PPARÎ³ Axis Mediates Insulin-Induced

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The mTORC1/4EBP1/PPAR# axis mediates insulin-induced lipogenesis by regulating lipogenic gene expression in bovine mammary epithelial cells Zhixin Guo, Xiaoou Cheng, Xue Feng, Keyu Zhao, Meng Zhang, Ruiyuan Yao, Yuhao Chen, Yanfeng Wang, Huifang Hao, and zhigang wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01411 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Agricultural and Food Chemistry

The mTORC1/4EBP1/PPARγ axis mediates insulin-induced lipogenesis by regulating lipogenic gene expression in bovine mammary epithelial cells

Zhixin Guo†#, Xiaoou Cheng†#, Xue Feng†, Keyu Zhao†, Meng Zhang†, Ruiyuan Yao†, Yuhao Chen†,‡, Yanfeng Wang†, Huifang Hao†*, Zhigang Wang†*

†

State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, 010021, China

‡

School of Life Sciences, Jining Normal University, Jining 012000, China

#Zhixin

Guo and Xiaoou Cheng contributed equally to this paper.

*Corresponding

author: Professor Zhigang Wang, Ph D Associate professor Huifang Hao, Ph D

School of Life Science, Inner Mongolia University, State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock No.235 Da Xue West Road, Hohhot 010021, Inner Mongolia, P.R. China Email: [email protected] (Zhigang Wang) or [email protected] (Huifang Hao) Telephone: +86-471-4992435

FAX: +86-471-4992435

1

ACS Paragon Plus Environment


1

ABSTRACT: 4EBP1 is a chief downstream factor of mTORC1, and PPARγ is a key

2

lipogenesis-related transcription factor. mTORC1 and PPARγ are associated with

3

lipid metabolism. However, it is unknown which effector protein connects mTORC1

4

and PPARγ. This study investigated the interaction between 4EBP1 with PPARγ as

5

part of the underlying mechanism by which insulin-induced lipid synthesis and

6

secretion are regulated by mTORC1 in primary bovine mammary epithelial cells

7

(pBMECs). Rapamycin, a specific inhibitor of mTORC1, downregulated 4EBP1

8

phosphorylation and the expression of PPARγ and the following lipogenic genes: lipin

9

1, DGAT1, ACC, and FAS. Rapamycin also decreased the levels of intracellular

10

triacylglycerol (TAG); 10 types of fatty acid; and the accumulation of TAG, palmitic

11

acid (PA), and stearic acid (SA) in the cell culture medium. Inactivation of mTORC1

12

by shRaptor or shRheb attenuated the synthesis and secretion of TAG, and PA. In

13

contrast, activation of mTORC1 by Rheb overexpression promoted 4EBP1

14

phosphorylation and PPARγ expression and upregulated the mRNA and protein levels

15

of lipin 1, DGAT1, ACC, and FAS, whereas the levels of intracellular and extracellular

16

TAG, PA, and SA also rose. Further, 4EBP1 interacted directly with PPARγ.

17

Inactivation of mTORC1 by shRaptor prevented the nuclear location of PPARγ.

18

These results demonstrate that mTORC1 regulates lipid synthesis and secretion by

19

inducing the expression of lipin 1, DGAT1, ACC, and FAS, which is likely mediated

20

by the 4EBP1/PPARγ axis. This finding constitutes a novel mechanism by which lipid

21

synthesis and secretion are regulated in pBMECs.

22

Keywords: mTORC1, 4EBP1, PPARγ, lipogenesis, insulin, bovine mammary 2


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epithelial cells

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 3



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■ Introduction

46

Cell metabolism is stimulated by growth signals. Mechanistic (formerly mammalian)

47

target of rapamycin (mTOR) complex 1 (mTORC1) is a growth signal-sensitive

48

multiprotein complex that integrates nutritional signals, growth factors, and energy

49

status to serve as the master regulator of cell growth and metabolism.1,2 mTORC1 is

50

sufficient to induce specific metabolic processes, including the accumulation of

51

triglycerides and de novo fatty acid biosynthesis.3-5 Raptor is unique to mTORC1 and

52

positively regulates mTORC1.6 Rheb is an upstream regulator of mTORC1 and

53

stimulates mTORC1 to govern metabolism in response to growth factors via 2

54

downstream effectors, S6K1 and 4EBP1.2,7

55

mTORC1 directs lipid synthesis through various effectors, including peroxisome

56

proliferator-activated receptors (PPARs) and sterol regulatory element-binding

57

proteins (SREBPs), which are 2 types of transcription factors that promote

58

adipogenesis and de novo FA synthesis.3,6,8 The PPARγ gene network controls the

59

expression of enzymes that are associated with milk fat synthesis in lactating

60

ruminants.3,9 The mTORC1/PPARγ pathway is crucial for fatty acid uptake and

61

synthesis in several types of cells10,11 and in ruminants and nonruminants.12-14

62

mTORC1 signaling regulates the activation of PPARs and, in turn, lipogenesis.

63

Although there are findings on the relationship between 4EBP1 and PPARγ, 15-18 few

64

studies have examined the interaction between 4EBP1 and PPARγ.

65

Primary bovine mammary epithelial cells (pBMECs) have been used widely as an

66

in vitro cellular model to study the synthesis of milk fat in the udder of dairy 4


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cows.19-21 Milk fat is the most variable component of milk—95% to 98% of which is

68

triacylglycerol (TAG).22,23 mTORC1 is involved in milk fat synthesis in cow 24,25 and

69

goat

70

expression in bovine24,28,29 and goat26,30,31 mammary epithelial cells, and such target

71

genes’ products as DGAT1, lipin1, ACC and FAS are the key rate-limiting enzymes in

72

lipogenesis. Although mTORC1 and PPARγ participate in lipogenic gene expression,

73

the mediator by which mTORC1 associates with PPARγ to regulate gene expression

74

is unknown.

26,27

mammary epithelial cells. PPARγ is essential to the lipogenic gene

75

To examine the role and mechanism of mTORC1/4EBP1/PPARγ axis in lipid

76

synthesis and secretion in pBMECs, we focused on the interaction of 4EBP1 with

77

PPARγ, the function of mTORC1 in PPARγ expression, lipogenic gene expression,

78

and lipid synthesis and secretion in pBMECs. The results of this study provide

79

insights into the precise mechanism by which lipid synthesis and secretion are

80

regulated in pBMECs.

81

■ MATERIALS AND METHODS

82

Ethics Statement. All experimental procedures with animals were conducted

83

according to the guidelines for the care and use of experimental animals that have

84

been established by the Inner Mongolia University Animal Care and Use Committee.

85

Primary BMEC cultures. Mammary tissues were obtained from Chinese Holstein

86

cows after being slaughtered on a commercial cattle slaughter farm. After the surgical

87

removal of mammary tissue from the slaughtered cow, it was placed in sterile,

88

ice-cold phosphate-buffered saline (PBS) that was supplemented with 300 U/mL 5



89

penicillin G and 100 mg/mL streptomycin (Sigma-Aldrich, Inc., USA) and

90

transported immediately to the laboratory. Mammary tissue was trimmed of visible fat

91

and connective tissue and washed with PBS several times until the solution turned

92

pellucid and was devoid of milk. Then, mammary tissue was cut into small pieces (1

93

× 1 × 1 mm3), primary cell cultures were established, and BMECs were isolated from

94

the primary cultures. Purified primary BMECs were cultured and maintained in

95

DMEM/F12 medium (Hyclone Laboratories, Inc., Logan, UT, USA) that contained

96

10% fetal bovine serum. Cells were cultured in 25 cm2 tissue culture flasks at 37°C in

97

humidified air with 5% CO2 as described.32,33 Morphology was examined by light

98

microscopy. Primary BMECs (pBMECs) that were in the logarithmic growth phase

99

were used in the experimental assays.

100

Reagents and antibodies. Rapamycin (Gene Operation, Ann Arbor, MI, USA) was

101

dissolved in ethanol (Sigma-Aldrich, Inc., USA) to a stock concentration of 50 mg/ml,

102

stored at -20°C, and diluted to the appropriate final concentration with culture

103

medium before use. The concentration of ethanol in the final solution did not exceed

104

0.5% (v/v) in any experiment.

105

The following primary antibodies were purchased and used: anti-Raptor, anti-ACC,

106

anti-p-4EBP1 (Thr37/46) (Cell Signaling Technology, Inc., Beverley, MA, USA),

107

anti-Rheb (Santa Cruz Biotechnology, Inc., CA, USA), anti-4EBP1, anti-PPARγ,

108

anti-lipin 1, anti-p-mTOR (Ser2448), anti-mTOR, anti-DGAT1, anti-FAS (Abcam,

109

Cambridge, UK), goat anti-rabbit IgG antibody labeled with FITC (Jackson

110

ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and anti-β-actin 6


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111

(Sigma-Aldrich, Inc., St. Louis, MO, USA). ECL anti-rabbit IgG-HRP and ECL

112

Anti-Mouse IgG-HRP were obtained from GE Healthcare (Buckinghamshire, UK).

113

ELISA. pBMECs were seeded into 6-well plates, incubated until 80% confluence,

114

and treated with the indicated conditions, including serum starvation followed by

115

insulin

116

pRNAT-U6.1/Neo-shRaptor

117

pIRES2-DsRed2-Rheb. For the Raptor or Rheb knocked down group, the same

118

number of cells was seeded in the plate after the transfected cells were selected with

119

G418 for 48 h to guarantee the equal cell numbers. Cell culture supernatants were

120

collected for measurement of extracellular triacylglycerol (TAG), palmitic acid (PA),

121

and stearic acid (SA) using ELISA kits (Beijing Winter Song Boye Biotechnology Co.

122

Ltd., Beijing, China) according to the manufacturer’s instructions. Cells were

123

harvested with trypsin and centrifuged to remove the supernatant, and cell lysates

124

were prepared through 5 freeze-thaw cycles. The protein concentration of the control

125

and treatment groups was standardized by adjusting the volume of the protein lysate.

126

An equal volume of each protein lysate was analyzed for TAG, PA, and SA by ELISA.

127

Absorbance at 450 nm and 630 nm was read on a Varioskan Flash Multimode Reader

128

(Thermo Fisher Scientific, Pittsburgh, PA, USA). All measurements were performed

129

in triplicate, and the mean value of the 3 independent measurements was used for

130

statistical analysis.

stimulation,

rapamycin

treatment,

or

and

transfection

pRNAT-U6.1/Neo-shRheb

with and

131

Western blot analysis. Western blot was used to detect the expression of indicated

132

proteins and phosphorylated proteins as previously described.34 Briefly, pBMECs 7



133

were harvested with trypsin, washed with cold PBS, and lysed in cell lysis buffer. The

134

component of lysis buffer include 50 mM Tris(pH 7.4) ，150 mM NaCl ，1% Triton

135

X-100 ，1% sodium deoxycholate ，0.1% SDS ，PMSF and phosphatase inhibitors.

136

Equal amounts (40 μg) of protein were electrophoresed on 10% (w/v) sodium dodecyl

137

sulfate-polyacrylamide gels, transferred to polyvinylidene ﬂuoride membranes, and

138

incubated with the primary antibody. Peroxidase-conjugated secondary antibody and

139

enhanced chemiluminescence (ECL) reagent were used to detect the signals with the

140

Western Blotting System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The

141

bands were quantified on a Gel-Pro Analyzer 4.0 (Media Cybernetics, USA).

142

Gas chromatography and mass spectroscopy. Control and rapamycin-treated

143

cells were collected and dissolved in 1 mL of lysis buffer. Fatty acid methyl esters

144

(FAME) were extracted twice with n-hexane at room temperature and evaporated to

145

dryness at 30 °C for 30 min then dissolved in n-hexane, and then separated in a gas

146

chromatography-mass spectrum (Shimadzu, GCMS-QP2010 ultra, Shimadzu, Japan)

147

using an Agilent HP-88 capillary-column (100 m × 0.25 mm × 0.20 μm, Agilent

148

Technologies, Santa Clara, CA, USA). The program was set to column temperature

149

60 °C for 1 min, with ramping of 40 °C/min up to 140 °C, and a hold for 10 min, 4

150

°C/min up to 240 °C, and a hold for 15 min. The injector temperature was 220 °C, and

151

the sample was 1 µL. The injection mode was split flow. External standards were

152

obtained from Sigma-Aldrich (Cat. No.18919-1AMP).

153

DNA constructs and in vitro transfection. Short hairpin (shRNA) Raptor 8


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RNA-silencing constructs (shRaptor) were designed and synthesized with the

155

sequence

156

5′-aaGAACTACACGCAGTACATCTTCAAGAGAGATGTACTGCGTGTAGTTCtt

157

-3′ to construct pRNAT-U6.1/Neo-shRaptor. shRNA Rheb RNA-silencing constructs

158

(shRheb)

159

5′-aaCGCGTTAGCAGAGTCTTGGATTCAAGAGATCCAAGACTCTGCTAACG

160

Ctt-3′. The DNA fragment that encoded shRheb was inserted into the

161

pRNAT-U6.1/Neo vector to generate pRNAT-U6.1/Neo-shRheb. Rheb cDNA was

162

PCR-amplified with forward (5'- ATGCCGCAGTCCAAGTCC-3') and reverse (5'-

163

TCACATCACCGAGCAGGAAG-3') primers, which were designed, based on the

164

Bos taurus Rheb sequence (GenBank Accession number NM 001031764.2). The Rheb

165

ORF PCR fragment was inserted into pIRES2-DsRed2 (Clontech Laboratories, Inc.,

166

Catalog No. 632420, CA, USA) to construct pIRES2-DsRed2-Rheb.

167

The

were

plasmids

designed

and

synthesized

pRNAT-U6.1/Neo-shRaptor,

with

the

following

sequence:

pRNAT-U6.1/Neo-shRheb,

and

168

pIRES2-DsRed2-Rheb were transfected into pBMECs using Lipofectamine TM2000

169

(Invitrogen, Carlsbad, New Mexico, USA) per the manufacturer’s instructions.

170

Transfectants were selected with G418 (Hyclone Laboratories, Inc. Logan, UT, USA)

171

for 48 h and imaged under a ZEISS AX10 fluorescence microscope (Carl Zeiss

172

Microscopy, LLC One Zeiss Drive, Thornwood, NY 10594 USA).

173 174

qPCR analysis. Quantitative real-time PCR (qPCR) was performed to determine

175

the abundance of PPARγ, DGAT1, Lipin1, ACC, and FAS mRNA in pBMECs from 9



176

the treatment and control groups. Cells were treated with 100 nM rapamycin for 8 h,

177

and total RNA was extracted from untreated and treated cells. To determine the levels

178

of Raptor and Rheb mRNA in transfectants and control cells, total RNA was extracted

179

from transfectants after selection with G418 for 48 h and control cells. The RNA

180

preparation method: Total RNA was prepared by RNAiso Plus reagent according the

181

manufacturer's instructions (9109, TaKaRa Co. Ltd., Dalian, China). Briefly, the cells

182

were washed with PBS and lysed by RNAiso Plus, and then the chloroform was

183

added to the cell lysates for homogenization, and the top liquid layer was transferred

184

to a new tube after centrifugation, and the isopropanol was added to the supernatant

185

and mixed well. Total RNA was precipitated by centrifugation and the pellet was

186

dissolved in RNase-free water.

187

mRNA was reverse-transcribed with oligo (dT)12–18 primer using the AMV 1st

188

Strand cDNA Synthesis Kit (Takara Co. Ltd., China). cDNA sequences were

189

amplified with the primers in Table S1. The KAPA SYBP® FAST qPCR Kit

190

Optimized for LightCycler® 480 (KAPA BIOSYSTEMS, Inc., Boston, MA, USA)

191

was used for the PCR, according to the manufacturer’s instructions. The program

192

comprised an initial denaturation step at 95°C for 5 min; 40 cycles of 95°C for 5 s,

193

54°C for 30 s, and 72°C for 20 s; and a final extension of 72°C for 10 min. Three

194

technical replicates were run in each experiment. 2-ΔΔCT values were calculated to

195

determine expression levels, and the qPCR results were analyzed by Student’s t-test to

196

compare the expression between untreated and treated groups. Three independent

197

experiments were performed. 10


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198


Coimmunoprecipitation

and

yeast

two-hybrid

screen.

For

the

199

coimmunoprecipitation, cells were collected, washed 3 times with cold PBS, and

200

dissolved in cell lysis buffer. Then, equivalent amounts of protein lysate were

201

incubated with anti-4EBP1 (1:50) or anti-His (1:50) (negative control) and inactive

202

resin (negative control) in Millipore catch-and-release spin columns. Equivalent

203

amounts of protein lysate were also evaluated by western blot as a positive control for

204

4EBP1 and PPARγ, per reported methods.35,36

205

A yeast two-hybrid screen was performed using the YeastmakerTM Yeast

206

Transformation System 2 and MatchmakerTM Gold Yeast Two-Hybrid System

207

(Clontech Laboratories, Inc., Mountain View, CA) per the manufacturer’s

208

instructions. Reagents for the synthetically defined plate and prototroph and

209

colorimetric screening were obtained from Clontech. The Y2HGold and Y187 yeast

210

strains were transformed with the plasmids pGBKT7-4EBP1 (expressing the bait

211

protein, GAL4BD-4EBP1) and pGADT7-PPARγ (expressing the prey protein,

212

GAL4AD-PPARγ), respectively. The transformed yeast strains that harbored the

213

plasmids

214

SD/-Leu/-Trp/X-a-Gal/AbA (DDO/X/A) plate, and the resulting blue colonies were

215

transferred to an SD/-Leu/-Trp/-His/X-α-Gal/AbA (TDO/X/A) plate and then to an

216

SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA (QDO/X/A) plate, followed by incubation for

217

3–6 days at 30°C. The yeast two-hybrid screen was performed as described.35,36

were

mated.

Diploid

cells

were

then

screened

on

an

218

Immunofluorescence staining. The cells were seeded on to the slide and incubated

219

overnight, then the cells were washed with PBS and fixed with the 4% 11



220

paraformaldehyde for 15 min. After treating with Triton X-100 for 10 min, the cells

221

were blocked with 1% BSA for 1 h. Then cells were incubated with primary antibody

222

for PPARγ at 4 ℃ for overnight, followed by incubation with a goat anti-rabbit IgG

223

antibody labeled with FITC for 1 h at room temperature. The DAPI was used to stain

224

the nucleus. Finally the slide was mounted with glycerinum and examined under laser

225

scanning confocal microscope (NIKON A1R, Nikon Corp., Tokyo, Japan).

226

Statistical Analyses. Statistical analyses were conducted using SPSS PASW

227

Statistics for Windows, v18.0 (SPSS Inc.: Chicago, IL, USA). Normally distributed

228

data were analyzed using standard parametric statistics and one-way ANOVA,

229

followed by Tukey’s method. Data are expressed as mean ± SD. The results are

230

presented as the average of at least 3 independent experiments. Western blot results

231

were quantified on a Gel-Pro Analyzer 4.0 (Media Cybernetics, USA). Statistical

232

significance was accepted when p≤ 0.05.

233 234

■ RESULTS

235

Insulin induces lipid synthesis and secretion and the expression of PPARγ and

236

lipogenic enzymes in pBMECs. To examine the insulin-induced synthesis and

237

secretion of TAG and FAs in pBMECs, the cells were treated with serum starvation

238

for 16 h, followed by insulin (5 μg/ml) for 12 h, and then, the intracellular and culture

239

medium concentrations of TAG, PA, and SA were determined by ELISA. The

240

activation of mTORC1 signaling and the expression of PPARγ, lipin 1, DGAT1, ACC,

241

and FAS were determined by western blot. 12


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The intracellular levels of TAG, PA, and SA decreased after serum starvation and

243

then rose after treatment with insulin (Figure 1A, B, C) (p＜0.05); the levels of TAG,

244

PA, and SA in the culture medium had the same trend (Figure 1D, E, F) (p ＜0.05).

245

These results indicate that insulin induces the synthesis and secretion of lipids after

246

serum starvation in pBMECs.

247

The phosphorylation of mTOR and 4EBP1 (Figure 2A) and the expression of

248

PPARγ, lipin 1, DGAT1, ACC, and FAS were inhibited by serum starvation and

249

activated by followed insulin stimulation (Figure 2B). These results suggest that

250

insulin-induced lipid synthesis is associated with mTORC1 signaling and PPARγ

251

expression and that lipin 1, DGAT1, ACC, and FAS are upregulated by insulin,

252

favoring TAG and FA synthesis and secretion.

253 254

Inactivation of mTORC1 attenuates lipid synthesis and secretion and

255

downregulates PPARγ and lipogenic genes in pBMECs. To validate the underlying

256

mechanism of lipid synthesis in pBMECs, we first treated pBMECs with rapamycin

257

(a specific inhibitor of mTORC1) at 100 nM for 8 h and measured the levels of

258

intracellular TAG by ELISA. Rapamycin decreased the intracellular levels of TAG

259

(Figure 3A) (p ＜ 0.01). Further, a total of 17 types of intracellular fatty acid were

260

assayed by GC-MS. The results showed that 10 types of FAs were decreased,

261

including PA (p＜0.01) and SA (p＜0.05) , while other 7 types were increased, and

262

total content of intracellular fatty acids declined in rapamycin treated cells (p＜0.01)

263

(Table 1). We also determined the levels of TAG, PA, and SA in the culture medium 13



264

by ELISA, which showed that all 3 declined significantly in response to rapamycin

265

(Figure 3B, C, D) (p ＜ 0.01). These data demonstrate that rapamycin inhibits lipid

266

synthesis and secretion in pBMECs.

267

Further, we examined mTORC1 activation by western blot, wherein mTORC1

268

signaling was inhibited by rapamycin (Figure 4A). Based on these results, we

269

hypothesized that the expression of PPARγ and lipogenic genes might be inhibited by

270

rapamycin. We examined the expression of PPARγ, lipin 1, DGAT1, ACC, and FAS

271

by RT-qPCR and western blot. We found that rapamycin inhibited mRNA and protein

272

levels of PPARγ (Figure 4B, C) and lipogenic genes' expression (lipin 1, DGAT1,

273

ACC, and FAS) (Figure 4D, E). These data suggest that mTORC1 signaling is

274

involved in lipid synthesis by regulating the expression of the transcription factor

275

PPARγ and lipogenic genes.

276

Next, to complement the data from rapamycin-treated cells, Raptor or Rheb was

277

knocked down by using targeting shRNA to inactivate mTORC1 (Figure S1A, B, C,

278

S2A, B, C). The effects of Raptor or Rheb silencing on extracellular and intracellular

279

TAG and PA were measured by ELISA. Silencing of Raptor or Rheb decreased the

280

levels of TAG and PA in cell lysates (Figure 5A, B, C, D) (p＜0.05) and cell culture

281

medium (Figure 5E, F, G, H) (p＜0.05), indicating that the synthesis and secretion of

282

TAG and PA are regulated by mTORC1 in pBMECs. Further, the mRNA levels of

283

PPARγ, lipin1, DGAT1, ACC and FAS were examined by RT-qPCR. mTORC1

284

activation and the expression of PPARγ and lipogenic enzymes were examined by

285

western blot. The results showed that mRNA level of the lipogenic genes was 14


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286

decreased by Raptor (Figure S1D) or Rheb silencing (Figure S2D), and the

287

phosphorylation of mTOR and 4EBP1 and the expression of PPARγ, lipin 1, DGAT1,

288

ACC, and FAS were reduced by Raptor (Figure 5I) or Rheb silencing (Figure 5J),

289

indicating that inhibition of PPARγ and lipogenic enzyme expression is due to the

290

inactivation of mTORC1. These results suggest that mTORC1 signaling governs lipid

291

secretion by controlling intracellular lipid synthesis.

292 293

Activation of mTORC1 promotes lipid synthesis and secretion and the

294

expression of PPARγ and lipogenic genes in pBMECs. To confirm that mTORC1

295

regulates the synthesis and secretion of triacylglycerol and fatty acids, mTORC1

296

activation was enhanced in pBMECs by overexpression of Rheb (Figure S3). The

297

levels of intracellular and extracellular TAG, PA, and SA were measured by ELISA,

298

and the phosphorylation of mTOR and 4EBP1 and the expression of PPARγ, lipin 1,

299

DGAT1, ACC, and FAS were measured by western blot and RT-qPCR.

300

Intracellular and extracellular levels of 3 types of lipid increased significantly due

301

to Rheb overexpression (Figure 6A, B, C, D, E, F) (p＜0.05). The phosphorylation of

302

mTOR and 4EBP1 were increased (Figure 7A), indicating that mTORC1 signaling

303

was activated by Rheb overexpression. PPARγ mRNA and protein levels were also

304

increased (Figure 7B, C), suggesting that PPARγ expression is associated with Rheb

305

overexpression or activated mTORC1 signaling. Moreover, the mRNA and protein

306

levels of lipogenic genes (lipin 1, DGAT1, ACC, and FAS) were upregulated as a

307

result of Rheb overexpression (Figure 7D, E). These data show that the expression of 15



308

lipogenic genes is regulated by mTORC1 signaling, a process that likely occurs

309

through PPARγ, and suggest that activated mTORC1 promotes the expression of

310

PPARγ and lipogenic genes and lipid synthesis and secretion in pBMECs.

311 312

4EBP1 interacts directly with PPARγ and mTORC1 inactivation prevents the

313

nuclear localization of PPARγ in pBMECs. Based on our western blot results,

314

activated mTORC1, which is stimulated by insulin or Rheb overexpression, increased

315

the phosphorylation of 4EBP1 and the levels of PPARγ and lipogenic enzymes

316

(Figure 2; Figure 7A, C, E), in contrast to inactive mTORC1 (Figure 4; Figure 5I, 5J).

317

These trends were consistent with lipid synthesis (Figure 1A, B, C; Figure 3A, Table

318

1; Figure 5A, B, C, D; Figure 6A, B, C) and secretion (Figure 1D, E, F; Figure 3B, C,

319

D; Figure 5E, F, G, H; Figure 6D, E, F). Thus, we reasoned that mTORC1 regulates

320

PPARγ and lipogenic gene expression through 4EBP1.

321

To test this hypothesis, coimmunoprecipitation (co-IP) assay was performed. The

322

4EBP1/PPARγ protein complex was detected by co-IP, whereas it was not detected in

323

the 2 negative control groups (Figure 8A), suggesting that 4EBP1 interacts directly or

324

indirectly with PPARγ in pBMECs. Next, a yeast two-hybrid screen was performed to

325

verify the interaction of 4EBP1 and PPARγ. The results showed that diploid cells of

326

mating yeast grew on the DDO plate (Figure 8B), and colonies were observed on

327

DDO/X/A (Figure 8C), TDO/X/A (Figure 8D), and QDO/X/A (Figure 8E) plates,

328

indicating that 4EBP1 associates directly with PPARγ.

329

The above data indicated that lipogenic genes expression regulated by 16


Page 16 of 45

Page 17 of 45


330

mTORC1/4EBP1/PPARγ axis, and 4EBP1 direct interacts with PPARγ, therefore, we

331

reasoned that mTORC1 activation affects the nuclear localization of PPARγ. To this

332

end, the immunofluorescence staining was used to confirm whether the inactivation of

333

mTORC1 could prevent the nuclear localization of PPARγ. The staining results

334

showed the nuclear localization of PPARγ was prevented in Raptor silencing cells,

335

compared with control (Figure 8F). These results indicate that mTORC1/4EBP1

336

pathway might regulate the nuclear localization of PPARγ to promote the lipogenesis.

337

It is well known that PPARγ regulates expression of target genes by connecting to

338

PPARγ response elements (PPREs) of target genes in nucleus. Based on these data,

339

we conclude that mTORC1 regulates insulin-induced lipogenic gene expression to

340

control lipid synthesis and secretion through the 4EBP1/PPARγ axis in pBMECs

341

(Figure 8G).

342

■ DISCUSSION

343

Over the past 2 decades, mTORC1 has been demonstrated to promote de novo lipid

344

synthesis through lipogenesis-related transcription factors, and 4EBP1 has been

345

shown to be associated with fat synthesis.16,37. PPARγ is a positive regulator of lipid

346

biosynthesis and governs the mRNA and protein levels of genes that participate in de

347

novo lipogenesis, resulting in the increased accumulation of fat.38,39 Li et al. (2014)

348

used an antibody against Raptor to precipitate associated proteins, which showed that

349

PPARγ interacts with Raptor,40 indicating that 4EBP1 coexists with PPARγ in a

350

protein complex. In the present study, we used coimmunoprecipitation and yeast

351

two-hybrid assay to show that 4EBP1 interacts directly with PPARγ and that the 17



352

mTORC1/4EBP1/PPARγ axis mediates insulin-induced lipogenesis in pBMECs.

353

Insulin stimulates lipogenesis in several cell types and tissues in vitro and in

354

vivo,41-43 and lipogenesis occurs in parallel with the activation of lipogenesis-related

355

transcription factors in response to insulin.41,43 In our study, the intracellular and

356

extracellular levels of TAG, PA, and SA were decreased as a result of serum

357

starvation, increased following treatment with insulin, which was accompanied by

358

fluctuations in mTORC1 activation and PPARγ and lipogenic gene expression in

359

pBMECs. In our previous study, docosahexaenoic acid (DHA) secretion was inhibited

360

by rapamycin in pBMECs,32 and another group found that mTOR signaling pathway

361

and PPARγ are involved in stearic acid-induced triglyceride secretion by dairy cow

362

mammary epithelial cells.22,44 In nonruminants, mTOR signaling mediates rotator cuff

363

fatty infiltration via PPARγ in rats45 and stimulates phosphatidylcholine synthesis to

364

promote triglyceride secretion in mice.46 In the present study, mTORC1 activation

365

was induced or impaired—for example, on stimulation with insulin, overexpression or

366

silencing by Rheb, silencing by Raptor, and administration of rapamycin. We found

367

that mTORC1 governs insulin-induced lipid synthesis and secretion of TAG, PA, and

368

SA. These data show that the mTORC1/4EBP1/PPARγ axis is critical in lipid

369

secretion in pBMECs.

370

In our study, mTORC1 activation-induced lipid synthesis and secretion was caused

371

by increased lipin 1, DGAT1, ACC, and FAS expression, and we have not checked the

372

changes of fatty acid oxidation yet after activation or inactivation of mTORC1 in

373

pBMECs. In fact, decreased mTORC1 in turn mediates an elevation of fatty acid 18


Page 18 of 45

Page 19 of 45


374

oxidation in mice,47 and it is probably that mTORC1-induced lipid accumulation is

375

caused by the co-operation of increased lipogenesis and suppressed fatty acid

376

oxidation.

377

Epithelial cells are the central component of mammary alveoli, which produce milk

378

during lactation. A lactating ruminant mammary cell model is useful for the study of

379

milk synthesis. In recent years, significant results have been obtained from bovine and

380

goat mammary epithelial cells, including the function of mTORC1 in cell

381

proliferation, milk synthesis, and secretion;48-50 potential novel functions of lysosomal

382

membrane proteins in lactation;51 the function of lipogenesis-related transcription

383

factors and lipogenic genes in lipid biosynthesis;52-54 and the effects of exogenous

384

conjugated linoleic acid and pathogenic or fungal toxins on lipogenic gene expression

385

and milk synthesis and secretion.55-57 4EBP1

interacts

387

mTORC1/4EBP1/PPARγ

axis—no

388

4EBP1/PPARγ in ruminant or nonruminant cells. Based on our study and other

389

reports, we propose a model in which the mTORC1 pathway mediates insulin

390

signaling to regulate lipid synthesis and secretion in bovine mammary epithelial cells.

391

Insulin associates with membrane receptors and then transmits its signal to mTORC1.

392

Activated mTORC1 then phosphorylates 4EBP1 and activates PPARγ, after which

393

PPARγ translocates into the nucleus and upregulates lipogenic gene expression, which

394

enhances lipid synthesis and secretion (Figure 8G). Although we have no direct

395

evidence to prove whether 4EBP1 and PPARγ bind in cytoplasm or in the nucleus in

386

Nevertheless,

directly such

with

findings

19


PPARγ have

been

to

form

the

reported

for


396

the present study, immunofluorescence assay demonstrated that inactivation of

397

mTORC1 prevented the nuclear localization of PPARγ. Further, it is well known that

398

4EBP1 mainly functions as a binding protein in cytoplasm, thus, 4EBP1 bind

399

cytosolic PPARγ is possible in pBMECs.

400

In summary, this study has provided evidence that 4EBP1 interacts directly with

401

PPARγ to form the mTORC1/4EBP1/PPARγ axis and that this axis is critical in

402

insulin-induced lipid synthesis and secretion in pBMECs. Inactivation of mTORC1

403

downregulates 4EBP1 phosphorylation and the expression of PPARγ and lipogenic

404

genes, including lipin 1, DGAT1, ACC, and FAS, and attenuates intracellular TAG and

405

FAs synthesis and the accumulation of TAG, PA, and SA in cell culture medium. In

406

contrast, activated mTORC1 stimulates the expression of PPARγ, lipin 1, DGAT1,

407

ACC, and FAS through 4EBP1 to promote lipid synthesis and secretion in pBMECs.

408

This study implicates a regulatory mechanism of mTORC1 that is related to lipid

409

synthesis and secretion via the 4EBP1/PPARγ axis in cells.

410 411

Abbreviations Used:

412

mTOR, mechanistic target of rapamycin; mTORC1, mTOR complex 1; Rheb, Ras

413

homolog enriched in brain; TAG, triacylglycerol; PA, palmitic acid; SA, stearic acid;

414

FAs, fatty acids; pBMECs, primary bovine mammary epithelial cells; PPAR,

415

peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding

416

protein; DGAT1, diacylglycerol acyltransferase 1; ACC, acetyl-CoA by acetyl-CoA

417

carboxylase; FAS, fatty acid synthase; PPREs, PPARγ response elements. 20


Page 20 of 45

Page 21 of 45


418 419 420

■ ASSOCIATED CONTENT Supporting Information

421

Supporting information is available free of charge on the ACS Publications website at

422

doi:

423

Primers that were used for real-time quantitative PCR, silencing of Raptor with

424

shRNA, silencing of Rheb with shRNA, and Rheb overexpression in pBMECs (PDF).

425

Supplementary figures were used for Silencing of Raptor with shRNA, Silencing of

426

Rheb with shRNA and overexpression of Rheb in pBMECs (PDF).

427 428

■ AUTHOR INFORMATION

429

Corresponding Authors

430

* Email: [email protected] Zhigang Wang

431

Telephone +86-471-4992435

432

FAX +86-471-4992435

433

School of Life Science, Inner Mongolia University, State Key Laboratory of Reproductive

434

Regulation & Breeding of Grassland Livestock

435

No.235 Da Xue West Road, Hohhot 010021, Inner Mongolia, China

436

* Email: [email protected] Huifang Hao

437

Telephone +86-471-4992435

438

FAX +86-471-4992435

439

School of Life Science, Inner Mongolia University 21



440

No.235 Da Xue West Road, Hohhot 010021, Inner Mongolia, China

441

ORCID

442

Zhigang Wang: 0000-0002-6818-2205

443

Huifang Hao: 0000-0002-9680-8308

444

Author Contributions

445

#Zhixin

Guo and Xiaoou Cheng contributed equally to this paper.

446 447

Acknowledgments

448

The authors thank Dr. Xiao Wang and his colleagues for insightful suggestions and

449

technical advice on primary BMEC cultures. We thank Ms. Guixiu Liu for generously

450

providing Chinese Holstein cow mammary tissue after slaughter on a commercial

451

cattle slaughter farm. We thank Dr. Ying Zhang and Dr. Haiyan Xu for their

452

generously help in GC-MS assay.

453 454

Funding

455

This work was supported by the Natural Sciences Foundation of China (NO.

456

31760675, 31860309) and the Science and Technology Major Project of Inner

457

Mongolia Autonomous Region of China to the State Key Laboratory of Reproductive

458

Regulation and Breeding of Grassland Livestock.

459 460

Notes

461

The authors declare no competing financial interest. 22


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462 463

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Table 1 Content of 17 types of intracellular fatty acid in control cells and rapamycin-treated cells ( %) Fatty acid

Controla

Rapamycinb

cris-10-Pentadecenoic acid (15:1) Palmitic acid (16:0) cris-10- Heptadecenoic acid (17:1) Stearic acid (18:0) Linolelaidic acid (18:2n6t) Arachidonic acid (20:4n6) Behenic acid (22:0) cis-13,16-Docosadienoic acid (22:2) Tricosanoic acid (23:0) Nervonic acid (24:1n9) Caproic acid (6:0) Palmitoleic acid (16:1) cis-11,14-Eicosadienoic acid (20:2) Eicosapentaenoic acid (20:5n3) Henicosanoic acid (21:0) Docosahexaenoic acid (22:6n3) Lignoceric acid (24:0) Total

9.54% 6.61% 10.23% 18.20% 5.67% 7.81% 0.85% 5.14% 1.39% 10.80% 0.70% 8.65% 3.65% 3.95% 0.65% 5.09% 1.09% 100%

7.55% 2.40%** 9.06% 12.93%* 1.67%** 6.57% 0.50% 3.25% 0.86% 8.90% 1.16% 13.84%** 8.61%** 7.44%* 0.70% 6.11% 1.15% 92.70%**

a: content of product in control group / total content in control group × 100% b: content of product in rapamycin-treated group / total content in control group × 100% （* p

PPARÎ³ Axis Mediates Insulin-Induced

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