Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergent-containing buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of M(r) and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).

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Physical activity during childhood and adolescence may influence the development of childhood obesity and cardiovascular disease later in life. Research focused prospectively on the effects of training on lipid levels in nonobese subjects, and studies using noninvasive measurements of subcutaneous and intraabdominal fat are lacking. It was hypothesized in nonobese sedentary adolescent males that a brief endurance-type exercise training intervention would reduce body fat and improve lipid profiles. Thirty-eight healthy, nonobese sedentary adolescent males (mean age 16 +/- 0.7 years old; 18 controls, 20 trained) completed a 5-week prospective, randomized, controlled study. Adiposity was measured using magnetic resonance images of the thigh and abdomen (subcutaneous abdominal adipose tissue [SAAT] and intraabdominal adipose tissue [IAAT]). Lipid measurements included serum triglycerides (TG), total cholesterol (TC), HDL and LDL cholesterol. There was no change in body weight in either control or training groups. Training led to small but significant reductions in thigh fat (-4.6 +/- 1.5%, p < 0.03) and SAAT% (1.7 +/- 0.8%, p < 0.02). There was no change in IAAT%. Unexpectedly in the control group there were significant increases in thigh fat (5.2 +/- 1.7%, p<0.01), SAAT% (1.8 +/- 0.6, p < 0.007) and IAAT% (4.5 +/- 1.1, p < 0.0007). Training-induced changes in adiposity were not accompanied by changes in circulating lipids. In nonobese adolescent males a brief period of endurance training led to reductions in body fat depots without weight change while body fat increased rapidly in the control group. Exercise training did not change lipid levels, the latter may require more sustained alterations in patterns of physical activity.