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Coumarins ameliorate diabetogenic action of dexamethasone via Akt activation and AMPK signaling in skeletal muscle
Journal of Pharmacological Sciences 139 (2019) 151e157
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Coumarins ameliorate diabetogenic action of dexamethasone via Akt activation and AMPK signaling in skeletal muscle Zejun Mo a, b, 1, Linghuan Li a, 1, Haiwen Yu a, Yingqi Wu a, Hanbing Li a, c, * a
Institute of Pharmacology, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, People's Republic of China c Section of Endocrinology, School of Medicine, Yale University, New Haven 06520, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2018 Received in revised form 29 December 2018 Accepted 9 January 2019 Available online 23 January 2019
Glucocorticoids are widely prescribed for lots of pathological conditions, however, can produce ‘Cushingoid’ side effects including central obesity, glucose intolerance, insulin resistance and so forth. Our study is intended to investigate the improving effects of coumarins on diabetogenic action of dexamethasone in vivo and in vitro and elucidate potential mechanisms. ICR mice treated with dexamethasone for 21 days exhibited decreased body weight, increased blood glucose and impaired glucose tolerance, which were prevented by fraxetin (40 mg/kg/day), esculin (40 mg/kg/day) and osthole (20 mg/ kg/day), respectively. Esculin, fraxetin and osthole also could promote glucose uptake in normal C2C12 myotubes, and improve insulin resistance in myotubes induced by dexamethasone. Western blotting results indicated that esculin, fraxetin and osthole could boost Akt activation, stimulate GLUT4 translocation, thus alleviate insulin resistance. Esculin and osthole also could activate AMPK, thereby phosphorylate TBC1D1 at Ser237, and consequently ameliorate diabetogenic action of dexamethasone. Our study indicates coumarins as potential anti-diabetic candidates or leading compounds for drug development. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Coumarin Osthole Esculin Fraxetin Insulin resistance Dexamethasone
1. Introduction The metabolic syndrome is a clustering of the following conditions including central obesity, hypertension, impaired glucose tolerance or diabetes, and dyslipidemia with high plasma concentrations of triglycerides and low levels of high-density lipoprotein cholesterol, which can contribute to cardiovascular risk.1 Insulin is a key hormone to clear the postprandial glucose load and maintain homeostasis of plasma glucose level. Insulin resistance is a progressive process of decreasing the sensitivity of insulin on target tissues, which is the pathological basis of metabolic syndrome.2 Glucocorticoids (GCs), such as dexamethasone and prednisolone, are commonly used as an anti-inflammatory, anti-allergic and immunosuppressive drug. However, the majority of these patients
* Corresponding author. Institute of Pharmacology, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China. Fax: þ86 571 88320535. E-mail addresses: [email protected], [email protected] (H. Li). Peer review under responsibility of Japanese Pharmacological Society. 1 Zejun Mo and Linghuan Li contributed equally to this paper.
suffer from ‘Cushingoid’ side effects,3 and chronic elevated GCs levels could cause central obesity and insulin resistance. Diabetes has been considered to be a common complication of chronic excess of GCs and it is an important factor contributing to the morbidity and mortality of the patients. GCs-induced diabetic like rodents displayed hyperglycaemia, dyslipidemia and impairment of insulin sensitivity in hepatic and peripheral tissues.4e6 In skeletal muscle, GCs can decrease insulin-mediated glucose uptake through impairing insulin signaling pathway, curtailing expression and activity of insulin receptor, insulin receptor substrate and PI3K.7,8 Furthermore, GCs lead to the accumulation of ectopic fat deposition in skeletal muscle and elevation of free fatty acid,9 which also can induce insulin resistance.10,11 In addition, local GCs metabolism via 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) plays an important role in contributing to adverse effects of excess GCs.12,13 Coumarins are famous as anticoagulant drugs, such as warfarin, dicoumarolum, acenocoumarol and so on. Recently, it has been discovered that some plants contain coumarins or some coumarin derivatives could improve insulin resistance and diabetic complications.14e17 For example, esculin and fraxetin reduced plasma glucose, diminished glucose-6-phosphatase activity and
https://doi.org/10.1016/j.jphs.2019.01.001 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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improved renal dysfunction.18e21 Scopoletin and osthole could ameliorate insulin resistance in HepG2 cells by upregulating PPARg2 expression,22,23 osthole also increased adiponectin release in fatty liver rats.24 Lee et al reported that osthole could enhance glucose uptake through activation of AMPK in skeletal muscle in vitro.25 However, in vivo effects and potential mechanisms of coumarins improving insulin resistance induced by GCs is lack of study. Thus, we investigated the effects of esculin, osthole and fraxetin upon insulin resistance in mice induced by dexamethasone, especially the effects of these coumarins on glucose disposal in skeletal muscle, which accounts for >80% of the entire body's glucose disposal under insulin stimulation,26 and potential mechanisms of these effects as well. 2. Materials and methods 2.1. Materials Esculin, osthole and fraxetin were purchased from Yongjian Pharmaceutical Company (Taizhou, China) with purity of 99.7%, 99.1% and 99.9%, respectively; insulin and dexamethasone were provided by Sigma (St. Louis, MO, USA); Metformin was from Shanghai Sine Pharma (Shanghai, China). The following items were purchased from the cited commercial sources: Akt antibody and Akt (phospho-Ser473) antibody, Anbo (San Francisco, USA); AMPK antibody and AMPK (phospho-Thr172) antibody, Santa Cruz (Dallas, USA); TBC1D1 antibody, Proteintech (Chicago, USA); TBC1D1 (phospho-Ser237) antibody, Millipore (Temecula, USA); GAPDH antibody, Beyotime (Shanghai, China); GLUT4 antibody and Goat anti-rabbit IgG, Boster (Wuhan, China). 2.2. Cell culture and differentiation The C2C12 cell line obtained from ATCC (Manassas, USA), were cultured in DMEM (Gibco, California, USA) with 10% (v/v) FBS (Hyclone, Logan, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 C. Cells were grown to 70e80% confluency, and then the medium was switched to DMEM with 2% (v/v) HS (Hyclone, Logan, USA) and changed every 2 days.27
and fraxetin (40 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 14 days; Osthole group: gum acacia (10 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 7 days and osthole (20 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 14 days; Metformin group: gum acacia (10 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 7 days and metformin (250 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 14 days. 2.5. Oral glucose tolerance test and intravenous glucose tolerance test Oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IvGTT) were performed after an overnight fast. For OGTT, the baseline blood glucose level was detected at 0 min, followed by an oral glucose load of 2 g/kg, and blood glucose levels was checked again at 15, 30, 60, 120 min. For IvGTT, The blood glucose were detected before glucose (1 g/kg, i.v.) injection, and the blood glucose levels were monitored at 1, 5, 10, 20, 50, 75min after glucose loading.29,30 Blood glucose was checked with using Onetouch Ultraeasy (Johnson, New Jersey, USA).The area under the curve for blood glucose was calculated by GraphPad Prism 5. 2.6. Cells treatment and detection of glucose uptake Cells were treated in the absence or presence of increasing dose dexamethasone over a time course of 24 h to select a suitable dose to induce cells to decrease the ability of glucose uptake. In set of experiments, cells were incubated with different concentrations (25, 50, 100 mM) of esculin, fraxetin and osthole for 24 h alone or along with dexamethasone stimulation. Cell viability was tested by MTT method and glucose uptake in the presence or absense of insulin (final concentration of 100 nM) was characterized by glucose consumption which was detected by the glucose oxidase/ peroxidase (GOD-POD) method.11,31 As for glucose consumption assay, the glucose concentration at 0 and 24 h in the culture medium was determined by the glucose oxidase assay kit (Jiancheng Bioengineering Institute, Nanjing, China), and the difference between the two concentrations was regarded as the amount of consumed glucose. 2.7. Western blotting
2.3. Animals Male Institute of Cancer Research (ICR) mice (7e8wk of age) were supplied by the Zhejiang Academy of Medical Sciences [License Number: SCXK (Zhe) 2014-0001]. The mice were maintained in sanitized polypropylene cages (6 per cage) under standard conditions of temperature (23 ± 2 C), relative humidity (55 ± 5%) and light 12/12 h light/dark cycle with free access to food and water. All animals were treated in compliance with the present institutional guidelines for animal care and use. 2.4. Dexamethasone-induced insulin resistance in mice and treatment After several days of adaptation, body weight and blood glucose were tested before treatment and randomized into 6 groups. The mice were treated for 21 days according to the following protocol as described previously4,28 with some modification: Control group: gum acacia (10 mg/kg/day, p.o.) for 21 days; Dexa group: gum acacia (10 mg/kg/day, p.o.)þdexamethasone (1 mg/kg/day, i.m.) for 21 days; Esculin group: gum acacia (10 mg/kg/day, p.o.)þdexamethasone (1 mg/kg/day, i.m.) for 7 days and esculin (40 mg/kg/day, p.o.)þ dexamethasone (1 mg/kg/day, i.m.) for 14 days; Fraxetin: gum acacia (10 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 7 days
Thirty minutes after being injected with insulin (0.5 IU/kg, i.p.),10 the mice of 6 groups were killed by cervical dislocation and gastrocnemius muscle was immediately isolated and homogenized in ice-cold lysis buffer to extract the protein. Lysates were centrifuged at 14,000 g for 5 min at 4 C and supernatants were collected. The protein concentration was determined by Bicinchoninic Acid protein assay kit with BSA as a standard (Beyotime, Shanghai, China). Protein samples were mixed with loading buffer and boiled for 5min, then resolved by SDS-PAGE and transferred onto PVDF membranes (Beyotime, Shanghai, China) in a semidry transfer system (Bio-Rad, Hercules, USA). PVDF membranes were blocked with freshly prepared 5% BSA or nonfat milk in Trisbuffered saline containing 0.05% Tween-20 surfactant for 60 min at room temperature, immunoblotted with the primary antibodies for 4 h at room temperature or overnight at 4 C, washed with TBST for 3 times and incubated with secondary antibodies for 2 h at room temperature, and finally visualized with enhanced chemiluminescence method. 2.8. Histopathological studies Skeletal muscles of experimental mice were fixed in 10% formalin, dehydrated with gradient ethanol, subsequently
Z. Mo et al. / Journal of Pharmacological Sciences 139 (2019) 151e157
embedded in paraffin, cut into 5 mm thickness using a microtome and stained with haematoxylin-eosin. Tissue structures were observed under light microscopy. 2.9. Statistical analysis Values were given as mean ± standard deviation (SD). Statistical significance of differences in measurements between samples was assessed by one-way analysis of variance (ANOVA) with Tukey's post hoc test using GraphPad Prism 5. P < 0.05 was considered statistically significant. 3. Results 3.1. Coumarins ameliorated diabetogenic action of dexamethasone in mice To investigate the effects of coumarins upon diabetogenic action of dexamethasone in mice, we tested fasting blood glucose and glucose tolerance in mice, with metformin as a positive control. The mice treated with dexamethasone (1 mg/kg/day, i.m.) exhibited a decrease of body weight at day 7, which became significant at day 14 and 21 compared with normal control group (p < 0.001) as shown in Table 1. Administration with esculin (40 mg/kg/day), fraxetin (40 mg/kg/day), osthole (20 mg/kg/day) or metformin (250 mg/kg/day) prevented the fall of body weight induced by dexamethasone (Table 1, p < 0.05). Meanwhile, the mice treated with dexamethasone exhibited an increase of blood glucose level at day 7, which became significant at day 15 and 22 compared with normal control group weight (p < 0.05 and p < 0.001, respectively). Treatment with esculin, fraxetin, osthole or metformin reduced the blood glucose level in mice. Dexamethasone impaired glucose tolerance, evidenced by delayed clearance of blood glucose after glucose load compared to normal control group (Fig. 1A, C). The AUC of Dexa group was significantly bigger than that of normal control group in IvGTT (Fig. 1D, p < 0.001), while not significantly in OGTT (Fig. 1B), similar with the report by Cummings,32 possibly due to less intestinal absorption by inhibiting the GLUT2 translocation to the apical membrane.33,34 Administration with esculin, fraxetin, osthole or metformin significantly improved glucose tolerance (p < 0.05). 3.2. Coumarins augmented glucose uptake in normal C2C12 myotubes or insulin-stimulated glucose uptake in dexamethasonetreated cells To validate the effects of coumarins on glucose disposal contributing to blood glucose homeostasis, we investigated the
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effect of coumarins upon glucose uptake in normal C2C12 myotubes and insulin-stimulated glucose uptake in dexamethasonetreated myotubes. As shown in Table 2A, dexamethasone (0.01, 0.1, 1 mM) dose-dependently inhibited glucose uptake in C2C12 myotubes, and only 1 mM dexamethasone decreased the viability of C2C12 myotubes (p < 0.05), so 0.1 mM dexamethasone was adopted to induce insulin resistance in myotubes. To investigate the impact of esculin, fraxetin and osthole on cell viability, myotubes were incubated with different concentrations (25, 50, 100 mM) of esculin, fraxetin or osthole for 24 h, and cell viability was detected by MTT method. As illustrated in Table 2B, esculin and fraxetin had no influence on cell viability, but 50 and 100 mM osthole significantly reduced cell viability. Then tests for glucose uptake displayed that 25 mM esculin and osthole stimulated glucose uptake around 1.6-fold in myotubes compared with normal control, which is similar to 100 nM insulin, whereas not significant in fraxetin group Table 2C. As indicated in Table 2D, esculin (50, 100 mM), fraxetin (100 mM) and osthole (25 mM) could prevent reduced insulin-stimulated glucose disposal in dexamethasonetreated C2C12 myotubes. 3.3. Coumarins improved histopathological changes of skeletal muscle In normal control, regular and tightly connected skeletal muscle fibres with regularly marshalled nucleus were observed. However, in dexamethasone-treated mice, the nucleus arrangement was irregular and there was a clear internal shift, which was improved to a certain extent by esculin, fraxetin, osthole and especially by metformin (Fig. 2). 3.4. Coumarins stimulated GLUT4 translocation in skeletal muscle In skeletal muscle, GLUT4 translocation to the plasma membrane is a key step for glucose uptake, thus we investigated the effects of esculin, osthole and fraxetin upon GLUT4 expression and translocation in skeletal muscle in dexamethasone-treated mice. As exhibited in Fig. 3A. Dexamethasone significantly decreased GLUT4 translocation manifested as the ratio of membranous GLUT4 to cell lysate GLUT4, which was prevented by esculin (p < 0.001), fraxetin (p < 0.05), osthole (p < 0.001), and metformin (p < 0.001), respectively. While the expression of cell lysate GLUT4 was not significantly different between these groups. 3.5. Coumarins promoted Akt activation in skeletal muscle Akt activation plays a crucial role in insulin signaling pathway. Therefore, we examined whether esculin, fraxetin and
Table 1 Effects of esculin (40 mg/kg/day), fraxetin (40 mg/kg/day) and osthole (20 mg/kg/day) upon body weight and blood glucose of mice treated with dexamethasone (Dexa), with metformin (250 mg/kg/day) as a positive control. Body weight of all mice and blood glucose of 6 mice per group at random were monitored every week to confirm the establishment of the model of insulin resistance induced by Dexa. Parameter
Control
Body Weight (g) Day 0 24.03 ± 1.11 Day 7 26.09 ± 0.69 Day 14 27.62 ± 0.70 Day 21 28.04 ± 0.70 Blood Glucose (mmol/L) Day 0 4.42 ± 0.64 Day 7 4.56 ± 0.48 Day 15 4.88 ± 0.45 Day 22 4.93 ± 0.79
Dexa þ Esculin
Dexa 24.49 24.03 23.42 22.48 4.71 4.87 5.69 6.59
± ± ± ±
± ± ± ±
1.99 1.47 1.30*** 1.20***
0.46 0.48 0.54* 0.62***
24.59 23.78 23.91 24.33 4.35 4.83 4.77 4.90
± ± ± ±
± ± ± ±
Dexa þ Fraxetin
2.50 1.95 1.77 1.63#
24.21 23.87 23.89 24.35
0.74 0.29 0.55# 0.25###
4.27 4.93 4.65 5.00
± ± ± ±
± ± ± ±
1.61 1.27 1.94 1.75#
1.01 0.55 0.28## 0.63##
Dexa þ Osthole 24.50 23.90 24.27 24.83 4.67 4.90 4.92 4.98
± ± ± ±
± ± ± ±
1.27 1.24 1.55 1.04##
0.58 0.26 0.72 0.76##
Dexa þ Metformin 24.31 23.68 23.93 24.34 4.69 4.83 4.85 4.97
± ± ± ±
± ± ± ±
2.42 1.48 1.18 1.61#
0.33 0.46 0.41# 0.64##
Data are expressed as mean ± SD. *p<0.05 vs Control; ***p<0.001 vs Control; #p<0.05 vs Dexa; ##p<0.01 vs Dexa; ###p<0.001 vs Dexa. n ¼ 12 for body weight, n ¼ 6 for blood glucose.
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Fig. 1. Effect esculin (40 mg/kg/day), fraxetin (40 mg/kg/day), osthole (20 mg/kg/day) and metformin (250 mg/kg/day) on OGTT and IvGTT in Dexa-treated mice. Mice were treated or not (control) with daily injection of Dexa (1 mg/kg, i.m.) for 7 days. Subsequently, mice received or not the indicated doses of metformin, esculin, fraxetin or osthole for 14 days along with injection of Dexa. At the end of the experiment, an oral glucose tolerance test and an intravenous glucose tolerance test were performed as described in Materials and Methods. A: OGTT, B: The AUC of OGTT. Values were given as mean ± SD. C: IvGTT, D: The AUC of IvGTT. Values were given as mean ± SD. n ¼ 6 ###p < 0.001 vs Control,*p < 0.05 vs Dexa; **p < 0.01 vs Dexa; ***p < 0.001 vs Dexa.
Table 2A Effects of coumarins upon glucose uptake and cell viability in C2C12 myotubes. A. Effects of different concentrations of Dexa on glucose uptake and cell viability in C2C12 myotubes.
Relative glucose uptake Relative cell viability
Control
Insulin
Dexa (mM) 0.01
0.10
1.00
100.00 ± 19.31 100.00 ± 3.43
160.93 ± 26.77### 105.07 ± 4.78
144.53 ± 31.77 101.01 ± 7.07
110.43 ± 30.68* 102.97 ± 5.00
103.30 ± 19.42** 93.33 ± 7.21*
Each value is expressed as means ± SD (n ¼ 6), ###p < 0.001 vs Control group, *p < 0.05, **p < 0.01 vs Insulin control group.
Table 2B Effects of different concentrations of esculin, osthole and fraxetion on cell viability in normal C2C12 myotubes. Parameter (%)
Coumarins (mM)
Control
100.00 ± 4.42 100.00 ± 7.04 100.00 ± 4.92
Esculin Fraxetin Osthole
25
50
100
101.76 ± 1.74 98.98 ± 13.05 97.77 ± 2.12
96.51 ± 5.27 98.63 ± 8.76 82.78 ± 12.36***
93.88 ± 3.79 98.09 ± 6.42 65.22 ± 24.57***
Each value is expressed as means ± SD. ***p < 0.001 vs Control group.
Table 2C Effects of different concentrations of esculin, osthole and fraxetion on glucose uptake in normal C2C12 myotubes. Parameter (%)
Control
Insulin
Esculin Fraxetin Osthole
100.00 ± 27.71 100.00 ± 17.57 100.00 ± 17.57
150.85 ± 16.90** 162.58 ± 13.82* 162.58 ± 13.82***
Coumarins (mM) 25
50
100
166.25 ± 25.16*** 99.75 ± 37.32 149.94 ± 19.67***
125.58 ± 25.93 122.39 ± 27.26 /
125.59 ± 24.98 120.82 ± 41.44 /
Each value is expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs Control group.
osthole could increase insulin-mediated Akt phosphorylation in skeletal muscle in dexamethasone-treated mice. As illustrated in Fig. 3B, dexamethasone significantly curtailed the
phosphorylation of Akt (p < 0.05), which was restored by esculin (p < 0.05), fraxetin (p < 0.05), osthole (p < 0.01) and metformin (p < 0.01).
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Table 2D Effects of different concentrations of esculin, osthole and fraxetin on insulin-stimulated glucose uptake in Dexa-treated C2C12 myotubes. Parameter (%)
Insulin-control
Dexa þ Coumarins (mM)
Dexa
25 Esculin Fraxetin Osthole
100.00 ± 24.00 100.00 ± 12.84 100.00 ± 20.67
**
50.07 ± 9.39 60.02 ± 15.66** 62.54 ± 12.69***
60.61 ± 16.63 51.40 ± 13.17 89.43 ± 25.85##
50 103.21 ± 13.44 59.34 ± 11.36 /
100 ###
85.00 ± 23.01## 94.87 ± 20.69# /
Each value is expressed as means ± SD. **p < 0.01, ***p < 0.001 vs Insulin-control group, #p < 0.05, ##p < 0.01, ###p < 0.001 vs Dexa group, n ¼ 6.
Fig. 2. Histological observation of skeletal tissues in experimental mice (HE staining). A: Normal control, B: Dexa-treated mice. C, D, E, F: esculin (40 mg/kg/day), fraxetin (40 mg/kg/ day), osthole (20 mg/kg/day) and metformin (250 mg/kg/day), respectively.
3.6. Esculin and osthole increased AMPK and TBC1D1 phosphorylation in skeletal muscle Metformin is an effective drug for type 2 diabetes treatment due to its ability to activate AMPK, another pathway to promote glucose disposal independently of insulin signaling.35 Given the effects of esculin, osthole and fraxetin to stimulate glucose uptake in normal C2C12 myotubes as shown in Table 2, we performed western blotting to investigate whether esculin, osthole or fraxetin could affect AMPK activation in skeletal muscle in dexamethasonetreated mice. As demonstrated in Fig. 3C, dexamethasone could suppress AMPK phosphorylation (p < 0.01), which was enhanced by esculin (p < 0.05), osthole (p < 0.05), and metformin (p < 0.05), whereas not significantly by fraxetin.
TBC1D1 phosphorylation at Ser237 is dependent on AMPK activation,36 thus we examined the effects of esculin, fraxetin and osthole on TBC1D1 phosphorylation at Ser237, a downstream substrate protein of AMPK. As predicted, esculin (p < 0.01), osthole (p < 0.001) and metformin (p < 0.001) could prevent from downregulating decreased TBC1D1 phosphorylation (p < 0.05) in skeletal muscle induced by dexamethasone, but not by fraxetin. 4. Discussion Insulin resistance is a key characteristic of the metabolic syndrome including central obesity, diabetes, dyslipidaemia and so forth, in which insulin-responsive tissues fail to respond to the normal level of insulin, to dispose blood glucose load and
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Fig. 3. Effects of Esculin, fraxetin and osthole on GLUT4 translocation, Akt phosphorylation at Ser473, AMPK and TBC1D1 phosphorylation in skeletal muscle in Dexa-treated mice. Mice were orally received with esculin (40 mg/kg/day), fraxetin (40 mg/kg/day), osthole (20 mg/kg/day) or metformin (250 mg/kg/day). Gastrocnemius muscles were isolated 30 min after insulin administration (0.5 IU/kg, i.p.), followed by lysis in homogenization buffer and processed for western analysis. Shown are representative immunoblots and densitometric quantification of membranous GLUT4 relative to cell lysate GLUT4 (A), phospho-Akt relative to total Akt (B), phospho-AMPK relative to total AMPK and phosphoTBC1D1 relative to total TBC1D1, respectively (C). The results expressed as mean ± SD (n ¼ 3). #p < 0.05, #p < 0.05, ###p < 0.001 vs Control, *p < 0.05, **p < 0.01, ***p < 0.001 vs Dexa.
synthesize glycogen effectively, leading to high blood glucose and hyperinsulinemia to compensate for the defect in activation of insulin signaling pathway.37 GCs has been broadly prescribed for clinical treatment, whereas chronic exposure or excess of GCs can produce undesired diabetogenic effects by impairing insulin signaling, which results in impaired glucose disposal and augmented glucose production.8 In this study, we treated ICR mice and C2C12 myotubes with dexamethasone to model insulin resistance in order to investigate whether coumarins could ameliorate diabetogenic action of dexamethasone. Dexamethasone reduced body weight, increased plasma glucose and impaired glucose tolerance in mice, in agreement with previous studies.38 Administration of esculin, fraxetin or osthole raised the body weight, decreased blood glucose, and improved the glucose tolerance, thus prevented this diabetogenic action of dexamethasone in mice. To validate these effects of coumarins, we performed in vitro glucose uptake test to explore if coumarins could promote glucose disposal in normal C2C12 myotubes and dexamethasone-treated myotubes. As shown in Table 2, esculin, fraxetin and osthole could facilitate glucose utilization in normal myotubes and insulin-stimulated glucose uptake in
dexamethasone-treated myotubes. To further elucidate the molecular mechanism of amelioration of insulin resistance by these coumarins, we collected skeletal muscle tissues and did western blotting and found that esculin, fraxetin and osthole could stimulate Akt activation and promote GLUT4 transport to the plasma membrane in skeletal muscle. IR/IRS/PI3K/Akt is an essential axis for insulin-mediated GLUT4 translocation to the cell surface.39 Akt activation plays an important role in insulin-mediated glucose uptake. Akt gene knockout mice exhibited high plasma glucose.40 In addition, esculin and osthole enhanced the phosphorylation of AMPK and TBC1D1. TBC1D1 is a Rab GTPase-activating protein, expressed abundantly in skeletal muscle,41 and can be phosphorylated at Ser 237 specifically by AMPK,42,43 which is consistent with previous literatures that the phosphorylation of TBC1D1 can be augmented by AICAR, an AMPK agonist, 14-3-3s can bind primarily to TBC1D1 phosphorylated at Ser 237, stabilizing Rab-GTP at an activated status, accelerating GLUT4 translocation to the cell membrane, and thus enhancing the glucose uptake in skeletal muscle.44e47 Taken together, our findings showed that esculin, fraxetin and osthole could promote glucose uptake in C2C12 myotubes in vitro
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and improve insulin resistance in mice induced by dexamethasone. Potential mechanisms of these actions include activating insulin signaling pathway and AMPK, facilitating GLUT4 translocation, leading to increased glucose disposal in skeletal muscle, while the precise target for anti-diabetic action and structure-activity relationship of coumarins remain to be elucidated. Together with our previous reports,48 coumarins could be potential anti-diabetic candidates or leading compound for drug development. Conflicts of interest None. Acknowledgments This research was supported by the Natural Science Foundation of Zhejiang Province (LY18H070004, LY14H310003) and the Key Program of School Foundation of Zhejiang University of Technology (2012XZ004). References 1. Rask-Madsen C, Kahn CR. Tissue-Specific insulin signaling, metabolic syndrome, and cardiovascular disease. Arterioscler Thromb Vasc Biol. 2012;32: 2052e2059. 2. Samuel VT, Shulman GI. The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest. 2016;126:12e22. 3. Morgan SA, Hassan-Smith ZK, Lavery GG. Mechanisms in endocrinology: tissue-specific activation of cortisol in Cushing's syndrome. Eur J Endocrinol. 2016;152:R83eR89. 4. Ghaisas M, Navghare V, Takawale A, Zope V, Tanwar M, Deshpande A. Effect of Tectona grandis Linn. on dexamethasone-induced insulin resistance in mice. J Ethnopharmacol. 2009;122:304e307. 5. Thomas C, Turner S, Jefferson W, Bailey C. Prevention of dexamethasoneinduced insulin resistance by metformin. Biochem Pharmacol. 1998;56: 1145e1150. 6. Patel SS, Udayabanu M. Urtica dioica, extract attenuates depressive like behavior and associative memory dysfunction in dexamethasone induced diabetic mice. Metab Brain Dis. 2014;29:121e130. 7. Geer EB, Islam J, Buettner C. Mechanisms of glucocorticoid-induced insulin resistance: focus on adipose tissue function and lipid metabolism. Endocrinol Metab Clin North Am. 2014;43:75e102. 8. Rafacho A, Ortsater H, Nadal A, Quesada I. Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes. J Endocrinol. 2014;223:R49eR62. n S, Rosengren V, Wolbert P, Sjo €holm Å, Ortsa €ter H. b-cell 9. Fransson L, Franze adaptation in a mouse model of glucocorticoid-induced metabolic syndrome. J Endocrinol. 2013;219:231e241. 10. Holland WL, Bikman BT, Wang LP, et al. Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acidinduced ceramide biosynthesis in mice. J Clin Invest. 2011;121:1858e1870. 11. Edirs S, Jiang L, Xin X, Aisa HA. Anti-diabetic effect and mechanism of Kursi Wufarikun Ziyabit, in L6 rat skeletal muscle cells. J Pharmacol Sci. 2018;137: 212e219. 12. Pereira C, Azevedo I, Monteiro R, Martins M. 11b-Hydroxysteroid dehydrogenase type 1: relevance of its modulation in the pathophysiology of obesity, the metabolic syndrome and type 2 diabetes mellitus. Diabetes Obes Metab. 2012;14:869e881. 13. Lee MJ, Pramyothin P, Karastergiou K, Fried SK. Deconstructing the roles of glucocorticoids in adipose tissue biology and the development of central obesity. Biochim Biophys Acta. 2014;1842:473e481. 14. Pandey J, Maurya R, Raykhera R, Srivastava MN, Yadav PP, Tamrakar AK. Murraya koenigii (L.) Spreng. ameliorates insulin resistance in dexamethasonetreated mice by enhancing peripheral insulin sensitivity. J Sci Food Agric. 2014;94:2282e2288. 15. Sur TK, Hazra AK, Bhattacharyya D, Hazra A. Antiradical and antidiabetic properties of standardized extract of Sunderban mangrove Rhizophora mucronata. Pharmacogn Mag. 2015;11:389e394. 16. Noipha K, Thongthoom T, Songsiang U, Boonyarat C, Yenjai C. Carbazoles and coumarins from Clausena harmandiana stimulate glucose uptake in L6 myotubes. Diabetes Res Clin Pract. 2010;90:e67ee71. 17. Li HB, Yao YF, Li LH. Coumarins as potential antidiabetic agents. J Pharm Pharmacol. 2017;69:1253e1264. 18. Wang YH, Liu YH, He GR, Lv Y, Du GH. Esculin improves dyslipidemia, inflammation and renal damage in streptozotocin-induced diabetic rats. BMC Compl Altern Med. 2015;15:402. 19. Kang KS, Lee W, Jung Y, et al. Protective effect of esculin on streptozotocininduced diabetic renal damage in mice. J Agric Food Chem. 2014;62:2069e2076.
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20. Murali R, Srinivasan S, Ashokkumar N. Antihyperglycemic effect of fraxetin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats. Biochimie. 2013;95:1848e1854. 21. Li JM, Zhang X, Wang X, Xie YC, Kong LD. Protective effects of cortex fraxini coumarines against oxonate-induced hyperuricemia and renal dysfunction in mice. Eur J Pharmacol. 2011;666:196e204. 22. Zhang W, Lee J, Kim Y, Kim I, Park J, Myung C. Amelioration of insulin resistance by scopoletin in high-glucose-induced, insulin-resistant HepG2 cells. Horm Metab Res. 2010;42:930e935. 23. Qi ZG, Zhao X, Zhong W, Xie ML. Osthole improves glucose and lipid metabolism via modulation of PPARalpha/gamma-mediated target gene expression in liver, adipose tissue, and skeletal muscle in fatty liver rats. Pharm Biol. 2016;54:882e888. 24. Qi Z, Xue J, Zhang Y, Wang H, Xie M. Osthole ameliorates insulin resistance by increment of adiponectin release in high-fat and high-sucrose-induced fatty liver rats. Planta Med. 2011;77:231e235. 25. Lee WH, Lin RJ, Lin SY, Chen YC, Lin HM, Liang YC. Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells. J Agric Food Chem. 2011;59:12874e12881. 26. DeFronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM. A balanced overview. Diabetes Care. 1992;15:318e368. 27. Li H, Yang Y, Mo Z, Ding Y, Jiang W. Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition. Braz J Med Biol Res. 2015;48:440e446. 28. Gholap S, Kar A. Efficacy of some plant extracts in regulating corticosteroidinduced hyperglycaemia in mice. Pharm Biol. 2008;41:315e318. n B. The high-fat dietefed mouse a model for studying 29. Winzell MS, Ahre mechanisms and treatment of impaired glucose tolerance and type 2 diabetes. Diabetes. 2004;53:S215eS219. n B. 30. Winzell MS, Wulff EM, Olsen GS, Sauerberg P, Gotfredsen CF, Ahre Improved insulin sensitivity and islet function after PPARd activation in diabetic db/db mice. Eur J Pharmacol. 2010;626:297e305. 31. Hayata K, Sakano K, Nishinaka S. Establishment of new highly insulin-sensitive cell lines and screening of compounds to facilitate glucose consumption. J Pharmacol Sci. 2008;108:348e354. 32. Cummings BP, Bremer AA, Kieffer TJ, D'Alessio D, Havel PJ. Investigation of the mechanisms contributing to the compensatory increase in insulin secretion during dexamethasone-induced insulin resistance in rhesus macaques. J Endocrinol. 2013;216:207e215. 33. Shepherd EJ, Helliwell PA, Mace OJ, Morgan EL, Patel N, Kellett GL. Stress and glucocorticoid inhibit apical GLUT2-trafficking and intestinal glucose absorption in rat small intestine. J Physiol. 2004;560:281e290. 34. Kellett GL, Brot-Laroche E. Apical GLUT2: a major pathway of intestinal sugar absorption. Diabetes. 2005;54:3056e3062. 35. Kim YB, Ciaraldi TP, Kong A, et al. Troglitazone but not metformin restores insulinstimulated phosphoinositide 3-kinase activity and increases p110b protein levels in skeletal muscle of type 2 diabetic subjects. Diabetes. 2002;51:443e448. 36. Cartee GD. Roles of TBC1D1 and TBC1D4 in insulin- and exercise-stimulated glucose transport of skeletal muscle. Diabetologia. 2015;58:19e30. 37. Czech MP. Insulin action and resistance in obesity and type 2 diabetes. Nat Med. 2017;37:804e814. 38. Dimitriadis G, Leighton B, ParryBillings M, et al. Effects of glucocorticoid excess on the sensitivity of glucose transport and metabolism to insulin in rat skeletal muscle. Biochem J. 1997;321:707e712. 39. Mackenzie RW, Elliott BT. Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the treatment of type 2 diabetes. Diabetes Metab Syndr Obes. 2014;7:55e64. 40. Garofalo RS, Orena SJ, Rafidi K, et al. Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKBb. J Clin Invest. 2003;112:197e208. 41. Vichaiwong K, Purohit S, An D, et al. Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle. Biochem J. 2010;431:311e320. 42. Liu K, Mei F, Wang Y, et al. Quercetin oppositely regulates insulin-mediated glucose disposal in skeletal muscle under normal and inflammatory conditions: the dual roles of AMPK activation. Mol Nutr Food Res. 2015;60: 551e565. 43. Chen S, Wasserman DH, MacKintosh C, Sakamoto K. Mice with AS160/TBC1D4Thr649Ala knockin mutation are glucose intolerant with reduced insulin sensitivity and altered GLUT4 trafficking. Cell Metab. 2011;13:68e79. 44. Chen S, Murphy J, Toth R, Campbell DG, Morrice NA, Mackintosh C. Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators. Biochem J. 2008;409:449e459. 45. Geraghty KM, Chen S, Harthill JE, et al. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. Biochem J. 2007;407:231e241. 46. Tan SX, Ng Y, Burchfield JG, et al. The Rab GTPase-activating protein TBC1D4/ AS160 contains an atypical phosphotyrosine-binding domain that interacts with plasma membrane phospholipids to facilitate GLUT4 trafficking in adipocytes. Mol Cell Biol. 2012;32:4946e4959. 47. Richter EA, Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 2013;93:993e1017. 48. Yao Y, Zhao X, Xin J, Wu Y, Li H. Coumarins improved type 2 diabetes induced by high-fat diet and streptozotocin in mice via antioxidation. Can J Physiol Pharmacol. 2018;96:765e771.
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Full Paper
Coumarins ameliorate diabetogenic action of dexamethasone via Akt activation and AMPK signaling in skeletal muscle Zejun Mo a, b, 1, Linghuan Li a, 1, Haiwen Yu a, Yingqi Wu a, Hanbing Li a, c, * a
Institute of Pharmacology, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, People's Republic of China c Section of Endocrinology, School of Medicine, Yale University, New Haven 06520, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2018 Received in revised form 29 December 2018 Accepted 9 January 2019 Available online 23 January 2019
Glucocorticoids are widely prescribed for lots of pathological conditions, however, can produce ‘Cushingoid’ side effects including central obesity, glucose intolerance, insulin resistance and so forth. Our study is intended to investigate the improving effects of coumarins on diabetogenic action of dexamethasone in vivo and in vitro and elucidate potential mechanisms. ICR mice treated with dexamethasone for 21 days exhibited decreased body weight, increased blood glucose and impaired glucose tolerance, which were prevented by fraxetin (40 mg/kg/day), esculin (40 mg/kg/day) and osthole (20 mg/ kg/day), respectively. Esculin, fraxetin and osthole also could promote glucose uptake in normal C2C12 myotubes, and improve insulin resistance in myotubes induced by dexamethasone. Western blotting results indicated that esculin, fraxetin and osthole could boost Akt activation, stimulate GLUT4 translocation, thus alleviate insulin resistance. Esculin and osthole also could activate AMPK, thereby phosphorylate TBC1D1 at Ser237, and consequently ameliorate diabetogenic action of dexamethasone. Our study indicates coumarins as potential anti-diabetic candidates or leading compounds for drug development. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Coumarin Osthole Esculin Fraxetin Insulin resistance Dexamethasone
1. Introduction The metabolic syndrome is a clustering of the following conditions including central obesity, hypertension, impaired glucose tolerance or diabetes, and dyslipidemia with high plasma concentrations of triglycerides and low levels of high-density lipoprotein cholesterol, which can contribute to cardiovascular risk.1 Insulin is a key hormone to clear the postprandial glucose load and maintain homeostasis of plasma glucose level. Insulin resistance is a progressive process of decreasing the sensitivity of insulin on target tissues, which is the pathological basis of metabolic syndrome.2 Glucocorticoids (GCs), such as dexamethasone and prednisolone, are commonly used as an anti-inflammatory, anti-allergic and immunosuppressive drug. However, the majority of these patients
* Corresponding author. Institute of Pharmacology, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China. Fax: þ86 571 88320535. E-mail addresses: [email protected], [email protected] (H. Li). Peer review under responsibility of Japanese Pharmacological Society. 1 Zejun Mo and Linghuan Li contributed equally to this paper.
suffer from ‘Cushingoid’ side effects,3 and chronic elevated GCs levels could cause central obesity and insulin resistance. Diabetes has been considered to be a common complication of chronic excess of GCs and it is an important factor contributing to the morbidity and mortality of the patients. GCs-induced diabetic like rodents displayed hyperglycaemia, dyslipidemia and impairment of insulin sensitivity in hepatic and peripheral tissues.4e6 In skeletal muscle, GCs can decrease insulin-mediated glucose uptake through impairing insulin signaling pathway, curtailing expression and activity of insulin receptor, insulin receptor substrate and PI3K.7,8 Furthermore, GCs lead to the accumulation of ectopic fat deposition in skeletal muscle and elevation of free fatty acid,9 which also can induce insulin resistance.10,11 In addition, local GCs metabolism via 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) plays an important role in contributing to adverse effects of excess GCs.12,13 Coumarins are famous as anticoagulant drugs, such as warfarin, dicoumarolum, acenocoumarol and so on. Recently, it has been discovered that some plants contain coumarins or some coumarin derivatives could improve insulin resistance and diabetic complications.14e17 For example, esculin and fraxetin reduced plasma glucose, diminished glucose-6-phosphatase activity and
https://doi.org/10.1016/j.jphs.2019.01.001 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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improved renal dysfunction.18e21 Scopoletin and osthole could ameliorate insulin resistance in HepG2 cells by upregulating PPARg2 expression,22,23 osthole also increased adiponectin release in fatty liver rats.24 Lee et al reported that osthole could enhance glucose uptake through activation of AMPK in skeletal muscle in vitro.25 However, in vivo effects and potential mechanisms of coumarins improving insulin resistance induced by GCs is lack of study. Thus, we investigated the effects of esculin, osthole and fraxetin upon insulin resistance in mice induced by dexamethasone, especially the effects of these coumarins on glucose disposal in skeletal muscle, which accounts for >80% of the entire body's glucose disposal under insulin stimulation,26 and potential mechanisms of these effects as well. 2. Materials and methods 2.1. Materials Esculin, osthole and fraxetin were purchased from Yongjian Pharmaceutical Company (Taizhou, China) with purity of 99.7%, 99.1% and 99.9%, respectively; insulin and dexamethasone were provided by Sigma (St. Louis, MO, USA); Metformin was from Shanghai Sine Pharma (Shanghai, China). The following items were purchased from the cited commercial sources: Akt antibody and Akt (phospho-Ser473) antibody, Anbo (San Francisco, USA); AMPK antibody and AMPK (phospho-Thr172) antibody, Santa Cruz (Dallas, USA); TBC1D1 antibody, Proteintech (Chicago, USA); TBC1D1 (phospho-Ser237) antibody, Millipore (Temecula, USA); GAPDH antibody, Beyotime (Shanghai, China); GLUT4 antibody and Goat anti-rabbit IgG, Boster (Wuhan, China). 2.2. Cell culture and differentiation The C2C12 cell line obtained from ATCC (Manassas, USA), were cultured in DMEM (Gibco, California, USA) with 10% (v/v) FBS (Hyclone, Logan, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 C. Cells were grown to 70e80% confluency, and then the medium was switched to DMEM with 2% (v/v) HS (Hyclone, Logan, USA) and changed every 2 days.27
and fraxetin (40 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 14 days; Osthole group: gum acacia (10 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 7 days and osthole (20 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 14 days; Metformin group: gum acacia (10 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 7 days and metformin (250 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 14 days. 2.5. Oral glucose tolerance test and intravenous glucose tolerance test Oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IvGTT) were performed after an overnight fast. For OGTT, the baseline blood glucose level was detected at 0 min, followed by an oral glucose load of 2 g/kg, and blood glucose levels was checked again at 15, 30, 60, 120 min. For IvGTT, The blood glucose were detected before glucose (1 g/kg, i.v.) injection, and the blood glucose levels were monitored at 1, 5, 10, 20, 50, 75min after glucose loading.29,30 Blood glucose was checked with using Onetouch Ultraeasy (Johnson, New Jersey, USA).The area under the curve for blood glucose was calculated by GraphPad Prism 5. 2.6. Cells treatment and detection of glucose uptake Cells were treated in the absence or presence of increasing dose dexamethasone over a time course of 24 h to select a suitable dose to induce cells to decrease the ability of glucose uptake. In set of experiments, cells were incubated with different concentrations (25, 50, 100 mM) of esculin, fraxetin and osthole for 24 h alone or along with dexamethasone stimulation. Cell viability was tested by MTT method and glucose uptake in the presence or absense of insulin (final concentration of 100 nM) was characterized by glucose consumption which was detected by the glucose oxidase/ peroxidase (GOD-POD) method.11,31 As for glucose consumption assay, the glucose concentration at 0 and 24 h in the culture medium was determined by the glucose oxidase assay kit (Jiancheng Bioengineering Institute, Nanjing, China), and the difference between the two concentrations was regarded as the amount of consumed glucose. 2.7. Western blotting
2.3. Animals Male Institute of Cancer Research (ICR) mice (7e8wk of age) were supplied by the Zhejiang Academy of Medical Sciences [License Number: SCXK (Zhe) 2014-0001]. The mice were maintained in sanitized polypropylene cages (6 per cage) under standard conditions of temperature (23 ± 2 C), relative humidity (55 ± 5%) and light 12/12 h light/dark cycle with free access to food and water. All animals were treated in compliance with the present institutional guidelines for animal care and use. 2.4. Dexamethasone-induced insulin resistance in mice and treatment After several days of adaptation, body weight and blood glucose were tested before treatment and randomized into 6 groups. The mice were treated for 21 days according to the following protocol as described previously4,28 with some modification: Control group: gum acacia (10 mg/kg/day, p.o.) for 21 days; Dexa group: gum acacia (10 mg/kg/day, p.o.)þdexamethasone (1 mg/kg/day, i.m.) for 21 days; Esculin group: gum acacia (10 mg/kg/day, p.o.)þdexamethasone (1 mg/kg/day, i.m.) for 7 days and esculin (40 mg/kg/day, p.o.)þ dexamethasone (1 mg/kg/day, i.m.) for 14 days; Fraxetin: gum acacia (10 mg/kg/day, p.o.) þ dexamethasone (1 mg/kg/day, i.m.) for 7 days
Thirty minutes after being injected with insulin (0.5 IU/kg, i.p.),10 the mice of 6 groups were killed by cervical dislocation and gastrocnemius muscle was immediately isolated and homogenized in ice-cold lysis buffer to extract the protein. Lysates were centrifuged at 14,000 g for 5 min at 4 C and supernatants were collected. The protein concentration was determined by Bicinchoninic Acid protein assay kit with BSA as a standard (Beyotime, Shanghai, China). Protein samples were mixed with loading buffer and boiled for 5min, then resolved by SDS-PAGE and transferred onto PVDF membranes (Beyotime, Shanghai, China) in a semidry transfer system (Bio-Rad, Hercules, USA). PVDF membranes were blocked with freshly prepared 5% BSA or nonfat milk in Trisbuffered saline containing 0.05% Tween-20 surfactant for 60 min at room temperature, immunoblotted with the primary antibodies for 4 h at room temperature or overnight at 4 C, washed with TBST for 3 times and incubated with secondary antibodies for 2 h at room temperature, and finally visualized with enhanced chemiluminescence method. 2.8. Histopathological studies Skeletal muscles of experimental mice were fixed in 10% formalin, dehydrated with gradient ethanol, subsequently
Z. Mo et al. / Journal of Pharmacological Sciences 139 (2019) 151e157
embedded in paraffin, cut into 5 mm thickness using a microtome and stained with haematoxylin-eosin. Tissue structures were observed under light microscopy. 2.9. Statistical analysis Values were given as mean ± standard deviation (SD). Statistical significance of differences in measurements between samples was assessed by one-way analysis of variance (ANOVA) with Tukey's post hoc test using GraphPad Prism 5. P < 0.05 was considered statistically significant. 3. Results 3.1. Coumarins ameliorated diabetogenic action of dexamethasone in mice To investigate the effects of coumarins upon diabetogenic action of dexamethasone in mice, we tested fasting blood glucose and glucose tolerance in mice, with metformin as a positive control. The mice treated with dexamethasone (1 mg/kg/day, i.m.) exhibited a decrease of body weight at day 7, which became significant at day 14 and 21 compared with normal control group (p < 0.001) as shown in Table 1. Administration with esculin (40 mg/kg/day), fraxetin (40 mg/kg/day), osthole (20 mg/kg/day) or metformin (250 mg/kg/day) prevented the fall of body weight induced by dexamethasone (Table 1, p < 0.05). Meanwhile, the mice treated with dexamethasone exhibited an increase of blood glucose level at day 7, which became significant at day 15 and 22 compared with normal control group weight (p < 0.05 and p < 0.001, respectively). Treatment with esculin, fraxetin, osthole or metformin reduced the blood glucose level in mice. Dexamethasone impaired glucose tolerance, evidenced by delayed clearance of blood glucose after glucose load compared to normal control group (Fig. 1A, C). The AUC of Dexa group was significantly bigger than that of normal control group in IvGTT (Fig. 1D, p < 0.001), while not significantly in OGTT (Fig. 1B), similar with the report by Cummings,32 possibly due to less intestinal absorption by inhibiting the GLUT2 translocation to the apical membrane.33,34 Administration with esculin, fraxetin, osthole or metformin significantly improved glucose tolerance (p < 0.05). 3.2. Coumarins augmented glucose uptake in normal C2C12 myotubes or insulin-stimulated glucose uptake in dexamethasonetreated cells To validate the effects of coumarins on glucose disposal contributing to blood glucose homeostasis, we investigated the
153
effect of coumarins upon glucose uptake in normal C2C12 myotubes and insulin-stimulated glucose uptake in dexamethasonetreated myotubes. As shown in Table 2A, dexamethasone (0.01, 0.1, 1 mM) dose-dependently inhibited glucose uptake in C2C12 myotubes, and only 1 mM dexamethasone decreased the viability of C2C12 myotubes (p < 0.05), so 0.1 mM dexamethasone was adopted to induce insulin resistance in myotubes. To investigate the impact of esculin, fraxetin and osthole on cell viability, myotubes were incubated with different concentrations (25, 50, 100 mM) of esculin, fraxetin or osthole for 24 h, and cell viability was detected by MTT method. As illustrated in Table 2B, esculin and fraxetin had no influence on cell viability, but 50 and 100 mM osthole significantly reduced cell viability. Then tests for glucose uptake displayed that 25 mM esculin and osthole stimulated glucose uptake around 1.6-fold in myotubes compared with normal control, which is similar to 100 nM insulin, whereas not significant in fraxetin group Table 2C. As indicated in Table 2D, esculin (50, 100 mM), fraxetin (100 mM) and osthole (25 mM) could prevent reduced insulin-stimulated glucose disposal in dexamethasonetreated C2C12 myotubes. 3.3. Coumarins improved histopathological changes of skeletal muscle In normal control, regular and tightly connected skeletal muscle fibres with regularly marshalled nucleus were observed. However, in dexamethasone-treated mice, the nucleus arrangement was irregular and there was a clear internal shift, which was improved to a certain extent by esculin, fraxetin, osthole and especially by metformin (Fig. 2). 3.4. Coumarins stimulated GLUT4 translocation in skeletal muscle In skeletal muscle, GLUT4 translocation to the plasma membrane is a key step for glucose uptake, thus we investigated the effects of esculin, osthole and fraxetin upon GLUT4 expression and translocation in skeletal muscle in dexamethasone-treated mice. As exhibited in Fig. 3A. Dexamethasone significantly decreased GLUT4 translocation manifested as the ratio of membranous GLUT4 to cell lysate GLUT4, which was prevented by esculin (p < 0.001), fraxetin (p < 0.05), osthole (p < 0.001), and metformin (p < 0.001), respectively. While the expression of cell lysate GLUT4 was not significantly different between these groups. 3.5. Coumarins promoted Akt activation in skeletal muscle Akt activation plays a crucial role in insulin signaling pathway. Therefore, we examined whether esculin, fraxetin and
Table 1 Effects of esculin (40 mg/kg/day), fraxetin (40 mg/kg/day) and osthole (20 mg/kg/day) upon body weight and blood glucose of mice treated with dexamethasone (Dexa), with metformin (250 mg/kg/day) as a positive control. Body weight of all mice and blood glucose of 6 mice per group at random were monitored every week to confirm the establishment of the model of insulin resistance induced by Dexa. Parameter
Control
Body Weight (g) Day 0 24.03 ± 1.11 Day 7 26.09 ± 0.69 Day 14 27.62 ± 0.70 Day 21 28.04 ± 0.70 Blood Glucose (mmol/L) Day 0 4.42 ± 0.64 Day 7 4.56 ± 0.48 Day 15 4.88 ± 0.45 Day 22 4.93 ± 0.79
Dexa þ Esculin
Dexa 24.49 24.03 23.42 22.48 4.71 4.87 5.69 6.59
± ± ± ±
± ± ± ±
1.99 1.47 1.30*** 1.20***
0.46 0.48 0.54* 0.62***
24.59 23.78 23.91 24.33 4.35 4.83 4.77 4.90
± ± ± ±
± ± ± ±
Dexa þ Fraxetin
2.50 1.95 1.77 1.63#
24.21 23.87 23.89 24.35
0.74 0.29 0.55# 0.25###
4.27 4.93 4.65 5.00
± ± ± ±
± ± ± ±
1.61 1.27 1.94 1.75#
1.01 0.55 0.28## 0.63##
Dexa þ Osthole 24.50 23.90 24.27 24.83 4.67 4.90 4.92 4.98
± ± ± ±
± ± ± ±
1.27 1.24 1.55 1.04##
0.58 0.26 0.72 0.76##
Dexa þ Metformin 24.31 23.68 23.93 24.34 4.69 4.83 4.85 4.97
± ± ± ±
± ± ± ±
2.42 1.48 1.18 1.61#
0.33 0.46 0.41# 0.64##
Data are expressed as mean ± SD. *p<0.05 vs Control; ***p<0.001 vs Control; #p<0.05 vs Dexa; ##p<0.01 vs Dexa; ###p<0.001 vs Dexa. n ¼ 12 for body weight, n ¼ 6 for blood glucose.
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Fig. 1. Effect esculin (40 mg/kg/day), fraxetin (40 mg/kg/day), osthole (20 mg/kg/day) and metformin (250 mg/kg/day) on OGTT and IvGTT in Dexa-treated mice. Mice were treated or not (control) with daily injection of Dexa (1 mg/kg, i.m.) for 7 days. Subsequently, mice received or not the indicated doses of metformin, esculin, fraxetin or osthole for 14 days along with injection of Dexa. At the end of the experiment, an oral glucose tolerance test and an intravenous glucose tolerance test were performed as described in Materials and Methods. A: OGTT, B: The AUC of OGTT. Values were given as mean ± SD. C: IvGTT, D: The AUC of IvGTT. Values were given as mean ± SD. n ¼ 6 ###p < 0.001 vs Control,*p < 0.05 vs Dexa; **p < 0.01 vs Dexa; ***p < 0.001 vs Dexa.
Table 2A Effects of coumarins upon glucose uptake and cell viability in C2C12 myotubes. A. Effects of different concentrations of Dexa on glucose uptake and cell viability in C2C12 myotubes.
Relative glucose uptake Relative cell viability
Control
Insulin
Dexa (mM) 0.01
0.10
1.00
100.00 ± 19.31 100.00 ± 3.43
160.93 ± 26.77### 105.07 ± 4.78
144.53 ± 31.77 101.01 ± 7.07
110.43 ± 30.68* 102.97 ± 5.00
103.30 ± 19.42** 93.33 ± 7.21*
Each value is expressed as means ± SD (n ¼ 6), ###p < 0.001 vs Control group, *p < 0.05, **p < 0.01 vs Insulin control group.
Table 2B Effects of different concentrations of esculin, osthole and fraxetion on cell viability in normal C2C12 myotubes. Parameter (%)
Coumarins (mM)
Control
100.00 ± 4.42 100.00 ± 7.04 100.00 ± 4.92
Esculin Fraxetin Osthole
25
50
100
101.76 ± 1.74 98.98 ± 13.05 97.77 ± 2.12
96.51 ± 5.27 98.63 ± 8.76 82.78 ± 12.36***
93.88 ± 3.79 98.09 ± 6.42 65.22 ± 24.57***
Each value is expressed as means ± SD. ***p < 0.001 vs Control group.
Table 2C Effects of different concentrations of esculin, osthole and fraxetion on glucose uptake in normal C2C12 myotubes. Parameter (%)
Control
Insulin
Esculin Fraxetin Osthole
100.00 ± 27.71 100.00 ± 17.57 100.00 ± 17.57
150.85 ± 16.90** 162.58 ± 13.82* 162.58 ± 13.82***
Coumarins (mM) 25
50
100
166.25 ± 25.16*** 99.75 ± 37.32 149.94 ± 19.67***
125.58 ± 25.93 122.39 ± 27.26 /
125.59 ± 24.98 120.82 ± 41.44 /
Each value is expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs Control group.
osthole could increase insulin-mediated Akt phosphorylation in skeletal muscle in dexamethasone-treated mice. As illustrated in Fig. 3B, dexamethasone significantly curtailed the
phosphorylation of Akt (p < 0.05), which was restored by esculin (p < 0.05), fraxetin (p < 0.05), osthole (p < 0.01) and metformin (p < 0.01).
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Table 2D Effects of different concentrations of esculin, osthole and fraxetin on insulin-stimulated glucose uptake in Dexa-treated C2C12 myotubes. Parameter (%)
Insulin-control
Dexa þ Coumarins (mM)
Dexa
25 Esculin Fraxetin Osthole
100.00 ± 24.00 100.00 ± 12.84 100.00 ± 20.67
**
50.07 ± 9.39 60.02 ± 15.66** 62.54 ± 12.69***
60.61 ± 16.63 51.40 ± 13.17 89.43 ± 25.85##
50 103.21 ± 13.44 59.34 ± 11.36 /
100 ###
85.00 ± 23.01## 94.87 ± 20.69# /
Each value is expressed as means ± SD. **p < 0.01, ***p < 0.001 vs Insulin-control group, #p < 0.05, ##p < 0.01, ###p < 0.001 vs Dexa group, n ¼ 6.
Fig. 2. Histological observation of skeletal tissues in experimental mice (HE staining). A: Normal control, B: Dexa-treated mice. C, D, E, F: esculin (40 mg/kg/day), fraxetin (40 mg/kg/ day), osthole (20 mg/kg/day) and metformin (250 mg/kg/day), respectively.
3.6. Esculin and osthole increased AMPK and TBC1D1 phosphorylation in skeletal muscle Metformin is an effective drug for type 2 diabetes treatment due to its ability to activate AMPK, another pathway to promote glucose disposal independently of insulin signaling.35 Given the effects of esculin, osthole and fraxetin to stimulate glucose uptake in normal C2C12 myotubes as shown in Table 2, we performed western blotting to investigate whether esculin, osthole or fraxetin could affect AMPK activation in skeletal muscle in dexamethasonetreated mice. As demonstrated in Fig. 3C, dexamethasone could suppress AMPK phosphorylation (p < 0.01), which was enhanced by esculin (p < 0.05), osthole (p < 0.05), and metformin (p < 0.05), whereas not significantly by fraxetin.
TBC1D1 phosphorylation at Ser237 is dependent on AMPK activation,36 thus we examined the effects of esculin, fraxetin and osthole on TBC1D1 phosphorylation at Ser237, a downstream substrate protein of AMPK. As predicted, esculin (p < 0.01), osthole (p < 0.001) and metformin (p < 0.001) could prevent from downregulating decreased TBC1D1 phosphorylation (p < 0.05) in skeletal muscle induced by dexamethasone, but not by fraxetin. 4. Discussion Insulin resistance is a key characteristic of the metabolic syndrome including central obesity, diabetes, dyslipidaemia and so forth, in which insulin-responsive tissues fail to respond to the normal level of insulin, to dispose blood glucose load and
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Fig. 3. Effects of Esculin, fraxetin and osthole on GLUT4 translocation, Akt phosphorylation at Ser473, AMPK and TBC1D1 phosphorylation in skeletal muscle in Dexa-treated mice. Mice were orally received with esculin (40 mg/kg/day), fraxetin (40 mg/kg/day), osthole (20 mg/kg/day) or metformin (250 mg/kg/day). Gastrocnemius muscles were isolated 30 min after insulin administration (0.5 IU/kg, i.p.), followed by lysis in homogenization buffer and processed for western analysis. Shown are representative immunoblots and densitometric quantification of membranous GLUT4 relative to cell lysate GLUT4 (A), phospho-Akt relative to total Akt (B), phospho-AMPK relative to total AMPK and phosphoTBC1D1 relative to total TBC1D1, respectively (C). The results expressed as mean ± SD (n ¼ 3). #p < 0.05, #p < 0.05, ###p < 0.001 vs Control, *p < 0.05, **p < 0.01, ***p < 0.001 vs Dexa.
synthesize glycogen effectively, leading to high blood glucose and hyperinsulinemia to compensate for the defect in activation of insulin signaling pathway.37 GCs has been broadly prescribed for clinical treatment, whereas chronic exposure or excess of GCs can produce undesired diabetogenic effects by impairing insulin signaling, which results in impaired glucose disposal and augmented glucose production.8 In this study, we treated ICR mice and C2C12 myotubes with dexamethasone to model insulin resistance in order to investigate whether coumarins could ameliorate diabetogenic action of dexamethasone. Dexamethasone reduced body weight, increased plasma glucose and impaired glucose tolerance in mice, in agreement with previous studies.38 Administration of esculin, fraxetin or osthole raised the body weight, decreased blood glucose, and improved the glucose tolerance, thus prevented this diabetogenic action of dexamethasone in mice. To validate these effects of coumarins, we performed in vitro glucose uptake test to explore if coumarins could promote glucose disposal in normal C2C12 myotubes and dexamethasone-treated myotubes. As shown in Table 2, esculin, fraxetin and osthole could facilitate glucose utilization in normal myotubes and insulin-stimulated glucose uptake in
dexamethasone-treated myotubes. To further elucidate the molecular mechanism of amelioration of insulin resistance by these coumarins, we collected skeletal muscle tissues and did western blotting and found that esculin, fraxetin and osthole could stimulate Akt activation and promote GLUT4 transport to the plasma membrane in skeletal muscle. IR/IRS/PI3K/Akt is an essential axis for insulin-mediated GLUT4 translocation to the cell surface.39 Akt activation plays an important role in insulin-mediated glucose uptake. Akt gene knockout mice exhibited high plasma glucose.40 In addition, esculin and osthole enhanced the phosphorylation of AMPK and TBC1D1. TBC1D1 is a Rab GTPase-activating protein, expressed abundantly in skeletal muscle,41 and can be phosphorylated at Ser 237 specifically by AMPK,42,43 which is consistent with previous literatures that the phosphorylation of TBC1D1 can be augmented by AICAR, an AMPK agonist, 14-3-3s can bind primarily to TBC1D1 phosphorylated at Ser 237, stabilizing Rab-GTP at an activated status, accelerating GLUT4 translocation to the cell membrane, and thus enhancing the glucose uptake in skeletal muscle.44e47 Taken together, our findings showed that esculin, fraxetin and osthole could promote glucose uptake in C2C12 myotubes in vitro
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and improve insulin resistance in mice induced by dexamethasone. Potential mechanisms of these actions include activating insulin signaling pathway and AMPK, facilitating GLUT4 translocation, leading to increased glucose disposal in skeletal muscle, while the precise target for anti-diabetic action and structure-activity relationship of coumarins remain to be elucidated. Together with our previous reports,48 coumarins could be potential anti-diabetic candidates or leading compound for drug development. Conflicts of interest None. Acknowledgments This research was supported by the Natural Science Foundation of Zhejiang Province (LY18H070004, LY14H310003) and the Key Program of School Foundation of Zhejiang University of Technology (2012XZ004). References 1. Rask-Madsen C, Kahn CR. Tissue-Specific insulin signaling, metabolic syndrome, and cardiovascular disease. Arterioscler Thromb Vasc Biol. 2012;32: 2052e2059. 2. Samuel VT, Shulman GI. The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest. 2016;126:12e22. 3. Morgan SA, Hassan-Smith ZK, Lavery GG. Mechanisms in endocrinology: tissue-specific activation of cortisol in Cushing's syndrome. Eur J Endocrinol. 2016;152:R83eR89. 4. Ghaisas M, Navghare V, Takawale A, Zope V, Tanwar M, Deshpande A. Effect of Tectona grandis Linn. on dexamethasone-induced insulin resistance in mice. J Ethnopharmacol. 2009;122:304e307. 5. Thomas C, Turner S, Jefferson W, Bailey C. Prevention of dexamethasoneinduced insulin resistance by metformin. Biochem Pharmacol. 1998;56: 1145e1150. 6. Patel SS, Udayabanu M. Urtica dioica, extract attenuates depressive like behavior and associative memory dysfunction in dexamethasone induced diabetic mice. Metab Brain Dis. 2014;29:121e130. 7. Geer EB, Islam J, Buettner C. Mechanisms of glucocorticoid-induced insulin resistance: focus on adipose tissue function and lipid metabolism. Endocrinol Metab Clin North Am. 2014;43:75e102. 8. Rafacho A, Ortsater H, Nadal A, Quesada I. Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes. J Endocrinol. 2014;223:R49eR62. n S, Rosengren V, Wolbert P, Sjo €holm Å, Ortsa €ter H. b-cell 9. Fransson L, Franze adaptation in a mouse model of glucocorticoid-induced metabolic syndrome. J Endocrinol. 2013;219:231e241. 10. Holland WL, Bikman BT, Wang LP, et al. Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acidinduced ceramide biosynthesis in mice. J Clin Invest. 2011;121:1858e1870. 11. Edirs S, Jiang L, Xin X, Aisa HA. Anti-diabetic effect and mechanism of Kursi Wufarikun Ziyabit, in L6 rat skeletal muscle cells. J Pharmacol Sci. 2018;137: 212e219. 12. Pereira C, Azevedo I, Monteiro R, Martins M. 11b-Hydroxysteroid dehydrogenase type 1: relevance of its modulation in the pathophysiology of obesity, the metabolic syndrome and type 2 diabetes mellitus. Diabetes Obes Metab. 2012;14:869e881. 13. Lee MJ, Pramyothin P, Karastergiou K, Fried SK. Deconstructing the roles of glucocorticoids in adipose tissue biology and the development of central obesity. Biochim Biophys Acta. 2014;1842:473e481. 14. Pandey J, Maurya R, Raykhera R, Srivastava MN, Yadav PP, Tamrakar AK. Murraya koenigii (L.) Spreng. ameliorates insulin resistance in dexamethasonetreated mice by enhancing peripheral insulin sensitivity. J Sci Food Agric. 2014;94:2282e2288. 15. Sur TK, Hazra AK, Bhattacharyya D, Hazra A. Antiradical and antidiabetic properties of standardized extract of Sunderban mangrove Rhizophora mucronata. Pharmacogn Mag. 2015;11:389e394. 16. Noipha K, Thongthoom T, Songsiang U, Boonyarat C, Yenjai C. Carbazoles and coumarins from Clausena harmandiana stimulate glucose uptake in L6 myotubes. Diabetes Res Clin Pract. 2010;90:e67ee71. 17. Li HB, Yao YF, Li LH. Coumarins as potential antidiabetic agents. J Pharm Pharmacol. 2017;69:1253e1264. 18. Wang YH, Liu YH, He GR, Lv Y, Du GH. Esculin improves dyslipidemia, inflammation and renal damage in streptozotocin-induced diabetic rats. BMC Compl Altern Med. 2015;15:402. 19. Kang KS, Lee W, Jung Y, et al. Protective effect of esculin on streptozotocininduced diabetic renal damage in mice. J Agric Food Chem. 2014;62:2069e2076.
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