Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid.

Abstract

Insulin binding to plasma membrane receptors results in the generation of substances that acutely mimic the actions of the hormone on certain target enzymes. Two such substances, which modulate the activity of the high-affinity cAMP phosphodiesterase (EC 3.1.4.17), have been purified from hepatic plasma membranes. The two have similar properties and activities but can be resolved by ion-exchange chromatography and high-voltage electrophoresis. They exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximately 1400. The generation of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol-specific phospholipase C purified from Staphylococcus aureus. This enzyme is known to selectively hydrolyze phosphatidylinositol and release from membranes several proteins that are covalently linked to phosphatidylinositol by a glycan anchor. Both enzyme-modulating substances appear to be generated by the phosphodiesterase cleavage of a phosphatidylinositol-containing glycolipid precursor that has been characterized by thin-layer chromatography. Some of the chemical properties of these substances have been examined. They appear to be related complex carbohydrate-phosphate substances containing glucosamine and inositol. These findings suggest that insulin may activate a selective phospholipase activity that hydrolyzes a membrane phospholipid, releasing a carbohydrate-containing molecule that regulates cAMP phosphodiesterase and perhaps other insulin-sensitive enzymes.The molecular events involved in the biological actions of insulin remain poorly understood. Several studies have suggested that, subsequent to the interaction of insulin with its receptor, one or more chemical mediators are generated that alter the activities of certain insulin-sensitive enzymes (1). The production of these substances has been detected in insulin-treated cells (2, 3). They can also be generated from membrane fractions in response to insulin (4-7), suggesting that all of the necessary components for this process are located in the plasma membrane.In this communication we report the purification and identification of two related carbohydrate-phosphate substances that are generated from liver plasma membranes in response to insulin. These substances modulate the activity of the high-affinity cAMP phosphodiesterase (PDE; EC 3.1.4.17) and may be responsible for the regulation of this enzyme by insulin. EXPERIMENTAL PROCEDURESMaterials. All reagents were from Sigma, with the exception of collagenase (Millipore); Generation of cAMP PDE Modulators by Insulin. These were generated from bovine liver particulate fractions by previously described methods (6)(7)(8) with modification. The particulate fraction (8) was prepared from 100 g of bovine liver and suspended in 500 ml of 50 mM ammonium bicarbonate, pH 7.4 (buffer A), at a final concentration of 2-4 mg of protein per ml. This solution was incubated with 10 nM insulin for 10 min at 370C and acidified to pH 4.0 with formi...

Malarial toxic antigens synergistically enhance insulin signalling

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Insulin‐induced decrease in 5′‐nucleotidase activity in skeletal muscle membranes

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