To obtain mice heterozygous for the Cc1 locus, C57BL/6J.Cc1
−/− mice [7, 12] were crossed with transgenic mice with liver-specific overexpression of wild-type rat Cc1 driven by ApoA-1 promoter (L-CC1) . These were backcrossed with C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) six times to obtain a progeny of Cc1
+/− with or without the transgene. Intercrossing this progeny produced several genotypes including Cc1
−/− with the transgene (Cc1
−/−xliver+) and control littermates: Cc1
−/− without the transgene (Cc1
+/+ without the transgene (Cc1
+/+) and Cc1
+/+ with the transgene (L-CC1). Genotypes were identified by PCR-based genotyping using ear genomic DNA and primers for the wild-type allele (exon 2), the knockout allele (the neomycin cassette between exon 7 and 9) and the rat transgene (exons 6 and 9) (see ESM Fig. 1).
Male mice were kept in a 12 h light/dark cycle under pathogen-free conditions and fed a standard diet (Harlan Laboratories, Teklad 2016, Haslett, MI, USA) ad libitum . In some experiments, mice were injected i.p. once daily with nicotinic acid (200 mg/kg body weight per day; Sigma-Aldrich, Saint Louis, MO, USA) for 2 days . Mice were placed in cages with Alpha-dri bedding (Shepherd Specialty Papers, Cincinnati, OH, USA) prior to food removal and phenotyping. The Institutional Animal Care and Utilization Committee approved all procedures. Animals and samples were not randomised. No data, samples or animals were excluded in analyses.
Whole body composition was assessed in awake mice by NMR technology (Bruker Minispec, Billerica, MA, USA). Magnetic resonance spectroscopy (1H-MRS; Echo Medical Systems, Houston, TX, USA) was used to determine fat and lean mass as percentage of total body weight, as previously described .
Retro-orbital venous blood was drawn at 11:00 hours from mice fasted overnight to measure plasma insulin (80-INSMSU-E01 ELISA kit; ALPCO, Salem, NH, USA), C-peptide (80-CPTMS-E01 ELISA kit; ALPCO), leptin (22-LEPHUU-E01 ELISA kit; ALPCO), NEFA (NEFA-C enzymatic colorimetric assay; Wako, Richmond, VA, USA), and plasma and hepatic triacylglycerol levels (Pointe Scientific, enzymatic colorimetric assays, Canton, MI, USA) [7, 12, 18].
Hyperinsulinaemic–euglycaemic clamp analysis
A 2 h hyperinsulinaemic–euglycaemic clamp was performed in awake overnight-fasted mice with primed and continuous infusion of human soluble insulin (Humulin, Lilly, Indianapolis, IN, USA) at a rate of 2.5 mU kg−1 min−1 . Glucose metabolism was estimated with a continuous infusion of 1850 Bq of [3-3H]glucose (Perkin-Elmer, Shelton, CT, USA) and then 3700 Bq throughout the clamp.
C75 treatment and daily food intake
Food was removed from individually housed mice at 15:00 hours and weighed for 4 days. At 18:00 hours, mice were injected i.p. with a daily dose of 30 mg/kg body weight of C75 (Calbiochem, EMD Bioscience, Bedford, MA, USA) dissolved in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA). Food was returned 5 min before the dark cycle began at 19:00 hours and the weight of ingested food/day was calculated.
Biotin-labelling of primary hepatocytes
Primary hepatocytes were isolated by perfusing livers with collagenase-type II (1 mg/ml) (Worthington, Lakewood, NJ, USA) and plated in six-well plates in complete Williams-E medium at 1 × 106 cells/well . After 24 h, cells were incubated in the absence or presence of 100 nmol/l insulin (Sigma-Aldrich) at 37°C for 5 min, followed by incubation with biotin (1 mg/ml) (Pierce, Rockford, IL, USA) in PBS for 30 min on ice. Cells were lysed in 1% Triton-X and subjected (30 μg proteins) to immunoprecipitation at 4°C with streptavidin (Pierce). Immunoprecipitates were centrifuged and analysed by 7% SDS-PAGE and immunoblotting with 1:1000 of insulin receptor alpha (IRα) antibody (N-20; Santa Cruz, Dallas, TX, USA) or a custom-made Ab3759 polyclonal antibody against purified mouse CEACAM1, as titrated [12, 19], followed by horseradish peroxidase-conjugated mouse anti-rabbit IgG antibody (Jackson Immunoresearch, West Grove, PA, USA) and subjected to enhanced chemiluminescence (ECL; Amersham Pharmacia, Sunnyvale, CA, USA).
Livers were homogenised, centrifuged and the supernatant fraction was added to a reaction mix containing 3700 Bq [14C]malonyl-CoA (Perkin-Elmer) and 25 nmol malonyl-CoA in the absence or presence of 500 μmol/l NADPH (Sigma-Aldrich) . The reaction was stopped with 1:1 chloroform:methanol solution and samples were centrifuged, butanol-extracted and counted. FASN activity was calculated as the cpm of [14C]-incorporated Bq/μg cell lysates.
Ex vivo palmitate oxidation
The liver was extracted, homogenised and incubated in 0.2 mmol/l of [1-14C]palmitate (185 × 105 Bq) (American Radiolabeled Chemicals, St Louis, MO, USA) and 2 mmol/l ATP . The reaction was terminated with perchloric acid to recover radioactive acid soluble metabolites. Trapped CO2 was measured by liquid scintillation. The oxidation rate was expressed as the sum of total and partial fatty acid oxidation expressed in nmol g−1 min−1.
Western blot analysis
This was achieved using 1:1000 polyclonal antibodies against phosphorylated insulin receptor beta (p-IRβ; phospho-Y1361), IR β (C18C4) (Abcam, Cambridge, MA, USA), phosphorylated Akt (p-Ser473 Akt), Akt, α-FASN (Cell Signaling, Danvers, MA, USA), custom-made rabbit polyclonal Ab2456 against the mouse CEACAM1 extracellular domain, as titrated , and custom-made rat CEACAM1 (αP3[Y488]) and phosphorylated CEACAM1 (α-p-CEACAM1) mouse antibodies (Bethyl Laboratories, Montgomery, TX, USA), as titrated [16, 21]. α-Actin, α-GAPDH and α-tubulin antibodies (Santa Cruz) were used at 1:5000 dilution for normalisation. Blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody, as per manufacturer’s titration, for analysis of transfected, knockout or siRNA-knockdown cells (GE Healthcare Life Sciences, Amersham, Sunnyvale, CA, USA), followed by ECL.
Quantitative real-time PCR
cDNA was synthesised using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and qPCR was performed using Fast SYBR Green Master Mix by the ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA)  and 10 μmol/l primers for Cc1 (mouse Cc1 [mCc1], rat Cc1 [rCc1] and total Cc1), Tnfα (Tnf), F4/80 (Adgre1), Il-1β (Il1b), Srebp-1c (Srebf1), Fasn, Fgf21, Cpt1α (Cpt1a), Pparα (Ppara), Ucp1, Cox2 (ESM Table 1). mRNA was normalised to Gapdh, unless otherwise stated.
Parametric data were analysed by one-way ANOVA with Tukey’s test for multiple comparisons. Nonparametric data were analysed by the Kruskal–Wallis test with Dunn’s correction for multiple pairwise comparisons. All data were expressed as means ± SEM using GraphPad Prism 6 software (www.graphpad.com, downloaded on 29 June 2016). A p value ≤ 0.05 was considered statistically significant.