ARTICLE IN PRESS Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

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Obesity associated Lyplal1 gene is regulated in diet induced obesity but not required for adipocyte differentiation Q1 Xinnuo Lei a,b, Mayson Callaway c, Hongyi Zhou b, Yi Yang a, Weiqin Chen b,* a b c

College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan Province 410128, China Department of Physiology, Georgia Regents University, Augusta, GA 30912, USA Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA

A R T I C L E

I N F O

Article history: Received 26 November 2014 Received in revised form 30 April 2015 Accepted 1 May 2015 Available online Keywords: Lyplal1 Obesity Adipocyte differentiation Fat distribution

A B S T R A C T

Obesity and its associated morbidities represent one of the major and most rapidly expanding health epidemics in the world. Recent genome-wide association studies (GWAS) have identified several variants in LYPLAL1 gene that are significantly associated with central obesity preferentially in females. However, the exact function of this gene in adipose tissue development and obesity remains completely uncharacterized. We found murine Lyplal1 gene demonstrated a depot and sex-specific expression profile in white adipose tissues (WAT), and was significantly reduced in the epididymal and retroperitoneal fats in a murine model of high fat diet induced obesity (DIO). Lyplal1 mRNA was mildly up-regulated during adipogenesis and enriched in mature adipocytes through a PPARγ-independent mechanism. However, overexpression and knockdown of Lyplal1 did not significantly perturb adipocyte differentiation, triacylglycerol accumulation and/or insulin signaling. These data highlight a depot-specific marked reduction of Lyplal1 transcripts in diet induced obesity but a dispensable role of Lyplal1 in adipose tissue development. © 2015 Published by Elsevier Ireland Ltd.

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1. Introduction Obesity has become a major public health problem worldwide. Other than chronic positive energy balance, genetic factors are well-known to contribute to the development of obesity. Recent genome-wide association studies (GWAS) have identified 32 gene variants influencing overall obesity as measured by body mass index (BMI), suggesting the key role of central regulation of overall adiposity (Speliotes et al., 2010; Thorleifsson et al., 2009; Willer et al., 2009). However, numerous epidemiologic studies have demonstrated that central obesity, measured by waist circumference or waist–hip ratio (WHR), has strong relationships to metabolic and cardiovascular diseases, independent of overall adiposity (Carey et al., 1997; Cassano et al., 1990; Seidell et al., 1991; Shuster et al., 2012). In the past 5 years, GWAS studies, including up to ~77,000 individuals, have identified genetic variants in 19 loci to be associated with measures of body composition (Chambers et al., 2008; Heid et al., 2010; Lindgren et al., 2009), the vast majority associating with BMI-adjusted WHR (Heid et al., 2010). These genes are thought to be mainly involved in regulating the specific processes of adipose

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* Corresponding author. Department of Physiology, Medical College of Georgia at Georgia Regents University, Augusta, GA 30912, USA. Tel.: +1 706 721 8706; fax: +1 706 721 7299. E-mail address: [email protected] (W. Chen).

tissue itself. However the physiologic role of most of the identified genes in regulating central obesity and fat distribution remains to be elucidated. Recently, several SNPs, namely single nucleotide polymorphism, near human Lysophospholipase-like 1 (LYPLAL1) gene were revealed to be significantly associated with fat distribution in a relatively sex-specific pattern. Among them, 3 SNPs including rs4846567 (Heid et al., 2010), rs2605100 (Lindgren et al., 2009) and rs2820443 (Randall et al., 2013) near LYPLAL1 gene are associated with increased WHR adjusted for BMI only in women not men. A genome-wide significance for rs11118316 at LYPLAL1 was also observed to associate with visceral adipose tissue/subcutaneous adipose tissue ratio in both men and women (Fox et al., 2012). Another SNP at rs12137855 near LYPLAL1 gene is strongly associated with nonalcoholic fatty liver disease (Speliotes et al., 2010). Meanwhile, the major G-allele of LYPLAL1 rs2605100 also associates with increased fasting serum insulin concentrations and insulin resistance as well as increased fasting serum triglyceride concentrations in the male gender (Bille et al., 2011). These data underscore an important interplay of LYPLAL1 locus with fat distribution and lipid metabolism. Human Lysophospholipase-like 1 (LYPLAL1) gene encodes lysophospholipase-like protein 1, a 26 kDa cytosol protein which belongs to a subclass of lysophospholipase family (Fischer and Pleiss, 2003). Sequence homology indicates this protein is closely related to human acyl protein thioesterase APT1 and has been proposed to

http://dx.doi.org/10.1016/j.mce.2015.05.001 0303-7207/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Xinnuo Lei, Mayson Callaway, Hongyi Zhou, Yi Yang, Weiqin Chen, Obesity associated Lyplal1 gene is regulated in diet induced obesity but not required for adipocyte differentiation, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.05.001

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ARTICLE IN PRESS X. Lei et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

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function as a potential triacylglycerol lipase in adipose tissue (Steinberg et al., 2007). Yet, biochemical studies and crystallization of human LYPLAL1 demonstrate that LYPLAL1 exhibits neither phospholipase nor triacylglycerol lipase activity (Burger et al., 2012). Despite the significant association of LYPLAL1 variants with fat distribution, the exact biological function and significance of LYPLAL1 in adipose tissue development and obesity remains largely unknown. More detailed analyses are required to pinpoint the specific causes underlying the observed association signals with central obesity. Therefore, the aim of the study was to shed some light in the role of LYPLAL1 in fat distribution and obesity. As a first step, our study was planned to evaluate whether murine Lypla1 exhibits fat depot and sex-specific mRNA expression and whether its transcript level correlates with obesity in different fat depots in an animal model of diet induced obesity. We further overexpressed and knocked down murine Lyplal1 in cultured cells to illustrate the potential role of Lyplal1 in regulating adipose tissue differentiation and triacylglycerol accumulation as well as whether Lyplal1 knockdown affects insulin signaling in mature adipocytes.

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2. Materials and methods 2.1. Mice Mice were maintained in a temperature-controlled facility with fixed 12-h-light and 12-h-dark cycles and free access to regular chow and water. Male and female C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, Maine). Six week old male C57BL/ 6J mice were allowed ad libitum access to low fat control diet (D12450B, 10% kcal from fat) or high fat diet (D12492, 60% kcal from fat, Research Diets, New Jersey, USA) for up to 12 weeks. All mice were sacrificed after 4 h fasting. All animal experiments were done using protocols approved by the IACUC of Georgia Regents University.

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2.2. Reagents and plasma biochemistry All drugs including insulin, dexamethasone, isobutylmethylxanthine (IBMX) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All cell culture reagents were from Fisher Scientific Inc. (Pittsburgh, PA). Blood glucose tests were performed with OneTouch UltraSmart blood glucose monitoring system (Life scan). Plasma nonesterified fatty acid (NEFA) (Wako Chemicals USA Inc., Richmond, VA), glycerol (Sigma-Aldrich), total cholesterol, total triacylglycerol levels (Fisher Scientific Inc.) were measured colorimetrically. Serum insulin (EMD Millipore, MA) was measured by enzyme-linked immunosorbent assays according to the manufacturer’s instructions.

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2.3. Cell culture and adipose tissue fractionation The 3T3-L1 cells, NIH-3T3 and C3H10T1/2 cells (American Type Culture Collection, Manassas, VA) were propagated in DMEM containing 10% fetal bovine serum (FBS). Adipocyte differentiation was initiated 2 d after confluence in DMEM containing 10% FBS, supplemented with standardized hormone cocktail (1 μM insulin, 0.5 mM IBMX, and 1 μM dexamethasone, heretofore referred to as DMI) and induced for 2 d and then supplemented with 1 μM insulin alone for another 2 d. After 4 d, cells were cultured in DMEM 10% FBS medium. Murine stromal vascular fraction and mature adipocytes were fractionated from epididymal WAT of 10 week old male C57BL/ 6J mice by collagenase digestion as described previously (Chen et al., 2009).

2.4. Generation of retroviral construct, retrovirus and lentivirus production and infection Murine Lyplal1 cDNA (NM_146106.2) was subcloned into pMSCVC-FLAG retroviral vector with puromycin selectable marker. pBABEpuro-PPAR gamma2 vector was obtained from Addgene (#8859). Retroviral packaging Bosc-23 (American Type Culture Collection) cells were cotransfected with the targeting plasmid and packaging vector pCL-eco (Imgenex, Sorrento Valley, CA). Forty-eight hours after transfection, the culture media containing the virus particles were collected and mixed with DMEM 10% FBS at 1:2 to infect 3T3L1 preadipocytes or NIH-3T3 fibroblasts in the presence of 8 μg/ mL polybrene. Cells were then selected with 2 μg/mL puromycin for at least 4 days as described previously (Jimenez et al., 2007). Lentivirus vector pLKO.1-puro harboring two short hairpin RNAs (shRNA) (CCGGGACTTAACATTCCAGCACATACTCGAGTATGTGCTGG AATGTTAAGTCTTTTTG and CCGGGCACGTGCTAAACCAAGACTTC TCGAGAAGTCTTGGTTTAGCACGTGCTTTTTG) against murine Lyplal1 were obtained from the TRC shRNA library (Sigma-Aldrich, St. Louis, MO). PLKO.1-puro expressing a scramble shRNA was obtained from Addgene (#1864). Lentiviral particles were produced by transient transfection of 293T cells with a packaging plasmid psPAX2 and envelope plasmid pMD2.G together with the shRNA expressing lentiviral vectors according to the manufacturer’s directions and purified using LENTI-XTM concentrator kit (Clontech Laboratories Inc., Mountain View, California). C3H10T1/2 cells were infected and selected under 2 μg/mL puromycin for 48 h and then expanded for adipocyte differentiation as described earlier. Experiments were repeated at least 2 times. Similar data were obtained from two different shRNAs against Lyplal1. Representative data were presented from one shRNA.

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2.5. Insulin signaling Differentiated mature adipocytes were starved in DMEM with 2% BSA media for 6 h before administration of 10 nM insulin for 5 and 15 minutes (min). Cells were then washed with cold PBS twice and harvested for immunoblot analyses.

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2.6. Real-time PCR analyses Total RNA was isolated from tissues or cultured cells with TRIzol (Invitrogen, Carlsbad, CA) and reverse transcribed using M-MLV reverse transcriptase and random primers (Invitrogen). Real-time quantitative RT-PCR was performed on the MX3500 real time detection system (Stratagene, La Jolla, CA) using iQ SYBR Green PCR reagent kit (Bio-Rad Laboratories, Hercules, CA). For tissue mRNA expression analysis, data were normalized to two house-keeping genes specified in the figure legend based on Genorm algorism (http://medgen.ugent.be/~jvdesomp/genorm/). The specificity of the Q2 PCR was verified by dissociation curve analysis.

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2.7. Triacylglycerol (TAG) content measurement and oil-red O staining At indicated days, cells were directly dissolved in PBS containing 1% triton X-100. The intracellular TAG in the adipocytes was measured with an Infinity Triglyceride assay kit (Thermo Scientific). The TAG content was normalized to the amount of cellular protein as determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA) and expressed as per milligram of protein. Oil-red O staining was performed as described previously (Jimenez et al., 2007) and photographed with camera or under microscopy.

Please cite this article in press as: Xinnuo Lei, Mayson Callaway, Hongyi Zhou, Yi Yang, Weiqin Chen, Obesity associated Lyplal1 gene is regulated in diet induced obesity but not required for adipocyte differentiation, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.05.001

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ARTICLE IN PRESS X. Lei et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

Table 1 Plasma biochemistry in male C57BL/6J mice after low and high fat diet feeding.

Total cell lysates were prepared with a lysis buffer containing 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 10% glycerol with freshly added protease inhibitor cocktail (Sigma-Aldrich). Same amount of proteins was loaded and immunoblot analysis was carried out according to the standard protocol. The following antibodies were used: GAPDH (Fisher Scientific), Flag (Sigma-Aldrich) and PPARγ2 (Millipore). Image J was used to quantify the band intensity. 2.9. Statistical analysis

HFD (n = 13)

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27.9 ± 0.7 152 ± 5.6 30.5 ± 2.4 105 ± 5.7 0.8 ± 0.06 1.0 ± 0.14

42.2 ± 1.4** 245 ± 14** 32.5 ± 2.2 183 ± 13** 0.86 ± 0.07 7.5 ± 0.9**

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Six week old male C57BL/6J mice were fed with 60% high fat diet (HFD) or its control low fat diet (LFD) for 12 weeks. Plasma biochemistry was analyzed 12 weeks after diet feeding under 4 h fasting states. Data were presented as means ± SEM. TAG, trigacyglycerol; NEFA, nonesterified free fatty acid; TC, total cholesterol. ** p < 0.005 between two groups.

B 1.2 1.0 0.8 0.6 0.4 0.2 0.0

##

*

**

**

2.0 1.5 1.0 0.5 0.0 M

F

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

sWAT

M

1.2 1.0 0.8 0.6 0.4 0.2 0.0 F

RpWAT

**

0

2

4 6 8 Feeding weeks

B eWAT

* HF

Fig. 1. Tissue distribution and sexual dimorphism of murine Lyplal1 expression. A: Lyplal1 mRNA expression in mouse tissues as determined by real-time RT-PCR in lung, small intestine (SI), liver, epididymal (eWAT) and subcutaneous (sWAT) white adipose tissue, adrenal gland (AD), brain (Brn), heart (Hrt), skeletal muscle (Skm), testis (Tes), kidney (Kid), and spleen (Spln) isolated from 4-h-fasted, 10-wk-old male C57BL/6J mice (each tissue contains one sample pooled from 3 mice). B: Lyplal1 mRNA expression in 4 different white adipose tissue depots as relative to RpWAT. RpWAT: retroperitoneal white adipose tissue, MsWAT: mesenteric white adipose tissue. n = 5 each. *p < 0.05, **p < 0.005 vs RpWAT; ##p < 0.005 vs eWAT. C: Lyplal1 mRNA expression in gonadal WAT (gWAT), sWAT and RpWAT of male (M) and female (F) 4 h fasted C57BL/6J mice (n = 10 each). Data were normalized to 36B4 and β-actin based on genome algorism. **p < 0.005.

12

2.5

sWAT

73

2.0 1.5 1.0 0.5 0.0

LF HF

E

D

F

10

C 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

LF

M

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LF HF

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*

Relative mRNA level

Relative mRNA level

2.5 gWAT

A

Relative mRNA level

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

R pW M A sW T A eW T AT sW AT

Lyplal1 (Relative mRNA level)

A

lowed by eWAT, subcutaneous (sWAT) and mesenteric (MsWAT) fat (Fig. 1B). Genetic variants near the LYPLAL1 gene demonstrate a sexspecific association with increased central obesity, we thus compared the Lyplal1 expression in gonadal (epididymal fat in male and periuterine fat in female), subcutaneous and retroperitoneal WAT of both genders. As shown in Fig. 1C, female mice had relatively higher expression of Lyplal1 in its peri-uterine fat as compared to male eWAT, while no differences were found in subcutaneous and retroperitoneal WAT between the two sexes (Fig. 1C). These data demonstrate a depot and sex-specific expression profile of Lyplal1 in murine adipose tissues.

Relative mRNA level

Murine Lyplal1 gene shares ~80% homology with human LYPLAL1 gene (http://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=full _report&list_uids=226791). Here, we first examined the tissue expression profile of murine Lyplal1 in male C57BL/6J mice. Lyplal1 gene is highly expressed in liver, epididymal white adipose tissue (eWAT), skeletal muscle (Skm) and kidney with relatively lower expression in small intestine (SI), lung, adrenal gland (AD) and spleen (Spln) (Fig. 1A). Since SNPs near the human LYPLAL1 gene influence fat distribution, we specifically analyzed the expression pattern of Lyplal1 among different fat depots. Lyplal1 has the highest gene expression in retroperitoneal fat (RpWAT, fat around kidney), fol-

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1.2 RpWAT 1.0 0.8 * 0.6 0.4 0.2 0.0 LF HF

Relative mRNA level

3.1. Tissue distribution and sexual dimorphism of murine Lyplal1 expression

C

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LFD (n = 10)

Body weight (g) Glucose (mg/dL) TAG (mg/dL) TC (mg/dL) NEFA (mM) Insulin (ng/mL)

Body weight (g)

3. Results

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Diet

Statistical analyses of the data were performed using a twotailed unpaired t test with equal variance. Data were presented as the means ± SEM with statistical significance set at a P value of

Obesity associated Lyplal1 gene is regulated in diet induced obesity but not required for adipocyte differentiation.

Obesity and its associated morbidities represent one of the major and most rapidly expanding health epidemics in the world. Recent genome-wide associa...
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