Multiple Comparisons of Glucokinase Activation Mechanisms of Five

Article pubs.acs.org/JAFC

Multiple Comparisons of Glucokinase Activation Mechanisms of Five Mulberry Bioactive Ingredients in Hepatocyte Hao He,†,‡ Wan-Guo Yu,†,‡ Jun-peng Yang,†,‡ Sheng Ge,*,§ and Yan-Hua Lu*,†,‡

Downloaded by NANYANG TECHNOLOGICAL UNIV on August 28, 2015 | http://pubs.acs.org Publication Date (Web): August 28, 2015 | doi: 10.1021/acs.jafc.5b02823

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State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China ‡ Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China § Clinical Nutrition Department, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai 200233, People’s Republic of China S Supporting Information *

ABSTRACT: Glucokinase (GK) activity, which is rapidly regulated by glucokinase regulatory protein (GKRP) in the liver, is crucial for blood glucose homeostasis. In this paper, the GK activation mechanisms of 1-deoxynojrimycin (DNJ), resveratrol (RES), oxyresveratrol (OXY), cyanidin-3-glucoside (C3G), and cyanidin-3-rutinoside (C3R) were compared. The results revealed that DNJ, RES, C3G, and C3R could differently improve glucose consumption and enhance intracellular GK activities. DNJ and RES significantly promoted GK translocation at 12.5 μM, whereas other ingredients showed moderate effects. DNJ, C3G, and C3R could rupture intramolecular hydrogen bonds of GK to accelerate its allosteric activation at early stage. RES and OXY could bind to a “hydrophobic pocket” on GK to stabilize the active GK at the final stage. Otherwise, RES, OXY, C3G, and C3R could interact with GKRP at the F1P binding site to promote GK dissociation and translocation. Enzymatic assay showed that RES (15−50 μM) and OXY (25−50 μM) could significantly enhance GK activities, which was caused by their binding properties with GK. Moreover, the most dramatic up-regulation effects on GK expression were observed in C3G and C3R groups. This work expounded the differences between GK activation mechanisms, and the new findings would help to develop new GK activators. KEYWORDS: glucokinase, glucokinase regulatory protein, mulberry, glucose homeostasis

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INTRODUCTION Elevated blood glucose after feeding, which is one of the earliest abnormalities of glucose homeostasis accompanied by diabetes, will increase the risk of diabetes complications. Nowadays, more and more therapies focus on improving postprandial and fasting glycemic status.1,2 Thus, much attention has been drawn to hepatic glucose metabolism.3 In the liver, glucokinase (GK) controls glucose disposal and glycogen synthesis;4,5 therefore, it needs to be regulated rapidly along with the elevated blood glucose after feeding.5 It can interact with glucokinase regulatory protein (GKRP) and form a GK−GKRP complex, which is simulated by fructose-6-phosphate (F6P) and reserved by fructose-1-phosphate (F1P).6 During the fasting−refeeding status, an inactive conformation of GK called the “super open” structure6−8 is changed, and the GK dissociates from GKRP followed with translocation to the cytoplasm in an activated status. This process can be promoted by glucokinase activators (GKAs). Unfortunately, recent warnings about the side effects (e.g., hypoglycemia) of antidiabetic drugs such as GKAs9−11 highlight the urgent demands for alternative and safer therapies, ideally through dietary polyphenols. Mulberry (Morus, Moraceae) plants are distributed extensively in China. Fruits and leaves from Morus have been used as medicinal herbs and nutritional food.12 It is reported that mulberry leaf tea can reduce postprandial hyperglycemia in type 2 diabetes patients,13 and dietary consumption of mulberry leaf © XXXX American Chemical Society

powder shows a significant reduction in blood glucose and increases expression of glycolysis-related genes.14,15 The hypoglycemic activity of mulberry resources is mainly due to its active ingredients, namely, 1-deoxynojrimycin (DNJ), cyanidin-3-glucoside (C3G), cyanidin-3-rutinoside (C3R), resveratrol (RES), and oxyresveratrol (OXY) (Figure 1). DNJ, an alkaloid component, is the main hypoglycemic ingredient of mulberry leaf. Pretreatment with DNJ could affect the activity, mRNA, and protein level of GK, meanwhile inhibiting glycogen-degrading enzymes such as amylo-1,6glucosidase.16,17 Park et al. and Ganjam et al.18,19 reported that RES could up-regulate the expression of gluconeogenic genes and down-regulate GK expression by deacetylating forkhead box O1 (FoxO1) in rats. However, other researchers confirmed that hexokinase and glucose-6-phosphatase were reverted to normal levels, and hepatic glycogen content was increased after RES treatment in rats.20,21 There is much confusion about RES’s activities based on these results. Otherwise, mulberry leaves also contain OXY, which is only found in a finite number of plants. Although only a few studies Special Issue: Phytochemicals in Food (ISPMF 2015) Received: June 7, 2015 Revised: August 13, 2015 Accepted: August 20, 2015

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DOI: 10.1021/acs.jafc.5b02823 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

(G8887) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell Culture. The HepG2 cell line was obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured in Dulbecco’s minimum essential medium (DMEM) containing 10% fetal bovine serum at 37 °C in a humidified atmosphere incubator with 5% CO2. The viability of the HepG2 cells was measured via MTT assay. Glucose Consumption (GC) and Glucokinase Activities. The GC analysis was performed according to a modified method of Zhang et al.25 After the cells reached 80%, the medium was removed and fresh DMEM containing different ingredients (25−100 μM) or metformin (100 μM) was added to each well. After 24 h, the medium was collected and the glucose concentrations were determined by a glucose assay kit. Then the cells were lysed by a cell lysis kit (Beyotime, Jiangsu, China) for total protein quantity and hepatic enzyme activity assays. To consider cell proliferation, GC was appraised by calculating the ratio of GC divided by protein contents (GC/mg protein). GK activity was determined as described by Slosberg et al.26 and estimated as differences between 0.5 and 100 mM glucose (nmol min−1 mg−1 of protein). Cell lysis solution and standard enzyme solution were used to determine the hepatic GK activity and direct effects of compounds on GK activities, respectively. Immunofluorescent Staining. The assay of immunofluorescent staining was based on a modified method described previously.10 After the HepG2 cells reached confluence on 12 mm coverslips in maintenance medium (10% FBS, 1 μg mL−1 insulin, 100 nM dexamethasone), cells were starved (4 h) followed by incubation with different concentrations of compounds (20 min). Glucose (10 μL of 25 mM) was then added and incubated for an additional 20 min at 37 °C. After that, cells were fixed in 4% paraformaldehyde for 20 min, followed by 10 min of permeabilizing (0.3% Triton X) and 1 h of blocking (1% BSA). Finally, anti-GK antibody and FITC-labeled antibody were used to discriminate GK. Each incubation step was followed by two washes with PBS for 5 min. Nucleus-to-cytoplasm ratio of GK translocation in HepG2 cells was captured and analyzed by Image-pro plus based on immunofluorescence images. Molecular Docking. The structures of human GK (PDB 1V4S) and GKRP (PDB 4BB9) were obtained from the RCSB protein Data Bank (http://www.pdb.org/pdb/home/home.do.) Five compounds of Morus, Moraceae (i.e., DNJ, RES, OXY, C3G, C3R), were chosen from the National Centre for Biotechnology Information (NCBI) PubChem compound database. Molecule files were downloaded and converted to Protein Data Bank (PDB) coordinates. The Autodock v4.2 program (Autodock, Autogrid) and Autodock tools (ADT) v1.5.4 were employed to analyze the interactions between ligands and target proteins. The analysis was carried out with the Lamarckian Genetic Algorithm (LGA) and extended over the whole GK and GKRP proteins using blinding docking in 250 individuals. After the analysis, a cluster was performed, and the most populated cluster with the lowest energy was deemed as the most reliable conformation. The details of interactions were displayed by using Pymol 0.99 and ligplus v1.4.5. Western Blot Analysis. After the cells were incubated with different ingredients (25−100 μM), total proteins were obtained and quantified as described above. Protein samples were first separated by sodium dodecyl sulfate (SDS)−polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride membranes (PVDF; Millipore, Bedford, MA, USA). After blocking with 5% milk in Tris-buffered saline overnight, GK and GKRP were discriminated by primary antibodies and AP-labeled antibody. Finally, the blots were detected in chromogenic reagent containing BCIP and NBT, followed by photographing. The band intensity was analyzed by Gel-pro analyzer v4.0, and protein contents were normalized by GAPDH. Statistical Analysis. All data were analyzed by using one-way analysis of variance (ANOVA) and expressed as the mean ± SD. A statistical difference of P < 0.05 was considered significant.

Figure 1. Chemical structures of 1-deoxynojirmycin, resveratrol, oxyresveratrol, cyanidin-3-glucoside, and cyanidin-3-rutinoside.

have focused on the antidiabetic effects of OXY, it may also be a potent antidiabetic drug because it is a structural analogue of RES and showed stronger α-glucosidase inhibitory effects than RES in our previous study.22 Moreover, mulberry fruits potentially exhibit hypoglycemic effect and contain many active ingredients including C3G, C3R, and RES.23 Guo et al. reported that C3G lowered fasting glucose level by promoting phosphorylation and nuclear exclusion of FoxO1, which could further up-regulate GK expression.24 Recent studies suggested that these five bioactive ingredients (DNJ, C3G, C3R, RES, and OXY) may show different regulatory activities on hepatic glucose metabolisms. However, previous studies on DNJ, C3G, and RES mainly dealt with their glucose-lowering effects and regulatory effects of enzyme activities. Few studies have focused on their mechanisms of GK activation, and there is no report indicating the differences of their activation mechanisms. In our laboratory, we had compared their contents in mulberry leaves and fruits from eight species in China, and variant α-glucosidase inhibitory effects and mechanisms were revealed.22,23 Which one of these compounds is the most effective GKA? Are their activities derived by different mechanisms? It is meaningful to illuminate these issues for the development of more effective antidiabetic drugs and efficacious utilization of mulberry fruits and leaves.

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MATERIALS AND METHODS

Materials. DNJ, C3G, and C3R were purchased from Shanghai Yaji Biotechnology Co., Ltd. (Shanghai, China). Resveratrol was purchased from Jianfeng-Natural Research and Development Co., Ltd. (Tianjin, China). Oxyresveratrol was isolated and purified in our labortory with individual purity of no less than 98% as described by Song et al.23 Polyclonal antibodies against human GK (sc-7908) and GKRP (sc11416) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT), 4,6-diamidino-2-phenylindole (DAPI), glucose-6-phosphate dehydrogenase (G6PDH), ATP, dithiothreitol (DTT), NAD, insulin, dexamethasone, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (H+L), and alkaline phosphatase (AP)-labeled goat anti-rabbit IgG (H+L) were purchased from Beyotime Institute of Biotechnology (Nanjing, China). Glucokinase B


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RESULTS Effects of Mulberry Ingredients on Glucose Consumption (GC) and Hepatic Enzyme Activities. On the basis of the results of the MTT assay, it is suggested that all ingredients at 25−100 μM had no cytotoxicity on HepG2 cells (Figure S1). As shown in Figure 2A, the GC of HepG2 cells

different degrees and their modulatory effects on GC could be attributed to their glucokinase regulatory role. Mulberry Ingredients Stimulate GK Translocation in HepG2. Specifically, GK in hepatocytes needed to separate from GKRP and translocate to the cytoplasm to show its activities in the feeding state. We next monitored the nucleocytoplasm translocation of GK. At the beginning, most GK located in the nucleus (green fluorescence). DNJ and RES could significantly decrease the nuclear GK fluorescence at 12.5 μM, and the green fluorescence in the nucleus was gradually decreased at higher concentration, which indicated GK translocation from the nucleus to the cytoplasm (Figure 3A,B). For OXY, C3G, and C3R, the changes of fluorescence intensity in nucleus were inconspicuous at 12.5 μM (Figure 3B and Figure S2). As shown in Figure 3C, DNJ and RES could significantly reduce the nuclear−cytoplasm ratio of GK at 12.5 μM, and the ratio was reduced in a dose-dependent manner. OXY, C3G, and C3R could significantly reduce the ratio only at 50 μM. These results suggested that they could activate GK by stimulating its nuclear−cytoplasm translocation. Mulberry Ingredients Induce GK Activation by Binding to GKRP or GK via Different Binding Properties. To clarify the possible mechanisms of activation, molecular docking was employed to analyze the interactions between ligands and target protein (GK and GKRP). As shown in Figure 4A, DNJ could bind to GKRP at a hydrophobic area formed by TYR136−ILE138, GLU162, PHE186, ASN373−GLN374 and GLU377, and two hydrogen bonds were formed with ILE138 and ASN373 (Figure 4E). Otherwise, RES was surrounded by ILE11, PRO29, GLU32, VAL110, GLY181, SER183, ARG215, LYS514, TRP517, and LEU529 in which ILE11 is essential for the formation of hydrogen bonds (Figure 4B,F). In this hydrophobic region, GLY181, SER183, LYS514, and TRP517 were part of an allosteric site of GKRP, which was essential for fructose-1-phosphate (F1P) binding.7 By binding to this site, RES mimicked the effect of F1P that directly induces GK dissociation from GKRP after feeding. OXY and C3G were also exposed to the allosteric site and formed hydrogen bonds with Ile11; however, they may not fully occupy the site because less key residue was found around them (Figure 4C,D,G,H). The conformations of C3R were scattered, and no populated cluster was found. These results suggested that RES, OXY, and C3G could bind to the allosteric site of GKRP and promote GK− GKRP dissociation by binding to GKRP As shown in Figure 5A, DNJ could bind to GK at the allosteric site formed by GLU96, TRP99, SER100, TYP215, GLU216, and ARG447, and hydrogen bonds were formed with GLU96, SER100, GLU216, and ARG447 (Figure 5E). According to a previous report,27 GLU216 and ARG447 played an important role in the conformational transition of GK between the active and inactive states. DNJ ruptured two pairs of hydrogen bonds between residues ARG447−ASP205 and GLU216−LYS458 at the early stage of the GK activation process. Figure 5B,C revealed that RES and OXY could bind to a “hydrophobic pocket” composed of TYR61, VAL62, VAL200, ALA201, MET202, VAL203, LEU451, VAL452, and ALA456, and hydrogen bonds were formed with ARG63 or THR65 (Figure 5F,G). On the basis of Zhang’s study,28 interactions between ASP158, ILE159, and this “hydrophobic pocket” provided the energy barrier for GK conformational change and served as a stabilizer of active GK at the final stage. However, in the OXY group, ASP158 was not found. C3G was also exposed to GLU216 and ARG447, but fewer hydrogen bonds were

Figure 2. Effects of mulberry ingredients on glucose consumption and hepatic glucokinase activities: (A) effects on glucose consumption; (B) effects on glucokinase activities in HepG2. Hepatocytes were incubated with DNJ, RES, OXY, C3G, and C3R (25−100 μM) for 24 h. Each value represents the mean ± SD of triplicate experiments. (∗) P < 0.05 and (∗∗) P < 0.01 as compared with control.

was improved by adding DNJ, RES, C3G, and C3R, whereas OXY showed moderate effects. DNJ improved GC significantly in a dose-dependent manner, and the most effective dose was 100 μM. RES at 25 μM could significantly improve GC. Unfortunately, GC treated with higher concentration RES continued to decline, and the most effective dose was 25 μM. C3G and C3R could significantly increase GC at 25 μM, but less difference was observed between different concentrations. As shown in Figure 2B, the trend of GK activities in DNJ, RES, and OXY groups was similar to GC. The most effective doses of DNJ and RES were 100 and 25 μM, respectively. GK activities were stimulated with the increasing concentration of DNJ and reduced with the increasing concentration of RES, whereas OXY did not show a dose-dependent effect. All doses of C3G and C3R significantly promoted GK activities, and the most effective dose was 100 μM. These results indicated that DNJ, C3G, C3R, and RES could improve GC of HepG2 cells at C


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Figure 3. Effects of mulberry ingredients on the GK translocation in HepG2 cells. After starving for 4 h, the cells were incubated with 12.5−50 μM mulberry ingredients: (A) DNJ; (B) RES + OXY for 20 min at 37 °C. (C) GK nuclear/cytoplasm ratio was calculated on the basis of the immunofluorescence images. Each value represents the mean ± SD of triplicate experiments. (∗) P < 0.05 and (∗∗) P < 0.01 as compared with control.

Effects of Mulberry Ingredients on GK and GKRP Expression. In this section, effects of mulberry ingredients on GK and GKRP expression were analyzed. The GK level was gradually up-regulated with the increasing DNJ concentration (Figure 7A,D). All doses of C3G significantly promoted GK expression in a dose-dependent manner. C3R showed similar effects at 50 and 100 μM. RES (50 and 100 μM) could significantly down-regulate the GK level, and OXY showed a more moderate effect than RES (Figure 7B,D). GKRP expressions were not affected by adding mulberry ingredients (Figure 7C).

found (Figure 5D,H). The conformations of C3R were still scattered and no populated cluster was found. These results suggested that DNJ and C3G promoted dissociation of the GK−GKRP complex, whereas RES stabilized the active state of GK. Effects of Mulberry Ingredients on GK Activities in Vitro. To exclude hormonal interference on hepatic GK, an in vitro enzymatic assay was employed. As shown in Figure 6, DNJ could slightly decrease GK activities, which was consistent with Li’s result,17 but the effect did not reach a significant level. RES and OXY could enhance GK activities (Figure 6) in a dose-dependent manner. RES at 15−50 μM could significantly increase GK activities by 11−39%. OXY at 25−50 μM could increase GK activities by 12−20%. According to the binding properties between ligands (RES and OXY) and GK, RES and OXY stabilized the active GK by binding to the “hydrophobic pocket”. As shown in Figure S3, both C3G and C3R had no significant effect on GK activity. These results suggested that RES and OXY could enhance GK activities.

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DISCUSSION Mulberry (Morus, Moraceae) plants are important economic crops for silkworm breeding in China, and more than 15 species of mulberry are widely planted. Preliminary experiments in vivo demonstrated that it can reduce postprandial hyperglycemia in type 2 diabetes patients.13 For further efficacious utilization of mulberry fruits and leaves, it would be very D


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Figure 4. Docking model predicted interaction details between GKRP and mulberry ingredients: (A, E) DNJ; (B, F) RES; (C, G) OXY; (D, H) C3G. The docking study was carried out by using Autodock tools (ADT) v1.5.4 and the Autodock v4.2 program. The details of interactions were displayed by using Pymol 0.99 and ligplus v1.4.5.

important to expound their biological activities systematically. In this paper, regulatory mechanisms of hepatic glucose metabolism and GK activation were taken into consideration.

Metabolic disturbance of glucose homeostasis is the main manifestation of diabetes. Nowadays, some new antidiabetic drugs take GK as a therapy target because it is especially E


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Figure 5. Docking model predicted interaction details between GK and mulberry ingredients: (A, E) DNJ; (B, F) RES; (C, G) OXY; (D, H) C3G. The docking study was carried out by using Autodock tools (ADT) v1.5.4 and the Autodock v4.2 program. The details of interactions were displayed by using Pymol 0.99 and ligplus v1.4.5.

important for glucose homeostasis.3,29,30 In the pancreas and brain, GK acts as a glucose sensor. However, GK in the liver needs to be activated rapidly to trigger disposal of postprandial

blood glucose in 0.5−2 h.31 During fasting−refeeding status, increasing F1P can bind to GKRP and change its conformation, which makes GK dissociate from GKRP. Meanwhile, two F


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According to our results, five bioactive ingredients, namely, DNJ, RES, OXY, C3G, and C3R, could promote glucose consumption of hepatocytes, which could partly be attributed to activation of GK. According to the results in this paper, their effects may derive by different mechanisms. For DNJ, the GC and GK activities were increased significantly in a dose-dependent manner, and the most effective dose was 100 μM. Docking results (Figure 5A,E) revealed that DNJ could bind to GK at the allosteric site, which contained GLU216 and ARG447 (from GK). According to Huang’s report,27 two hydrogen bonds (ASP205−ARG447 and GLU216−LYS458) of GK were ruptured at the beginning of GK activation to provide energy for the conformational transition of GK. It was suggested that DNJ could cut off these hydrogen bonds and accelerate the conformational transition. Otherwise, binding site of DNJ in GKRP had not been reported before (Figure 4A,E). Moreover, the GK activity was slightly decreased in enzymatic assay (Figure 6), and DNJ significantly up-regulated the GK level (Figure 7D), which was consistent with Li’s result.17 These results suggested that DNJ could promote the conformational transition of GK for dissociation and up-regulating the GK level in hepatocytes. Compared with the DNJ group, the underlying mechanisms of RES were more complicated. The GC and GK activities were increased significantly in the presence of 25 μM RES. However, the promoting effects on GC and GK declined gradually at 50

Figure 6. Effects of DNJ, RES, and OXY on GK activities in vitro. Mulberry ingredients at different concentrations were incubated with the GK enzymes for 15 min before assay. Each value represents a percentage of control and the mean ± SD of triplicate experiments. (∗) P < 0.05 and (∗∗) P < 0.01 as compared with control.

hydrogen bonds of GK (ASP205−ARG447 and GLU216− LYS458) are ruptured, which changes the inactive structure of GK called the superopen state. At the end of activation, a “hydrophobic pocket” was formed to stabilize active GK.

Figure 7. Effects of ingredients on the expression of GK and GKRP. After incubating with different ingredients, the cells were lysed, and GK, GKRP, and GAPDH were analyzed by Western blotting. The relative band intensity was analyzed by Gel-pro analyzer v4.0, and protein contents were normalized by GAPDH. Each value represents the mean ± SD of triplicate experiments. (∗) P < 0.05 and (∗∗) P < 0.01 as compared with control. G


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Journal of Agricultural and Food Chemistry and 100 μM. Elevated GC at 25 μM may be driven by two types of binding properties. On the one hand, RES could bind to GK at the “hydrophobic pocket” composed of TYR61, VAL62, VAL200, ALA201, MET202, VAL203, LEU451, VAL452, and ALA456 (Figure 5B,F) to stabilize the active GK. The hydrophobic pocket was formed at the final stage of GK activation, and the interaction between ASP158, ILE159 and this hydrophobic pocket provided the energy barrier for GK conformational change and served as a stabilizer for active GK.27,28 RES may strengthen the interaction and stabilize the active GK, which was validated in the enzymatic assay. On the other hand, RES could bind to GKRP at an allosteric site that contained GLY181, SER183, LYS514, TRP517 (Figure 4B,F) and accelerate GK dissociation from GKRP. This allosteric site belonged to the F1P binding site. By binding to the allosteric site, F1P, which was an activator of GK, could stabilize the inactive conformation of GKRP with lower affinity for GK.7 Therefore, it was suggested that RES mimicked the role of F1P and dose-dependently increased translocation of GK. Otherwise, the decline of promotion effects at 50−100 μM may also be driven by two types of mechanisms. First, according to Ganjam and Park’s studies,18,19 RES promoted deacetylation of the forkhead transcription factor (FoxO1) and led to nuclear localization of FoxO1 following by the suppression of GK expression. Second, it was reported that dysfunction of GKRP could disrupt the regulation of GK translocation from cytoplasm to nuclear and subsequently lead to degradation of GK in GKRP mutant mice.32 RES bound to GKRP may result in dysfunction of GKRP, which caused the degradation of GK. However, this hypothesis needs to be further investigated. On the basis of the data above, RES at low dose promoted translocation of GK from nuclear to cytoplasm and stabilized the active GK; RES at high dose reduced the intracellular level of GK. As a structural analogue of RES, OXY could also expose GKRP at the F1P binding site and GK at the hydrophobic pocket. However, OXY did not fully occupy the site of F1P because only LYS514 and TRP517 (from GKRP) were found (Figure 4C,G). Moreover, on the basis of the interaction details (Figure 5C,G) between GK and OXY, ASP158 was not found around OXY, which may indicate weaker effects of OXY on GK activities. It was suggested that the weaker activation effect on GK and the down-regulation of GK level may maintain the intracellular GK activities in a homeostasis state. Therefore, aqn inconspicuous effect of OXY on GC was observed. For C3G, both interactions between C3G, GK, and GKRP promoted the translocation of GK from the nuclear to the cytoplasm. A significant increase of GK level (Figure 7D) in the C3G group may be attributed to its promoting effect on nuclear exclusion and phosphorylation of FoxO1.24 All of these effects of C3G significantly increased intracellular GK activities and GC. During the experiment, C3R showed effects similar to those with C3G, but no populated cluster was found in the docking results. We therefore hypothesized that the activities of C3R could partially be attributed to its hydrolyzing product C3G. In this paper, the effects and mechanisms of five mulberry bioactive ingredients (DNJ, RES, OXY, C3G, and C3R) on GK activation were investigated, and variant effects on GC between different groups could be attributed to their effects on GK activities. After treatment with mulberry ingredients, the GK levels in liver cells were significantly changed. However, their potential mechanisms on GK expression were still not clear. In the liver, GK expression is regulated by insulin33 and mainly

relies on the fasting and refeeding states. Many factors, including FoxO1,34 sterol regulatory element binding protein (SREBP1c),35,36 hepatic nuclear factor-4-α (HNF4α),37 and upstream stimulatory factor 1 (USF1),38 are involved in GK gene expression. Moreover, 6-phosphofructo-2-kinase/fructose2,6-biphosphatase (PFK2/FBPase2) could bind GK in the cytoplasm, affecting nuclear to cytoplasm translocation and stabilizing active GK.39,40 Signaling interference such as AMPactivated protein kinase (AMPK) activities correlated inversely with GK translocation.41 Effects of mulberry ingredients on these factors and subsequent GK gene expression and activation will be taken into consideration in our future research. The current GKA research continues to progress. Unfortunately, therapeutic approaches focusing on GK activation are often associated with an unfavorable risk of hypoglycemia.42 Among recent candidates, some design strategies emerge. Liverselective GKAs such as GKM-001, PF-04937319, and GK1-399 are in phase II.43 More recently, two compounds, AMG1694 and AMG3969, are attractive in a recent paper. They specifically disrupt the liver-specific GK−GKRP interaction and avoid hypoglycemic risk. Meanwhile, an unknown binding pocket of GKRP containing ILE11, GLY181, MET213, and ARG525 has been found.44,45 Recently, works on GK−GKRP disruptors, a novel approach for diabetes treatment, are becoming extremely important. In summary, we have compared the hepatic GK regulation effects of five bioactive ingredients (DNJ, RES, OXY, C3G, and C3R), and their impacts on the GK−GKRP complex are further elaborated. This work suggests that they may be used as GK activators to improve postprandial glucose disposal. Dietary intake of mulberry fruits and leaves that are rich in these ingredients can effectively improve diabetes mellitus. Additionally, the potential mechanisms of mulberry ingredients on GK expression need to be investigated in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02823. Effects of mulberry ingredients on HepG2 cell viability, effects of C3G and C3R on GK translocation in HepG2 cells, and effects of C3G and C3R on GK activities in vitro (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(Y.-H.L.) Mail: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Box 283, 130 Meilong Road, Shanghai 200237, People’s Republic of China. E-mail: [email protected]. Phone: +86-2164251185. Fax: +86-21-64251185. *(S.G.) Mail: Clinical Nutrition Department, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, People’s Republic of China. Email: [email protected]. Phone: +86-21-24058352. Funding

This work was supported by the Fundamental Research Funds for the Central Universities (WF1113010) and partially supported by the National High Technology Research and Development Program of China (2013AA092901) and the H


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National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED GK, glucokinase; GKRP, glucokinase regulatory protein; DNJ, 1-deoxynojrimycin; C3G, cyanidin-3-glucoside; C3R, cyanidin3-rutinoside; RES, resveratrol; OXY, oxyresveratrol; GKAs, glucokinase activators; FoxO1, forkhead box O1; DMEM, Dulbecco’s minimum essential medium; PMSF, phenylmethanesulfonyl fluoride


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