Treatment of insulin resistance

Abstract

Methods and compositions are provided for the treatment of insulin-resistance through the inhibition of protein kinase C-mediated phosphorylation of the amino acid residue Ser 1270 of the insulin receptor. Methods for testing candidate compounds suitable for inhibition of serine-phosphorylation by protein kinase C are also provided.

What is claimed is:  
     
         1 . A protein kinase C antagonist characterized by activity in inhibition of protein kinase C-mediated phosphorylation of serine residue 1270 of an insulin receptor.  
     
     
         2 . The protein kinase C antagonist of  claim 1 , wherein the antagonist comprises a three-dimensional structural motif of a protein kinase C substrate, the substrate containing a peptide motif of an amino acid sequence comprising serine residue 1270 of a human insulin receptor, with the proviso that the antagonist is not a native human insulin receptor.  
     
     
         3 . The protein kinase C antagonist of  claim 1 , wherein the activity of the antagonist maintains an insulin-stimulated insulin receptor activity selected from the group consisting of: autophosphorylation activity, tyrosine kinase activity, phosphatidylinositol kinase activity, and insulin-stimulated DNA synthesis.  
     
     
         4 . The protein kinase C antagonist of  claim 1 , wherein the activity is mediated by antagonist interaction with protein kinase C.  
     
     
         5 . The protein kinase C antagonist of  claim 4 , wherein the antagonist interaction inactivates protein kinase C.  
     
     
         6 . The protein kinase C antagonist of  claim 4 , wherein the antagonist interaction is at the protein kinase C catalytic binding site.  
     
     
         7 . The protein kinase C antagonist of  claim 1 , wherein the inhibitory activity is mediated by antagonist interaction with an insulin receptor.  
     
     
         8 . The protein kinase C antagonist of  claim 7 , wherein the interaction with an insulin receptor is binding to an insulin receptor peptide motif.  
     
     
         9 . The protein kinase C antagonist of  claim 8 , wherein the peptide motif contains serine residue 1270 of the insulin receptor.  
     
     
         10 . The protein kinase C antagonist of  claim 1 , wherein the antagonist is a peptidomimetic compound.  
     
     
         11 . The protein kinase C antagonist of  claim 1 , wherein the antagonist is a peptide.  
     
     
         12 . The protein kinase C antagonist of  claim 11 , wherein the peptide has the amino acid sequence DDLHPSFPEVS (SEQ ID NO: 1).  
     
     
         13 . A purified polynucleotide encoding a polypeptide having the amino acid sequence DDLHPSFPEVS (SEQ ID NO: 1), with the proviso that polynucleotide does not encode a native insulin receptor.  
     
     
         14 . The purified polynucleotide of  claim 13 , wherein said nucleotide encodes the amino acid sequence DDLHPSFPEVS (SEQ ID NO: 1).  
     
     
         15 . The purified polynucleotide of  claim 14 , wherein said nucleotide has the sequence SEQ ID NO: 2.  
     
     
         16 . A construct comprising the polynucleotide of  claim 13 .  
     
     
         17 . A transformed host cell comprising the construct of  claim 16 .  
     
     
         18 . The host cell of  claim 17 , where the cell is prokaryotic.  
     
     
         19 . The host cell of  claim 17 , where the cell is eukaryotic.  
     
     
         20 . A method of testing a candidate compound for protein kinase C antagonist activity comprising: 
 contacting the candidate compound with protein kinase C and a substrate for protein kinase C, and detecting a level of phosphorylation of the substrate, wherein the substrate has an insulin receptor amino acid sequence, the amino acid sequence comprising serine residue 1270 of the insulin receptor.    
     
     
         21 . The method of  claim 20 , wherein detecting is by incorporation of a radionuclide.  
     
     
         22 . The method of  claim 20 , wherein the level of phosphorylation of the substrate is detected by detecting a level of insulin receptor function selected from the group consisting of: autophosphorylation activity, tyrosine kinase activity, phosphatidylinositol kinase activity, and insulin-stimulated DNA synthesis.  
     
     
         23 . The method of  claim 20 , wherein said candidate compound is a peptidomimetic compound.  
     
     
         24 . The method of  claim 20 , wherein said candidate compound is a peptide.  
     
     
         25 . The method of  claim 20 , wherein said contacting is performed in a cell-free system.  
     
     
         26 . The method of  claim 20 , wherein the substrate is a polypeptide comprising the amino acid sequence DDLHPSFPEVS (SEQ ID NO: 1).  
     
     
         27 . The method  claim 26 , wherein said substrate is an insulin receptor.  
     
     
         28 . The method of  claim 27 , wherein the insulin receptor is expressed in a mammalian cell.  
     
     
         29 . The method of  claim 28 , wherein said contacting is by microinjection.  
     
     
         30 . A PKC antagonist identified by the method of  claim 20 .  
     
     
         31 . A therapeutic composition comprising: 
 1) a protein kinase C antagonist of  claim 1;  and    2) a physiologically-acceptable carrier.    
     
     
         32 . A method of treating insulin resistance comprising administering to an insulin-resistant patient an amount of a therapeutic composition of  claim 31 , said amount being effective to inhibit protein kinase C phosphorylation of insulin receptors of the patient.  
     
     
         33 . The method of  claim 32 , wherein the insulin-resistant patient is susceptible to non-insulin dependent diabetes mellitus.  
     
     
         34 . The method of  claim 32 , wherein the insulin-resistant patient has non-insulin dependent diabetes mellitus.  
     
     
         35 . A purified polynucleotide encoding a serine phosphorylation-resistant human insulin receptor.  
     
     
         36 . The polynucleotide of  claim 35 , wherein the serine phosphorylation-resistant insulin receptor contains an amino acid substitution at residue position 1270 relative to the amino acid sequence of native human insulin receptor.  
     
     
         37 . The polynucleotide of  claim 35 , wherein the serine phosphorylation-resistant insulin receptor contains alanine at residue position 1270.  
     
     
         38 . A construct comprising the polynucleotide of  claim 35 .  
     
     
         39 . A transformed host cell comprising the polynucleotide of  claim 35 .  
     
     
         40 . A method of treating a patient having insulin resistance comprising: 
 genetically transforming cells of a patient with a construct comprising the polynucleotide of  claim 35 , and a eukaryotic promoter sequence operably linked to the polynucleotide.    
     
     
         41 . The method of  claim 40 , wherein the cells are skeletal muscle cells.