Determination of fat tissue infiltration in human skeletal muscle biopsies

Abstract

The aim of this study was to develop an assay to determine the level of fat tissue infiltration in muscle biopsies obtained from healthy humans. For this purpose, a muscle biopsy and subcutaneous adipose tissue were obtained from the musculus vastus lateralis in 14 healthy men (age = 33.1 ± 2 yr, height = 175.9 ± 1.7 cm, body mass = 81.2 ± 3.8 kg, body fat = 22.5 ± 1.9%). Protein extracts were prepared from the muscle biopsies and subcutaneous adipose tissue. Then, 50 μg of skeletal muscle protein extracts were run together with subcutaneous adipose tissue protein extract samples containing 1, 2, 3, 4, 5 μg of protein in the same gel. The Western blots were performed using a polyclonal rabbit anti-perilipin A antibody and normalized by alpha-tubulin optical density. From the band optical densities obtained for perilipin a standard curve was calculated by linear regression (all curves had a r2 value 0.98). The corresponding equation was used to calculate the amount of protein from fat origin present in each muscle biopsy. The muscle samples contained 1.18±0.13 micrograms of protein coming from adipose tissue in each 50 μg of protein muscle extract. This perilipin A band density corresponded to a level of adipose tissue protein contamination of 2.4±0.2% in the protein extract from the muscle biopsies. Thus, perilipin A content, a protein exclusive of adipocytes, can be used to determine the degree of adipose tissue infiltration in human skeletal muscle biopsies.