Hypoxylonol F Isolated from Annulohypoxylon annulatum Improves Insulin Secretion by Regulating Pancreatic β-cell Metabolism

Insulin plays a key role in glucose homeostasis and is hence used to treat hyperglycemia, the main characteristic of diabetes mellitus. Annulohypoxylon annulatum is an inedible ball-shaped wood-rotting fungus, and hypoxylon F is one of the major compounds of A. annulatum. The aim of this study is to evaluate the effects of hypoxylonol F isolated from A. annulatum on insulin secretion in INS-1 pancreatic β-cells and demonstrate the molecular mechanisms involved. Glucose-stimulated insulin secretion (GSIS) values were evaluated using a rat insulin ELISA kit. Moreover, the expression of proteins related to pancreatic β-cell metabolism and insulin secretion was evaluated using Western blotting. Hypoxylonol F isolated from A. annulatum was found to significantly enhance glucose-stimulated insulin secretion without inducing cytotoxicity. Additionally, hypoxylonol F enhanced insulin receptor substrate-2 (IRS-2) levels and activated the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway. Interestingly, it also modulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and pancreatic and duodenal homeobox 1 (PDX-1). Our findings showed that A. annulatum and its bioactive compounds are capable of improving insulin secretion by pancreatic β-cells. This suggests that A. annulatum can be used as a therapeutic agent to treat diabetes.


Introduction
Diabetes is a common chronic metabolic disease that affects millions of people worldwide and is closely related to modern lifestyle [1]. Hyperglycemia is the hallmark of diabetes and is caused by impaired insulin synthesis or secretion [2]. Pancreatic β-cells located in the islet of Langerhans play a critical role in insulin synthesis and secretion, which controls energy metabolism [3]. Typically, the elevation in blood glucose levels after a meal stimulates insulin secretion [4]. However, when insulin secretion is inadequate to meet the metabolic demand, blood glucose levels remain high, resulting in diabetes [5,6]. 1 H and 13 C, respectively) in acetone-d 6 . Chemical shifts in the proton and carbon spectra measured in acetone-d 6 were reported in reference to residual solvent peaks at 2.05 and 29.9 ppm, respectively. Ultra-high-performance liquid chromatography (UPLC) ESI mass spectrometry was performed on a Shimadzu LCMS-2020 system (Shimadzu, Kyoto, Japan). High-resolution mass spectra were acquired using a JEOL JMS-700 mass spectrometer (JEOL Ltd, Tokyo, Japan) under electron impact or fast atom bombardment (FAB) conditions at the Korea Basic Science Institute.

Fungal Material
Mushrooms collected from Yeongok-myeon, Gangneung city, Korea, were identified as A. annulatum. A voucher specimen (MCO-NP-I-0026) was deposited at the Library of Natural Products Research Institute, Korea Institute of Science and Technology. The genus Annulohypoxylon is a member of the Xylariaceae family that has brown to dark brown and phaseoliform single-cell ascospores with a conspicuous full germ slit. Samples from dead wood were washed thoroughly to eliminate extraneous material, lyophilized, and stored in a refrigerator at −15 • C until use.

Extraction and Isolation
The lyophilized A. annulatum (280 g) was extracted twice with methanol (3 L) at room temperature and filtered. The methanolic extract (ca. 35 g) was suspended in water and then successively partitioned with normal hexane (Hex), ethyl acetate (EA), and normal butanol (BuOH), yielding 5.4, 7.1, and 8.2 g of residue, respectively. To identify active ingredients responsible for insulin secretion, each fraction was evaluated for glucose-stimulated insulin secretion (GSIS) using a rat insulin ELISA kit. The active fraction, the EA-soluble fraction, was first separated by reversed-phase HPLC (Phenomenex C18 column, 250 × 21.2 mm, 10 µm) eluting water (A) and MeCN (B), both containing 0.1% formic acid, at a flow rate of 20 mL/min, using gradient solvent systems (50% B over 5 min, 50-100% B over 50 min, 100% B over 5 min) with a 230 nm UV detector to yield three sub-fractions (A-C). Further purification of each sub-fraction (A, B, and C) was carried out using semi-preparative HPLC with a column fitted with a Phenomenex column (C6-ph, 250 × 10 mm, 5 µm) and RI detector and eluted with 25% aqueous MeOH, at a flow rate of 4 mL/min, to afford pure compounds 1 (t R 12.9 min, 682 mg, purity 96.4%), 2 (t R 13.8 min, 407 mg, purity 95.3%), and 3 (t R 16.0 min, 588 mg, purity 94.5%).
Ultra-high-performance liquid chromatography (UPLC) ESI mass spectrometry was performed on a Shimadzu LCMS-2020 system (Shimadzu, Kyoto, Japan). High-resolution mass spectra were acquired using a JEOL JMS-700 mass spectrometer (JEOL Ltd, Tokyo, Japan) under electron impact or fast atom bombardment (FAB) conditions at the Korea Basic Science Institute.

Fungal Material
Mushrooms collected from Yeongok-myeon, Gangneung city, Korea, were identified as A. annulatum. A voucher specimen (MCO-NP-I-0026) was deposited at the Library of Natural Products Research Institute, Korea Institute of Science and Technology. The genus Annulohypoxylon is a member of the Xylariaceae family that has brown to dark brown and phaseoliform single-cell ascospores with a conspicuous full germ slit. Samples from dead wood were washed thoroughly to eliminate extraneous material, lyophilized, and stored in a refrigerator at −15 °C until use.

Cell Viability Assay
Cell viability was evaluated using the Ez-Cytox cell viability detection kit (Daeil Lab Service Co., Seoul, Korea) [25]. INS-1 cells were seeded (1 × 10 4 cells/well) in 96-well plates. After incubation for 24 h, the cells were treated with test compounds for 24 h and then incubated with Ez-Cytox reagent (10 µL/well) for 2 h. Following incubation, absorbance values were measured at 450 nm using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The cell viability of the control (untreated cells) was regarded as 100%.
The human hepatocellular carcinoma cell lines HepG2 and Hep3B, human cervical carcinoma cell line HeLa, human breast carcinoma cell lines MCF7 and MDA-MB-231, and human glioblastoma cell line T98G were purchased from the American Type Culture Collection (ATCC). The cells were routinely grown in DMEM (Gibco) and RPMI1640 (Gibco), supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C in a humidified atmosphere with 5% CO 2 .

Insulin Secretion Assay
Glucose-stimulated insulin secretion (GSIS) was evaluated using a rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Gunma, Shibukaw, Japan). INS-1 cells were seeded (5 × 10 5 cells/well) in 12-well plates. After incubation for 24 h, each well was washed twice with Krebs-Ringer bicarbonate HEPES buffer (KRBB, 4.8 mM KCl, 129 mM NaCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 2.5 mM CaCl 2 , 10 mM HEPES, 5 mM NaHCO3, and 0.1% BSA, pH 7.4) and 2.8 mM glucose. Before treatment, the cells were allowed to starve in fresh KRBB. After incubation for 2 h, the cells were treated with KRBB containing test samples and gliclazide (positive control), and then KRBB containing basal (2.8 mM) and stimulating (16.7 mM) glucose concentrations was added to each well. After incubation for 1 h, the supernatants from each well were collected and centrifuged at 12,000 rpm and 4 • C for 10 min and then GSIS was assessed using a rat insulin ELISA kit according to the manufacturer's instructions. The glucose stimulation index (GSI) was calculated by dividing the insulin level at the stimulating (16.7 mM) glucose concentration by the insulin level at the basal (2.8 mM) glucose concentration and was compared with the control (untreated glucose-stimulated cells).

Western Blot Analysis
The expression of proteins related to pancreatic β-cell metabolism was evaluated using Western blot analysis [26]. INS-1 cells were seeded (4 × 10 5 cells/well) in 6-well plates. After incubation for 24 h, the cells were treated with test samples for 24 h and then lysed with RIPA buffer (Cell Signaling, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride on ice at 4 • C. Cell lysates were collected and centrifuged at 6000 rpm for 2 min at 4 • C. The supernatants were collected and the concentration of each protein was determined using the Pierce™ BCA protein assay kit (Thermo Scientific, Carlsbad, CA, USA).

Statistical Analysis
Statistical significance was determined using the One-Way Analysis of Variance (ANOVA) and multiple comparisons with a Bonferroni correction. p values of less than 0.05 indicated statistical significance. All analyses were performed using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Effect of Compounds 1-3 Isolated from A. annulatum on Glucose-Stimulated Insulin Secretion
To determine the non-toxic dose ranges of compounds 1-3, we assessed the cytotoxic effect of various concentrations of compounds 1-3 on INS-1 cells. As shown in Figure 2, compounds 1-3 at 1, 2.5, and 5 µM show no toxic effects. Additionally, as shown in Figure 3, compounds 1-3 lead to an increase in GSI even at 1 µM. The GSI levels are 6.1 ± 0.1, 10.6 ± 0.3, and 6.8 ± 0.2 for compounds 1-3 at 5 µM, respectively. Among the three compounds, hypoxylonol F (2) leads to the strongest increase in GSI in a dose-dependent manner ( Figure 3B).
Based on insulin secretion assays, hypoxylonol F (2) from A. annulatum stimulates insulin secretion in INS-1 cells without inducing cytotoxicity. Therefore, further mechanistic studies were carried out using hypoxylonol F (2).

Effect of Compounds 1-3 Isolated from A. annulatum on Glucose-Stimulated Insulin Secretion
To determine the non-toxic dose ranges of compounds 1-3, we assessed the cytotoxic effect of various concentrations of compounds 1-3 on INS-1 cells. As shown in Figure 2, compounds 1-3 at 1, 2.5, and 5 µM show no toxic effects. Additionally, as shown in Figure 3, compounds 1-3 lead to an increase in GSI even at 1 µM. The GSI levels are 6.1 ± 0.1, 10.6 ± 0.3, and 6.8 ± 0.2 for compounds 1-3 at 5 µM, respectively. Among the three compounds, hypoxylonol F (2) leads to the strongest increase in GSI in a dose-dependent manner ( Figure 3B).
Based on insulin secretion assays, hypoxylonol F (2) from A. annulatum stimulates insulin secretion in INS-1 cells without inducing cytotoxicity. Therefore, further mechanistic studies were carried out using hypoxylonol F (2).

Discussion
In the present study, we investigated the insulin secretory effects of A. annulatum and its bioactive compounds and the underlying mechanisms to find a favorable alternative therapy for diabetes.
To determine insulin secretory function, we assessed the effects of A. annulatum and its fractions on GSIS in INS-1 cells. The MeOH extract, EA fraction, and water fraction led to an increase in GSIS in a dose-dependent manner in INS-1 cells without inducing cytotoxicity. Compounds 1-3 (hypoxylonol C (1), hypoxylonol F (2), and BNT (3)) isolated from A. annulatum also led to an increase in GSIS in a dose-dependent manner in INS-1 cells without inducing cytotoxicity. In response to high blood glucose concentrations, insulin secretion may be influenced by various factors related to insulin synthesis, insulin secretion from secretory granules, and pancreatic β-cell metabolism [28,29]. IRS2-PI3K-Akt signaling plays a key role in pancreatic β-cell metabolism influencing insulin synthesis [30]. The IR and its main intracellular tyrosine kinase substrates, insulin receptor substrate IRS-1 and IRS-2, are involved in growth, function, and insulin secretion in pancreatic β-cells [8].

Discussion
In the present study, we investigated the insulin secretory effects of A. annulatum and its bioactive compounds and the underlying mechanisms to find a favorable alternative therapy for diabetes.
To determine insulin secretory function, we assessed the effects of A. annulatum and its fractions on GSIS in INS-1 cells. The MeOH extract, EA fraction, and water fraction led to an increase in GSIS in a dose-dependent manner in INS-1 cells without inducing cytotoxicity. Compounds 1-3 (hypoxylonol C (1), hypoxylonol F (2), and BNT (3)) isolated from A. annulatum also led to an increase in GSIS in a dose-dependent manner in INS-1 cells without inducing cytotoxicity. In response to high blood glucose concentrations, insulin secretion may be influenced by various factors related to insulin synthesis, insulin secretion from secretory granules, and pancreatic β-cell metabolism [28,29]. IRS2-PI3K-Akt signaling plays a key role in pancreatic β-cell metabolism influencing insulin synthesis [30]. The IR and its main intracellular tyrosine kinase substrates, insulin receptor substrate IRS-1 and IRS-2, are involved in growth, function, and insulin secretion in pancreatic β-cells [8].
Some studies have also indicated that phosphorylation of the PI3K/Akt pathway inactivates FOXO1-dependent gene expression. FOXO1 leads to the inhibition of pancreatic β-cell growth and acts as a transcriptional brake, leading to transcriptional restraint in the expression of PPARγ and PDX-1 [4,10]. In contrast, overexpression of PPARγ increases the nuclear translocation of PDX-1 [9][10][11]38]. PPARγ is a known regulator of glucose and lipid homeostasis, inflammation, and cellular proliferation and differentiation [9]. PDX-1, a transcription factor, plays an important role in normal pancreatic development, insulin secretion, and β-cell survival and function. Previous studies have suggested that the FOXO1/PPARγ-mediated pathway plays a crucial role in pancreatic β-cell survival and function in diabetic rats [38]. In line with previous studies, hypoxylonol F (2) caused an increase in PPARγ and PDX-1 protein expression.
Consequently, our findings revealed that A. annulatum and its bioactive compounds are capable of improving insulin secretion by regulating IRS-2, PI3K, Akt (Ser473), PPARγ, and PDX-1, which are indispensable for maintaining the normal function of pancreatic β-cells. Our study suggests that A. annulatum and its compounds can prevent or delay the development of diabetes.

Conclusions
In conclusion, this in vitro study provided the first knowledge that hypoxylonol F (2) isolated from A. annulatum significantly promoted GSIS without toxicity in pancreatic β-cells. Our study suggested that IRS-2, PI3K, Akt, PPARγ, and PDX-1 played a major role in this effect. Although further studies are needed to elucidate the molecular mechanism by which hypoxylonol F (2) promotes GSIS, there is potential to use hypoxylonol F (2) as a naturally occurring antidiabetic agent.

Conflicts of Interest:
The authors declare no conflict of interest.