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ÓSCAR ESCRIBANO Y COLS. AN. R. ACAD. NAC. FARM.

2.7. Real-time quantitative PCR for IR isoforms

The pancreatic islets were isolated from 6-month-old control and
iLIRKO mice as previously described (10). IR isoforms expression in
isolated islets was analyzed by qPCR, using Taqman probes (Applied
Biosystems, NJ). The comparative threshold cycle (Ct) method was
used to calculate the relative expression. For gene expression
quantification, the target genes values were normalized to the
expression of the endogenous reference (18S). Thus, the amount of
target, normalized to 18S and relative to the control is given by 2-??Ct
[?Ct = Ct (Target gene) - Ct (18S); ??Ct = ?Ct for any sample - ?Ct
for the control].

2.8. Reconstitution of IRKO immortalized beta cells with
2.8. IR A or B iosoforms b retroviral infection

IRKO beta cells were generated in our laboratory as previously
described (17). From these cells we reconstituted and characterized
the expression of the IR-A (Rec A) and IR-B (Rec B) generating new
cell lines as previously described (18).

2.9. Measurement of glucose uptake in beta cells

Cells were cultured to 80% confluence in 10% FBS-DMEM and
then serum and glucose starved for 4-6 h. After that, 10 nM Insulin
or 10 nM IGF-1 were added to the wells for 30 min. Glucose uptake
was measured by incubating cells with 2-deoxy-D-[1-3H]glucose for
the last 10 min, in triplicate dishes from six independent experiments
as previously described (19).

2.10. Cell viability assays

Cells were plated in 12-multiwell plates and cultured in 10% FBS-
DMEM until 40-50% of confluence was reached. Then cells were
serum starved for 4 h, and then treated with 10nM Insulin, 10 nM
IGF-1 or both during 24 hours. Then, the cells were washed with
PBS and stained with violet crystal as described (17).

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