The Effects of Chronic Exercise on UCP1 Gene in Human White Adipose Tissue

The investigators obtained three (baseline, post-exercise, post-de-training) subcutaneous fat biopsies from 32 healthy men who undertook three different eight week exercise programs [aerobic (9 participants), resistance (8 participants), combined (aerobic+resistance, 8 participants)] and a non-exercise group (7 participants), followed by an eight week de-training period. Exercise intensity was set at 65% of peak oxygen consumption (VO2peak) and 1 repetition maximum (1RM) throughout the exercise period, while the content of the exercise programs based on a previous study that examined the PGC1a gene, which is thought that increases UCP1 in human white adipose tissue (WAT). Exercise performed in three local gyms in Trikala, Thessaly, Greece, each for every exercise group to avoid cross-contamination between participants, while the exercise supervisors were blinded to the aim of the study. Measurements for anthropometry, body composition, resting energy expenditure (REE), and subcutaneous fat biopsy were obtained at baseline, post-exercise (week 8) and post de-training (week 16) periods while diet data were randomly collected for two weekdays and one weekend day at baseline, 8th and 16th week. VO2peak and 1RM were measured at baseline, 4th (to adjust the required exercise intensity at 65%), 8th and 16th week.

Anthropometry The participants visited the laboratory between 07:00 and 09:00 am and the following anthropometry measures took part: Height was measured using a Seca (Hamburg, Germany) device and weight using a scale (KERN & Sohn GmbH, Version 5.3, Germany) while waist-to-hip ratio was measured using a tape measure and blood pressure through an acoustic method using an Aneroid sphygmomanometer. Percent body fat and fat-free mass were measured via bioelectrical impedance using a body composition monitor (Fresenius Medical Care AG & Co. KGaA D-61346 Bad Hamburg, Germany).

Fat biopsies All biopsies were executed by an experienced surgeon following a previous methodology via a non-diathermy method. The participants underwent a subcutaneous fat biopsy after at least an eight hour fast and they were instructed to refrain from exercise, alcohol, and passive smoking in 72 hours prior the biopsy procedure in order to minimize the risk for misleading results. Each participant was positioned on a surgical bed in a supine position. The site of the incision was disinfected and a 10 ml of xylocaine 2%-no adrenaline was injected in the region of the incision for local anaesthesia. An incision on the skin and subcutaneous tissue until adipose tissue was revealed, was executed approximately 3-5 centimetres nearby the navel while the incision length was approximately 2-2.5 centimetres. Subsequently, the subcutaneous tissue was removed with an operating scissors and when the adipose tissue became visible nearly 500 milligrams of adipose tissue was captured and removed. The collected adipose tissue was immediately immersed in liquid nitrogen of -190° Celsius. For the final deposition the samples were placed in Eppendorfs and they were deposited in a freezer at -80° Celsius until analyses.

UCP1 mRNA analysis The investigators who performed the gene and protein expression analyses were blinded to the aim of the study. Total RNA was extracted from adipose tissue biopsies using RNeasy Lipid Tissue mini kit (QIAGEN) following the manufacturer's protocol. First-strand cDNAs were synthesized from equal amounts of total RNA using random primers and M-MLV reverse transcriptase (Promega). Quantitative real time polymerase chain reaction for the UCP1 gene was performed using Sybr Green fluorophore. The change in fluorescence at every cycle was monitored and a threshold cycle above background for each reaction was calculated. A melt curve analysis was performed following every run to ensure a single amplified product for every reaction. All reactions were carried out in at least duplicated for every sample. 18S rRNA gene was constantly expressed under all experimental conditions and was then used as a reference gene for normalization given that this gene was suggested as the most appropriate one for normalization of UCP1 mRNA.

UCP1 protein analysis Subcutaneous adipose tissue was homogenized in RIPA Lysis Buffer with protease inhibitors (Sigma-Aldrich, Milan, Italy), centrifuged at 800g for 10 minutes at 4° Celsius and then the middle layer was collected. Equal amounts (50 micrograms) of proteins were separated on 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane. UCP1 from subcutaneous adipose tissue samples were detected by primary antibodies, respectively rabbit polyclonal anti-human UCP1 (1:1000, Sigma-Aldrich, Milan, Italy) and mouse monoclonal anti-human β-actin (1:5000, Sigma-Aldrich, Milan, Italy). Secondary antibodies were peroxidase-conjugated anti-rabbit IgG for UCP1 and anti-mouse IgG for β-actin. Human adipocytes that treated with 100uM menthol were used as positive controls for UCP1 protein expression according to previous methodology. Western blotting analysis was performed using Immobilion Western Chemiluminescent HRP Substrate (Millipore) and detection was made using photographic films. The images have been analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density.