Threonine Kinase 1 Regulates de Novo Fatty Acid

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Akt Serine/Threonine Kinase 1 Regulates De Novo Fatty Acid Synthesis through mTOR/SREBP1 Axis in Dairy Goat Mammary Epithelial Cells Tianying Zhang, Jiangtao Huang, Yongqing Yi, Xueying Zhang, Juan J. Loor, Yanhong Cao, Huaiping Shi, and Jun Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05305 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

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Journal of Agricultural and Food Chemistry

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Akt Serine/Threonine Kinase 1 Regulates De Novo Fatty Acid Synthesis through

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mTOR/SREBP1 Axis in Dairy Goat Mammary Epithelial Cells

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Tianying Zhang†, Jiangtao Huang†, Yongqing Yi†, Xueying Zhang†, Juan J. Loor‡,

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Yanhong Cao#, Huaiping Shi*† and Jun Luo*†

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Running title: Goat Akt1 Regulates Lipid Biosynthesis

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† Shanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal

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Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, PR

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China

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‡ Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division

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of Nutritional Sciences, University of Illinois, Urbana 61801, USA

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# Guangxi Institute of Animal Science, Nanning, Guangxi, 535001, China

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* Email: [email protected], * Email: [email protected].

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ABSTRACT

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Akt serine/threonine kinase acts as a central mediator in the PI3K/Akt signaling

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pathway, regulating a series of biological processes. In lipid metabolism, Akt

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activation regulates a series of gene expressions, including genes related to

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intracellular fatty acid synthesis. However, the regulatory mechanisms of Akt in dairy

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goat mammary lipid metabolism have not been elaborated. In this study, the coding

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sequences of goat Akt1 gene were cloned and analyzed. Gene expression of Akt1 in

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different lactation stages was also investigated. For In vitro studies, a eukaryotic

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expression vector of Akt1 was constructed and transfected to goat mammary epithelial

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cells (GMECs), and specific inhibitors of Akt/mTOR signaling were applied to

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GMECs. Results showed that Akt1 protein was highly conserved, and its mRNA was

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highly expressed in mid-lactation. In vitro studies indicated that Akt1 phosphorylation

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activated mTOR and subsequently enhanced sterol regulatory element binding protein

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1 (SREBP1), thus increased intracellular triacylglycerol content. Inhibition of

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Akt/mTOR signaling down-regulated the gene expression of lipogenic genes. Overall,

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Akt1 plays an important role in regulating de novo fatty acid synthesis in goat

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mammary epithelial cells, and this process may probably be through mTOR/SREBP1

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axis.

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Key words: Akt1; mammary; lipid; goat

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INTRODUCTION

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Akt

serine/threonine

kinase,

which

is

a

central

mediator

of

the

38

phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway, regulates a series

39

of biological processes including glucose transport, glycolysis, protein synthesis,

40

lipogenesis, cell survival, and cell-cycle progression.1 Akt comprises three isoforms,

41

termed Akt1/2/3 or PKBα/β/γ, which are highly conserved in domain structure.2

42

Typically, insulin and growth factors bind to their respective receptors to activate

43

PI3K and then phosphorylate Akt.3 Akt activation is required for phosphorylation at

44

the threonine 308 and serine 473 sites, and phosphorylation of the serine 473 site is

45

usually deemed as an indispensable part for full Akt1 activation.4

46

Goat milk is known as a nutritious food with a high content of short to

47

medium-chain fatty acids.5 In ruminant mammary gland, most of the short to

48

medium-chain fatty acids are synthesized through de novo lipogenic pathways.6 Akt

49

activation regulates a series of gene expressions in fatty acid synthesis, including fatty

50

acid synthase (FASN), acetyl-CoA carboxylase alpha (ACACA), stearoyl-CoA

51

desaturase 1 (SCD1), and sterol regulatory element binding transcription factor 1

52

(SREBF1; protein name: sterol regulatory element binding protein 1, SREBP1),

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promotes processing of SREBP1 protein, and increases the concentration of cellular

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fatty acids and phosphoglycerides.7-8 Also, Akt regulation in lipogenesis is related to

55

activation of mammalian target of rapamycin (mTOR), the downstream atypical

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serine/threonine protein kinase that belongs to the PI3K-related kinase family.4 In

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lactating murine mammary tissues, the function of Akt1 is involved in maintaining 3


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lactation and milk composition, and the loss of Akt1 results in a failure in regulating

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cellular metabolism, including lipid synthesis.9 In contrast, side effects of constitutive

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Akt1 activation include higher lipid content in mammary epithelial cells during the

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transition from pregnancy to lactation, and higher viscosity of the milk secretion.10

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Although previous studies in mice models9-10 have shown the functions of Akt1

63

in mammary lipid metabolism, few studies have focused on its regulatory mechanism

64

in dairy goats. In the present work, goat Akt1 gene was cloned and in vitro studies

65

performed on goat mammary epithelial cells (GMECs), aimed at investigating the

66

roles of Akt1 on goat mammary fatty acid metabolism. MATERIALS AND METHODS

67 68

Ethics Statement

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Experiments were conducted under the approval of the Northwest A&F

70

University (China) and the Institutional Animal Use and Care Committee (permit

71

number: 15-516, date: 2015-9-13). All animals received humane care as outlined in

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the Guide for the Care and Use of Experimental Animals of the National Institutes of

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Health. All surgeries were designed to minimize suffering.

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Animals, Mammary Tissue Collection and RNA Extraction

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Mammary tissue samples were collected from healthy five herds of female

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Xinong Saanen goats (average of about 57 kg) during early-lactation, peak-lactation,

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mid-lactation, late-lactation (15, 60, 120, and 270 d after parturition, respectively) and

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dry-period (60 d prior to parturition), then tissues were frozen immediately in liquid

79

nitrogen after washing with diethylpyrocarbonate-treated PBS, as previous methods 4



80

described.11-12 Total RNA from each sample was extracted using TRIzol reagent

81

(Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's instructions.

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First-strand cDNA from each sample was synthesized from 1 µg of purified total

83

RNA using the PrimeScript RT kit with gDNA (Takara Bio Inc., Shiga, Japan).

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Primer Design and Cloning of Goat Akt1 Gene

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Primers were designed based on the sequence of Xinong Saanen goat Akt1 gene

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from previous de novo RNA sequencing;13 primer sequences for amplification were

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listed in Table 1. PCR reactions were performed using Xinong Saanen goat mammary

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tissue cDNA as templates. The resulting PCR reaction fragments were cloned into

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pMD®-19T plasmid vectors (Takara Bio Inc., Shiga, Japan), sequenced at a

90

commercial facility (Invitrogen Corp., Shanghai, China), and the positive plasmids

91

were conserved.

92

Sequence Analysis of Goat Akt1

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A phylogenetic tree based on Akt1 protein sequences from 8 representative

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mammalian species was constructed using MEGA7 software according to the

95

neighbor-joining method. The secondary stricter of goat Akt1 was predicted using

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GOR4

97

Protein tertiary structures of 4 representative species were predicted using Phyre2

98

(http://www.sbg.bio.ic.ac.uk/phyre2).

99

Vector Construction of pcDNA3.1-Akt1

(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html).

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Primers containing HindIII and EcoRI restriction enzyme cutting sites were

101

designed (Table 1) using pMD® -19T-Akt1 plasmid as template. Fragments of Akt1, 5


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after cutting by HindIII and EcoRI enzymes (Takara Bio Inc., Shiga, Japan), were

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cloned into the pcDNA3.1 double enzyme cutting fragment using the same enzymes,

104

the product sequenced at a commercial facility (Invitrogen Corp., Shanghai, China),

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and then the positive plasmids were conserved.

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Preparation of Inhibitors

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The specific Akt inhibitor MK-2206 and mTORC1 inhibitor rapamycin (Selleck

108

Chemicals, Houston, TX, USA), were dissolved in dimethylsulfoxide (DMSO,

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Sigma-Aldrich, Inc., St. Louis, MO, USA) to produce 10 mM stock solutions that

110

were stored at -80°C.4 Prior to applications in cell treatments, stock solutions were

111

diluted to working concentrations.

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Cell Culture and Treatments

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The cell isolation and cell culture were according to the methods of Zhu et al.

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and Zhang et.al.14-15 In brief, GMECs were isolated from Xinong Saanen goats at

115

mid-lactation and cultured at 37°C in a humidiﬁed atmosphere with 5% CO2. The

116

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

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1 µg/mL hydrocortisone (Sigma-Aldrich, Inc., St. Louis, MO, USA), 5 µg/mL insulin,

118

100 U/mL penicillin and 100 mg/mL streptomycin (Harbin Pharmaceutical Group Co.,

119

Ltd., Harbin, China), 5 mM sodium acetate (Sigma-Aldrich, Inc., St. Louis, MO,

120

USA), 10 ng/mL epidermal growth factor 1 (EGF-1, Invitrogen Corp., Carlsbad, CA,

121

USA), and 10% fetal bovine serum (Gibco Laboratories, Gaithersburg, MD, USA).

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For the final treatment, the growth medium was changed to serum-free medium,

123

which is EGF-free, serum-free and containing 2 µg/mL prolactin (Sigma-Aldrich, Inc., 6



124

St. Louis, MO, USA). For Akt1 overexpression, cells were transfected with

125

pcDNA3.1-(+) vehicle or pcDNA3.1-Akt1 plasmid for 5 h according to the

126

manufacturers’ instructions (Invitrogen Corp., Carlsbad, CA, USA). Then, after the

127

medium was changed to serum-free medium, the cells were incubated for 48 h. For

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MK-2206 inhibition assays, cells were treated with serum-free medium for 12 h

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followed by incubation with DMSO or MK-2206 (500 nM) for 8 h. For rapamycin

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inhibition assays, cells were treated with serum-free medium for 12 h followed by

131

incubation with DMSO or rapamycin (10 nM) for 8 h.

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Triglyceride Content Assay

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GMECs were grown in 60 mm culture plates until ~80% confluent before

134

applying treatments. TG content was assayed using a commercial kit (Triglyceride

135

Content Assay Kit, Applygen Technologies Inc., Beijing, China), according to the

136

manufacturer’s instructions. Data were normalized with protein concentrations

137

assayed with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc.,

138

Waltham, MA, USA).

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GMECs RNA Extraction

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GMECs were grown in 12-well culture plates until ~80% confluence before

141

applying treatments. Total RNA was extracted using an RNAprep Pure Cell kit

142

(Tiangen Biotech Co., Ltd., Beijing, China). The first-strand cDNA from each

143

treatment was synthesized from 500 ng of purified total RNA using a PrimeScript RT

144

kit (Takara Bio Inc., Shiga, Japan), according to manufacturer's instructions.

145

Real-time Quantitative PCR 7


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Real-time

quantitative

PCR

(RT-qPCR) was performed according to

147

manufacturer's instructions using SYBR green (SYBR® Premix Ex Taq™ II, Perfect

148

Real Time; Takara Bio Inc., Shiga, Japan). Ubiquitously expressed transcript (UXT)

149

and ribosomal protein S9 (RPS9) were used as internal control genes, all qPCR

150

reactions performed in a Bio-RadCFX96 sequence detector (Bio-Rad Laboratories,

151

Inc., Hercules, CA, USA), and data normalized to internal controls. Data were

152

analyzed using the relative quantiﬁcation (2-∆∆Ct) method. Primers for real-time

153

quantitative PCR were shown in Table 2 and Supplemental Table 1.

154

Western Blot

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GMECs were grown in 60 mm culture plates until ~80% confluence before

156

applying treatments. Cellular protein was harvested after cells were lysed with RIPA

157

buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) containing

158

protease and phosphatase inhibitor cocktail tablets (Roche Molecular Biochemicals,

159

Mannheim, Germany). Cell lysate of each sample was extracted, collected, and then

160

boiled with Lane Marker Loading Buffer (Beijing CoWin Biotech Co., Ltd., Beijing,

161

China), according to manufacturer’s instructions. Western blots were conducted

162

according to the methods of Xu et.al and Gou et.al.16-17 In brief, total protein of each

163

treatment was separated by SDS-PAGE and the blot transferred to a nitrocellulose

164

membrane (Pall Life Sciences Corp., Port Washington, NY, USA) by Trans-Blot SD

165

semi-dry transfer cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Then, the

166

membrane was blocked using 5% skim milk prior to incubation with primary

167

antibodies. Monoclonal rabbit anti-p-Akt1 Ser473 (~60 KDa; 1:2000 dilutions, 4060; 8



168

Cell Signaling Technology Inc., Danvers, MA, USA), monoclonal rabbit anti-Akt1

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(~60 KDa; 1:1000 dilution, ab32505; Abcam, Inc., Cambridge, UK), polyclonal rabbit

170

anti-p-mTOR Ser 2448 (~289 KDa; 1:800 dilution, AF1665; R&D Systems, Inc.,

171

Minnesota, MN, USA), polyclonal rabbit anti-mTOR (~289 KDa; 1:1000 dilution,

172

2972; Cell Signaling Technology Inc., Danvers, MA, USA), monoclonal mouse

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anti-SREBP1 (60~70 KDa; 1:1000 dilution, ab3259; Abcam, Inc., Cambridge, UK),

174

and monoclonal mouse anti-β-Actin (~45 KDa; 1:4000 dilution; Beijing CoWin

175

Biotech Co., Ltd., Beijing, China) were used as primary antibodies. Polyclonal goat

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anti-mouse IgG coupled to horse radish peroxidase (HRP; 1:4000 dilution, CW0102S;

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Beijing CoWin Biotech Co., Ltd., Beijing, China) and polyclonal goat anti-rabbit IgG

178

coupled to HRP (1:4000 dilution, CW0103S; Beijing CoWin Biotech Co., Ltd.,

179

Beijing, China) were used as secondary antibodies. Signals were detected using a

180

chemiluminescent ECL western blot detection system (Bio-Rad Laboratories, Inc.,

181

Hercules, CA, USA). The intensity of indicated band was quantified by densitometry

182

using ImageJ software (http://imagej.nih.gov/ij/), and relative protein abundance was

183

normalized to total kinase or β-actin.

184

Statistical Analysis

185

Each experiment included at least three biological replicates and results were

186

expressed as means ± standard error of means. Statistical significance was analyzed

187

by SPSS19.0 (SPSS Inc., Chicago, IL). Student’s t-test (unpaired and two-tailed) was

188

applied when only two groups were compared. For multiple comparisons, data were

189

analyzed by One-way analysis of variance (ANOVA) using the general linear model 9


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190

procedure and mean separation was performed via Tukey test. Statistical significance

191

was declared at P < 0.05. RESULTS

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Molecular Cloning and Sequence Analysis of Goat Akt1

194

The cDNA of goat Akt1 was amplified with specific primers, and the gel

195

electrophoresis was shown in Figure 1A. The length of goat Akt1 was 2015 bp, and

196

coding sequence was 1443 bp long, encoding 480 amino acids (Gene Bank ID:

197

KX889091). The potential evolutionary pathways of Akt1 protein among different

198

species was investigated by examining Akt1 from 8 representative species, including

199

Capra

200

(NM_001159776.1), Bos taurus (AY781100.1), Equus caballus (XM_001492713.5),

201

Homo sapiens (NM_005163.2), Mus musculus (NM_009652.3), and Rattus

202

norvegicus (NM_033230.2), and a phylogenetic tree was constructed based on the

203

neighbor-joining method. The results showed that goat Akt1 was most closely related

204

to Ovis aries, followed by Sus scrofa (Figure 1B).

hircus

(KX889091),

Ovis

aries

(NM_001161857.1),

Sus

scrofa

205

The secondary structure of goat Akt1 protein was examined, and the tertiary

206

structures of Akt1 protein from goat, sheep, human, and cow were compared. The

207

results showed that goat Akt1 protein contained 178 alpha-helical, 84 extended-strand,

208

and 218 random-coil amino acid regions (Figure 1C). The spatial structures of these

209

Akt1 proteins shared high similarity among different species (Figure 1D).

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Expression Analysis of Goat Akt isoforms in Different Lactation Stages

211

The relationship between Akt isoforms expression and lactation stage in dairy 10



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goats was revealed by studying the abundance of mRNA expression. The expression

213

level of Akt1 was ~21-fold higher in mid-lactation compared with dry period,

214

followed by ~5-fold higher in peak-lactation (P < 0.05, Figure 2). The third and fourth

215

expression levels were early-lactation and late-lactation, respectively, while there

216

were no significance between them (P > 0.05, Figure 2). The expression level of Akt2

217

was highest in early-lactation (~2.5-fold higher compared with dry period, P < 0.05,

218

Supplemental Figure 1B), and the expression level of Akt3 was highest in

219

mid-lactation (~3.7-fold higher compared with dry period, P < 0.05, Supplemental

220

Figure 1B).

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Akt1 Regulates Lipogenic Genes in GMECs

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To investigate the fatty acid metabolism regulatory of Akt1 in goat mammary

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gland, a eukaryotic expression vector of Akt1 (pcDNA3.1-Akt1) was constructed and

224

transfected into GMECs. Akt1 mRNA abundance was upregulated over 60-fold in the

225

pcDNA3.1-Akt1 group, compared with the pcDNA3.1-(+) group (P < 0.01, Figure

226

3A). The mRNA expression related to fatty acid uptake and activation, including

227

CD36 molecule (CD36), fatty acid binding protein 3 (FABP3), acyl-CoA synthetase

228

long-chain family member 1 (ACSL1), and acyl-CoA synthetase short-chain family

229

member 2 (ACSS2); de novo fatty acid synthesis and desaturation, including FASN,

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ACACA and SCD1 were analyzed. The results revealed an evident upregulation of

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FABP3, FASN, ACACA, and SREBP1 (Figure 3B and C, P < 0.05). Genes related to

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TG

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1-acylglycerol-3-phosphate

synthesis,

including

glycerin-3-phosphat-acyltransferase O-acyltransferase 11


6

1

(GPAM), (AGPAT6),

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diacylglycerolacyltransferase 1 (DGAT1), and diacylglycerolacyltransferase 2

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(DGAT2) were upregulated (Figure 3D, P < 0.05).

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MK-2206 is a novel, specific Akt1 inhibitor,4 and the physiological effects of

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inhibiting Akt1 activity in GMECs were subsequently investigated. By analyzing the

238

genes related to fatty acid uptake and activation, de novo fatty acid and TG synthesis,

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we found a dramatic downregulation of FABP3 (Figure 4A, P < 0.01), and the mRNA

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abundance of FASN, SCD1 and SREBF1 were significantly downregulated compared

241

with the control (Figure 4B, P < 0.05).

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Overexpression Akt1 increased TG Accumulation in GMECs

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We subsequently examined the intracellular lipid accumulation in GMECs after

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transfected with pcDNA3.1 plasmids. The results indicated that Akt1 overexpression

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increased intracellular TG content (Figure 5A, P < 0.05). The oil red O result showed

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an obviously accumulation of neutral lipids in GMECs after overexpression of Akt1,

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compared with the control group (Figure 5B).

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Akt1 Regulates mTOR Phosphorylation and nSREBP1 abundance in GMECs

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To clear out the detailed molecular mechanism of Akt1 in regulation of lipid

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synthesis, we examined the phosphorylation of Akt/mTOR signaling. Overexpression

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of Akt1 upregulated phosphorylation of Akt1 at Ser473, as well as the total Akt1

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abundance (Figure 6A, P < 0.05). And as a consequence, downstream mTOR

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phosphorylation at Ser2448 was increased (Figure 6A, P < 0.05). The nuclear form of

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SREBP1 (nSREBP1), which is the active fragment, was up-regulated (Figure 6A, P

Threonine Kinase 1 Regulates de Novo Fatty Acid

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