The blood levels of triglyceride, cholesterol, non-esterified fatty acid (Wako, Osaka, Japan), adiponectin (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan), 3-hydroxybutyric acid (Abbott Laboratories, IL, USA) and insulin, (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan) were determined with commercial assay kit according to the manufacturer’s instructions.
Non esterified fatty acid
Manufactured by Fujifilm
Sourced in Japan, United States, Spain, Germany
Non-esterified fatty acids are a class of lipid molecules that are not bound to other molecules. They play a crucial role in various biological processes and are commonly used in laboratory equipment and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Omegawax 250 column, by Merck Group (2 mentions)
Minispec lf90ii, by Bruker (2 mentions)
Adiponectin, by R&D Systems (2 mentions)
Infinity cholesterol, by Thermo Fisher Scientific (2 mentions)
β hydroxybutyrate, by Thermo Fisher Scientific (2 mentions)
Infinity triglyceride reagent, by Thermo Fisher Scientific (2 mentions)
Saccharin, by Merck Group (1 mentions)
Quinine hydrochloride, by Merck Group (1 mentions)
Leptin, by Merck Group (1 mentions)
Glycerol, by Cayman Chemical (1 mentions)
Cholecystokinin, by Phoenix Pharmaceuticals (1 mentions)
Total gip, by Merck Group (1 mentions)
Total glp 1, by Merck Group (1 mentions)
Intralipid, by Merck Group (1 mentions)
Insulin, by ALPCO (1 mentions)
Triacylglyceride reagent, by Merck Group (1 mentions)
Immulite 1000, by Siemens (1 mentions)
Free glycerol, by Merck Group (1 mentions)
Cholesterol e, by Fujifilm (1 mentions)
Glucose meter, by Bayer (1 mentions)
23 protocols using non esterified fatty acid
1
Metabolic Marker Determination Protocol
2
Taste and Lipid Metabolism Assessment
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As taste stimuli, we used a “sweet solution” consisting of a mixture of 0.125% saccharin and 3% glucose (both Sigma-Aldrich, St Louis, MO) or a “bitter solution” consisting of 0.15% (0.0038 M) quinine hydrochloride (Sigma-Aldrich). The saccharin-glucose mixture is avidly ingested by rats [12] (link); the 0.15% quinine is strongly disliked–tasting it elicits negative hedonic responses (i.e., gapes and chin rubs) and it is almost completely avoided in two-bottle preference tests [7] (link). As an intragastric fat load, 20% intralipid was purchased from Sigma-Aldrich (Cat. No. I-141). Radioactive 14C-triolein was purchased from American Radiolabeled Chemicals Inc (St Louis, MO) and stored at −20°C until use.
The following enzymatic colorimetric kits or ELISA kits were used for the assay of blood components; triglycerides, ketones, glycerol and glucose from Cayman Chemical Co. (Ann Arbor, MI); non-esterified fatty acid from Wako Diagnostics (Richmond, VA); insulin from Alpco Diagnostics (Windham, NH); total GIP, total GLP-1 and leptin from Millipore (Billerica, MA); peptide YY and cholecystokinin from Phoenix Pharmaceuticals (Belmont, CA).
The following enzymatic colorimetric kits or ELISA kits were used for the assay of blood components; triglycerides, ketones, glycerol and glucose from Cayman Chemical Co. (Ann Arbor, MI); non-esterified fatty acid from Wako Diagnostics (Richmond, VA); insulin from Alpco Diagnostics (Windham, NH); total GIP, total GLP-1 and leptin from Millipore (Billerica, MA); peptide YY and cholecystokinin from Phoenix Pharmaceuticals (Belmont, CA).
3
Metabolic Biomarkers Measurement Protocol
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Plasma glucose (Thermo Scientific, Waltham, MA), non-esterified fatty acid (Wako Chemicals USA, Richmond, VA, USA), triacylglyceride (Triacylglyceride reagent; Sigma Aldrich), and total‐ and high-density lipoprotein (HDL; Cholesterol E and HDL-Cholesterol E; Wako Chemicals USA) cholesterol concentrations were measured using commercially available colorimetric assay kits. Plasma insulin concentration was measured with a chemiluminescent immunoassay (Siemens IMMULITE 1000, Flanders NJ). Matsuda insulin sensitivity index (ISI) was calculated as described by Matsuda and DeFronzo (1999) (link).
4
Quantitative Liver Lipid Analysis
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Plasma concentrations of triglycerides (Sigma) and non-esterified fatty acid (Wako Diagnostics) were measured using commercial assay kits. Liver triglyceride was extracted and measured as previously described 58 (link). Body fat and lean mass were measured using an NMR analyzer (Minispec LF90II, Bruker Optics). For liver lipid analysis, lipids were extracted by the method of Bligh-Dyer in the presence of an internal standard (T21:0 TAG, 10 nmol/mg protein) and separated on silica gel 60-Å plates that were developed with a nonpolar acidic mobile phase (70:30:1, v/v/v, hexane/ethyl ether/acetic acid). Briefly, spots corresponding to TAG were visualized with 0.01% rhodamine 6G and identified with TAG standard. The bands were scraped, extracted, and treated with acidic methanol. Quantitative GC analysis of resulting FA methyl esters was conducted (Hewlett-Packard 5890 GC; Hewlett-Packard, Palo Alto, CA, USA) with a 30-m×0.32 mm Omegawax 250 column (Sigma) coupled with a flame ionization detector.
5
Metabolic Profiling in Mice
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Blood tail vein glucose was measured using a glucose meter (Bayer, Leverkusen, Germany). Glucose tolerance tests or insulin tolerance tests were performed by intraperitoneal injection of 1 or 2 g kg−1 body weight glucose or 0.5 IU kg−1 body weight insulin after 16-hour or 5-hour fasting, respectively. Plasma triglyceride (ThermoFisher), Cholesterol E, nonesterified fatty acid (Wako, Osaka, Japan), free glycerol (Sigma) were measured using colorimetric assays according to the manufacturer’s protocol. Plasma insulin (Mercodia, Uppsala, Sweden) and adiponectin (R&D system, MN) were assessed by ELISA. Hepatic and adipose lipids were extracted by the Folch method, and measured by colorimetric assays as above. Mice were individually housed in metabolic chambers (CLAMS, Columbus Instrument) in order to assess metabolic activity. Mice were acclimated for 48 h before data were collected. Mice had free access to food and water for recordings. Oxygen consumption, carbon dioxide production, and RER were calculated at 20 min intervals44 (link).
6
Quantitative Liver Lipid Analysis
7
Quantifying Metabolic Markers in Insulin Signaling
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Plasma concentrations of free glycerol and triglycerides (Sigma), β-hydroxybutyrate (Stanbio Laboratory), and nonesterified fatty acid (Wako Diagnostics) were measured using commercial assay kits. Liver triglyceride was extracted and measured as previously described (38 (link)). Plasma insulin was measured using an ELISA kit (CrystalChem). Glucose and insulin tolerance tests were performed as previously described (39 (link)). For insulin signaling studies, mice were fed HFD for 8 weeks before receiving a single dose of intravenous injection of saline or insulin (1.5 units/kg). Tissues were rapidly dissected 10 min after injection for immunoblotting analyses.
For gene expression analysis, total RNA from WAT was extracted using a commercial kit from Invitrogen. Total RNA from other tissues and cultured cells was extracted using the TRIzol method. For qPCR analysis, equal amount of RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase followed by qPCR reactions using SYBR Green (Life Technologies). Relative abundance of mRNA was normalized to ribosomal protein 36B4. Adipose tissue and liver gene expression was analyzed using specific primers (Supplementary Table 1 ). Statistical significance was determined by Student t test.
For gene expression analysis, total RNA from WAT was extracted using a commercial kit from Invitrogen. Total RNA from other tissues and cultured cells was extracted using the TRIzol method. For qPCR analysis, equal amount of RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase followed by qPCR reactions using SYBR Green (Life Technologies). Relative abundance of mRNA was normalized to ribosomal protein 36B4. Adipose tissue and liver gene expression was analyzed using specific primers (
8
Plasma Metabolite Measurements
9
Hepatic Lipid Extraction and Quantification
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Lipids were extracted from liver samples using Folch solution composed of a 2:1 (vol/vol) mixture of chloroform/methanol as described previously56 (link). Lipids were solubilized in 10% Triton X-100 before evaporation. Non-esterified fatty acid (Fujifilm/Wako, Lexington, MA, USA), cholesterol (Stanbio, Boerne, TX, USA), and triglyceride content (Stanbio, Boerne, TX, USA) were measured in hepatic extracts and plasma samples using colorimetric assays.
10
Liver Lipid Biomarker Measurement Protocol
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Liver triglycerides were measured using an enzymatic assay (#T7532-120, Pointe Scientific, Canton MI). Plasma was stored at -80 °C until further processing. Plasma was diluted 1:20 in saline in order to measure triglycerides (#TR22421, Infinity Triglyceride Reagent, Thermo Scientific, Waltham, MA). Cholesterol (Infinity Cholesterol, #TR13421, Thermo Scientific, Waltham, MS), total bile acids (#BQ 092A-EALD, BQkits Diagnostics, San Diego, CA), β-hydroxybutyrate (#SBHR-100, Fisher Scientific, Waltham, MA), and non-esterified fatty acids (Wako Diagnostics, Richmond, VA) were measured using enzymatic assays. TGFβ measurements were made using a standard ELISA (#MB100B, R&D Systems, Minneapolis, MN). Estradiol and progesterone assays were performed by the Vanderbilt Hormone Assay and Analytical Services Core (Vanderbilt University, Nashville, TN).
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