meal type: MUFA time: 4h background: Caucasian gender: male age: 64 years
Treatment protocol
The design of the study was a randomized cross-over design with three different treatments. On four occasions, subjects arrived at the university after an overnight fast. On the first day, subjects completed anthropometric measurements, a fasting blood sample was collected, and a hyperinsulinaemic–euglycaemic clamp was performed to measure insulin sensitivity. On the other three days (2-3 weeks between measurements, in randomized order), subjects were studied under baseline conditions and for 4 hours after the ingestion of a liquid high-fat mixed-meal, which was high either in saturated fatty acids (SFA), mono-unsaturated fatty acids (MUFA), or polyunsaturated fatty acids (PUFA).
Growth protocol
Ten insulin resistant men (age 50–70 years; BMI 29-39 kg/m2; HOMAIR >2.5) participated in a single-blinded randomized, crossover study. Exclusion criteria were weight change of >3 kg within the last 3 months before the study; diabetes; chronic inflammatory conditions; kidney or liver dysfunction; use of hypolipidemic or anti-inflammatory medication; use of β-blockers; use of aspirin >1 per week; highly-trained athletes; and alcohol abuse. All subjects were informed about the nature of the study and written informed consent was obtained before study participation. The local Medical Ethical Committee of Maastricht University Medical Centre approved the study protocol.
Extracted molecule
total RNA
Extraction protocol
Skeletal muscle biopsies were taken at baseline (after placing the three catheters and before the background blood sampling) and at the end of the postprandial measurement period (t=240mins). Biopsies were obtained from the vastus lateralis muscle under local anesthesia of the skin and fascia using the Bergström method with suction, cleaned from any visible fat and blood, immediately frozen in isopentane at its melting point, and stored at −80ºC until analysis.
Label
biotin
Label protocol
The Affymetrix GeneChip WT Sense Target Labeling and Control Reagents kit (P/N 900652) was used for the preparation of labelled cDNA from 100ng of total RNA without rRNA reduction. A detailed description can be found in the User Manual, Chapter 3 (P/N 701880, revision 5).
Hybridization protocol
Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 fluidics station using the protocol FS450_0001, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.16.0).
