All animals were housed and tested in accordance with European (Directive on the Protection of Animals used for Scientific Purposes, 2010/63/EU) and UK Home Office regulations, experiments were approved by the University of Aberdeen Ethics Board and performed in accordance with the Animal (Scientific Procedures) Act 1986 and following Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The transgenic PLB4 and WT lines were generated and bred as previously described , experimenters were not blinded to genotype. All mice were housed in single sex groups, unless food and water consumption was being measured. Physiological assessments were performed in male mice at 3, 4, 5 and 8 months of age. All mice were killed by neck dislocation. Five-hour-fasted mice aged 3 and 8 months were used for liver assays, and all postmortem molecular analyses of signalling pathways (western blotting and quantitative real-time PCR [qPCR]) were performed using samples from 8-month-old mice. Glucose-stimulated insulin secretion (GSIS) was determined in 4-month-old mice at 0, 15 and 30 min time points during glucose tolerance tests (GTTs). Blood leptin content was determined from baseline reading at 3, 4 and 8 months in a 5 h fasted state. EchoMRI scans (EchoMRI, Houston, TX, USA) were performed on mice at ages 4, 5 and 8 months. Additional 6-month-old mice were used for lipidomic analysis and 18FDG-PET imaging. Body weights were recorded and food and water consumption (in g) were measured for 2 weeks in 5- and 8-month-old male mice.
Tail blood glucose was determined using an AlphaTRAK glucometer (Berkshire, UK). GTTs were performed (as described in [6, 20]) in 5-h fasted male mice at age 3 (WT, n = 4; PLB4 n = 7), 4 (WT, n = 8; PLB4, n = 7), 5 (WT, n = 9; PLB4, n = 15) and 8 (WT, n = 4; PLB4 n = 4) months. Fasting blood glucose was determined at baseline (time 0) before i.p. injection of bolus glucose (20%; 2 g/kg body weight). Blood glucose clearance was assessed at 15, 30, 60 and 90 min post injection in 3-, 5- and 8-month-old mice; and at 0, 15 and 30 min post injection in 4-month-old mice.
Tail- or trunk-derived blood was collected from 5-h fasted mice aged 3 (WT, n = 6; PLB4, n = 8), 4 (WT, n = 7; PLB4, n = 8), 5 (WT, n = 9; PLB4, n = 12) and 8 (WT, n = 8; PLB4, n = 10) months into serum separator microtubes; serum was used for insulin and leptin determination (Insulin ELISA, Millipore, Darmstadt, Germany; leptin ELISA, CrystalChem, Zaandam, the Netherlands). A multiplex assay (customer-designed mouse metabolic hormone Magnetic Multiplex Assay; Merck Millipore, Darmstadt, Germany) was used for the simultaneous quantification of leptin, amylin (active form), C-peptide 2, glucose-dependent insulinotropic peptide (GIP; total), pancreatic polypeptide, peptide tyrosine tyrosine (PYY), IL-6 and resistin following the manufacturer’s instructions. Triacylglycerol and glycogen assays were used as previously described .
Comparative lipidomics plasma and brain analyses
Frozen plasma and forebrain samples from 6-month-old male mice (WT, n = 8; PLB4, n = 8) were used for global lipid analysis using liquid chromatography–mass spectroscopy (LC–MS). Plasma lipids were extracted from samples according to the Folch method. Lipids were solvent extracted in methanol/chloroform (2:1 vol./vol.). The lipid extracts were subsequently analysed by LC–MS in positive and negative ion modes with a C18 column and a water/acetonitrile/isopropanol gradient using an Exactive Orbitrap system (Thermo Scientific, Hemel Hempstead, UK) . Lipidomic datasets were processed using Progenesis QI software (version 2.0, Non-Linear Dynamics, Newcastle upon Tyne, UK) and searched against LIPID MAPS (www.lipidmaps.org/) and the Human Metabolome Database (www.hmdb.ca/).
Brain and upper body PET/CT imaging
Six-month-old female mice (WT, n = 10; PLB4, n = 9) were imaged using an Argus GE dual ring PET/CT scanner (Sedecal, Madrid, Spain) using our published protocol .
All tissues were lysed in RIPA buffer [as described in . Tissue from 5-h fasted mice was immunoblotted using rabbit polyclonal antibodies diluted 1:1000 in TRIS-buffered saline containing Tween-20, 5% BSA and 0.05% sodium azide. Antibodies raised against human BACE1 (C-terminal 485–501; no. 195111, Calbiochem, UK); phospho-protein kinase B (p-AktSer473; no. 4060), total Akt (Akt; no. 9272), phospho-glycogen synthase kinase-3β (p-GSK-3βSer21/9; no. 9331), phospho-ribosomal protein S6 (p-rpS6Ser235–236; no. 4858), total rpS6 (rpS6; no. 2217), phospho-S6 kinase (p-S6KThr389; no. 9234), total S6 kinase (S6K; no. 2708), phospho-mammalian target of rapamycin (p-mTORSer2448; no. 5536), mTOR (no. 2983), phospho-glycogen synthase (p-GSSer641; no. 3891), phospho-eukaryotic translation initiation factor 2α (p-eIF2αSer51; no. 3398), total eIF2α (no. 5324), protein kinase RNA-like ER kinase (PERK; no. 5683; all obtained from Cell Signaling Technology, Leiden, the Netherlands); insulin receptor β (IRβ; C-19, no. Sc-711; 1:500 dilution), apolipoprotein (ApoE; no. Sc-6384), mouse monoclonal protein tyrosine phosphatase 1B (PTP1B; no. Sc-14021), C/EBP homologous protein (CHOP; also known as GAD153, no. Sc-7351; Santa Cruz, Heidelberg, Germany); and retinol-binding protein 4 (RBP4; no. A0040, Dako, Glastrup, Denmark) were used following manufacturer’s instructions and as described before [6, 13]. Coomassie Blue (PhastGel Blue R; GE Healthcare, Watford, UK) was used as a loading control [13, 22].
RNA was extracted from frozen hypothalamic and cortical samples using TRI Reagent, as previously described . Target genes (hBACE1 and mouse Pomc, Mc4R, Npy, LepR, Ptp1b, Chop, Cd11 and Cd68) were amplified by qPCR using GoTaq Master Mix (Promega, Madison, WI, USA). The geometric mean of three commonly used reference mRNAs (Ywhaz, Nono and Actb) was used to normalise data.
Prism 5 (GraphPad, La Jolla, CA, USA) was used for statistical analyses, and data are expressed as the means ± SEM. All molecular and genetic data were adjusted to loading controls or housekeeping genes and calculated relative to WT. Two-tailed t tests with Welch’s correction were used for unbiased comparisons between transgenic mice and controls. Group analyses used two-way ANOVA (with repeated measures where appropriate), followed by Bonferroni post hoc tests.