Islet isolation and INS-1E beta cell line culture
Diabetes-prone male P. obesus (Hebrew University Colony, Harlan, Jerusalem, Israel), aged 2.5–3.5 months, were fed a low-energy (9.96 kJ/g) diet (Koffolk, Petach-Tikva, Israel), which maintains normoglycaemia (3–5 mmol/l in this species). Diabetes, defined by random blood glucose levels >8.3 mmol/l, was induced by feeding a high-energy diet (14.23 kJ/g; Teklad Global Diets, Boston, MA, USA) . Islets were isolated from normoglycaemic and diabetic P. obesus by collagenase digestion (Collagenase P; Roche Diagnostics, Mannheim, Germany) as described . The islets were used after repeated washes with Hanks’ balanced salt solution. They were cultured in suspension in RPMI 1640 medium (Biological Industries, Beit-Haemek, Israel) with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l l-glutamine (Biological Industries) and different glucose concentrations. Animal use was approved by the Institutional Animal Care and Use Committee of the Hebrew University and the Hadassah Medical Organization.
INS-1E cells were grown in RPMI 1640 medium containing different glucose concentrations as indicated, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mmol/l sodium pyruvate, 2 mmol/l l-glutamine, 10 mmol/l HEPES and 50 nmol/l 2-mercaptoethanol.
Islets from P. obesus were incubated overnight with medium containing 3.3 or 22.2 mmol/l glucose with and without 250 μmol/l diazoxide. INS-1E cells were incubated overnight with 3.3 mmol/l glucose, then transferred for 2 h to medium containing 11.1 and 22.2 mmol/l glucose with and without 500 nmol/l human insulin (Humulin R; Eli Lilly, Fegersheim, France) or the insulin secretagogues KCl (40 mmol/l), 3-isobutyl-1-methylxanthine (IBMX; 0.5 mmol/l), exendin 4 (100 nmol/l; Bachem, Torrance, CA, USA) and palmitate (0.5 mmol/l). In experiments on the effect of secreted insulin on TXNIP protein levels, INS-1E cells were treated with different insulin secretagogues and palmitate with and without diazoxide or 20 μmol/l of the phosphoinositide 3-kinase (PI3 kinase) inhibitor LY294002.
The role of nitric oxide (NO) in the regulation of insulin signalling and TXNIP levels was studied using the NO donor S-nitroso-glutathione (GSNO) and the NO synthase inhibitor l-NG-nitro-l-arginine methyl ester (L-NAME).
Islet and INS-1E cell extracts were used for quantitative real-time PCR and western blot analyses. Medium was collected for insulin measurement by RIA. All reagents except for insulin and exendin 4 were purchased from Sigma (Rehovot, Israel).
Vector design and virus production
The small interfering RNA (siRNA) sequence targeted against Txnip corresponded to bases 864–886 of the mRNA (accession number NM_001008767). It was generated by annealing of the sense and antisense oligonucleotides: 5′-CGATCTGAGTGAGCACTTTGGTCTGGCTTTCAAGAGAAGCCAGACCAAAGTGCTCACTCAGTTTTTGAT and 5′-ATCAAAAACTGAGTGAGCACTTTGGTCTGGCTTCTCTTGAAAGCCAGACCAAAGTGCTCACTCAGAT (the Txnip siRNA sequences are underlined).
The annealed oligonucleotides were inserted into the plasmid pBS-H1 digested with ClaI and EcoRV downstream to the human H1 promoter to produce the plasmid pBS-H1-shRNA.TXNIP. The Txnip silencing cassette was introduced into the lentiviral vector pSIN18.cPPT.hEF1_p.EGFP.WPRE producing the reporter enhanced green fluorescent protein (EGFP) using the XhoI and EcoRV restriction sites. The plasmids pBS-H1 and the lentiviral vector pSIN18.cPPT.hEF1_p.EGFP.WPRE were kindly provided by Dr M. Gropp (Hadassah Human Embryonic Stem Cell Research Center, Goldyne Savad Institute of Gene Therapy, Hadassah University Medical Center, Jerusalem, Israel).
Transduction of INS-1E cells and generation of Txnip knockdown cells
INS-1E cells were plated in six-well plates and grown overnight to approximately 70% confluence. The cell medium was exchanged for concentrated virus-containing medium for 18–24 h and then replaced with fresh culture medium. Transduced INS-1E cells were plated at low density. EGFP-producing colonies were picked, replated and propagated to give a polyclonal cell line expressing the Txnip silencing cassette.
Quantitative real-time RT-PCR
RNA was extracted from INS-1E cells using Trireagent (Biolab, Jerusalem, Israel); samples of 1 μg total RNA were reverse transcribed using Moloney murine leukaemia virus reverse transcriptase (Promega, Madison, WI, USA). Quantitative real-time RT-PCR for Txnip was performed on a Prism 7000 Sequence Detection System using the TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA). All samples were analysed in triplicate and corrected for the 18S ribosomal subunit (Applied Biosystems) used as an internal control.
Transient transfection experiments
INS-1E cells were plated in 24-well plates and grown overnight to approximately 70% confluence. Cells were co-transfected with a reporter construct encoding the human TXNIP promoter region 1,777 bp upstream of the ATG start codon (kindly provided by A. Shalev, University of Wisconsin–Madison, Madison, WI, USA) and pRL-TK control plasmid expressing the luciferase reporter gene (Promega). Transfection was performed in serum-free RPMI medium using Lipofectamine (Invitrogen, Philadelphia, PA, USA) according to the manufacturer’s instructions. Six hours after transfection, the medium was replaced and transcriptional activity was assessed after overnight incubation in medium containing different glucose concentrations. Luciferase activity was determined using a luciferase assay kit (Promega).
Western blot analysis
Expression of TXNIP protein and thioredoxin was studied by a standard western blot technique using monoclonal rabbit antibodies against TXNIP (MBL International, Woburn, MA, USA) and thioredoxin (kindly provided by Dr M. Chevion, Hebrew University, Jerusalem, Israel). Activation of insulin signalling was assessed by analysing the expression and serine 473 phosphorylation of protein kinase B (PKB)/Akt using polyclonal rabbit antibodies (Cell Signaling Technology, Beverly, MA, USA). Molecular activation of apoptosis was assessed by measurement of cleaved vs total caspase 3 using polyclonal goat anti-rabbit antibodies (Cell Signaling Technology). X-ray film densitometry was used for quantification (ImageMaster VDS-CL; Amersham Pharmacia Biotech, Little Chalfont, UK).
Immunoreactivity of P. obesus and rat (INS-1E) insulin was determined using commercial human and rat RIA kits (Linco Research, St Charles, MI, USA). The routine intra-assay coefficient of variation was 4–6% and the interassay coefficient of variation was 6–10%.
Cells were plated in 96-well plates and grown in RPMI 1640 medium containing 11.1 mmol/l glucose until they reached 70% confluence. The cells were then cultured at 11.1 or 27.8 mmol/l glucose for 48 h. The cells were lysed and oligonucleosomes in the cytosol indicative of apoptosis-induced DNA degradation were quantified using the cell death ELISAPLUS assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
Data presentation and statistical analysis
Data are shown as mean±SEM. The statistical significance of differences between groups was determined by one-way ANOVA followed by the Newman–Keuls test using the InStat statistical program (Graphpad Software, San Diego, CA, USA). A paired-sample t test was used when the difference between a reference (taken as 100%) and the test was analysed. A p value of less than 0.05 was considered significant.