Genotypes of yeast strains
Strain
|
Genotype
|
Source
|
By4741
|
MAT a his3∆ leu2∆ met15∆ ura3∆
|
This study
|
ZHY02
|
MAT alpha ade2::HisG his3 leu2 met15∆::ADE2 trp1∆63 ho∆::SCW11pr-CRE-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP-HPHMX
|
Tyler lab [43]
|
BJY02
|
MAT alpha ade2:: His G his3 leu2 met15∆::ADE2 trp1∆63 ho∆::SCW11pr-CRE-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP-HPHMX fob1::kan
|
Tyler lab [43]
|
DCY05
|
MATa his3∆ leu2∆ met15∆ ura3∆ GFP-PNC1
|
This study
|
gtr1∆
|
MATa his3∆ leu2∆ met15∆ ura3∆ gtr1::KAN
|
Taiwan Yeast Bioresource Center
|
HSY002 (pGAL-GTR1)
|
MATa his3∆ leu2∆ met15∆ ura3∆ pGal-GFP-GTR1::KAN
|
This study
|
To overexpress GTR1, we used a plasmid generated by Longtine et al that replaced the GTR1 promoter with a regulated promoter by galactose with a GFP tag[44].
General experimental procedure
Optical rotations of compounds were measured on a JASCO P-1010 polarimeter using a 10-cm cell. UV spectra were recorded on a Hitachi UV-2010 spectrophotometer, and IR spectra were recorded on a JASCO FT-IR-4000 spectrometer. NMR spectra were recorded on a Bruker AVANCE-400. Proton and carbon NMR spectra were measured on a 400-MHz instrument. Mass spectrometry data were obtained on a Finnigan TSQ 700 mass spectrometer. HPLC separations were performed on a HITACHI L-2130 HPLC equipped with a HITACHI L-2400 refractive index detector.
Chemicals
Peptone was obtained from OXOID (LP0037; Basingstoke, UK), and yeast extract was obtained from BD (212750; Sparks, MD, USA). DMSO (D8418), estradiol (E2758-1G) and ADH (A7001-15KU) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and TCA was obtained from Nippon Shiyaku Kogyo (Osaka, Japan).
TCM Materials
All of the TCMs were supplied and authenticated by the Chuang Song Zong pharmaceutical company (Pingtung, Taiwan). A voucher specimen (CGU-PC-1) was deposited in the herbarium of Chang Gung University, Taoyuan, Taiwan.
Extraction and Isolation of P. corylifoliaThe dried fruits (5.4 kg) of P. corylifolia were repeatedly extracted with ethanol (11 L × 4) and extracted 5 times with ethanol at 70°C for 4 hr (11 L × 5). The combined crude extracts (1.4 kg) were partitioned sequentially between H2O (842.38 g) and n-hexane (557.62 g). The wet n-hexane layer was separated by silica gel chromatography using sequential mixtures of hexane and ethyl acetate. The 20:1=H:E mixture eluted 6; the 10:1=H:E mixture eluted (in order of elution) 1, 2, and 7; the 7:1=H:E mixture eluted (in order of elution) 5, 8, 9, 11, 13, and 18; the 5:1=H:E mixture eluted (in order of elution) 12, 14, 15, 16, and 17; and the 1:1=H:E mixture eluted (in order of elution) 3, 4, and 10.
Mother enrichment program (MEP)
The liquid aging assay was performed as previously described [12]. Briefly, cells were cultured at 30°C in YEPD medium overnight, and the cultures were diluted to A600=0.25 and recovered to the log phase. Cultures were counted and inoculated in culture tubes at a cell density of 2 X 103 cells/ml. The mixtures contained 17 β-estradiol at a final concentration of 1 µM, and they were incubated in a roller drum at 50 rpm and 30°C for 60 hr. Each group was monitored by harvesting samples at the indicated time points. The collected samples were washed and plated in solid media, and the viability is reported as CFUs per 500 μl.
Micromanipulation assay
The micromanipulation of the yeast cells was carried out as described previously [45]. Prior to analysis, strains were plated onto fresh solid medium and grown for 2 days at 30°C. Single colonies were grown in YEPD medium overnight at 30°C, and a small number of cells were then plated onto a fresh YPD plate for lifespan analysis. After overnight growth on the lifespan plates, the cells were arrayed on the plate using a micromanipulator and allowed to grow for approximately 3 hr. Virgin daughter cells were selected and subjected to lifespan analysis. In the lifespan experiments, the plates were incubated at 30°C during the day and stored at 4°C overnight. Each experiment consisted of more than 70 mother cells and was independently repeated 3 times.
Immunoblotting
Yeast protein was prepared as previously described [46]. The samples were loaded in 10% SDS-polyacrylamide gel. The following primary antibodies were used for immunoblotting at a 1:1000 dilution: anti-Pgk1 (ab113687; Abcam, UK) and anti-GFP (G1544; Sigma-Aldrich; St. Louis). Secondary antibodies were obtained from Sigma-Aldrich (St. Louis) and used at a 1:100000 dilutions. Images were acquired with a Wealtec KETA-CL imaging system.
Intracellular NAD+ content
Cells were harvested (4*108 per group), washed with 50% DMSO and water, pelleted and stored at -80°C. The extractions were performed with 250 μl of 1 M formic acid saturated with butanol. After incubation for 30 min on ice, 62.5 μl of 100% TCA (W/V) was added to each sample, and the samples were then incubated on ice for an additional 15 min. The samples were pelleted by centrifugation at 17000 g for 5 min, the supernatant was transferred to another Eppendorf tube, the pellets were washed with 125 μl of 20% TCA, and the material was repelleted by centrifugation. The combined supernatants were used for the following tests. For analysis, each sample was assembled in reaction buffer (100 μl of extract, 400 μl of 360 mM Tris, 240 mM lysine, pH 9.7, 0.24% (v/v) EtOH; the control group had 5 μl of water, and the ADH group had 5 μl of 5 mg/ml alcohol dehydrogenase). After 5 min at room temperature, the absorbance of each sample was measured at 340 nm. The NAD+ content of the cells in each sample was determined relative to the water group (as the basal NADH level) and the alcohol ADH catalytic group.
Fluorescence microscopy
Yeast cells were recovered twice to the log phase in YEPD. For the Msn2 relocation assay, Msn2-GFP cells were cultured in 1 ml of culture liquid in a microtube for 1.5 hr in a rolling drum. The samples were washed with 100 mM HEPES and stained with Hoechst (#33342; Sigma-Aldrich; St. Louis, MO, USA) containing 3.7% formaldehyde for 5 min at room temperature. The samples were washed twice with HEPES and spotted onto slides for observation. The Pnc1-GFP strain was harvested, and 2*107 cells were washed twice with YEP. Each group was collected for further analysis by a Nikon ECLIPSE Ni-U plus fluorescence microscope equipped with a 100x oil objective. Images were acquired with a DS-U3 CCD camera and controlled using NIS-Element BR 4.0 software.
Molecular docking analysis
The crystal structure of yeast Gtr1-Gtr2 was used for analysis (PBD ID: 3R7W). The docking analysis was performed using BIOVIA Discovery Studio v19.1.0.18287. The protein was prepared, and the ligand was minimized before docking. Docking was performed using the standard protocol.
Chemical shift experiment
NMR binding experiments were carried out with peptide substrates, which included the GTR1 peptide Ile-Gly-Thr-Ser-Ile-Trp-Asp-Glu-Ser-Leu. 1H NMR spectra were recorded on a 400-MHz Bruker DRX spectrometer equipped with a TXI cryoprobe at 25°C. Data sets were the average of 100 scans. All NMR spectra were collected in the presence of peptide, corylin, and 99.9% DMSO-d6.
Cell culture
HUVECs were kindly provided by Professor Shu-Huei Wang of College of Medical, National Taiwan University Department of Anatomy and Cell Biology (Taiwan). HUVECs were cultured in M199 supplemented with HEPES, ECGS (Millipore), heparin, NaHCO3, L-glutamine, sodium pyruvate, and FBS (20% final concentration). The cells were grown in an atmosphere of 5% CO2 at 37°C and sub-cultured by trypsinization with trypsin-EDTA (Lonza). Cells were seeded at 8*104 in 3.5-cm culture dishes and passaged such that the monolayers never exceeded 90% confluency. A sample was collected in every passage for cell extraction, RNA extraction and SA-β-gal staining. The cells were propagated until senescence, and cell numbers were determined when sub-cultured. Population doublings (PDs) were estimated using the following equation: PDL = 3.32 (log (total viable cells at harvest/total viable cells at seeding).
SA-β-gal staining
HUVECs were treated with or without corylin. Then, when the cells reached 90% confluency, they were washed twice with phosphate-buffered saline (PBS) and fixed for 5 min with 2% formaldehyde and 0.2% glutaraldehyde. The cells were then incubated at 37 °C for 18 hr with a staining solution (40 mmol/L citric acid, sodium phosphate, pH 6.0, 1 mg/mL 5-bromo-4-chloro-3-isolyl-β-D-galactoside (X-gal, Sigma), 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L NaCl, and 2 mmol/L MgCl2). Senescence-associated (SA)-β-gal-positive cells were observed by microscopy, and over 300 cells were counted in three independent fields.
RNA sequencing
RNA was collected from HUVECs and extracted by TRIzol (T9424, Sigma, USA). The extracted RNA samples were sent to Genomics (Taipei, Taiwan) for analysis, and a library was constructed. Briefly, after quality control of raw reads, mRNA was purified using reverse transcriptase and a random primer to synthesize single-strand cDNA, and dUTP was used in place of dTTP to generate double-stranded cDNA. A single ‘A’ nucleotide was added to the 3’ end of ds cDNAs. Then, multiple indexing adapters were ligated to the 5’ and 3’ ends of ds cDNA. PCR was used to selectively amplify the DNA fragments with adapters on both ends. The library was validated on an Agilent 2100 Bioanalyzer and Real-Time PCR System. The gene ratio and expression change were summarized by Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway data and Gene Ontology (p-value <0.05, log-2> 2).
Animals
C57BL/6 mice (34 weeks of age) were provided by the National Laboratory Animal Center (NLAC), NAR Labs, Taiwan. All mice were housed in individual cages and maintained at room temperature at 23 ± 1°C with a 12-hr dark/light cycle. All animal procedures were approved by Chang Guan University Animal Care Center (IACUC protocol no. CGU15-150). After reaching 40 weeks of age, the mice were randomly divided into two groups: Group I (HFD), fed a HFD with 54% fat (Table 1) (n=30), and Group II (HFD/C), fed a HFD containing corylin (1 g corylin/1 kg HFD) (n=30). Corylin (purity ≥ 98 %) was purchased from Shanghai BS Bio-Tech Co., Ltd., China.
|
HFD
|
HFD/C
|
|
|
g/kg diet
|
Source
|
Casein
|
254
|
254
|
Sigma
|
Cellulose
|
61
|
61
|
Sigma
|
Sucrose
|
321
|
321
|
General Stores
|
Soybean oil
|
10
|
10
|
General Stores
|
Unsalted butter
|
290
|
290
|
General Stores
|
AIN-93G Mineral mixture
|
44.5
|
44.5
|
MP Biomedicals
|
Ain-93 Vitamin mixture
|
12.5
|
12.5
|
Dyets, Inc.
|
L-Cystine
|
4
|
4
|
Sigma
|
Choline bitartrate
|
3
|
3
|
Sigma
|
Corylin
|
-
|
1
|
Shanghai BS Bio-Tech
|
kcal/g
|
5.016
|
|
|
|
|
CHO calorie/total calories (%)
|
25.7
|
|
Fat calorie/total calories (%)
|
54.0
|
|
Protein calorie/total calories (%)
|
20.3
|
|
Pharmacokinetic analysis
Blood samples were collected at 1, 3, 6, 9, 12 and 15 hr after corylin oral gavage, and serum samples were then separated. Serum samples (100 μl) were mixed with ice-cold acetonitrile (150 μl) at 4°C for 1 hr and then centrifuged for 15 min at 15,000 × g and 4°C. After centrifugation, the supernatant (150 μl) was harvested in a new Eppendorf tube, and 150 μl of ddH2O was added. The sample was analyzed by an LC/FTMS system.
Fasting blood glucose level
To determent the blood glucose, mice was fasting for 16 hr. The blood samples were obtained by cutting the tip of tail and the blood glucose was measured by ACCU-CHEK (Roche).
The rotarod test
Mice were tested on a rotarod at 81 weeks of age. The mice were habituated for 3 days, during which they were placed on the rotarod at a constant speed (4 rpm) and had to remain on the rotarod for 1 min every day. Testing on each day consisted of 5 trials with a 10 min rest between each trial. The acceleration trial began with the rotarod set at an initial rate of 4 rpm, accelerating to a maximum of 40 rpm within 5 min. The constant trial began with the rotarod set at a rate of 4 rpm and lasted for a maximum of 200 s. The latency to falling was recorded, and the average latency to falling was calculated for each trial.
Rearing behavior
Rearing behavior was analyzed in obese mice fed a HFD with or without corylin for 35 weeks (75 weeks of age) using the OxyletPro System. Before the metabolic rate was monitored, the mice were individually caged for 24 hr to acclimate to the system.
Serum parameter analysis
The serum LDL, TG and total cholesterol levels were measured using specific reagent kits (Fortress Diagnostics, Antrim, Northern Ireland). The serum glucose and high-density lipoprotein (HDL) levels were measured using specific reagent kits (Randox, Antrim UK). Insulin was measured by ELISA (Mercodia, Uppsala, Sweden). HOMA-IR was calculated as the fasting insulin level (mU/L) ×blood glucose level (mmol/L)/22.5. The serum AST and CK levels were measured using a Fuji Dri-Chem 4000i analyzer (Fujifilm, Tokyo, Japan).