intra-testicular lactate concentration, and

193

lactate dehydrogenase (LDH) enzyme activity measurement

8|Page

194

Testicular glucose and lactate were estimated by using commercially available biochemical

195

estimation kits (Autospan, India; and Sigma Aldrich, St. Louis, MO, USA; respectively).

196

Testicular LDH was measured using a commercially available LDH (P-L) KIT (Coral Clinical

197

Systems, India).

198

2.10. Evaluation of testicular antioxidant enzymes activity

199

The anti-oxidative enzyme activities for superoxide dismutase (SOD), catalase (CAT), and

200

glutathione peroxidase (GPx) in the testicular homogenate of control and treated mice were

201

determined according to procedures described [33-35], with minor modifications [36]. Lipid

202

peroxidation assay by measuring level of thiobarbituric acid (TBARS) in the testicular

203

homogenate of control and adiponectin-treated T2D mice was performed in accordance with the

204

procedure described previously [37] with minor modifications as published [27].

205

2.11. Statistical analysis and correlation studies

206

The significance of the differences between groups was measured by using one-way analysis of

207

variance (ANOVA) followed by Bonferroni's post-hoc test using software SPSS, version 12

208

(SPSS Inc., IBM, Chicago, IL, United States) to compare the data from different groups. The

209

correlation studies were performed by linear regression analysis using Pearson's coefficient

210

method to find out the effect of adiponectin in improving testicular activities of T2D mice. To

211

achieve this, changes in various spermatogenic, steroidogenic, metabolic, and antioxidative

212

parameters were correlated with each other in vehicle-treated control mice, T2D mice, and T2D

213

mice treated with adiponectin. The differences were considered significant if P < 0.05. Data were

214

expressed as mean ± SEM.

215

3. Results

216

3.1. Changes in the expression of adiponectin, AdipoR1, AdipoR2 proteins, and

217

accumulation of white adipose tissue in HFD/STZ-induced T2D mice.

9|Page

218

In order to find out the possible role of adiponectin in diabetes, expression of testicular

219

adiponectin and its receptors (AdipoR1 and AdipoR2) proteins were evaluated using

220

immunohistochemistry and western blotting of T2D and control mice. Immunohistochemical

221

study showed a distinct change in the expression of testicular adiponectin and its receptors

222

protein T2D mice compare to the control mice. Both adiponectin (Figure 1a A-B) and AdipoR1

223

(Figure 1a B-C) proteins were localized in the interstitial cells mainly Leydig cells, whereas

224

AdipoR2 (Figure 1a D-E) protein was localized particularly in the round spermatid present in the

225

seminiferous tubules of the mice testes. The intensity of immunostaining of all the three proteins

226

declined distinctly in T2D mice compared to vehicle-treated control mice. The testicular

227

expression of adiponectin, AdipoR1, and AdipoR2 proteins declined significantly approximately

228

~53.7%, 87.5%, and 56.5% respectively in the diabetic mice compared to the control mice (Fig

229

1b). The increased accumulation of abdominal white adipose tissue (WAT) was observed in T2D

230

mice compared to the control mice (Figure 2).

231

3.2. Effect of systemic administration of adiponectin to HFD/STZ-induced T2D mice on:

232

This study was undertaken to find out the possible effect of adiponectin-treatment to T2D mice.

233 234 235

3.2.1. Changes in body mass, testis mass, fasting serum insulin, fasting serum glucose, HOMA-IR, and insulin sensitivity.

236

This study evaluated the changes in metabolic markers in diabetic mice treated with adiponectin.

237

The body mass increased significantly (p<0.05) by 26.72 % whereas testis mass declined

238

significantly by 40.59 % in T2D mice compared to control mice. The T2D mice treated with

239

adiponectin in both ST (2 weeks) and LT (4 weeks) showed significant decrease in body mass by

240

12.05 % and 17.47 % respectively while a significant increase in testis mass by 16.21 % and 50.9

241

% respectively compared to the untreated diabetic mice (Figure 3A-B). The results also showed

242

a significant increase in fasting serum insulin by 213.1 % and fasting serum glucose by 453.33 % 10 | P a g e

243

in T2D mice compared to control mice. The treatment of T2D mice with adiponectin for both ST

244

and LT showed significant decline in fasting serum insulin (by 83.77 % and 74.76 %) and fasting

245

glucose levels (by 36.47 % and 58.34 %) respectively compared to untreated T2D mice (Figure

246

3C-D). The insulin sensitivity decreased significantly by 94.46 % whereas HOMA-IR value

247

increased significantly by 1666.67 % in T2D mice compared to the control mice. The T2D mice

248

treated with adiponectin for both ST and LT showed significant increase in insulin sensitivity by

249

909.52 % and 804.76 % respectively while significantly decreased value of HOMA-IR by 89.73

250

% and 89.52 % respectively compared to untreated T2D mice (Figure 3E-F).

251

3.2.2.

252

The testes of control mice showed healthy and compactly arranged seminiferous tubules with

253

active spermatogenesis, and Leydig cells appeared healthy. The testes of T2D mice showed

254

regressive ST as indicated by decreased tubular diameter, increased apoptotic changes in the

255

germ cells, decreased germinal epithelium height, the presence of vacuoles, and widening of

256

intertubular spaces. Large lumen contained deformed elongated spermatids. The T2D mice when

257

treated with adiponectin for both ST and LT duration showed regenerative changes such as the

258

reappearance of complete spermatogenic cell layers, increased tubular diameter, increased

259

thickness of the germinal epithelium height, decreased rate of apoptosis, increased proliferation

260

of germ cells, and compactly arranged seminiferous tubules (Figure 4).

261

3.2.3. Changes in the expression of spermatogenic marker proteins for cell proliferation

262

(proliferating cell nuclear antigen; PCNA), cell survival (B cell lymphoma factor-2; Bcl2),

263

and cellular apoptosis (cysteine-aspartic proteases-3; caspase-3)

264

In order to assess the effect of adiponectin on spermatogenic activity; changes in the rate of cell

265

proliferation (expression of PCNA), cell survival (expression of Bcl2), and apoptosis (expression

266

of caspase-3) were evaluated in the testis of T2D mice compared with the control mice. The

Histomorphological changes in the testis

11 | P a g e

267

PCNA immunostaining was performed to detect mitotically-active type A spermatogonia with

268

maximum expression at S-phase cells and finally counted number of PCNA positive cells per

269

seminiferous tubules which is a useful marker for assessing germ cell kinetics and

270

spermatogenesis [23, 38]. Bcl-2 is a cell survival protein best known for its roles in inhibiting

271

apoptosis [39]. Caspase-3 protein is utilized as a marker of testicular germ cell apoptosis [40,

272

41]. Immunohistochemical study showed PCNA positive immunostaining particularly in the type

273

A spermatogonial (Sg) cells of the seminiferous tubule of the testis of all experimental groups.

274

The testes of T2D mice showed significant (P<0.05) decline in the number of PCNA-positive

275

cells (by 29.79%) compared to the control mice. The treatment of T2D mice with adiponectin for

276

both ST and LT showed significant increase in number of PCNA positive cells by 27.27% and

277

39.39% respectively compared to untreated diabetic mice (Figure 5A). In addition, the results

278

also showed a significant decrease in the expression of testicular PCNA by 20.23% and testicular

279

Bcl2 by 15.15% while significant increased expression of testicular caspase-3 by 25.64% in T2D

280

mice compared to control mice. The T2D mice treated with adiponectin for both ST and LT

281

duration showed significantly increased expression of both PCNA (by 45.28% and 17%) and

282

Bcl2 (by 53.18% and 31.18%) respectively while significant decline in the expression of

283

caspase-3 protein by 44.76% and 83.35% respectively compared to the untreated T2D mice

284

(Figure 5B).

285 286

3.2.4. Changes in the expression of testicular steroidogenic markers (StAR, 3β-HSD)

287

proteins and serum testosterone level

288

This study examined the changes in steroid synthesis in diabetic mice and diabetic mice treated

289

with adiponectin. We have evaluated the expression of testicular steroidogenic acute regulatory

290

protein (StAR) protein and testicular testosterone making enzyme 3β-HSD, along with the levels

291

of the sex steroid hormones (testosterone) in serum which is considered pivotal for several

12 | P a g e

292

events that control spermatogenesis, including testicular metabolism. The changes in

293

steroidogenic markers (StAR and 3β-HSD) proteins were studied by western blot followed by

294

densitometric analysis in the testis of all experimental groups. The testes of T2D mice showed

295

significant (P<0.05) decline in the expression of StAR protein by 83.97% and 3β-HSD protein

296

by 70.45% compared to control mice. The T2D mice treated with adiponectin for both ST and

297

LT durations showed significant increase in the expression of StAR protein (by 497.59% and

298

869.48%) and 3β-HSD proteins (by 21.56% and 180.99%) respectively compared to the

299

untreated-T2D mice (Figure 6 A-B). The circulating testosterone concentration declined

300

significantly in the T2D mice by 80.35% compared to the control mice. The T2D mice treated

301

with adiponectin for both ST and LT durations showed a significantly increased concentration of

302

circulating testosterone by 88.89% and 122.96% respectively compared to the untreated diabetic

303

mice (Figure 6C).

304

3.2.5. Changes in the expression of metabolic markers (AdipoR1, IR, GLUT8, glucose and

305

lactate levels, LDH enzyme activity), antioxidant enzymes (SOD, Catalase, GPx, and

306

TBARS) activity and lipid peroxidation (TBARS level)

307

Our study assessed the changes in testicular glycolytic profile and antioxidant markers in

308

diabetic mice and following treatment with adiponectin. To study testicular glycolytic profile,

309

we have studied its main substrate viz. glucose. In the testis, glucose uptake initiated via insulin

310

sensitizing action particularly through AdipoR1 and IR mediated GLUT8 transporter signaling.

311

The glucose through glycolysis converted to pyruvate and LDH acts on pyruvate to convert it to

312

lactate in the Sertoli cells. The lactate is a critical “fuel” for developing germ cells. The testes of

313

T2D mice showed significant (P<0.05) decline in the expression of testicular AdipoR1 by

314

92.29%, IR by 34.16%, and GLUT8 by 67.35% compared to the control mice. The T2D mice

315

when treated with adiponectin for both ST and LT durations, showed significantly increased

316

expression of testicular AdipoR1 (by 997.43% and 1602.27%), IR (by 155.24% and 151.85%), 13 | P a g e

317

and GLUT8 (by 133.04% and 244.59%) respectively compared with the untreated T2D mice

318

(Figure 7A). In addition, T2D mice also showed a significant decline in the concentration of

319

intra-testicular glucose concentration by 57.64%, testicular LDH activity by 54.55%, and intra-

320

testicular lactate concentration by 65.86% compared to the control mice. Further, T2D mice

321

treated with adiponectin for both the duration (ST and LT) showed significant increased

322

concentration of intra-testicular glucose level (by 74.06% and 152.82%), LDH activity (by

323

186.67% and 326.67%), as well as testicular lactate level (by 93.72% and 263.12%) respectively

324

compared to the untreated T2D mice (Figure 7B,C).

325

The T2D mice showed a significant (P<0.05) decline in the testicular activity of SOD by

326

48.28%, catalase by 71.95%, and GPx by 60.37% while enhanced testicular TBARS level by

327

111.36% compared to the control mice. Treatment of T2D mice with adiponectin for each

328

different (ST and LT) duration showed a significant increased activity of testicular SOD (by

329

197.59% and 133.56%), catalase (by 515.63% and 468.28%), and GPx (by 321% and 229.78%)

330

respectively while significant decrease in the concentration of TBARS by 26.64% and 44.02%

331

respectively compared to untreated T2D mice (Figure 8 A-D).

332

3.2.6. Correlation between changes in various testicular metabolic markers (AdipoR1, IR,

333

GLUT8, glucose, LDH, and lactate levels), spermatogenic markers (PCNA, Bcl2, and

334

Caspase-3), testicular antioxidant enzymes (SOD, Catalase, and GPx), and serum

335

parameters (testosterone, fasting glucose, and fasting insulin) in different treatment groups.

336

In order to find out the effect of adiponectin in improving testicular activities of T2D mice,

337

correlation analysis was performed. To achieve this, changes in various spermatogenic,

338

steroidogenic, metabolic, and antioxidative parameters were correlated with each other in

339

vehicle-treated control mice, T2D mice, and T2D mice treated with adiponectin. The results of

340

correlation studies are summarized in Table 2. The changes in testicular AdipoR1, IR, GLUT8,

341

intra-testicular glucose and lactate levels, and LDH showed a significant positive correlation 14 | P a g e

342

with each other’s and with the activity of antioxidant enzymes (SOD, Catalase, and GPx).

343

Further, testicular PCNA and Bcl2 showed positive correlation with the testicular antioxidant

344

enzymes activity. The serum testosterone showed positive correlation with the testicular

345

AdipoR1, GLUT8, glucose and lactate levels among all the treatment groups.

346

4. Discussion

347

The present study aimed to identify factors that can reverse the testicular dysfunctions of T2D

348

mice. Current studies were conducted on the mice fed with an HFD and treated with a single

349

high dose of STZ to induce true T2D-like features. The HFD/STZ-induced T2D mice showed

350

features such as increased body mass, hyperglycemia, hyperinsulinemia, insulin resistance

351

(increased HOMA-IR and decreased insulin sensitivity), and increased oxidative stress as

352

typically found in a patient with T2D. The T2D mice showed a significant increase in body mass

353

as a result of increased accumulation of white adipose tissue. The increased accumulation of

354

white adipose tissue (obesity) has been associated with the development of insulin resistance in

355

T2D mice as previously suggested [42, 43]. Insulin resistance is an important characteristic of

356

T2D animals [44], which consequently may be responsible for the development of

357

hypogonadism in obese T2D male [45]. This finding is in accordance with the earlier study

358

showed a marked reduction in body weight in obese male resulted in a reversal of both insulin

359

resistance and hypogonadism [46]. Further studies showed hypogonadism with obesity is mainly

360

attributed to insulin resistance [47]. A decline in testosterone synthesis is a major factor

361

responsible for hypogonadism. However, the mechanism by which T2D-associated decline in

362

testosterone level causes hypogonadism is not fully elucidated. In a recent study, T2D-associated

363

decrease testosterone levels was shown to induce abnormal metabolic changes in Sertoli cells

364

resulting in decrease conversion to lactate from glucose. Low testosterone dependent metabolic

365

alterations reported in Sertoli cells may be responsible for reduced metabolic support of

366

spermatogenesis which contribute to hypogonadism [13]. The biosynthesis of testosterone in

15 | P a g e

367

Leydig cells is under the primary regulation of LH with modulating (positive and negative)

368

effects of local autocrine and paracrine factors [48]. It has been suggested that diminished

369

testosterone production in T2D may be attributed to the reduced stimulatory effect of insulin

370

(due to insulin resistance) on Leydig cells [49]. It has also been suggested that insulin has an

371

important role in the maintenance of the LH receptor and that declined testosterone synthesis

372

may be due to insulin resistance associated down-regulation of LH receptor in Leydig cells in the

373

diabetic rat [26]. These findings thus suggest a close interrelationship between obesity, insulin

374

resistance, and testosterone deficiency in T2D.

375

In order to reveal the factor responsible for the insulin resistance in T2D, this study evaluated the

376

changes in the localization and concentration of adiponectin and its receptors proteins in the

377

testes of T2D mice. The results of the present study substantiated our recent reports showing the

378

presence of adiponectin and AdipoR1 mainly on the Leydig cells in the testes of the adult mice

379

[11]. Furthermore, in this study, the Western blot analyses revealed a significant decline in the

380

expression of adiponectin and its receptor proteins in the testes of T2D mice. Simultaneously

381

with the decline in the level of adiponectin receptors, this study also showed down-regulation of

382

IR (suggesting insulin resistance condition, confirmed by increased HOMA-IR) and decreased

383

circulating levels of testosterone in the T2D mice, as compared with the control mice. The results

384

of the present study thus suggest that a decline in testicular adiponectin synthesis and action may

385

be responsible for the induction of insulin resistance and decreased testosterone synthesis in T2D

386

mice. Similar to the present finding, the circulating adiponectin levels were decreased

387

significantly in the diabetic monkey and human as compared with the normal control [50, 51]

388

and adiponectin receptor knockout mice showed insulin resistance, increased oxidative stress,

389

and glucose intolerance conditions [52]. The earlier study also showed a decreased adiponectin

390

level in T2D rats with insulin resistance [53]. Furthermore, it has been suggested that a higher

391

concentration of adiponectin may protect the development of T2D by improving insulin

392

sensitivity (or preventing insulin resistance) [54]. 16 | P a g e

393

Intriguingly, the T2D mice supplemented with adiponectin showed a significantly increased

394

expression of adiponectin-receptors (AdipoR1) simultaneously with increased expression of IR

395

and GLUT8 proteins in the testis compared with the untreated T2D mice. This finding suggests

396

that adiponectin treatment in T2D mice may be responsible for reversing insulin resistance by

397

up-regulation of insulin receptor (sensitivity), which consequently may be responsible for

398

insulin-mediated increased uptake of glucose due to increased expression of GLUT8 protein in

399

the testis. This finding is further supported by the significant positive correlation between the

400

increased expression of adiponectin-receptor protein with the increased expression level of

401

insulin receptor (r = 0.74) and GLUT8 (r = 0.97) proteins and also with the increased intra-

402

testicular concentration of glucose (r = 0.87) in the testis of T2D mice treated with adiponectin.

403

GLUT8 is known as major glucose transporter associated specifically with insulin-dependent

404

transport of glucose in the testis [55, 56]. In accordance with the earlier research [14], our study

405

also showed a significant positive correlation between the increase in GLUT8 protein with

406

increase in glucose concentration in the testis of mice (r=0.87). It is now well recognized that a

407

sufficient amount of glucose is mandatory for active spermatogenesis [10, 23]. The testicular

408

glucose undergoes a series of metabolic changes to produce lactate from pyruvate, which finally

409

provides energy (ATP) to post-meiotic germ cells for active proliferation and differentiation

410

during spermatogenesis [5, 57]. In our recent study, testes treated in vitro with adiponectin was

411

shown to increase activity of LDH enzyme which converted glucose to lactate in the Sertoli cells

412

[11]. These findings are further supported by the significant positive correlation found between

413

increased expression of GLUT8 (r = 0.70) protein and intra-testicular concentration of glucose (r

414

= 0.52) with the increase in the expression of PCNA in the testis of T2D mice treated with

415

adiponectin. This study thus provided evidence for the first time suggesting the important role of

416

adiponectin in increasing transport of glucose by promoting insulin sensitivity in the testis and

417

resulted in increased synthesis of lactate required for active spermatogenesis.

17 | P a g e

418

In the present study, the T2D mice showed regressive changes in the testes as demonstrated by

419

the decreased diameter of seminiferous tubule and reduced germinal epithelial cell height. These

420

morphological changes occur mainly due to an increased rate of apoptotic cell death in the testes

421

of T2D mice. This is in agreement with the earlier finding showing an increased rate of apoptosis

422

of the germ cells as the leading cause of impaired testicular functions in diabetic mice [8]. It has

423

previously been demonstrated that increased production of oxidative stress triggers the process

424

of apoptosis by enhancing the expression of caspase-3 and inhibiting the expression of Bcl2 in

425

the testes of T2D [58]. The oxidative stress is also capable of impairing the Leydig cells

426

steroidogenic activity [59]. Our results further showed a significant correlation between

427

decreased testicular antioxidant enzymes activity with serum testosterone levels in T2D mice

428

compared to control mice (Table 2). The decreased production of testosterone is a well-known

429

factor responsible for testicular impairment in T2D [13]. The T2D mice treated exogenously

430

with adiponectin showed a marked improvement in testicular histology as indicated by decreased

431

rate of apoptosis due to decreased expression of apoptotic (caspase-3) markers proteins, together

432

with increased rate of germ cells proliferation due to increased expression of cell proliferation

433

(PCNA) and cell survival (Bcl2) markers as compared with the untreated T2D mice. The

434

presence of adiponectin receptors on germ cells of active adult testis further supports the

435

stimulatory effect of adiponectin on germ cell proliferation [11, 60]. The adiponectin treatment

436

of T2D showed a significant increase in circulating testosterone together with increased

437

expression of steroidogenic factors, StAR, and 3β-HSD proteins, in the testis. The present study

438

on T2D mice treated with adiponectin for short and long durations also showed significant

439

increases in antioxidant enzyme (SOD, catalase, and GPx) activity, with a significant decline in

440

oxidative factors (lipid peroxidation or TBARS level). The stimulatory effect of adiponectin on

441

testosterone synthesis may also be due to the stimulatory effect of adiponectin on catalase and

442

GPx enzymes contained in Leydig cells. Our findings together with the earlier studies thus

443

clearly suggest the important role of adiponectin in improving the testicular activities by 18 | P a g e

444

suppressing oxidative stress as well as by stimulating testosterone synthesis in the testis of T2D

445

mice.

446

In brief, this study was undertaken to elucidate the significance of adiponectin in ameliorating

447

T2D-mediated testicular dysfunction in mice. The testes of HFD/STZ-induced T2D mice

448

exhibited a distinct decline in the expression of adiponectin and its receptors proteins compared

449

to vehicle-treated control mice. The decline in the synthesis and action (due to decreased adipoR

450

level) of adiponectin may be responsible for the development of insulin resistance (as indicated

451

by increased HOMA-IR value) in the testes of T2D. Furthermore, in current study, the

452

exogenous administration of adiponectin to T2D mice showed a significant increase in the

453

expression of insulin receptor proteins (suggesting a decrease in insulin resistance) compared

454

with the testes of the untreated T2D mice. The adiponectin-induced increased IR mediated

455

insulin action increases expression of Glut8 protein simultaneously with increased in intra-

456

testicular level of glucose and production of lactate in the testes of T2D mice. These findings are

457

further supported by the significant positive correlation between increased expression of IR with

458

the expression of GLUT8 (r = 0.74) protein together with the increased concentration of intra-

459

testicular glucose (r = 0.75) and lactate (r = 0.74) in the testis of T2D mice treated with

460

adiponectin. In conclusion, the adiponectin-induced increased transport of glucose and lactate

461

and a marked reduction in oxidative stress are two major pathways by which adiponectin revert

462

testicular dysfunction in T2D mice.

463 464

Authors Contributions

465

MC has designed, performed all the experiments, and analyzed the data, and wrote full draft of

466

the manuscript. AK has critically revised and AR has corrected the manuscript. PSB has

467

synthesized the adiponectin peptide. All authors read and approved the manuscript.

468 469

Conflict of interest 19 | P a g e

470

The authors declare that there is no conflict of interest.

471 472

Acknowledgments

473

Mayank Choubey greatly acknowledges financial assistance in the form of a Senior Research

474

Fellowship

475

RBMH/FW/2018/1), New Delhi, India. The authors would like to thank Ms. Shaina Brown from

476

Applied Research Centre of Arkansas, Little Rock, AR, United States for helping in editing the

477

manuscript.

(SRF)

from

Indian

Council

of

Medical

Research

(ICMR

File

No.

478

479

Funding

480

This work did not receive any specific research grant.

481

482

483 484

References [1]. Hurrle S, Hsu WH, 2017. The etiology of oxidative stress in insulin resistance. Biomedical Journal; 40, 257-262.

485

[2]. Silink M, 2002. Childhood diabetes: A global perspective. Horm Res.; 57, 1-5.

486

[3]. Rosenbloom AL, Joe JR, Young RS, Winter WE, 1999. Emerging epidemic of type 2

487 488 489

diabetes in youth. Diabetes Care; 22(2): 345-54. [4]. Tangvarasittichai S, 2015. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World J Diabetes; 6(3): 456-80. doi: 10.4239/wjd.v6.i3.456.

490

[5]. Alves MG, Martins AD, Rato L, Moreira PI, Socorro S, Oliveira PF, 2013. Molecular

491

mechanisms beyond glucose transport in diabetes-related male infertility. Biochim Biophys

492

Acta; 1832(5): 626-35. doi: 10.1016/j.bbadis.2013.01.011.

20 | P a g e

493 494

[6]. Hempel R, Onopa R, Convit A, 2012. Type 2 Diabetes Affects Brain Health Differentially in Men and Women. Diabetes Metab Res Rev.; 28(1): 76–83. doi: 10.1002/dmrr.1230.

495

[7]. Long L, Qiu H, Cai B, Chen N, Lu X, Zheng S, Ye X, Li Y, 2018. Hyperglycemia induced

496

testicular damage in type 2 diabetes mellitus rats exhibiting microcirculation impairments

497

associated with vascular endothelial growth factor decreased via PI3K/Akt pathway.

498

Oncotarget; 9(4): 5321-5336. doi: 10.18632/oncotarget.23915.

499

[8]. Maresch CC, Stute DC, Alves MG, Oliveira PF, de Kretser DM, Linn T, 2018. Diabetes-

500

induced hyperglycemia impairs male reproductive function: a systematic review. Hum

501

Reprod Update; 24(1): 86-105. doi: 10.1093/humupd/dmx033.

502 503 504

[9]. Wallach E, Steinberger E, 1978. The Etiology and Pathophysiology of Testicular Dysfunction in Man. Fertility and Sterility; 29(5): 481-491. [10].

Rato L, Alves, MG, Socorro S, Duarte AI, Cavaco JE, Oliveira PF, 2012. Metabolic

505

regulation is important for spermatogenesis. Nature Reviews Urol; 9:330–33877.

506

doi:10.1038/nrurol.2012.77.

507

[11].

Choubey M, Ranjan A, Bora PS, Baltazar F, Krishna A, 2019a. Direct actions of

508

adiponectin on changes in reproductive, metabolic, and anti-oxidative enzymes status in the

509

testis of adult mice. Gen. Comp. Endocrinol; 279: 1-11. doi: 10.1016/j.ygcen.2018.06.002.

510

[12].

Morgan DH, Ghribi O, Hui L, Geiger JD, Chen X, 2014. Cholesterol-enriched diet

511

disrupts the blood-testis barrier in rabbits. Am J Physiol Endocrinol Metab; 307(12): E1125-

512

30. doi: 10.1152/ajpendo.00416.2014.

513

[13].

Rato L, Alves MG, Dias TR, Cavaco JE, Oliveira PF, 2015. Testicular metabolic

514

reprogramming in neonatal streptozotocin-induced type 2 diabetic rats impairs glycolytic

515

flux

516

10.1155/2015/973142.

and

promotes

glycogen

synthesis.

J

Diabetes

Res;

2015:973142.

doi:

21 | P a g e

517

[14].

Banerjee A, Anuradha, Mukherjee K, Krishna A, 2014. Testicular glucose and its

518

transporter GLUT 8 as a marker of age-dependent variation and its role in steroidogenesis in

519

mice. J. Exp. Zool. Part A; 321:490–502. doi: 10.1002/jez.1881.

520

[15].

Carosa E, Radico C, Giansante N, Rossi S, D'Adamo F, Di Stasi SM, Lenzi A,

521

Jannini EA, 2005. Ontogenetic profile and thyroid hormone regulation of type-1 and type-8

522

glucose transporters in rat Sertoli cells, Int J Androl.; 28(2): 99-106.

523 524 525

[16].

Gomez O, Romero A, Terrado J, Mesonero JE, 2006. Differential expression of

glucose transporter SLC2A8 during mouse spermatogenesis. Reproduction; 131, 63–70. [17].

Doege H, Schürmann A, Bahrenberg G, Brauers A, Joost HG, 2000. GLUT8, a novel

526

member of the sugar transport facilitator family with glucose transport activity. J Biol

527

Chem; 275(21):16275-80. doi: 10.1074/jbc.275.21.16275

528 529 530

[18].

Chen Y, Nagpal ML, Lin T, 2003. Expression and regulation of glucose transporter 8

in rat Leydig cells. J Endourol 179:63–72. [19].

Schoeller EL, Albanna G, Frolova AI, Moley KH, 2012. Insulin rescues impaired

531

spermatogenesis via the hypothalamic-pituitary-gonadal axis in Akita diabetic mice and

532

restores male fertility. Diabetes; 61(7): 1869-78. doi: 10.2337/db11-1527.

533 534 535

[20].

Jangir RN, Jain GC, 2014. Diabetes mellitus induced impairment of male

reproductive functions: a review. Curr Diabetes Rev; 10(3): 147-57. [21].

Singh A, Choubey M, Bora P, Krishna A, 2018. Adiponectin and chemerin: contrary

536

adipokines in regulating reproduction and metabolic disorders. Reprod Sci; 25(10):1462-

537

1473. doi: 10.1177/1933719118770547.

538 539 540

[22].

Martin LJ, 2014. Implications of adiponectin in linking metabolism to testicular

function. Endocrine; 46(1): 16-28. doi: 10.1007/s12020-013-0102-0. [23].

Choubey M, Ranjan A, Bora PS, Baltazar F, Martin LJ, Krishna A, 2019b. Role of

541

adiponectin as a modulator of testicular function during aging in mice. Biochim Biophys

542

Acta Mol Basis Dis; 1865(2): 413-427. doi: 10.1016/j.bbadis.2018.11.019. 22 | P a g e

543

[24].

Guo Z, Yan X, Wang L, Wu J, Jing X, Liu J, 2016. Effect of Telmisartan or Insulin

544

on the Expression of Adiponectin and its Receptors in the Testis of Streptozotocin-Induced

545

Diabetic Rats. Horm Metab Res;48(6): 404-12. doi: 10.1055/s-0042-101549.

546

[25].

Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T,

547

Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, UchLabela S, Takekawa S,

548

Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K,

549

Kitamura T, Shimizu T, Nagai R, Kadowaki T, 2003. Cloning of adiponectin receptors that

550

mediate

551

http://dx.doi.org/10.1038/nature01683.1.

552

[26].

antidiabetic

metabolic

effects.

Nature;

423:762–769.

Ahn SW, Gang GT, Kim YD, Ahn RS, Harris RA, Lee CH, Choi HS, 2013. Insulin

553

directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in

554

testicular Leydig cells. J Biol Chem; 288(22): 15937-46. doi: 10.1074/jbc.M113.451773.

555

[27].

Verma R, Samanta R, Krishna A, 2019. Comparative Effects of Estrogen and

556

Phytoestrogen, Genistein on Testicular Activities of Streptozotocin-Induced Type 2 Diabetic

557

Mice. Reprod Sci.; 26(9):1294-1306. doi: 10.1177/1933719118815576.

558

[28].

Shibata S, Tada Y, Hau CS, Mitsui A, Kamata M, Asano Y, Sugaya M, Kadono T,

559

Masamoto Y, Kurokawa M, Yamauchi T, Kubota N, Kadowaki T, Sato S, 2015.

560

Adiponectin regulates psoriasiform skin inflammation by suppressing IL-17 production

561

from γδ-T cells. Nat Commun; 6:7687.

562

[29].

Wang Y, Wang X, Guo Y, Bian Y, Bai R, Liang B, Xiao C, 2017. Effect of

563

adiponectin on macrophage reverse cholesterol transport in adiponectin-/- mice and its

564

mechanism. Exp Ther Med; 13(6): 2757-2762. doi: 10.3892/etm.2017.4321.

565

[30].

Van-Dijk TH, Laskewitz AJ, Grefhorst A, Boer TS, Bloks VW, Kuipers F, Groen

566

AK, Reijngoud DJ, 2013. A novel approach to monitor glucose metabolism using stable

567

isotopically labelled glucose in longitudinal studies in mice. Lab Anim; 47(2):79-88. doi:

568

10.1177/0023677212473714. 23 | P a g e

569

[31].

Russell LD, Ren HP, Sinha Hikim I, Schulze W, Sinha Hikim AP, 1990. A

570

comparative study in twelve mammalian species of volume densities, volumes, and

571

numerical densities of selected testis components, emphasizing those related to the Sertoli

572

cell. Am J Anat; 188:21-30.

573

[32].

Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram

574

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem; 72, 248-

575

54.

576

[33].

Das K, Samanta L, Chainy GBN, 2000. A modified spectrophotometric assay of

577

superoxide dismutase using nitrite formation by superoxide radicals. Indian Journal of

578

Biochemistry and Biophysics; 37, 201–204.

579 580 581 582 583

[34].

Aebi H, 1974. Catalase. In: Methods of Enzymatic Analysis (ed. HU Bergmeyer),

Academic Press, New York; pp. 673–684. [35].

Mantha SV, Prasad M, Kalra J, Prasad K, 1993. Antioxidant enzymes in

hypercholesterolemia and effects of vitamin E in rabbits. Atherosclerosis; 101:135-44. [36].

Ranjan A, Choubey M, Yada T, Krishna A, 2019. Direct effects of neuropeptide

584

nesfatin-1 on testicular spermatogenesis and steroidogenesis of the adult mice. Gen Comp

585

Endocrinol. 15; 271: 49-60. doi: 10.1016/j.ygcen.2018.10.022.

586 587 588

[37].

Ohkawa H, Ohishi N, Yagi K, 1979. Assay for lipid peroxides in animal tissues by

thiobarbituric acid reaction. Anal Biochem; 95, 35–358. [38].

Tousson E, Ali EM, Ibrahim W, Mansour MA, 2011. Proliferating cell nuclear

589

antigen as a molecular biomarker for spermatogenesis in PTU-induced hypothyroidism of

590

rats. Reproductive Science; 18(7):679-86. doi: 10.1177/1933719110395401.

591 592

[39].

Cory S, Adams JM, 2002. The Bcl2 family: regulators of the cellular life-or-death

switch. Nat Rev Cancer; 2(9):647-56.

24 | P a g e

593

[40].

Kim JM1, Ghosh SR, Weil AC, Zirkin BR, 2001. Caspase-3 and caspase-activated

594

deoxyribonuclease are associated with testicular germ cell apoptosis resulting from reduced

595

intratesticular testosterone. Endocrinology; 142(9):3809-16.

596

[41].

Almeida C, Correia S, Rocha E, Alves A, Ferraz L, Silva J, Sousa M, Barros A,

597

2013. Caspase signalling pathways in human spermatogenesis. J Assist Reprod Genet;

598

30(4):487-95. doi: 10.1007/s10815-013-9938-8.

599 600 601 602

[42].

DuPlessis SS, Cabler S, McAlister DA, Sabanegh E, Agarwal A, 2010. The effect of

obesity on sperm disorders and male infertility. Nature Reviews Urology; 7: 153 – 161. [43].

Akingbemi BT, 2013. Adiponectin receptors in energy homeostasis and obesity

pathogenesis. Prog Mol Biol Transl Sci; 114:317-42.

603

[44].

Taylor R, 2012. Insulin Resistance and Type 2 Diabetes. Diabetes; 61(4): 778–779.

604

[45].

Contreras PH, Serrano FG, Salgado AM, Vigil P, 2018. Insulin sensitivity and

605

testicular function in a cohort of adult males suspected of being insulin-resistant. Front Med

606

(Lausanne); 5: 190. doi: 10.3389/fmed.2018.00190.

607

[46].

Corona G, Rastrelli G, Monami M, Saad F, Luconi M, Lucchese M, Facchiano E,

608

Sforza A, Forti G, Mannucci E, Maggi M, 2013. Body weight loss reverts obesity-associated

609

hypogonadotropic hypogonadism: a systematic review and meta-analysis. Eur J Endocrinol;

610

168(6): 829-43. doi: 10.1530/EJE-12-0955.

611

[47].

Ghanayem BI, Bai R, Kissling GE, Travlos G, Hoffler U, 2010. Diet-induced obesity

612

in male mice is associated with reduced fertility and potentiation of acrylamide-induced

613

reproductive toxicity. Biol Reprod; 82(1): 96–104.

614 615 616

[48].

Saez JM, 1994. Leydig cells: endocrine, paracrine, and autocrine regulation. Endocr

Rev; 15(5): 574-626. [49].

Ballester J, Muñoz MC, Domínguez J, Rigau T, Guinovart JJ, Rodríguez-Gil JE,

617

2004. Insulin-dependent diabetes affects testicular function by FSH- and LH-linked

618

mechanisms. J Androl; 25(5): 706-19. 25 | P a g e

619

[50].

Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H,

620

Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K,

621

Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y. Plasma

622

concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients.

623

Arterioscler Thromb Vasc Biol; 20(6):1595-9.

624

[51].

Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K,

625

Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K,

626

Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y, 1999. Paradoxical

627

decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res

628

Commun; 257(1):79-83.

629

[52].

Yamauchi T, Nio Y, Maki T, Kobayashi M, Takazawa T, Iwabu M, Okada-Iwabu

630

M, Kawamoto S, Kubota N, Kubota T, Ito Y, Kamon J, Tsuchida A, Kumagai K, Kozono H,

631

Hada Y, Ogata H, Tokuyama K, Tsunoda M, Ide T, Murakami K, Awazawa M, Takamoto I,

632

Froguel P, Hara K, Tobe K, Nagai R, Ueki K, Kadowaki T, 2007. Targeted disruption of

633

AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat

634

Med.; 13(3):332-9.

635

[53].

Abdella NA, Mojiminiyi OA, 2018. Clinical Applications of Adiponectin

636

Measurements in Type 2 Diabetes Mellitus: Screening, Diagnosis, and Marker of Diabetes

637

Control. Dis Markers; 2018:1-6. doi: 10.1155/2018/5187940.

638

[54].

Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K, 2006. Adiponectin

639

and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin

640

Invest.; 116(7): 1784-92.

641 642 643 644

[55].

Gómez O, Romero A, Terrado J, Mesonero JE, 2006. Differential expression of

glucose transporter GLUT8 during mouse spermatogenesis. Reproduction; 131(1):63-70. [56].

Kim ST, Moley KH, 2007. The expression of GLUT8, GLUT9a, and GLUT9b in the

mouse testis and sperm. Reprod Sci; 14:445–455. 26 | P a g e

645 646 647

[57].

Erkkilä K, Aito H, Aalto K, Pentikäinen V, Dunkel L, 2002. Lactate inhibits germ

cell apoptosis in the human testis. Mol Hum Reprod; 8(2):109-17. [58].

Tang XY, Zhang Q, Dai DZ, Ying HJ, Wang QJ, Dai Y, 2008. Effects of strontium

648

fructose 1,6-diphosphate on expression of apoptosis-related genes and oxidative stress in

649

testes of diabetic rats. Int J Urol; 15(3):251-6. doi: 10.1111/j.1442-2042.2007.01980.x.

650

[59].

Wang Y, Chen F, Ye L, Zirkin B, Chen H, 2017. Steroidogenesis in Leydig cells:

651

effects of aging and environmental factors. Reproduction; 154(4):R111-R122. doi:

652

10.1530/REP-17-0064.

653

[60].

Bjursell M, Ahnmark A, Bohlooly-Y M, William-Olsson L, Rhedin M, Peng XR,

654

Ploj K, Gerdin AK, Arnerup G, Elmgren A, Berg AL, Oscarsson J, Lindén D, 2007.

655

Opposing effects of adiponectin receptors 1 and 2 on energy metabolism. Diabetes;

656

56(3):583-93.

657

658

List of Tables

659

Table 1. Details of the antibodies used for Immunohistochemistry and Immunoblotting

660

experiments.

661

Table 2. Correlation studies of control, T2D, and adiponectin treated T2D mice between the

662

different serum and testicular metabolic, spermatogenic, and antioxidative parameters. *

663

denotes data were correlated significantly; Values are significantly different at p < 0.05. NS, not

664

significant.

665

666

Figures Legends

27 | P a g e

667

Figure 1. (a) Representative images showing immunohistochemical localization of adiponectin

668

and its receptors (AdipoR1 and AdipoR2) in the testes of vehicle-treated control (A, C & E) and

669

high fat diet (HFD)/streptozotocin (STZ)-induced type 2 diabetic (T2D) mice (B, D & F).

670

Intense immunostaining of adiponectin and AdipoR1 were observed in the interstitium

671

particularly in the Leydig cells (Lc) (marked with black arrowhead) of the control testis whereas

672

intense AdipoR2 positive staining was observed in the seminiferous tubules (ST) particularly in

673

the round spermatids (SR) (marked with red arrowhead) as compared to the testis of HFD/STZ-

674

induced T2D mice. (b) The densitometric analysis of the Immunoblots showed a significant

675

decrease in the expression of adiponectin, AdipoR1, and AdipoR2 proteins in the testis of T2D

676

mice as compared to the normal control mice testes.

677

Figure 2. Representative images showing accumulation of white adipose tissue (WAT) in the

678

abdominal region of vehicle-treated control and HFD/STZ-induced T2D mice. WAT is

679

indicated by black arrowhead while testis is indicated by a white arrowhead.

680

Figure 3. Changes in the (A) body mass; (B) testis mass; (C) fasting serum insulin; (D) fasting

681

glucose; (E) insulin sensitivity; and (F) homeostatic model assessment value of insulin

682

resistance (HOMA-IR) in 1. Vehicle-treated control mice; 2. Diabetic mice; 3. Diabetic mice

683

treated with adiponectin (50 µg/animal/day) for 2 weeks (DT_APN_ST); and 4. Diabetic mice

684

treated with adiponectin (50 µg/animal/day) for 4 weeks (DT_APN_LT). Values are represented

685

as the mean ± standard error of the mean (SEM). * and $ showed significant (P < .05) difference

686

in the level in the diabetic group as compared with the control group. $ and # showed significant

687

(P < .05) difference in the treated groups as compared with the diabetic group.

688

Figure 4.a, Histological analysis of the testis of control (A) vehicle-treated control mice; (B)

689

diabetic mice; (C) diabetic mice treated with adiponectin (50 µg/animal/day) for 2 weeks

690

(DT_APN_ST); and (D) diabetic mice treated with adiponectin (50 µg/animal/day) for 4 weeks

28 | P a g e

691

(DT_APN_LT) along with variation in the seminiferous tubule diameter and germinal

692

epithelium height. In the testes of control mice (Figure 4.b), seminiferous tubules (ST) were

693

found to be occupied with spermatogenic cells. Few spermatogenic cells like preleptotene

694

spermatocyte (Pl) and pachytene spermatocyte (P) at the basement side and round spermatids

695

(SR) and elongated spermatids (SE) at the luminal side of the tubule, were observed in

696

histological sections. Testicular sections of the diabetic mice (Figure 4B), showed loosening of

697

germinal epithelium (LGE) along with increased vacuoles formation in the seminiferous

698

tubules. Values are represented as the mean ± standard error of the mean (SEM). * and $

699

showed significant (P < .05) difference in the level in the control, diabetic, and APN-treated

700

diabetic group.

701

Figure 5. A. Immunohistochemical staining and proliferating cell nuclear antigen (PCNA) cell

702

counting in the testes of (a) vehicle-treated control mice; (b) HFD/STZ-induced type 2 diabetic

703

mice; (c) Diabetic mice treated with adiponectin (50 µg/animal/day) for 2 weeks

704

(DT_APN_ST); and (d) Diabetic mice treated with adiponectin (50 µg/animal/day) for 4 weeks

705

(DT_APN_LT). B. Immunoblot and densitometric analysis of PCNA, Bcl2, and caspase-3

706

proteins in the testis of (a) vehicle-treated control mice; (b) HFD/STZ-induced type 2 diabetic

707

mice; (c) Diabetic mice treated with adiponectin (50 µg/animal/day) for 2 weeks

708

(DT_APN_ST); and (d) Diabetic mice treated with adiponectin (50 µg/animal/day) for 4 weeks

709

(DT_APN_LT). Data were represented as the mean ± SEM. ($) showed significant (P < .05)

710

difference in the proteins level of PCNA, Bcl2, and caspase-3 in the diabetic group as compared

711

with the control. (*) showed significant (P< .05) difference in the level of PCNA, Bcl2, and

712

caspase-3 proteins in both the duration of adiponectin-treated diabetic groups when compared to

713

the diabetic group alone.

714

Figure 6. (A,B). Immunoblot and densitometric analysis of StAR and 3β-HSD proteins in the

715

testis of (a) vehicle-treated control mice; (b) HFD/STZ-induced type 2 diabetic mice; (c) 29 | P a g e

716

diabetic mice treated with adiponectin (50 µg/animal/day) for 2 weeks (DT_APN_ST); and (d)

717

diabetic mice treated with adiponectin (50 µg/animal/day) for 4 weeks (DT_APN_LT). Figure

718

6.C. Changes in the concentration of serum testosterone in (a) vehicle-treated control mice; (b)

719

HFD/STZ-induced type 2 diabetic mice; (c) diabetic mice treated with adiponectin (50

720

µg/animal/day) for 2 weeks (DT_APN_ST); and (d) diabetic mice treated with adiponectin (50

721

µg/animal/day) for 4 weeks (DT_APN_LT). Data were represented as the mean ± SEM. (*) and

722

($) showed significant (P < .05) difference in the proteins level of StAR, 3β-HSD, and

723

testosterone in diabetic group as compared with the control. ($) and (#) showed significant (P <

724

.05) difference in the level of StAR, 3β-HSD, and testosterone in both adiponectin-treated

725

diabetic groups when compared to the diabetic group alone.

726

Figure 7. (A) Western blot and densitometric analysis of adiponectin receptor 1 (AdipoR1),

727

Insulin receptor (IR), and glucose transporter 8 (GLUT8) proteins in the testis of (a) vehicle-

728

treated control mice; (b) HFD/STZ-induced type 2 diabetic mice; (c) diabetic mice treated with

729

adiponectin (50 µg/animal/day) for 2 weeks (DT_APN_ST); and (d) diabetic mice treated with

730

adiponectin (50 µg/animal/day) for 4 weeks (DT_APN_LT). Figure 7. (B-C) Intra-testicular

731

concentration of glucose, lactate, and lactate dehydrogenase (LDH) activity in (a) vehicle-

732

treated control mice; (b) HFD/STZ-induced type 2 diabetic mice; (c) diabetic mice treated with

733

adiponectin (50 µg/animal/day) for 2 weeks (DT_APN_ST); and (d) diabetic mice treated with

734

adiponectin (50 µg/animal/day) for 4 weeks (DT_APN_LT). Data were represented as the mean

735

± SEM. ($) showed significant (P < .05) difference in the proteins level of AdipoR1, IR,

736

GLUT8, testicular glucose, lactate, and LDH in diabetic group as compared with the control

737

group (*) and (#) showed significant (P < 0.05) difference in the level of AdipoR1, IR, GLUT8,

738

testicular glucose, lactate, and LDH in both the duration of adiponectin-treated diabetic groups

739

when compared to the diabetic group alone.

30 | P a g e

740

Figure 8. (A-D), Changes in the activity of testicular SOD, catalase, GPx, and TBARS in

741

vehicle control and T2D mice before and after adiponectin treatment for 2 weeks

742

(DT_APN_ST) and 4 weeks (DT_APN_LT) duration, respectively. Values are represented as

743

the mean ± SEM. (*) and ($) showed significant (P < 0.05) difference in the diabetic group as

744

compared to the control group. (*) and (#) showed significant (P < 0.05) difference in the

745

treated group when compared to the diabetic group.

31 | P a g e

Table 1. Details of the antibodies used for Immunohistochemistry and Immunoblotting experiments.

S.No.

Antibody

Species raised in; Monoclonal/Polyclonal

Source

1

Adiponectin

Rabbit; Polyclonal

Sigma-Aldrich Co. LLC., USA (A6354) Sigma-Aldrich Co. LLC., USA (A6354) Santa Cruz Biotechnology Inc., CA, USA (N-20, SC 99183) Thermo Fisher Scientific Inc. (PA1-1071) Santa Cruz Biotechnology Inc., CA, USA (C-19, SC 711) Santa Cruz (Biotechnology Inc., CA, USA) (H-60, SC-30108) Thermo Fisher Scientific Inc. (PA1-38424) Thermo Fisher Scientific Inc. (PA1-38424) Santa Cruz Biotechnology Inc., CA, USA (N-20, SC492) Santa Cruz Biotechnology Inc., CA, USA (E-8, SC-7272) Santa Cruz Biotechnology Inc., CA, USA (N-20, SC-25806) Santa Cruz Biotechnology Inc., CA, USA (N-20, SC-28206) Sigma-Aldrich Co. LLC., USA (A2228)

2

AdipoR1

Rabbit; Polyclonal

3

AdipoR2

Rabbit; Polyclonal

4

IR

Rabbit; Polyclonal

5

GLUT8

Rabbit; Polyclonal

6

PCNA

Rabbit; Polyclonal

7

Bcl2

Rabbit; Polyclonal

8

Caspase-3

Rabbit; Polyclonal

9

StAR

Rabbit; Polyclonal

10

3β-HSD

Rabbit; Polyclonal

11

β-actin

Rabbit; Monoclonal HRP-tagged

Concentration (used for Western blot) 1:500 1:50 (IHC) 1:300 1:50 (IHC) 1:100 1:25 (IHC) 1:1000 1:500 1:1600 1:200 (IHC) 1:1000 1:250 1:1600 1:500 1:100000

Table 2. Correlation studies of control, T2D, and adiponectin treated T2D mice between the different serum and testicular metabolic, spermatogenic, and antioxidative parameters. * denotes data were correlated significantly; Values are significantly different at p < 0.05. NS, not significant.

Testicular AdipoR1

Testicular IR

Testicular GLUT8

Intratesticular glucose

Testicular LDH activity

Intratesticular lactate

SOD

Catalase

GPx

Testicular AdipoR1

-

r = 0.74*

r = 0.97*

r = 0.87*

r = 0.82*

r = 0.94*

r = 0.81*

r = 0.72*

r = 0.74*

Testicular IR

-

-

r = 0.74*

r = 0.75*

r = 0.92*

r = 0.74*

r = 0.90*

r = 0.94*

r = 0.90*

Testicular GLUT8

-

-

-

r = 0.87*

r = 0.82*

r = 0.97*

r = 0.85*

r = 0.70*

r = 0.75*

Intra-testicular glucose

-

-

-

-

r = 0.84*

r = 0.91*

r = 0.76*

r = 0.66*

r = 0.62*

Testicular LDH activity

-

-

-

-

-

r = 0.87*

r = 0.85*

r = 0.82*

r = 0.76*

Intra-testicular lactate

-

-

-

-

-

-

r = 0.85*

r = 0.70*

r = 0.70*

PCNA

r = 0.692*

r = 0.66*

r = 0.705*

r = 0.52*

NS

r = 0.571*

r = 0.76*

r = 0.75*

r = 0.85*

Bcl2

NS

r = 0.86*

NS

NS

r = 0.65*

NS

r = 0.84*

r = 0.93*

r = 0.93*

Caspase-3

r = -0.78*

r = -0.92*

r = -0.78*

r = -0.84*

r = -0.99*

r = -0.84*

r = -0.83*

r = -0.80*

r = -0.74*

Serum testosterone

r = 0.61*

NS

r = 0.61*

r = 0.60*

NS

r = 0.52*

NS

NS

NS

Serum glucose

r = -0.90*

r = -0.60*

r = -0.91*

r = -0.71*

r = -0.61*

r = -0.83*

r = -0.80*

r = -0.69*

r = -0.77*

Serum insulin

r = -0.86*

r = -0.83*

r = -0.87*

r = -0.70*

r = -0.75*

r = -0.80*

r = -0.94*

r = -0.90*

r = -0.96*

Highlights •

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) proteins were significantly declined in the testis of HFD/STZ-induced T2D mice.

•

Exogenous administration of adiponectin to T2D mice ameliorates testicular dysfunctions.

•

Treatment of adiponectin to T2D mice improves testicular activity by increasing expression of insulin receptor-mediated increased transport of energy substrate (glucose and lactate) and a marked reduction in oxidative stress.