The effect of caloric restriction on the lateral diffusion constant of hepatocyte membrane proteins in C57BL/6 male mice of different ages: FRAP studies on liver smears.

The lateral mobility of proteins in hepatocyte plasma membranes was compared in calorically restricted and ad libitum (AL)-fed C57BL/6 male mice in age groups from 7 to 28 months. Caloric restriction was achieved by means of the every-other-day (EOD) feeding regimen, maintained for various periods from 1 to 15 months. Protein lateral diffusion constant (D) in hepatocyte membranes was measured by means of fluorescence recovery after photobleaching (FRAP) in liver smears. The peroxide-induced autofluorescence (PIAF) was utilized as a fluorescent label. A mild (1 mM for 10 min) H2O2 treatment of liver smears produces oxidation of riboflavin that is bound to all proteins of the cell membrane. Using this technique, the average lateral diffusion constant (D) and the fractional recovery (FR) of these proteins can be measured. EOD feeding resulted in a significant decrease in body weights and also a significant increase in the values of D in all age groups after 1 month of EOD feeding. After 3.5 months of EOD feeding a further increase of D was observed (up to about 15%). Nevertheless no further change in D occurred if the EOD feeding was maintained for 6.5 or even 15 months. The negative linear age correlation of D observed in the AL-fed animals was present also under the EOD feeding; however, the whole regression equation shifted towards higher values. These experiments indicate that caloric restriction influences the lateral diffusion constant of membrane proteins in hepatocytes. The results are interpreted as a result of an increased protein turnover caused by the caloric restriction.