Expression of Vesicle Nucleotide Transporter in Pancreatic Cells and its Role in ATP Accumulation in Secretory Granules

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 834-867-Islet Biology
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-847
Jessica Catherine Geisler1, Hongxia Chao2, John David Castle3 and Chien Li*2
1University of Virginia, Charlottesville, VA, 2Univ of Virginia, Charlottesville, VA, 3University of virginia, Charlottesville, VA
ATP plays a critical role in regulating pancreatic β cell function. Intracellularly, ATP regulates insulin secretion by binding and closing ATP-sensitive potassium (KATP) channels and also facilitates exocytosis. Further, it has been shown that ATP accumulates in insulin secretory granules and is co-secreted with insulin upon stimulation; extracellular ATP in turn can serve as a signal to regulate β cell function via purinergic receptors. The mechanism by which ATP accumulates in insulin granules has remained unclear. Vesicular nucleotide transporter (VNUT) has recently been identified in adrenal chromaffin cells where it is responsible for transporting ATP into secretory vesicles for storage and release. We have shown that VNUT is expressed in mouse pancreas, particularly in pancreatic islets. Moreover, suppression of VNUT expression in β cells greatly reduced ATP secretion whereas overexpression increased it. Immunofluorescence microscopy shows that VNUT and insulin colocalize suggesting that VNUT resides in insulin granules and regulates vesicular ATP uptake and secretion. In the present study, we used a combination of velocity sedimentation and sucrose density gradient centrifugation to assess the extent to which VNUT in β cells is concentrated in the granules. Consistent with immunofluorescent staining, VNUT predominantly fractionates with granules marked by insulin immunoreactivity and does not distribute appreciably with other organelles including ER, mitochondria, lysosomes and low-density membranes (containing endosomes and Golgi). Finally, neither knockdown of VNUT expression nor VNUT overexpression in MIN6 cells altered total cellular ATP content even though these manipulations, respectively, reduced and enhanced vesicular ATP accumulation as judged by quinacrine staining. Altered VNUT expression also affected the expression of a number of genes that function in glucose sensing and insulin secretion including glucose transporter 2 and glucokinase. This result raises the possibility that VNUT plays a critical role in modulating cellular energy homeostasis by regulating the levels of ATP in a non-diffusible compartment like insulin granules.

Nothing to Disclose: JCG, HC, JDC, CL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH Grant DK078049 awarded to CL.