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VOL. 71 (4), 765-782, 2005 OLD HORMONES OF THE INSULIN...

Abnormal levels of proinsulin/insulin signalling
cause changes in programmed cell death

Since classic binding-competition and autoradiography studies
had revealed that the chick embryo had insulin receptors and
IGF-I receptors from the stage of gastrulation onwards (1,18), we
suspected that the proteins of the family had previously unknown
roles in early development. We focused our attention on the process
of programmed cell death, generally occurring as apoptosis, which is
relevant to multiple morphogenetic events in development (43)
including the closure of the neural tube (Fig. 4A). Using the
technique of Terminal Deoxynucleotidyltransferase-mediated UTP
Nick End Labelling (TUNEL), we could quantify the number of cells
of a chick embryo during neurulation that undergo apoptosis, in
vivo and in culture (Fig. 4B, C). When the embryonic endogenous
proinsulin signal was interfered by using antisense oligonucleotides
against proinsulin mRNA (25) or the insulin receptor mRNA (19),
there was an increase in the number of cells dying of apoptosis
(Fig. 4). Preventing cell death, thus, appeared as an important
function of embryonic proinsulin. The modulation of the chaperone
heat shock cognate 70 by proinsulin/insulin was found to correlate
with the prevention of apoptosis (10).

We had known for some years that correct proinsulin/insulin
signalling was needed to continue normal development beyond
neurulation since insulin or insulin receptor antibodies caused a
certain level of malformed or dead embryos between E2 and E4 of
development (12). Opposite pharmacological experiments involving
the addition of excess insulin or proinsulin also caused abnormalities
and death (13).

In a similar way, it was found critical during neurulation to
maintain the proinsulin signal within physiological limits. An excess
of exogenously added proinsulin caused abnormalities that affected
the neuropore, neural tube, and optic vesicles in the neurulating
embryo (Table I), concomitant with a decrease in natural apoptosis
(42). A tightly regulated proinsulin gene expression, therefore, is
required to maintain the level of cell survival/cell death also strictly
regulated in the embryo. This suggests an early important role for a
protein prohormone previously thought to have little activity.

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