Dual Beneficial Effects of α-Spinasterol Isolated from Aster pseudoglehnii on Glucose Uptake in Skeletal Muscle Cells and Glucose-Stimulated Insulin Secretion in Pancreatic β-Cells

Herein, we determined whether α-Spinasterol, a stigmastane-type phytosterol isolated from Aster pseudoglehnii, potentially impacts glucose uptake and glucose-stimulated insulin secretion in skeletal muscle cells and pancreatic β-cells, respectively. We observed that A. pseudoglehnii and its fractions enhanced glucose uptake, with no toxic effects on C2C12 cells, with the n-hexane fraction exhibiting the most potent effect. α-Spinasterol, isolated from the n-hexane fraction, enhanced glucose uptake with no toxic effects on C2C12 cells. Additionally, α-Spinasterol increased the expression of associated proteins, including insulin receptor substrate-1, AMP-activated protein kinase, and glucose transporter type 4, as determined by Western blotting. Furthermore, α-Spinasterol enhanced insulin secretion in response to high glucose concentrations, with no toxic effects on INS-1 cells; this effect was superior to that demonstrated by gliclazide (positive control), commonly prescribed to treat type 2 diabetes (T2D). α-Spinasterol enhanced the expression of associated proteins, including insulin receptor substrate-2, peroxisome proliferator-activated receptor γ, and pancreatic and duodenal homeobox 1, as determined using Western blotting. The insulin secretory effect of α-Spinasterol was enhanced by a K+ channel blocker and L-type Ca2+ channel agonist and was suppressed by a K+ channel activator and L-type Ca2+ channel blocker. α-Spinasterol isolated from A. pseudoglehnii may improve hyperglycemia by improving glucose uptake into skeletal muscle cells and enhancing insulin secretion in pancreatic β-cells. Accordingly, α-Spinasterol could be a potential candidate for anti-T2D therapy.


Introduction
Type 2 diabetes (T2D) is a steadily growing metabolic disease worldwide, characterized by a decline in insulin secretion from pancreatic β-cells and impaired insulin function in insulin-target tissues, such as skeletal muscle [1]. Skeletal muscle is a glucose-metabolic organ, where 70-80% of insulin-dependent glucose uptake reportedly occurs after glucose intake [2]. During the pathogenesis of T2D, insulin resistance in skeletal muscles is considered one of the main characteristics. A previous study has revealed that limonene isolated from citrus fruits could induce glucose uptake via the Akt pathway in C2C12 skeletal muscle cells [3]. Myrtenal in pepper, eucalyptus, cumin, and mint has been shown to induce glucose uptake by upregulating Akt and glucose transporter type 4 (GLUT-4) in C2C12 cells [4,5]. Tricin, found in cereals, can reportedly induce glucose uptake by upregulating insulin receptor substrate-1 (IRS-1) and Akt [6]. Based on the above literature, Akt, GLUT-4, and IRS-1 play a central role in enhancing glucose uptake in skeletal muscle cells.
A decline in insulin secretion from pancreatic β-cells has also been confirmed as an important characteristic in the pathogenesis of T2D [7]. Glucose-stimulated insulin secretion (GSIS) is characteristic of differentiated pancreatic β-cells [8]. Robust GSIS after food consumption suppresses hyperglycemia. In particular, a decline in GSIS under insulinresistant conditions may accelerate the transition to T2D [9]. A previous study has found that hypoxylonol F isolated from Annulohypoxylon annulatum increased GSIS by upregulating peroxisome proliferator-activated receptor γ (PPARγ) in the rodent insulin-secreting β-cell line, INS-1 cells [10]. In addition, Panax ginseng berry extracts can reportedly increase GSIS by upregulating insulin receptor substrate-2 (IRS-2) and pancreatic and duodenal homeobox 1 (PDX-1) in INS-1 cells [11]. Given the accumulated evidence, PPARγ, IRS-2, and PDX-1 appear to play a central role in improving GSIS in insulin-secreting β-cells.
Despite the presence of a long list of antidiabetic drugs in the pharmaceutical market, the search for plant-derived natural products is still needed. Because T2D drugs have limitations in that they have unresolved side effects, plant-derived drugs are needed [12]. Although the antidiabetic effects of several natural products have been reported, limited reports are available regarding natural products that exhibit dual activities on both glucose uptake in insulin target cells and GSIS in insulin-secreting cells.
Aster pseudoglehnii is a perennial plant endemic to Korea and native to Ulleung Island. The young leaves and stems of A. pseudoglehnii are widely consumed in Korean cuisines. This plant has been used as a cold antipyretic, antitussive, and expectorant for tonsillitis [13]. It has been reported that A. pseudoglehnii has anti-adipogenic effects in vivo and in vitro [14] and ameliorates scopolamine-induced memory and cognitive impairment in mice [15]. However, the antidiabetic effects of A. pseudoglehnii remain unexplored. Thus, we determined whether α-Spinasterol, a stigmastane-type phytosterol isolated from A. pseudoglehnii, potentially impacts glucose uptake and glucose-stimulated insulin secretion in skeletal muscle cells and pancreatic β-cells, respectively.

Isolation and Identification of α-Spinasterol
A 70% EtOH extract of A. pseudoglehnii significantly enhanced glucose uptake in C2C12 cells ( Figure 1). A bioassay-guided fractionation led to the isolation of an active compound from an n-hexane fraction of the 70% EtOH extract. The compound was isolated as a white powder and its liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) data exhibited an [M+1] + peak at m/z 413.0 ( Figure S1). The 1 H nuclear magnetic resonance (NMR) spectroscopic data of the compound displayed three doublet methyl signals at δ H 1.02 (3H, d, J = 6. , and an oxygenated methine proton at δ H 3.59 (1H, ddd, J =11.0, 6.5, 4.0 Hz, H-3) were detected. The 13 C NMR exhibited 29 signals, including six methyl carbons (δ C 21.5, 21.2, 19.1, 13.1, 12.3, and 12.1), four sp 2 carbons, (δ C 139. 6, 138.3, 129.5, and 117.5), and an oxygenated sp 3 carbon (δ C 71.1) ( Figure S3), inferring that this compound is a stigmastane-type phytosterol with two olefinic groups. The chemical structure of the compound was identified as α-Spinasterol by analysis of the MS and 1D NMR data and by comparison with those reported in the literature [16].

Effect of α-Spinasterol on Glucose Uptake in Skeletal Muscle Cells
All concentrations of the extract and fractions were non-toxic toward C2C12 cells ( Figure 1A-D). We then confirmed whether the extract and fractions could increase glucose uptake activity at non-toxic concentrations. As shown in Figure 1E, the 70% EtOH extract of A. pseudoglehnii increased glucose uptake activity, presented as fold induction. The n-hexane fraction showed the most potent enhancing effect ( Figure 1F). α-Spinasterol was isolated from the n-hexane fraction ( Figure 2A). All examined α-Spinasterol concentrations were non-toxic toward C2C12 cells ( Figure 2B). We then confirmed whether non-toxic α-Spinasterol concentrations could increase glucose uptake activity. As shown in Figure 2C, α-Spinasterol increased glucose uptake activity, presented as fold induction.

Effect of α-Spinasterol on Glucose Uptake in Skeletal Muscle Cells
All concentrations of the extract and fractions were non-toxic toward C2C12 cells ( Figure 1A-D). We then confirmed whether the extract and fractions could increase glucose uptake activity at non-toxic concentrations. As shown in Figure 1E, the 70% EtOH extract of A. pseudoglehnii increased glucose uptake activity, presented as fold induction. The n-hexane fraction showed the most potent enhancing effect ( Figure 1F).

Effect of α-Spinasterol on the Protein Expression of P-IRS-1, IRS-1, P-AMPK, AMPK, and GLUT-4
Treatment with 5 and 10 μM α-Spinasterol increased protein expression of GLUT-4 and phosphorylation levels of IRS-1 and AMPK in C2C12 cells when compared with the untreated controls ( Figure 3).

Effect of α-Spinasterol on GSIS
α-Spinasterol was found to be non-toxic toward INS-1 cells ( Figure 4A). As shown in Figure 4B, C, α-Spinasterol increased GSIS, expressed as the glucose-stimulated index (GSI). These values were superior to those of gliclazide (the positive control). These results suggested that α-Spinasterol enhanced insulin secretion in response to high glucose while exhibiting no toxic effects on INS-1 cells.

Effect of α-Spinasterol on GSIS
α-Spinasterol was found to be non-toxic toward INS-1 cells ( Figure 4A). As shown in Figure 4B, C, α-Spinasterol increased GSIS, expressed as the glucose-stimulated index (GSI). These values were superior to those of gliclazide (the positive control). These results suggested that α-Spinasterol enhanced insulin secretion in response to high glucose while exhibiting no toxic effects on INS-1 cells.   As shown in Figure 4D, α-Spinasterol increased the glucose-dependent ATP/ADP ratio. In addition, we evaluated the ability of α-Spinasterol to modulate K + and Ca 2+ channels. As shown in Figure 4E, F, α-Spinasterol-enhanced GSIS was enhanced by Bay K 8644 (L-type Ca 2+ channel agonist) and glibenclamide (K + channel blocker), whereas it was abrogated by nifedipine (L-type Ca 2+ channel blocker) and diazoxide (K + channel activator).
2.5. Effect of α-Spinasterol on the Protein Expression of P-IRS-2, IRS-2 (Ser731), PPARγ, and PDX-1 Compared with the untreated controls, INS-1 cells treated with 5 and 10 µM α-Spinasterol showed increased protein expression of PPARγ, PDX-1, and phosphorylation levels of IRS-2 ( Figure 5). Figure 6 presents a schematic illustration of the proposed mechanisms underlying the effects of α-Spinasterol on GSIS and glucose uptake in pancreatic β-cells and skeletal muscle cells, respectively.
Plants 2022, 11, x FOR PEER REVIEW levels of IRS-2 ( Figure 5). Figure 6 presents a schematic illustration of the proposed anisms underlying the effects of α-Spinasterol on GSIS and glucose uptake in pan β-cells and skeletal muscle cells, respectively.

Discussion
In the present study, our findings revealed that a 70% aqueous EtOH extract of the whole plants of A. pseudoglehnii significantly enhanced glucose uptake in C2C12 cells. Bioassay-guided isolation led to the isolation of α-Spinasterol as an active compound in the 70% EtOH extract. To the best of our knowledge, the presence of α-Spinasterol in A. pseudoglehnii is reported for the first time in this work. α-Spinasterol is a stigmastane-type phytosterol, which exhibits various pharmacological activities such as protective effects on diabetic nephropathy and benign prostatic hyperplasia [17,18], anti-inflammatory effects [18][19][20], and antibacterial effects [21]. However, the antidiabetic effects of α-Spinasterol remain unexplored. In addition, α-Spinasterol increased glucose uptake, which appeared to depend on the phosphorylation of IRS-1 and AMPK and increased GLUT-4 expression. In the postprandial state, skeletal muscle is the main site for glucose absorption, taking up glucose via an insulin-regulated glucose transporter such as GLUT-4. Thus, insulin-stimulated glucose uptake in the skeletal muscle depends on GLUT-4 expression at the plasma membrane [22]. Phosphorylation of IRS-1 and AMPK positively regulates the activation of GLUT-4 [23]. Therefore, upregulation of GLUT-4 in association with phosphorylation of IRS-1 and AMPK indicated that α-Spinasterol improved glucose uptake by translocating GLUT-4 to the skeletal muscle plasma membrane. In addition, we investigated the effect of α-Spinasterol on GSIS. We observed that α-Spinasterol increased GSIS, exhibiting a superior effect on GSI values than gliclazide (positive control), a drug commonly prescribed to treat T2D. Moreover, treatment with α-Spinasterol increased the ATP/ADP ratio. Glibenclamide and Bay K 8644 enhanced the α-Spinasterol-enhanced GSIS, which was suppressed by nifedipine and diazoxide. Thus, the increased ATP/ADP ratio indicated that the effect of α-Spinasterol on GSIS could be attributed to the closure of ATP-sensitive K + (KATP) channels and Ca2 + influx. It has been previously documented that an elevated ATP/ADP ratio plays an essential role in the closure of ATP-sensitive K + (KATP) channels and Ca2 + influx to promote insulin secretion [24]. Previous studies have

Discussion
In the present study, our findings revealed that a 70% aqueous EtOH extract of the whole plants of A. pseudoglehnii significantly enhanced glucose uptake in C2C12 cells. Bioassay-guided isolation led to the isolation of α-Spinasterol as an active compound in the 70% EtOH extract. To the best of our knowledge, the presence of α-Spinasterol in A. pseudoglehnii is reported for the first time in this work. α-Spinasterol is a stigmastane-type phytosterol, which exhibits various pharmacological activities such as protective effects on diabetic nephropathy and benign prostatic hyperplasia [17,18], anti-inflammatory effects [18][19][20], and antibacterial effects [21]. However, the antidiabetic effects of α-Spinasterol remain unexplored. In addition, α-Spinasterol increased glucose uptake, which appeared to depend on the phosphorylation of IRS-1 and AMPK and increased GLUT-4 expression. In the postprandial state, skeletal muscle is the main site for glucose absorption, taking up glucose via an insulin-regulated glucose transporter such as GLUT-4. Thus, insulin-stimulated glucose uptake in the skeletal muscle depends on GLUT-4 expression at the plasma membrane [22]. Phosphorylation of IRS-1 and AMPK positively regulates the activation of GLUT-4 [23]. Therefore, upregulation of GLUT-4 in association with phosphorylation of IRS-1 and AMPK indicated that α-Spinasterol improved glucose uptake by translocating GLUT-4 to the skeletal muscle plasma membrane. In addition, we investigated the effect of α-Spinasterol on GSIS. We observed that α-Spinasterol increased GSIS, exhibiting a superior effect on GSI values than gliclazide (positive control), a drug commonly prescribed to treat T2D. Moreover, treatment with α-Spinasterol increased the ATP/ADP ratio. Glibenclamide and Bay K 8644 enhanced the α-Spinasterol-enhanced GSIS, which was suppressed by nifedipine and diazoxide. Thus, the increased ATP/ADP ratio indicated that the effect of α-Spinasterol on GSIS could be attributed to the closure of ATP-sensitive K + (K ATP ) channels and Ca2 + influx. It has been previously documented that an elevated ATP/ADP ratio plays an essential role in the closure of ATP-sensitive K + (K ATP ) channels and Ca2 + influx to promote insulin secretion [24]. Previous studies have reported that glibenclamide and Bay K 8644 stimulate insulin secretion in pancreatic β-cells. Conversely, diazoxide and nifedipine decrease insulin secretion in pancreatic β-cells [25][26][27][28].
Herein, INS-1 cells presented increased expression levels of IRS-2, PPARγ, and PDX-1 following treatment with α-Spinasterol. According to previous literature, treatment with PPARγ agonists can increase GSIS in mouse islets, rat islets, and INS-1 cells [29][30][31]. These results suggest that PPARγ activation could increase insulin secretion in pancreatic β-cells. Moreover, knockdown of IRS-2 and PDX-1 in mice has been shown to decrease GSIS [29,32,33]. Accumulating evidence suggests that upregulated protein expression of IRS-2, PPARγ, and PDX-1 may be responsible for an increase in GSIS in pancreatic β-cells. Therefore, improved GSIS after treatment with α-Spinasterol could be related to the increased protein expression of IRS-2, PPARγ, and PDX-1. α-Spinasterol isolated from A. pseudoglehnii may improve hyperglycemia by improving glucose uptake into skeletal muscle cells and enhancing insulin secretion in pancreatic β-cells. α-Spinasterol appears to be a promising, naturally-derived compound for treating T2D, warranting further preclinical investigation. In skeletal muscle, androgen receptor stimulation by 5α-dihydrotestosterone has been shown to cause an increase in glucose uptake in association with phosphorylation of GLUT-4 and Akt [34]. In pancreatic β-cells, activation of the androgen receptor and the estrogen receptor has been shown to cause an increase in GSIS [35,36]. Since α-Spinasterol is a sterol compound, these previous studies suggested the possibility that α-Spinasterol may exert an antidiabetic effect by acting on steroid nuclear receptors, including the androgen receptor and estrogen receptor. However, the α-Spinasterol action on the androgen receptor and the estrogen receptor has not been reported, thus future studies expanding on this study are needed. In addition, in vivo investigations, such as the oral glucose tolerance test, insulin sensitivity, gut sucrose content, gut perfusion, disaccharidase enzyme activity, and gut motility, are required to validate in vitro findings in Type 2 diabetes mellitus.

Cell Viability Assay
C2C12 cells and INS-1 cells were incubated with treatment samples in 96-well plates overnight. After termination of treatment, cell viability was determined using the Ez-Cytox cell viability detection kit from Daeil Lab Service Co. (Seoul, Korea) according to the manufacturer's instructions. Ez-Cytox reagent was added to each well and incubated for 2 h at 37 • C. The absorbance of the colored product was measured at 490 nm using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

ADP/ATP Ratio Assay
INS-1 cells were incubated with KRBB containing α-Spinasterol in 12-well plates for 2 h, followed by incubation in KRBB containing 2.8 mM and 16.7 mM glucose for 1 h. After termination of treatment, the ADP/ATP ratio assay kit was used to examine the ADP/ATP ratio in cell lysates (Sigma-Aldrich), according to the supplier's instructions.

Statistical Analysis
Statistical significance was performed using one-way analysis of variance (ANOVA), with the Bonferroni correction for multiple comparisons. All data represent the mean ± standard error of the mean (S.E.M.). Statistical significance was set at * p < 0.01 and # p < 0.05. All analyses were performed using SPSS Statistics, ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Conclusions
The present study demonstrated that α-Spinasterol isolated from whole plants of A. pseudoglehnii improve glucose uptake in C2C12 cells and induce GSIS in INS-1 cells in vitro. In addition, α-Spinasterol-treated C2C12 cells showed increased protein expression of GLUT-4, as well as elevated levels of phosphorylated IRS-1 and AMPK, which were demonstrated to possess important roles in mediating these effects. In addition, α-Spinasterol enhanced GSIS in INS-1 cells by altering the intracellular ATP/ADP ratio and modulating K + and Ca 2+ channels. α-Spinasterol-treated INS-1 cells demonstrated increased protein expression of PPARγ and PDX-1 and phosphorylation levels of IRS-2, exhibiting important roles in mediating these effects. Further studies, including animal experiments, are necessary to comprehensively elucidate additional mechanisms of action. Accordingly, these results indicate that treatment with α-Spinasterol may be useful in developing novel anti-T2D strategies.