Elm adiponectin

Lipid biomarkers including low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), non-esterified fatty acids (NEFA), TC, and TG were measured using colorimetric assays following the manufacturer’s instruction (Wako Diagnostics, USA). Plasma insulin levels were measured using a mouse insulin ELISA kit (EZRMI-13 K, EMD Millipore Co, Billerica, MA, USA). Adiponectin was measured using a mouse enzyme-linked immunosorbent assays (ELISA) kit (ELM-Adiponectin, RayBiotech, USA). Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP1), and hormones (leptin and resistin) were measured using a Bio-Plex kit (Biorad, USA). The cytokine assay kit was not sensitive enough to detect the inflammatory cytokines in most of the samples. Therefore, values of these cytokines IL-6, TNF-α, and MCP-1 were excluded.

Insulin was measured in duplicates using ultra-sensitive mouse ELISA kit (90080, Crystal Chem, Downers Grove, IL) according to manufacturer’s instructions.
Adiponectin was measured using a commercial available ELISA kit according to manufacturer’s protocol (ELM-Adiponectin, RayBiotech, Norcross, GA).
For liver triglycerides measurements, 50 mg liver was weighted and added 125 ul ethanolic KOH in microfuge tubes. Samples were incubated overnight and added 175 ul H₂O:EtOH (1:1), centrifuged 5 min at 5000 rpm and the supernantant was moved to new tubes. 100 ul EtOH was added, vortexed and 200 ul was moved to new tubes and added 215 ul 1M MgCl₂. Samples were centrifuged, moved to new tubes and measured for triglycerides.
Free fatty acids were measured by a commercial available kit according to the supplied instructions (NEFA-HA(2), Wako, Neuss, Germany).