L glutamine

CRISPR/Cas9 gene-edited NIH3T3 cells expressing endogenous levels of EHD1 with GFP fused to the C-terminus were plated on fibronectin-coated coverslips and grown for 4 h at 37°C in 5% CO2 in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), 1x Penicillin Streptomycin (Gibco, 15140–122), 50 mg of Normocin (InvivoGen, NOL-40-09), and 2 mM L-Glutamine (Gibco, 25030–081). The cells were then treated with HA-tagged EHD4 in pcDNA 3.1 (+) (Invitrogen, V79020) for 72 h at 37°C in 5% CO2 in DMEM/High Glucose containing 10% heat inactivated Fetal Bovine Serum, and 2 mM L-Glutamine, using FuGene 6 Transfection Reagent (Promega, E2691), following the manufacturer’s protocol.

DRGs from adult male Sprague-Dawley rats were dissected and dissociated as described previously (Calcutt et al., 2017 (link); Sabbir et al., 2018 (link)) and cultured in defined Hams F12 media containing 10mM D-glucose (N4888, Sigma, St Louis, MO, USA) supplemented with modified Bottenstein's N2 additives without insulin (0.1 mg/ml TF, 20 nM progesterone, 100 μM putrescine, 30 nM sodium selenite, 0.1 mg/ml BSA; all additives were from Sigma, St Louis, MO, USA) (Akude et al., 2011 (link); Roy Chowdhury et al., 2012 (link); Saleh et al., 2013 (link); Calcutt et al., 2017 (link)). In all experiments, the media was also supplemented with 0.146 g/L L-glutamine, a low dose cocktail of neurotrophic factors (0.1 ng/ml NGF, 1.0 ng/ml GDNF and 1 ng/ml NT-3 – all from Promega, Madison, WI, USA), 0.1 nM insulin and 1X antibiotic antimycotic solution (A5955, Sigma).

Prior to bioluminescence analysis of Bmal1-dLuc fibroblast cultures on 35 mm dishes, growth medium containing control or experimental treatments (IL-6 or TNFα recombinant proteins, neutralizing antibodies, palmitate, or BSA) was removed. Cultures were rinsed, placed in DMEM recording medium containing 15uM forskolin, 25 mM HEPES, 292 µg/ml L-glutamine, 100units/ml penicillin, 100 μg/ml streptomycin and 10uM luciferin (Promega) and then sealed airtight with sterile glass coverslips and sterile silicon grease. The temporal patterns of Bmal1-dLuc bioluminescence were analyzed using an automated 32-channel luminometer (LumiCycle; Actimetrics) housed in a standard culture incubator at 35 °C. Bioluminescence from individual cultures was continuously recorded for ~70 sec at intervals of 10 min and analyzed using the LumiCycle Analysis program. As described previously^{5 (link),36 (link)}, rhythm parameters (period, amplitude) were determined from baseline-subtracted data using the damped sine fit and Levenberg–Marquardt algorithm (Y(t) = A*sin (2πft + ϕ)*e^−t/τ + C). The amplitude of phase shifts in response to treatment with IL-6 or TNFα recombinant proteins, or palmitate was determined by measuring the time difference between the peaks of the Bmal1-dLuc rhythms during the third cycle in PBS, BSA or BSA/vehicle (PBS) controls and experimental treatment groups.