Cell-autonomous programming of rat adipose tissue insulin signalling proteins by maternal nutrition

All studies were approved by the University of Cambridge Animal Welfare and Ethical Review Board, and conducted according to the Home Office Animals (Scientific Procedures, UK) Act 1986. Stock Wistar Han rats (Rattus norvegicus; Crl:WI[Han] strain) were purchased from Charles River (Tranent, UK) and dams were produced by in-house breeding of stock animals. Virgin female rats weighing 240–260 g were individually housed in a specific pathogen free (SPF) environment in individually ventilated cages with environmental enrichment and maintained at 22°C on a 12 h light:dark cycle. After mating, the day on which virginal plugs were expelled was taken as day 1 of gestation. Pregnant dams were given ad libitum access to control (20% protein, wt/vol.) or an isoenergetic low protein (8% protein, wt/vol.) diet [14]. At 48 h after delivery, litters were reduced at random to eight pups: four males and four females. Lactating dams were maintained on experimental diets. Twenty-one-day-old pups were weaned onto standard chow (LAD1 diet, Special Diets Services, Witham, UK), fed ad libitum and maintained in pairs in SPF cages with environmental enrichment. Two 14-month-old males from each litter were fasted overnight. The next day, their body weights were recorded, blood was taken for glucose measurement and plasma was obtained for biochemical analysis. The rats were then killed by CO₂ asphyxiation, and individual fat depots were collected and weighed. The intra-abdominal (mesenteric) fat depot was collected from around the intestines, retroperitoneal (perirenal) fat was collected from around the kidneys and epididymal (gonadal) fat was collected from near the testes. WAT samples were snap-frozen and stored at −80°C.

Fasting glucose concentrations were measured in tail blood using a blood glucose analyser (AlphaTRAK, Abbott, Maidenhead, UK)). Fasting plasma insulin and leptin concentrations were determined by ELISA (ultrasensitive rat insulin EIA, Mercodia, Uppsala, Sweden; rat leptin ELISA, Crystal Chem, Downers Grove, IL, USA) and lipid content was measured using a Folch assay [15]. Lipid analysis was performed by the Core Biochemical Assay Laboratory (Cambridge, UK).

Epididymal WAT was fixed in paraformaldehyde, processed on a 14-h cycle (VIP6, Sakula, Torrance, CA, USA), embedded in paraffin, cut into 5 μm sections, placed onto Superfrost microscope slides (VWR, Lutterworth, UK) using synthetic mountant (Thermo Scientific, Hemel Hempstead, UK) and stained with haematoxylin and eosin [16]. Images were digitally captured (at ×10 magnification) using an inverted light microscope (Olympus BX41, Olympus, Southend-on-Sea, UK). One slide was prepared for each rat and five fields of view per slide (whole cells only in field of view) were analysed and quantified using Cell^P software (Olympus, Shinjuku, Tokyo, Japan) (n = 6 rats per group).

Total RNA was extracted using Direct-zol RNA MiniPrep kits (Zymo Research, Irvine, CA, USA) with an inclusion of a DNAse digestion step. RNA purity and concentration was determined by spectrophotometric analysis (NanoDrop ND-1000, Thermo Scientific, UK), and RNA integrity was determined by agarose gel electrophoresis. cDNA was synthesised using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Warrington, UK). Gene expression was assessed by quantitative (q)RT-PCR using custom designed primers (Sigma-Aldrich, Haverhill, UK and SYBR Green (Applied Biosystems; primer sequences are shown in Table 1). Analysis of melting curves confirmed the absence of primer dimers (StepOnePlus Real-Time PCR System, Applied Biosystems). Genomic rat DNA (Novagen, Madison, WI, USA) was serially diluted to generate standard curves. Several housekeeping genes were tested: Gapdh, Ppia and Ywhaz in epididymal fat; and Gapdh, Hprt, Nono, Ppia, Actb, Ywhaz, Rpl and Gusb in differentiated adipocytes. Expression of mRNA was normalised to Gapdh and Ywhaz expression because these genes had the least variable expression that was not affected by the maternal diet. Expression of the Cebpa gene (encoding CCAAT/enhancer-binding protein alpha) was determined using TaqMan gene expression assay ID Rn00560963_s1 and TaqMan reagents (Applied Biosystems).

Putative miRNAs targeting the 3′ untranslated regions (UTRs) of Irs1, Pik3cb (gene encoding the catalytic subunit of PI3K [p110β]), Pik3r1 (gene encoding the regulatory subunit of PI3K [p85α]) and Akt1 were identified using the TargetScan [17], microRNA.org [18], miRTarBase [19] and miRanda/mirSVR [20] tools. Candidate miRNAs were ranked according to their conservation across species, the strength of the predicted interaction, whether the mRNA target site was within a proximal or distal location, and their expression in WAT. Total RNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcriptase Kit (Applied Biosystems) and miRNA-specific primers (TaqMan MicroRNA Assays): miR-19a-3p, ID 000395; miR-25-3p, ID 000403; miR-30a-5p, ID 000417; miR-93-5p, ID 001090; miR-126, ID 002228; miR-128a, ID 002216; miR-130a-3p, ID 000454; miR-145, ID 002278; miR-222, ID 002276; miR-301a-3p, ID 000528; miR-320-3p, ID 002277; and miR-335-5p, ID 000546 (Applied Biosystems). qRT-PCR was performed using a TaqMan Universal PCR Master Mix No AmpErase UNG kit (Applied Biosystems). Standard curves were generated using serial dilutions of pooled sample cDNA.

Western Blotting was done as previously described [21]. Primary antibodies used were: anti-IRS-1 and anti-PI3K p85α subunit (Millipore, Lake Placid, NY, USA); anti-Akt1, anti-Akt2, anti-phospho-Akt Ser473, anti-phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2) Thr202/Tyr204, anti-phospho c-Jun N-terminal kinases 1 and 2 (JNK1/2) Thr183/Tyr185 and anti-phospho-p38 mitogen-activated protein kinase (MAPK) Thr180/Tyr182 (Cell Signaling, Danvers, MA, USA); anti-insulin receptor beta (IRβ), anti-PI3K p110β subunit, anti-protein kinase C ζ (PKCζ) and anti-suppressor of cytokine signalling 1 (SOCS1; Santa Cruz Biotechnology, Heidelberg, Germany); and anti-GLUT4 (Abcam, Cambridge, UK). Peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were used (Jackson ImmunoResearch, Stratech, Newmarket, UK).

Epididymal fat was dissected and collected into Hanks Basic Salt Solution (HBSS; all chemicals from Sigma-Aldrich). For each pre-adipocyte preparation, 7 g fat was chopped, placed into 30 ml digestion solution (45 mg collagenase type II and 675 mg BSA in HBSS), shaken at 180 rev/min for 40–50 min at 37°C, strained through a 100 μm mesh (BD Falcon, Tewksbury, MA, USA) and placed on ice for 20 min. The upper layer containing mature adipocytes was then removed. The top two-thirds was mixed with growth medium (high glucose DMEM supplemented with 10% newborn calf serum, 1% penicillin-streptomycin and 200 μmol/l l-glutamine). The stromal–vascular fraction was centrifuged and re-suspended in growth medium. Following a second centrifugation, the pellets were re-suspended in 5 ml erythrocyte lysis buffer (150 mmol/l ammonium chloride, 10 mmol/l potassium bicarbonate, 0.1 mmol/l EDTA, pH 8.0) for 5 min. Cells were pelleted, re-suspended in growth medium, counted and plated at a density of 1 × 10⁴ per cm² into 75 ml flasks. The culture medium was supplemented with an adipogenic cocktail (30 μmol/l insulin [Actrapid, Novo Nordisk, Gatwick, UK], 150 μmol/l sodium ascorbate) daily for the first 3 days and then every other day until harvesting on day 11.

Data were analysed using the Student’s t test and corrected for multiple comparisons (Holm–Šidák method; GraphPad Prism 6, La Jolla, CA, USA). The adipocyte area is presented as median values (interquartile range [IQR]) and was analysed using the Mann–Whitney U test. SOCS1 data were analysed using unpaired t-tests with Welch’s correction. Protein content, protein phosphorylation and mRNA expression data are the percentage of control ± SEM; all other data are the means ± SEM. n values refer to the number of litters. A p value of <0.05 was considered statistically significant.