Animals
The procedures for animal care and experiments were approved by the Institutional Animal Care and Use Committees of Seoul National University (SNU-170417-23-2).
Six-week-old male C57BL/6 mice (wildtype and NAG-1 Tg) were used in this study. The animals were housed in a temperature-controlled (21 ± 2°C) room with a 12-h dark/light cycle. The mice had free access to food and water.
All mice were maintained on HFD (60% from fat, D12492, Research Diets, Inc., New Brunswick, NJ) throughout the experimental period and the study was carried out in compliance with the ARRIVE guidelines.
Experimental design
Mice were grouped into the following two groups: WT mice and NAG-1 Tg mice (n = 5 for each group). After three weeks of maintenance on HFD, the mice were and intraperitoneally injected with STZ (40 mg/kg bodyweight; #13104, Cayman Chemical, MI, USA) after 8 h of fasting. The mice were continued to be maintained on HFD (Fig. 1). At week 10, the mice were again administered with STZ. The levels of FBG were analyzed to confirm diabetes in mice at week 11.
Measurements of bodyweight and fasting FBG and insulin levels
Bodyweight and FBG were initially measured at week 6 and monitored every week thereafter until the end of the experimental period. The FBG levels were measured using the fresh blood samples collected from the mouse tail vein with Accu-Chek® Performa (Roche Diabetes Care, Indianapolis, IN, USA). The fasting serum insulin levels were determined using the ultra-sensitive mouse insulin ELISA kit (#90080, Crystal Chem, IL, USA), following the manufacturer’s instructions.
IPGTT
The mice were allowed to fast for 18 h and subjected to IPGTT on week 11. Mice were intraperitoneally injected with fresh glucose solution (2 g/kg bodyweight, A2494001, Gibco, Gaithersburg, MD, USA) and the blood glucose level was measured at 0 (baseline),15, 30, 60, and 120 min post-glucose injection using Accu-Chek® Performa. The AUC value of insulin was calculated using GraphPad Prism software (GraphPad Prism 8 Software Inc., San Diego, CA, USA).
IPITT
At week 12, the mice were allowed to fast for 6 h before the experiment. The serum glucose and insulin levels were measured using Accu-Chek® Performa at baseline (0 min) and after intraperitoneal injection of insulin (0.75 U/kg bodyweight) (I0516, Sigma-Aldrich, St. Louis, MO, USA). The blood glucose levels and serum insulin levels were also determined at 15, 30, 60, and 120 min post-injection. Insulin resistance was calculated using the following formula: HOMA-IR index = glucose level × (serum insulin level/22.5)
Histological analysis
The pancreas, liver, muscle, iWAT, eWAT, and BAT were fixed with paraformaldehyde and subjected to H&E staining. The tissues were fixed in 10% neutral formalin, embedded in paraffin, sectioned (thickness: 5 µm), and stained with H&E. The images were captured using Panoramic SCAN (3DHISTECH, Budapest, Hungary).
Immunohistochemistry
The tissue blocks were fixed in 10% formalin, embedded in paraffin, sectioned to 5-µm-thick sections. The sections were incubated in citrate buffer for 1 min in a microwave (Immunobioscience, Mukilteo, WA, USA) to retrieve the antigens. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline for 1 h at room temperature. Immunoreactivity was detected using an ultra-sensitive ABC staining kit (Thermo Scientific, Rockford, IL, USA), following the manufacturer’s instructions. The sections were incubated with rabbit polyclonal anti-cleaved caspase-3 (#9661S, Cell Signaling Technology, Beverly, MA, USA) antibody at 4°C overnight. The negative control samples were incubated with secondary antibodies but not primary antibody. The sections were incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate (ImmPACT® DAB kit, VECTOR Laboratory, Burlingame, CA, USA) at room temperature for 45 s and counterstained with hematoxylin (VECTOR Laboratory, Burlingame, CA, USA) for 30 s. Coverslip-mounted sections were observed using the Panoramic SCAN slide scanner.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from tissues using the RNeasy mini kit (QIAGEN, Hilden, Germany), following the manufacturer’ s instructions. The 1 µg of RNA was reverse transcribed using Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Following by amplifying DNA by PCR using MiniAmp Plus Thermal Cycler (A37835, Applied Biosystems, Marsiling Industrial Estate, Singapore) with GoTaq® Green PCR Master Mix (Promega, Madison, WI, USA). The primers are human NAG-1 (F: 5’-CTCCAGATTCCGAGAGTTGC-3’ and R: 5’-AGAGATACGCAGGTGCAGGT-3’) and mouse β-actin (F: 5’-GGCTGTATTCCCCTCCATCG-3’ and R: 5’-CCAGTTGGTAACAATGCCATGT-3’). The thermal cycling conditions were as follows: initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min, then final extension at 72°C for 5 min. The products were electrophoresed on 1.5% agarose gel and were visualized under UV light using Alliance Q9 mini (Cambridge, UK).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from tissues using the RNeasy mini kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. RNA (1 µg) was reverse transcribe into cDNA using the first strand cDNA synthesis kit (K1612, Thermo Scientific™, Waltham, MA, USA) in a MiniAmp Plus Thermal Cycler (A37835, Applied Biosystems, Marsiling Industrial Estate, Singapore). The primers are used for qRT-PCR analysis are shown in Table 1. The relative level of each RNA was measured using qRT-PCR with SYBR Green reagents (PowerUp™ SYBR™ Green Master Mix, A25741, Applied Biosystems, Thermo Scientific™) in QuantStudio™ 1 real-time PCR system (Applied Biosystems, Marsiling Industrial Estate, Singapore). The expression level of the target gene was normalized to that of Gapdh (housekeeping gene). The relative gene expression was calculated using the comparative Ct (2−ΔΔCt) method.
Table 1
Real-time PCR primers used in this study.
|
Forward primer (5’->3’)
|
Reverse primer (5’->3’)
|
Adiponectin
|
ATCTGGAGGTGGGAGACCAA
|
GGGCTATGGGTAGTTGCAGT
|
Leptin
|
AGGTAGGGATGGGTAGAGCC
|
GTGTGCTGCTTGGGAGTTTC
|
GLUT4
|
GGTGTGGTCAATACGGTCTTCAC
|
AGCAGAGCCACGGTCATCAAGA
|
PI3K
|
CAAACCACCCAAGCCCACTACT
|
CCATCAGCAGTGTCTCGGAGTT
|
Ptpn1
|
GCGCTTCTCCTACCTGGCTGTCAT
|
ACGTGCTCGGGTGGAAGGTCTA
|
IRS-1
|
TGTCACCCAGTGGTAGTTGCTC
|
CTCTCAACAGGAGGTTTGGCATG
|
AKT
|
GGACTACTTGCACTCCGAGAAG
|
CATAGTGGCACCGTCCTTGATC
|
GSK3β
|
CATAGTGGCACCGTCCTTGATC
|
CCAACTGATCCACACCACTGTC
|
PPARγ
|
GTACTGTCGGTTTCAGAAGTGCC
|
ATCTCCGCCAACAGCTTCTCCT
|
FOXO1
|
CTACGAGTGGATGGTGAAGAGC
|
CCAGTTCCTTCATTCTGCACTCG
|
AS160
|
GCCAACAGTCTTGCCTCAGAGA
|
CGTCTTCGGAACTGTGGAGAGT
|
mTORC1
|
CTTCCTATCCGTCTTGGCAGAC
|
CTCCAGACAGATGGCAATCAGG
|
mTORC2
|
CAGTGTGAGGTCCTTTCCATCC
|
GCCATAGATGCTTGCGACTGTG
|
NLRP3
|
TGCTCTTCACTGCTATCAAGCCCT
|
ACAAGCCTTTGCTCCAGACCCTAT
|
IL-18
|
TGGTTCCATGCTTTCTGGACTCCT
|
TTCCTGGGCCAAGAGGAAGTGATT
|
IL-1β
|
TGGACCTTCCAGGATGAGGACA
|
GTTCATCTCGGAGCCTGTAGTG
|
ASC
|
CTGCTCAGAGTACAGCCAGAAC
|
CTGTCCTTCAGTCAGCACACTG
|
Caspase-1
|
GGCACATTTCCAGGACTGACTG
|
GCAAGACGTGTACGAGTGGTTG
|
GAPDH
|
CATCACTGCCACCCAGAAGACTG
|
CATCACTGCCACCCAGAAGACTG
|
Statistical analysis
Data processing and analysis were performed using GraphPad Prism 8.0.1. All experimental data are represented as the mean ± standard error of the mean for parametric data. The means between two groups were analyzed using the Student's t-test. The data of NAG-1 Tg mice were compared with those of controls (WT mice). The differences were considered significant at *p < 0.05, **p < 0.01, or ***p < 0.001.