It is well recognized that the binding of insulin to its cell membrane receptor and the resulting activation of receptor tyrosine kinase are a prerequisite for the action of insulin on cells. It is completely unknown, however, how phosphorylation of the insulin receptor effects the translocation of glucose transporters into the cell membrane or the activation or inhibition of insulin-sensitive enzymes in the cytoplasm, mitochondria, and nucleus of the cell. To bridge this gap, numerous substances with a potential second messenger role have been investigated, but none of them has completely fulfilled all the conditions for being the insulin mediator. This lack of understanding of the mechanism of action of insulin in cells under normal conditions is the underlying cause of the lack of understanding of the biochemical basis of postreceptor insulin resistance, which is observed in several disease states as well as during recovery from a severe trauma or injury. The proposed project is intended to study a potential biochemical mechanism producing postreceptor insulin resistance and through this work contribute not only to understanding the biochemical basis of postreceptor insulin resistance but also the cellular mechanism of action of insulin in general. The proposed project is an extension of our recent finding that insulin resistance in skeletal muscle and liver is associated with elevated tissue concentrations of 1,2-diacylglycerol and ceramide. The project is intended to test the hypothesis that a long- term elevation of tissue 1,2-diacylglycerol and ceramide produces insulin resistance in the tissue. Studies will be performed in both insulin- sensitive and insulin-resistant tissues in vivo and in vitro to determine the activity of protein kinase C and the state of phosphorylation or dephosphorylation of specific phosphoproteins which may participate in insulin signalling in cells. The investigation will also evaluate the role of protein phosphatases-1 and -2A, which may reverse the actions of protein kinase C, and determine whether ceramide and its degradation product, sphingosine, can produce insulin resistance at concentrations actually measured in insulin resistant tissues in vivo.
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