Methods for Producing Enteroendocrine Cells That Make and Secrete Insulin

Abstract

Methods are described for producing enteroendocrine cells that make and secrete insulin in a mammal by blocking the expression or biological activity of one or more forkhead box O (Foxo) proteins or biologically active fragments or variants thereof.

What is claimed is: 
     
         1 . A high-throughput screening cell-based assay for identification of agents that induce mammalian gut noninsulin-producing enteroendocrine progenitor cells to express insulin, comprising
 a. isolating a population of cells comprising gut noninsulin-producing enteroendocrine progenitor cells and incubating them under conditions suitable for the production and secretion of insulin,   b. providing a control and a test population of noninsulin-producing enteroendocrine progenitor cells,   c. contacting the test population with the a test agent,   d. determining the level of insulin expression in the control and the test population, and   e. selecting the test agent if the level of insulin in the test population is significantly higher than the level in the control.   
     
     
         2 . The assay of  claim 1 , wherein a gene encoding the insulin in the gut noninsulin-producing enteroendocrine cells is operatively linked to a protein or peptide that can be visualized. 
     
     
         3 . The assay of  claim 2 , wherein the protein or peptide is a fluorescent protein. 
     
     
         4 . The assay of  claim 3 , the protein or peptide is a member selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, urease, and beta galactosidase. 
     
     
         5 . The assay of  claim 1 , wherein the agent is therapeutically effective to treat intolerance disease or disorder associated with impaired pancreatic function. 
     
     
         6 . The assay of  claim 1 , wherein a library of agents is screened. 
     
     
         7 . The assay of  claim 1 , wherein the agent reduces expression of Foxo1, Foxo3 or Foxo4 or combinations thereof. 
     
     
         8 . A method for identifying an agent that causes noninsulin-producing enteroendocrine cells to differentiate into insulin-producing cells, comprising:
 a. providing a test animal and a control animal both of which have impaired pancreatic function   b. administering the agent to the test animal,   c. determining the level of an indicia of insulin-producing cells in both animals, which indicia is selected from the group consisting of serum insulin, insulin sensitivity, and glucose tolerance, and   d. identifying the agent as an agent that causes noninsulin-producing enteroendocrine cells to differentiate into insulin-producing cells if the indicium is significantly increased in the test animal compared to the control animal.   
     
     
         9 . The method of  claim 8 , wherein the agent is effective to treat a disease or disorder associated with impaired pancreatic function. 
     
     
         10 . A method of identifying an agent that is effective in treating or preventing a disease or condition associated with impaired pancreatic function, comprising:
 a. providing an animal that has the disease or condition;   b. determining the amount of Foxo1, Foxo2, Foxo3 or Foxo4 expression in a pre-treatment biological sample taken from the animal;   c. administering the agent to the animal; and   d. determining the amount of Foxo1, Foxo3 or Foxo4 expression in a post-treatment biological sample taken from the animal; wherein if the amount in the post-treatment biological sample is significantly lower than the amount of in the pre-treatment sample, the agent is identified as an agent that is effective in treating or preventing the disease or condition.   
     
     
         11 . The method of  claim 10 , wherein the biological sample is serum or a sample of enteroendocrine cells.