Nefa hr 2 kit

250 mg of STD-, HSD-fed or 500 mg of HFD- and WD-fed adipose tissue was freshly isolated after extraction pieces for punch biopsies, transferred into 37 °C KRBH+ (Krebs-Ringer-Bicarbonate-HEPES; 20 mM HEPES, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, 1 g/l glucose, 2% BSA), minced, and digested with fresh collagenase type II (C6885, Sigma) for 30 min in a shaking water bath @ 37°C with 120 rpm disrupting tissue by pipetting every 7 min. Digested adipocytes were filtered through a 250 μm cell strainer (ThermoFisher). After 5 min for layer formation (adipocyte toplayer and sublayer buffer), buffer was withdrawn, adipocytes were washed with KRBH+ and after layer formation buffer was aspirated again. Adipocyte were appropriately adjusted with KRBH+ to the sample with lowest cell weight and adipocyte solution was plated in a 96-well plate (V-shape, Sarstedt). Triplicates were stimulated with 0, 1, 10, 25, 50 nM isoprenaline (Sigma) on a plate shaker with 300 rpm for 3.5 h at 37°C. 10 min before end, shaking was stopped to allow layer formation again and buffer was collected and stored at −80°C for analysis of free fatty acid (FFA) secretion in duplicates via NEFA-HR(2) kit (Wako).

Blood glucose concentrations were measured using a handheld glucometer (Auvon). Plasma FGF-21, insulin, corticosterone, and VEGF concentrations were measured by ELISA (R&D Systems, Mercodia, Abcam, and Sigma, respectively), and total plasma bile acids using a colorimetric assay (Abcam). Kidney PC activity was measured enzymatically^{54 (link)}, and plasma NEFA using the Wako NEFA-HR(2) kit. Bicarbonate and transaminase (ALT, AST) concentrations were measured by COBAS, and tissue triglyceride concentrations measured enzymatically using the method of Bligh and Dyer^{55 (link)}. Acetyl-^{56 (link)} and long-chain acyl-CoA concentrations^{36 (link)} were measured by LC-MS/MS while β-OHB by GC/MS^{57 (link)} as previously described. Sections of the right medial lobe of the liver were obtained from mice with NASH, fixed in 10% neutral buffered formalin, and stained with hematoxylin & eosin. Samples were examined and images captured by a blinded investigator using an Olympus BX51 multi-headed brightfield microscope (Yale Liver Center Morphology Core). Whole kidneys were obtained from mice with kidney cancer, fixed in 10% neutral buffered formalin, and stained with hematoxylin & eosin. Sections from the center of the kidney cortex were stained with hematoxylin & eosin, examined and images captured by a blinded investigator using an Olympus BX51 microscope.

e2f1^−/− or WT MEF adipocyte-like cells were incubated without or with TNF (10 ng/ml) + IL1B (10 ng/ml) treatment for 20 h in DMEM 10% FCS. Next, cells were washed 3 times with PBS and incubated with Krebs-Ringer buffer (KRBH; 135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.4 mM K₂HPO₄, 1 mM CaCl₂, 20 mM HEPES, pH 7.4) for 1 h. Glycerol release was measured in KRBH buffer using Free Glycerol reagent (Sigma-Aldrich, F6428) and free fatty acids release was measured by NEFA-HR2 kit (Wako Chemicals, 999-34691). Results were normalized to mg protein cell lysate.