Method for modulating insulin production
View Patent ↗The present invention provides screening methods for the detection of agents that affect various aspects of β-cell biology, particularly insulin gene expression. Screening methods are also provided for detection of agents that affect β-cell differentiation from progenitor cells. Additionally, agents identified using such methods are provided and are useful for increasing insulin gene expression and reducing lipotoxicity.
1. A method of screening for an agent that reduces lipotoxicity in a subject having Type I or Type II diabetes comprising:
a) transfecting a cell with a vector comprising an insulin promoter operably linked to a nucleic acid encoding a reporter protein, wherein the reporter protein is a fluorescent protein;
b) contacting the cell with a candidate agent; and
c) detecting the reporter protein, thereby identifying an agent that reduces lipotoxicity.
2. The method of claim 1 , where the cell is derived from a human fetal islet cell.
3. The method of claim 1 , wherein the cell is further contacted with a fatty acid.
4. The method of claim 3 , wherein the fatty acid is palmitate.
5. The method of claim 1 , wherein the subject is a human.
6. The method of claim 1 , wherein the cell is transfected in solution and then cultured in a microtiter plate.
7. The method of claim 6 , wherein the cells are culture 4 days before detection.
8. The method of claim 1 , wherein the fluorescent protein is green fluorescent protein (GFP) or enhanced green fluorescent protein (eGFP).
9. The method of claim 1 , wherein detection of the reporter protein is performed using fluorescence microscopy.
10. The method of claim 1 , wherein the screening is performed in a high throughput format.
11. A method of screening for an agent that reduce lipotoxicity in a subject having Type I or Type II diabetes comprising:
a) transfecting a cell with a vector comprising an insulin promoter operably linked to a nucleic acid encoding a reporter protein, wherein the reporter protein is green fluorescent protein (GFP) or enhanced green fluorescent protein (eGFP);
b) contacting the cell with a candidate agent; and
c) detecting the reporter protein, thereby identifying an agent that reduces lipotoxicity.