Total RNA from isolated islets were extracted using the RNAiso Plus (Takara, Dalian, China) and reverse-transcribed into cDNA using the Prime Script RT reagent kit (Takara, Dalian, China). Data for triplex real-time PCR assay were collected with an AB 7500 Real time PCR system (Applied Biosystems, Inc. United States). Reactions were performed with SYBR Green PCR Master Mix (Takara, Dalian, China) under following thermal cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Conventional PCR reactions were performed in a 20 μl reaction mixture containing 0.5 μmol/L primers and 12.5 mg/L cDNA. Relative gene expression data were analyzed in the 2-ΔΔCT method, and the results were expressed as the expression fold over the control group. In exception, since there is no CETP mRNA expression in WT mice, the relative CETP mRNA of adipose tissue from aP2-CETPTg mice and NFR-CETPTg mice and of islets from NFR-CETPTg mice were expressed as 2^-ΔCT. The following primer sequences were used:
CETP310-A, 5′-TAGTGTTTACAGCCCTCATGAACAGCAAAG- 3′;
CETP310-B, 5′-CTCCATCTCCGTACTCCTAACCCAACTTCC- 3′.
Ki-67 forward, 5′-ATCATTGACCGCTCCTTTAGGT- 3′;
Ki-67 reverse, 5′-GCTCGCCTTGATGGTTCCT- 3′.
Pdx1 forward, 5′-GATGAAATCCACCAAAGCTCA- 3′;
Pdx1 reverse, 5′-AGAATTCCTTCTCCAGCTCCA- 3′.
BETA2 forward, 5′-ACTCCAAGACCCAGAAACTGTC- 3′;
BETA2 reverse, 5′-ACTGGTAGGAGTAGGGATGCAC- 3′.
IL-1β forward, 5′- TGCAGAGTTCCCCAACTGGTACATC- 3′;
IL-1β reverse, 5′- GTGCTGCCTAATGTCCCCTTGAATC- 3.
TNF-α forward, 5′- CCCTCACACTCAGATCATCTTCT- 3′;
TNF-α reverse, 5′- GCTACGACGTGGGCTACAG- 3′.
IL-6 forward, 5′- CCAAGAGGTGAGTGCTTCCC- 3′;
IL-6 reverse, 5′- CTGTTGTTCAGACTCTCTCCCT- 3′.
IL-10 forward, 5′- GCTCTTACTGACTGGCATGAG- 3′;
IL-10 reverse, 5′- CGCAGCTCTAGGAGCATGTG- 3′.
CHOP forward, 5′-CATACACCACCACACCTGAAAG- 3′;
CHOP reverse, 5′-CCGTTTCCTAGTTCTTCCTTGC- 3′.
Atf6 forward, 5′- GACTCACCCATCCGAGTTGTG- 3′;
Atf6 reverse, 5′- CTCCCAGTCTTCATCTGGTCC- 3′.
Xbp1 forward, 5′- AGCAGCAAGTGGTGGATTTG- 3′;
Xbp1 reverse, 5′- GAGTTTTCTCCCGTAAAAGCTGA- 3′.
Ldlr forward, 5′-CTGTTCCCA CCTCTGTTT AC- 3′;
Ldlr reverse, 5′- AGTGAG ATACGGCGA ATA GA- 3′.
HMGCS forward, 5′-TGG GAC CAA CCT TCT ACC TC- 3′;
HMGCS reverse, 5′-CAT CAA GGA CAG CTC ACC AG- 3′.
Srebp2 forward, 5′-TAACCCCTTGACTTCCTTGCT- 3′;
Srebp2 reverse, 5′-TGCTCTTAGCCTCATCCTCCA- 3′.
Histology and immunohistochemistry
Pancreata were isolated, fixed with 10 % buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin for histological analysis. Pancreatic sections were scanned by Zeiss microscope at ×200 magnifications and islet size was analyzed with AxioVision software. For analysis of pancreatic islet apoptosis, double staining of in situ cell death detection fluorescence and insulin fluorescent immunohistochemistry were performed. The following primary antibodies were used: Rabbit anti-insulin polyclonal antibody, 1:50 (Proteintech Group, Inc. Chicago, USA); Secondary antibodies DyLight594 rabbit anti-goat IgG[H+L], 1:300 (MultiSciences Biotech, Hangzhou, China). The beta cell apoptosis was assessed by transferase-mediated dUTP nick-end labeling (TUNEL) assay (keyGEN, Nanjing, China) according to the manufacturer’s protocol. For the determination of the overall rate of apoptosis, each section stained for insulin, TUNEL, and DAPI were imaged at 400 magnification (40 × objective), and the total number of TUNEL-positive cells per field were quantified. To determine the frequency of beta cell apoptosis, the number of cells positive for TUNEL was quantified in each islet and expressed as percentage of the total number of beta cells (n = ~4000). Beta cells were counted in 16 week old of age male mice per genotype (n = 4–5).