The pivotal role of protein kinase C zeta (PKCzeta) in insulin- and AMP-activated protein kinase (AMPK)-mediated glucose uptake in muscle cells

Cellular Signalling 22 (2010) 1513–1522

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

The pivotal role of protein kinase C zeta (PKCzeta) in insulin- and AMP-activated protein kinase (AMPK)-mediated glucose uptake in muscle cells Li-Zhong Liu 1, Stanley C.K. Cheung 1, Lin-Lin Lan, Stanley K.S. Ho, Juliana C.N. Chan, Peter C.Y. Tong ⁎ Department of Medicine and Therapeutics, Hong Kong Institute of Diabetes and Obesity, Li Ka Shing Institute of Health, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, N.T., Hong Kong, China

a r t i c l e

i n f o

Article history: Received 12 March 2010 Received in revised form 28 May 2010 Accepted 29 May 2010 Available online 4 June 2010 Keywords: Signal integration PKCzeta AMPK PKB Insulin Glucose homeostasis

a b s t r a c t Insulin and AMP-activated protein kinase (AMPK) signal pathways are involved in the regulation of glucose uptake. The integration of signals between these two pathways to maintain glucose homeostasis remains elusive. In this work, stimulation of insulin and berberine conferred a glucose uptake or surface glucose transporter 4 (GLUT4) translocation that was less than simple summation of their effects in insulin-sensitive muscle cells. Using specific inhibitors to key kinases of both pathways and PKCzeta small interference RNA, protein kinase C zeta (PKCzeta) was found to regulate insulin-stimulated protein kinase B (PKB) activation and inhibit AMPK activity on dorsal cell surface. In the presence of berberine, PKCzeta controlled AMPK activation and AMPK blocked PKB activity in perinuclear region. The inhibition effect of PKCzeta on AMPK activation or the arrestment of PKB activity by AMPK still existed in basal condition. These results suggest that there is antagonistic regulation between insulin and AMPK signal pathways, which is mediated by the switch roles of PKCzeta. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Insulin is a key anabolic hormone in promoting glycogen, lipid and protein synthesis and inhibiting their degradations. Glucose uptake is precisely regulated by insulin through recruitment of GLUT4 to the cell surface [1,2]. It is well established that activation of GLUT4 translocation by insulin requires signal transduction via insulin receptor, insulin receptor substrates, phosphatidylinositol 3-kinase (PI3K), PKB/Akt [3,4] and atypical protein kinase C (aPKC) [5–8]. The AMPK system acts as a sensor of cellular energy that is conserved in eukaryotic cells. Once activated, AMPK switches on catabolic pathways that generate ATP by lipolysis and glycolysis, while turns off ATP-consuming processes such as biosynthesis and cell growth and proliferation [9,10]. Given that the antagonistic relationship between the insulin-induced anabolic and AMPK-mediated catabolic pathways, it is intriguing to find that these two pathways both stimulate glucose uptake in certain cell types. In skeletal muscle, both insulin [1] and AMPK [11] activation stimulate glucose uptake via GLUT4 translocation to the cell surface. It has been reported that AS160, a protein with Rab-GTPase-activating protein (Rab-GAP) domain that is involved in regulation of GLUT4 translocation, may be the convergence of these two pathways [12,13]. In patients with

Abbreviations: PKCzeta, protein kinase C zeta; AMPK, AMP-activated protein kinase; PKB, protein kinase B; GLUT4, glucose transporter 4. ⁎ Corresponding author. Tel.: +852 37636052; fax: +852 2144 6365. E-mail address: [email protected] (P.C.Y. Tong). 1 These authors contribute equally to this work. 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.05.020

type 2 diabetes where insulin resistance is present, activation of AMPK by exercise or pharmacological agents such as metformin [14] or thiazolidinediones [15] improves glycemic control. Berberine, an isoquinoline alkaloid isolated from Chinese herbs Coptidis Rhizoma or Cortex Phellodendri, has been demonstrated to possess insulinsensitizing effect in diabetic condition through activating AMPK [16,17]. In this work, we examined the interaction and integration between insulin- and AMPK-signal transductions in mediating glucose uptake in rat skeletal muscle cells. Specific inhibitors to key enzymes of the two pathways and small interference RNA of PKCzeta were used to delineate the effect on activation of kinases and subsequent glucose uptake. We reported that PKCzeta played a key role in modulating activity of PKB and AMPK following stimulation by insulin and berberine, respectively. 2. Materials and methods 2.1. Reagents Tissue culture medium, serum, and other tissue culture regents were purchased from Gibco™ Invitrogen Corporation (Grand Island, NY, USA). Soluble insulin (Actrapid) was purchased from Novo Nordisk (Bagsværd, Denmark). Antibodies against phosphorylation of Thr410 PKCzeta, Thr308/Ser473 of PKB and PKB substrate were purchased from Cell Signaling Technology (Beverly, MA) or from Santa Cruz Biotechnology. Polyclonal antibodies against PKCzeta (C20) and PKB (H136), monoclonal anti-c-myc antibody, goat anti-

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rabbit and goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bio-Rad Protein Assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Enhanced chemical luminescence (ECL) and 2-deoxy-[3H] D-glucose were purchased from Amersham Pharmacia Biotech UK Limited (Little Chalfont, Buckinghamshire, England). OptiPhase “HiSafe” 2 Scintillation Solution was purchased from Wallac Scintillation Product (Wallac Oy, Turku, Finland). Phenylmethylsulfonyl fluoride and PKB inhibitor API2 were purchased from Calbiochem (La Jolla CA). Dithiothreitol (DTT), orthovanadate, β-mercaptoethanol, bovine serum albumin (BSA), cell permeable PKCζ pseudo-substrate myristoyl trifluoroacetate, compound C, Wortmnnin, O-phenylenediamine dihydrochloride (OPD), protein G and phosphate-bufferedsaline (PBS) were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibody for AS160 was purchased from Upstate (Hampshire, UK).

(3 × 15 min) TBST. Proteins were visualized by enhanced chemiluminescence (ECL). 2.6. RNA interference The following small interfering RNA (siRNA) oligonucleotides were used to knockdown the expression of endogenous PKCzeta: Invitrogen, Rat Steath Selected 3 set, catalog number RSS303768, RSS303769 and RSS303770; and Stealth RNAi Negative Control Med GC, catalog number 12935300, was used as scrambled control. L6 cells were transfected with these siRNAs using Lipofectamine™ RNAiMAX reagent (Invitrogen) according to the manufacturer's instruction. siRNAs were introduced into the cells by reverse transfection at the beginning of subculture and transfected again at the start of day 4 of differentiation by forward transfection, then cells were maintained for another 48 or 72 h until experimentation.

2.2. Cell culture L6 muscle cells expressing c-myc epitope tagged GLUT4 (L6myc cells) [18,19] were maintained in myoblast monolayer culture in α-MEM containing 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) antibiotic–antimyocotic solution (100 U/ml penicillin G, 10 μg/ml streptomycin, and 25 mg/ml amphotericin B) in an atmosphere of 5% CO2 at 37 °C. For differentiation into myotubes, myoblasts were plated in medium containing 2% (vol/vol) FBS at 104 cells/ml to allow spontaneous myoblast fusion. Medium was changed every 48 h and myotubes were ready for experiment 6–8 days after plating. 2.3. 2-deoxy-[3H] deoxyglucose uptake After serum deprivation, L6 myotubes were left untreated or treated with 100 nM insulin for different times at 37 °C. After this period, cells were washed three times with glucose-free HEPES-buffered saline solution (140 mmol/L NaCl, 20 mmol/L Na-HEPES [pH 7.4], 2.5 mmol/L MgSO4, 5 mmol/L KCl, 1 mmol/L, CaCl2). Glucose uptake was measured as described previously by using 2-deoxy-[3H] deoxyglucose [20]. Each condition was assayed in triplicate. 2.4. Densitometric assay of surface GLUT4myc After serum deprivation, L6 myotubes were left untreated or treated with 100 nM insulin for various times at 37 °C. Cells were washed three times with ice-cold PBS, followed by blocking with 5% (vol/vol) goat serum in PBS for 10 min. Cells were incubated with anti-myc monoclonal antibody in HEPES-buffered RPMI containing 3% (vol/vol) goat serum for 60 min at 4 °C before fixation with 3% (vol/vol) formaldehyde in PBS for 3 min. Cells were incubated with 100 mM glycine in PBS at 4 °C for 10 min followed by horseradish peroxidaseconjugated (HRP conjugated) goat anti-mouse IgG (1:1000) in PBS containing 3% goat serum for 60 min. To quantify the amount of bound antibody, OPD reagent was added at room temperature for up to 30 min and the reaction stopped by adding 3 M hydrochloric acid. An aliquot of the reaction was removed for measuring the absorbance at 492 nm [21]. 2.5. Western blot Western blot was resolved by SDS-PAGE as described everywhere [22] and after cell lysis, aliquots proteins were separated by SDS-PAGE (10% polyacrylamide). Thereafter proteins were electrophoretically transferred to polyvinylidine difluoride membrane and block in 5% BSA and 0.05% Tween 20 in Tris-buffered saline (TBST) for 1.5 h at room temperature. Membranes were incubated overnight at 4 °C with indicated first antibodies. Membranes were washed (3 × 5 min) in TBST, and incubated with horseradish peroxidase-conjugated IgG for 0.5 h at room temperature, followed by additional washes in

Fig. 1. Effect of berberine on insulin-induced glucose uptake in muscle cells. L6myc cells were prepared on 12-well plates for measurement of glucose uptake (A) or 24-well plates for measurement of surface GLUT4 (B) as described in Materials and Methods. 3 h serum-starved L6myc cells were left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h. For the treatment of berberine plus insulin, cells were first incubated with 5 μg/ml berberine for 50 min and followed by adding 100 nM insulin for another 10 min. Data means ± SE of five to eight experiments performed in triplicate. #P b 0.05, compared with basal. For protein detection (C), 3 h serum-starved L6myc cells were left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h. For the treatment of berberine plus insulin, cells were first incubated with 5 μg/ml berberine for 50 min and followed by adding 100 nM insulin for another 10 min. Then 25 μg whole cell lysis protein was subjected to 8% SDS-PAGE and immunoblotted with indicated antibodies.

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2.7. Immunostaining L6 GLUT4myc muscle cells were grown to the stage of myotubes on 25-mm-diameter glass coverslips placed in six-well plates. Myotubes are characterized by multinucleation. Myotubes were deprived of serum for 3 h and treated with 100 nmol/l insulin for 5 min at 37 °C. Myotubes were fixed with 3% (vol/vol) paraformaldehyde in PBS for 20 min and then washed with 0.1 mol/l glycine in PBS for 10 min, permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 3 min, and then washed with PBS. Cells were first incubated for 1 h at room temperature with primary antibodies in 0.1% (wt/vol) BSA at a dilution of 1:100, then washed with

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PBS and subsequently incubated with either Alexa-conjugated goat antirabbit or anti-mouse secondary antibodies, respectively, at a dilution of 1:250 for 1 h at room temperature. Cell monolayers were washed further with PBS and mounted in ProLong Antifade solution onto glass slides. 3. Results 3.1. Effect of berberine on insulin-induced glucose uptake in muscle cells It has been reported that berberine exhibits anti-diabetic effect in insulin-resistant state [23,24]. Whether berberine improves insulin-

Fig. 2. Kinases detection in the presence of insulin and/or berberine. 3 h serum-starved L6myc cells were left untreated or treated with 100 nM insulin for 10 min or 5 ug/ml berberine for 1 h. For the treatment of berberine plus insulin, cells were first incubated with 5 μg/ml berberine for 50 min and followed by adding 100 nM insulin for another 10 min. Then 25 μg whole cell lysis protein was subjected to 8% SDS-PAGE and immunoblotted with indicated antibodies (A, B, D, E and F). The same membrane was first blotted for phosphorylation followed by stripping and re-blotted for protein. For the recruitment of p85 to IRS1, total IRS1 protein was first immunoprecipitated with anti-IRS1 antibody from unstimulated and insulin/berberinetreated cells, then detected with p85 antibody (A). The amount of phosphorylated PKCzeta (p-PKCzeta) and PKCzeta or phosphorylated AS160 (p-AS160) and AS160 were quantified by densitometric scanning and analyzed with Bio-Rad Molecular Analysis software. Pixel intensity was normalized and a value of 1 was assigned to the basal condition (B and E). For PKCzeta activity measurement (D), PKCzeta was immunoprecipitated with anti-PKCzeta antibody from unstimulated and insulin/berberine-treated cells, and then PKCzeta activity was measured as described in Materials and Methods. PKCzeta activity was expressed as fold of basal. Data means± SE of three experiments performed in triplicate. *P b 0.05, compared with basal.

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induced glucose disposal in insulin-sensitive muscle cells is not clear. Here, compared with basal, acute treatment with insulin and/or berberine improved glucose uptake (Fig. 1A) and GLUT4 translocation (Fig. 1B) significantly. However, insulin plus berberine did not provide an additive or synergistic effect. There was no significant change of GLUT1 and GLUT4 expressions upon acute treatment of insulin or berberine (Fig. 1C).

3.2. The participation of kinases in insulin- and berberine-induced glucose uptakes The above results suggested that there was antagonistic interaction between insulin- and berberine-induced pathways of glucose uptake. To investigate the molecular mechanism beyond, key kinases involved in insulin signaling pathway and AMPK-signaling pathway were studied. Insulin increased tyrosine phosphorylation of IRS1, the recruitment of p85 to IRS1 and the phosphorylation of Thr308/Ser473 on PKB, whereas berberine exhibited some inhibition effect on PKB (Fig. 2D). Intriguingly, PKCzeta activity increased with the treatment of insulin and/or berberine (Fig. 2B and C). Furthermore, costimulation of berberine and insulin exerted an additive increase in PKCzeta activity, causing an increase in the phosphorylation of Thr410 on PKCzeta. Thus, the Thr410 phosphorylation could be used as a marker to label PKCzeta activity upon insulin or berberine stimulation. Acute treatment with insulin or berberine increased the phosphorylation of AS160, but insulin plus berberine did not provide an additive or synergistic effect (Fig. 2E). AMPK activity increased

with the treatment of berberine but its activity decreased by insulin, which was further demonstrated by reduction of phosphorylation of ACC, one of the substrates of AMPK (Fig. 2F).

3.3. Effect of inhibitors of kinases on insulin and/or berberine-induced glucose uptake Next, we evaluated the contribution of key kinases to insulin and/ or berberine-induced glucose uptake by employing specific inhibitors. First, cells were pretreated with kinases inhibitors, thereafter, stimulated with insulin or berberine respectively. In Fig. 3A, insulininduced glucose uptake decreased dramatically with the preincubation of PI3K inhibitor wortmannin (WM), PKCzeta inhibitor pseudosubstrate (PS) and PKB inhibitor (API2). AMPK inhibitor compound C did not block the effect of insulin. On the other hand, only PS and compound C eliminated berberine-induced glucose uptake, whereas WM and API2 had no effect. The results indicated that PI3 kinase and PKB were involved only in insulin-mediated signal transduction whereas PKCzeta participated in both insulin and berberine-induced glucose uptakes. In contrast, AMPK was involved only in berberine-induced glucose uptake. Second, cells were pretreated with kinase inhibitors followed by stimulation of insulin plus berberine. In cells pretreated with WM and API2, glucose uptake was increased by berberine (Fig. 3B). Glucose uptake was stimulated by insulin in cells pretreated with compound C which abrogated berberine-mediated glucose transport. Only PKCzeta inhibitor PS decreased insulin- or berberine-induced glucose uptake.

Fig. 3. Effect of kinases inhibitors on insulin and/or berberine-induced glucose uptake. L6myc cells were prepared on 12-well plates for measurement of glucose uptake as described in Materials and Methods. 3 h serum-starved L6myc cells were serum-starved for 3 h. Cells were left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h. For the treatment of berberine plus insulin, cells were first incubated with 5 μg/ml berberine for 50 min and followed by adding 100 nM insulin for another 10 min. For inhibitors groups, cells were first treated with kinases inhibitors (100 nM WM, 10 μM PS, 20 μM API2 or 20 μM compound C) as indicated for 20 min, then treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h, or treated with 5 μg/ml berberine for 50 min and followed by adding 100 nM insulin for another 10 min. Glucose uptake was showed as fold of basal, a value of 1 was assigned to the basal condition. Data means ± SE of three experiments performed in triplicate. *P b 0.05, compared with insulin or/and berberine stimulation.

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3.4. Effect of kinases inhibitors on insulin or AMPK signal transduction pathways In the presence of WM and PS, phosphorylation of PKCzeta and PKB by insulin was reduced. API2 had no effect on PKCzeta phosphorylation, supporting the notion that PKB was located downstream of PKCzeta. In contrast, the inhibition of PI3K by WM

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had no effect on berberine-induced PKCzeta phosphorylation, which was abrogated by PS only (Fig. 4). Insulin-induced phosphorylation of PKB decreased upon pretreatment of WM, PS and API2, whereas compound C had no inhibition effect. PKB phosphorylation increased when AMPK was inhibited by compound C. Insulin-mediated phosphorylation of AS160 was reduced by pre-treatment of WM, PS and API2 but not compound

Fig. 4. Effect of kinases inhibitors on insulin or berberine signal transduction. L6myc cells were serum-starved for 3 h. Cells were left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h; for inhibition groups, cells were pretreated with 100 nM WM, 10 μM PS, 20 μM API2 or 20 μM comp C respectively for 20 min, followed by adding 100 nM insulin and incubated for another 10 min or adding 5 μg/ml berberine and incubated for another 60 min. Then 20 μg whole cell lysis protein was subjected to 8% SDS-PAGE and immunoblotted with indicated antibodies. The same membrane was first blotted for phosphorylation followed by stripping and re-blotted for protein. Actin bands were shown as internal marker. The bar charts below the blots were the quantitative analysis. The protein bands were quantified by densitometric scanning and analyzed with Bio-Rad Molecular Analysis software. Pixel density of phosphorylation band was corrected after dividing by the corresponding protein level and showed as fold of control; a value of 1 was assigned to the control. The data is representative of 3 independent experiments. Data are means ± SE, *P b 0.01 vs. Ins control or #P b 0.01 vs. BBR control by t-test.

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Fig. 5. Antagonistic effect of PKCzeta and AMPK in basal state. (A) L6 myotubes were serum-starved for 3 h. Cells were left untreated or treated with 20 μM PS or 20 μM Comp C respectively for 30min. 20 μg whole cell lysis protein was subjected to 10% SDS-PAGE and immunoblotted with indicated antibodies. The same membrane was first blotted for phosphorylation followed by stripping and re-blotted for protein. Actin bands were shown as internal marker. The bar charts below the blots were the quantitative analysis. The protein bands were quantified by densitometric scanning and analyzed with Bio-Rad Molecular Analysis software. Pixel density of phosphorylation band was corrected after dividing by the corresponding protein level and showed as fold of control; a value of 1 was assigned to the control. The data is representative of 3 independent experiments. Data are means ± SE, *P b 0.01 vs. control by t-test. (B) L6myc cells were prepared on 12-well plates for measurement of glucose uptake. After serum-starved for 3 h, cells were left untreated or treated with 20 μM PS or 20 μM comp C respectively for 30 min. Then glucose uptake was measured as described in Materials and Methods. Glucose uptake was expressed as fold of control, a value of 1 was assigned to the control. Data means ± SE of five experiments performed in triplicate.

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C. In contrast, WM or API2 did not affect berberine-induced AS160 phosphorylation. Following inhibition of PKCzeta and AMPK by PS and compound C, respectively, phosphorylation of AS160 by berberine was reduced significantly. The phosphorylation of AMPK in muscle cells was reduced by insulin. The inhibitory effect of insulin on AMPK was abrogated by pre-incubation with WM. Despite the presence of insulin, AMPK activity increased significantly when PKCzeta activity was inhibited by PS. The increase in AMPK activity was accompanied by the ACC phosphorylation. These results suggested that insulin inhibits AMPK via PI3K and its downstream targets. Upon berberine treatment, AMPK activity and phosphorylation of ACC increased significantly. WM and API2 failed to prevent berberine-induced activation of AMPK and ACC. When AMPK activity was blocked by compound C, phosphorylation of ACC was reduced, indicating suppression of AMPK activity within the myotubes. Intriguingly, treatment with PS reduced berberine-induced phosphorylation of AMPK and ACC. Taken together, these results demonstrated that 1) PKCzeta regulated PKB activation and inhibited AMPK activity in insulin signaling pathway; 2) PKCzeta participated in the modulation of AMPK activation in the presence of berberine. 3.5. Antagonistic effect of PKCzeta and AMPK in basal state At baseline, preincubation with PS led to reduction in phosphorylation of PKCzeta and PKB and simultaneously increased phosphorylation of AMPK. Intriguingly, phosphorylation at Ser473 and Thr308 positions of PKB increased with reduction of AMPK activity by compound C (Fig. 5A). The effect of compound C did not mediate through PKCzeta which activity was not altered. Despite these changes in kinases activity brought by kinase inhibitors at baseline, there was no change in glucose uptake in L6 myotubes (Fig. 5B). 3.6. Roles of PKCzeta in regulating PKB and AMPK activities The above results based on pharmacological inhibitors showed that PKCzeta could regulate PKB and AMPK activation upon insulin or berberine treatment respectively. To confirm the function of PKCzeta further, PKCzeta siRNA was transiently transfected into L6 muscle cells. The PKCzeta protein level and the phosphorylation at Thr410 were decreased by approximately 50% with siRNA pools (RSS303768, RSS303769 and RSS303770 from Invitrogen) specific to PKCzeta gene (siPKCzeta), compared with the cells transfected with RNAi negative control (scrambled). Knock down of PKCzeta attenuated insulininduced phosphorylation of PKB at Ser473 and Thr308 and AS160, but AMPK and ACC phosphorylations increased at the same time. Berberine-induced AMPK activation was decreased by PKCzeta RNA interference, and consequently the phosphorylation of ACC was reduced too (Fig. 6A and B). The introduction of PKCzeta siRNA also inhibited insulin- and berberine-stimulated glucose uptake by 45 ± 22% and 42 ± 24% respectively (Fig. 6C). 3.7. Spatial interaction between PKCzeta, PKB and AMPK We have previously reported that insulin causes reorganization of PKCzeta on the cell surface and co-localization with actin-remodeling [25]. In the present study, immunostaining results demonstrated the translocation and co-localization of PKCzeta and PKB on the dorsal cell surface upon insulin stimulation (Fig. 7A). Intriguingly, AMPK was found to be present in the new actin structure with PKCzeta after insulin treatment (Fig. 7B). Upon berberine stimulation, there was colocalization between PKCzeta, PKB and AMPK in the cytoplasm (Fig. 7C and D). These results show that specific redistribution of kinases into new actin structures or in the cytosol took place following insulin or berberine stimulation, respectively.

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4. Discussion Insulin signaling pathway is activated when nutrients are available, whereas AMPK pathway is activated when cells are under metabolic stress. Once activated, both of these two pathways lead to glucose uptake although the fate of glucose is different after glucose enters into cells. Within the muscle cells, the integrated regulation of these two pathways related to glucose homeostasis remains undefined. In the present study, we examined the impact of insulin and berberine on enzymes thought to be important in regulation of glucose uptake. We demonstrated that there were antagonistic interactions between insulin and AMPK signal pathways. PKCzeta played an important pivot role in modulating activation of kinases involved in both insulin- and AMPK-signaling pathways. Berberine has been shown exhibiting insulin-sensitizing effect in insulin-resistant state, but little is known in normal/insulin-sensitive condition. Here, insulin plus berberine did not provide an additive or synergistic effect on glucose uptake. When cells were treated with insulin plus berberine, propagation of insulin signals remained normal. Intriguingly, basal or berberine-stimulated AMPK activity decreased in the presence of insulin, and berberine could not antagonize the effect of insulin on the phosphorylation of IRS1 and PKB. These results suggested that there was a negative regulation of AMPK activity by insulin signals. In ischemic cardiac muscle, insulin had been shown to inhibit AMPK activity via PI3K pathway [26,27]. The application of kinases inhibitor and RNA interference in the present study confirmed that PKCzeta inhibited AMPK activity in both basal and insulin-stimulated conditions. Given that AMPK pathway were switched on in the event of energy stress, insulin signaling pathway would therefore exert a negative effect on AMPK activation via PKCzeta when glucose was abundant as in the fed state. In conditions where there was cellular energy depletion, AMPK pathway would be activated for regulation of energy balance at the cellular, as well as the whole body level. Under these conditions, the anabolic action of insulin is blocked following activation of AMPK and its downstream molecules. Previous finding suggested that PKCzeta located downstream of AMPK in C2C12 skeletal muscle cells [28]. Our results on the inhibitory effect of PKCzeta inhibitor PS on berberineinduced AMPK activity, the phosphorylation of ACC/AS160 and the final glucose uptake supported the notion that PKCzeta may locate upstream of AMPK, and control AMPK activation upon berberine treatment. Since the phosphorylation of threonine172 on AMPK might fail to reflect the real-time activity of AMPK [11], the phosphorylation of ACC, one of the AMPK substrates, should be detected together with AMPK activity. Here, we showed the inhibition of PKCzeta with its specific inhibitors or siRNA prevented berberine-induced AMPK activation and ACC phosphorylation simultaneously. Thus, berberine-induced PKCzeta activation regulates AMPK activity positively rather than inhibit AMPK. Obviously, berberine can activate a new group of PKCzeta, which is different from those of basal or stimulated by insulin, suggesting that PKCzeta can be activated by different signals and carry out different functions. In insulin-resistant state, the activation of PKCzeta by insulin is defective [29–31], thus the impaired PKCzeta loses its inhibition on AMPK. Since the mechanism of AMPK mediated glucose uptake is PI3 kinase-independent (Fig. 3), activation of AMPK may bypass the insulin signal pathway [11,16] and improve glucose uptake. Protein Kinase B is one of the AS160 kinases and is activated by insulin via PKCzeta. PKCzeta inhibition attenuated not only PKCzeta and PKB activities, but also eliminated AS160 phosphorylation simultaneously. Although PKB inhibitor blocked AS160 phosphorylation and insulin-induced glucose uptake, it had no inhibition effect on PKCzeta. These results suggested that PKCzeta was involved in insulin-induced PKB activation. Our observations also supported the finding that identified PKCzeta as an adaptor to interact with PKB and mediate PKB phosphorylation by PKD2 on Ser472 [22]. These results

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Fig. 7. Spatial interaction between PKCzeta, PKB and AMPK. L6myc cells were serum-starved 3 h and then left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h at 37 °C. Afterward, the cells were fixed, permeabilized, and double stained with indicated actibodies as described in Materials and Methods. Scale Bar: 10 μM. The images are representative of three experiments.

were consistent with McConkey's work, which reported that there was crosstalk between PKCzeta, PKB and cAMP-induced taurocholate uptake and Ntcp translocation are mediated via the PI3K/PKCzeta/PKB signaling pathway [32]. Inactivation of PKB by AMPK has ever been reported in differentiated hippocampal neurons and neuroblastoma SH-SY5Y cells [33]. In the present study, we demonstrated that presence of berberine reduced the activation of PKB. Furthermore, PKB activity recovered when AMPK was inhibited by compound C in both basal and berberine-stimulated states. These results suggested that AMPK regulated PKB activity negatively and weakened insulin effect. The antagonistic effect of AMPK on insulin signal transduction makes it possible to deliver the catabolic signal correctly. In this condition, the insulin-induced anabolic pathway could be blocked whereas the catabolic pathway is opened by berberine. In the present study, the inhibitory effect of PKCzeta on AMPK in the presence of insulin, and the attenuation of PKB by AMPK in the presence of berberine suggested that there might be physiological associations between these kinases. The immunostaining work confirmed the co-localization of these kinases following insulin or berberine stimulation. There was significant difference in the spatial distribution of these kinases following insulin or berberine stimulation. In the presence of insulin, PKCzeta, PKB and AMPK were re-

distributed to actin-remodeling like structure. In contrast, there was enrichment of kinases in the perinuclear region of the cytosol following berberine treatment. We hypothesize that PKCzeta regulates PKB and AMPK activation on the dorsal cell surface in the presence of insulin whereas berberine may enhance PKCzetamediated AMPK activation or AMPK-induced PKB inhibition in the cytosol. The difference in re-localization of these kinases supports the multifunctional roles of PKCzeta and AMPK in insulin- and berberineinduced signal transductions. There is one more thing that needs to be noted, in basal state, although AMPK activity increases upon PKCzeta inhibition or PKB activity recovers through AMPK inhibition, the increased kinases activity cannot bring more glucose uptake. The reason may be is that the recovered kinase activity is not as same as the one induced by insulin or berberine. Sakoda et al. also reported wide-type AMPK transfection significantly increased AMPK activity in muscle but did not enhance glucose uptake [34]. Thus, the recovered kinases in these processes may play the regulation roles rather than contribute to glucose uptake directly. In conclusion, there is antagonistic regulation between insulin and AMPK signal pathways (Fig. 8). Insulin signal plays the predominant role in regulating glucose transport through PKCzeta-PKB axis and inhibits AMPK activity via PKCzeta. Berberine promotes glucose uptake through

Fig. 6. Roles of PKCzeta in regulating PKB and AMPK activities. L6 myc cells were plated in medium containing 2% (vol/vol) FBS at 104 cells/ml to allow spontaneous myoblast fusion. PKCzeta siRNAs or scrambled control was transfected into cells as described in Materials and Methods. 48 or 72 h later when cells are ready for experimentation, L6 myotubes were serum-starved for 3 h. (A) Cells were left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h. Then 20 μg whole cell lysis protein was subjected to 10% SDS-PAGE and immunoblotted with indicated antibodies. Actin bands were shown as internal marker. (B) Insulin and berberine-induced phosphorylation levels of PKCzeta, PKB, AS160, AMPK and ACC were quantified by densitometric scanning and analyzed with Bio-Rad Molecular Analysis software, and results were expressed as fold of changes relative to scrambled basal, a value of 1 was assigned to the scrambled basal. The data is representative of 3 independent experiments. Data are means ± SE, *P b 0.01 vs. corresponding scrambled Ins or BBR treatment by t-test. (C) Transfected cells were left untreated or treated with 100 nM insulin for 10 min or 5 μg/ml berberine for 1 h, and then glucose uptake was measured as described in Materials and Methods. Glucose uptake was expressed as fold of basal; a value of 1 was assigned to the scrambled basal. Data means ± SE of three experiments performed in triplicate.

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Fig. 8. Hypothetical model of the regulation role of PKCzeta and AMPK between insulin and berberine-induced signal transductions. Insulin activates PKCzeta and PKB through PI3 kinase. PKCzeta locates upstream of AMPK and mediates berberine-induced AMPK activation. Once activated, these two signal pathways may cause phosphorylation of AS160, leading to GLUT4 translocation to the plasma membrane and glucose uptake. PKCzeta antagonizes AMPK activity in basal and insulin-stimulated condition whereas AMPK suppresses PKB activity naturally or in the presence of berberine. Furthermore, insulin activated PKCzeta/PKB exhibits their roles on dorsal cell surface whereas berberine-activated PKCzeta/AMPK functions in the cytoplasm.

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