Download The involvement of protein kinase C in glucose

428s Biochemical Society Transactions ( 1 993) 21
The involvement of protein kinase C in glucose-stimulated
insulin secretion
CONTROL
SHANTA J. PERSAUD AND PETER M. JONES
I OLIGO-TREATED
I
Biomedical Sciences Division, King’s College London, Campden
Hill Road, Kensington, London, W8 7AH, U.K.
Protein kinase C (PKC) is a family of serine/threonine
kinases, with multiple isoforms, which is thought to play
important roles in cellular responses to external signals [l]. The
phospholipid-, diacylglycerollphorbol ester- and Ca*+-sensitive
isoforms of PKC, a and S, have been identified in B-cells of
pancreatic islets and direct activation of PKC with phorbol esters
results in the stimulation of insulin secretion [reviewed in 21.
This family of enzymes has been implicated in the insulin
secretory responses of islets to receptor-mediated agonists, such
as acetylcholine and its analogue carbachol, and to
cholecystokinin [2,3]. However, the role of PKC in the
stimulation of insulin secretion by the major physiological
secretagogue, glucose, is far from clear. The activation of many
isoforms of PKC is thought to be accompanied by translocation
of the enzyme from a predominantly cytosolic location to a
membrane-associated compartment. Assays of totai PKC activity
in cytosolic and membrane fractions have been unable to show
translocation of PKC in response to glucose under conditions
where glucose stimulated insulin secretion and where 4BPMA,
a phorbol ester, stimulated translocation [4,5]. Conversely, an
immunological approach has indicated that the a isoform of PKC
becomes associated with B-cell membranes upon short-term (15
minutes) stimulation with glucose [6].
We have investigated the involvement of PKC in glucoseinduced insulin secretion using three separate approaches. Islet
PKC can be depleted by prolonged (- 20 hours) exposure to the
tumour promoting phorbol ester, 4RPMA [7]. After phorbol
ester-induced PKC down-regulation, islets did not show a
secretory response to 500nM 4BPMA (control: 313f57% basal,
P<0.05; PKC-depleted: 117&18% basal, P>0.2, n=7), but
were fully responsive to 20mM glucose (control: 2.69k0.24
ng/islet/h; PKC-depleted: 3.31 k0.22, P<O.Ol vs secretion at
2mM glucose, n = 8) suggesting that 4BPMA-sensitive isoforms
of PKC are not required for glucose-stimulated insulin secretion.
However, staurosporine (SP), a potent inhibitor of PKC activity,
caused a marked, but incomplete, inhibition of the secretory
response to glucose (2mM glucose, 0.12&0.0lng/islet/h; 20mM
glucose, 2.55k0.35; +100nM SP, 1.23k0.13, PCO.01 vs
secretion at both 2mM and 20mM glucose, n=9). This may not
necessarily point to an involvement of PKC in glucose-induced
insulin secretion since SP interacts with the ATP-binding site of
kinase catalytic domains and inhibits kinases distinct from PKC
in several cell types including islets [8]. To further resolve the
involvement of PKC in glucose-stimulated insulin release, islets
were treated with antisense oligonucleotides directed against
nucleotide sequences encompassing the initiation codons of the
mRNAs for the a and B isoforms of PKC. A requirement for
PKC isozymes in insulin-stimulated glucose transport in
adipocytes has recently been identified using this approach [9].
In our experiments, treatment of islets with the antisense
oligonucleotides (phosphorothioate derivatives; 8pM of each) for
20 hours produced a marked reduction in PKC activity (65+3%
decrease, determined by phosphorylation of exogenous substrate)
and in PKC expression (61% decrease, determined by western
blotting of fractionated islet proteins and immunoblotting with an
anti-PKC antibody), but no change in B-cell insulin content
(control: 44.7k4.1 pg/cell; oligonucleotide-treated: 40.3k3.1).
Antisense oligonucleotide-induced PKC depletion was coupled to
2
2
20
Glucose (mM)
Figure 1. Insulin secretion from islets depleted of PKCa and
I? by antisense oligonucleotide treatment.
Islets in which PKC had been depleted by exposure to 8pM of
oligonucleotides antisense to PKCa and PKCB showed a
significant secretory response to 20mM glucose (P<O.Ol vs
2mM glucose), but were unresponsive to acute (one hour)
exposure to 4RPMA (500nM; hatched bars). 4RPMA did
stimulate insulin secretion from control islets whkh had been
maintained in the absence of antisense oligonucleotides (P < 0.01
vs 2mM glucose). Bars show meansfSEM, n=7.
a loss of insulin secretory responses to 4RPMA at both 2mM and
20mM glucose, but islets still showed a significant secretory
response to an elevation in glucose concentration from 2 to
20mM (Figure 1).
These results suggest that activation of the phorbol estersensitive isozymes of PKC which have been identified in B-cells
( a and B) are not required for B-cells to secrete insulin in
response to glucose. The involvement of the phorbol esterinsensitive PKC isozyme. {, which may be expressed in islets of
Langerhans [lo] can not be deduced from the present studies,
but its selective depletion by the antisense oligonucleotide
strategy should yield useful information.
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