Detection of amplifiable mRNA extracellular to insulin-producing cells: potential for predicting beta cell mass and function.
BACKGROUND
Detecting extracellular nucleic acids in the serum/plasma of cancer patients may help in cancer diagnosis. We investigated whether extracellular mRNAs are reproducibly detectable in conditioned medium (CM) from insulin-producing cell cultures and if their presence and amounts are indicative of cell number and/or function.


METHODS
We isolated mRNA from medium conditioned by the culture of several insulin-producing cell types: MIN6(L) (glucose-responsive), MIN6(H) (glucose-nonresponsive), and MIN6 B1 murine beta cells and monkey kidney fibroblast cells engineered to produce human preproinsulin (PPI) (Vero-PPI). We used reverse transcription-PCR analyses to evaluate the occurrence of several mRNAs and investigated whether the presence and amounts of the various extracellular mRNAs are associated with cell mass and/or function.


RESULTS
Reproducible amplification of mRNAs encoded by Pdx1, Npy, Egr1, Pld1, Chgb, Ins1, Ins2, and Actb from MIN6(L), MIN6(H), and MIN6 B1 cells and their CM suggests that beta cells transcribe and release these mRNAs into their culture environment. Similarly, PPI mRNA was detected in samples of Vero-PPI cells and CM. The amounts of some mRNAs reflected the numbers and functional status (i.e., glucose responsiveness vs nonresponsiveness) of the cells conditioning the medium. Although Pax4 mRNA was detected in the MIN6 B1 cell line, the fact that this transcript was not amplifiable from the corresponding CM suggested that mRNA release was selective.


CONCLUSIONS
mRNAs may be secreted from insulin-producing cells, are reproducibly detected in the extracellular environment, and may have potential as extracellular biomarkers for assessing beta cell mass and function.
Rani
Sweta
S
National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland.
Clynes
Martin
M
O'Driscoll
Lorraine
L
D000818:Animals; D002452:Cell Count; D002460:Cell Line; D002522:Chlorocebus aethiops; D017077:Culture Media, Conditioned; D003851:Deoxyribonucleases / Q000737:chemistry; D005110:Extracellular Space / Q000737:chemistry*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D011384:Proinsulin / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000737:chemistry
D017077:Culture Media, Conditioned; D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D011384:Proinsulin; D003851:Deoxyribonucleases; D012260:Ribonucleases; D005947:Glucose