Chemicals
All chemicals were purchased from Sigma (USA) or Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless otherwise stated.
Gene construct
Based on the nucleotide sequence of the Pai gene (GenBank accession number AX062088) from Propionibacterium acnes, the DNA sequence was optimized with high frequency codons in mammals using the OptimumGeneTM algorithm software (GenScript, USA). The Kozak consensus sequence (GCCACC) and a glycine codon (GGA) were added immediately upstream and downstream of the start codon (ATG), respectively. Meanwhile, and 6 x His affinity tag sequence was also added immediately upstream of the terminal codon (TAA). Then the codon-optimized Pai gene was synthesized and inserted into the pCAGGS shuttle vector (Gift from Dr. Timothy J. Ley, The Washington University) with an EcoR I (Fermentas, USA) digestion to generate the pCAGGS-Pai expression vector, in which the Pai gene was under the control of the cytomegalovirus enhancer and the chicken beta-actin promoter. Finally, the linear 3.5-kb tg cassettes digested with Sal I + Hind III (Fermentas) were purified using a QIA quick Gel Extraction Kit (Qiagen) and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) to a final concentration of 2 µg/mL for microinjection.
Animals and treatments
ICR mice at 6–8 weeks of age were obtained from the Institute of Laboratory Animal Science, the Chinese Academy of Medical Sciences (Beijing, China) and maintained in a light-controlled room (14L:10D, lights on at 0500 h) at a temperature of 22–25°C. All of the mice procedures in the current study were approved by the Committee for Experimental Animals of China Agricultural University and Yangzhou University. During the entire experimental period, all mice were normal raised on a 10% kcal% fat diet except the obese mice.
Transgenic mice were produced by pronuclear microinjection according to the normal procedure. In brief, fertilized eggs from ICR matings were collected from superovulated females at 7–8 week of age and released from the cumulus layers by treatment with HEPES-buffered CZB medium containing 300 IU/ml hyaluronidase. Embryos with an obvious polar body and two pronuclei were used for DNA microinjection. Fifteen to twenty embryos were transferred into each oviduct of 0.5 day-post-coitum pseudopregnant females. Pregnant recipients were then housed individually and allowed to carry the pregnancies to term.
Female offspring from eight matings of wild-type (wt) female x transgenic (tg) male were divided into two groups at random in the subsequent experiments. In details, pups from four matings contained 9 wt and 12 tg females were fed with a 10% kcal% fat diet until analysis at age of 7 weeks. Pups from the other four matings contained 12 wt and 8 tg females were first fed with a 10% kcal% fat diets, and then switch to 60% kcal% high-fat diets at age of 8 weeks for another 10 weeks. At the end of the trials, blood was obtained from the heart under Avertin anesthesia. All organs, scapular brown fat, and abdominal fat (consisting of retroperitoneal fat and peripheral white fat from the kidney and gonad) were weighed or maintained at 80°C until further analysis.
Transgene detection
Genomic DNA was extracted from the tail tissues of pups utilizing a standard phenol-chloroform method and dissolved in TE buffer (pH 8.0) for nucleic acid analysis. The presence of the transgene was first assayed by PCR amplification using a set of primers (F: 5’-taa cca tgt tca tgc ctt ctt c-3’, R: 5’-cac ctt gtt gta gtg tcc gtt t-3’) and amplified the 514-bp fragments that spanned the Pai gene. PCR amplification was performed as follows: 94°C for 5 min; 30 cycles at 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min; and a final extension at 72°C for 10 min.
Southern blot was performed according to standard procedures. In brief, genomic DNA (10 µg) was digested with EcoR I enzyme (Fermentas) for 12–16 h at 37°C and subjected to overnight 0.8% agarose gel electrophoresis followed by transferring to Hybond-N+ membrane (Amersham, UK). The 671-bp Pai fragments as a probe were amplified using specific primers (F: 5’-tga tga ccc tca cca gat-3, R: 5’-gaa caa agg ttg gct ata aa-3’) and DIG-labeled dNTP mix (Roche, Germany). The membrane was subsequently hybridized with labeled probes according to standard protocols, using storage phosphor autoradiography (GE Healthcare, USA) for 5 d to obtain an autoradiographical image.
RNA analysis
Offspring from wt females mated with tg males were used for following analysis. Briefly, total RNA was extracted from each fresh tissue using RNAiso Plus (TaKaRa, Japan) and treated with DNase I (TaKaRa). The purified RNA was used for first-strand cDNA synthesis, and reverse transcription was performed using an M-MLV reverse transcriptase (Promega, Madison, USA) with oligo-dT primers according to the manufacturer’s instructions. To avoid genomic contamination, reactions were also performed in the absence of reverse transcriptase for each RNA sample tested. The resulting cDNA was then used for PCR amplification with the Pai specific primers (F: 5’-gga ttc cac gat tac acc a-3’, R: 5’-ggg tca tca gcc cat cta-3’) that produced an 878-bp fragment. As a control, a 352-bp fragment of the Hprt gene (F: 5’-cct gct gga tat cat taa agc act g-3’, R: 5’-gtc aag ggc ata tcc aac aac aaa c-3’) was amplified under the same conditions. PCR amplification was performed as follows: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min; and a final extension at 72°C for 10 min.
The real-time PCR was used to determine the Pai gene transcription with the SYBR® Premix Ex Taq™ II kit (TaKaRa) following the manufacturer’s protocol. The cDNA was used for real-time PCR amplification using the specific primer pairs to the respective genes of Pai (F: 5’-tga cga gcg gga ata ctt ta-3’, R: 5’-gag ggt cat cag ccc atc ta-3’) and Gapdh (F: 5’-gaa cat cat ccc tgc atc c-3’, R: 5’-cca gtg agc ttc ccg ttc a-3’). The assay was performed using an ABI Prism VII7 sequence detection system (Applied Biosystems, USA) at 95°C, 2 min, 1 cycle; 40 cycles of 95°C, 5 sec, 60°C, 32 sec. Each reaction performed as three replicates was measured at least three times. The relative transcript level was determined using the Eq. 2-ΔCt, where Ct is the PCR cycle number at which the accumulated fluorescence signal in each reaction crosses a threshold above background and ΔCt = CtPai – CtGapdh.
Northern blot
Total RNAs (20 µg) was run under denaturing conditions in an agarose-formaldehyde gel and transferred to the Hybond-N+ membrane (Amersham). The 878-bp Pai-specific fragments were amplified using labeled dNTP mix (Roche) and used as probes. The membrane was subsequently hybridized with the DIG-labeled probes according to standard protocols and obtained an autoradiographical image using storage phosphor autoradiography (GE Healthcare, USA).
Western blot
Proteins from each tissue were manually homogenized with radio-immunoprecipitation assay buffer (Beijing Kangwei Shiji Biotechnol Ltd, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using a bicinchoninic acid assay reagent (Beijing Kangwei Shiji Biotech Ltd) according to the manufacturer’s instructions. Twenty microgram of protein samples were first separated by SDS-PAGE and transferred to PVDF membrane (Abcam, ab133411). Then the membrane was incubated with monoclonal anti-His-tag antibody (1:1000, Santa Cruz Biotechnology, sc-57598) at 4°C overnight, followed by incubation with goat anti-mouse IgG-HRP antibody (1:5000, Santa Cruz Biotechnology, sc-2005) at room temperature for 2 h. The antigen was finally visualized using enhanced chemiluminescence substrate SuperSignal™ West Femto (ThermoFisher, 34094) and analyzed by a gel imager (Bio-Rad, USA, GelDoc XR+).
Gas chromatography
Each sample homogenized by grinding in liquid nitrogen was collected in a glass methylation tube for direct fatty acid analysis as described as in our previous study (Li et al. 2015). Fatty acid methyl esters were separated on a fused-silica capillary column (HP-88, 100 m, J & W 112-88A7, Agilent Technologies, USA) and quantified using a fully automated 7890 Network GC System with a flame ionization detector (Agilent). Peaks were identified by comparison with fatty acid standards (Sigma, 47885-U and O5632), and the area percentage for all resolved peaks was analyzed using GC ChemStation Software (Agilent Technologies).
Blood sampling and analysis
The serum samples were analyzed using an automatic analyzer (Toshiba FR-120, Toshiba Co., Ltd., Japan) immediately or within two days. The measurements of total cholesterol (TC) and triglycerides (TG) were performed by the cholesterol oxidase endpoint method using the CHO Assay Kit (Baiding Biotech Co., Beijing, China) and by the enzymatic method using the TG Assay Kit (Baiding Co.).
Histological analysis
Histologic analysis was performed following the standard procedure. In brief, the liver and abdominal adipose tissues were fixed by immersion in 4% formaldehyde in PBS at 4°C and then dehydrated before paraffin embedding. The tissue sections were dewaxed in xylene followed by rehydration, and histological details were examined after staining with hematoxylin-eosin. Formalin-fixed samples were embedded in paraffin, sliced into 5 µm sections (Leica CM1950), mounted, and stained with hematoxylin and eosin. Stained slides were examined with a Olympus microscope equipped with a digital camera.
To estimate the adipocyte size, each cell area was modified following the method as described by Chen and Farese (Chen and Farese, 2002). In detail, six slices from each mouse abdominal WAT were random chosen for image analysis. Firstly, three photos were random taken from non-overlapping microscope fields of every slice under the 20 x objective, then the cross-sectional area of each adipocyte on a photo were calculated one by one using ImageJ software (Version 2.0.0) and the means of the top 100 cell areas per picture represented the value of a microscope sampling field. Finally, the means of 18 values per mouse WAT (3 fields x 6 slices) looked upon as the average cellular cross-sectional area of this WAT sample for statistical analysis.
Statistical analysis
One-way analysis of variance was performed on all data and the statistical significance was assessed by Student’s t-test. All values are presented as the means ± SD. Differences of p < 0.05 were considered to be significant.