Pcpt camp

At 48 h post-seeding, cells were incubated in serum-free medium for 4 h, washed twice with PBS, and incubated for 2 h with DMEM containing glutamine, sodium lactate, and sodium pyruvate, in the presence or absence of pCPT-cAMP (100 μM, Sigma) and/or 10 nM insulin. After 2 h, the supernatant was collected, centrifuged twice (200 × g, 3 min, 340 × g, 3 min); 50 μL of the supernatant were used for glucose quantification (Thermo). The attached hepatocytes were washed once with PBS and frozen in −80 °C.

R28 cells were purchased from Kerafast, Inc. Boston, MA. R28 cells from passages 67 to 70 were cultured in DMEM with low glucose (Sigma, cat# D5523) supplemented with 10% fetal calf serum (Cytiva HyClone, cat# CSH3007303) and 1% penicillin-streptomycin (Gibco, cat# 15070063), 7.5% sodium bicarbonate (Sigma, cat# S8761), 1% MEM non-essential amino acids (Gibco, cat# 11130051) as described previously [33 (link)]. R28 cells were differentiated overnight in complete medium supplemented with 250 μM pCPT-cAMP (Sigma, cat# C3912) on laminin-coated plates. The next day, cells were transduced with adenoviral vectors containing either A2 or RFP for 6 h at 5, 10, and 20 multiplicities of infection (MOI), as previously described [7 (link)]. The final MOI of 20 was chosen according to previously published studies and preliminary experiments [7 (link)]. After 6 h, the media was changed to normal complete growth media for 24 h. The following day, cells were treated with vehicle or 1 µM ABH or 5 mM L-Glutamate for 16–18 h and subjected to Western blot and Seahorse XFe flux analysis.

Cultured mouse hepatocytes were serum-starved overnight and washed with PBS with MgCl₂ and CaCl₂ (Sigma-Aldrich). Hepatocytes were exposed to glucose-free DMEM (Sigma-Aldrich) (20 mM sodium lactate, 2 mM sodium pyruvate, 2 mM L-glutamine, 15 mM HEPES) with or without 0.1 mM pCPT-cAMP (Sigma-Aldrich) for 11 hours. Media was collected and glucose concentrations were measured via Autokit Glucose enzymatic assay (FUJIFILM Medical Systems Inc.) and normalized to protein content determined via Bicinchoninic Acid assay.

Infected salivary glands from tsetse flies with WT and two FHR^−/− clones were used to infect BALB/c mice^{61 (link)}. The experiments designed for this study were carried out according to the UK Animals (Scientific) Procedures Act under a licence (30/3046) from the UK Home Office and approved by the University of Bristol ethics committee. To adapt BSFs to culture, aliquots of infected mouse blood were transferred into modified HMI-11 media^{66 (link)} with 1% (w/v) methylcellulose, 10% HI FCS^{67 (link)} and cultured at 37 °C with 5% CO₂. Cells were maintained between 1 × 10⁵ to 1 × 10⁶ cells/ml and all experiments were performed when the cells were in mid-log phase growth of 5 × 10⁵ to 1 × 10⁶ cells/ml. BSF cells were adapted to standard culture by twofold dilution every 2–3 days or more regularly as necessary, along with an incremental decrease in methylcellulose.
The induction of stumpy BSFs in culture was performed by following an established protocol^{42 (link),43 (link)}; 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt (pCPT-cAMP, Sigma) was added to cultured slender BSFs (100 μM) and cells were collected or analysed 24 h later.

Glucagon, pCPT-cAMP, insulin, LPS, and MG132 were obtained from Sigma-Aldrich; H-89, DMOG, and tunicamycin were from Enzo Life Sciences (Farmingdale, NY, USA), Merck Calbiochem (Darmstadt, Germany), and Wako Pure Chemical Industries (Osaka, Japan), respectively. Recombinant human TNF-α and murine IL-6 were from Promega (Madison, WI, USA) and Peprotech (Rocky Hill, NJ, USA), respectively. Antibodies used in this study were obtained from Novus Biologicals (Littleton, CO, USA), Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnology (Dallas, TX, USA), Merck Millipore (Billerica, MA, USA), Sigma-Aldrich, Abcam (Cambridge, UK), BD Biosciences (San Jose, CA, USA), and Transgenic (Fukuoka, Japan), and are listed in Supplementary Table S1.

After primary hepatocytes adhered to the plates, the culture medium was replaced with serum-free medium (M199 supplemented with 1% BSA and 1% PS) and cells were starved overnight. Glucose-producing solution, i.e., phenol red-free, glucose-free DMEM, containing substrates needed for gluconeogenesis (20 mmol/L lactate, 2 mmol/L pyruvate, 2 mmol/L L-glutamine, and 15 mmol/L HEPES) was prepared. Then, primary hepatocytes were incubated at 37°C for 6 h with a glucose-producing solution with, or without 0.1 mmol/L pCPT-cAMP (Sigma Aldrich, St. Louis, MO, United States) and 1 μmol/L dexamethasone (Sigma Aldrich). Two-hundred microliter of the culture medium were then collected, and the glucose concentration was measured using a colorimetric assay kit (glucose oxidase method; E1010; Applygen, Beijing, China).