Inhibition of C1-Ten PTPase activity reduces insulin resistance through IRS-1 and AMPK pathways

Insulin resistance causes type 2 diabetes; therefore, increasing insulin sensitivity is a therapeutic approach against type 2 diabetes. Activating AMP-activated protein kinase (AMPK) is an effective approach for treating diabetes, and reduced insulin receptor substrate-1 (IRS-1) protein levels have been suggested as a molecular mechanism causing insulin resistance. Thus, dual targeting of AMPK and IRS-1 might provide an ideal way to treat diabetes. We found that 15,16-dihydrotanshinone I (DHTS), as a C1-Ten protein tyrosine phosphatase inhibitor, increased IRS-1 stability, improved glucose tolerance and reduced muscle atrophy. Identification of DHTS as a C1-Ten inhibitor revealed a new function of C1-Ten in AMPK inhibition, possibly through regulation of IRS-1. These findings suggest that C1-Ten inhibition by DHTS could provide a novel therapeutic strategy for insulin resistance-associated metabolic syndrome through dual targeting of IRS-1 and AMPK.


Results
Identification of DHTS as an inhibitor of C1-Ten PTPase. To identify chemical inhibitors for C1-Ten PTPase, we employed an in vitro screening system. Active C1-Ten protein was purified from stable FresStyle293-F cells expressing 3 × FLAG tagged-C1-Ten (full-length, wild-type C1-Ten; Supplementary Fig. S1A). Next, we screened a natural product library (502 compounds) for their ability to inhibit C1-Ten. Seven compounds -15,16-dihydrotanshinone I (DHTS), tanshinone IIA, cryptotanshinone, β-lapachone, shikonin, plumbagin, and streptonigrin -showed inhibitory effects against C1-Ten PTPase activity (Fig. 1A). Interestingly, all of these compounds are naphthoquinone derivatives, with 1,2-naphthoquinone derivatives exhibiting more effective inhibition of C1-Ten PTPase activity than 1,4-naphthoquinone (Fig. 1A, Supplementary Fig. S1B). As DHTS showed the most potent inhibitory effect, the compound was selected for further study. The IC 50 value of DHTS was 4.3 µM (Fig. 1B). PTPases are readily inactivated by reactive oxygen species (ROS) through the oxidation of catalytic cysteine 12 . To investigate whether DHTS inhibits C1-Ten through ROS generation, we cotreated L6 myoblasts with N-acetyl-L-cysteine (NAC) and DHTS (Fig. 1C). Both ROS-generating agents (H 2 O 2 and Menadione) and DHTS strongly induced IRS-1 tyrosine phosphorylation. Although NAC treatment prevented H 2 O 2 and Menadione-induced IRS-1 tyrosine phosphorylation, DHTS-induced IRS-1 tyrosine phosphorylation remained in the presence of NAC. Next, we measured the intracellular ROS levels in cells using flow cytometry (Fig. 1D). DHTS did not generate ROS, excluding the possibility of ROS involvement in DHTS-mediated C1-Ten inhibition. Instead, we observed that DHTS inhibited the interaction between C1-Ten and IRS-1 ( Supplementary  Fig. S1C). Therefore, we conclude that DHTS inhibits C1-Ten PTPase activity in a ROS-independent manner. DHTS improves diabetic phenotypes in db/db mice. To validate the functional effect of DHTS on C1-Ten, we first examined whether DHTS could improve pathological phenotypes in type 2 diabetes. Insulin resistance impairs glucose tolerance and induces muscle atrophy in type 2 diabetes, and Akt activation is often impaired in insulin-resistant muscle 13,14 . Consistent with previous findings 3 , C1-Ten levels were higher in the skeletal muscle of db/db mice, and Akt phosphorylation levels were lower in these tissues than in those from control (db/m+) mice ( Fig. 2A). Administration of DHTS in db/db mice restored Akt phosphorylation to the levels observed in control mice ( Fig. 2A), and improved oral glucose tolerance (Fig. 2B). Akt activation is important for glucose disposal in skeletal muscle; dexamethasone has been shown to induce insulin resistance in skeletal muscle by upregulating C1-Ten and decreasing Akt activation ( Supplementary Fig. S2A) 3 . To validate the role of DHTS in glucose use of muscle cells, glucose uptake was measured in L6 myotubes under dexamethasone-induced insulin-resistant conditions. DHTS increased glucose uptake in a dose-and time-dependent manner in differentiated L6 myotubes (Fig. 2C, Supplementary Fig. S2B). Furthermore, we used a homogeneous population of differentiated myocytes derived from primary prepared EDL myoblasts to investigate whether DHTS stimulates glucose uptake in the primary cells as in the cell lines. Incubation of primary myocytes from EDL muscles with DHTS stimulated glucose uptake ( Supplementary Fig. S2C).
Next, we assessed the effects of DHTS on muscle atrophy in db/db mice. DHTS treatment caused a 30% increase in the mean cross-sectional area and a rightward shift in the distribution of myofiber cross-sectional area in db/db mice (Fig. 2D). Upregulated C1-Ten causes muscle atrophy by activating FoxO, thus inducing a high-fidelity marker of muscle atrophy, muscle-specific E3 ligase (muscle RING finger 1 [MuRF-1]) 3 . We investigated whether DHTS could prevent MuRF-1 induction by inhibiting FoxO (inducing FoxO phosphorylation). The results show that DHTS induced FoxO phosphorylation and prevented MuRF-1 expression in L6 myotubes, despite the dexamethasone-induced increase in C1-Ten (Fig. 2E), suggesting that DHTS blocks the action of C1-Ten. Taken together, we demonstrated that DHTS improves glucose metabolism and muscle atrophy in the insulin-resistant state using in vitro and in vivo models. DHTS increases IRS-1 stability. C1-Ten reduces IRS-1 protein levels via its PTPase activity 3 . Therefore, a potential mechanism for the observed improvement in diabetic phenotypes in this study might be that DHTS increased IRS-1 levels. IRS-1 is a key protein that transduces insulin signals in skeletal muscle and white adipose tissue (WAT) 15 . We revealed that both of these tissues from db/db mice had relatively low IRS-1 protein levels but relatively high C1-Ten levels (Fig. 3A,B), supporting the notion that the mechanism of insulin resistance is controlled at the point of IRS-1 levels. DHTS treatment significantly restored IRS-1 levels without affecting C1-Ten levels in skeletal muscle (Fig. 3A) and WAT from db/db mice (Fig. 3B). To verify whether DHTS can regulate IRS-1 protein stability through C1-Ten inhibition, we treated L6 myoblasts and myotubes with DHTS after silencing C1-Ten. As in vivo, DHTS increased IRS-1 protein levels, phenocopying the C1-Ten knockdown effects; however, DHTS did not further increase IRS-1 levels in the absence of C1-Ten in myoblasts (Fig. 3C) and myotubes ( Supplementary Fig. S3A). These results indicate that DHTS regulates IRS-1 levels via C1-Ten, supporting the role of DHTS in the direct inhibition of C1-Ten activity in vitro.
To characterize DHTS in IRS-1/Akt signaling further, we induced insulin resistance in L6 myotubes by exposing them to dexamethasone for 24 h. As reported previously 3,16 , dexamethasone reduced IRS-1 protein levels. However, this decrease in IRS-1 levels was blocked by DHTS incubation without altering Irs-1 mRNA levels ( Fig. 3D,E, Supplementary Fig. S3B), suggesting that DHTS affects IRS-1 protein stability. Increased IRS-1 protein levels were sustained up to 4 h (Fig. 3E). As IRS-1 is upstream of Akt, DHTS also enhanced Akt phosphorylation (Fig. 3D,E), consistent with DHTS-induced Akt activation in the skeletal muscle of db/db mice ( Fig. 2A). Previously, we reported that C1-Ten-mediated IRS-1 Y612 dephosphorylation reduced IRS-1 stability. We used L6 myoblasts because myoblasts display a slower rate of IRS-1 turnover than myotubes, thereby providing the opportunity to monitor phosphorylation before its degradation occurred 3 . DHTS potentiated IRS-1 Y612 phosphorylation in a DHTS dose-dependent manner with increased Akt activation in L6 myoblasts (Fig. 3F). A general PTPase inhibitor, sodium orthovanadate (NaV), strongly potentiated insulin receptor tyrosine phosphorylation without increasing IRS-1 protein levels; however, DHTS exhibited stronger effects toward IRS-1 protein levels  with almost no effect on insulin receptor tyrosine phosphorylation (Fig. 3G), implying that the DHTS-mediated increase in IRS-1 signaling was not mediated through general PTPase inhibition. These results demonstrate that DHTS restores IRS-1 stability and its downstream Akt signaling via inhibition of C1-Ten PTPase activity through a mechanism that differs from the action of a general PTPase inhibitor.

DHTS targets both IRS-1 and AMPK pathways.
We verified the potential antidiabetic property of the C1-Ten inhibitor, DHTS. Metformin is the most widely used first-line antidiabetic drug, which prevents insulin resistance by targeting the AMPK pathway 17,18 . In addition, pharmacological activation of AMPK can potentiate insulin signaling via PI3K/Akt activation 19 . Therefore, we compared the effects on insulin signaling of AMPK agonists (metformin or 5-aminoimidazole-4-carboxamide ribonucleotide, AICAR) and DHTS. As expected,  AICAR-or metformin-induced AMPK activation increased Akt phosphorylation, but to a lesser extent than did DHTS under dexamethasone-induced insulin-resistant conditions (Fig. 4A). Surprisingly, DHTS induced AMPK activation comparable to the levels achieved by AICAR or metformin (Fig. 4A). Next, we examined the involvement of AMPK upstream kinases such as liver kinase B1 (LKB1) or Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) in DHTS-mediated AMPK activation. First, we used HeLa cells, which do not express LKB1 protein (Supplementary Fig. S4A). DHTS-induced AMPK phosphorylation was also observed in HeLa cells. Next, cotreatment with CaMKK inhibitor STO-609 did not block DHTS-induced AMPK phosphorylation ( Supplementary Fig. S4B). These results suggest that DHTS-mediated AMPK activation occurs in an LKB1and CaMKK-independent manner. Instead of well-known AMPK upstream kinases, a novel pathway might be involved in this process. DHTS indeed increased AMPK phosphorylation in L6 myotubes in a dose-dependent manner (Fig. 4B). Moreover, DHTS administration increased AMPK activation in the skeletal muscle of db/db mice in vivo (Fig. 4C), as well as in isolated skeletal muscle of wild-type mice ex vivo (Fig. 4D, Supplementary  Fig. S4C). In contrast, the AMPK agonist itself did not increase IRS-1 levels (Fig. 4A). These data indicate that AMPK activation per se is not the cause of enhanced IRS-1 stability by DHTS. To verify this, we incubated L6 myotubes with DHTS in the presence of the AMPK inhibitor, compound C. Although compound C efficiently blocked DHTS-induced AMPK activation, it did not inhibit the DHTS-mediated increase in IRS-1 levels and Akt phosphorylation (Fig. 4E), suggesting that DHTS restored the IRS-1/Akt pathway in an AMPK-independent manner.
Since DHTS mediated the activation of AMPK, we used siRNA knockdown of C1-Ten to test whether C1-Ten could inhibit AMPK. We observed AMPK activation in L6 myotubes expressing C1-Ten siRNA (Fig. 4F). Conversely, C1-Ten overexpression downregulated AMPK phosphorylation (Fig. 4G). Additionally, C1-Ten overexpression in HEK293 cells led to inhibition of the AMPK pathway and insulin-induced Akt activation (Fig. 4H). These results suggest that DHTS sensitizes insulin signaling by increasing IRS-1 stability and activating AMPK through C1-Ten inhibition.

IRS-1 Y612 regulates both Akt and AMPK pathways. Knowing that DHTS-induced AMPK activation
is not upstream of IRS-1/Akt signaling, we investigated whether IRS-1 Y612 phosphorylation could affect AMPK activation. Previously, we reported that the IRS-1 Y612F mutant mimics the effect of C1-Ten overexpression in HEK293 cells 3 . IRS-1 Y612F overexpression inhibited not only Akt activation but also AMPK activation (Fig. 5A), indicating that modulation of IRS-1 phosphorylation status itself is capable of regulating AMPK activity. IRS-1 Y612 is critical for PI3K activity 20 ; thus, to determine whether PI3K activity is involved in modulating AMPK activation, myotubes were incubated with DHTS in the presence of the PI3K inhibitor, LY294002. LY294002 efficiently blocked DHTS-induced Akt activation without altering AMPK phosphorylation or IRS-1 protein levels (Fig. 5B), indicating that DHTS-induced AMPK activation is independent of DHTS-induced PI3K/Akt activation. Taken together, IRS-1 phosphorylation at the Y612 site is critical in both Akt activation and AMPK activation, emphasizing the roles of C1-Ten and its inhibitor, DHTS, in insulin resistance (Fig. 5C).

Discussion
In this study, we identified DHTS as a C1-Ten PTPase inhibitor that improves glucose tolerance and muscle atrophy in db/db mice. DHTS increased IRS-1 stability, which activates Akt via C1-Ten inhibition. This identification of DHTS as a C1-Ten inhibitor revealed a novel role of C1-Ten in AMPK inhibition via IRS-1 Y612 dephosphorylation. Therefore, inhibition of C1-Ten-mediated IRS-1 degradation suggests a novel therapeutic strategy for insulin-resistance-associated metabolic syndrome via dual targeting of IRS-1/Akt and IRS-1/AMPK.
PTPases are highly vulnerable to oxidative and electrophilic inactivation due to the low pKa of their catalytic cysteines 12 . Reactive organic compounds, such as quinones, can generate ROS or directly impair PTPase activity either by thiolate oxidation or covalent modification of catalytic and regulatory residues. However, NAC treatment did not block DHTS-induced IRS-1 tyrosine phosphorylation (Fig. 1C), and DHTS increased IRS-1 tyrosine phosphorylation without ROS generation (Fig. 1D). Accordingly, the DHTS-induced C1-Ten inhibition was independent of ROS. Instead, we observed that DHTS inhibited the interaction between C1-Ten and IRS-1 in vitro ( Supplementary Fig. S1C). In the case of PTP1B, 1,2-naphthoquinone (1,2-NQ) inactivates PTP1B through covalent attachment to the reactive cysteine in the active site 21 . DHTS may share a similar inhibition mechanism with 1,2-NQ since DHTS is a derivative of naphthoquinone. It is likely that covalent modification of DHTS would interrupt its accessibility to substrates. Although all of the active compounds identified in the inhibitor screening were naphthoquinone derivatives (Fig. 1A), the inhibitory activities of the compounds were different. In addition, DHTS has more propensity towards IRS-1, in contrast to general PTPase inhibitors, sodium orthovanadate, or PTP1B inhibitors with strong effects on insulin receptor phosphorylation (Fig. 3G).
According to the NCBI Gene Expression Omnibus (GEO) database, C1-Ten mRNA levels are upregulated in human skeletal muscle in insulin-resistant individuals (GDS ID: GDS3715), which is consistent with the results in diabetic mice 3 . However, in-depth analysis of C1-Ten expression levels in subjects with diabetes or other pathological conditions is still needed to understand the role of C1-Ten in those conditions. How C1-Ten expression is phosphorylation in L6 myoblasts. Indicated concentrations of DHTS were administered to L6 myoblasts for 1 h at 48 h post-seeding. Data are presented as the mean ± SEM (n = 4); **P < 0.01 and ***P < 0.001. (G) Comparison of DHTS with NaV for the effects on insulin signaling. L6 myotubes were incubated with 200 nM dexamethasone for 24 h, followed by incubation with DMSO (NT), DHTS (10 μM), or NaV (1 mM) for 1 h. Data are presented as the mean ± SEM (n = 4); ***P < 0.001; NS, not significant.  increased in the insulin-resistant condition remains elusive. A detailed mechanism for the upregulation of C1-Ten should be elucidated in further studies.

ScIenTIfIc
Insulin resistance in skeletal muscle plays a pivotal role in the pathogenesis of type 2 diabetes because muscle is the largest tissue of the human body, and 80% of whole-body insulin-stimulated glucose uptake occurs in muscle 22 . Impaired responsiveness to insulin typically occurs at the level of IRS-1, and this defect in IRS-1-mediated signaling has been observed in type 2 diabetes 1-5 . Therefore, maintenance of an appropriate IRS-1 level is essential for proper insulin signaling. A number of E3 ligases regulate IRS-1 stability and have been implicated in muscle atrophy and glucose intolerance 8,9,11,23 ; however, inhibition of E3 ligase may result in unexpected side effects because of its non-canonical catalytic site and its broad-spectrum substrate specificity. To date, there are no known chemical inhibitors that regulate IRS-1 protein levels. Approaches other than E3 ligase targeting are necessary to prevent insulin resistance via IRS-1 regulation.
Along with IRS-1, AMPK is another attractive therapeutic target molecule. AMPK activation results in improved insulin sensitivity and maintenance of glucose homeostasis. In addition, accumulating evidence in mice and humans has highlighted the role of AMPK activation against diabetes 24,25 . Although AMPK activity and protein expression levels are generally normal in type 2 diabetes 26,27 , pharmacological activation of AMPK improves insulin sensitivity by stimulating glucose uptake and lowering blood glucose levels. Two classes of antidiabetic drugs, the biguanides (i.e., metformin) and the thiazolidinediones (i.e., rosiglitazone), improve insulin action by activating AMPK 17,28 . In addition, recent papers have shown that AMPK activation in skeletal muscle is a potent therapeutic approach for the treatment of diabetes. It was shown that new compounds (PF-739 and MK-8722) lower blood glucose levels through skeletal muscle AMPK activation in rodents and in non-human primates 29,30 . Therefore, the modulation of these two pathways (IRS-1 and AMPK) together may be an effective way to improve insulin sensitivity.
Although AMPK activation and increasing insulin sensitivity seem to be effective therapeutic approaches for insulin resistance, AMPK activation is the representative catabolic pathway and insulin is the representative anabolic hormone. However, many studies have suggested a close interconnection between the two pathways. AMPK sensitizes insulin signaling 19,31 via the attenuation of mTORC1-mediated negative feedback towards IRS-1 32,33 or by positively regulating PI3K/Akt 34 . A recent modeling study raised the possibility that the anabolic component, IRS, positively regulates the catabolic regulator, AMPK 35 , but no mechanism has been elucidated. Our study is the first to propose that IRS-1, and more specifically IRS-1 Y612 phosphorylation, might act upstream of AMPK. How IRS-1 Y612 phosphorylation regulates AMPK activation still remains to be elucidated. However, our findings provide proof-of-concept for targeting C1-Ten with DHTS, a 1,2-naphthoquinone derivative, as a potential therapeutic strategy for insulin resistance resulting from an imbalance between anabolism and catabolism.

Recombinant Adenovirus.
Adenovirus expressing C1-Ten WT was generated through homologous recombination between a linearized transfer vector pAd-Track-CMV vector carrying Flag-C1-Ten WT and the adenoviral backbone vector pAd-Easy as described previously 3 . The virus encoded the green fluorescent protein (GFP) transcribed from a second independent CMV promoter to monitor viral infection efficiency. Adenovirus coding for GFP alone (pAd-GFP) was used as a control. L6 myotubes were infected with adenoviruses for 48 h.
Western blotting. Frozen tissues and harvested cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM Na 3 VO 4 , 20 mM NaF, 10 mM glycerophosphate, 1 mM PMSF, 10% glycerol, 1% Triton X-100, 0.2% sodium deoxycholate, and protease inhibitor cocktail. Tissue samples were homogenized using the Tissue Lyser II (Qiagen, Hilden, Germany). The lysates were centrifuged at 14,000 rpm for 15 min. The resultant supernatants were subjected to SDS-PAGE on 6-16% gradient gels, followed by Western blot analysis. In brief, the proteins were transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). After blocking with 5% skim milk in TTBS buffer, the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Signals were detected by enhanced chemiluminescence (ECL; Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time PCR analysis. Total RNA was isolated from L6 myotubes using TRIzol reagent (iNtRON Biotechnology, Sungnam, South Korea), and 3 μg of RNA was reverse transcribed to cDNA with M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative PCR analysis was performed using SYBR Green I Nucleic Acid Gel Stain (Invitrogen) and a C1000 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The PCR was carried out in a final volume of 20 μL. The relative quantity of mRNA was calculated using the comparative Ct method after normalization to GAPDH. The sequences of the primers used in PCR are as follows: IRS-1: 5′-GTGAACCTAAGTCCCAACCATAAC-3′ and 5′-CCGGCAGCCTTGAGTGTCT-3′ C1-Ten: 5′-CTCGGTGGAGTTTGTCTTCTCCTC-3′ and 5′-GCTGGTTGAAGTTTTCATAGGAGTC-3′ GAPDH: 5′-CCATGACAACTTTGGCATCG-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′ Glucose uptake. Glucose uptake assays were performed as described previously 36 . Briefly, differentiated L6 myotubes were treated with DHTS for the indicated times or concentrations. After washing twice with Krebs buffer, the cells were incubated with 2-deoxy[ 14 C] glucose for 10 min. The reaction was terminated by washing three times with ice-cold phosphate-buffered saline (PBS) containing 25 mM D-glucose. The cells were lysed using a solution of 0.1 N NaOH and 0.1% SDS. Glucose levels were measured by liquid scintillation counter.
Ex vivo glucose uptake. Primary EDL muscles were obtained from the forelimbs of 3-to 4-week-old littermate pups as described previously 37 . Dissected and minced muscle was enzymatically disaggregated in PBS containing 1.5 U/mL dispase II and 1.4 U/mL collagenase D (Roche, Penzberg, Germany) at 37 °C, and triturated with a 10-mL pipette every 5 min for 20 min. Cells were filtered through 70-µm mesh (BD, Seoul, South Korea) and collected by pelleting at 1,000 rpm for 5 min. The cell pellet was dissociated in F10 medium (Invitrogen) supplemented with 10 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA) and 10% fetal calf serum (Hyclone, Logan, UT, USA). Finally, cells were pre-plated twice onto non-collagen-coated plates for 1 h each to deplete the fibroblasts. After cells reached confluence, differentiation was induced by incubation in DMEM supplemented with 2% FBS for 2 days. Cells were washed twice with PBS and starved in serum-free low-glucose DMEM for 3 h. Cells were incubated with KRB (20 mM HEPES [pH 7.4], 130 mM NaCl, 1.4 mM KCl, 1 mM CaCl 2 , 1.2 mM MgSO 4 , and 1.2 mM KH 2 PO 4 ), and incubated with the indicated compounds at 37 °C. The uptake assay was initiated by adding 2-deoxy-d(H 3 )-glucose (2-DG) to each well and incubating at 37 °C for 15 min. The reaction was terminated by washing with ice-cold PBS. The cells were lysed in 10% SDS and mixed with a scintillation cocktail to measure radioactivity. The experiment was approved by the Korea University Institutional Animal Care Use Committee (KOREA-2017-0087-C1) and was performed in accordance with the guidelines and regulations of the IACUC.