Impaired insulin receptor phosphorylation in skeletal muscle membranes of db/db mice: the use of a novel skeletal muscle plasma membrane preparation to compare insulin binding and stimulation of receptor phosphorylation

A method has been developed to isolate skeletal muscle plasma membranes from mice in good yield without harsh extraction procedures. The method involves perfusion of mouse hindquarters with a calcium-deficient buffer containing collagenase and hyaluronidase. This is followed by gentle disruption, filtration, and differential centrifugations. The entire procedure takes about six hours and the yield is approximately 4 mg. protein from 10 g. equivalent of hindquarter muscle. The preparation contained predominantly plasma membranes based on specific activities of marker enzymes, electron microscopic data, and specific binding sites for insulin and a -adrenergic ligand. Studies using such preparations from lean, 4-5 week old and 12-20 week old db/db mice showed marked reduction in the phosphorylation of the 95 kDa subunit of the insulin receptor of the obese mice with no change in insulin binding. In addition, there was a progressive reduction in insulin sensitivity in stimulating receptor phosphorylation in the db/db mice.