Glycerol

Assessments of clinical chemistry parameters were performed using an AU480 Automated Chemistry Analyzer (Beckman Coulter, Brea, CA, USA) and specific kits for free fatty acids (Wako Chemicals, Neuss, Germany), glycerol (Randox Laboratories, Crumlin, UK) as well as all other parameters covered by the present study (Beckman Coulter, Brea, CA, USA)^{126 (link)}. A set of 21 parameters consisting of specific metabolite levels, electrolyte concentrations and enzyme activities was measured in fed mice: albumin, α-amylase, alkaline phosphatase (AP), aspartate-aminotransferase (ASAT/GOT), alanine-aminotransferase (ALAT/GPT), calcium, cholesterol, chloride, creatinine, fructosamine, glucose, iron, lactate, lactate dehydrogenase (LDH), phosphate, potassium, sodium, total protein, triglycerides, urea and unsaturated iron-binding capacity. In addition, a selection of parameters (cholesterol, glucose, glycerol, HDL-cholesterol, non-esterified fatty acids (NEFA), non-HDL cholesterol and triglycerides) was measured in fasted animals after 4 (Ghrhr study), 6 (IF study) or 16–18 h (mTOR study, C57BL/6J baseline study) of food deprivation. Plasma insulin levels were measured using a commercial kit based on immuno-electrofluorescence (Mesoscale Discovery, Rockville, Maryland, USA) or ELISA technology (Mercodia, Uppsala, Sweden).

Heparinized plasma was prepared from blood collected by cardiac puncture at the termination and stored at −80 °C prior to analysis. Insulin concentrations were analyzed by DRG Ultrasensitive Mouse Insulin ELISA kit (DRG Diagnostics, Germany). Plasma alanine aminotransferase, total cholesterol, LDL-cholesterol, triacylglycerides (TAG), glucose (MaxMat, France), non-esterified fatty acids (NEFA), HDL-cholesterol, total bile acids (Dialab, Austria), hydroxy-butyrate (OH-butyrate), glycerol (Randox, UK) and lactate (Sentinel Diagnostics, Italy) concentrations were analyzed by conventional kits using a MaxMat PL II analyzer (MAXMAT S.A., Montpellier, France).

Plasma glucose levels were measured by glucose oxidase assay (GLU GOD, Erba-Lachema, Brno, Czech Republic) and TAG, cholesterol, HDL-cholesterol (Erba-Lachema, Brno, Czech Republic), and glycerol (Randox Laboratories Ltd., Crumlin, UK) concentrations were quantified using standard enzymatic methods. Non-esterified fatty acid levels (NEFA) were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Plasma insulin concentrations were measured using a rat insulin enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden). MCP-1 and leptin concentrations were measured by rat multiplex enzyme-linked immunosorbent assay kit (Milliplex: RADPKMAG-80K, Merck KGaA, Darmstadt, Germany).