E3 Ubiquitin Ligase, WWP1, Interacts with AMPKα2 and Down-regulates Its Expression in Skeletal Muscle C2C12 Cells*

Background: The role of the ubiquitin-proteasome pathway under high glucose conditions is unclear. Results: AMPKα2 interacts with WWP1, and its expression is down-regulated by the ubiquitin proteasome pathway in high glucose culture conditions in C2C12 cells. Conclusion: WWP1 down-regulates AMPKα2 expression through direct interaction in high glucose culture conditions of skeletal muscle C2C12 cells. Significance: The ubiquitin proteasome pathway may involve high glucose-induced AMPKα2 down-regulation. It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.

It is known that the activity of AMP-activated protein kinase (AMPK␣2) was depressed under high glucose conditions. However, whether protein expression of AMPK␣2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPK␣2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPK␣2 downregulation. Endogenous AMPK␣2 ubiquitination was detected by immunoprecipitation of AMPK␣2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPK␣2-interacting protein in skeletal muscle cells. Interaction between AMPK␣2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucoseinduced AMPK␣2 down-regulation. The overexpression of WWP1 down-regulated AMPK␣2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPK␣2 was downregulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPK␣2 under high glucose culture conditions via the ubiquitin-proteasome pathway.
The metabolic syndrome is a combination of metabolic risk factors, including obesity, insulin resistance, hyperglycemia, and dyslipidemia, and it increases the risk of developing cardiovascular disease and diabetes. Maintaining energy balance depends on the efficiency of tightly regulated mechanisms of energy intake and expenditure. Hyperglycemia is a condition in which an excess amount of glucose circulates in the blood plasma. Glucose homeostasis is maintained by a balance between hepatic glucose production and glucose uptake by peripheral tissues. Activation of AMP-activated protein kinase (AMPK) 2 enhances glucose uptake through the translocation of the glucose transporter 4 (GLUT4) to the cell membrane and also through regulation of Glut4 gene expression (1)(2)(3). The AMPK activator enhances glucose uptake into muscle by increasing cell surface GLUT4 levels (4 -6). AMPK may regulate GLUT4 endocytosis (7,8) and GLUT4 translocation (9,10). Thus, AMPK is a well-accepted therapeutic target for the metabolic syndrome and type 2 diabetes (11,12).
AMPK is an enzyme that plays a role in cellular energy homeostasis. AMPK, a heterotrimeric complex comprised of a catalytic subunit and two regulatory subunits, is activated when cellular energy is depleted (13). Activated AMPK switches on ATP-generating catabolic pathways, including glucose and fatty acid oxidation (14 -16), while it simultaneously switches off ATP-consuming anabolic pathways (cholesterol, fatty acid, and triacylglycerol synthesis) (17). Thus, AMPK plays as a counter-regulatory mechanism and restores the AMP:ATP ratio. In addition to its roles in energy homeostasis, AMPK has also been shown to be regulated by ubiquitination (18). This fact suggests that the expression level of AMPK may play an important role in in specific biological conditions; however, the molecular link by which ubiquitination acts in high glucose conditions is obscure. It is reported that elevated glucose downregulated the activity of AMPK in pancreatic cells (19) and rat skeletal muscle (20).
Protein ubiquitination is a mechanism for targeting proteins for rapid proteasomal degradation (21). Ubiquitin is attached covalently to target proteins in a series of reactions driven by ubiquitin ligase. E1 ligases load ubiquitin onto E2 ligases, which in turn modify target proteins. Substrate specificity is generated by E3 ligases, which serve as adaptors. Recent studies demon-* This study was supported by the National Research Foundation of Korea strated that the ubiquitin proteasome system may regulate AMPK activity. The de-ubiquitination enzyme USP9X interacts with NUAK1 (AMPK-related kinase 5) (18), implicating a role of AMPK in ubiquitination. This group identified Cidea (cell death-inducing DFFA-like effector a) as an E3 ubiquitin ligase for AMPK (22). Although the basic regulation of AMPK activity by kinases has been delineated, regulation by posttranslational modifications such as ubiquitination remains to be determined.
In this study, we found that AMPK␣2 directly interacted with E3 ubiquitin ligase WWP1. We also showed that the expression level of AMPK␣2 was down-regulated in C2C12 cells cultured under high glucose conditions, and further demonstrated that the ubiquitination is involved in high glucose-induced AMPK␣2 down-regulation. These findings provide novel insights into the manner in which high glucose contributes to insulin resistance through down-regulation of AMPK␣2 in skeletal muscle cells.
Cell Culture and Differentiation-Mouse myoblast C2C12 and human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in an incubator with 5% CO 2 . Cells were grown in culture medium consisting of 500 l of DMEM (GIBCO TM , Auckland, NZ) containing 0.584 g/liter of L-glutamate and 4.5 g/liter of glucose, and mixed with 500 ml of F-12 medium containing 0.146 g/liter of L-glutamate, 1.8 g/liter of glucose, 100 g/ml of gentamicin, 2.5 g/liter of sodium carbonate, and 10% heat-inactivated FBS. L6 myoblasts were maintained in cell monolayers in ␣-MEM supplemented with 10% FBS, 50 units of penicillin per ml, and 50 g of streptomycin per ml in a 5% CO 2 humidified atmosphere at 37°C. Two days after the myoblasts achieved confluence, differentiation to myotubes was induced by incubating the cells for 6ϳ7 days in ␣-MEM supplemented with 2% FBS, which was changed every 2 days. The monolayer myotubes were finally serum-starved in DMEM for 2 h, then used in the following experiments.
Animal Tissue-Male C57BLKS/J-db/db mice and wild-type C57BL/6J mice were obtained from the Central Lab. Animal Inc. (Seoul, Korea) at age 6 weeks. All mice were maintained under standard light (12 hours light/dark), temperature (22 Ϯ 2°C), and humidity (40 Ϯ 1 0%) conditions. The mice were anesthetized with dry ice, and quadriceps muscle was prepared. All procedures were approved by the Institutional Animal Care and Utilization Committee for Medical Science of Korea University.
Immunoblot Analysis-Cells were grown on 6-well plates and serum-starved for 24 h prior to treatment with the indicated agents. Following treatment of the cells, the media was aspirated, and the cells were washed twice in ice-cold PBS and lysed in 100 l of lysis buffer. The samples were then briefly sonicated, heated for 5 min at 95°C, and centrifuged for 5 min. The supernatants were electrophoresed on SDS-PAGE (8%) gels, and transferred to polyvinylidene difluoride membranes. The blots were incubated overnight at room temperature with primary antibodies and then washed six times in Tris-buffered saline/0.1% Tween 20 prior to 1 h incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. The blots were then visualized via ECL (Amersham Biosciences, Buckinghamshire, UK). In some cases, the blots were stripped and reprobed using other antibodies.
Immunoprecipitation-The amount of proteins from C212 cells or Flag-WWP1-transfected HEK293 cells was determined by the Bradford method. Cellular protein (100 g) was mixed with 1 g of anti-Flag or anti-AMPK antibodies and incubated overnight at 4°C. Then, 10 l of protein A-Sepharose (Amersham, Uppsala, Sweden) was added to these samples and incubated for another 3 h at 4°C. After the incubation, samples were washed three times with wash buffer (25 mM HEPES, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride, 1 M leupeptin, 1 M pepstatin, and 1 M aprotinin A, pH 7.2). The washed samples were resuspended in SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% [v/v] glycerol, 4% [w/v] SDS, 100 mM dithiothreitol, and 0.1% [w/v] bromphenol blue), and heated at 100°C for 5 min prior to electrophoresis.
Silencing AMPK␣2 and WWP1-C2C12 cells were seeded in 6-well plates and allowed to grow to 70% confluence for 24 h. Transient transfections were performed with transfection reagent (Lipofectamine 2000; Invitrogen) according to the manufacturer's protocol. Briefly, both AMPK␣2 siRNA (NM_001013367; Dharmacon, CO), WWP1 siRNA (Genolution Pharmaceuticals, Inc.) and non-targeted control siRNAs (Non-targeting pool; Dharmacon CO) were designed. 5 l of siRNA and 5 l of transfection reagent (Lipofectamine 2000) were each diluted with 95 l of reduced serum media (Opti-MEM; Invitrogen), then mixed. The mixtures were allowed to incubate for 30 min at room temperature and then were added dropwise to each culture well containing 800 l of reduced serum media (Opti-MEM; final siRNA concentration, 100 nM). Four hours after transfection, the medium was changed with fresh complete medium. The cells were cultivated for 24 h and lysed, and expression of AMPK␣2 protein was assayed with Western blotting.
Immunohistochemistry-Seven micrometer-thick cryosections from db/db and control mouse quadriceps femoris muscle were fixed with 4% freshly depolymerized paraformaldehyde for 15 min at 4°C. After blocking with normal goat serum, sections were immunostained with antibodies against AMPK␣ for overnight at room temperature. After extensive washing with phosphate-buffered saline (PBS), sections were incubated with FITC-labeled secondary antibody.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-
First-strand cDNA synthesis was performed using 1 g of total RNA isolated from C2C12 at 55°C for 20 min using the Thermoscript II one-step RT-PCR Kit (Invitrogen, Paisly, UK). Amplification of cDNA was carried out in the same tube using the Gene Amp System 9700 thermocycler (Applied Biosystems, Warrington, UK). Heating to 94°C for 5 min inactivated the reverse transcriptase. The following PCR conditions were used: 27 cycles of 30 s at 94°C, 30 s at 56°C, and 30 s at 72°C, followed by 7 min at 72°C. The number of PCR cycles used was optimized to ensure amplification at the exponential phase. 10-l samples from each RT-PCR product were removed and analyzed by agarose gel electrophoresis. Bands were stained with ethidium bromide and visualized under ultraviolet light. Band intensity quantification was determined by a gel documentation system (Gene Genius, Syngene, UK). The following primers were used: WWP1-sense (5Ј-TCC TGG TGA CGG AAG AAA AC-3Ј) and WWP1-antisense (5Ј-AAC CAG GTG TCT TTG CCA AC-3Ј); and AMPK␣2-sense (5Ј-CGC CTC TAG TCC TCC ATC AG-3Ј) and AMPK␣2-antisense (5Ј-CAG CTG TGC TGG AAT CAA AA-3Ј); and ␤-actin-sense (5Ј-CAG GAG GAG CAA TGA TCT TGA-3Ј) and ␤-actin antisense (5Ј-ACT ACC TCA TGA AGA TCC TCA-3Ј).
GST Pull-down Assay-GST fusion proteins containing different domains of AMPK␣2 were engineered by PCR. The fusion proteins were expressed in Escherichia coli bacteria by incubation with 0.2 mm isopropyl ␤-D-galactopyranoside for 6 h at 37°C. The bacteria were lysed by sonication, and the fusion proteins were purified with glutathione-agarose beads with buffer A followed by immunoblotting with anti-WWP1 antibody.
Glucose Uptake-Glucose uptake was measured using the 2-deoxy-D-[ 3 H]glucose. Briefly, L6 cells were serum-starved overnight before each experiment, and glucose uptake was measured on day 7. Medium was replaced in the morning (serum-free medium), and cells were exposed to drugs for 1 h. Cells were washed twice in warm PBS before media and drugs were placed in DMEM devoid of glucose for 20 min. 2-Deoxy-D-[ 3 H]glucose (50 nM) was added for 15 min at 37°C, and the reactions were terminated by washing twice in ice-cold PBS. Cells were lysed in 10% SDS or 50 mM NaOH, and samples were transferred to scintillation vials with scintillant and allowed to sit at room temperature for 1 h before being counted.
Plasmid Construction of Flag-wwp1-The open reading frames of human WWP1 was cloned into the Pcmv-Tag2C vector (Stratagene) between EcoRI and SalI sites. cDNA from Hela cell line was amplified by PCR (forward primer, 5Ј-C GGA ATT CGA ATG GCC ACT GCT TCA CCA AG-3Ј; reverse primer 5Ј-A CGC GTC GAC TCA TTC TTG TCC AAA TCC CTC TG-3Ј). Kpn1-and Sac1-digested products were ligated into a linearized pCMV-Tag2C vector. The construct was verified by direct sequencing.
Data Analysis-Data are expressed as the means Ϯ S.E. Image Gauge (version 3.12; Fujifilm, Tokyo, Japan) was used for analysis of band intensity. One-way ANOVA was used followed by a Holm-Sidak multiple-range test for comparison between groups. p values Ͻ0.05 were considered statistically significant.

AMPK␣2 Down-regulated in C2C12 Cells Cultured under
High Glucose Conditions-High glucose concentrations are known to have detrimental effects on many cells types. To gain insight into the effect of high glucose conditions on skeletal muscle system, we evaluated the effect of high glucose concentration on the expression of AMPK␣2 in skeletal muscle C2C12 cells. The expression of AMPK␣2 significantly down regulated in high glucose concentration in cell culture medium. Mannitol is used as the isotonic control. L-Glucose is used as the D-glucose specificity control. The level of AMPK␣2 was not affected by these two agents, suggesting that AMPK␣2 down-regulation is D-glucose-specific (Fig. 1A). We also assessed the effects of high concentrations of glucose in time dependence. Exposure to 30 mM concentration of glucose for 3 days dramatically down-regulated AMPK␣2 expression (Fig. 1B). Together, these results demonstrate that high glucose conditions down-regulates AMPK␣2 expression in skeletal muscle cells.
Proteasome Degradation Pathway Is Involved in High Glucose-induced AMPK␣2 Degradation-To check the involvement of proteasome degradation in AMPK␣2 down-regulation, we analyzed the effect of proteasome inhibition on AMPK␣2 expression. MG132, a proteasome inhibitor, blocked high glucose-induced AMPK␣2 down-regulation ( Fig. 2A). This effect was also observed by another proteasome inhibitor, lactacystin (Fig. 2B), suggesting the involvement of the ubiquitin-proteasome pathway. Next, we tested whether AMPK␣2 was ubiquitinated in cells. We immunoprecipitated AMPK␣2 from cell lysates, and then analyzed with antibody against ubiquitin. As can be seen, the multi-ubiquitination of AMPK␣2 was detected in the immunoprecipitated sample (Fig. 2C). These results demonstrate that proteasome pathway is involved in high glucose-mediated AMPK␣2 down-regulation.
AMPK␣2 Interacts with WWP1-To explain the mechanism of high glucose-mediated AMPK␣2 down-regulation, we tried to identify AMPK␣2-interacting protein. To identify protein  FEBRUARY 15, 2013 • VOLUME 288 • NUMBER 7 that interacts with the catalytic domain of AMPK␣2, we carried out a YTH screen of a skeletal muscle C2C12 cDNA library. In our results, WWP1, a E3 ubiquitin ligase, was identified as an AMPK␣2-interacting protein. To determine whether WWP1 interacts with AMPK␣2 in mammalian cells, Flag-AMPK␣2 was transfected into HEK293 cells. Lysates from transfected cells were immunoprecipitated with antibody against Flag. A band corresponding to WWP1 was detected in the immunoprecipitates, demonstrating interaction (Fig. 3A). Moreover, to assess in vivo interaction, we performed immunoprecipitation of skeletal muscle C2C12 cell protein using an AMPK␣2 antibody, followed by Western blotting with an antibody to WWP1. AMPK␣2 was co-immunoprecipitated with WWP1 from endogenous C2C12 cells (Fig. 3B). Immunoprecipitation of Flag-WWP1-transfected HEK293 cells using a Flag antibody showed that AMPK␣2 was co-immunoprecipitated with WWP1 (Fig. 3C). These results demonstrated that the interaction detected in the YTH also occurs in vivo and in vitro.

WWP1 Interacts with AMPK␣2 and Down-regulates Its Expression
Expression Level of AMPK␣2 Is Regulated by Expression Status of WWP1-To investigate the relationship between WWP1 and AMPK␣2, we first examined the effect of WWP1 knockdown on high glucose-induced AMPK␣2 down-regulation. High glucose-induced down-regulation of AMPK␣2 was blocked in WWP1 knockdown conditions. However, AMPK␣2 down-regulation was apparent in scramble siRNA, implying that WWP1 is important to down-regulate AMPK␣2 (Fig. 4A). Interestingly, the anti-WWP1 antibody recognized two protein bands in the C2C12 cell lysates. The intensity of the two bands changed in similar patterns, indicating that WWP1 may be recognized by both protein bands. Next, to further study the function of WWP1, we transiently transfected with Flag-WWP1. The level of AMPK␣2 was down-regulated in cells in which WWP1 was overexpressed. This down-regulation was blocked by treatment with proteasome inhibitor (Fig. 4B), suggesting that ubiquitin-proteasome pathway is critical for AMPK␣2 down-regulation. To determine the interaction domain, we constructed GST-fused whole AMPK␣2, C-terminal AMPK␣2, and N-terminal AMPK␣2. We incubated GST fusion proteins with cell lysates from C2C12 cells. Targeted AMPK␣2 proteins (asterisk) were detected by Coomassie Blue staining. Immunoblotting with anti-WWP1 antibody showed that C-terminal AMPK␣2 interacted with WWP1 (Fig. 4C). Interaction between AMPK␣2 and WWP1 was enhanced by high glucose incubation (Fig. 4D). Together, these results indicate that WWP1 play an important role in AMPK␣2 down-regulation in high glucose culture conditions. WWP1 Increased in High Glucose Conditions-To gain insight into the role of WWP1 in high glucose culture conditions, we examined the effect of high glucose concentrations on expression of WWP1 both in protein and mRNA levels. When cells were cultured with high concentrations of glucose, Western blot analysis showed that the level of WWP1 protein expression increased in dose-dependently (Fig. 5A). In experiments with RT-PCR, mRNA levels of WWP1 were also increased by incubation of C2C12 cells under a 30 mM concentration glucose condition (Fig.  5B). Together, these results suggest that WWP1 may be regulated by glucose concentration status.
AMPK␣2 Decreased in db/db Mouse-To examine the effect of high glucose on AMPK␣2 in vivo, we used the type 2 diabetes animal model, db/db mouse. Proteins of the quadriceps from wild type and db/db mice were prepared. Each group had 5 mice. The non-fasting blood glucose levels in db/db mice were significantly higher than those of wild type mice (data not shown). Insulin levels of db/db mice were also increased (data not shown). Hyperinsulinemic-hyperglycemia of db/db mice indicates that these animals show insulin resistance, a primary characteristic of type 2 diabetes. The expression of AMPK␣2 was down-regulated in quadriceps muscle of these mice (Fig.  6A). Furthermore, the level of AMPK␣2 mRNA was also downregulated (Fig. 6B). To confirm whether AMPK␣2 is regulated at the transcriptional level, we performed RT-PCR experiments in C2C12 cells. The level of AMPK␣2 mRNA was slightly down-regulated at 12 h after incubation with high glucose con-  centration, suggesting that AMPK␣2 may be regulated by transcriptional level (Fig. 6C). Immunostaining of db/db mouse and wild type mouse identified AMPK␣2 in quadriceps muscle. In hyperglycemic db/db mouse, the level of AMPK␣2 was downregulated compared with euglycemic wild type mouse. (Fig.  6D). The results from the diabetic animal model were correlated with biochemical data. These results indicate that the WWP1 may play a key function in AMPK␣2 down-regulation under high glucose conditions.
Expression of WWP1 Suppressed the Metformin-induced Glucose Uptake-Finally, to provide physiological relevance of WWP1, we investigated the effect of WWP1 expression on glu-cose uptake. Metformin-induced glucose uptake was blocked in WWP1 overexpression (Fig. 7A). To gain insight into the role of high glucose-mediated AMPK␣2 down-regulation, we examined the effect of metformin on glucose uptake under high glucose culture condition. Attenuation of glucose uptake under high glucose culture conditions was recovered by treatment with metformin, a first choice for the treatment of type 2 diabetes (Fig. 7B). These results indicated that expression of WWP1 is important for metformin-mediated glucose uptake through regulating AMPK␣2 expression.

DISCUSSION
The principal finding of this study was that WWP1 is involved in high glucose-induced AMPK␣2 down-regulation. Specifically, we demonstrated that direct interaction between AMPK␣2 and WWP1 is instrumental in AMPK␣2 down-regulation.
The anti-diabetic role of AMPK has previously been evaluated in conjunction with its activity. Dysfunction of AMPK activity may lead to metabolic syndrome. There is correlation between the low activation state of AMPK with metabolic disorders associated with insulin resistance and obesity (23,24). AMPK activity is also suppressed in muscle and liver by sustained hyperglycemia (25). Moreover, many agents that are useful in treating diabetes, including TZDs (26,27), metformin (28) have been shown to stimulate AMPK activity. The contribution of AMPK activity to the anti-diabetic role has raised questions as to which aspect of the protein expression of AMPK may be relevant to its metabolic role. In the present study, we have established that high glucose culture conditions down-regu- The anti-␤-actin antibody was a protein loading control. C, C2C12 cells were prepared and incubated with GST fusion proteins of various AMPK␣2. Formed protein complexes were isolated by glutathione beads, washed three times with washing buffer, and analyzed by SDS/PAGE and subsequent Coomassie staining. The pull-down sample was also immunoblotted with anti-WWP1 antibody. D, C2C12 cells were exposed to 30 mM glucose incubation for 48 h. The cell lysates (1 mg) of normal and high glucose conditions C2C12 cells were immunoprecipitated with anti-AMPK␣2 antibody. The immunoprecipitates were analyzed via Western blotting for anti-WWP1 and AMPK␣2 antibodies. were analyzed via Western blotting for anti-WWP1 antibody. The anti-␤-actin antibody was a protein loading control. B, total mRNA was prepared for these cells after 30 mM glucose incubation, and RT-PCR was conducted using specific WWP1 primers. The PCR product was then gel-run in 1% agarose, and visualized in UV. ␤-Actin was employed as a positive control. FEBRUARY 15, 2013 • VOLUME 288 • NUMBER 7 lated AMPK expression. Additionally, we have demonstrated that WWP1 down-regulated AMPK␣2 through direct interaction. The relationship of AMPK with ubiquitination has been suggested. Collectively, our results indicate that AMPK may down-regulate in high glucose culture conditions via the ubiquitination pathway.

WWP1 Interacts with AMPK␣2 and Down-regulates Its Expression
The main goal of the present study was to ascertain whether or not AMPK␣2 is directly regulated by high glucose, and if so, to determine which mechanisms are involved in this process. Our data have revealed a novel role for WWP1 of AMPK␣2 down-regulation in high glucose conditions. Our identification of an AMPK␣2-WWP1 interaction in high glucose-treated skeletal muscle cells led us to hypothesize that WWP1 might play a role in the high glucose-mediated down-regulation of AMPK␣2. Indeed, this is the first report of a link between AMPK␣2 and WWP1 in skeletal muscle cells. It is thus tempting to speculate that WWP1-mediated regulation of AMPK␣2 plays a role in diabetes, since AMPK␣2 expression has been implicated in insulin resistance. Overall, although the mechanism by which high glucose influences AMPK␣2 remains unknown, the findings in this report suggest that high glucose may down-regulate AMPK␣2 expression through the WWP1 as part of the process of high glucose-mediated insulin resistance. In the present study, we further demonstrated that high FIGURE 6. AMPK␣2 down-regulated in db/db mouse. A, Western blot analysis of quadriceps muscles of wild type and db/db mice. The tissue lysates (40 g) were analyzed via Western blotting for anti-AMPK␣2 antibody. The anti-␤-actin antibody was a protein loading control. Densitometry analysis (n ϭ 5) of values are means Ϯ S.E. values of the ratios of densities (AMPK␣2/␤-actin). *, p Ͻ 0.05 versus wild type mouse. B, RT-PCR analysis of quadriceps muscles of wild type and db/db mice. Total mRNA was prepared for these mice, and RT-PCR was conducted using specific AMPK␣2 primers. The PCR product was then gel-run in 2% agarose, and visualized in UV. ␤-Actin RNA was employed as a positive control. Densitometry analysis (n ϭ 5) of values are means Ϯ S.E. values of the ratios of densities (AMPK␣2 mRNA/␤-actin mRNA). *, p Ͻ 0.05 versus wild type mouse. C, total mRNA was prepared for these cells after 30 mM glucose incubation, and RT-PCR was conducted using specific AMPK␣2 primers. The PCR product was run in 1% agarose gel, and visualized in UV. ␤-Actin was employed as a positive control. D, histochemistry of the quadriceps muscle of wild type and db/db mice. Each cryosection was stained with anti-AMPK␣2 antibody. glucose induces expression of WWP1, and overexpression of WWP1 decreased AMPK␣2 expression. In addition, wild type WWP1 blocks metformin-mediated GLUT4 translocation. These results demonstrate that WWP1 has an important role in glucose homeostasis through regulating AMPK␣2 expression. Regulation of AMPK by ubiquitination has been suggested (29,30). The AMPK kinases NUAK1 and MARK4 are polyubiquitinated, and expression of NUAK and MARK4 mutants did not interact with the de-ubiquitinating enzyme USP9X de-ubiquitinating enzyme (DUB), thus resulting in AMPK activity (18). This result implies an important role for de-ubiquitination in AMPK activity regulation and further raises the question of what E3 ubiquitin ligases are involved. Because E3 ubiquitin ligases function to recognize specific substrates and to transfer ubiquitin to target protein, the specificity of the ubiquitin process is found in the E3 ubiquitin ligases. In the present study, we found an interaction of AMPK␣2 with E3 ubiquitin ligase WWP1. This interaction is presumably required for high glucose-mediated AMPK␣2 down-regulation. Combined with the recent report of interaction between proteasome itself and AMPK (31), protein-protein interaction has become a model for ubiquitin-mediated AMPK regulation.
On the basis of our present results, we propose a novel mechanism to explain how the expression of AMPK is down-regulated in high glucose culture conditions. This study revealed that AMPK␣2 is down-regulated in hyperglycemic animal db/db mice, while its interaction partner WWP1 is up-regulated. In conclusion, WWP1 appears to be involved in insulin resistance through suppressing AMPK␣2 expression. In conclusion, the present study supports the hypothesis that WWP1 may have an important role in hyperglycemic situations, such as diabetes and insulin resistance, through direct interaction with AMPK␣2 and down-regulation of its expression.