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Diabetes, Vol 49, Issue 3 424-430, Copyright © 2000 by American Diabetes Association
Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion
HE Hohmeier, H Mulder, G Chen, R Henkel-Rieger, M Prentki and CB Newgard
BetaGene, Inc., Dallas, Texas, USA.
The biochemical mechanisms involved in regulation of insulin secretion are
not completely understood. The rat INS-1 cell line has been used to gain
insight in this area because it secretes insulin in response to glucose
concentrations in the physiological range. However, the magnitude of the
response is far less than that seen in freshly isolated rat islets. In the
current study, we have stably transfected INS-1 cells with a plasmid
containing the human proinsulin gene. After antibiotic selection and clonal
expansion, 67% of the resultant clones were found to be poorly responsive
to glucose in terms of insulin secretion (< or =2-fold stimulation by 15
mmol/l compared with 3 mmol/l glucose), 17% of the clones were moderately
responsive (2- to 5-fold stimulation), and 16% were strongly responsive (5-
to 13-fold stimulation). The differences in responsiveness could not be
ascribed to differences in insulin content. Detailed analysis of one of the
strongly responsive lines (832/13) revealed that its potent response to
glucose (average of 10-fold) was stable over 66 population doublings
(approximately 7.5 months of tissue culture) with half-maximal stimulation
at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose,
insulin secretion was potentiated significantly by 100 pmol/l
isobutylmethylxanthine (320%), 1 mmol/l oleate/palmitate (77%), and 50
nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect.
Glucose-stimulated insulin secretion was also potentiated by the
sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15
mmol/l glucose) and was abolished by diazoxide, which demonstrates the
operation of the ATP-sensitive K+ channel (K(ATP)) in 832/13 cells.
Moreover, when the K(ATP) channel was bypassed by incubation of cells in
depolarizing K+ (35 mmol/l), insulin secretion was more effectively
stimulated by glucose in 832/13 cells than in parental INS-1 cells, which
demonstrates the presence of a K(ATP) channel-independent pathway of
glucose sensing. We conclude that clonal selection of INS-1 cells allows
isolation of cell lines that exhibit markedly enhanced and stable
responsiveness to glucose and several of its known potentiators. These
lines may be attractive new vehicles for studies of beta-cell function.

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October 1, 2003;
144(10):
4433 - 4445.
[Abstract]
[Full Text]
[PDF]
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