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, CONTACT FOR REAGENTS AND RESOURCE SHARING Further information and requests for reagents may be directed to, and will be fulfilled by, the Lead Contact, Miguel P. Soares (mpsoares@igc.gulbenkian.pt)
, Alb Cre Fth D/D , and ROSA26ER T2 Fth lox/lox mice were generated by intercrossing C57BL/6 Fth lox/lox mice, 2012.
, Deletion of the G6pc1 lox/lox allele in Alb Cre ER T2 G6pc1 D/D mice was achieved by Tamoxifen administration (i.p.; 50mg/kg in 100 mL corn oil/5% ethanol for 5 consecutive days). Deletion was confirmed by western blot and genomic PCR of liver extracts (Figure 5A). All animal protocols used were approved by the Instituto Gulbenkian de Ciê ncia ethical committee and the ''Ó rga?o Responsa´vel pelo Bem-estar dos Animais (ORBEA)'' and consequently licenced by the Direcca?o Geral de Alimentaç a?o e Veterina´ria (DGAV), ROSA26 Cre ER T2 (Jackson Laboratories; stock 008463) mice, respectively. Conditional deletion (D) of Fth lox/lox allele in Mx1 Cre Fth D/D mice was achieved at 5 weeks of age by Poly(I:C)(InvivoGen Europe, 2011.
, Sigma Ref: A3641) in sterile 0.9% saline was administered (i.p.; 200 mL in saline) 24 hr before or 6 hr after CLP. Vehicle (0.9% saline) or bovine serum albumin (BSA, Sigma Ref: A3059) at the same dosage were administered to control mice. D-glucose (Sigma Ref: G-8270) (200 mL 2 mg/g BW in water) or Pyruvate (Sigma Ref: P4562) (200 mL 4 mg/g BW in water) were administered via gavage twice daily for 4 days starting 12 hr after CLP and then once daily for 3 days. N-acetylcystein (NAC) was administered (i.p.; 200 mL, 15mg/kg in sterile PBS) twice daily for 12 consecutive days starting 48 hr before CLP. Butylated Hydroxianisole (BHA; Sigma, ref: B1253) was administered (gavage.; 100 mL, 50mg/kg in corn oil) twice daily for 12 consecutive days starting 48 hr before CLP. Hemin (Frontier Scientific Ref: H651-9) or empty protoporphyrin (Frontier Scientific Ref: P562) were administered to C57BL/6, Tlr4 À/À and Ifnr1 À/À (i.p.; 30 mg/kg) or to Fth lox/lox and Mx1 Cre Fth D/D (i.p.; 25-30 mg/kg), Procedures were performed routinely at the same time frame, starting at 9 AM. CLP consisted of 20%-30% cecum ligation and double puncture with a 23 Gauge (G) needle. Severe CLP consisted of Pharmacologic Approaches Apoferritin: Purified horse spleen ferritin (Sigma Ref: F4503) or apoferritin, 2010.
, Heparanized blood was obtained by intracardial puncture. Serial dilutions were plated onto TrypticaseSoy Agar II with 5% Sheep Blood plates (Becton Dickinson Ref: 254053) and incubated (24 hr at 37 C) in air 5% CO 2 (aerobes) or in an air tight container equipped with the GasPak anaerobe container system, Anaerobic conditions were confirmed in all experiments using BBL Dry Anaerobic Indicator Strips, p.271051, 260678.
, Liver samples were harvested, fixed in 10% buffered formaldehyde for 48 hr at room temperature, then embedded in paraffin and stained with Hematoxylin & Eosin, essentially as described, Cell, vol.169, p.4, 2012.
, Temperature was recorded continuously and kept stable. The apex of the left ventricle was punctured with a 27 G needle using the open chest approach and a FTS-1912B-8018; pressure-volume conductance catheter (Scisense, London, Canada) was inserted in the left ventricle. Mice were allowed to stabilize for 3-10 min. Baseline values, values with varying preload caused by inferior vena cava clamps using a blunt forceps and aortic pressures, were recorded with the Scisense pressure-volume control unit FV896B and analyzed using the Labscribe2 (Labscribe, iWorx Systems, USA) software. The machine was calibrated with internal and cuvette calibration, Cardiovascular Function Cardiovascular function was measured using pressure-volume conductance catheter technique, 2008.
, Alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN), creatinephosphokinase (CPK)
, CA, USA) and Insulin (Mouse Ultrasensitive Insulin ELISA, ALPCO, USA) serum concentrations were measured by ELISA according to manufacturer instructions. Alternatively, serological parameters were determined by DNATech
, Tolerance Tests Oral glucose tolerance test (oGTT), pyruvate tolerance test (PTT) and insulin tolerance test (ITT) were performed in fasted mice (14 hr) while the glucagon challenge test was performed without fasting. Glucose (Sigma) was administered orally (2 mg/g BW in 200 mL 0.9% saline)
, 16 mg/g of BW) were administered intraperitoneally (i.p.). Tests were performed in before and 48 hr after CLP. Cell Culture HepG2 cells were grown in low glucose (1g/L) DMEM (GIBCOâ Ref: 21885-108), 10% heat inactivated FBS, 1% Penicilin/Streptomycin (GIBCOâ Ref: 15140-122). Before seeding
, R&D Systems Europe Ref: 210-TA-20) was added in media (50 ng/mL; 4 hr) and cells were collected. When indicated cells were pre-treated with N-acetyl-L-cysteine (NAC; Sigma, Ref: A9165; 7.5mM in media; 3 hr) before and during heme treatment in HBSS and subsequently throughout the experiment. When indicated cells were pre-treated Deferoxamine (DFO, Sigma Ref: D9533; 60 mM; 16 hr) before and during heme treatment in HBSS and subsequently throughout the experiment. At 80%-90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs, 5mg/ml in 20mM acetic) acid. At 100% confluence cells were treated with heme, vol.40, 2003.
, Luciferase Assays HepG2 cells were seeded onto 6-well plates and transfected 24h thereafter (60%-80% confluency) with pGL2B-G6PC (containing À1320/+60 from the rat G6pc1 promoter
, ) using Lipofectamine LTX Reagent with PLUS Reagent (Invitrogen, Thermo Scientific), according to manufacturer instructions. A Renilla Luciferase expressing vector, 2002.
, Forty-eight hours thereafter cells were treated with heme (40 mM, 1h) in HBSS, and subsequently exposed to human recombinant TNF (50 ng/mL; 4 hr) in culture medium and collected for luciferase activity. Alternatively cells were treated with heme (40 mM, 1h) in HBSS, exposed to human recombinant TNF (50 ng/mL; 3 hr) in culture medium and collected for luciferase activity, 12 hr after exposure to TNF. Luciferase activity was determined using the Luciferase assay system Dual-Glo (Promega) was used according to manufacturer's intructions. Luminescence was measured using a microplate reader (Victor3 Multilabel Readers, Perkin Elmer). Firefly Luciferase was normalized to Renilla Luciferase activity and expressed as relative light units (RLU) using pGL2B activity as background, France) was co-transfected as transfection control, 2002.
, All livers were fixed with 10% buffered formalin and embedded in paraffin. Paraffin sections were used for immunohistochemistry. Briefly, for antigen retrieval slides were placed in citrate buffer and heated to sub-boiling conditions for 6 min, Immunohistochemistry Livers were harvested 12 hr after severe CLP
, Data are shown as mean and SD from 3 mice per genotype. (b) Blood glucose levels in Fth lox/lox (n = 3) and Mx1 Cre Fth D/D (n = 3) mice in the first 12 hr after CLP. Mean ± SD, data from 1 experiment. (c) Blood glucose levels in C57BL/6 mice in the first 12 hr after CLP (n = 7) or control sham operation (n = 12). Mean ± SD, data from 3-4 independent experiments. (d) Total heme levels in the peritoneal cavity in C57BL/6 mice before (n = 4) and 3 hr after CLP (n = 3). (e) Blood glucose levels in C57BL/6 mice in the first 8 hr after heme (n = 15) or protoporphyrin IX (PPIX) (n = 10) administration (i.p.; 30 mg/kg BW). Mean ± SEM, data from 3-5 independent experiments. (f) Blood glucose levels in Fth lox/lox (n = 8) and Mx1 Cre Fth D/D (n = 9) in the first 8 hr after heme administration (i.p.; 25 to 30 mg/kg BW). Mean ± SD from 2 independent experiments. (g) Blood glucose levels in C57BL/6 Tlr4 +/+ (n = 13) and Tlr4 À/À (n = 9) mice in the first 8 hr after heme administration (i, Fth lox/lox and Mx1 Cre Fth D/D mice before and after CLP
, Mean ± SD from 2 independent experiments. (i) Oral glucose tolerance test (oGTT), (j) pyruvate tolerance test (PTT) and (k) glucagon challenge test (GCT) in Fth lox/lox and Mx1 Cre Fth D/D mice. Data are shown as mean ± SEM, pooled from 3 independent experiments with n = 3 mice per genotype per experiment. (l) insulin tolerance test (ITT) in Fth lox/lox and Mx1 Cre Fth D/D mice. Data are shown as mean ± SD, from 1 experiment with n = 5 mice per genotype. (m) Plasma insulin levels in Fth lox/lox and Mx1 Cre Fth D/D mice after overnight fasting. Data are shown as mean ± SD from 4-5 mice per genotype
, , p.48