Plasmids, cells and adenovirus preparation
The construct pcDNA3 CA-Akt was a kind gift from J. R. Woodgett (University of Toronto). Dominant negative (DN) Smad2 was kindly provided by R. Derynck (University of California, San Francisco). ALK7 cDNAs, including wild-type (Wt), constitutively active (CA; the threonine-194 was replaced with aspartic acid) and DN (the lysine-222 was replaced with arginine) forms of ALK7, were made as reported previously [20, 21]. MIN6 and INS-1 cells were a kind gift from M. Wheeler (University of Toronto); InRIG9 cells  were provided by D. Drucker (University of Toronto). The culture medium for MIN6 and INS-1 cells was RPMI 1640 (Invitrogen, Carlsbad, CA, USA) containing 10 mmol/l HEPES, 10% fetal bovine serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, 1 mol/l sodium pyruvate, 50 μM 2-mercaptoethanol and 10 mol/l NaOH; DMEM was used for INR1G9 cells  and acinar AR42J cells . Cells were grown in monolayer cultures and were maintained at 37°C in a humidified atmosphere with 5% CO2. In studies involving serum starvation, serum was replaced with 0.1% BSA in the medium.
Adenoviral ALK7 constructs were made using the AdEasy system (Cloncancer.org) . Briefly, different forms of ALK7 plasmids were first subcloned into a shuttle vector (pAdTrack-CMV). After linearising by digestion with PmeI, the plasmids were cotransformed into E. coli BJ5183 cells with an adenoviral backbone plasmid (pAdEasy-1). Recombinants were selected for kanamycin resistance and were further confirmed by restriction enzyme analysis with BglII. After linearisation with PacI, the recombinant plasmids were transfected into adenovirus packaging HEK293 cells. The viruses were purified as described previously .
Insulin concentrations were measured using an insulin RIA kit (Linco Research, St Louis, MO, USA) as previously described . INS-1 cells grown in 24-well plates to 85–90% confluency were rinsed twice and incubated with Krebs–Ringer bicarbonate buffer (KRB) containing 115 mol/l NaCl, 5 mol/l KCl, 24 mol/l NaHCO3, 2.5 mol/l CaCl2, 1 mol/l MgCl2, 10 mol/l HEPES and 0.1% BSA for 60 min. Cells were incubated in KRB in the presence of various concentrations of glucose (dextrose; Merck, Darmstadt, Germany) as indicated. After 30 min of incubation, the media were collected and insulin levels measured by RIA using a rat insulin RIA kit (Linco Research, St Charles, MO, USA) according to the manufacturers instructions. The insulin secretion was normalised to the cellular protein content. Protein was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).
Adenoviral infection and transient transfections
INS-1 cells were incubated with adenovirus for 2 h, rinsed, and placed in complete medium to recover for an additional 22 h. For some experiments, 6 h after adenoviral infection of the INS-1 cells, cells were washed and transfected (2.5 μg per well on a six-well plate) with control pcDNA3, DN-Smad2, CA-Akt or DN-ALK7 plasmids using Lipofectamine 2000 Plus (Invitrogen, Mississauga, ON, Canada) for an additional 16 h according to the manufacture’s instructions. Pilot studies were done to evaluate the transfection efficiency. Lipofectamine yielded an 80–85% transfection rate in the beta cells, as evaluated by expression of green fluorescent protein (GFP).
Rat islets were isolated from the pancreas of male Sprague–Dawley rats (weight 150–200 g), obtained from Charles River Canada (Montreal, QC, Canada), using collagenase digestion and dispersion into single islet cells as described previously . Single islet cells were seeded onto glass coverslips coated with poly-d-lysine (Sigma Chemical, St Louis, MO, USA) and allowed to adhere prior to use in immunofluorescence experiments. For some experiments, islets were placed into 10 cm tissue culture plates and were hand-picked for use in mRNA or protein extraction studies.
Total RNA was extracted using Trizol (Invitrogen) following the manufacturer’s instructions. One hundred nanograms of template was used to analyse the expression of ALK7, using the One-step RT-PCR kit (Qiagen, Valencia, CA, USA) in a final volume of 25 μl containing Qiagen RT-PCR buffer and 2 μl of Qiagen One-step RT-PCR enzyme mix, 400 μmol/l of each dNTP and 0.48 μmol/l of forward and reverse primers specific to ALK7. The sequences of the primer pairs were as follows: (1) ALK7: forward, 5′- CCTCTGGATCTGGCTCTGGTCTAC-3′; reverse, 5′-GTCCGCTATGGCACAAGTTTCAC-3′. 2) BCL2-associated X protein (BAX): forward, 5′-AGACAGGGGCCCTTTTGCTTC-3′; reverse, 5’-TGCAGCTCCATGTTACTGTCC-3’. RT-PCR was performed at 50°C for 30 min, and 95°C for 15 min followed by 35 cycles of PCR at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min, and culminating in extension for 15 min at 72°C. Products were separated on an agarose gel and visualised using ethidium bromide.
Western blot analysis
Cells were lysed in RIPA (radio-immunoprecipitation assay) lysis buffer containing the protease inhibitors phenylmethylsulphonylfluoride (PMSF) (1 mol/l) and EDTA (1 mol/l); Na3VO4 (1 mol/l); and NaF (1 mol/l). Rat brain or pancreatic tissue (50–100 mg total wet weight) was minced and homogenised in RIPA lysis buffer. About 20 μg of protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes using semi-dry transfer (Bio-Rad Laboratories). The membranes were probed with primary poly- or monoclonal anti-ALK7 antibodies (1:1,000), anti-phospho-Smad2 (1:250; R and D Systems, Minneapolis, MN, USA), anti-Akt and anti-phospho-Akt (1:1,000), anti-cleaved caspase-3 (1:1,000; Cell Signaling, Mississauga, ON, Canada) or anti-beta-actin (1:2,000; Sigma Chemical), visualised with horseradish peroxidase-conjugated secondary antibodies using electrochemiluminescence (ECL) Plus detection (Amersham, Mississauga, ON, Canada), and analysed by densitometry.
Islet cells or cell lines were fixed using 4% paraformaldehyde and permeabilised using 0.1% Triton X-100 in PBS, and then blocked (3% bovine serum albumin in PBS, 1 h) prior to overnight incubation with the appropriate antibodies: mouse or goat anti-ALK7 IgG (1:500; R and D Systems), guinea-pig anti-insulin (1:1,000) or rabbit anti-glucagon (1:1,000; DakoCytomation, Mississauga, ON, Canada). The corresponding fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibodies were used prior to visualisation using confocal microscopy (Zeiss LSM 510).
Proliferation and cell death assays
Cell growth and death were determined by cell counting using a light microscope and Trypan Blue exclusion, which discriminates dead cells (stained) from living cells (non-stained) . Cell proliferation was measured in non-infected cells or cells infected with various adenoviral ALK7 (Ad-ALK7) or Ad-GFP (control) vectors using 3H-thymidine (Perkin-Elmer, Boston, MA, USA) incorporation, by incubating the cells with the culture medium containing 3H-methylthymidine for 4 h, as described previously .
Twenty-four hours after viral infection, INS-1 cell apoptosis was determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) using the In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. For DNA degradation analysis, floating and adherent cells were combined and centrifuged at 400 g for 5 min and rinsed twice with PBS. The pellet was resuspended in 0.2 ml lysis buffer (100 mol/l NaCl, 10 mol/l Tris [pH 8.0], 1 mol/l EDTA, 0.5% SDS, 0.2 mg/ml proteinase K, 200 μg/ml RNAse A) and incubated at 37°C for 2 h. DNA was then extracted twice with phenol/chloroform and then with chloroform alone. The DNA was then precipitated and washed in 70% ethanol prior to resuspension in 1 mol/l EDTA, 10 mol/l Tris–Cl (pH 8.0) to a final concentration of 20 μg/ml. The DNA fragments were resolved using a 1.5% agarose gel. Naive INS-1 cells treated with H2O2 (50 μmol/l, 24 h) were used as a positive control.
Detection of active form of caspase-3
The INS-1 cells infected with various adenoviral vectors and/or transfected with various vectors were permeabilised and probed using anti-active caspase-3 antibody (1:1,000; Cell Signaling) as described above.