Tambulin from Zanthoxylum armatum acutely potentiates the glucose-induced insulin secretion via KATP-independent Ca2+-dependent amplifying pathway

Biomedicine & Pharmacotherapy 120 (2019) 109348

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Original article

Tambulin from Zanthoxylum armatum acutely potentiates the glucoseinduced insulin secretion via KATP-independent Ca2+-dependent amplifying pathway

T

Abdul Hameeda,c,1, Sayed Ali Razaa,1, M. Israr Khana, Janaki Barald, Achyut Adhikarib,d, Mohammad Nur-e-Alame, Sarfaraz Ahmede, Adnan J. Al-Rehailye, Sajda Ashrafa,b, Zaheer Ul-Haqa,b, Rahman M. Hafizura,⁎ a

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan c Centre for Advanced Drug Research (CADR), COMSATS University Islamabad (CUI), Abbottabad 22060, Pakistan d Central Department of Chemistry, Tribhuvan University, Kathmandu, Nepal e Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box. 2457, Riyadh 11451, Saudi Arabia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tambulin Insulin secretion Mice islets cAMP Ca2+ channel KATPchannel Protein kinase A

Tambulin, a flavonol isolated from Zanthoxylum armatum, showed potent insulin secretory activity in our preliminary anti-diabetic screening. Here, we explored the insulin secretory mechanism(s) of tambulin focusing in glucose-dependent, KATP ‒ and Ca2+‒channels dependent, and cAMP-PKA pathways. Mice islets and MIN6 cells were incubated with tambulin in the presence of pharmacological agonists/antagonists and the secreted insulin was measured using mouse insulin ELISA kit. The intracellular cAMP was measured by an acetylation cAMP ELISA kit. Tambulin (200 μM) showed potent insulin secretory activity only at stimulatory glucose (11–25 mM) concentrations; however, no change in insulin release was observed at basal glucose both in mice islets and MIN6 cells. Notably, in the presence of diazoxide, a KATP channel opener; the incomplete inhibition of tambulininduced insulin secretion was observed whereas, complete inhibition was found using verapamil, an L-type Ca2+ channel blocker. Furthermore, the insulinotropic potential of tambulin was amplified in tolbutamide treated, and depolarized islets suggest tambulin’s target other than tolbutamide. Tambulin showed no additive effect in the IBMX-induced intracellular cAMP; whereas, exerted an additive effect in the IBMX-induced insulin secretion. Furthermore, tambulin-induced insulin secretion was dramatically inhibited by PKA inhibitor (H-89), while moderate inhibition was found by using PKC inhibitor (calphostin C). Molecular docking studies also showed the best binding affinities of tambulin with PKA suggest the PKA dependent signaling cascade is involved more in tambulin-induced insulin secretion. Based on these findings, it is concluded that tambulin stimulates insulin secretion in a Ca2+ channel-dependent but KATP channel-independent manner, most likely by activating the cAMP-PKA pathway.

1. Introduction Diabetes, a life-style related disease has been affecting millions of people worldwide. Generally, the concomitant occurrence of diabetes and obesity is leading to the so-called diabesity [1]. However, in Asia, it

emerges as non-obese type 2 diabetes characterized by predominant insulin secretory defects [2]. Currently, tolbutamide, a member of sulfonylureas family used as insulin secretagogue and have been at the forefront of the current therapeutic strategies for type 2 diabetes; however, reported with the increased risk of hypoglycemia. Due to this

Abbreviations: PKA, protein kinase A; Epac2, exchange protein directly activated by cAMP2; PKC, protein kinase C; MIN6, mouse insulinoma pancreatic β-cells; IBMX, 3-isobutyl-1-methylxanthine; TLC, thin-layer chromatography; UV, ultraviolet; IR, infrared ⁎ Corresponding author at: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan. E-mail address: hafi[email protected] (R.M. Hafizur). 1 Co-first authors. https://doi.org/10.1016/j.biopha.2019.109348 Received 23 April 2019; Received in revised form 30 July 2019; Accepted 7 August 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

study, we evaluated tambulin in the context of the glucose-dependent insulinotropic mechanism(s), using pharmacological and biochemical approaches in the isolated mice islets and MIN6 cells. Furthermore, we elucidated the insulinotropic effect of tambulin through modulation of the glucose-dependent signaling cascade in a co-operative manner with Ca2+, independent of the KATP channel. We compared the tambulininduced insulin secretion with reference drug, tolbutamide to address the drug-induced hypoglycemia, thereby identifying a better therapeutic alternative. We demonstrated that tambulin possess insulin secretory potential and may be employed as an alternative therapeutic option in diabetes.

characteristic, inappropriate monitoring in the hospitals at night shifts in diabetic patients during aggressive treatment with subsequent sulfonylureas administration often lead to sudden hypoglycemic shock and sometimes death [3,4]. Therefore, here we are addressing the imperative need of new insulin secretory candidates that amplify insulin secretion using only stimulatory glucose. Stimulus-secretion coupling is an essential biologic event in pancreatic β cells and is regulated by numerous ionic and nonionic signaling pathways also known as the KATP-dependent and -independent pathways. Glucose-stimulated insulin secretion is the principle mechanism in β-cells for insulin secretion to prevailing blood glucose levels through glucose metabolism. When glucose rise in the β cell, the rate of glycolysis is increased, ultimately leading to the generation of ATP (or ATP/ADP ratio). An increase in the ATP/ADP ratio provides the functional link between a glucose stimulus and insulin secretion. This event causes the closure of ATP-sensitive K+ (KATP) channels, depolarization of the β cell membrane, activation of the voltage-dependent Ca2+ channels (VDCCs), thereby allowing the intracellular concentration of Ca2+([Ca2+]i), which leads to fusion of insulin-containing secretory granules with the plasma membrane and the first phase insulin secretion followed by sustained second phase of insulin secretion is held when the granules from the readily releasable pool are converted to the immediately releasable pool, an ATP-dependent process termed “priming”. Most of the signals involved in this process also come from glucose mitochondrial metabolism, comprising the amplifying pathways [5,6,27]. Natural compounds are proposed to modulate GSIS by regulating the activity of several ion channels involved in KATP-dependent insulin secretion as well as steps distal to channel modulation; therefore, we tested tambulin-mediated insulin secretion in KATP-dependent and –independent manner. The signaling molecules thus far implicated in the stimulus-induced signaling include adenylyl cyclase/cAMP/PKA/Epac2, PI3-K/ERK/ MAPK, and PLC/PKC. Of these, only cAMP/PKA signaling has been firmly established as a mediator of the insulinotropic effect. PKA phosphorylation has been implicated in the regulation of KATP channels, VDCCs, Kv channels, NSCCs, intracellular Ca2+ stores, docking and fusion of vesicles, and intracellular energy homeostasis. However, recent studies have shown that cAMP also potentiates insulin secretion by PKA‐independent mechanisms involving the cAMP‐binding protein Epac2 (exchange protein activated by cAMP2) [7–11]. Elucidation of the mechanism by which compounds exert its clinically relevant effects and the contribution of various signaling pathways and effector targets remains an important task toward developing effective therapies for type 2 diabetes. Recently, glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-4 (DPP4) inhibitors have been developed as new antidiabetic drugs. GLP-1 has been shown to improve mainly the first phase of insulin secretion by glucose challenge in type 2 diabetic patients. This effect of GLP-1 may be mediated in part by Epac2A/Rap1 signaling, which is involved primarily in the potentiation of the first phase of glucose-stimulated insulin secretion. These drugs all act through cAMP signaling in pancreatic β-cells. The cAMP-PKA is thought to involve in the second phase whereas cAMP-Epac2 in the first phase of insulin secretion. Thus, cAMP signaling is critical not only for potentiating of glucose-stimulated insulin secretion but also for induction of glucose responsiveness [6]. The identification of natural insulin secretagogue(s) and their roles in the glucose-stimulated insulin secretion is of great clinical importance. Tambulin, isolated from the Zanthoxylem armatum, was evaluated for the insulin secretory activity. The Zanthoxylem armatum and its constituents have been reported in several studies for the important medicinal activities such as potent antioxidant activity, significant antitumor activity [12,13] and have been traditionally used by the local people in Nepal for diabetes [14]. However, the molecular details of tambulin, a major compound isolated from Zanthoxylem armatum, in the context of insulin secretion has not yet been investigated. In the present

2. Materials and methods 2.1. Plant materials The fruits of Zanthoxylem armatum were taken from district palpa, Nepal, during November 2013 at approximately 2000 m of altitude. The Plant material was identified in the National Herbarium and Plant Resources, Ministry of Forests and Soil Conservation, Godawari, Nepal. 2.2. Tambulin isolation Air-dried fruits of Zanthoxylem armatum (2.15 kg) were powdered using a grinder and extracted with methanol (5.0 L) three times. The concentrated methanolic extract (330.0 g), after evaporation of the solvent, was dissolved in acetone (500.0 mL). The acetone soluble part (170.0 g) was evaporated and dissolved in distilled water. Water-insoluble part (50 g) was selected based on TLC and subjected to silica gel column chromatography using EtOAc/hexanes as an eluting agent. This yielded compound 1 at the polarity of 10% EtOAc/hexanes. A small impurity in the compound which was seen in the 13C-NMR spectrum was removed by passing through LH-20 Sephadex column, using methanol as an eluting agent. The compound obtained from LH-20 Sephadex column was seen perfectly pure in 13C-NMR spectra. 2.3. The structure elucidation of tambulin Tambulin (3,5-dihydroxy-7,8-dimethoxy-2-(4-methoxyphenyl) chromen-4-one) was obtained as a yellow powder. The EI-MS spectrum of tambulin showed molecular ion [M+] at m/z 344, and base peak at m/z 329. The molecular formula C18H16O7 was determined from HREIMS spectrum which showed [M+] at m/z 344.0906 (Calcd for C18H16O7 = 344.0896), and 13C-NMR (BB and DEPT) spectra. The IR spectrum displayed absorptions at 3327 (OH), 1651 (aromatic), and 1556 (olefinic) cm−1. The UV spectrum showed absorptions at 367, 325, and 273 nm. The 1H-NMR spectrum exhibited resonances for three singlets at δ 3.89, 3.90, and 3.98 were attributed for protons of methoxy groups attached to C-4′, C-7, and C-8, respectively. A downfield singlet resonated at δ 6.51 was ascribed to H-6. Similarly, two downfield ortho coupled doublets at δ 7.13 d (J3′,2′/5′, 6′=9.0 Hz) and 8.27 d (J2′,3′/6′, 5′=9.0 Hz), were assigned to H-3′/H-5′ and H-2′/H-6′ respectively. Two exchangeable protons appeared at δ 11.58 and 6.55 were due to hydroxyl protons attached to C-5 and C-3, respectively. The 13C-NMR spectra (Broad-band decoupled and DEPT) displayed the resonances for all eighteen carbons including three methyl, five methine, and ten quaternary carbons. Structure of compound was further confirmed from 2D-NMR spectra (COSY, HSQC, HMBC, and NOESY) (Supplementary Figs.1–7). Position of hydroxyl and methoxy groups was assigned with the help of the HMBC correlations. The HMBC between protons and carbons resonated at δ 3.88 and δ 162.2 (C-4′), 3.92 and δ 130.3 (C-8), and 3.94 and 159.6 (C-7) indicated the position of methoxy groups in the compound. The key HMBC correlations in tambulin are shown in Fig. S1. 2

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

2.4. Chemicals

2.9. Cell culture, insulin secretion, and immunocytochemical analysis

The 3-isobutyl-1-methylxanthine (IBMX), tolbutamide, calphostin C, Collagenase V, diazoxide, verapamil, and H-89 were purchased from Sigma (St. Louis, MO, USA). Mouse insulin ELISA kit was purchased from Crystal Chem Inc. (IL, USA). cAMP ELISA kit was obtained from Abcam (Cambridge, UK). SQ22536 and calphostin C were obtained from Merck Millipore (Darmstadt, Germany). Tambulin was obtained as a yellow powder from 10% EtOAc/hexanes.

MIN6 cells were kindly provided by Dr. Jun-Ichi Miyazaki (Osaka University, Japan). MIN6 cells were cultured, used for the insulin secretory and immunocytochemical analysis as described previously [16,17]. For insulin staining intensity, 3-D surface plot analysis of insulin immunostained MIN6 cells was made using ImageJ software ver. 1.50i. 2.10. The cell viability assay of tambulin

2.5. Animal studies

For the evaluation of the cell viability, isolated islets incubated in the presence and absence of tambulin were disintegrated into single cells and were then treated with trypan excluding blue for 15 min. Additionally, the cytotoxic effect of tambulin was determined in MIN6 cells by using MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide) assay as described by Mosmann T [18].

Male BALB/c mice were used from the animal house of the International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan. The animals were kept under the standard laboratory conditions (25 ± 2 °C; 50–55% humidity; 12-h light, 12-h dark cycle). The mice were provided with a clean environment and access to water and food ad libitum. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and with prior approval from the Animal Use Committee of the International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan (Protocol number: 2015-0020).

2.11. Molecular docking studies In silico investigation was carried out to rationalize the binding mechanism of tambulin with PKA. The atomic coordinates of PKA available in the protein data bank under the accession code: 4MX3 [19], was optimized using the protocol as reported previously [20]. The chemical structure of tambulin was generated by builder module implemented in MOE [21]. The compound was charged and minimized by MMFF94x force field [22] and saved into mol2 format. For docking simulation, default postprocessing protocol with rigid receptor scheme and GBVI/WSA dG scoring function was used to estimate the binding free energy in Kcal/mol. 100 conformations were generated and saved in mdb format. The top-ranked pose of the compound was selected based on binding free energy.

2.6. Mice pancreatic islet isolation The fresh islets from male BALB/c mice (30–40 g) were isolated as previously described [15,16]. Briefly, mice pancreas was exposed and distended with 3 ml collagenase solution (1 mg/ml) administered via the common bile duct. The pancreas was extracted and immediately digested in collagenase solution (37 °C; 15 min), washed in Hank’s Balanced Salt Solution and the medium-sized (80–120 μm) islets were manually picked under NIKON SMZ-745 stereomicroscope.

2.12. Statistical analysis All statistical analysis was performed by SPSS 12.0 Statistical Package for Windows (SPSS, Inc., Chicago, IL, USA). Q-Q probability was used to analyze the data distribution. All values were expressed as Mean ± S.E.M. Comparisons were made using an unpaired t-test and one-way ANOVA with Dunnet’s and Bonferroni post hocs tests, as appropriate. P values < 0.05 were considered statistically significant.

2.7. Insulin secretion The freshly isolated islets were pre-incubated for 45 min at 37 °C in Krebs-Ringer bicarbonate buffer (KRBB) with 3 mM glucose. After preincubation, batches of three islets were incubated for 60 min at 37 °C in KRBB with 3 mM (basal) and 17 mM (stimulatory) glucose supplemented with the test substance(s) as follow. The freshly isolated mice islets in a group of three (3) were treated as follows: various concentrations of tambulin, 0, 10, 50, 100, 200, and 400 μM in the presence of 3 and 17 mM glucose; different glucose concentrations (3, 6, 11.2, 17, and 20 mM) with or without tambulin (200 μM) and tolbutamide (200 μM); 17 mM glucose plus 50 μM diazoxide, with or without tambulin; 17 mM glucose plus 25 mM KCl with or without tambulin; 17 mM glucose plus 200 μM verapamil, with or without tambulin; 17 mM glucose plus 100 μM isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor with or without tambulin; 17 mM glucose plus 50 μM H-89, a PKA inhibitor, with or without tambulin; 17 mM glucose plus 25 μM SQ22536, an adenylate cyclase inhibitor with or without tambulin. Following incubation, the secreted insulin was quantified with mouse insulin ELISA kit.

3. Results 3.1. Effects of tambulin on the glucose-stimulated insulin secretion (GSIS) We tested tambulin (Fig. 1) for the concentration-dependent (10–400 μM) effect on insulin secretion of statically incubated isolated mouse islets and MIN6 cells. Tambulin markedly increased insulin level at stimulatory glucose (17 mM) but no effect on insulin secretion in the presence of basal glucose (3 mM). Furthermore, tambulin-induced insulin secretion occurs in a dose-dependent manner where the optimum

2.8. Intracellular cAMP assay Islets in groups of 9 were incubated in KRBB supplemented with 17 mM glucose for 60 min in the absence or presence of tambulin with or without IBMX (100 μM). Following incubation, media was removed, washed with KRBB and 300 μL 0.1 N HCl was added to the islet pellets. After sonication, cAMP contents of the islets were measured using an acetylation enzyme immunoassay system (Abcam).

Fig. 1. The structure of tambulin. 3

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

Fig. 2. Effect of tambulin (TM) on the glucose-induced insulin secretion in isolated mice islets and MIN6 cells. (A) TM stimulated glucose-dependent insulin secretion in isolated mice islets. TM was co-incubated at 0, 10, 50, 100, 200, and 400 μM concentrations with isolated islets at 17 mM 3 mM glucose. (B) Following incubation in the presence or absence of TM, insulin immunocytochemistry (a–d), 3-D surface plot analysis (e–h) as drawn from the immunochemical data, and fluorescence intensity was estimated in MIN6 cells after insulin secretion at basal (2 mM) and stimulatory (20 mM) glucose concentrations (i). For fluorescence intensity measurements 5–7 fields were selected per image from 3 to 5 independent experiments for representative images. Groups of 3 size-matched islets (Fig. 2A) or 5 × 105 MIN6 cells (Fig. 2B) were incubated at 37 °C for 60 min in KRB buffer containing basal or stimulatory glucose in the absence or presence of TM at the indicated concentration. All data points are an average of n = 3 and n = 4 separate experiments for mice islets and MIN6 cells. *P < 0.05, **P < 0.01, ***P < 0.001 significant change over the respective control values.

4

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

concentration. Interestingly, the insulin secretion induced by tambulin was higher than the tolbutamide-induced insulin secretion at 17 mM glucose concentrations. Furthermore, tambulin exerted significant (P < 0.01) additive effect in the tolbutamide-induced insulin secretion (69.51 ± 2.6 vs. 34.57 ± 2.05 ng/islet/h) (Fig. 3B). 3.2. Tambulin-induced insulin secretory effect independent of KATP channels The KATP channel is well-studied therapeutic target in the β-cell for sulfonylureas; however, they cause sulfonylurea-induced hypoglycemia which is one of the major rationales for the present study. We found that the insulin secretory potential of tambulin was decreased but not completely (28.75 ± 2.9 vs. 56.62 ± 2.2 ng/islet/h) by diazoxide at 50 μM and partially persisted its insulinotropic effect at stimulatory glucose concentration (Fig. 4A). We found that the tambulin-induced insulin secretion was further amplified (71.25 ± 2.8 vs. 55.07 ± 2.6 ng/islet/h), in the depolarized islets with KCl (25 mM) and in the presence of diazoxide (Fig. 4B). These findings in conjunction with no effect of tambulin at basal glucose collectively suggest that tambulin may act indirectly via the K-ATP channel-dependent influx of Ca2+ in tambulin-induced insulin exocytosis. 3.3. Roles of tambulin in the Ca2+-dependent insulin secretion Here, we investigated whether tambulin has any modulatory effect on the subcellular Ca2+ concentration. Based on our experimental observations, we obtained numerous findings, first at stimulatory glucose using verapamil, tambulin-induced insulin secretion was significantly (P < 0.001) inhibited to almost basal level (3.75 ± 0.3 vs. 57.25 ± 3.8 ng/islet/h) (Fig. 4C). Second, removing the extracellular Ca2+, tambulin-induced insulin secretion was significantly (P < 0.001) inhibited (7.75 ± 1.3 vs. 61.21 ± 2.8 ng/islet/h) (Fig. 4D). Third, at stimulatory glucose concentration, no complete inhibition in the tambulin-induced insulin secretion (28.75 ± 2.9 vs. 56.62 ± 2.2 ng/islet/h) was observed even after using diazoxide (Fig. 4A). Fourth, tambulin showed no effect on insulin release at basal glucose (Fig. 2A–B; Fig. 3A). Fifth, tambulin showed an additive effect in the tolbutamide-induced insulin secretion (Fig. 3B). Sixth, tambulin amplification of insulin secretion was further enhanced in the depolarized islets (Fig. 4B). These exclusive findings suggest that tambulin exets glucose-induced insulin secretion via KATP-independent Ca2+-dependent amplifying pathway.

Fig. 3. TM exerts glucose-dependent insulinotropic effect different from sulfonylurea. (A) Comparative effect of TM and TB on the insulin secretion from mice islets at different glucose concentrations. Insulin secretion was induced by 200 μM TM (●), 200 μM TB (▲) and glucose (control) alone (○). (B) Islets were incubated in 17 mM glucose in the presence or absence of TM and/or TB at the indicated concentrations. Values are mean ± S.E.M. for 3–5 independent experiments each in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, †P < 0.05, significant changes when compared with the control values.

increase (about 2.7-fold; P < 0.001) was found at 200 μM (Fig. 2A). Tambulin significantly enhanced glucose-stimulated insulin secretion in MIN6 cells (Fig. 2Ba–h) that was further justified by the immunocytochemical findings (Fig. 2Ba–i). We observed that as insulin secretion increases, the fluorescence of insulin immunostained MIN6 cells decreases and this inverse co-relation of insulin vs. fluorescence was used as the indicator of insulin expression (Fig. 2B a, c, e, and g). We found that fluorescence intensity significantly reduced in MIN6 cells co-incubated with tambulin at 20 mM glucose (Fig. 2Bg). However, no pronounced effect in the fluorescence intensity was observed at 2 mM glucose (Fig. 2Bc). Also, the surface plot analysis revealed that decrease in the insulin intensity in conjunction with insulin staining observed at the peripheries of cells, which showed that the insulin is being surged out of the cells (Fig. 2B b, d, f, and h). Moreover, the insulinotropic effects of tambulin were evaluated and compared to a reference standard, tolbutamide, a sulfonylurea in the context of their mode of action (Fig. 3A) at different glucose concentrations (3, 6, 11.2, 17, and 20 mM). In contrast to the comparator, the sulfonylurea tolbutamide, tambulin did not enhance insulin secretion both at 3 and 6 mM glucose concentrations but significantly stimulated insulin secretion at 11.2 mM (about 3.2-fold; P < 0.001), 17 mM (about 2.1-fold; P < 0.001) and 25 mM (about 1.8-fold; P < 0.001) glucose concentrations. In contrast, tolbutamide exhibited an insulinotropic effect each at 3, 6, 11.2, and 17 mM glucose

3.4. cAMP-PKA mediated tambulin-induced insulin secretion We further explored the cAMP-PKA, the important player of amplifying pathway, for tambulin-induced insulin secretion. Based on the cAMP ELISA assay, we found that tambulin enhanced the intracellular cAMP (Fig. 5A). We further investigated whether the increase in the intracellular cAMP is either via activating adenylate cyclase or inhibition of cAMP hydrolysis by inhibition of phosphodiesterase. Using IBMX, a phosphodiesterase inhibitor, we found that tambulin showed no additive effect in IBMX-induced intracellular cAMP contents (Fig. 6A). In contrast to cAMP, tambulin exerted additive effect (87.55 ± 3.3 vs. 61.37 ± 4.1 ng/islet/h) in the IBMX-induced insulin secretion (Fig. 5B). Furthermore, we determined the active role of the PKA signaling pathway in the tambulin-induced insulin secretion by using a nullifying approach, that what if the role of PKA is excluded. We found that in the presence of H89, a PKA inhibitor (50 μM), the insulinotropic activity of tambulin was significantly inhibited at 17 mM glucose (30.21 ± 1.2 ng/islet/h) compared the tambulin-induced insulin secretion alone (57.75 ± 3.4 ng/islet/h) (Fig. 5D). In contrast to PKA, lesser inhibition in tambulin-induced insulin secretion was observed by using SQ22536 (Fig. 5C). These findings suggest that the cAMP/PKA signaling pathway is contributing a major role in the 5

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

Fig. 4. TM exerts the glucose-dependent insulinotropic effect -independent of KATP channels and Ca2+ dependent. (A) Effect of TM on insulin secretion in mice islets using diazoxide (KATP channels opener), (B) depolarized by KCl in the presence of diazoxide and (C) verapamil, an L-type Ca2+ channels blocker (C) Ca0 buffer, without extracellular Ca2+. Mice islets were incubated in 16.7 mM glucose in the presence or absence of TM with or without diazoxide, KCl. verapamil used at the concentration as indicated and Ca2+ 0 buffer. Values are mean ± S.E.M. from 3 to 5 independent experiments each in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs. insulin secretion in the absence of diazoxide and/or KCl.

insulin secretion [16,23–26]. Based on numerous findings, in the current study, we demonstrate that tambulin acts on targets other than tolbutamide. First, tambulin showed no effect on insulin secretion in basal glucose conditions. Secondly, tambulin showed an additive effect in tolbutamide-induced insulin secretion. Third, the insulin secretory potential of tambulin was not completely inhibited by diazoxide. Fourth, the insulin secretory potential of tambulin was further amplified in depolarized islets. Taken together, these findings suggest that though K+ current is integral to glucose-dependent insulin secretion; however, is bypassed in tambulininduced insulin secretion as shown in previous studies [5]. Our findings are in strong agreement with Efanov et al. 2001 [28], where they reported a similar study of imidazoline-induced insulin secretion. The intracellular Ca2+ concentration integrated with signaling cascades that significantly modulate the insulin secretory activity [8,11]. In our study, we identified the involvement of L-type Ca2+ channels using verapamil, a Ca2+ channels blocker. The tambulin-induced insulin secretion, in the presence of verapamil and/or removing the extracellular Ca2+, was significantly inhibited. Our data of Ca2+ and K+ currents are in strong agreement with a study performed by Skelin et al., [11], where they have shown that the amplifying pathway works in K-ATP independent Ca2+ channels dependent manner. Based on these observations, we describe that a) TM-induced insulin secretion works in insulin amplifying pathways rather than insulin triggering pathway, b) Glucose-stimulated insulin secretion works in insulin triggering pathway followed by insulin amplifying pathway, and c) TMinduced insulin secretion and glucose-stimulated insulin secretion works simultaneously, unifying the triggering and amplifying pathway thereby potentiates acute insulin secretion. These new findings distinguish our studies from previously reported insulin secretory activity of other flavonoids [31–33].

tambulin-induced insulin secretion. In vitro PKA-dependent insulin secretory potential of tambulin was also correlated with in silico studies, which showed a good correlation between the experimental and theoretical results. The Molecular docking simulation was performed to investigate the binding potential of tambulin by targeting the regulatory domain of PKA. The analysis of the docking results indicated that the compound was well accommodated in the binding site of PKA with docking score -6.437. As shown in Fig. 6AB., tambulin stabilized by making the number of hydrophilic and hydrophobic interactions with PKA leads to stable conformation of tambulin. Both hydroxyl groups of benzopyran-4-one ring involved in hydrogen bond interactions with Val313 and Ala334 at a distance of 2.54 and 3.02 Å. Similarly, the carbonyl and methoxy group of the compound involved in hydrogen bond interaction with Arg333 (2.75 Å), Ala334 (3.52 Å) and Ser373 (3.45 Å), respectively. The compound was further stabilized by making hydrophobic interactions with Val300, Ile325, Ala334, and Tyr371.

4. Discussion In this study, we have shown the possible mechanism of tambulin in stimulating glucose-induced insulin secretion. Based on the insulin secretory assay and immunocytochemical analysis, the established data suggests that tambulin works exclusively in higher glucose concentrations; however not at basal glucose concentrations. This characteristic speculated pharmacological significance of lowering the risk of sulfonylurea-induced hypoglycemia as reported previously [3,4]. Furthermore, the tambulin-induced insulin secretion data alone, and in comparison, to that of the tolbutamide, indicate that tambulin is more potent and least toxic. Also, there are several studies which reported natural molecules and their significant roles in glucose-stimulated 6

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

Fig. 5. TM stimulates cAMP-dependent PKA activity. (A) Raised in the intracellular cAMP and (B) insulin secretion by TM in the presence or absence of IBMX. Inhibition of TM-induced insulin secretion in the presence or absence of (C) SQ22536, an adenylate cyclase inhibitor and (D) H-89, a PKA inhibitor. Islets were incubated in 16.7 mM glucose in the presence or absence of TM and/or IBMX, SQ22536, H-89 at the indicated concentrations. Values are mean ± S.E.M. for 3–5 independent experiments each in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, significant changes when compared with addition of IBMX and TM alone. ns. not significant.

incubated with tambulin at different glucose concentrations coupled with surface plot analysis of insulin staining only at cells peripheries indicate that tambulin is probably affecting the second phase of insulin secretion exhibiting amplifying pathway by trafficking insulin granules pools to be fused with plasma membrane followed by insulin exocytosis. Our findings are in strong agreement with the mechanism of amplifying insulin secretion described by MLL de la Vega et al., where they reported the rapid first phase of glucose-stimulated insulin secretion, caused by a triggering pathway and/or KATP-dependent mechanism, followed by a sustained second phase due to an amplifying pathway or KATP-independent mechanism [30]. Thus, overall computational modeling and in vitro studies concluded the mechanism of tambulin via Ca2+ and PKA signaling pathway with no direct involvement of KATP channel. Collectively, these promising findings suggest tambulin as a possible drug candidate against diabetes.

Numerous earlier studies have identified the cAMP-PKA signaling cascade as one of the major pathways that work in synchronized fashion with Ca2+ for the regulation of insulin secretion and glucose homeostasis [2,7]. The cAMP stimulates insulin secretion in PKA- and Epac2-dependent phosphorylation that leads to accelerate insulin production increase the sensitivity of the insulin granules to Ca2+ coupling with glucose, and eventually lead to triggered insulin exocytosis [7,8]. Previously, cAMP-PKA has been investigated for glycemic control type 2 diabetes therapies [10]. Our results demonstrated that tambulin showed significant insulinotropic effect through the PKA signaling pathway. First, according to the reported data, PKA has a predominant role in the overall effect of cAMP on insulin exocytosis [7–9]. Second, tambulin showed significant additive effect in IBMX-induced insulin secretion; whereas, no additive effect in IBMX-induced cAMP level. Third, tambulin-induced insulin secretion was inhibited more by using H-89, protein kinase A inhibitor compared to SQ22536, an adenylate cyclase inhibitor. These findings were further validated by our molecular docking studies where we found the best binding affinities of tambulin with PKA. Furthermore, docking studies revealed the partial interaction of tambulin with PKC binding pockets (data not shown); however, this does not necessarily mean to be involved in tambulininduced insulin secretion. These findings suggest that tambulin may have the insulinotropic effect specifically on the modulation of a cAMPdependent PKA signaling cascade that leads to the activation of downstream mediators through phosphorylation, coupled with glucose for insulin secretion. Our results are justified by similar studies performed by the different research group, where they found other flavonoids, namely genistein and eriodictyol enhance insulin secretion in pancreatic β-Cells through cAMP-dependent PKA pathway [16,23,29]. Furthermore, the immunocytochemical findings in MIN6 cells

Author contributions A.H. and S.A.R. designed the islets experiments, conducted the research, cell culture experiments and preparation of MIN6 cells for immunocytochemical analysis, generated and validated the data, analyzed the data and wrote the manuscript. M.I.K. contributed in islet isolation experiments and data analysis, critically reviewed/edited and revised the manuscript. J.B., A.A., J.B., M.N., S.A., and A.J.A., were involved in isolation, purification and structure elucidation of tambulin. S.A. and Z.U.H. did molecular docking studies. R.M.H. designed the work, analyzed the data, and closely supervised and monitored all aspects of this study from conception of the idea to submission of the paper.

7

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

Fig. 6. Molecular docking representation of TM with PKA, (A) 3-D, (B) 2-D. The predicted binding mode of tambulin showed the interactions with key residues of PKA.

Declaration of Competing Interest

Appendix A. Supplementary data

We wish to declare that there are no known conflicts of interest with the research reported that could have influenced the outcome.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109348. References

Acknowledgments [1] D.R. Leitner, G. Frühbeck, V. Yumuk, K. Schindler, D. Micic, E. Woodward, H. Toplak, Obesity and type 2 diabetes: two diseases with a need for combined treatment strategies - EASO can lead the way, Obes. Facts 10 (5) (2017) 483–492. [2] Asian Association for the Study of Diabetes. Promoting research for better diabetes care in Asia: Kyoto declaration on diabetes, J. Diab. Invest. 4 (2) (2013) 222. [3] P.E. Cryer, Glycemic goals in diabetes: trade-off between glycemic control and iatrogenic hypoglycemia, Diabetes 63 (7) (2014) 2188–2195. [4] A.J. Scheen, Investigational insulin secretagogues for type 2 diabetes, Expert Opin. Investig. Drugs 25 (4) (2016) 405–422. [5] P. Rorsman, F.M. Ashcroft, Pancreatic beta-cell electrical activity and insulin

This work was supported by a grant from the Higher Education Commission (HEC), Pakistan [HEC/R&D/NRPU/2017/8544]. We are grateful to Prof. Jun-Ichi Miyazaki from the Division of Medicine, Graduate School of Medicine, Osaka University, Japan, for providing MIN6 cells used in this research. The authors thank the Deanship of Scientific Research at King Saud University, Saudi Arabia for funding through the research group project no. RGP-1438-043. 8

Biomedicine & Pharmacotherapy 120 (2019) 109348

A. Hameed, et al.

secretion: of mice and men, Physiol. Rev. 98 (2018) 117–214. [6] S. Seino, T. Shibasaki, K. Minami, Dynamics of insulin secretion and the clinical implications for obesity and diabetes, J. Clin. Invest. 121 (6) (2011) 2118–2125. [7] I. Alenkvist, N. Gandasi, S. Barg, A. Tengholm, Recruitment of epac2A to insulin granule docking sites regulates priming for exocytosis, Diabetes 66 (10) (2017) 2610–2622. [8] Q. Ni, A. Ganesan, N. Aye-Han, X. Gao, M. Allen, A. Levchenko, J. Zhang, Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit, Nat. Chem. Biol. 7 (1) (2010) 34–40. [9] H. Yang, L. Yang, Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy, J. Mol. Endocrinol. 57 (2) (2016) R93–R108. [10] T. Shibasaki, H. Takahashi, T. Miki, Y. Sunaga, K. Matsumura, M. Yamanaka, C. Zhang, A. Tamamoto, T. Satoh, J. Miyazaki, Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP, Proc. Natl. Acad. Sci. 104 (49) (2007) 19333–19338. [11] S. Masa, R. Marjan, cAMP increases the sensitivity of exocytosis to Ca2+ primarily through protein kinase A in mouse pancreatic beta cells, Cell Calcium 49 (2) (2011) 89–99. [12] B.H. Babu, H.N. Jayaram, N.K. Mary, B.S. Shylesh, J. Padikkala, In vitro free radical scavenging, cytotoxic and antitumor activity of tambulin - A flavone from Zanthoxylum alatum, Amala Res. Bull. 22 (2002) 90–98. [13] K.P. Devkota, J. Wilson, C.J. Henrich, J.B. Mcmahon, K.M. Reilly, J.A. Beutler, Isobutyl hydroxy amides from the pericarp of Nepalese Zanthoxylum armatum inhibit NF1-defective tumor cell line growth, J. Nat. Prod. 76 (1) (2013) 59–63. [14] T.P. Singh, O.M. Singh, Phytochemical and pharmacological profile of Zanthoxylum armatum DC.-An overview, Indian J. Nat. Prod. Resour. 2 (3) (2011) 275–285. [15] R.M. Hafizur, A. Hameed, M. Shukrana, S.A. Raza, S. Chishti, N. Kabir, R.A. Siddiqui, Cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and by stimulating insulin secretion in vitro, Phytotomedicine 22 (2) (2015) 297–300. [16] A. Hameed, R.M. Hafizur, N. Hussain, S. Raza, M. Rehman, S. Ashraf, Z. Ul-Haq, F. Khan, G. Abbas, M.I. Choudhary, Eriodictyol stimulates insulin secretion through cAMP/PKA signaling pathway in mice islets, Eur. J. Pharmacol. 820 (2018) 245–255. [17] J. Miyazaki, K. Araki, E. Yamato, H. Ikegami, T. Asano, Y. Shibasaki, Y. Oka, K. Yamamura, Establishment of a pancreatic β-cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms, Endocrinology 127 (1) (1990) 126–132. [18] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983) 55–63. [19] J.G. Bruystens, J. Wu, A. Fortezzo, A.P. Kornev, D.K. Blumenthal, S.S. Taylor, PKA RIα homodimer structure reveals an intermolecular interface with implications for

[20]

[21] [22] [23]

[24]

[25]

[26]

[27] [28]

[29]

[30]

[31] [32]

[33]

9

cooperative cAMP binding and Carney complex disease, Structure 22 (1) (2014) 59–69 7. A. Hameed, S. Ashraf, M.I. Khan, R.M. Hafizur, Z. Ul-Haq, Protein kinase A-dependent insulinotropic effect of selected flavonoids, Int. J. Biol. Macromol. 119 (2018) 149–156. Molecular Operating Environment (MOE), Chemical Computing Group Inc. 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015 (2013). T.A. Halgren, Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94, Wiley Online Libr. 17 (1996) 490–519. D. Liu, W. Zhen, Z. Yang, J.D. Carter, H. Si, K.A. Reynolds, Genistein acutely stimulates insulin secretion in pancreatic β-Cells through A cAMP-dependent protein kinase A pathway, Diabetes 55 (4) (2006) 1043–1050. G. Bardy, A. Virsolvy, J.F. Quignard, M.A. Ravier, G. Bertrand, S. Dalle, G. Cros, R. Magous, S. Richard, C. Oiry, Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells, Br. J. Pharmacol. 169 (5) (2013) 1102–1113. A.M. Efanov, S.V. Zaitsev, H. Mest, A. Raap, I.B. Appelskog, O. Larsson, P. Berggren, S. Efendic, The novel imidazoline compound BL11282 potentiates glucose-induced insulin secretion in pancreatic β-cells in the absence of modulation of K-ATP channel activity, Diabetes 50 (4) (2001) 797–802. Z. Orfi, F. Waczek, F. Baska, I. Szabadkai, R. Torka, J. Hartmann, L. Orfi, A. Ullrich, Novel members of quinoline compound family enhance insulin secretion in RIN5AH beta cells and rat pancreatic islet microtissue, Sci. Rep. 7 (2017) 44073. P. Rorsman, F.M. Ashcroft, Pancreatic beta-cell electrical activity and insulin secretion: of mice and men, Physiol. Rev. 98 (1) (2018) 117–214. A.M. Efanov, S.V. Zaitsev, H. Mest, A. Raap, I.B. Appelskog, O. Larsson, P. Berggren, S. Efendic, The novel imidazoline compound BL11282 potentiates glucose-induced insulin secretion in pancreatic β-cells in the absence of modulation of KATP channel activity, Diabetes 50 (4) (2001) 797–802. A. Hameed, S. Ashraf, M.I. Khan, R.M. Hafizur, Z. Ul-Haq, Protein kinase A-dependent insulinotropic effect of selected flavonoids, Int. J. Biol. Macromol. 119 (2018) 149–156. M. la Vega-Monroy, C. Fernandez-Meji, Beta-cell function and failure in type 1 diabetes. Type 1 diabetes - pathogenesis, Genet. Immunother. (2011), https://doi. org/10.5772/22089. S.A. Aherne, N.M. O’Brien, Dietary flavonols: chemistry, food content, and metabolism, Nutrition 18 (1) (2002) 75–81. M. Pinent, A. Castell, I. Baiges, G. Montagut, L. Arola, A. Ardévol, Bioactivity of flavonoids on insulin‐secreting cells, Comp. Rev. Food Sci. Food Saf. 7 (4) (2008) 299–308. J.A. Chemler, L.T. Lock, M.A. Koffas, E.S. Tzanakakis, Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion, App. Microbiol. Biotechnol. 77 (4) (2007) 797–807.