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Knockdown of UDP‐glucose dehydrogenase in INS‐1 832/13 cells inhibits insulin release (767.12)
Author(s) -
Perez Jaritza,
MacDonald Michael
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.767.12
Subject(s) - gene knockdown , insulin , islet , cell culture , medicine , endocrinology , glucokinase , small hairpin rna , biology , chemistry , genetics
Our unpublished mass spectrometry studies of pancreatic islet insulin‐producing cells, as well as a published study by others, showed that several nucleotide‐sugar compounds increased with glucose stimulation. These compounds included ADP‐glucose (not known to be present in mammals), UDP‐glucose and GDP‐mannose. That these compounds increased with glucose stimulation suggests they might play a role in insulin secretion. We focused our study on two enzymes that use UDP‐glucose as a substrate: UDP‐glucose Dehydrogenase (UGDH) and UDP‐glucose glycoprotein glucosyltransferase (UGGT1). Results: Transcripts of both genes were detected in human pancreatic islets and in the glucose‐responsive INS‐1 832/13 cell line. Human and rat islets showed six‐fold more UGDH mRNA than liver. Enzyme activity of UGDH was detected in rat liver, but was below the level of detection in the INS‐1 832/13 cell line. Two shRNAs targeting the UGDH gene significantly knocked down the mRNA of the gene in INS‐1 832/13‐derived cell lines. Knockdown of the UGDH mRNA was associated with 38% and 22% decrease in glucose‐stimulated insulin release in two UGDH shRNA / INS‐1 832/13‐derived cell lines. Cell lines with knockdown of UGGT1 are currently being generated. Conclusions: Based on enzyme activity assay, the concentration of UGDH in INS‐1 832/13 cells is very low. However an inhibition of insulin release was observed in two separate UGDH knockdown cell lines, and it is unlikely that the inhibition of insulin release was due to an off target shRNA effect. Further studies are needed to establish whether UGDH and UGGT1 play a role in insulin release in INS‐1 832/13 cells. Acknowledgements: NIH / NIDDK STEP‐UP Program award to JMP and NIH Grant DK28348 to MJM. Grant Funding Source : Supported by NIH / NIDDK STEP‐UP Program award to JMP and NIH Grant DK28348 to MJM