Generation of β-V59M mice.
ROSA mice were generated using standard gene-targeting techniques. The Kcnj11
gene (ENSMUSG00000070561) encoding Kir6.2 was amplified by PCR from mouse genomic DNA and cloned into the pGEM-T Easy Vector (Promega). The Kir6.2-V59M mutant was generated using the QuikChange Site-Directed Mutagenesis Kit (QIAGEN) and ligated into the AscI site of the STOP-EGFP-ROSA targeting vector (34
The STOP-EGFP-ROSA plasmid (Figure B) consists of a 5′ homology arm to the ROSA26 locus followed by an adenoviral splice acceptor signal (SA). Downstream of the SA site, a loxP-flanked neomycin resistance cassette containing a strong transcriptional STOP sequence not only confers resistance to G418 but also prevents expression of the transgene prior to Cre excision. Downstream of the neomycin resistance cassette lies the cDNA for Kir6.2-V59M and an FLP recombinase target site–flanked IRES-GFP expression cassette, followed by a 3′ homology sequence to the ROSA26 locus.
The targeting vector (Figure B) was linearized and electroporated into V6.5 (50% C57BL/6, 50% 129/sv) embryonic stem cells. Recombinant ES cells were identified by Southern blotting, using a 5′-ROSA probe (700 bp EcoRI/PacI fragment from the A-04 vector; ref. 35
) external to the targeting vector (Figure E). Positive ES clones were injected into CB20 blastocysts to generate ROSA
Mice were backcrossed to C57BL/6 mice for more than 2 generations. In order to express the Kir6.2-V59M transgene specifically in pancreatic β cells, ROSA
mice were mated with mice expressing Cre recombinase under the control of the rat insulin promoter (RIP-Cre mice; provided by Pedro Herrera, University of Geneva Medical School, Geneva, Switzerland). Littermates were used for all studies.
All experiments were conducted in accordance with the 1986 UK Animals (Scientific Procedures) Act and University of Oxford ethical guidelines, and all animal studies were approved by the Ethics Committee of the University of Oxford. Mice were housed in same-sex littermate groups of 2 to 8 in a temperature- and humidity-controlled room on a 12-hour light/12-hour dark cycle (lights on at 6 am). Regular chow food (Teklad Global 2019 Rodent containing 55% carbohydrate, 19% protein, and 9% fat; Harlan Teklad) was freely available except where it is indicated mice were fasted overnight. Mice had ad libitum access to water at all times. They were weighed every 4 days. Because V59M mice produced copious urine after a few weeks of life, breeding pairs and offspring were maintained on high-absorbency bedding. All mice were genotyped by PCR.
Genotypes were identified by PCR using genomic DNA isolated from ear biopsies (DNeasy Blood and Tissue kit; QIAGEN). The presence of the Kir6.2-V59M mutant gene was confirmed using the following set of primers: P1, 5′-AAAGTCGCTCTGAGTTGTTATC-3′; P2, 5′-GATATGAAGTACTGGGCTCTT-3′; and P3, 5′-GCATCGCCTTCTATCGCCT-3′. These amplify a 590-bp amplicon from the WT ROSA26 allele but a 460-bp product from the targeted allele.
The presence of the RIP-Cre gene was confirmed by the amplification of an approximately 230-bp product, using forward ACGAGTGATGAGGTTCGCA and reverse ATGTTTAGCTGGCCCAAATGT primers. For both genes, PCR conditions were 94°C for 3 minutes, followed by 45 cycles of 94°C for 30 seconds, 57°C for 45 seconds, 72°C for 1 minute 30 seconds. This was followed by a final extension of 72°C for 10 minutes.
Pancreatic islet and β cell isolation.
Mice were killed by cervical dislocation. Pancreata were removed and islets isolated by liberase digestion and handpicking. For electrophysiology, isolated islets were dispersed into single cells by incubation in calcium-free Hank’s solution (137 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 1 mM NaH2PO4, 4.2 mM NaHCO3, 10 mM HEPES [pH 7.4 with NaOH], 1 mM EGTA, and 2.5 mM glucose) and trituration in pancreatic islet medium containing 11 mM glucose (hCell Technology) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in this medium at 37°C in a humidified atmosphere of 5% CO2 in air and used 1–2 days after isolation. For islet perifusion studies, the perifusate was collected every 0.5 or 2 minutes. The dead-space time of the perifusion system was approximately 2 minutes and has not been corrected for in the figures.
RNA extraction and cDNA synthesis.
Total RNA was extracted from approximately 100 isolated islets using RNeasy Mini Kit (QIAGEN), including an on-column DNase digestion step to remove traces of genomic DNA. RNA concentration was determined using a NanoDrop Spectrophotometer (Thermo Scientific) and its quality checked with the Agilent Bioanalyzer. Equal quantities of total RNA were reverse transcribed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The reaction cycle consisted of 10 minutes of incubation at 25°C followed by 30 minutes of incubation at 48°C. Samples were subsequently stored at –20°C.
Mouse Kir6.2 transcript was amplified by PCR using cDNA prepared from islets isolated from either V59M or WT 5-week-old mice. Primers used were as follows: forward, 5′-ATGCTGTCCCGAAAGGGCAT-3′; and reverse, 5′-TGGTCTGGTGGCTCATCGCC-3′. After 40 cycles (95°C for 30 seconds; 60°C for 30 seconds; 72°C for 1 minute), PCR products were digested with BtsCI restriction enzyme (New England BioLabs) for approximately 1 hour at 50°C and digested PCR fragments visualized on a 2% agarose gel. Sequences of primers used for GFP amplification were as follows: forward, 5′-GAGGTGAAGTTCGAGGGCGAC-3′; and reverse, 5′-CAGGACCATGTGATCGCGCTT-3′.
Real-time quantitative PCR.
Quantitative real-time PCR was performed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using either SYBR Green or TaqMan chemistries. For SYBR Green detection, primers were designed using Primer Express Software 2.0 (Applied Biosystems) based on Ensembl sequence data. Sequences of the primers used were as follows: for unspliced Xbp1, forward, 5′-CAGCACTCAGACTATGTGCA-3′, and reverse, 5′-GTCCATGGGAAGATGTTCTGG-3′; for spliced Xbp1 (36
), forward, 5′-CTGAGTCCGAATCAGGTGCAG-3′, and reverse, 5′-GTCCATGGGAAGATGTTCTGG-3′; for Chop, forward, 5′-AGCCTGGTATGAGGATCTGCAG-3′, and reverse, 5′-GGTCAAGAGTAGTGAAGGTTTTTGATTC-3′; for Insulin 1, forward, 5′-AGACCATCAGCAAGCAGGTCA-3′, and reverse, 5′-AAGTGCACCAACAGGGCC-3′; and for Gapdh, forward, 5′-AGCGAGACCCCACTAACATC-3′, and reverse, 5′-GGTTCACACCCATCACAAAC-3′.
All reactions were performed in triplicate in a final volume of 25 μl containing 12.5 μl Power SYBR Green Master Mix, 300 nM of each primer, and 50 ng of cDNA template. cDNA was amplified by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Gene expression was determined by the standard curve method and normalized to GAPDH.
Mouse Kir6.2 and SUR1 mRNA levels were determined using predesigned TaqMan Gene Expression assays (assays Mm 00440050-s1 and Mm 00803450-m1, respectively) (Applied Biosystems) according to the manufacturer’s instructions and normalized to the housekeeping gene β-2-microglobulin (assay Mm 00437762-m1).
Measurements of blood glucose, insulin, and glucagon levels.
Five-week-old mice were fasted for 16 hours. They were then weighed, and a blood sample was collected by cardiac puncture under terminal anesthesia (Euthatal; Merial). Plasma glucose level was determined using a GM9 Glucose Analyser (Analox Instruments). Plasma insulin and glucagon levels were measured using a mouse endocrine LINCOplex kit (LINCO Research Inc.) and a Bio-Plex 200 System (Bio-Rad), according to the manufacturers’ instructions. For determination of total insulin content, the pancreas was removed and incubated overnight in acidified alcohol. Insulin was measured using a Mercodia Ultrasensitive Mouse ELISA Kit.
Isolated islets were incubated overnight in pancreatic islet medium (hCell Technology). They were then placed on top of 8-μm pore diameter filter membranes mounted inside Swinnex filter holder chambers (Millipore) and perifused at 37°C at a rate of 1 ml/min–1 with Krebs-Ringer-HEPES buffer (KRH): 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1 mM KH2PO4, 1.2 mM MgSO4, 10 mM HEPES, and 20 mM NaHCO3, pH 7.4 with NaOH, plus 0.2% BSA and glucose as indicated. After 40 minutes preincubation in 2 mM glucose KRH, islets were perifused for 10 minutes in 20 mM glucose KRH and subsequently for 20 minutes with 20 mM glucose KRH containing 0.5 mM tolbutamide. Insulin was measured using a Mercodia Ultrasensitive Mouse ELISA Kit.
Macroscopic currents were recorded from inside-out or cell-attached membrane patches at –60 mV, filtered at 5 kHz, and digitized at 20 kHz. The pipette contained 140 mM KCl, 10 mM HEPES (pH 7.2 with KOH), 1.1 mM MgCl2, and 2.6 mM CaCl2. The intracellular (bath) solution contained 107 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 11 mM EGTA, and 10 mM HEPES (pH 7.2 with KOH) plus MgATP as indicated. Whole-cell currents were recorded in the perforated patch configuration in response to –10 mV steps from –70 mV at 21–23°C. Currents were normalized to cell capacitance to correct for differences in cell size. The pipette solution contained 76 mM K2SO4, 10 mM NaCl, 10 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.35 with KOH, plus 0.24 mg/ml amphotericin. The bath solution contained 137 mM NaCl, 5.6 mM KCl, 10 mM HEPES, pH 7.4 with NaOH, 2.6 mM CaCl2, and 1.1 mM MgCl2. Recordings were initiated 30 minutes after exposure to substrate-free solutions.
ATP concentration-response curves were fit with the following equation:
I/Ic = a + (1 – a)/(1+ ([ATP]/IC50)h) (Equation 1)
where I and Ic are the current amplitude in the presence and absence of nucleotide, respectively, IC50 is the ATP concentration ([ATP]) at which inhibition is half maximal, h is the slope factor, and a is the fraction of unblocked current at saturating [ATP]. To control for current rundown, Ic was taken as the mean of the conductance in control solution before and after ATP application.
Intracellular calcium measurements.
For microfluorimetry, islets were loaded for 30 minutes at room temperature with 3 μM fura 2–AM (Invitrogen) and 0.01% pluronic acid in extracellular buffer containing 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl2
, 1 mM MgCl2
, and 5 mM HEPES, pH 7.4 with NaOH. [Ca2+
was measured ratiometrically as described previously (37
) using a PTI microfluorimetry system and FeliX32, version 1.1 (Photon Technology International).
For confocal calcium imaging, islets were loaded with 4 μM fluo 4–AM (Invitrogen) for 2 hours at room temperature in extracellular buffer. Changes in [Ca2+]i were recorded at 37°C by laser scanning confocal microscopy using an LSM 510 Meta system (Zeiss). Individual cells were selected as “regions of interest” with the LSM software, and their calcium responses to the various stimuli were identified as changes in fluo 4 emission intensity at 500–550 nm upon excitation with the 488-nm line of an argon laser.
Immunohistochemistry and morphometric analysis.
Pancreatic specimens from 5-week-old female mice were fixed overnight in 10% formaldehyde in PBS at 4°C and wax embedded. Dewaxed and rehydrated sections (5-μm thick) were stained with antibodies against insulin (guinea pig), glucagon (mouse; Dako), somatostatin (rabbit; Sera Laboratories International), and pancreatic polypeptide (rabbit; Lilly). For confocal microscopy, insulin-positive cells were detected using a secondary antibody conjugated with fluorescein (goat anti-GP; Vector Laboratories), and glucagon-positive cells were identified with a TRITC-conjugated antibody (horse anti-mouse; Vector Laboratories).
For assessment of islet architecture (shape), β cells were stained brown using an anti-GP HRP-coupled antibody and 3,3′-diaminobenzidine (DAB) reagent and then counterstained with hematoxylin. For quantitative morphometric analysis, β cells were stained red with Fast Red Substrate (Sigma-Aldrich) and alkaline-phosphatase anti-GP secondary antibody (Sigma-Aldrich), and α, δ, and pancreatic polypeptide–positive cells were stained brown with HRP-coupled antibodies against mouse (glucagon; Vector Laboratories) and rabbit (somatostatin and pancreatic polypeptide; Dako) and DAB reagent (Sigma-Aldrich). Images of islets and the entire pancreas were taken with an Olympus digital camera and analyzed using AxioVision 4.6 software (Zeiss). For quantitation of β cell area, sections 100 μm distant from each other were analyzed for 3 animals of each genotype. A total of approximately 60 islets, distributed over 5 sections, were analyzed per mouse. β cell area was calculated by subtracting non-β cell area from total islet area. Data are expressed as islet density (islet number/pancreas area) and β cell/islet proportion (β cell area/islet area). Analysis was performed blinded to the mouse genotype.
Unless specified, data are mean ± SEM of the indicated number of experiments.