Insulin-like Effect of Clostridial Phospholipase C, Neuraminidase, and Other Bacterial Factors on Brown Fat Cells

Abstract

Abstract Incubation of brown fat cells with a preparation of neuraminidase from Clostridium perfringens at a concentration of 50 µg per ml stimulated basal glucose oxidation and abolished any further response of the cells to insulin and cysteine. High concentrations (125 or 250 µg per ml) of this neuraminidase preparation were toxic to both white and brown fat cells. Preparations of neuraminidase from Vibrio cholerae or Lee stain influenza virus did not affect glucose metabolism in either the absence or presence of insulin or cysteine. The effect of the C. perfringens neuraminidase appears to be due to contamination with a potent insulin-like agent. Crude preparations of the enzyme produced an insulin-like effect at a concentration of 0.1 µg per ml. Chemical analysis of the sialic acid content of both brown and white fat cells indicated that most of the sialic acid present in adipose tissue is removed during preparation of the cells by digestion with crude collagenase. A partially purified preparation of C. perfringens phospholipase C stimulated basal glucose metabolism at a concentration of 1 µg per ml in brown fat cells and inhibited the response to insulin and cysteine at a concentration of 5 µg per ml. Insulin-like activity was similarly observed with 10 µg per ml of a purified preparation of clostridial phospholipase C. The ability to stimulate glucose conversion to carbon dioxide by the purified phospholipase C was blocked by 1 mm EDTA or Mg-EDTA while that of the partially purified preparation was enhanced. The addition of egg lipoprotein (10 µl per ml) to the medium, enhanced the insulin-like activity of high concentrations of crude preparations of clostridial phospholipase C and neuraminidase. Treatment of brown fat cells with concentrations of trypsin comparable to those of C. perfringens neuraminidase completely inhibited the stimulation of glucose metabolism by insulin but had little effect on that due to cysteine. Basal glucose oxidation was unaffected by trypsin treatment. White and brown fat cells treated with trypsin did not recover their responsiveness to insulin after four hours of incubation. Our findings indicate that neuraminidase treatment of fat cells does not mimic the action of trypsin and that some commercially available preparations of neuraminidase and phospholipase C from C. perfringens contain insulin-like impurities.