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A Novel Insulin Receptor-Binding Protein from Momordica charantia Enhances Glucose Uptake and Glucose Clearance in Vitro and in Vivo through Triggering Insulin Receptor Signaling Pathway Hsin-Yi Lo,†,‡ Tin-Yun Ho,†,‡ Chia-Cheng Li,§ Jaw-Chyun Chen,∥ Jau-Jin Liu,⊥ and Chien-Yun Hsiang*,⊥ †

Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan Graduate Institute of Cancer Biology, China Medical University, Taichung 40402, Taiwan ∥ Department of Medicinal Botany and Healthcare, Da-Yeh University, Changhua 51591, Taiwan ⊥ Department of Microbiology, China Medical University, Taichung 40402, Taiwan §

S Supporting Information *

ABSTRACT: Diabetes, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principal mediator of glucose homeostasis. In a previous study, we identified a trypsin inhibitor, named Momordica charantia insulin receptor (IR)binding protein (mcIRBP) in this study, that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-cross-linking coupled with mass spectrometry showed that three regions (17−21, 34−40, and 59−66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cysteine-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by (5.87 ± 0.45)-fold, increased the amount of phospho-IR protein by (1.31 ± 0.03)-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3L1 cells by (1.36 ± 0.12)-fold. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9 ± 3.2% and 10.8 ± 3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggest that mcIRBP is a novel IRBP that binds to sites different from the insulin-binding sites on IR and stimulates both the glucose uptake in cells and the glucose clearance in mice. KEYWORDS: Momordica charantia, diabetes, insulin receptor-binding protein, glucose homeostasis

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INTRODUCTION Diabetes mellitus is a chronic metabolic disorder that has a significant impact on world health. It is also a growing medical problem in developed countries that affects 6% of the population in the U.S. and 3% of the population in Northern Europe. In the U.S., diabetes is the leading cause of renal disease, nontraumatic lower extremity amputations, and blindness. Therefore, with an increasing incidence worldwide, diabetes will likely continue to be a leading cause of morbidity and mortality in the future.1,2 Diabetes is characterized by hyperglycemia that is attributed to defective insulin secretion, resistance to insulin action, or a combination of both. On the basis of the pathogenic process that leads to hyperglycemia, diabetes is classified into type 1 diabetes (insulin-dependent) and type 2 diabetes (non-insulindependent).2 Clinical studies have demonstrated that hyperglycemia is an important etiologic factor leading to diabetic complications, such as retinopathy, nephropathy, and neuropathy. For examples, Diabetes Control and Complications Trial demonstrates that tight glucose control and lowered glycated hemoglobin in patients with type 1 diabetes significantly reduce the development and the progression of chronic diabetic complications.3 Long-term follow-up of these patients demonstrates the beneficial effects on macrovascular outcomes.4 Other studies on patients with type 2 diabetes also indicate a reduced © XXXX American Chemical Society

risk of microvascular complications with improved long-term glycemic control.5 These findings conclusively indicate that an effective control of blood glucose level is a key step to prevent or reverse diabetic complications and to improve the life quality of patients with diabetes. There are several drugs currently available for diabetes. Insulin is the well-known drug for the treatment of type 1 and type 2 diabetes. Insulin is a 51-residue peptide hormone that maintains the glucose homeostasis in the body. Insulin binding to insulin receptor (IR) stimulates the autophosphorylation of IR, leading to the activation of phosphoinositide-3-kinase (PI3K) and Akt, the translocation of glucose transporter 4 (GLUT4), and the subsequent glucose uptake of adipocytes and skeletal muscle. 6 In addition to insulin, insulin secretagogues, peroxisome proliferator-activated receptors (PPAR) agonists, alpha glucosidase inhibitors, glucagon-like peptide 1 analogues, and dipeptidyl peptidase 4 inhibitors have also been used for type 2 diabetes.7 Although these drugs are used for diabetes, insulin is the only medicine for the treatment Received: September 17, 2013 Revised: August 13, 2014 Accepted: August 21, 2014

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dx.doi.org/10.1021/jf5002099 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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of type 1 diabetes. Moreover, insulin is also the only drug that triggers insulin signaling pathway via binding to IR. Momordica charantia Linn. (MC) is a common vegetable that displays antidiabetic potential.8−12 In a previous study, we have demonstrated that an aqueous extract of MC stimulates the insulin signaling pathway and displays a hypoglycemic effect in mice. By proteomic and docking analyses, we have found that trypsin inhibitor might interact with IR.13 Trypsin inhibitor, also named as bitter gourd trypsin inhibitor or MC trypsin inhibitor, has been isolated from MC, and its amino acid sequences have been identified.14 Trypsin inhibitor consists of 68 amino acid residues and inhibits not only trypsin but also subtilisin Carlsberg. In this study, we renamed this protein as MC IR-binding protein (mcIRBP) because of its novel function on glucose homeostasis. The physical and functional interactions between mcIRBP and IR were clearly elucidated here. Recombinant mcIRBP was purified from Escherichia coli (E. coli) overexpression system. The physical and functional interactions between mcIRBP and IR were analyzed by photo-cross-linking coupled with mass spectrometry (MS), IR-binding assay, and IR kinase activity assay. Furthermore, the hypoglycemic effect and mechanism of mcIRBP were evaluated in normal and type 1 diabetic mice. Our data showed that mcIRBP bound to the sites different from the insulin-binding sites on IR and consequently enhanced the uptake of glucose in cells and the clearance of glucose in mice.

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MATERIALS AND METHODS

Chemicals. Chemicals, unless indicated, were purchased from Sigma (St. Louis, MO). SuperScript III, RPMI-1640, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) and calf serum were purchased from Hyclone (Logan, UT). [125I]Insulin, [γ-32P]ATP, and 2-[1,2-3H(N)]-deoxy-D-glucose were purchased from PerkinElmer (Boston, MA). Rabbit monoclonal antibodies against IR, phospho-IR, phosphoinositide-dependent kinase 1 (PDK1), phospho-PDK1, Akt, phospho-Akt (Ser473), and phospho-Akt (Thr308) were purchased from Cell Signaling (Beverly, MA). Mouse monoclonal antibody against β-actin was purchased from Santa Cruz (Dallas, TX). Cloning of mcIRBP Gene. To clone the mcIRBP cDNA, total RNA was extracted from MC, reverse transcribed by SuperScript III, and amplified with P1 (5′-GATCAAGCTTATGTGTCAGGGGAAGTCGTCGTGGCCGCAG-3′) and M1 (5′-GATCGAGCTCTCAACCGATGGTGGGGGGGCGGGCGACGAT-3′) primers. The resulting 216-bp mcIRBP cDNA fragment was inserted into HindIII and SacI sites of pBluescript II KS(−) vector to create pBKS-mcIRBP. pBKS-mcIRBP was further amplified with P2 (5′-GATCACATGTGTCAGGGGAAGTCGTCGTGG-3′) and T7 (5′-AATACGACTCACTATAG-3′) primers. The 242-bp mcIRBP cDNA fragment was then inserted into NcoI and SacI sites of pET-28a(+) vector to create pETmcIRBP (Figure 1A). The plasmid DNA, which was created in this study, was confirmed as an in-frame construction by sequencing. The template sequences for primer design are originated from GenBank (accession number: 2111250B). The protein sequences of mcIRBP have been deposited in GenBank (accession number: AGU04661.1). Expression and Purification of Recombinant mcIRBP. Recombinant mcIRBP was expressed in E. coli BL21(DE3) pLysS strain, which was transformed with pET-mcIRBP. Expression and purification of recombinant mcIRBP were performed as described previously.15 Molecular mass of mcIRBP was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE), and the concentration of mcIRBP was quantified by a Bradford method (Bio-Rad, Hercules, CA). Photo-Cross-Linking Coupled with MS. Photoinduced crosslinking was performed as described previously.16 Briefly, mcIRBP was mixed with IR in a molar ratio of 10:1 in 10 mM sodium phosphate buffer (pH 7.4). To a total volume of 18 μL of protein solution were

Figure 1. Cloning, expression, and purification of recombinant mcIRBP. (A) Map of pET-mcIRBP. The diagram represents the location of mcIRBP gene in the pET-28a(+) vector. Closed arrow represents mcIRBP gene. (B) SDS−PAGE analysis. The preparations of uninduced E. coli containing pET-mcIRBP (lane C), isopropyl-β-Dthiogalactoside-induced E. coli containing pET-mcIRBP (lane I), and purified mcIRBP (lane P) were analyzed by 15% SDS−PAGE and stained by Coomassie Brilliant Blue. The molecular masses of protein standards (lane M) are shown at the left. Recombinant mcIRBP is indicated by the arrow. then added 1 μL of tris(bipyridyl)Ru(II) (RuBpy) solution (1 mM RuBpy in 10 mM sodium phosphate buffer, pH 7.4) and 1 μL of ammonium persulfate (APS) solution (20 mM APS in 10 mM sodium phosphate buffer, pH 7.4). The reaction mixture was exposed to ultraviolet light for 30 s and quenched by adding 1 μL of 1 M dithiothreitol. Protein solution was further identified using an Ultimate capillary liquid chromatography system (LC Packings, Amsterdam, The Netherlands) coupled to a QSTARXL quadrupole-time-of-flight MS (Applied Biosystem/MDS Sciex, Foster City, CA). Docking Calculations. The PatchDock was used for the prediction of ligand-binding sites.17 The PDB files of IR (PDB code 3LOH), mcIRBP (PDB code 1VBW), and insulin (PDB code 1JCO) were obtained from Protein Data Bank (http://www.rcsb.org/pdb/). The PDBQ files of these proteins were derived from PDB2PQR version 1.8 (http://nbcr-222.ucsd.edu/pdb2pqr_1.8/).18 Cell Culture. IM-9 myeloma cells, HepG2 hepatocytes, and 3T3L1 preadipocytes were purchased from Bioresource Collection Research Center (Hsinchu, Taiwan). IM-9 cells were maintained in B



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another 10 min. Finally, cells were solubilized in 0.1% SDS and the radioactivity uptaken by cells was measured by scintillation counter. Animals. BALB/c mice were obtained from National Laboratory Animal Center (Taipei, Taiwan). Mouse experiments were conducted under ethics approval from China Medical University Animal Care and Use Committee (Permit Number: 101-188-N). Type I diabetic mice were induced by streptozotocin.21 Briefly, male BALB/c mice (6 weeks old) were injected intraperitoneally with streptozotocin (200 mg/kg in 200 μL of normal saline). Two weeks later, mice were bled via tail veins after 4 h starvation, and the blood glucose was measured by glucose oxidase method using a glucometer (ACCU-CHEK Advantage, Roche Diagnostics, Basel, Switzerland). Glucose Tolerance Assay. Normal and type I diabetic mice were fasted for 18 and 4 h, respectively. mcIRBP (2.5 nmol/kg) was then given intraperitoneally 15 min before intraperitoneal administration of glucose solution (4 g/kg for normal mice and 1 g/kg for diabetic mice).22 Blood samples were collected from tails at 15 min before and at 15, 30, 60, 90, 120, 150, 180, 240, and 270 min after glucose challenge. Blood glucose was measured by glucose oxidase method using a glucometer. Data were also expressed as area under the curve (AUC). Mice were sacrificed 5 h after glucose challenge, and muscles were collected for microarray analysis. Microarray Analysis. Five hours after glucose challenge, mice were sacrificed and muscle tissues were collected for microarray analysis. Total RNAs were extracted from muscle tissues using RNeasy Mini kit (Qiagen, Valencia, CA). Total RNA from three mice was pooled, and three microarray replicates for each pooled RNA were performed. Microarray analysis was performed as described previously.23−25 Briefly, fluorescence-labeled RNA targets were hybridized to Mouse Whole Genome OneArray (Phalanx Biotech Group, Hsinchu, Taiwan). The Cy5 fluorescent intensity of each spot was analyzed by Genepix 4.1 software (Molecular Devices, Sunnyvale, CA). The signal intensity of each spot was corrected and further normalized by R program in the limma package.26 Fold changes of genes were calculated by dividing the normalized signal intensities of genes in mcIRBP-treated tissues by those in untreated tissues. We used the geneSetTest function to test which biological pathways were affected by mcIRBP. This function computes a p-value to test the hypothesis that the selected genes in a pathway tend to be differentially expressed. Kyoto Encyclopedia of Genes and Genomes pathways (http://www.genome.jp/kegg/pathway.html) was extracted and used for this analysis. Microarray data are MIAME compliant, and raw data have been deposited in the Gene Expression Omnibus (accession number: GSE39689). Statistical Analysis. Data were presented as mean ± standard error. Student’s t test was used for comparisons between two experiments. A value of p < 0.05 was considered statistically significant.

RPMI-1640 supplemented with 10% FBS. HepG2 cells were maintained in DMEM supplemented with 10% FBS. 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% heat-inactivated calf serum. All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. IR-Binding Assays. The binding of mcIRBP to IR was measured by IR-binding assay, IR kinase activity assay, and enzyme-linked immunosorbent assay (ELISA). IR-binding assay was performed as described previously.19 Briefly, IM-9 cells (1.2 × 106 cells/mL) were incubated with different amounts of mcIRBP for 15 min at 25 °C. 125Ilabeled insulin (60,000 cpm/mL of cells) was then added and incubated for 90 min at 16 °C. Cells were centrifuged at 3000g for 10 min, and the supernatant containing unbound ligands was removed. The radioactivity of cell pellet containing bound 125I-labeled insulin was measured by scintillation counter (Beckman Coulter, Fullerton, CA). IR kinase activity assay was performed as described previously.13 Briefly, mixtures containing IR and various amounts of mcIRBP in kinase buffer (25 mM HEPES, pH 7.6, 25 mM MgCl2, 100 μM ATP, 100 μM sodium orthovanadate, 2.5 mg/mL poly(Glu,Tyr), 25 μCi/ mL [γ-32P]ATP) were incubated at 30 °C for 10 min and spotted on chromatography papers. Poly(Glu,Tyr) was precipitated on papers by soaking papers in 10% trichloroacetic acid solution, and the radioactivity incorporated into the precipitated poly(Glu,Tyr) was counted by scintillation counter. For ELISA, HepG2 cells were treated with various amounts of mcIRBP, incubated at 37 °C for 10 min, and solubilized in lysis buffer (1% NP-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin) on ice for 15 min. After the centrifugation at 2000g for 5 min, the supernatant was collected, and the protein concentration was quantified. ELISA was performed with the DuoSet IC Human Phospho-IR set (R&D Systems, Minneapolis, MN). Briefly, microtiter plates (MaxiSorp, Nunc, Denmark) were coated with antibody against phospho-IR at 4 °C overnight, incubated with cellular proteins (250 μg) at room temperature for 2 h, and then incubated with horseradish peroxidase-conjugated antibody against phospho-tyrosine at room temperature for 2 h. Following four washes, chromogenic substrate tetramethylbenzidine was added, incubated at room temperature for 20 min, and stopped with 2 N H2SO4. The absorbance at 450 nm was measured in an ELISA plate reader. Western Blot Analysis. HepG2 cells were cultured in 25 cm2 flasks, treated with various amounts of mcIRBP for 30 min, collected using cell scrapers, and lysed with sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol). HepG2 cells were also treated with LY294002 (50 μM) to block the PI3K activity. Proteins (10 μg) were separated by 10% SDS−PAGE and transferred electrophoretically to nitrocellulose membranes. Membranes were then probed with anti-IR, anti-phospho-IR, anti-PDK1, anti-phosphoPDK1, anti-Akt, anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), and anti-β-actin antibodies. The bound antibody was detected with horseradish peroxidase-conjugated anti-rabbit antibody followed by chemiluminescence and X-ray film exposure. Adipocyte Differentiation and Glucose Uptake Assay. The differentiation of 3T3-L1 preadipocytes was induced by the addition of 0.5 mM 3-isobutyl-1-methylxanthine, 0.5 μM dexamethasone, 10 μg/ mL insulin, and 10% FBS in DMEM for 3 days. The aforementioned medium was then replaced with 10% FBS/DMEM containing 10 μg/ mL insulin. After another 3 days, the medium was subsequently replaced with fresh culture medium (10% FBS/DMEM), which was changed every 2 days until the cells were fully differentiated, judged by oil red O staining. Fully differentiated 3T3-L1 adipocytes were used for glucose uptake assay. Glucose uptake assay was performed as previously described.20 Briefly, 3T3-L1 adipocytes were cultured in 24well plates. After a 4.5 h starvation, adipocytes were incubated in Krebs−Ringer bicarbonate buffer (118 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM Na2HPO4, 2% bovine serum albumin, 0.5 mM glucose, 25 mM NaHCO3, pH 7.4) without or with various amounts of mcIRBP for 30 min at 37 °C. [3H]-2-Deoxy-Dglucose (0.1 μCi) was then added to adipocytes and incubated for

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RESULTS

Expression and Purification of mcIRBP. In our previous study, we identified a trypsin inhibitor, named as mcIRBP here, that might interact with IR. The mcIRBP gene was therefore amplified by PCR and cloned for expression. mcIRBP was expressed and purified from E. coli BL21 (DE3) pLysS strain, which was transformed with pET-mcIRBP. After the induction with isopropyl-β-D-thiogalactoside, mcIRBP with molecular mass of approximately 7 kDa was observed by SDS−PAGE analysis (Figure 1B). The amount of purified mcIRBP recovered was approximately 2 mg per 100 mL of bacterial culture. Analysis of the Physical Interaction between IR and mcIRBP. Photo-cross-linking coupled with MS was performed to analyze the physical interaction between IR and mcIRBP. mcIRBP was cross-linked with IR by ultraviolet light oxidized Ru2+, and the cross-linked proteins were further identified by LC-MS/MS. We mixed mcIRBP and IR in the molar ratio of 1:1 and 10:1 and found that the cross-linking efficiency at 10:1 C



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Figure 2. Physical interaction between mcIRBP and IR. (A) Docking structure of IR and mcIRBP. The IR ectodomain monomer is shown in a schematic presentation. L1, CR, L2, fibronectin domains, and α-CT segment are colored purple, pink, green, orange, and gray, respectively. mcIRBP is shown in an atomic sphere representation. The 17−21, 34−40, and 59−66 amino acid residues of mcIRBP are colored blue, yellow, and light green, respectively. The remaining regions of mcIRBP are colored violet. (B) Close-up view of mcIRBP complexes with IR. The 17−21, 34−40, and 59−66 amino acid residues of mcIRBP and the amino acid residues located on L1, CR, and L2 regions of IR are shown in atomic sphere representations. The remaining regions of mcIRBP and IR are represented by stick styles. D



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was better (data not shown). A total of 2,731 peptide fragments was identified by MS/MS, and 1,518 fragments were chosen after excluding the repeated fragments. We further picked up the fragments that had ≥3 consecutive amino acid sequences of mcIRBP on the central region and ≥2 consecutive amino acid sequences of IR on either terminus. A total of 146 fragments fulfilling aforementioned criteria were selected. We found that three regions located on 17−21, 34−40, and 59−66 residues of mcIRBP physically interacted with the amino acid residues located on leucine-rich repeat domains and cysteine-rich (CR) region of IR. We further built the docking structures of IR and mcIRBP based on photo-cross-linking data. As shown in Figure 2A, the crystal structure of IR ectodomain monomer formed an inverted “V” layout of which one side consists of leucine-rich repeat domains (L1 and L2) and CR region, and another side is composed of fibronectin domains and α-CT segment. mcIRBP interacted with L1/CR/L2 segments on IR, and the docking score between mcIRBP and IR was 14,636. In comparison with mcIRBP, insulin, as expected, interacted with the α-CT segment on IR, with a docking score of 14,134 (Supporting Information, Figure S1).27 Close-up view of mcIRBP and IR showed that three regions located on 17−21, 34−40, and 59− 66 residues of mcIRBP were close to the amino acid residues located on the L1, CR, and L2 regions of IR (Figure 2B). These data suggested that mcIRBP was a novel IRBP that bound to the sites different from the insulin-binding sites on IR. We analyzed the binding behavior of mcIRBP on IR by incubating cells with various amounts of mcIRBP, radioisotopelabeled insulin (hot insulin), and/or non-radioisotope-labeled insulin (cold insulin). As shown in Figure 3A, when more hot insulin was added to a fixed amount of IR, an increasing fraction of IR was occupied by hot insulin. However, when more cold insulin was added to a fixed amount of IR and hot insulin, a decreasing fraction of IR was occupied by hot insulin (Figure 3B). These results indicated that cold insulin competed with hot insulin and blocked the insulin-binding sites on IR. Then, we measured the IR-binding behavior of mcIRBP by incubating IM-9 cells with various amounts of mcIRBP and 6 pM of hot insulin. As shown in Figure 3C, an increasing fraction of IR was occupied by hot insulin when an increasing amount of mcIRBP was added to a fixed amount of IR and hot insulin. These data suggested that mcIRBP bound to a site other than the insulin-binding sites on IR and enhanced the binding of hot insulin to IR. However, the amount of bound hot insulin in the presence of 10 nM mcIRBP was dramatically decreased by the addition of an excessive amount (1 μM) of cold insulin, suggesting that the mcIRBP-dependent enhancement of insulin binding on IR was specifically blocked and abolished by cold insulin. These results indicated that mcIRBP might bind to the allosteric site of IR and further enhance the effectivity of IR on the insulin binding. mcIRBP Enhanced the Kinase Activity and Autophosphorylation of IR. IR is a transmembrane protein that exhibits tyrosine kinase activity. Insulin binding to IR stimulates the intrinsic tyrosine kinase activity, leading to the autophosphorylation of IR.6 Therefore, we analyzed the kinase activity of IR by kinase activity assay and evaluated the level of autophosphorylated IR by ELISA. Figure 4A shows that the addition of insulin stimulated the kinase activity of IR by (7.35 ± 0.89)-fold. mcIRBP also enhanced the kinase activity of IR in a dose-dependent manner. Moreover, the maximal enhancement [(5.87 ± 0.45)-fold] of kinase activity was achieved in the

Figure 3. IR-binding assay of mcIRBP. IM-9 cells were treated with various amounts of 125I-labeled insulin (A), 6 pM 125I-labeled insulin and various amounts of insulin (B), or various amounts of mcIRBP, 6 pM 125I-labeled insulin, and/or 1 μM insulin (C). The reaction was incubated for 90 min at 16 °C, and the radioactivity of bound 125Ilabeled insulin was counted by scintillation counter. Values are mean ± standard error (n = 3).

presence of 5 μM mcIRBP. Figure 4B shows that insulin increased the level of phosphorylated IR in HepG2 cells, as compared with mock. mcIRBP significantly stimulated the autophosphorylation of IR in a dose-dependent manner. Moreover, the maximal increase [(1.31 ± 0.03)-fold] of phosphorylated IR was achieved by 5 μM mcIRBP. These findings indicated that mcIRBP bound to IR, activated the kinase activity of IR, and sequentially stimulated the autophosphorylation of IR. mcIRBP Affected IR/PI3K/Akt Signaling Pathway. We further performed Western blot to analyze the IR-downstream signaling pathway affected by mcIRBP. As shown in Figure 5A, insulin and mcIRBP stimulated the phosphorylation of IR, which was consistent with aforementioned data (Figure 4). Insulin enhanced the phosphorylations of PDK1 and Akt at Ser473 and Thr308, as compared with mock. mcIRBP also increased the amounts of phosphorylated PDK1 and Akt in a dose-dependent manner. However, LY294002, a PI3K inhibitor, inhibited the mcIRBP-induced phosphorylation of E



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Figure 5. Effect of mcIRBP on the insulin signaling pathway in HepG2 cells and the glucose uptake of 3T3-L1 adipocytes. (A) Western blot. HepG2 cells were treated with 1 μM insulin or various amounts of mcIRBP for 30 min. LY294002 (50 μM) was used to inhibit PI3K activity. IR, phosphorylated IR, PDK1, phosphorylated PDK1, Akt, phosphorylated Akt (Ser473), and phosphorylated Akt (Thr308) were then detected by Western blot and visualized by chemiluminescence. Similar results are obtained in three independent experiments. Photos are representative images. (B) Glucose uptake assay. Fully differentiated 3T3-L1 adipocytes were treated with 1 nM insulin or various amounts of mcIRBP. Thirty minutes later, [3H]-2-deoxy-D-glucose was added and the radioactivity uptaken by cells was counted. Data are expressed as relative radioactivity, which is presented as the comparison with the radioactivity relative to mock. Values are mean ± standard error (n = 3). *p < 0.05, compared with mock.

Figure 4. Effect of mcIRBP on the kinase activity and autophosphorylation of IR. (A) Kinase activity assay. IR was mixed with 0.5 μM insulin or various amounts of mcIRBP in the presence of [γ-32P]ATP and poly(Glu,Tyr). The radioactivity (dpm) incorporated into the precipitated poly(Glu,Tyr) was counted by scintillation counter. (B) ELISA. HepG2 cells were treated with 0.5 μM insulin or various amounts of mcIRBP. Ten minutes later, cellular proteins were collected and the phosphorylated IR was quantitated by ELISA. Values are mean ± standard error (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, compared with no treatment.

Akt. These findings suggested that mcIRBP bound to IR, stimulated the kinase activity of IR, and sequentially activated the PI3K/PDK1/Akt pathway. mcIRBP Stimulated the Uptake of Glucose in 3T3-L1 Adipocytes. PI3K has a pivotal role in the metabolic action of insulin. Activation of PI3K stimulates the activation of Akt and subsequently stimulates the translocation of GLUT4 to the cell surface, a crucial event for glucose uptake by skeletal muscle and fat.28 Therefore, we further evaluated the effect of mcIRBP on glucose uptake in 3T3-L1 adipocytes. As shown in Figure 5B, insulin and mcIRBP significantly enhanced the uptake of glucose in 3T3-L1 by (1.63 ± 0.20)-fold and (1.36 ± 0.12)fold, respectively. The enhancement displayed a dose-dependent manner, and the maximal enhancement was achieved by 1 μM mcIRBP. These data suggested that mcIRBP bound to IR, stimulated PI3K/PDK1/Akt pathway, and, in turn, increased the uptake of glucose in adipocytes. mcIRBP Displayed the Hypoglycemic Effect in Normal and Diabetic Mice. To analyze whether mcIRBP was capable of lowering the blood glucose levels in sera, we evaluated the hypoglycemic effect of mcIRBP by glucose tolerance assay in normal and diabetic mice. As shown in Figure 6, fasting blood glucose levels of normal and diabetic mice were approximately 40 mg/dL and 450 mg/dL, respectively. After glucose challenge, the blood glucose concentrations were dramatically

increased at 30 min. The mock group displayed a poor glucose clearance, while intraperitoneal administration of mcIRBP resulted in a more rapid clearance of glucose in both normal and diabetic mice. The AUC was significantly decreased by 20.9 ± 3.2% and 10.8 ± 3.6% in mcIRBP-treated normal and diabetic mice, respectively. The AUC was significantly decreased by 34.7 ± 2.1% in insulin-treated diabetic mice. These data indicated that mcIRBP exhibited hypoglycemic effects in mice. Signal Transduction Pathways of mcIRBP on Glucose Metabolism in Normal and Diabetic Mice. Microarray analysis was performed to investigate whether insulin signaling pathway and glucose metabolism were affected by mcIRBP in mice. Although muscle and adipose tissues are dominant tissues for glucose uptake, we chose muscle tissues only to analyze the gene expression profiles affected by mcIRBP because of the similar pathway pattern between muscle and adipose tissues in our previous study.13 In a total of 29,922 genes, 636 and 1,523 genes with fold changes ≥2 or ≤−2 in normal and diabetic mice, respectively, were selected and tested for the biological pathways by geneSetTest function. Top 20 upregulated and downregulated genes are listed in Table S1 and Table S2 (Supporting Information). A total of 108 pathways and 95 F



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Figure 6. Hypoglycemic effects of mcIRBP in normal and type 1 diabetic mice by glucose tolerance assay. Normal (A) and type 1 diabetic mice (B) were administrated mcIRBP (2.5 nmol/kg) or insulin (2.5 nmol/kg) intraperitoneally. Fifteen minutes later, glucose was administrated intraperitoneally, blood samples were collected at intervals, and blood glucose levels were measured by the glucometer. AUC of glucose tolerance assay is shown at the right. Values are mean ± standard error (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, compared with mock.

pathways was significantly affected by mcIRBP in normal and diabetic mice, respectively (Supporting Information, Table S3 and Table S4). The information on genes for each of the significant pathways listed in Table S3 and Table S4 (Supporting Information) is shown in Table S5 and Table S6 (Supporting Information), respectively. As shown in Table 1, insulin signaling pathway and glucose metabolism, such as glycolysis/gluconeogenesis, citrate cycle, pentose phosphate pathway, and pyruvate metabolism, were significantly regulated by mcIRBP in both normal and diabetic mice. Moreover, PPAR signaling pathway, adipocytokine signaling pathway, leptin signaling pathway, and fatty acid metabolism were also significantly regulated by mcIRBP. These findings suggested that administration of mcIRBP triggered the insulin signaling pathway and subsequently affected the glucose and lipid metabolism in mice.

Table 1. Pathways Significantly Affected by mcIRBP in Normal and Diabetic Mice p valueb (obsd/totalc) for mcIRBP-treated mice

pathway

a

normal

insulin signaling pathway PPAR signaling pathway adipocytokine signaling pathway leptin signaling pathway glycolysis/ gluconeogenesis citrate cycle

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DISCUSSION In this study, we identified that a novel protein, mcIRBP, from MC directly bound to IR and triggered the insulin signaling transduction pathway in vitro and in vivo. Recently, the interaction of insulin with its primary binding site on IR was reported.27 IR is a disulfide-linked homodimer, and each IR monomer contains two different insulin binding sites. Insulin binds asymmetrically to two different sites in IR dimer via bivalent growth hormone-receptor-binding mechanism.29 The primary binding site with high affinity is located on both the αCT segment of one IR monomer and the central β-sheet of L1 of another IR monomer. The second site is located on both fibronectin domains and L2 segment of each IR monomer.

pentose phosphate pathway pyruvate metabolism fatty acid metabolism

diabetic

obsd/totalc for insulintreated tissue human skeletal muscled

L6 skeletal muscle cellse

4.06 × 10−7 (16/132) 1.02 × 10−10 (23/72) 0.000898 (11/69)

2.48 × 10−6 (24/132) 6.92 × 10−7 (26/72) 0.000173 (14/69)

4/132

29/132

2/72

2/72

1/69

11/69

0.000206 (6/25) 2.27 × 10−6 (15/54) 6.86 × 10−6 (5/26) 0.000209 (11/25)

0.000448 (11/25) 3.53 × 10−5 (19/54) 1.80 × 10−9 (17/26) 0.000516 (6/25)

1/25

0/25

5/54

3/54

0/26

0/26

3/25

3/25

0.001912 (10/39) 4.57 × 10−7 (11/42)

2.06 × 10−7 (19/39) 5.61 × 10−8 (23/42)

2/39

0/39

2/42

0/42

a

Pathways associated with glucose and lipid metabolism are shown. bp values were calculated by the geneSetTest function implemented in the limma package. cObsd/total means the number of selected genes/ the number of total genes in this pathway. dMicroarray data were cited from Rome et al.39 eMicroarray data were cited from Di Camillo et al.40 G



Article

dephosphorylate IR.39 Because LY294002 inhibits PI3K and PI3K-downstream Akt activities, we proposed that the decreased amount of phosphorylated IR in the presence of insulin resulted from the increased amount of unphosphorylated PTP1B. The hypoglycemic effect of mcIRBP was analyzed by glucose tolerance assays in normal and diabetic mice. The AUC was significantly decreased by 20.9% in mcIRBP-treated normal mice, while the AUC was significantly decreased by 10.8% and 34.7% in diabetic mice treated with mcIRBP and insulin, respectively. Although the hypoglycemic effects of mcIRBP and insulin displayed a significant manner, mcIRBP was less effective than insulin in blood glucose-lowering ability. Photocross-linking assay suggested that mcIRBP interacted with L1/ CR/L2 region on IR, on opposite site of the primary binding site of insulin on IR. IR-binding assay further showed that mcIRBP cooperated with insulin for binding to IR. Therefore, we proposed that the mcIRBP-binding site on IR was minor, in comparison with primary insulin-binding site on IR, in stimulating the IR signaling pathway and subsequently the clearance of glucose. That might explain why the blood glucoselowering ability of mcIRBP was less effective than that of insulin. We performed microarray analysis to study gene expression profiles of mcIRBP in normal and diabetic mice. Microarray profiles of insulin in human skeletal muscle and rat L6 skeletal muscle myoblasts have been reported.40,41 Their data show that insulin regulates several genes involved in intermediary and energy metabolism, transcriptional and translational regulation, cell cycle, cytoskeleton, vesicle traffic, and immune response. By pathway analysis, we found the consistency of gene expression profiling between mcIRBP and insulin. Among mcIRBPregulated pathways, the majority of pathway was associated with glucose, lipid, and energy metabolism. There were 20 and 45 differentially expressed genes in insulin-treated human skeletal muscle and insulin-stimulated rat skeletal muscle myoblasts, respectively, involved in glucose and lipid metabolism pathways (Table 1). The dominant affected pathway in insulin-treated human skeletal muscle, insulinstimulated rat skeletal muscle myoblasts, and mcIRBP-treated mouse muscle was insulin signaling pathway. Moreover, pathways involved in transcriptional and translational regulation, such as ribosome, proteasome, RNA polymerase, SNARE, and protein transport, were affected by mcIRBP. Cell cycle- and cytoskeleton-associated pathways, such as tight junction, focal adhesion, cell cycle, regulation of actin cytoskeleton, gap junction, apoptosis, and cell adhesion molecules, were also regulated by mcIRBP. Additionally, mcIRBP significantly affected four immune response-associated pathways, namely, complement and coagulation cascades, leukocyte transendothelial migration, antigen processing and presentation, and Toll-like receptor signaling pathway, and five signaling pathways, namely, PPAR, insulin, adipocytokine, mitogen-activated protein kinase, and gonadotropin-releasing hormone signaling pathways (Supporting Information, Table S3 and Table S4). These findings suggested that although mcIRBP bound to the sites different from the insulin-binding sites on IR, mcIRBP stimulated the similar biological pathways as insulin in mice. In conclusion, our findings suggested that mcIRBP was a novel IR-binding polypeptide that bound to the sites different from the insulin-binding sites on IR. After binding to IR, mcIRBP activated IR-downstream signaling transduction path-

Docking structure of IR and insulin in this study displayed that insulin interacted with two different sites with different docking scores on IR, as described previously.27 Analysis of the physical interaction between IR and mcIRBP by docking and photocross-linking showed that mcIRBP interacted with L1/CR/L2 region on IR, on opposite site of the primary binding site of insulin on IR. Moreover, IR-binding assay showed that the binding behavior of mcIRBP and insulin on IR was cooperative. These data suggested that mcIRBP was a novel IR-binding protein that bound to the sites different from the insulinbinding sites on IR. Additionally, IR is a tyrosine kinase and, in the basal state, the kinase domain of IR is wrapped up by the juxtamembrane regions proximal to the catalytic domain. Upon binding to insulin, rearrangement within the ectodomain of IR results in the removal of the juxtamembrane region, the release of tyrosine kinase domain, and the trans-phosphorylation of IR.30 Binding of mcIRBP to IR might induce the conformational change and release the kinase domain. This might explain why the incubation of mcIRBP and IR in tubes resulted in the activation of tyrosine kinase activity of IR. IR significantly plays an important role in glucose homeostasis. Upon binding to insulin, tyrosine kinase activity of IR is activated, leading to the activation of PI3K/Akt and the subsequent translocation of GLUT4. Defects in the IRmediated signaling pathways in tissues from diabetic patients or animal models also emphasize the importance of the IR pathway in the regulation of glucose metabolism.6,29 In this study, we found that mcIRBP bound to IR, affected IR/PI3K/ Akt pathway and, in turn, stimulated glucose uptake in 3T3-L1 cells. The hypoglycemic mechanism of mcIRBP was similar to that of insulin but was different from those of other glucoselowering agents. For example, thiazolidinediones, such as troglitazone, rosiglitazone, and pioglitazone, bind to PPAR-γ, resulting in the enhancement of fatty acid storage, the increase of adiponectin levels, and the subsequent reduction of insulin resistance. Biguanides, such as metformin, improve insulin sensitivity and lower blood glucose levels mainly through the activation of AMP-activated protein kinase pathway.7 In addition, some compounds exhibit hypoglycemic effects by affecting the PI3K/Akt pathway. For example, herbal compounds, such as ginsenoside Rb1, 6-gingerol, tea catechins, honokiol, and embelin, stimulate the phosphorylations of PI3K and Akt, leading to the translocation of GLUT4.31−34 Cellular proteins, such as nexillin (a cardiomyopathy-associated F-actin binding protein), apelin (a cytokine mainly secreted by adipocytes), and obestatin (ghrelin gene-derived peptide), affect glucose homeostasis by activating the PI3K/Akt pathway.35−38 However, none of these compounds or proteins targets to IR even though they activated the PI3K/Akt pathway. These data suggested the unique mechanism of mcIRBP on the regulation of glucose homeostasis. Furthermore, mcIRBP stimulated the phosphorylation of Akt proteins at both Ser473 and Thr308. Because mammalian target of rapamycin complex 2 (mTORC2) induces the phosphorylation of Akt at Ser473 and PDK1 activates Akt activity by phosphorylating Akt at Thr308,28 we suggested that mcIRBP might also activate mTORC2 activity downstream from IR. In addition, in Figure 5A, we found that PI3K inhibitor LY294002 inhibited mcIRBPinduced phosphorylation of IR. Previous study indicates that PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the IR. However, phosphorylation of PTP1B by Akt negatively modulates its phosphatase activity and impairs it ability to H



Article

Nathan, D. M. DCCT/EDIC Research Group. The effect of intensive glycemic treatment on coronary artery calcification in type 1 diabetic participants of the Diabetes Control and Complications Trial/ Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study. Diabetes 2006, 55, 3556−3565. (5) UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998, 352, 837−853. (6) Saltiel, A. R.; Kahn, C. R. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001, 414, 799−806. (7) Powers, A. C. Diabetes mellitus. In Harrison’s principles of internal medicine; Longo, D. L., Fauci, A. S., Kasper, D. L., Hauser, S. L., Jameson, J. L., Loscalzo, J., Eds.; The McGraw-Hill Companies, Inc.: New York, 2012; pp 2275−2304. (8) Broadhurst, C. L.; Polansky, M. M.; Anderson, R. A. Insulin-like biological activity of culinary and medicinal plant aqueous extracts in vitro. J. Agric. Food Chem. 2000, 48, 849−852. (9) Horax, R.; Hettiarachchy, N.; Over, K.; Chen, P.; Gbur, E. Extraction, fractionation and characterization of bitter melon seed proteins. J. Agric. Food Chem. 2010, 58, 1892−1897. (10) Hsu, C.; Hsieh, C. L.; Kuo, Y. H.; Huang, C. J. Isolation and identification of cucurbitane-type triterpenoids with partial agonist/ antagonist potential for estrogen receptors from Momordica charantia. J. Agric. Food Chem. 2011, 59, 4553−4561. (11) Chaturvedi, P. Antidiabetic potentials of Momordica charantia: multiple mechanisms behind the effects. J. Med. Food 2012, 15, 101− 107. (12) Wang, X.; Sun, W.; Cao, J.; Qu, H.; Bi, X.; Zhao, Y. Structures of new triterpenoids and cytotoxicity activities of the isolated major compounds from the fruit of Momordica charantia L. J. Agric. Food Chem. 2012, 60, 3927−3933. (13) Lo, H. Y.; Ho, T. Y.; Lin, C.; Li, C. C.; Hsiang, C. Y. Momordica charantia and its novel polypeptide regulate glucose homeostasis in mice via binding to insulin receptor. J. Agric. Food Chem. 2013, 61, 2461−2468. (14) Miura, S.; Funatsu, G. Isolation and amino acid sequences of two trypsin inhibitors from the seeds of bitter gourd (Momordica charantia). Biosci., Biotechnol., Biochem. 1995, 59, 469−473. (15) Chen, J. C.; Ho, T. Y.; Chang, Y. S.; Wu, S. L.; Li, C. C.; Hsiang, C. Y. Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses. J. Ethnopharmacol. 2009, 121, 372−378. (16) Rahimi, F.; Maiti, P.; Bitan, G. Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides. J. Visualized Exp. 2009, 23, 1071. (17) Schneidman-Duhovny, D.; Inbar, Y.; Nussinov, R.; Wolfson, H. J. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res. 2005, 33, W363−W367. (18) Dolinsky, T. J.; Nielsen, J. E.; McCammon, J. A.; Baker, N. A. PDB2PQR: an automated pipeline for the setup, execution, and analysis of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004, 32, W665−W667. (19) Gauguin, L.; Delaine, C.; Alvino, C. L.; McNeil, K. A.; Wallace, J. C.; Forbes, B. E.; De Meyts, P. Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I. J. Biol. Chem. 2008, 283, 20821−20829. (20) Chen, C. C.; Hsiang, C. Y.; Chiang, A. N.; Lo, H. Y.; Li, C. I. Peroxisome proliferator-activated receptor gamma transactivationmediated potentation of glucose uptake by Bai-Hu-Tang. J. Ethnopharmacol. 2008, 118, 46−50. (21) Hayashi, K.; Kojima, R.; Ito, M. Strain differences in the diabetogenic activity of streptozotocin in mice. Biol. Pharm. Bull. 2006, 29, 1110−1119. (22) Zhao, L.; Ye, H.; Li, D.; Lao, X.; Li, J.; Wang, Z.; Xiao, L.; Wu, Z.; Huang, J. Glucagon-like peptide-1(1−37) can enhance blood glucose homeostasis in mice. Regul. Pept. 2012, 178, 1−5. (23) Li, C. C.; Hsiang, C. Y.; Lo, H. Y.; Pai, F. T.; Wu, S. L.; Ho, T. Y. Genipin inhibits lipopolysaccharide-induced acute systemic

way, regulated genes involved in glucose and lipid metabolism, and, in turn, enhanced the glucose uptake in cells and the clearance of glucose in normal and diabetic mice.

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ASSOCIATED CONTENT

S Supporting Information *

Figure depicting docking structure of IR and insulin and tables of the top 20 upregulated and downregulated genes by mcIRBP in normal mice and diabetic mice, pathways significantly affected by mcIRBP in normal mice and type 1 diabetic mice, information of genes for each of the significant pathways affected by mcIRBP in normal mice and type 1 diabetic mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Department of Microbiology, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886 4 22053366 ext 2163. Fax: +886 4 22053764. E-mail: cyhsiang@ mail.cmu.edu.tw. Author Contributions ‡

These authors contributed equally to this work.

Funding

This work was supported by grants from National Research Program for Biopharmaceuticals (NSC101-2325-B-039-007, NSC102-2325-B-039-007, and MOST103-2325-B-039-002), Ministry of Science and Technology (NSC101-2320-B-039034-MY3, NSC101-3114-Y-466-002, NSC102-2632-B-039001-MY3, and MOST103-2815-C-039-044-B), Council of Agriculture (103AS-14.3.2-ST-a1), China Medical University (CMU101-S-21, CMU101-AWARD-09, CMU102-NSC-04, and CMU103-SR-44), and CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED APS, ammonium persulfate; AUC, area under the curve; CR, cysteine-rich; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; E. coli, Escherichia coli; FBS, fetal bovine serum; GLUT4, glucose transporter 4; IR, insulin receptor; IRBP, IR-binding protein; mTORC2, mammalian target of rapamycin complex 2; MS, mass spectrometry; MC, Momordica charantia Linn.; PPAR, peroxisome proliferator-activated receptors; PI3K, phosphoinositide-3-kinase; PDK1, phosphoinositide-dependent kinase 1; SDS−PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

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REFERENCES

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