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J. A. Haley Veteran’s Hospital Research Service, and Departments of Internal Medicine and Biochemistry/Molecular Biology, University of South Florida College of Medicine, Tampa, Florida 33612
Address all correspondence and requests for reprints to: Robert V. Farese, M.D., Research Service (VAR 151), J. A. Haley VA Hospital, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, 
) and
novel (
, 
, 
) PKCs at relatively low concentrations
(EC50s, approximately 20–100 nM) and the
atypical PKC-
at higher concentrations (EC50, 1–4
µM) (1). RO 31-8220 also inhibits the PKC-related kinase
(PRK-1), also known as protein kinase N (PKN), at concentrations
similar to those that inhibit conventional and novel PKCs (2). In
addition to inhibiting PKC, RO 31-8220 has recently been reported to
activate c-Jun N-terminal kinase (JNK) in rat-1 fibroblasts
(3). JNK is a member of the mitogen-activated protein kinase (MAPK)
superfamily, and, like other MAPKs such as extracellular response
kinases, ERK1 and ERK2, JNK is activated by analogous MAPK kinases
(MEKs) and MEK kinases (MEKKs) (4). We noted in studies of insulin action that RO 31-8220 provokes increases in glycogen synthase (GS) activity in rat adipose tissue (5). We initially postulated that the activation of GS might be due to an inhibition of basally active PKC, which directly phosphorylates and thereby inhibits GS activity (6). However, the activation of JNK by anisomycin has been found to result in GS activation (7). Moreover, insulin activates JNK in skeletal muscle, and it was postulated that JNK might play a role in insulin stimulation of GS (7). Accordingly, it seemed plausible that the activation of GS by RO 31-8220 may be due, not to inhibition of PKC, but rather to the activation of JNK, or, for that matter, other related MAP kinases, [e.g. both JNK and ERK1/2, presumably via their downstream effectors, ribosomal S6 kinases-2 and 3 (RSK2/3) (RSK2 is also referred to as MAPK-activated kinase 1) activate GS (see Refs. 7, 8, 9)].
In addition to modulating effects of PKC, JNK, and ERK1/2 on GS, recent findings suggest that protein kinase B (PKB) may function as a positive regulator of GS, particularly during insulin action (10, 11, 12). PKB, in turn, appears to be largely regulated through insulin-induced increases in phosphatidylinositol (PI) 3-kinase activity (13, 14, 15), presumably via its lipid products, PI-3,4,5-(PO4)3 and PI-3,4-(PO4)2, and a 3-phosphoinositide-dependent-protein kinase (PDK-1) (16, 17, 18). In this regard, RO 31-8220 does not inhibit PI 3-kinase or insulin-induced activation of PI 3-kinase (19), but effects of RO 31-8220 on PKB activation have not been reported.
Presently, we compared potential mechanisms whereby RO 31-8220 and insulin activate GS in two widely used, insulin-sensitive cell types, i.e. rat adipocytes, and L6 myotubes. Our findings suggested that: 1) JNK, rather than PKC, PKB, or ERK1/2, may play an important role in the activation of GS by RO 31-8220; and 2) JNK does not appear to be important for insulin-induced activation of GS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L6 myotubes were cultured as described previously (21) and used for studies of glucose incorporation into glycogen and GS activation, essentially as described above in adipocyte experiments.
PKB activation
PKB activation was assessed either by 1) a shift in its
electrophoretic migration [reflective of phosphorylation status—see
Ref. 22 ] as measured by Western analysis [using antibodies from
Upstate Biotechnology, Inc. (UBI; Lake Placid, NY)] of
cellular extracts, following resolution of PKB by SDS-PAGE, and
electrolytic transfer of PKB to nitrocellulose membranes, or 2)
measurement of immunoprecipitable PKB enzyme activity, in most cases
using a PKB assay kit obtained from UBI. For immunoprecipitation
assays, adipocytes were washed and lysed by sonication in Buffer A
containing 50 mM Tris/HCl (pH 7.5), 1 mM EDTA,
1 mM EGTA, 1 mM Na3VO4,
0.1% 2-mercaptoethanol, 50 mM NaF, 5 mM
Na4P2O7, 10 mM
sodium-ß-glycerophosphate, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 30 µg/ml leupeptin, and 1.5
µM Microcystin. Postnuclear supernatant (minus the fat
cake) was collected by centrifugation at 1,000 x g for
10 min, after which, 1% Triton X-100 was added, and, after standing
for 30 min at 4 C, insoluble material was removed by centrifugation at
10,000 x g for 10 min. Cell lysate containing 600 µg
protein was incubated with 5 µl immunoaffinity-purified sheep IGG
antihuman Akt1/PKB pleckstrin homology domain, which was coupled to
protein G-agarose and suspended in Buffer A. After incubation for 90
min at 4 C with constant rotation, the PKB/antibody/protein G-agarose
complex was collected by centrifugation and washed three times with
Buffer A containing 0.5 M NaCl, then washed twice with
Buffer B [50 mM Tris/HCl (pH 7.5), 0.03% Brij-35, 0.1
mM EGTA and 0.1% 2-mercaptoethanol], and twice with assay
dilution buffer [20 mM MOPS
(3-[N-morpholino propane sulfonic acid) (pH 7.2), 25
mM sodium ß-glycerophosphate, 5 mM EGTA, 1
mM Na3VO4 and 1 mM
dithiothreitol (DTT)]. Immunoprecipitates were then incubated for 10
min at 30 C in the presence of 10 µM PKA inhibitor
peptide (TYADFIASGRTGRRNAI), 113 µM ATP, 17
mM MgCl2, 15 mM MOPS (pH
7.2), 18 mM sodium ß-glycerophosphate, 4 mM
EGTA, 0.75 mM Na3VO4, 0.75
mM DTT, 10 µCi [-32P]ATP (DuPont NEN) and 100 µM Akt/PKB specific substrate
(RPRAATF, related to the sequence surrounding the serine-9
phosphorylation site of GSK3-ß), in a final volume of 40 µl. After
incubation, the reaction mixture was pulse-spun to remove the
PKB/antibody/protein G-agarose immunocomplex, and the supernatant was
removed and added to 20 µl 40% trichloroacetic acid, mixed, and
incubated for 5 min at room temperature. An aliquot (40 µl) of this
mixture was then transferred to P81 phosphocellulose paper, washed four
times with 0.85% phosphoric acid and once with acetone, and counted
for 32P radioactivity. Blank values were determined by
using preimmune serum instead of anti-PKB antiserum, or by conducting
assays in the absence of substrate (in both cases, blank values were
approximately 10% of the insulin-stimulated level of PKB activity). In
a few experiments, PKB was assayed by using polyclonal antiserum
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA) for immunoprecipitation and cross-tide (GRPRTSSFAEG; from UBI) as
substrate; relative effects of insulin, RO 31-8220 and the PKC-
pseudosubstrate in this PKB assay were similar to those observed with
the UBI PKB assay kit described above, except that the total level of
32P incorporation was considerably less.
JNK activation
Enzyme activity of immunoprecipitable JNK was determined as
described (3). In brief, adipocyte lysates [in 20 mM
Tris/HCl (pH 7.6), 250 mM NaCl, 3 mM EDTA, 3
mM EGTA, 1 mM DTT, 10 mM sodium
ß-glycerophosphate, 2 mM Na3VO4,
2 mM NaF, 2 mM
Na4P2O7, 10 µg/ml aprotinin, 20
µg/ml leupeptin, 1 µM Microcystin-LR, and 0.5% Triton
X-100] were subjected to immunoprecipitation (500 µg protein/5 µl
antiserum) with rabbit polyclonal antiserum raised against a JNK-1
epitope (amino acids 368–384; obtained from Santa Cruz Biotechnology, Inc.), after which, precipitates were washed and
then incubated for 15 min at 30 C in buffer containing 20
mM HEPES (pH 7.5), 20 mM sodium
ß-glycerophosphate, 10 mM MgCl2, 500
µM Na3VO4, 500 µM
NaF, 10 µM ATP, 5 µCi [-32P]ATP and
1.5 µg c-Jun peptide complexed with
glutathione-S-transferase (GST) (Santa Cruz Biotechnology, Inc.). After incubation, the assay mixture was boiled for 5 min,
and the c-Jun-GST fusion protein was purified by SDS-PAGE
and quantified for 32P in a phosphorimager from
Bio-Rad Laboratories, Inc. (Hercules, CA).
ERK1/2 activation
ERK1/2 activity was measured by two methods. First, the activity
of ERK1/2 (along with other MAP kinases) was measured in cytosolic
extracts (approximately 5 µl containing 5 µg protein) as described
(5, 23), using either 50 µg myelin basic protein (MBP) or a more
specific substrate, i.e. 200 µM epidermal
growth factor receptor (EGFR) peptide (amino acids, 662–681;
Quality Controlled Biochemicals, Inc., Hopkington, MA) in
50 µl buffer containing 25 mM ß-glycerophosphate (pH
7.3), 0.5 mM DTT, 1.25 mM EGTA, 0.5
mM Na vanadate, 10 mM MgCl2, 1
µM okadaic acid, and 0.1 mM
[-32P]ATP (1.5 x 106 dpm/nmol).
After incubation for 10 min at 30 C, aliquots were placed on p81 filter
paper, washed with 1% H3PO4 and counted for
radioactivity. As reported previously (5, 23), results with both
substrates are similar and are reflective of pp42/44-dependent
(i.e. ERK1 and 2) phosphorylation of MBP, as determined by
assays conducted in MBP-containing, renatured SDS-PAGE gels. As a
second method, ERK2 was immunoprecipitated from cytosolic extracts (in
Buffer A supplemented with 0.15 M NaCl 1% Triton X-100,
and 0.5% Nonidet) with a polyclonal antiserum (Santa Cruz Biotechnology, Inc.), collected on protein AG-agarose beads,
washed and assayed (conditions as described above) using MBP as
substrate, which was collected on p81 filter paper, washed with 1%
H3PO4, and counted. Results with the ERK2
immunoprecipitation assay were similar to those observed with simple
cytosolic assays of total MAPK activity, except that relative insulin
effects were greater in the ERK2 immunoprecipitation assay, perhaps
reflecting a greater specificity of this assay, and lower relative
basal values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As
seen in Fig. 2, RO 31-8220 stimulated GS
activity in both rat adipocytes and L6 myotubes, and, using maximally
effective concentrations of both agonists, insulin effects on GS
appeared to be additive to those of RO 31-8220. In keeping with the
apparently additive effects of RO 31-8220 and insulin on GS activity,
the mechanisms used by these agonists were found to be decidedly
different, as evidenced by the fact that wortmannin, the PI 3-kinase
inhibitor, inhibited the effects of insulin, but not those of RO
31-8220, on GS (Fig. 1).
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); of further note, insulin
effects on glucose incorporation into glycogen appeared to be additive
to, or synergistic with, those of RO 31-8220. On the other hand,
somewhat different from alterations in GS activity, insulin effects on
labeled glucose incorporation into glycogen were greater than those of
RO 31-8220, irrespective of the concentration of RO 31-8220 that was
used: this difference may reflect the participation of factors other
than GS on labeled glucose incorporation into glycogen. It may also be
noted that glucose incorporation into glycogen tended to diminish at
higher concentrations of RO 31-8220, particularly in L6 myotubes; this
diminution most likely reflects the fact that glucose transport is
inhibited at higher concentrations of RO 31-8220 (see 19, 21, and
discussion below), and this inhibition of glucose transport probably
imposes a greater limitation on overall glucose utilization in L6
myotubes than in adipocytes incubated in 5 mM glucose.
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); these findings are
in concert with the reported failure of PKC inhibitors other than RO
31-8220 to activate JNK (3), confirm the specificity of the stimulatory
effects of RO 31-8220 on glycogen synthesis, and provide clear evidence
that simple inhibition of PKC cannot account for the activation of JNK,
GS, and overall glycogen synthesis by RO 31-8220.
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Studies of JNK activation
In keeping with findings in rat-1 fibroblasts (3), RO 31-8220
provoked 2- to 3-fold increases in the enzymatic activity of
immunoprecipitable JNK in rat adipocytes (Fig. 5, A and B) and L6 myotubes (Fig. 5C)
(note that this activation of JNK is indirect, as direct addition of RO
31-8220 to the immunoprecipitates was without effect on JNK
activity—data not shown). Also, similar to findings in rat skeletal
muscle (7), insulin provoked rapid (maximal within 1 min and sustained
at comparable levels at 15 min) increases in immunoprecipitable JNK
enzyme activity in rat adipocytes (Fig. 5B) and L6 myotubes (Fig. 5C):
however, it is important to note that these effects of insulin on JNK
activity appeared to be slightly less than, and clearly not additive
to, those of RO 31-8220.
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), and 2) ERK2, as measured in
specific immunoprecipitates of rat adipocytes (Fig. 7). RO 31-8220 also inhibited
insulin-induced activation of immunoprecipitable ERK2 in intact
adipocytes (Fig. 7). It should be noted that the inhibition of MAPK and
ERK2 in vitro was observed at RO 31-8220 concentrations
(EC50 of approximately 1–3 µM) that were
higher than those required to inhibit conventional and novel PKC’s
(see 1). Like RO 31-8220, GF109203X, another structurally related
bisindolemaleimide PKC inhibitor, inhibited total MAPK activity, but
this inhibition was less than that observed with RO 31-8220,
i.e. 50% inhibition at a GF109203X concentration of 50
µM (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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From the above observations, it may be surmised that the activation of GS by RO 31-8220 cannot be explained by changes in the activity of either ERK1/2 or PKB. Also, because other PKC inhibitors did not affect glycogen synthesis, it seems clear that the activating effects of RO 31-8220 on GS cannot be explained by inhibitory effects of RO 31-8220 on basal PKC activity. Finally, unlike insulin, activating effects of RO 31-8220 on GS were independent of PI 3-kinase. By exclusion, therefore, the JNK pathway seemed to be the most likely candidate to explain the stimulatory effects of RO 31-8220 on GS activation. In keeping with this possibility, anisomycin, another activator of JNK (3, 7) and GS in skeletal muscle (7), was found to activate glycogen synthesis in rat adipocytes, albeit much less effectively than RO 31-8220. Nevertheless, in the absence of a specific JNK inhibitor, our postulation that JNK was responsible for activation of GS can only be considered as tentative. In addition, we cannot exclude the possibility that factors other than JNK may have participated in the activation of GS during RO 31-8220 treatment, and further studies will be required to see if JNK fully accounts for GS activation during RO 31-8220 treatment.
Whereas JNK appeared to be the most likely candidate to explain the stimulatory effects of RO 31-8220 on GS, our findings also suggested that JNK activation was unlikely to serve as a major mechanism for stimulatory effects of insulin on glycogen metabolism. Indeed, although insulin provoked increases in JNK activity in both rat adipocytes and L6 myotubes, these insulin-induced increases were not additive to increases in JNK activity that were provoked by RO 31-8220. Accordingly, because insulin effects on both GS and glucose incorporation into glycogen were clearly additive to those of RO 31-8220, it follows that a factor other than JNK is more likely to serve as the major effector for insulin effects on GS and net glycogen synthesis.
It was surprising to find that RO 31-8220 directly inhibited total MAPK and ERK2 at concentrations that were comparable to those that inhibit atypical PKCs. Although we did not make an extensive study of other PKC inhibitors, we found that another commonly used bisindolemaleimide PKC inhibitor, GF109203X, inhibited total MAPK, but only at concentrations considerably above those that were found to be effective for RO 31-8220. In this regard, it may be noted that both inhibitors inhibit various PKCs at similar concentrations, with conventional PKCs being most sensitive (24, 25).
The presently observed inhibitory effects of RO 31-8220 on total MAPK
and ERK2 activity in vitro, and their activation by insulin
in intact adipocytes and L6 myotubes, appear to be opposite of what may
be expected from the findings of Beltman et al. (3), who
reported that RO 31-8220 potentiated the activation of ERK by epidermal
growth factor (EGF) in rat-1 fibroblasts. This difference, however, may
in part reflect the fact that ERK1/2 can be activated by several
mechanisms, e.g. via the well-studied GRB2/SOS/Raf/MEKK
pathway and via a less defined pathway requiring PI 3-kinase. In rat
adipocytes and L6 cells, based upon wortmannin sensitivity, it appears
that PI 3-kinase is required for insulin-stimulated MAPK activation (5, 26). Moreover, we have recently found (unpublished observations) that,
in addition to RO 31-8220 (present results), other more specific
inhibitors of PKC- (e.g. the PKC- pseudosubstrate and
transient expression of kinase-inactive PKC-), as well as the MEK
inhibitor, PD94008 block insulin effects on the activation of
immunoprecipitable ERK2 in rat adipocytes; it therefore seems likely
that insulin effects on ERK2 in these cells are mediated largely via PI
3-kinase, PKC-, MEKK and MEK; whether or not ras functions upstream
of PI 3-kinase is presently under study. In contrast, assuming that 5
µM RO 31-8220 at least partially inhibited PKC- in
rat-1 fibroblast studies of Beltman et al. (3), it may be
surmised that effects of EGF on ERK are not dependent upon PKC-, and
perhaps PI 3-kinase, in rat-1 fibroblasts. On the other hand, it may
also be surmised that 5 µM RO 31-8220 did not strongly
inhibit ERK in the studies of Beltman et al. (3). Whatever
the explanation for differences in mechanisms used by insulin and EGF
to activate ERK1/2, it should be noted that we have observed that RO
31-8220 directly inhibits ERK1/2 not only in rat adipocytes and L6
myotubes (present data), but also in rat skeletal muscle (data not
shown).
As discussed above, the activation of JNK and GS by RO 31-8220 could
not be explained by inhibition of PKC, as other structurally related
bisindolemaleimide PKC inhibitors, such as GF109203X, do not activate
either JNK or GS. These differences suggest that the activation of JNK
and GS by RO 31-8220 is dependent upon molecular determinants that are
not necessarily shared by other bisindolemaleimides and are distinct
from determinants that are responsible for inhibition of the kinase
activity of PKC, PRK1/PKN, and ERK1/2. This suggests that it may be
possible to devise compounds that could serve as effective activators
of JNK and GS but lack significant inhibitory effects on PKC, PRK1/PKN,
or ERK. Such compounds could be useful in stimulating glycogen
synthesis, which is known to be defective in poorly controlled diabetes
mellitus. Along these lines, in preliminary studies, we have found
that, at certain, but not higher doses, RO 31-8220 treatment in
vivo partially reverses the hyperglycemia observed in type II
diabetic Goto-Kakizaki (GK) rats (unpublished observations). We have
also found that low µM (2, 3, 4, 5) concentrations of RO
31-8220 enhance GS activity and stimulate total glucose uptake in rat
adipocytes (incubated in 5 mM glucose), despite causing
mild inhibition of 2-deoxyglucose uptake. Although these preliminary
findings are intriguing, it should also be noted that, as shown in
studies in which soleus muscles of normal and GK-diabetic rats were
incubated in vitro, 20 µM RO 31-8220 increased
labeled glucose incorporation into glycogen, without increasing total
glucose uptake (27); the latter failure probably reflects the fact that
this concentration causes a moderate inhibition of 2-deoxyglucose
uptake (i.e. glucose transport) (27). Moreover, at slightly
higher concentrations of RO 31-8220, glucose transport effects of
insulin are markedly inhibited in soleus muscles (27), presumably
reflecting a requirement for PKC- (see 21). Similarly, as alluded to
above, at higher doses of RO 31-8220 treatment in vivo,
serum glucose levels in GK rats increased (unpublished observations),
most likely reflecting inhibition of glucose transport. Thus, the
margin of safety for RO 31-8220 did not appear to be suitable for
treating diabetes mellitus; on the other hand, as discussed above, it
is theoretically possible to devise an agent that activates JNK but
does not inhibit PKC-, and such an agent may be useful for improving
glycogen synthesis and overall glucose homeostasis.
It is important to note that RO 31-8220 did not directly activate JNK, and we presently have no insight into the mechanism(s) whereby RO 31-8220 activates JNK. Accordingly, it is presently uncertain what cellular receptor is responsible for the initial signaling that results in subsequent JNK activation during RO 31-8220 action. Along these lines, JNK has been found to be activated by several kinase cascades that involve MEKs and MEKKs (4), at least one of which may be activated by small G proteins, including Rac1 and Cdc42hs (28) that, in this regard, operate via p21-activated kinase (PAK). Whether or not RO 31-8220 activates JNK through these pathways remains to be evaluated.
In summary, we presently found that the PKC inhibitor, RO 31-8220, (a) inhibited ERK1/2, as well as PKC, signaling pathways, (b) had no effect on PKB activity, and (c) activated JNK in rat adipocytes and L6 myotubes. The activation of JNK appeared to be the most logical candidate to explain the activating effects of RO 31-8220 on GS. In contrast, insulin-induced activation of GS appeared to occur independently of alterations in JNK activity.
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Footnotes |
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Received September 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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as a downstream
effector of phosphatidylinositol 3-kinase during insulin stimulation in
rat adipocytes. Potential role in glucose transport. J Biol Chem 272:30075–30082S and insulin on
the activation of Rho, phosphatidylinositol 3-kinase, and protein
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and noninvolvement of diacylglycerol-sensitive PKCs in
insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138:4721–4731This article has been cited by other articles:
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