Labassay cholesterol

Serum was collected for the measurement of plasma lipids and plasma proteins. 500-700 µl of blood were collected from each mouse and centrifuged at 3.500× g for 10 min at room temperature. Blood serum was transferred to a fresh 1.5 ml tube and snap-frozen in liquid nitrogen. Cholesterol was measured using LabAssay Cholesterol (Fuji Film, Japan) following the manufacturer’s instructions.
The Olink Mouse Exploratory Panel was used to measure circulating proteins. Serum was prepared according to the manufacturer’s instructions (Olink, Sweden) and measured with the Mouse Exploratory 96 panel. Protein concentration was determined using the proximity extension assay (PEA) technology described elsewhere (18 (link)). Briefly, oligonucleotide-labeled antibodies bind to their target proteins where they come into proximity with other labeled antibodies that bind to the same target. The oligonucleotides hybridize in proximity and form a basis for qPCR and quantification. The number of qPCR cycles stands in relation to the protein concentration and gives the arbitrary unit normalized protein expression (“NPX”) as the readout. To examine changes in the secretome in the hyperlipidemic mouse model vs. mice fed a normal chow (NC) diet, two-sideded t-test was performed, followed by enrichment analysis of the significant regulated proteins (FDR cutoff= 0.1).

Plasma triacylglycerol and total cholesterol levels were determined using a LabAssay™ Triglyceride Kit (Wako) and a LabAssay™ Cholesterol (Wako), respectively. To analyze fecal fatty acids, the total lipids in feces were extracted as described above without adding an internal standard, after which they were saponified using 0.3 N of NaOH (Wako) in 90% MeOH (Wako). The fatty acids were extracted using n-hexane (Wako), and the organic layer was dried using N₂ gas. The residues were dissolved in 2-propanol (Wako), and fatty acid concentrations were measured using a LabAssay™ NEFA (Wako).

Blood glucose was assessed using a portable glucometer with compatible glucose test strips (OneTouch^®Ultra^®, LifeScan, Milpitas, CA, USA). Plasma cholesterol (LabAssay™ Cholesterol, Wako, Tokyo, Japan), non-esterified fatty acids (NEFAs) (LabAssay™ NEFA, Wako, Tokyo, Japan), triglyceride (TG) (LabAssay™ Triglyceride, Wako, Tokyo, Japan), and insulin (Insulin ELISA KIT (RTU), Shibayagi, Gunma, Japan) were measured using commercial assay kits following manufacturer’s instructions.

Blood glucose concentrations were measured using a One Touch Ultra device (LifeScan, Milpitas, CA, USA). The concentrations of plasma triglycerides (LabAssay Triglyceride; Wako), total cholesterol (LabAssay Cholesterol; Wako), non-esterified fatty acid (LabAssay NEFA; Wako), Peptide YY (Mouse/Rat PYY ELISA Kit, Wako Pure Chemical Co. Ltd., Osaka, Japan), GLP-1 (active) ELISA kit (Merck Millipore, Billerica, MA, USA), and insulin ELISA Kit (Shibayagi, Gunma, Japan) were measured according to manufacturer instructions. For GLP-1 measurement, plasma samples and culture media were treated with a DPP-IV inhibitor (Merck Millipore, Billerica, MA, USA) to prevent degradation of active GLP-1. Bacterial endotoxin levels were evaluated by a limulus amebocyte lysate chromogenic endpoint assay, according to the manufacturer’s instructions (Hycult Biotech, Wayne, PA, USA).

Blood was collected from mice fasted for 6 h by cardiac puncture into EDTA coated tubes and plasma was obtained by centrifugation at 3000r.p.m/20 min/4 °C. Plasma insulin was measured by Ultra-Sensitive Mouse Insulin ELISA kit (Crystal Chem), ALT activity by kinetic colorimetric assay (Sigma–Aldrich), free fatty acids by NEFA-HR(2) assay (Wako Chemicals), cholesterol by LabAssay Cholesterol (Wako Chemicals) and triglycerides by Cobas TRIGB kit (Roche/Hitachi), all following manufacturer instructions. Absorbance was measured by SynergyMx Plate reader and data analyzed by Gen5 v3.08 software (BioTek). CTHRC1 in human plasma was determined by sandwich ELISA following kit instructions (MMCRI)^{55 (link)} We have validated the antibody by overexpression of CTHRC1 in HEK-293T cells, which do not express this protein, and determining CTHRC1 levels in culture media. Importantly, we also spiked recombinant CTHRC1 protein into plasma of patients with undetected circulating CTHRC1.

Serum triglyceride and serum total cholesterol were measured by LabAssay Triglyceride (Wako Chemicals, Richmond, VA, USA) and by LabAssay Cholesterol (Wako Chemicals, Richmond, VA, USA), respectively. Plasma insulin was measured by Ultra Sensitive Rat Insulin ELISA Kit (Morinaga Institute of Biological Science, Yokohama, Japan).