Knockout of the predominant conventional PKC isoform, PKC, in ...

Am J Physiol Endocrinol Metab 297: E340–E348, 2009. First published May 19, 2009; doi:10.1152/ajpendo.90610.2008.

Knockout of the predominant conventional PKC isoform, PKC␣, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake Thomas E. Jensen,1 Stine J. Maarbjerg,1 Adam J. Rose,1 Michael Leitges,2 and Erik A. Richter1 1

Molecular Physiology Group, Copenhagen Muscle Research Centre, Department of Exercise and Sport Sciences, Section of Human Physiology, University of Copenhagen, Copenhagen, Denmark; and 2The Biotechnology Centre of Oslo, University of Oslo, Blindern, Oslo, Norway Submitted 20 July 2008; accepted in final form 15 May 2009

Jensen TE, Maarbjerg SJ, Rose AJ, Leitges M, Richter EA. Knockout of the predominant conventional PKC isoform, PKC␣, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake. Am J Physiol Endocrinol Metab 297: E340 –E348, 2009. First published May 19, 2009; doi:10.1152/ajpendo.90610.2008.—Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Go¨-6976, and Go¨-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited contraction-stimulated glucose uptake by 50 –100%, whereas both Go¨ compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMP-activated protein kinase (AMPK) and eukaryotic elongation factor 2 phosphorylation was unaffected by the blockers. PKC␣ was estimated to account for ⬃97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKC␣ knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Go¨-6983 on contractioninduced glucose uptake were similar in muscles lacking PKC␣ and in the wild type. It can be concluded that PKC␣, representing ⬃97% of cPKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions. protein kinase C

C (PKC) superfamily is divided into three subfamilies, conventional (c) (␣, ␤I, ␤II, and ␥ isoforms), novel (n) (␦, ε, ␪, and ␩ isoforms), and atypical PKCs (␨ and ␭/␫ isoforms), based on differences in structure and responsiveness to the second messengers, Ca2⫹ and diacylglycerol (DAG) (37). Skeletal muscle contraction increases intracellular Ca2⫹ and has been demonstrated to increase DAG following in situ contraction in rat muscle (11), although controversial (54). This induces a rapid translocation of cPKC isoforms, synergistically activated by Ca2⫹ and DAG (36), from a cytosolic to a particulate fraction (11, 40), suggesting their activation. Because glucose uptake in cell culture and isolated rodent muscles is stimulated by either the DAG-mimicking phorbol TRADITIONALLY, THE PROTEIN KINASE

Address for reprint requests and other correspondence: E. A. Richter, Molecular Physiology Group, Copenhagen Muscle Research Centre, Dept. of Exercise and Sport Sciences, Section of Human Physiology, Univ. of Copenhagen, Universitetsparken 13, DK-2100 Denmark (e-mail: [email protected]). E340

esters (17, 24, 59) or compounds that increase intracellular Ca2⫹ (21, 24, 58), cPKC isoforms have long been proposed to mediate the observed contraction-stimulation of skeletal muscle glucose uptake. In support of this, pharmacological inhibition of cPKC (20, 56) or chronic downregulation of cPKC and nPKC isoforms (10) inhibits contraction-stimulated glucose uptake in incubated and perfused rat muscles. Furthermore, a range of studies in muscle cell culture suggests a crucial role of cPKCs in regulating translocation of GLUT4, the major insulin- and contraction-responsive glucose transporter isoform, using pharmacological inhibition or mutated forms of various cPKC isoforms (8, 13, 24, 39, 55). Meanwhile, the lack of PKC inhibitor specificity is a well-described problem (3, 14), often making interpretations problematic. Also, extrapolation of findings from muscle cell culture is difficult because muscle cell culture does not faithfully mirror fully differentiated skeletal muscle architecture (31). In addition, some studies indicate that the relative PKC isoform expression differs markedly between cell culture (24) and mature muscle (38). The current study was undertaken to investigate the role of cPKC in fully differentiated mouse skeletal muscle using three different PKC blockers as well as PKC␣ knockout (KO) mice that display an ⬇97% decrease in cPKC expression. MATERIALS AND METHODS

Animals. Generation and characterization of the PKC␣ KO mice on a 129S2/Sv background have been previously published (32). PKC␣ male KO (Bred by Michael Leitges, Medical School, Hannover, Germany) were paired with age-matched wild-type male 129S2/Sv mice (Charles River Laboratories, Wiga, Germany) and kept in the same animal stables on standard chow diet and a 12:12-h light-dark cycle for 2–3 wk before experimentation to minimize effects of environment. Male wild-type 129S2/Sv mice (Charles River Laboratories) were used for all other experiments. All mice were 12–18 wk old when experiments were performed. Experiments were approved by the Danish Animal Experimental Inspectorate and complied with the “European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes.” Muscle incubation. Soleus or extensor digitorum longus (EDL) muscles were obtained from fed anesthetized wild-type or PKC␣ KO mice (6 mg of pentobarbital/100 g body wt) and suspended at resting tension (4 –5 mN) in incubation chambers (Multi Myograph system; Danish Myo-Technology, Aarhus, DK) in Krebs-Ringer-Henseleit buffer supplemented with 2 mM pyruvate and 8 mM mannitol at 30°C (23). Muscles were preincubated for 1 h for contraction experiments and 30 min for insulin experiments with various PKC inhibitors [calphostin C (Calbiochem, Nottingham, UK): 10 ␮M, 0.16% dimethyl sulfoxide (DMSO); Go¨-6976 (Calbiochem): 20 ␮M, 0.09% DMSO; Go¨-6983 (Calbiochem): 10 ␮M, 0.3% DMSO; or control DMSO (0.3%)] before measuring glucose uptake or signaling. Muscle contractions were induced following 1 h preincubation by electrical

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EFFECT OF PKC␣ ON CONTRACTION-STIMULATED GLUCOSE UPTAKE

stimulation with 1-s trains (0.2 ms, 100 Hz) every 15 s for 10 min. Force development was measured during all incubations by a force transducer hooked to one end of the muscles. For insulin treatment (10,000 ␮U/ml for 30 min), media were changed to insulin media with or without inhibitors or DMSO following 30 min preincubation. For phorbol 12-myristate 13-acetate (PMA) stimulation, 10 or 20 ␮M PMA dissolved in DMSO or corresponding control DMSO (1 or 2%) was added for 40 min following 20 min preincubation. Following stimulation, glucose uptake was evaluated by measuring accumulation of 2-[3H]deoxyglucose (2-[3H]DG) for 10 min, with [14C]mannitol as the extracellular space marker (23). Muscle analyses. Basal or stimulated muscles were quick-frozen by immersion in liquid nitrogen, processed into lysates, and subjected to standard SDS-PAGE immunoblotting techniques (22), using the following total protein or phospho-specific antibodies: PKC␣ (sc-8393; Santa Cruz Biotechnology, Santa Cruz, CA), PKC␤I (sc-209; Santa Cruz), PKC␤II (sc-210; Santa Cruz), PKC␥ (sc-211; Santa Cruz), PKC␪ (610089; BD Biosciences Pharmingen, San Diego, CA), PKC␭ (sc-216; Santa Cruz), hexokinase II (HXK23-A; Alpha Diagnostic international, San Antonio, TX), GLUT4 (P14672; Chemicon International, Temecula, CA), PKC␣/␤II Thr638/641 (no. 9375; Cell Signaling Technology, Beverly, MA), AMP-activated protein kinase (AMPK) Thr172 (no. 2531; Cell Signaling Technology), EF2 Thr56 (no. 2331; Cell Signaling Technology), extracellular signalregulated kinase (ERK) 1 Thr202/Tyr204-ERK2 Thr185/Tyr187 (no. 9101; Cell Signaling Technology), or phospho-Ser PKC substrate (R/K)X(S*)(hydrophobic)(R/K) (no. 2261; Cell Signaling Technology) recognizing a exercise-sensitive band in human muscle around 50 kDa (42). For quantitative blotting, an increasing amount of recombinant PKC␣ (P-1782; Sigma-Aldrich), PKC␤I (P-1787; SigmaAldrich), PKC␤II (P-3287; Sigma-Aldrich), and PKC␥ (P-9542; SigmaAldrich) diluted in sample buffer was compared with wild-type 129S2/Sv mouse brain and quadriceps. In vitro PKC␣ translocation. As a positive control for PKC␣ translocation, three mouse quadriceps muscle lysates were generated in standard lysis buffer (42) without NaCl or Nonidet P-40. Next, to one

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aliquot of each sample, CaCl2 was added to raise free Ca2⫹ concentration to 10 ␮M as described (http://www-leland.stanford.edu/⬃cpatston/ webmaxc/webmaxcE.htm), adding the same volume of double-distilled H2O to the control aliquot. Samples were incubated for 30 min on ice followed by fractionation as previously described (42) to obtain cytosolic and particulate fractions and subjected to immunoblotting. Statistical analysis. Results are means ⫾ SE. Statistical testing was performed using an unpaired t-test or ANOVA with Tukey’s honest significant difference post hoc test. An underlined symbol denotes bars that meet the criteria represented by that symbol. Statistical evaluation was performed using SPSS 15.0 for Windows. The significance level was set at ␣ ⫽ 0.05. RESULTS

The use of several and ideally structurally unrelated pharmacological inhibitors has been advocated when testing the involvement of a given protein in a biological process (14). Here, to assess the involvement of cPKC isoforms in glucose uptake, three PKC inhibitors [Go¨-6976 (35), Go¨-6983 (16), and calphostin C (28)] were chosen based on their relatively selective inhibition of cPKC isoforms and/or use in previous ex vivo incubation studies. As predicted, all three compounds inhibited contraction-stimulated glucose uptake (Fig. 1, A and B). Both Go¨ compounds, but not calphostin C, also inhibited insulinstimulated glucose uptake (Fig. 1, C and D). None of the inhibitors affected tension development during stimulation (data not shown). The phosphorylation of a 50-kDa protein recognized by an antibody raised against the cPKC consensus phosphorylation sequence was higher in EDL, but not soleus, with contraction compared with basal (Fig. 2A, left and right, representative blots shown at the bottom). Noteworthy, soleus DMSO control contraction samples showed both higher (Fig. 2, see represen-

Fig. 1. Conventional (c) protein kinase C (PKC) inhibitor effects on glucose uptake. Ex vivo basal vs. contraction (100 Hz, 1s/15s, 10 min) stimulated 2-deoxyglucose (2-DG) uptake in soleus (A) and extensor digitorum longus (EDL; B) mouse muscles preincubated with either dimethyl sulfoxide (DMSO) alone or with 20 ␮M Go¨6976 (n ⫽ 5–9), 10 ␮M Go¨-6983 (n ⫽ 7), or 10 ␮M calphostin C (CalC; n ⫽ 5–7) for 1 h. C: maximal insulin (10,000 ␮U/ml, 30 min)-stimulated glucose uptake in soleus (C) and EDL (D) mouse muscles preincubated with inhibitors as above for 30 min. P ⬍ 0.05 (†), 0.01 (††), and 0.001 (†††), inhibitor effect using Tukey’s post hoc test.

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Fig. 2. cPKC inhibitor signaling. Ex vivo basal vs. contraction (100 Hz, 1 s/15 s, 10 min)-stimulated cPKC substrate of 50 kDa phosphorylation (arrowhead) (A), AMPactivated protein kinase (AMPK) Thr172 phosphorylation (B), and EF2 Thr56 phosphorylation in soleus (left) and EDL (right) (C), n ⫽ 4 – 8 for all blots. P ⬍ 0.05 (*) and 0.001 (***), contraction main effect. P ⬍ 0.05 (†), 0.01 (††), and 0.001 (†††), inhibitor main effect. Representative blots are shown at bottom.

tative blot) and lower 50-kDa band phosphorylation compared with the corresponding basal samples, whereas all EDL samples were clearly higher with contraction compared with basal. All three inhibitors reduced the phosphorylation of the 50-kDa band in soleus, albeit with varying potency (Go¨-6983/Go¨6976 ⬎ calphostin C), while only the Go¨ compounds (Go¨6983 ⬎ Go¨-6976) reduced the phosphorylation of the 50-kDa band in EDL (Fig. 2A, right and left). To evaluate the specificity of the inhibitors, the phosphorylation of two non-cPKC contraction-responsive proteins, AMPK and eukaryotic elongation factor (eEF) 2, was measured. Neither AMPK phosphorAJP-Endocrinol Metab • VOL

ylation (Fig. 2B, left and right) nor eEF2 phosphorylation (Fig. 2C, left and right) was affected by the inhibitors. Noteworthy, the relatively low activation of AMPK in response to the current contraction protocol in soleus from the 129/Sv strain is different from that previously observed in soleus from the C57Bl/6 strain (22). When the immunoblotting signal in muscle lysates was normalized to that obtained with recombinant cPKC isoform proteins, PKC␣ was by far the predominant cPKC isoform in mouse skeletal muscle, accounting for close to 97% of total cPKC expression (Fig. 3). Therefore, to further test whether

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Fig. 3. PKC␣ is the predominant cPKC isoform in mouse skeletal muscle. Increasing amounts of recombinant cPKC proteins together with 1 ␮g mouse brain lysate (MB) and 40 ␮g of five different mouse quadriceps muscle lysates were immunoblotted with cPKC isoform-specific antibodies. Blots are shown on right.

cPKCs are required for contraction-stimulated glucose uptake, PKC␣ wild-type and KO mice were obtained to evaluate contraction-stimulated glucose uptake. In PKC␣ KO mouse muscles, PKC␣ was not detectable (Fig. 4A), and only a quantitatively small increase was found in the PKC␤II splice variant in relation to total cPKC expression in soleus muscle (Fig. 4B, compare with relative expression in Fig. 3). No genotype differences were found in other measured

PKC isoforms (Fig. 4, C–E). Contraction-stimulated glucose uptake measured ex vivo did not differ between wild-type and PKC␣ KO mice (Fig. 5, A and B). It was also tested whether Go¨-6983 and calphostin C had a more potent effect in PKC␣ KO mice compared with the wild type, but both compounds inhibited contraction-stimulated glucose uptake with similar potency irrespective of genotype (Fig. 5, A and B). Neither basal and contraction-stimulated AMPK Thr172 phosphoryla-

Fig. 4. PKC isoform expression in wild-type and PKC␣ knockout (KO) soleus (SOL) and EDL muscles. A: PKC␣ expression, n ⫽ 10/bar. ND, not detectable. B: PKC␤II splice variant expression, n ⫽ 9/bar. C: PKC␦ expression, n ⫽ 9 –10/bar. D: PKC␪ expression, n ⫽ 9 –10/ bar. E: PKC␭ expression, n ⫽ 7/bar. P ⬍ 0.05, genotype difference using Student’s t-test. Representative blots are shown at bottom right.


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Fig. 5. Contraction-stimulated glucose uptake and non-cPKC signaling is normal in PKC␣ KO muscles. Soleus (A) and EDL (B) ex vivo contraction (100 Hz, 1 s/15 s, 10 min)-stimulated 2-DG uptake in wild-type and PKC␣ KO muscles preincubated with either DMSO alone (n ⫽ 7) or with 20 ␮M Go¨-6976 (n ⫽ 5–9), 10 ␮M Go¨-6983 (n ⫽ 7), or 10 ␮M calphostin C (n ⫽ 5–7) for 1 h. AMPK Thr172 phosphorylation (C) and EF2 Thr56 phosphorylation (D) in wild-type and PKC␣ KO soleus and EDL, n ⫽ 9 –10 for signaling. P ⬍ 0.01 (††) and 0.001 (†††), contraction ⫻ inhibitor interaction. P ⬍ 0.05 (*), 0.01 (**), and 0.001 (***), contraction main effect.

tion (Fig. 5C) nor eEF2 Thr56 phosphorylation (Fig. 5D) differed between genotypes. Contraction is a composite and most likely redundant signaling process to increase glucose uptake (43). Therefore, the effect of the c/nPKC-activating phorbol ester, PMA, was also AJP-Endocrinol Metab • VOL

assessed in wild-type and PKC␣ KO mice. In general, glucose uptake in isolated wild-type 129S2/Sv mouse muscles responded poorly to 10 ␮M PMA, showing only a 20% increase in soleus muscles (Fig. 6A) and no increase in EDL, even at 20 ␮M (data not shown). Still, in PKC␣ KO soleus muscles,

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whether muscles were extracted in the presence or absence of metal ion chelators EGTA and EDTA (data not shown). Indeed, the majority of PKC␣ was localized to the particulate or cytosolic fraction when samples were extracted in the absence or presence of chelators, respectively, regardless of whether muscles were rested or contracted (data not shown). Also, because raising the lysate Ca2⫹ concentration to 10 ␮M in vitro causes a clear translocation of PKC␣ to membranes (Fig. 7), we believe that PKC␣ was likely active during our current contraction and exercise interventions but that the use of Ca2⫹ chelator-containing buffers may have reversed cPKC localization below a detectable level. DISCUSSION

cPKCs have been proposed as vital mediators of contractionstimulated glucose uptake in muscle cell culture and using pharmacological inhibitors in rodent muscles (8, 13, 20, 24, 39, 55, 56). In the current study, three different cPKC inhibitors were shown to reduce contraction-stimulated glucose uptake in fully differentiated mouse muscles. However, the knockout of PKC␣, estimated to reduce total muscle cPKC expression by 97%, did not affect contraction or PMA-stimulated glucose uptake. This suggests that the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or contraction signaling through other PKC isoforms or non-cPKC pathways could have compensated in the absence of PKC␣. All cPKC inhibitors used in this study reduced contractionstimulated glucose uptake, suggesting the involvement of cPKCs in contraction-stimulated glucose uptake. The use of three different inhibitors of cPKC makes an unspecific offtarget effect less likely but still cannot formally prove the involvement of cPKC. Noteworthy, Go¨-6976 was recently stated to inhibit glucose uptake in signaling-independent giant sarcolemmal vesicle preparations, suggesting a direct effect on Fig. 6. Phorbol 12-myristate 13-acetate (PMA)-stimulated glucose uptake is normal in PKC␣ KO muscles. A: ex vivo PMA-stimulated (10 ␮M, 40 min) 2-DG uptake in wild-type and PKC␣ KO soleus muscles, n ⫽ 4. **P ⬍ 0.01. PMA main effect. Soleus (B) and EDL (C) ex vivo PMA-stimulated (10 ␮M, 40 min) extracellular signal-regulated kinase (ERK) 1 Thr202/Tyr204 (arrow on top in representative blot)/ERK2 Thr185/Tyr187 (arrow in bottom of representative blot) phosphorylation; n ⫽ 6. P ⬍ 0.05 (*) and 0.001 (***) vs. basal.

PMA-stimulated glucose uptake was not lower than in the wild type (Fig. 6A), showing that PMA does not require PKC␣ to increase glucose uptake. As a positive control for PMA stimulation of soleus and EDL muscles, PMA was shown to stimulate ERK1 (upper band) phosphorylation in EDL and ERK2 (lower band) phosphorylation in both muscles (Fig. 6, B and C). Neither GLUT4 nor hexokinase II expression differed between genotypes (data not shown). PKC␣/␤II phosphorylation was detectable in wild-type, but not PKC␣ KO, soleus and EDL and did not respond to contraction (data not shown). cPKC are allosterically activated by Ca2⫹ and DAG-dependent interaction with membranes (37). Therefore, attempts were made to use cPKC translocation from cytosol to membranes as an index of contraction activation of cPKC, as previously described (11, 40). However, consistent with an earlier report in human muscle (42), no movement of cPKC to membranes could be detected in contracted rodent muscles AJP-Endocrinol Metab • VOL

Fig. 7. PKC␣, but not ␪, translocates to cellular membranes with increased Ca2⫹ concentration in vitro. CaCl2 (10 ␮M) or double-distilled H2O was added to mouse skeletal muscle homogenates for 30 min, ultracentrifuged into a cytosol and total cellular membrane (TCM) fraction, and blotted for PKC␣ and -␪ as indicated, n ⫽ 3/condition. P ⬍ 0.01 (**) and 0.001 (***) using an unpaired Student’s t-test. Representative blot shown on right.

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GLUT4 (33). Therefore, glucose uptake measured in the presence of Go¨-6976 in particular should be interpreted with caution. In addition, the fact that the inhibitors worked equally well in wild-type and PKC␣ KO muscle suggests that their effect may not involve cPKC although it cannot be excluded that the remaining PKC isoforms, although quantitatively much less important, may in fact be involved in contractioninduced glucose uptake. Calphostin C inhibition of PKC is known to be phorbol ester competitive (28). Hence, all proteins containing the phorbol ester/DAG-binding C-1 domain are potential targets of calphostin C, including chimaerin Rac GTPase-activating proteins, Ras guanyl-releasing proteins, DAG kinases, and protein kinase D (PKD) (12). Interestingly, Ihlemann and coworkers (20), as well as the present study, identified calphostin C as a specific inhibitor of contraction, but not insulin-stimulated glucose uptake, in incubated rodent muscles. From the current study, however, it would seem that other cPKC inhibitors are not contraction-specific, making the exact target of calphostin C unclear. Among the C-1 domain-containing proteins, PKD is another candidate for mediating contraction-stimulated glucose uptake. PKD was originally identified as a novel member of the PKC superfamily, but more recent structural analyses of the kinase domain have shown higher structural similarity to Ca2⫹/calmodulin-dependent kinases than to PKCs (44). PKD is expressed as three isoforms in mammalian mouse skeletal muscle, (PKD1 also known as PKCv, PKD2, or PKD3), of which PKD1 seems the most highly expressed (26). Recently, PKD inhibition by the broadly selective Ser/Thr kinase inhibitor staurosporine was shown to correlate with inhibited contraction and oligomycin-stimulated glucose uptake and GLUT4 translocation in a perfused mouse heart model (33). Furthermore, muscle-specific transgenic overexpression of PKD1 in mouse skeletal muscle enhances fatigue resistance and causes switching from glycolytic type II to oxidative type I fibers by a histone deacetylase phosphorylation-dependent increase in myocyte enhancer factor 2 activity (26), likely involving activation of peroxisome proliferator-activated receptor-␥ coactivator 1␣ (1). Based on these observations, PKD could be an important regulator of both acute and chronic glucose metabolism. PMA preincubation in the present study, despite its ability to enter muscle as judged by stimulation of ERK signaling, elicited only a 20 –30% higher glucose uptake in 129S2/Sv mouse soleus and no increase in EDL, even at 20 ␮M PMA. Previous studies in different mouse strains using other phorbol ester compounds have found just below a doubling of 2-DG uptake in Swiss albino soleus using 1 ␮M 12-O-tetradecanoylphorbol 13-acetate for 30 min (49) and an ⬃50% increase in C57BL/10 EDL using phorbol 12,13-didecanoate for 15 min (34). In stark contrast to these modest increases in mouse muscle, rat muscle incubation studies have shown that both PMA and another phorbol ester compound, 12-deoxyphorbol13-phenylacetate 20-acetate, causes 2.5- to 3.5-fold higher glucose uptake in incubated glycolytic epitroclearis muscles but has no effect in oxidative soleus muscle (17, 59). Apart from the obvious species difference, the disagreement regarding the phorbol ester effect in incubated mouse muscles may relate to variations in mouse strain responsiveness to phorbol esters or the different phorbol ester compounds used. Of note, AJP-Endocrinol Metab • VOL

the species difference is unlikely to be related to differences in PKC isoform expression, since this seems to be conserved between mice (present study) and rats (38). c/nPKC isoforms have been proposed to inhibit insulin signaling and glucose uptake, with excessive c/nPKC activation by lipid metabolites contributing to lipid-induced insulin resistance (45, 46). In agreement, knockout of either PKC␣ (32) or PKC␤ (48) enhances insulin signaling and glucose uptake, with the former acting on the insulin receptor and insulin receptor substrate (IRS)1-phosphatidylinositol 3-kinase (PI 3-kinase) signaling (6, 9, 32) while the latter appears to act downstream of PI 3-kinase (48). Similarly, mice lacking PKC␪, the most abundant PKC isoform in skeletal muscle (38), are completely protected from acute lipid-induced decreases in IRS-1-PI 3-kinase signaling and insulin-stimulated glucose uptake in vivo (25), although this is controversial (47). Somewhat at odds with this paradigm, some studies, including the present, have reported that pharmacological c/nPKC inhibition lowers insulin-stimulated glucose uptake in various models (7, 8, 19, 20, 24, 57) while others report no reduction in insulinstimulated glucose uptake using various methods to inhibit c/nPKCs (4, 5, 10, 27, 55). It is possible that cPKCs are required both to stimulate and limit insulin-stimulated glucose uptake in muscle. Alternatively, this could reflect the unspecific nature of many commonly used PKC inhibitors in addition to differences in the models used. Supporting this notion in the present study, both Go¨ compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake ex vivo, showing an inconsistency between the inhibitors. Overall, the clear findings of enhanced, not reduced, glucose uptake in skeletal muscle of the various c/nPKC KO mouse models supports that c/nPKCs are not essential for skeletal muscle insulin stimulation of glucose uptake, at least when knocking out single isoforms. It is an apparent paradox that muscle contractions appear to activate c/nPKC (11, 17) yet prior contraction does not inhibit insulin signaling and actually increases submaximal insulinstimulated glucose uptake in the hours postcontraction (41). Possible explanations could include c/nPKC movement to distinct sites or in different amounts when translocated by insulin (13, 60), high fat, or exercise. A promising molecular link is AS160/TBC1D4, which may regulate insulin- and contraction-stimulated glucose uptake in skeletal muscle (29). Hence, AS160/TBC1D4 phosphorylation, measured using a phospho-protein kinase B substrate (PAS) antibody, is increased by PMA and Ca2⫹-increasing stimuli in L6 myoblasts (51) and appears to remain elevated in the hours after exercise in rats and humans (2, 18, 52). However, transgenic mice lacking ␣2-AMPK catalytic activity or the ␥3-AMPK regulatory subunit display greatly reduced contraction-stimulated PAS phosphorylation in EDL muscle (30, 53), indicating that PKCs play at most a minor role in PAS phosphorylation. Meanwhile, it was recently shown that the majority of the PAS signal around 160 kDa in glycolytic EDL muscle actually stems from TBC1D1, another potential regulator of GLUT4 trafficking, whereas AS160/TBC1D4 predominates in the oxidative soleus muscle (50). Also, the PAS antibody does not recognize all phosphorylations on AS160/TBC1D4 (15). Thus, it is currently unknown whether c/nPKC is a quantitatively important regulator of AS160/TBC1D4 phosphorylation, in particular in the more oxidative muscles.

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In summary, contraction-stimulated glucose uptake is consistently inhibited by cPKC inhibitors, yet PKC␣ KO, reducing total muscle cPKC expression by 97%, does not affect PMA or contraction-stimulated glucose uptake ex vivo. This likely reflects that the effect of the PKC blockers on glucose uptake is either nonspecific, or contraction signaling through other PKC isoforms or non-cPKC pathways could have compensated in the absence of PKC␣. ACKNOWLEDGMENTS Betina Bolmgren is acknowledged for expert technical assistance with the in vivo glucose uptake assay. Thomas Alsted and Jens Bjarke Kobberø are thanked for their work on cPKC translocation in vitro. GRANTS This study was supported by The Copenhagen Muscle Research Centre, the Novo Nordisk Research Foundation, The Danish Diabetes Association, an Integrated Project (LSHM-CT-2004-005272) funded by the European Commission, The Lundbeck Foundation, and The Danish Medical and Natural Sciences Research Councils. A. J. Rose was supported by postdoctoral grants from the European Commission and from the Carlsberg Foundation. REFERENCES 1. Akimoto T, Li P, Yan Z. Functional interaction of regulatory factors with the Pgc-1␣ promoter in response to exercise by in vivo imaging. Am J Physiol Cell Physiol 295: C288 –C292, 2008. 2. Arias EB, Kim J, Funai K, Cartee GD. Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle. Am J Physiol Endocrinol Metab 292: E1191–E1200, 2007. 3. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P. The selectivity of protein kinase inhibitors: a further update. Biochem J 408: 297–315, 2007. 4. Bandyopadhyay G, Standaert ML, Galloway L, Moscat J, Farese RV. Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138: 4721– 4731, 1997. 5. Bandyopadhyay G, Standaert ML, Zhao L, Yu B, Avignon A, Galloway L, Karnam P, Moscat J, Farese RV. Activation of protein kinase C (alpha, beta, and zeta) by insulin in 3T3/L1 cells. Transfection studies suggest a role for PKC-zeta in glucose transport. J Biol Chem 272: 2551–2558, 1997. 6. Caruso M, Miele C, Oriente F, Maitan A, Bifulco G, Andreozzi F, Condorelli G, Formisano P, Beguinot F. In L6 skeletal muscle cells, glucose induces cytosolic translocation of protein kinase C-alpha and trans-activates the insulin receptor kinase. J Biol Chem 274: 28637–28644, 1999. 7. Chen J, Lu G, Wang QJ. Protein kinase C-independent effects of protein kinase D3 in glucose transport in L6 myotubes. Mol Pharmacol 67: 152–162, 2005. 8. Chernogubova E, Cannon B, Bengtsson T. Norepinephrine increases glucose transport in brown adipocytes via beta3-adrenoceptors through a cAMP, PKA, and PI3-kinase-dependent pathway stimulating conventional and novel PKCs. Endocrinology 145: 269 –280, 2004. 9. Cipok M, ga-Mizrachi S, Bak A, Feurstein T, Steinhart R, Brodie C, Sampson SR. Protein kinase Calpha regulates insulin receptor signaling in skeletal muscle. Biochem Biophys Res Commun 345: 817– 824, 2006. 10. Cleland PJ, Abel KC, Rattigan S, Clark MG. Long-term treatment of isolated rat soleus muscle with phorbol ester leads to loss of contractioninduced glucose transport. Biochem J 267: 659 – 663, 1990. 11. Cleland PJ, Appleby GJ, Rattigan S, Clark MG. Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport. J Biol Chem 264: 17704 –17711, 1989. 12. Colon-Gonzalez F, Kazanietz MG. C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochim Biophys Acta 1761: 827– 837, 2006. 13. Condorelli G, Vigliotta G, Trencia A, Maitan MA, Caruso M, Miele C, Oriente F, Santopietro S, Formisano P, Beguinot F. Protein kinase C (PKC)-alpha activation inhibits PKC-zeta and mediates the action of PED/PEA-15 on glucose transport in the L6 skeletal muscle cells. Diabetes 50: 1244 –1252, 2001. AJP-Endocrinol Metab • VOL

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14. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000. 15. Geraghty KM, Chen S, Harthill JE, Ibrahim AF, Toth R, Morrice NA, Vandermoere F, Moorhead GB, Hardie DG, MacKintosh C. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. Biochem J 407: 231–241, 2007. 16. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett 392: 77– 80, 1996. 17. Henriksen EJ, Rodnick KJ, Holloszy JO. Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium J Biol Chem 264: 21536 –21543, 1989 (published erratum appears in J Biol Chem 265: 5917, 1990). 18. Howlett KF, Mathews A, Garnham A, Sakamoto K. The effect of exercise and insulin on AS160 phosphorylation and 14-3-3 binding capacity in human skeletal muscle. Am J Physiol Endocrinol Metab 294: E401–E407, 2008. 19. Hutchinson T, Bengtsson DS. AMP-activated protein kinase activation by adrenoceptors in L6 skeletal muscle cells: mediation by ␣1-adrenoceptors causing glucose uptake. Diabetes 55: 682– 690, 2006. 20. Ihlemann J, Galbo H, Ploug T. Calphostin C is an inhibitor of contraction, but not insulin-stimulated glucose transport, in skeletal muscle. Acta Physiol Scand 167: 69 –75, 1999. 21. Jensen TE, Rose AJ, Hellsten Y, Wojtaszewski JF, Richter EA. Caffeine-induced Ca2⫹ release increases AMPK-dependent glucose uptake in rodent soleus muscle. Am J Physiol Endocrinol Metab 293: E286 –E292, 2007. 22. Jensen TE, Rose AJ, Jorgensen SB, Brandt N, Schjerling P, Wojtaszewski JF, Richter EA. Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction. Am J Physiol Endocrinol Metab 292: E1308 –E1317, 2007. 23. Jørgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, Schjerling P, Vaulont S, Richter EA, Wojtaszewski JFP. Knockout of the alpha2 but not alpha1 5’-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle. J Biol Chem 279: 1070 –1079, 2004. 24. Khayat ZA, Tsakiridis T, Ueyama A, Somwar R, Ebina Y, Klip A. Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2⫹ and cPKC. Am J Physiol Cell Physiol 275: C1487–C1497, 1998. 25. Kim JK, Fillmore JJ, Sunshine MJ, Albrecht B, Higashimori T, Kim DW, Liu ZX, Soos TJ, Cline GW, O’Brien WR, Littman DR, Shulman GI. PKC-theta knockout mice are protected from fat-induced insulin resistance. J Clin Invest 114: 823– 827, 2004. 26. Kim MS, Fielitz J, McAnally J, Shelton JM, Lemon DD, McKinsey TA, Richardson JA, Bassel-Duby EN, Olson R. Protein kinase D1 stimulates MEF2 activity in skeletal muscle and enhances muscle performance. Mol Cell Biol 28: 3600 –3609, 2008. 27. Klip A, Ramlal T. Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture. Biochem J 242: 131–136, 1987. 28. Kobayashi E, Nakano H, Morimoto M, Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 159: 548 –553, 1989. 29. Kramer HF, Witczak CA, Taylor EB, Fujii N, Hirshman MF, Goodyear LJ. AS160 regulates insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle. J Biol Chem 281: 31478 –31485, 2006. 30. Kramer HF, Witczak CA, Fujii N, Jessen N, Taylor EB, Arnolds DE, Sakamoto K, Hirshman MF, Goodyear LJ. Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle. Diabetes 55: 2067–2076, 2006. 31. Lauritzen HP, Galbo H, Brandauer J, Goodyear LJ, Ploug T. Large GLUT4 vesicles are stationary while locally and reversibly depleted during transient insulin stimulation of skeletal muscle of living mice: imaging analysis of GLUT4-enhanced green fluorescent protein vesicle dynamics. Diabetes 57: 315–324, 2008.

297 • AUGUST 2009 •

www.ajpendo.org

E348


32. Leitges M, Plomann M, Standaert ML, Bandyopadhyay G, Sajan MP, Kanoh Y, Farese RV. Knockout of PKC alpha enhances insulin signaling through PI3K. Mol Endocrinol 16: 847– 858, 2002. 33. Luiken JJ, Vertommen D, Coort SL, Habets DD, El HM, Pelsers MM, Viollet B, Bonen A, Hue L, Rider MH, Glatz JF. Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4mediated glucose uptake, independent of AMPK. Cell Signal 20: 543–556, 2008. 34. MacLennan PA, McArdle A, Edwards RH. Acute effects of phorbol esters on the protein-synthetic rate and carbohydrate metabolism of normal and mdx mouse muscles. Biochem J 275: 477– 483, 1991. 35. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J Biol Chem 268: 9194 – 9197, 1993. 36. Newton AC. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 101: 2353–2364, 2001. 37. Newton AC. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J 370: 361–371, 2003. 38. Osada S, Mizuno K, Saido TC, Suzuki K, Kuroki T, Ohno S. A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle. Mol Cell Biol 12: 3930 –3938, 1992. 39. Patel N, Khayat ZA, Ruderman NB, Klip A. Dissociation of 5’ AMPactivated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2⫹ and protein kinase C inhibition. Biochem Biophys Res Commun 285: 1066 –1070, 2001. 40. Richter EA, Cleland PJ, Rattigan S, Clark MG. Contraction-associated translocation of protein kinase C in rat skeletal muscle. FEBS Lett 217: 232–236, 1987. 41. Richter EA, Garetto LP, Goodman MN, Ruderman NB. Muscle glucose metabolism following exercise in the rat: increased sensitivity to insulin. J Clin Invest 69: 785–793, 1982. 42. Rose AJ, Michell BJ, Kemp BE, Hargreaves M. Effect of exercise on protein kinase C activity and localization in human skeletal muscle. J Physiol 561: 861– 870, 2004. 43. Rose AJ, Richter EA. Skeletal muscle glucose uptake during exercise: how is it regulated? Physiology 20: 260 –270, 2005. 44. Rykx A, De Kimpe L, Mikhalap S, Vantus T, Seufferlein T, Vandenheede JR, Van Lint J. Protein kinase D: a family affair. FEBS Lett 546: 81– 86, 2003. 45. Sampson Cooper SR, DR. Specific protein kinase C isoforms as transducers and modulators of insulin signaling. Mol Genet Metab 89: 32– 47, 2006. 46. Schmitz-Peiffer C. Protein kinase C and lipid-induced insulin resistance in skeletal muscle. Ann NY Acad Sci 967: 146 –157, 2002. 47. Serra C, Federici M, Buongiorno A, Senni MI, Morelli S, Segratella E, Pascuccio M, Tiveron C, Mattei E, Tatangelo L, Lauro R, Molinaro M, Giaccari A, Bouche M. Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity. J Cell Physiol 196: 89 –97, 2003.


48. Standaert ML, Bandyopadhyay G, Galloway L, Soto J, Ono Y, Kikkawa U, Farese RV, Leitges M. Effects of knockout of the protein kinase C beta gene on glucose transport and glucose homeostasis. Endocrinology 140: 4470 – 4477, 1999. 49. Tanti JF, Rochet N, Gremeaux T, van OE, Le Marchand-Brustel, Y. Insulin-stimulated glucose transport in muscle. Evidence for a proteinkinase-C-dependent component which is unaltered in insulin-resistant mice. Biochem J 258: 141–146, 1989. 50. Taylor EB, An D, Kramer HF, Yu H, Fujii NL, Roeckl KS, Bowles N, Hirshman MF, Xie J, Feener EP, Goodyear LJ. Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle. J Biol Chem 283: 9787–96, 2008. 51. Thong FS, Bilan PJ, Klip A. The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic. Diabetes 56: 414 – 423, 2007. 52. Treebak JT, Frosig C, Pehmoller C, Chen S, Maarbjerg SJ, Brandt N, MacKintosh C, Zierath JR, Hardie DG, Kiens B, Richter EA, Pilegaard H, Wojtaszewski JF. Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle. Diabetologia 52: 891–900, 2009. 53. Treebak JT, Glund S, Deshmukh A, Klein DK, Long YC, Jensen TE, Jorgensen SB, Viollet B, Andersson L, Neumann D, Wallimann T, Richter EA, Chibalin AV, Zierath JR, Wojtaszewski JF. AMPKmediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits. Diabetes 55: 2051–2058, 2006. 54. Turinsky J, Bayly BP, O’Sullivan DM. 1,2-Diacylglycerol and ceramide levels in rat skeletal muscle and liver in vivo. Studies with insulin, exercise, muscle denervation, and vasopressin. J Biol Chem 265: 7933– 7938, 1990. 55. Wijesekara N, Tung A, Thong FS, Klip A. Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis, independently of AMPK. Am J Physiol Endocrinol Metab 290: E1276 –E1286, 2006. 56. Wojtaszewski JF, Laustsen JL, Derave W, Richter EA. Hypoxia and contractions do not utilize the same signaling mechanism in stimulating skeletal muscle glucose transport. Biochim Biophys Acta 1380: 396 – 404, 1998. 57. Wright DC, Fick CA, Olesen JB, Craig BW. Evidence for the involvement of a phospholipase C-protein kinase C signaling pathway in insulin stimulated glucose transport in skeletal muscle. Life Sci 73: 61–71, 2003. 58. Wright DC, Geiger PC, Holloszy JO, Han DH. Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2⫹-dependent mechanism in slow-twitch rat soleus muscle. Am J Physiol Endocrinol Metab 288: E1062–E1066, 2005. 59. Wright DC, Geiger PC, Rheinheimer MJ, Han DH, Holloszy JO. Phorbol esters affect skeletal muscle glucose transport in a fiber typespecific manner. Am J Physiol Endocrinol Metab 287: E305–E309, 2004. 60. Yamada K, Avignon A, Standaert ML, Cooper DR, Spencer B, Farese RV. Effects of insulin on the translocation of protein kinase C-theta and other protein kinase C isoforms in rat skeletal muscles. Biochem J 308: 177–180, 1995.

297 • AUGUST 2009 •

www.ajpendo.org