(Received for publication, May 29, 1996, and in revised form, November 1, 1996)
From the Department of Endocrinology and Metabolism,
Hebrew University-Hadassah Medical Center, 91120 Jerusalem, Israel and
the § Department of Molecular Pharmacology, Stanford
University School of Medicine, Stanford, California 94035
The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds
to a specific eceptor for
ctivated
inase (RACK) (Mochly-Rosen, D. (1995)
Science 268, 247-251) that provides an anchoring site in
close proximity to the isoenzyme's specific substrate. Pancreatic islet cells contain at least six PKC isoenzymes (Knutson, K. L., and
Hoenig, M. (1994) Endocrinology 135, 881-886). Although
PKC activation enhances insulin release, the specific function of each
isoenzyme is unknown. Here we show that following stimulation with
glucose,
PKC and
PKC translocate to the cell's periphery, while
PKC and
PKC translocate to perinuclear sites.
C2-4, a peptide
derived from the RACK1-binding site in the C2 domain of
PKC,
inhibits translocation of
PKC and reduces insulin response to
glucose. Likewise,
V1-2, an
PKC-derived peptide containing the
site for its specific RACK, inhibits translocation of
PKC and
reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both
PKC and
PKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of
PKC but not
PKC and lowers insulin response by 50%. These findings illustrate the unique ability
of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.
Protein kinase C is a family of 11 lipid-dependent
serine/threonine kinases involved in a wide spectrum of signal
transduction (7, 8). Upon activation, PKC1
isoenzymes translocate to new cellular sites, including the plasma membrane (9, 10), cytoskeletal elements (11, 12), and the nucleus (13,
14), as well as other subcellular compartments (15). Many cells are
known to contain several isoenzymes (16, 17), each localizing to a
different cellular site upon stimulation (18). The multiplicity of
isoforms of a single enzyme renders the analysis of enzyme-function
relationship difficult. Recent work revealed that activated PKC
isoenzymes bind anchoring proteins termed RACKs (1-3), believed to be
positioned in close proximity to the isoenzyme's substrate. It was
further shown that the functional specificity of the PKC isoenzyme is
determined, in part, by the differential localization of the
isoenzyme-specific RACKs (19). The RACK for PKC, RACK1, has been
cloned, and at least part of its binding site on
PKC has been mapped
to a short sequence within the C2 domain (1).
C2-4, a nonopeptide
derived from this region, inhibits phorbol ester-induced translocation
of the C2-containing isoenzymes but not the translocation of C2-less
isoenzymes such as
- and
PKC when tested in intact cells (1). A
short peptide derived from the V1 region of
PKC,
V1-2, was
similarly shown to inhibit the translocation of
PKC, but not
-,
-, and
PKC (20). Furthermore, these isozyme-specific inhibitors
blocked the specific function of individual isoenzymes; for example,
V1-2, but not
C2-4, inhibited phorbol 12-myristate
13-acetate-induced regulation of the contraction rate in intact
cardiomyocytes. Here we use these novel PKC isozyme-specific inhibitors
to determine that PKC activation is part of the signals involved in the
regulation of glucose-induced insulin secretion and to identify the
specific isoenzymes that mediate this glucose effect.
Islets obtained from 200-g male Sprague-Dawley rats were
cultured for 3-5 days in glass chamber slides coated with
extracellular matrix of bovine corneal endothelial origin (21). When
more than 75% of the islet cells spread out to form a monolayer, the media were replaced with modified Krebs Ringer solution (KRB) (22)
containing either 2.5 or 20 mM glucose. Following a brief wash, the islets were fixed in cold acetone, blocked with 1% normal goat serum for 1 h, and treated overnight with the specific
anti-PKC isoenzyme antibody (Research and Diagnostic Antibodies,
Berkeley, CA). Fluorescein isothiocyanate-linked goat-anti-rabbit IgG
(Sigma Israel Chemicals, Rehovot, Israel) was applied
for 2 h, and the slides were mounted in 90% glycerol, 10%
phosphate-buffered saline, 0.1% sodium azide, 3%
diazabicyclo[2.2.2]octane, pH 9.0, for microscopic imaging.
Histochemical imaging was conducted on a PhioBos 1000 confocal
microscope (Sarastro Inc., Ypsilanti, MI), equipped with Zeiss
Universal Optics and argon laser illumination. The anti-PKC antisera
each exhibited a single band of the appropriate size on Western blot
(10 µg of rat brain or rat islet homogenate/lane). No bands were
observed when the antibodies were preincubated with excessive amounts
of their corresponding antigen derived from the V5 region of the
isoenzyme (PKC-epsilon-(728-737) or
PKC--(Tyr663-(664-672)), Research and Diagnostic
Antibodies, Berkeley, CA). Preincubation of anti-
PKC with excess
PKC-
-(Tyr663-(664-672)) and anti
PKC with excess
PKC-epsilon-(728-737) completely abolished the fluorescence images of
the glucose-dependent isoenzyme translocation. The cells
were identified as
-cells by counterstaining with
tetramethylrhodamine B isothiocyanate-conjugated rabbit anti-guinea pig
IgG and guinea pig anti-insulin serum (Dako Corp., Carpinteria, CA).
Freshly isolated islets were used for insulin release studies. Islets were preincubated for 1 h in KRB-BSA buffer containing 2.5 mM glucose, then transferred, 1-2 islets per tube, for an additional 60-min incubation in KRB-BSA buffer containing either 2.5 mM or 20 mM glucose as described previously (22). When calcium-free buffer was used, 5 mM EGTA was added to the calcium-free KRB-BSA buffer during the test period.
Peptides were synthesized at the Hebrew University School of Medicine Intradepartmental Equipment Services. Peptide introduction into islet cells was achieved by transient permeabilization ("skinning") (4). Ten-minute skinning, followed by a 1-h incubation of isolated rat islets with Krebs buffer containing 2.5 mM glucose, had no effect on islet viability as examined by trypan blue exclusion or by the exhibition of intact islet secretory response to glucose in either batch or perifusion studies.
Insulin release was measured by radioimmunoassay using specific guinea pig anti-rat insulin antiserum (Linco Research, St Charles, MO) and rat insulin standard (Novo Research Institute, Bagsvaerd, Denmark) (22). Data presented are mean net insulin values after subtraction of non-stimulated level (2.5 mM glucose). Statistical significance was determined by paired, non-parametric comparison to control or to control skinned islet, using the Wilcoxon test.
The role of PKC as an amplifier of the glucose-generated signal to
release insulin has been well established (5, 23-25). Six PKC
isoenzymes, ,
,
,
,
, and
, were found thus far in
rat pancreatic islets of Langerhans (5, 6, 25). The specific function
of the individual isoenzymes remains unknown. We used adult rat islet
cultures (21) to assess the direct effect of glucose on PKC isoenzymes
in intact islet cells. Site-specific translocation could be
demonstrated for
-,
-,
-, and
PKC (Fig. 1).
Confocal microscopy imaging revealed that
PKC and
PKC
redistributed following glucose stimulation to the cell's periphery,
PKC concentrated in an asymmetric structure in perinuclear region
(possibly the Golgi apparatus), and
PKC concentrated as a ring
around the cell nucleus (Fig. 1).
Isoenzyme is a member of the calcium-sensitive cPKC subfamily (7,
8). Alterations in cytosolic calcium levels play a prominent role in
-cell stimulus-secretion coupling for insulin release (for review,
see, for example, Ref. 26). We therefore examined whether modulation of
the calcium-sensitive PKC subtypes affects glucose-induced insulin
release in rat islets.
C2-4 (Ser-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr) is a nonopeptide derived from the binding site (amino acids 218-226) of
IIPKC to RACK1 (19), a well conserved sequence within the C2
domain of members of the cPKC subfamily. Introduction of
C2-4 into
islet monolayers by means of transient permeabilization at low
temperature (skinning) (4) abolished the glucose-induced translocation
of
PKC to the cell's periphery (Fig. 2c),
whereas
C2-4-s, a scrambled control peptide
(Trp-Asn-Pro-Glu-Ser-Leu-Asn-Thr), did not prevent translocation
(Fig. 2d).
Moreover, administration of C2-4 into freshly isolated islets
resulted in 35% reduction in the insulin response to glucose stimulation (Fig. 2e); introduction of the scrambled analog,
C2-4-s, had no inhibitory effect on insulin secretion. To rule out
the possibility that the partial inhibition of the insulin response was
the result of unequal penetration of the peptide, freshly isolated
islets in suspension were skinned in the absence or presence of 10 µM
C2-4 and subsequently incubated with 20 mM glucose. At the end of a 60-min stimulation, the islets
were fixed in paraformaldehyde, dehydrated in ethanol, and further
treated in propylene oxide Surre mixture. Following resin
polymerization, 5- and 10-µm slices were prepared and stained with
anti-
PKC antibodies. Confocal imaging revealed uniform inhibition of
the glucose-induced isoenzyme translocation throughout the sections
(not shown), indicating homogeneous penetration of the octapeptide
throughout the islet.
C2-4 has been shown to be equally effective
against all members of the classical PKC subfamily (19). While
PKC
often has been reported to be the predominant PKC isoenzyme in islet
-cells,
PKC exhibits only scant expression in these cells (5);
we, however, assume that all cPKC isoenzymes were equally inhibited by
C2-4. The fact that maximally effective concentrations of
C2-4
had only a partial inhibitory effect on glucose-induced insulin release
may therefore indicate that in addition to the cPKC subfamily, other
PKC isoenzymes or non-PKC stimulus amplifiers are involved in the
glucose-generated stimulus-secretion coupling.
Despite the pivotal role of cytosolic calcium mobilization in the
control of insulin release, data are accumulating supporting the role
of additional, calcium-independent signals in the modulation of insulin
secretion (27-29). We therefore examined the role of PKC, a
calcium-insensitive PKC isoenzyme in glucose-induced insulin secretion
utilizing
V1-2, the octapeptide (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr) derived from the RACK-binding site of
PKC (amino acids 14-21 within
the V1 region of the isoenzyme) (20). Introduction of
V1-2 into
islet monolayers by means of skinning abolished glucose-induced translocation of
PKC (Fig. 3c); the
scrambled analog
V1-2-s (Leu-Ser-Glu-Thr-Lys-Pro-Ala-Val) had no
effect (Fig. 3d). Moreover, introduction of
V1-2 into
freshly isolated islets in suspension inhibited the insulin response to
glucose by 40% (Fig. 3e), whereas
V1-2-s, the scrambled
analog, had no inhibitory effect (Fig. 3e). In histochemical
studies,
V1-2 had no effect on glucose-dependent translocation of
PKC and, vice versa,
C2-4 had no effect on the
translocation of
PKC (not shown).
5 µM was found to be a maximally effective concentration
for both C2-4 and
PKC in 1-h incubations, never exceeding 43%
inhibition of glucose-induced insulin release. The effect of the two
peptides was additive at that concentration in fresh islets incubated
for 60 min at 20 mM glucose (Fig.
4b). The fact that both peptides together
inhibited only 67% of the glucose-mediated insulin release suggests
that
PKC and
PKC are each independently involved in one of
several distal coupling systems (see below). Stimulation of islet
monolayer in the absence of Ca2+ (and in the presence of
EGTA) abolished glucose-dependent translocation of
PKC
but not of
PKC (Fig. 4a). It also diminished the
glucose-induced insulin response in fresh islets by 85% (Fig.
4b), providing further evidence that
PKC but not
PKC
is involved in the calcium-mediated regulatory signal to release
insulin. However, addition of mannoheptulose, an inhibitor of glucose
metabolism, completely abolished the glucose-induced translocation of
both
- and
PKC isoenzymes (Fig. 4a) as well as the
islet insulin response to the sugar (Fig. 4b), indicating that the glucose-stimulatory coupling signal for the activation of PKC
is linked to glycolysis, the primary signal for
-cell insulin
response (26).
The islet -cell coupling mechanism regulating the insulin secretory
response to glucose is a complex sequence of metabolic events (for
review, see, for example, Ref. 26), leading to a unique multiphasic
dynamic of hormonal secretion (for review, see Ref. 30). While the
primary coupling signal originates from glucose metabolism, leading to
calcium mobilization and activation of poorly defined
calcium-dependent pathways, the same primary signal also
activates numerous distal potentiating pathways, some calcium-dependent, others calcium-independent (27-29).
Since insulin secretion is strongly modulated by the duration of
glucose stimulation (30), it is possible that different PKC isoenzymes
have distinct roles in the different phases of release. This subject is
presently under investigation. Islets coupling signals originating from activation of adenylate cyclase, phospholipase C, and phospholipase A2 are among the more thoroughly investigated potentiating
signals involved in the modulation of the insulin response to glucose (31-33). Messengers generated from these key metabolic pathways are
known to activate multiple PKA, PKC, and CaM kinases, each controlling
a specific amplifying branch of the insulin stimulus-secretion coupling
pathway, resulting in the multiphasic dynamics of hormonal secretion in
response to glucose stimulus (30, 31). The identification of PKC
isoenzyme-specific binding proteins offers novel tools to resolve the
specific contribution of each isoform to this complex of interrelated
signals. Furthermore, RACK-binding translocation inhibitors should
prove to be valuable tools in resolving the specific function of the
individual PKC isoenzyme in cells expressing multiple forms of the
enzyme as well as in identifying their specific substrates.