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RO 31-8220 Activates c-Jun N-Terminal Kinase and Glycogen Synthase in Rat Adipocytes and L6 Myotubes. Comparison to Actions of Insulin¹

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.

	Abstract

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The activation of c-Jun N-terminal kinase (JNK) by insulin and anisomycin has been reported to result in increases in glycogen synthase (GS) activity in rat skeletal muscle (Moxham et al., J Biol Chem, 1996, 271:30765-30773). In addition, the protein kinase C (PKC) inhibitor, RO 31-8220, has been reported to activate JNK in rat-1 fibroblasts (Beltman et al., J Biol Chem, 1996, 271:27018-27024). Presently, we found that the RO 31-8220, as well as insulin, activated JNK and GS and stimulated glucose incorporation into glycogen in rat adipocytes and L6 myotubes. In contrast to activation of JNK, RO 31-8220 inhibited extracellular response kinases 1 and 2 (ERK1/2) and had no significant effects on protein kinase B (PKB). Stimulatory effects of RO 31-8220 on JNK and glycogen metabolism were not explained by PKC inhibition, as other PKC inhibitors were without effect on glucose incorporation into glycogen and have no effect on JNK (Beltman et al., J Biol Chem, 1996, 271:27018). Insulin, on the other hand, activated JNK, as well as PKB and ERK1/2. However, stimulatory effects of insulin on GS and glucose incorporation into glycogen appeared to be fully intact and additive to those of RO 31-8220, despite the fact that insulin did not provoke additive increases in JNK activity above those observed with RO 31-8220 alone. Our findings suggest that JNK serves to activate GS during the action of RO 31-8220 in rat adipocytes and L6 myotubes; insulin, on the other hand, appears to activate GS largely independently of JNK.

	Introduction

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RO 31-8220 IS a bisindolemaleimide derivative of staurosporine that is commonly used to inhibit protein kinase C (PKC). RO 31-8220 inhibits conventional (, 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.

	Materials and Methods

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Incubation conditions and studies of glycogen metabolism
As described (5, 19), rat adipocytes were prepared by collagenase digestion of epididymal fat pads of 250 g male Sprague Dawley rats (Zivic-Miller Laboratories, Inc., Portersville, PA or Harlan Industries, Indianapolis, IN), and washed and suspended in Krebs Ringer phosphate (KRP) buffer containing 1% BSA. To study glucose incorporation into glycogen in intact cells, adipocytes were equilibrated in KRP medium containing 5 mM glucose and various PKC inhibitors [RO 31-8220, GF 109203X, and GO 6976 (all from Alexis Laboratories, San Diego, CA); and CG 53353 (kindly provided by Drs. A. Suter and D. Fabbro, Ciba Geigy Corp., Berne, Switzerland)] as indicated, and then incubated for 60 min with [U-¹⁴C]D-glucose or [6-³H]D-glucose (DuPont NEN, Boston, MA) (results with these isotopes were interchangeable), with or without 10 nM insulin as indicated, following which, incorporation of labeled glucose into glycogen was measured as described (20). To examine the activation of GS, as described previously (5), adipocytes were equilibrated for 30 min in KRP medium containing indicated (optimal for observing changes in GS) concentrations of glucose and inhibitors, and then treated with or without 10 nM insulin for 15 min, following which, incorporation of UDP-[U-¹⁴C]D-glucose (DuPont NEN, Boston, MA) into glycogen was measured in cell-free extracts incubated in the presence of 0 or 10 mM glucose-6 phosphate (G6P) to determine the fractional ratio of GS activity (i.e. -G6P/+G6P).

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.

	Results

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Studies of glycogen metabolism
In studies of rat adipocytes, maximal increases in GS activity were observed during treatment with 10 µM RO 31-8220 and 10 nM insulin (Fig. 1). 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).

View larger version (11K): [in this window] [in a new window]   Figure 1. Effects of RO 31-8220, insulin, and wortmannin on GS activity in rat adipocytes. Cells were incubated for 30 min with indicated concentrations of RO 31-8220 or wortmannin and then treated with or without indicated concentrations of insulin for 15 min. Cells were then harvested, homogenized, and examined for GS activity in the presence of 0 or 10 mM glucose-6-phosphate (G6P). Shown here are mean ± SE values of fractional ratios (minus G6P/plus G6P) observed in (n) determinations.

View larger version (10K): [in this window] [in a new window]   Figure 2. Effects of RO 31-8220 on basal and insulin-stimulated GS activity in rat adipocytes (left) and L6 myotubes (right). Cells were incubated for 30 min with indicated concentrations of glucose, with or without 10 µM RO 31-8220, and then incubated for 15 min with or without maximally effective concentrations of insulin (10 nM in adipocytes and 100 nM in myotubes) as indicated. Cells were then harvested, homogenized, and examined for GS activity in the presence of 0 or 10 mM glucose-6-phosphate (G6P). Shown here are mean ± SE values of fractional ratios (-G6P/+G6P) observed in three to six experiments. Asterisks indicate P < 0.025 (t test).

View larger version (6K): [in this window] [in a new window]   Figure 3. Effects of RO 31-8220 on incorporation of labeled glucose into glycogen in control and insulin-treated rat adipocytes and L6 myotubes. Cells were incubated for 30 min with indicated concentrations of RO 31-8220 in KRP medium containing 5 mM glucose and then incubated for 60 min in the presence or absence of 10 nM (adipocyte) or 100 nM (L6 myotubes) insulin, along with [U-14C]D-glucose (2 µCi per 0.5 ml of a 10% adipocyte suspension in each tube) or [6-3H]D-glucose (5 µCi per well of L6 myotubes). Values are mean ± SE values of four determinations in a representative experiments. Similar results were observed in five separate experiments.

View larger version (7K): [in this window] [in a new window]   Figure 4. Effects of insulin and various PKC inhibitors on glucose incorporation into glycogen in rat adipocytes. Adipocytes were equilibrated for 30 min in KRP medium containing 5 mM glucose and indicated inhibitors (10 µM each of RO 31-8220, GF 109203X, CG 53353, and GO 6976), and then incubated for 60 min with or without 10 nM insulin along with [6-3H]D-glucose (5 µCi/0.5 ml 10% adipocyte suspension). Values are mean ± SE of four determinations. P was determined by t test.

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.

View larger version (0K): [in this window] [in a new window]   Figure 5. Effects of RO 31-8220 and insulin on enzymatic activity of JNK in rat adipocytes and L6 myotubes. Cells were incubated first for 30 min in the presence of indicated concentrations of RO 31-8220 in A, or 10 µM RO 31-8220 in B, or 5 µM RO 31-8220 in C, and then for 1 min in the absence or presence of insulin (10 nM for adipocytes and 100 nM for L6 myotubes), following which, JNK was immunoprecipitated and assayed for ability to phosphorylate c-Jun-GST peptide (resolved by SDS-PAGE) as described in Materials and Methods. Representative autoradiograms showing phosphorylation of c-Jun-GST are shown at right. Bargrams shown at left depict mean ± SE of (n) determinations. Asterisks indicate P < 0.05, vs. control (t test).

View larger version (6K): [in this window] [in a new window]   Figure 6. Effects of RO 31-8220 and GF109203X on MAPK activity in vitro in cytosolic extracts of control and insulin-treated rat adipocytes (A) and/or L6 myotubes (B). RO 31-8220 was added directly to in vitro assays of cytosols obtained from control and insulin-treated (10 nM or 100 nM x 10 min, respectively) adipocytes and L6 myotubes. MBP was used as substrate in these assays, but similar effects of insulin and RO 31-8220 were obtained with EGFR peptide as substrate. See Materials and Methods and references 5 and 23 for further details of the assay. Values are means of four determinations in representative experiments.

View larger version (5K): [in this window] [in a new window]   Figure 7. Direct and indirect effects of RO 31-8220 on Immunoprecipitable ERK2 activity in rat adipocytes. A and B, Adipocytes were incubated for 15 min with 20 µM RO 31-8220 in panel A, and for 15 min with indicated concentrations of RO 31-8220 in panel B, and then treated with or without 10 nM insulin for indicated times in panel A, or for 10 min in panel B. After incubation, ERK2 was immunoprecipitated and assayed. C, Adipocytes were incubated for 10 min with or without (control) 10 nM insulin, following which, ERK2 was immunoprecipitated and assayed with indicated concentrations of RO 31-8220 added in vitro. Values are mean ± SE of three to four determinations.

	Discussion

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It seems clear that the PKC inhibitor, RO 31-8220, alters the activity of a number of signaling pathways. In addition to inhibiting PKC and the PKC-related kinase, PRK-1/PKN (see 1, 2), we presently found that RO 31-8220 inhibited total MAPK and ERK2 in rat adipocytes and L6 myotubes. Also, somewhat paradoxically, in both cell types, RO 31-8220 activated JNK, which is in many respects analogous to ERK1/2. This activation of JNK could not be simply explained by inhibitory effects of RO 31-8220 on PKC because other PKC inhibitors, including the bisindolemaleimide, GF109203X, do not activate JNK (3). In contrast to alterations in the activity of JNK and ERK1/2, RO 31-8220 (in doses up to 20 µM) did not alter PKB activity in intact rat adipocytes.

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.

	Footnotes

 
¹ This work was supported by funds from the Department of Veterans Affairs Merit Review Program and National Institutes of Health Grant DK-38079.

Received September 10, 1998.

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