Adult, inbred male Wistar–Furth rats purchased from Scanbur (Sollentuna, Sweden) were used. The animals had free access to pelleted food and autoclaved tap water. All experimental procedures were approved by the Animal Ethics Committee for Uppsala University.
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise mentioned.
Islets were isolated by using collagenase digestion followed by separation on a density gradient. Briefly, under deep anaesthesia with sodium pentobarbital (Apoteket, Gothenburg, Sweden; 200 mg/kg i.p.), a laparatomy was performed and the pancreas exposed. After ligation at the ampulla of Vater, 5 ml collagenase solution (from Clostridium histolyticum; F. Hoffman-La Roche, Basel, Switzerland) was injected into the pancreas via the common bile duct. The animal was killed and the pancreas dissected from surrounding tissues, removed and incubated in a water bath for approximately 16 min at 37°C. The islets were separated by a density gradient (Histopaque-1077) centrifugation at 900×g for 22 min. After washing, islets were handpicked and groups of 150 islets were maintained free-floating at 37°C (air/CO2; 95:5) in culture medium (CM) consisting of RPMI 1640 supplemented with l-glutamine (2 mmol/l), benzylpenicillin (100,000 U/l; Hoffman-La Roche), streptomycin (0.1 g/l; Hoffman-La Roche) and 10% (vol./vol.) FCS for 4-6 days. CM was changed every 48 h.
Isolation of islet endothelial cells
Outgrowth of islet stromal cells on a collagen matrix was stimulated using a modification of a previously described protocol . Briefly, 20 hand-picked, apparently clean islets were transferred onto a collagen matrix (1.8 mg collagen type 1/ml; Nutacon, Leimuden, the Netherlands) in a 24 well culture dish. Islets were cultured at 37°C (air/CO2; 95:5) in 1 ml endothelial CM (ECM), this being CM with 20% (vol./vol.) FCS and with 100 μg endothelial cell growth supplement/ml.
Expanding cells were detached before reaching confluence with 0.25% (wt/vol.) trypsin–EDTA solution for <5 min at room temperature. The suspension was washed twice in CM. The endothelial cells were extracted from the cell suspension by a Dynabead (Dynal Biotech, Oslo, Norway) method previously described . By the use of Bandeiraea (Griffonia) simplicifolia (BS-1)-coated Dynabeads, endothelial cells were separated from contaminating cells, generating a purity of >90% .
Purity and characterisation of endothelial cell preparations
As a control of endothelial cell purity, samples of endothelial cells were trypsinised, centrifuged onto poly-l-lysine-coated glass slides, fixed in 4% (vol./vol.) paraformaldehyde for 2 h, and subsequently dehydrated. The slides were stained to detect endothelium with a rat anti-CD31 antibody (HyCult Biotechnology, Uden, the Netherlands) using the EnVision+ system (Dako, Glostrup, Denmark). Antigen retrieval was performed by boiling for 20 min with Target Retrieval Solution (pH 9.0; Dako) in a microwave oven (750 W). The slides were counterstained with haematoxylin. After staining, the fraction of CD31-positive cells was manually counted in a light microscope (×400). Isolated islet endothelial cells were also cultured on collagen-coated glass slides (1.8 mg collagen type 1/ml; Nutacon) and fixed in zinc fixative (Becton Dickinson Biosciences, San Jose, CA, USA [BD]) for 30 min. After incubation with primary antibodies against CD31 (Chemicon, Chandlers Ford, UK), vascular endothelial cadherin (VE-cadherin; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and alpha smooth muscle actin (α-SMA) the slides were washed and incubated with goat-anti-mouse Alexa 568 (Molecular Probes, Eugene, OR, USA). The slides were then washed, stained with Hoechst, and mounted with Fluoromount-G (Southern Biotechnology, Birmingham, AL, USA). Rat mesenchymal stem cells (MSC) and rat brain microvascular endothelial cells (RBMVEC; Cell Applications, San Diego, CA, USA) were used as negative and positive controls, respectively.
To further characterise the isolated endothelial cells, uptake of fluorescently labelled dioctadecyl indocarbocyanine acetylated LDL (DiI-Ac-LDL; Invitrogen, Lidingö, Sweden) was investigated as endothelial cells have an increased metabolism of Ac-LDL in comparison with supportive stromal cells . Cells cultured on collagen-coated glass slides (1.8 mg collagen type 1/ml; Nutacon) were incubated with 20 μg/ml DiI-Ac-LDL for 4 h at 37°C. The slides were subsequently washed, fixed in zinc fixative (BD), stained with Hoechst and mounted with Fluoromount G (Southern Biotechnology, Birmingham, AL, USA). Rat MSC and human dermal microvascular endothelial cells (HDMEC; PromoCell, Heidelberg, Germany) were used as negative and positive controls, respectively. The cells were analysed using a confocal microscope (C-1/TE200U; Nikon, Tokyo, Japan).
Dynabead-purified endothelial cells were cultured for 2 days in ECM as described above. At this time point the endothelial cells were in exponential growth, but had not reached confluence. The wells were washed with CM to remove all ECM and fresh CM was then added to the wells. The medium was collected 24 h later and centrifuged for 2 min at 600 g to remove cells. The endothelium-conditioned medium, hereafter termed ECCM, was then stored at −70°C.
ECCM with cyclooxygenase 2 (COX-2) inhibition
The endothelial cells were treated as mentioned above, but the CM contained 1 μmol/l of the COX-2 inhibitor rofecoxib (4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone) (Kemprotec, Middlesbrough, UK) or DMSO as vehicle control. The medium was collected, centrifuged for 2 min at 600×g to remove cells and stored at −70°C.
Islet incubation in ECCM
Twenty islets were precultured for 4–7 days in CM, and subsequently cultured in 1 ml of control CM or ECCM in a 24 well culture dish for 24 h. The reason for the pre-culture period was to minimise remnant islet endothelial cells, as studies have shown that ~90% of the intra-islet endothelial cells disappear after 4 days in culture [6, 19].
Glucose-stimulated insulin release
Groups of ten islets were transferred in triplicates to glass vials containing 250 μl Krebs–Ringer bicarbonate buffer supplemented with 10 mmol/l HEPES and 2 mg/ml BSA (MP Biomedicals, Irvine, CA, USA) (hereafter referred to as KRBH buffer). The KRBH buffer contained 1.67 mmol/l d-glucose during the first hour of incubation at 37°C (O2/CO2; 95:5). The medium was removed and replaced by 250 μl KRBH supplemented with 16.7 mmol/l glucose and the incubation continued for a second hour. The medium was retrieved and stored at −20°C. To measure the total insulin content and DNA content, the islets were harvested, pooled in groups of 30, and homogenised by sonication in 200 μl redistilled water. A fraction of the homogenate was mixed with acid–ethanol (0.18 mol/l HCl in 95% [vol./vol.] ethanol), from which insulin was extracted overnight at 4°C. Samples were then stored at −20°C until insulin and DNA measurement (see below). In separate experiments, soluble laminin (LM)-111 (α1β1γ1; according to new LM nomenclature ) (30 μg/ml) (BD), neutralising antibodies to thrombospondin-1 (TSP-1; 1 μg/ml) (LabVision, Fremont, CA, USA), hepatocyte growth factor (HGF; 1 μg/ml) (R&D Systems, Minneapolis, MN, USA) or the β1-chain of LM (18 μg/ml; Clone 3E5, Catalogue number MAB2041; Millipore, Solna, Sweden), or the endothelin A (ETA) and endothelin B (ETB) receptor antagonists BQ-123 (10 μmol/l) and BQ-788 (1 μmol/l) (Bachem UK, St Helens, UK), were added to the high-glucose medium.
After appropriate dilution, insulin concentrations in incubation medium and homogenates were determined by a commercial insulin ELISA (Mercodia, Uppsala, Sweden).
After appropriate dilution, the homogenates were used for DNA measurements by fluorophotometry (PicoGreen dsDNA Quantitation Kit; Molecular Probes).
Glucose oxidation rate
Islet glucose oxidation rates were determined according to a previously described method . Briefly, triplicates of ten islets were transferred to glass vials containing 100 μl KRBH supplemented with d-[U-14C]glucose (GE Healthcare Life Sciences, Chalfont St Giles, UK) and non-radioactive d-glucose to a final glucose concentration of 16.7 mmol/l and a specific radioactivity of 18.5 × 106 Bq/mmol. The 14CO2 produced was captured with Hyamine. After incubation for 90 min at 37°C (O2/CO2; 95:5), the oxidation was terminated by injection of 100 μl 0.05 mmol/l antimycin A into the vials. The 14CO2 generated by cell metabolism was released by the addition of 100 μl 0.4 mmol/l NaH2PO4 (pH 6.0) during a 120 min incubation. The radioactivity in the samples was then measured by liquid scintillation counting.
Islet (pro)insulin biosynthesis and total protein biosynthesis
Groups of ten islets were incubated in duplicate for 2 h in 100 μl Krebs–Ringer buffer containing 2 mg/ml BSA, 16.7 mmol/l glucose and 1.85 × 109 Bq/mmol l-[3,4,5-3H]leucine (American Radiolabeled Chemicals, St Louis, MO, USA) at 37°C in (air/CO2; 95:5). After incubation the islets were washed in Hanks’ balanced salt solution containing 10 mmol/l non-radioactive leucine, and then sonicated in 200 μl redistilled water. The islet homogenates were frozen before immunoprecipitation.
Islet intracellular degradation of insulin was determined using a pulse-chase method. The islet proteins were labelled by maintaining about 150 islets in the presence of 5.5 mmol/l glucose and 1.5 GBq/l l-[3,4,5-3H]leucine (American Radiolabeled Chemicals), yielding a specific radioactivity of 3.3 GBq/mmol in the medium during the 72 h incubation. The islets were then washed twice in non-radioactive medium, and duplicate groups of 15 islets were homogenised by sonication (22 kHz, 50 W) for 10 s in 200 μl of a 50 mmol/l glycine buffer pH 8.8 containing 2.5 g/l BSA. The remaining islets were divided into two aliquots, which were incubated for 24 h without radioactivity in CM or ECCM. After the chase period, duplicates of 15 islets from both groups were homogenised by sonication in 200 μl glycine–BSA–buffer, and 100 μl portions of the incubation medium were also retrieved. The islet homogenates and incubation medium were frozen before immunoprecipitation.
Portions of 10 μl from the islet homogenates and the 100 μl medium samples from the islet incubations were mixed with 100 μl of a 50 mmol/l glycine buffer supplemented with 2.5 g/l BSA and 1 ml/l Triton X-100, and in duplicates supplied with 10 μl of guinea pig anti-human insulin serum (Fitzgerald Industries International, Concord, MA, USA) or 10 μl normal guinea pig serum (Harlan Sera-Lab, Loughborough, UK). The immune binding was allowed to proceed for 1 h at room temperature. Subsequently 50 μl rProtein-A-Sepharose Fast Flow suspended in glycine–BSA–Triton X-100 buffer to a concentration of 110 ml/l was added and the material was tumbled for 15 min at room temperature and then centrifuged for 3 min at 4,000×g. The Sepharose sediment with the anti-insulin–insulin complex was washed twice with 500 μl glycine–BSA–Triton X-100 buffer and finally resuspended in 500 μl 1 mol/l acetic acid with 2.5 g/l BSA. After addition of 4 ml Ultima Gold scintillation fluid, the radioactivity was determined by a liquid-scintillation spectrometer (Wallac System 1400; Turku, Finland) using external standardisation. Non-specific binding was corrected for by subtracting the radioactivity bound by normal guinea pig serum from that precipitated by immune serum. For the analysis of total protein biosynthesis rate, two 10 μl aliquots were taken from the water homogenate samples of sonicated islets and transferred to Eppendorf tubes. Glycine solution (250 μl) was added to dilute the homogenate and 250 μl of 10% (wt/vol.) trichloroacetic acid was added to precipitate the proteins. The samples were mixed vigorously and centrifuged (10 min at 600 g). The supernatant fraction was removed and the pellets dissolved in 2 × 250 μl of 0.15 mol/l NaOH and transferred to scintillation vials before 4 ml Ultima Gold scintillation liquid was added and the samples counted in a liquid scintillation counter.
Gene expression analysis
Isolation of total RNA was performed with an RNeasy Micro Kit (Qiagen, Hilden, Germany) and all the samples were treated with RNase-free DNase (Qiagen). Conversion of total RNA to cDNA was performed with reverse transcription system (Promega, Madison, WI, USA) using oligo (dT)15 primers. The LightCycler System (Hoffman-La Roche), and detection with SYBR Green (SYBR Green JumpStart Taq ReadyMix), was used to amplify and analyse generated cDNA. TATA box binding protein (GenBank accession number D01034; CyberGene, Stockholm, Sweden), 5′-ACC CTT CAC CAA TGA CTC CTA TG-3′, 5′-ATG ATG ACT GCA GCA AAT CGC-3′, was used as housekeeping gene. PCR amplifications were performed in a total volume of 10 μl, containing 1 μl of cDNA, 1 μl of each primer (Tebu-Bio, Roskilde, Denmark or MWG Biotech, Edersberg, Germany) (see Electronic supplementary material [ESM] Table 1), 4 μl CYBR Green JumpStart Taq ReadyMix and RNase-free water added to the final volume. Each RT-PCR run started with a denaturation at 94°C for 10 s. Cycling variables were 94°C, 10 s; 57°C, 15 s; and 72°C, 8 s.
For multiple comparisons, repeated measurements of ANOVA with Bonferroni’s post hoc test was used. When only two groups were compared, Student’s paired or unpaired t test was applied as appropriate.