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«Molecules 2015, 20, 4516-4529; doi:10.3390/molecules20034516 OPEN ACCESS molecules ISSN 1420-3049 Article ...»

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Molecules 2015, 20, 4516-4529; doi:10.3390/molecules20034516

OPEN ACCESS

molecules

ISSN 1420-3049

www.mdpi.com/journal/molecules

Article

N-Hydroxycinnamide Derivatives of Osthole Ameliorate

Hyperglycemia through Activation of AMPK and p38 MAPK

Wei-Hwa Lee 1,†, Hsueh-Hsia Wu 2,†, Wei-Jan Huang 3, Yi-Ning Li 2, Ren-Jye Lin 4,5,

Shyr-Yi Lin 4,5 and Yu-Chih Liang 2,6,* Department of Pathology, Shuang Ho Hospital, Taipei Medical University, 291 Zhongzheng Rd., New Taipei City 23561, Taiwan; E-Mail: whlpath97616@shh.org.tw School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan;

E-Mails: wuhh@tmu.edu.tw (H.-H.W.); m120097071@tmu.edu.tw (Y.-N.L.) Graduate Institute of Pharmacognosy Science, College of Pharmacy, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan; E-Mail: wjhuang@tmu.edu.tw Department of Primary Care Medicine, Taipei Medical University Hospital, 252 Wuxing St., Taipei 11031, Taiwan; E-Mails: linrenjye@tmu.edu.tw (R.-J.L.); sylin@tmu.edu.tw (S.-Y.L.) Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, 252 Wuxing St., Taipei 11031, Taiwan † These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: ycliang@tmu.edu.tw;

Tel.: +886-2-2736-1661 (ext. 3318); Fax: +886-2-2739-3447.

Academic Editor: Derek J. McPhee Received: 20 January 2015 / Accepted: 5 March 2015 / Published: 11 March 2015 Abstract: Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK.

The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly Molecules 2015, 20 4517 reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes.

Keywords: osthole; N-hydroxycinnamide; AMP-activated protein kinase (AMPK); p38 MAPK; glucose uptake; skeletal muscle; streptozotocin

1. Introduction AMP-activated protein kinase (AMPK), a serine/threonine kinase, plays a central role in regulating glucose and lipid metabolism and functions as a fuel gauge [1]. In skeletal muscles, the liver, and adipose tissues, activation of AMPK can increase metabolism, insulin sensitivity, and gene expression, and all of these effects are beneficial in preventing type 2 diabetes. Skeletal muscles have a primarily important role in maintaining normal glucose homeostasis, and type 2 diabetes is characterized by insulin resistance in skeletal muscles [2]. Glucose uptake is mainly mediated by glucose transporter 4 (GLUT4) and plays a crucial step in glucose utilization by insulin stimulation and muscle contractions [3]. Activation of phospatidylinositol-3 kinase (PI3K)/Akt and AMPK signal pathways, respectively, lead to increased insulin- and contraction-stimulated GLUT4 translocation in muscles [4].

Previous studies found that many antihyperglycemic chemicals can activate AMPK through different mechanisms, such as antidiabetic drugs, such as metformin [5] and troglitazone [6], and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) [7], as well as natural polyphenols, such as resveratrol [8], curcumin [9], and epigallocatechin gallate [10].

p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family, and has wide-spectrum roles in controlling energy metabolism of adipose tissues, skeletal muscles, islet cells, and the liver [11]. Activation of p38 MAPK participates in stimulation of glucose uptake by both insulin and contraction stimuli in skeletal muscles [12]. As AMPK activators, many natural and synthesized chemicals are also able to increase glucose uptake and improve hyperglycemia through activation of p38 MAPK in different experimental models. In db/db diabetic mice, the synthesized compound, biaryl-4-carbonitrile, increased the phosphorylation of AMPK and p38 MAPK and ameliorated glucose uptake through the AMPK/p38 MAPK pathway [13]. The natural compound, capsaicin, also stimulated the phosphorylation of AMPK and p38 MAPK and subsequently increased glucose uptake by skeletal muscle cells [14].





Osthole is extracted from the Chinese herbs Cnidium monnieri and Angelica pubescens, and exhibits many biological activities such as antitumor [15], anti-inflammatory [16], antiosteoporosis [17], and antihyperlipidemic functions [18]. Recently, we found that osthole can ameliorate hyperglycemia Molecules 2015, 20 4518 in both db/db mice and mice with streptozocin (STZ)-induced diabetes, and the underlying mechanisms involved in these effects are associated with activation of AMPK and peroxisome proliferator-activated receptors α/γ (PPARα/γ) [19,20]. To improve the antihyperglycemic activity of osthole, OHC-4p and OHC-2m were semi-synthesized from osthole in this study. OHC-4p and OHC-2m were compared to osthole to evaluate their antihyperglycemic activities in skeletal muscle cells and understand the underlying molecular mechanisms.

2. Results and Discussion

2.1. OHC-4p and OHC-2m Were Superior to Osthole in Activating AMPK and Increasing Glucose Uptake in Skeletal Muscle Cells A series of N-hydroxycinnamide derivatives of osthole and a osthole-derived phenyl-Nhydroxycarboxamate (pOHCON) were synthesized (Figure 1b–d), and their ability to activate AMPK in differentiated L6 skeletal muscle cells was examined. As shown in Figure 1a, OHC-2p, OHC-4p, and OHC-2m exhibited greater abilities than the other osthole derivatives in increasing AMPK phosphorylation. The N-hydroxycinnamide had no any effect on the induction of AMPK phosphorylation (data not shown). Next, a pilot animal study was performed, and we found that OHC-4p and OHC-2m were able to improve hyperglycemia in mice with STZ-induced diabetes, while OHC-2p was not. Therefore, OHC-4p and OHC-2m were used in the subsequent experiments of this study. Compounds OHC-4p and OHC-2m were structurally characterized as para-osthole N-hydroxycinnamide with a chain-length of four carbons and meta-osthole N-hydroxycinnamide with a chain-length of two carbons, respectively (Figure 1d).

Figure 1. Cont.Molecules 2015, 20 4519

Figure 1. Screening of a series of N-hydroxycinnamide derivatives of osthole on the phosphorylation of AMPK and the chemical structures of (b) osthole and (c) osthole-derived phenyl-N-hydroxycarboxamate (pOHCON), and (d) various kinds of osthole-derived N-hydroxycinnamides.

(a) Cells were treated with 10 µM of various kinds of osthole derivatives for 3 h. Total cell lysates were used to determine phosphorylation levels of AMPK by western blotting. Metformin (Met) was used as a positive control.

Next, the abilities of OHC-4p and OHC-2m to activate AMPK and glucose uptake in differentiated L6 skeletal muscle cells were compared to the parental chemical, osthole. OHC-4p and OHC-2m produced greater increases in the phosphorylation of AMPK and its downstream mediator, acetyl-CoA carboxylase (ACC), as well as glucose uptake in differentiated L6 skeletal muscle cells compared to osthole (Figure 2a,b). Both OHC-4p and OHC-2m dose-dependently increased glucose uptake (Figure 2c). At 1~10 µM of OHC-4p and OHC-2m, we found no significant cytotoxicity during 24 h of treatment of differentiated L6 skeletal muscle cells. These results suggest that OHC-4p and OHC-2m are superior to osthole in activating AMPK and increasing glucose uptake by skeletal muscle cells.

2.2. OHC-4p and OHC-2m Stimulated Glucose Uptake that Depended on Activation of AMPK and p38 MAPK in Skeletal Muscle Cells It is known that activation of AMPK plays an important role in increasing glucose uptake [1].

OHC-4p and OHC-2m treatments significantly induced the phosphorylation of AMPK and ACC in differentiated L6 skeletal muscle cells (Figure 3a). Two maximal responses were seen at 3 and 24 h with both OHC-4p and OHC-2m treatments. There was no significant change in the AMPK phosphorylation during the culture period in the cells without drug treatment (Supplementary Figure S1).

In addition, OHC-4p and OHC-2m treatments dose-dependently increased the phosphorylation of AMPK and ACC at 3 and 24 h (Figure 3b,c). To confirm the involvement of AMPK in OHC-4p- and OHC-2m-stimulated glucose uptake by L6 skeletal muscle cells, we treated cells with the selective AMPK inhibitor, compound C. As shown in the lower panel of Figure 3d, 10 µM compound C indeed decreased phosphorylation levels of AMPK and ACC in both OHC-4p- and OHC-2m-treated cells.

Moreover, OHC-4p- and OHC-2m-induced glucose uptake levels were significantly reversed by compound C treatment (Figure 3d, lower panel).

Molecules 2015, 20 4520 Figure 2. Comparisons of glucose uptake and AMP-activated protein kinase (AMPK) activation in differentiated L6 skeletal muscle cells between osthole and osthole derivatives.

(a) Cells were treated with 1 or 10 µM of osthole, OHC-4p, or OHC-2m for 3 h. Total cell lysates were used to determine the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC) by western blotting. (b,c) Cells were incubated with (b) 10 µM of osthole, OHC-4p, or OHC-2m for 3 h, and (c) various concentrations of OHC-4p or OHC-2m for 3 h, and then 2-NBDG uptake was determined as described in the Experimental Section. Data are presented as the mean ± S.D. of three independent experiments.

* p 0.05 vs. the control; # p 0.05 vs. osthole treatment. Insulin (1 µM) was used as a positive control. p-ACC, phosphorylated ACC; p-AMPK, phosphorylated AMPK; t-ACC, total protein of ACC; t-AMPK, total protein of AMPK. Os, osthole.

Figure 3. Cont.Molecules 2015, 20 4521

Figure 3. Involvement of AMP-activated protein kinase (AMPK) in glucose uptake induced by OHC-4p and OHC-2m in differentiated L6 skeletal muscle cells.

(a) Cells were treated with 10 µM of OHC-4p or OHC-2m for different time periods; (b,c) Cells were treated with various concentrations of OHC-4p or OHC-2m for (b) 3 or (c) 24 h. Total cell lysates were used to determine the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC) by western blotting. Metformin (Met) was used as a positive control.

(d) Cells were treated with 10 µM OHC-4p or OHC-2m in the presence or absence of compound C (Comp. C, 10 µM) for 3 h and then 2-NBDG uptake was determined as described in the Experimental Section. Data are presented as the mean ± S.D. of three independent experiments. * p 0.05 vs. the control; # p 0.05 vs. OHC-4p alone or OHC-2m alone. p-ACC, phosphorylated ACC; p-AMPK, phosphorylated AMPK; t-ACC, total protein of ACC; t-AMPK, total protein of AMPK; p-Akt, phosphorylated Akt; t-Akt, total protein of Akt; Os, osthole.

A previous study also demonstrated that p38 MAPK activation is involved in stimulating glucose uptake in response to insulin and muscle contractions [13]. We next investigated whether OHC-4p and OHC-2m stimulated glucose uptake through activation of p38 MAPK. After treatment with OHC-4p or OHC-2m, p38 MAPK phosphorylation reached a peak at 3 h and persisted from 12 to 24 h (Figure 4a).

Molecules 2015, 20 4522 There was no significant change in the p38 phosphorylation during the culture period in the cells without drug treatment (Supplementary Figure S1). OHC-4p and OHC-2m also dose-dependently increased p38 MAPK phosphorylation at 3 and 24 h (Figure 4b,c). Moreover, OHC-4p- and OHC-2m-increased glucose uptake was inhibited by the p38 MAPK inhibitor, SB203580 (Figure 4d, lower panel).

Figure 4. Involvement of p38 mitogen-activated protein kinase (MAPK) in the glucose uptake induced by OHC-4p and OHC-2m in differentiated L6 skeletal muscle cells.

(a) Cells were treated with 10 µM of OHC-4p or OHC-2m for different time periods; (b,c) Cells were treated with various concentrations of OHC-4p and OHC-2m for (b) 3 or (c) 24 h. Total cell lysates were used to determine phosphorylation levels of p38 MAPK by western blotting. (d) Cells were treated with 10 µM OHC-4p or OHC-2m in the presence or absence of SB203580 (SB, 10 µM) for 3 h, and then 2-NBDG uptake was determined as described in the Experimental Section. Data are presented as the mean ± S.D. of three independent experiments. * p 0.05 vs. the control; # p 0.05 vs. OHC-4p alone or OHC-2m alone. p-p38 MAPK, phosphorylated p38 MAPK.

–  –  –

activation of both AMPK and p38 MAPK, and the subsequent increase in GLUT4 translocation in skeletal muscles.

Figure 5. Effects of OHC-4p and OHC-2m on glucose transporter (GLUT) 4 translocation in differentiated L6 skeletal muscle cells.

Cells were treated with 1 or 10 µM of OHC-4p or OHC-2m for 3 h. Equal amount of proteins (50 µg) from plasma membrane fractions were used to determine GLUT4 and GLUT1 (internal control) expression by western blotting.

2.3. OHC-4p and OHC-2m Improved Hyperglycemia in Mice with STZ-Induced Diabetes

–  –  –

* # #

–  –  –

Figure 6. Effects of OHC-4p and OHC-2m on blood glucose levels of mice with streptozotocin (STZ)-induced diabetes mellitus.



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