Total cholesterol

Plasma insulin, IL-6, TNFα, IL-10, leptin, and MCP1 (eBioscience, USA) were measured by ELISA. Plasma and liver triacylglycerol (TG), non-esterified fatty acids (NEFA) and total cholesterol (Wako Chemicals, Richmond, USA) levels were measured enzymatically.

Food quantity was measured for 3 consecutive days to determine mean daily food intake. Tissue and blood collection was carried out at 32 weeks of age. Serum triglyceride, total cholesterol (Wako Pure Chemicals, Osaka, Japan) and NEFA levels (Eiken Chemicals, Tochigi, Japan) were measured using enzymatic method. Glucose level was determined using Glutest Ace (Sanwa Kagaku, Nagoya, Japan). Insulin and leptin levels were measured by enzyme-immuno-assay (Morinaga Institute of Biological Science, Yokohama, Japan). Human regular insulin (Humalin R; Novo Nordisk, Bagsvaerd, Denmark) was used for ITT. Serum LCN2 levels were determined by enzyme-linked immunosorbent assay (BioPorto Diagnostics, Hellerup, Denmark).
Blood pressure was determined as mean of 6 consecutive measurements by indirect tail-cuff method with MK-2000ST (Muromachi Kikai, Tokyo, Japan).
To extract catecholamine, BAT samples were weighed and homogenized on ice in 10 volumes of 0.4 N HClO₄ buffer (containing 2 g/L ethylenediamine tetraacetate-2Na and 20 mg/L ascorbic acid). Supernatant was collected after centrifugation at 3500 rpm for 20 min at 4 °C. Noradrenaline and adrenaline levels in urine or BAT were determined by high-performance liquid chromatography–electrochemical detection (HLC-725CAII, Tosoh Bioscience, Tokyo)^{23 (link),51 (link)}.

Blood samples were collected in EDTA-containing tubes, and centrifuged at 3000 rpm for 5 min to obtain plasma, which was stored at −20 °C for analysis. Plasma Total cholesterol and triglycerides were determined using enzymatic kits (Total cholesterol, Wako (LabAssay); triglycerides (Sigma)) according to the manufacturers’ instructions.

Glucose concentrations were measured by a hexokinase method (Randox Laboratories, Co. Antrim, UK). NEFA, total cholesterol, HDL-cholesterol, triacylglycerol (TG) and liver function tests (LFTs) were measured by colorimetric methods (NEFA: Wako Chemicals, Neuss, Germany; total cholesterol: Olympus Diagnostics, Watford, UK; TG: Alpha Laboratories, Eastleigh, UK; LFTs: Abbott Diagnostics, Maidenhead, UK). Serum samples for insulin and C-peptide were analysed by ELISA (Mercodia, Uppsala, Sweden).

Mice were denied access to food for 16 h before the measurements. 30-μl blood samples were collected from the tail vein in accordance with the guidelines for animal experiments of the University of Tokyo. Fasting blood glucose was measured with an automatic glucometer (Glutest Ace. Sanwa Chemical Co., Japan). Plasma levels of insulin (Morinaga Co., Ltd., Japan), triglyceride, total cholesterol, free fatty acids and high-density lipoprotein (Wako Pure Chemical Industries, Ltd., Japan) were assayed by enzymatic methods. Plasma total adiponectin levels (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and plasma HMW adiponectin levels (ALPCO Diagnostics., USA) were measured with ELISA kits.