Novel mechanism for identifying drugs for the treatment of type II diabetes

Abstract

Insulin resistance is a central feature of type II diabetes and other diseases, and may affect every tissue of the body, including the pancreatic beta cell. Insulin signaling is mediated by a complex network of diverging and converging pathways, with alternative proteins and isoforms at almost every step in the process. We have previously shown that insulin activates the transcription of its own gene by signaling through Insulin Receptor A type (Ex11−), PI3 kinase and p70 s6 kinase. When studying the mechanisms underlying the glucose-stimulated activation of the glucokinase gene in pancreatic beta cells, we now demonstrate that also here secreted insulin is a key-factor. In contrast to the insulin gene, transcription of the glucokinase gene is promoted by signaling via Insulin Receptor B type (Ex11+) and protein kinase B (c-Akt). These data provide the first evidence for selectivity in insulin action via the two isoforms of the Insulin Receptor, A type (Ex11−) and B type (Ex11+), and reinforce the concept of the beta cell being an important target of insulin action.

1 . An Insulin Receptor isoform-specific readout system comprising: 
 a) measurement of insulin promoter activation as an indication of Insulin Receptor isoform A activation and/or    b) measurement of βGK promoter activation as an indication of Insulin Receptor isoform B activation.    
     
     
         2 . An Insulin Receptor isoform-specific readout system according to  claim 1 , wherein measurement of promoter activation is performed by measurement of reporter gene expression.  
     
     
         3 . An Insulin Receptor isoform-specific readout system according to  claim 1  or  2 , characterised by 
 a) transfecting a suitable cultured or isolated cell with at least one genetical element comprising a suitable reporter gene under control of either an insulin promoter or a βGK promoter,  
 b) exposing said cell to a test substance in an amount sufficient to influence a receptor activation, and  
 c) measuring the selective influence of said test substance on insulin promoter activation and/or βGK promoter activation by measuring the level of reporter gene expression.  
 
     
     
         4 . An Insulin Receptor isoform-specific readout system according to any of the previous claims, wherein measurement of reporter gene expression is performed by measurement of fluorescence.  
     
     
         5 . An Insulin Receptor isoform-specific readout system according to any of the previous claims, wherein the measurement of insulin promoter activation is performed by measurement of DsRed fluorescence.  
     
     
         6 . An Insulin Receptor isoform-specific readout system according to any of the previous claims, wherein the measurement of βGK promoter activation is performed by measurement of GFP fluorescence.  
     
     
         7 . An Insulin Receptor isoform-specific readout system according to any of claims  2 - 6 , characterised by transfecting a suitable cell with at least one plasmid selected from the group consisting of prIns1.DsRed and prβGK.GFP.  
     
     
         8 . An Insulin Receptor isoform-specific readout system according to any of claims  2 - 7 , wherein said suitable cell is a cell in or from a cell culture, or a cell in or from a primary cell culture, or a cell that is isolated from a sample, or a combination thereof.  
     
     
         9 . An Insulin Receptor isoform-specific readout system according to  claim 8 , wherein said suitable cell is a mammalian cell.  
     
     
         10 . An Insulin Receptor isoform-specific readout system according to  claim 9 , wherein said suitable cell is a human cell.  
     
     
         11 . An Insulin Receptor isoform-specific readout system according to any of claims  8 - 10 , wherein said suitable cell is a pancreatic beta cell.  
     
     
         12 . A plasmid selected from the group consisting of prIns1.DsRed and prβGK.GFP.  
     
     
         13 . An isolated transfected cell expressing the gene product of at least one genetical element comprising a suitable reporter gene under control of 
 a) an insulin promoter, or    b) a βGK promoter,    said cell being selected from the group consisting of transfected isolated pancreatic islets cells or insulin-producing HIT-T15 cells.    
     
     
         14 . An isolated transfected cell, expressing the gene product of a plasmid selected from the group consisting of prIns1.DsRed and prβGK.GFP, said cell being selected from the group consisting of transfected isolated pancreatic islets cells or insulin-producing HIT-T15 cells.  
     
     
         15 . A stable transfected pancreatic beta cell line expressing the gene product of at least one genetical element comprising a suitable reporter gene under control of either an insulin promoter or a βGK promoter.  
     
     
         16 . A stable transfected pancreatic beta cell line expressing the gene product of a plasmid selected from the group consisting of prIns1.DsRed and prβGK.GFP.  
     
     
         17 . A transfected primary pancreatic beta cell culture expressing the gene product of at east one genetical element comprising a suitable reporter gene under control of either an insulin promoter or a βGK promoter.  
     
     
         18 . A transfected primary pancreatic beta cell culture expressing the gene product of a plasmid selected from the group consisting of prIns1.DsRed and prβGK.GFP.  
     
     
         19 . A method for determining the selectivity of a drug, chemical substance and/or a chemical composition for Insulin Receptor isoform A versus Insulin Receptor isoform B, comprising measuring the ratio between insulin promoter activation and βGK promoter activation that is induced by providing said drug, chemical substance and/or a chemical composition.  
     
     
         20 . A method for determining the selectivity of a drug, chemical substance and/or a chemical composition for Insulin Receptor isoform A versus Insulin Receptor isoform B, comprising measuring the ratio between insulin promoter activation and βGK promoter activation that is induced by providing said drug, chemical substance and/or chemical composition, characterised by 
 a) transfecting a suitable cultured or isolated cell with at least one genetical element comprising a suitable reporter gene under control of either an insulin promoter or a βGK promoter,  
 b) exposing said cell to a test substance in an amount sufficient to influence receptor activation, and  
 c) measuring the selective influence of said test substance on insulin promoter activation and/or βGK promoter activation by measuring the level of reporter gene expression.  
 
     
     
         21 . A method of treatment of Type II Diabetes comprising administering a drug selective for Insulin Receptor isoform B to a patient in need thereof.  
     
     
         22 . A method of treatment of Type II Diabetes comprising administering a drug selective for Insulin Receptor isoform A to a patient in need thereof.  
     
     
         23 . A method of treatment of Type II Diabetes according to  claim 21  or  22 , wherein said respective Insulin Receptor isoform is activated.  
     
     
         24 . A method of treatment of Type II Diabetes according to  claim 21  or  22 , wherein the activation of said respective Insulin Receptor isoform is inhibited.  
     
     
         25 . A method for identifying a drug capable of stimulating βGK transcription comprising the steps of testing whether it activates cellular signalling through 
 a) Insulin Receptor isoform B, and/or  
 b) PI3 class II-like kinase/protein kinase B (PKB/c-Akt).  
 
     
     
         26 . A method for identifying a drug capable of stimulating insulin transcription comprising the steps of testing whether it activates cellular signalling through 
 a) Insulin Receptor isoform A, and/or    b) PI3 class Ia/p70s6 kinase.    
     
     
         27 . A drug identified by a method according to  claim 25  or  26 .  
     
     
         28 . Use of a drug identified by a method according to  claim 25  or  26  for treating Type II Diabetes.  
     
     
         29 . Use of a drug identified by a method according to  claim 25  or  26  for the manufacture of a pharmaceutical composition for the treatment of Type II Diabetes.  
     
     
         30 . A method of treatment of Type II Diabetes comprising administering a drug capable of stimulating βGK transcription in pancreatic beta cells to a patient in need thereof.  
     
     
         31 . A method of treatment of Type II Diabetes comprising administering a drug capable of stimulating insulin transcription in pancreatic beta cells to a patient in need thereof.  
     
     
         32 . Use of a plasmid selected from the group consisting of prIns1.DsRed and prβGK.GFP for measuring Insulin Receptor isoform-specific activation.  
     
     
         33 . Use of an isolated transfected cell or a stable transfected cell line, or a transfected primary cell culture according to any of claims  13 - 18  for measuring an Insulin Receptor isoform-specific activation.  
     
     
         34 . A pharmaceutical composition comprising a compound, the compound being selective for Insulin Receptor isoform B and a market authorisation, the market authorisation being based on an application for market authorisation comprising data showing selectivity for Insulin Receptor isoform B.  
     
     
         35 . A pharmaceutical composition according to  claim 31 , wherein the indication is Type II diabetes.