Alismatis Rhizoma Triterpenes Alleviate High-Fat Diet-Induced Insulin Resistance in Skeletal Muscle of Mice

Alismatis rhizoma (AR), which is the dried rhizome of Alisma orientale (Sam.) Juz. (Alismataceae), is an important component of many famous Chinese formulas for hypoglycemic. This study aimed to evaluate the insulin resistance (IR) alleviating effects of AR triterpenes (ART) and ART component compatibility (ARTC, the mixture of 16-oxo-alisol A, 16-oxo-alisol A 23-acetate, 16-oxo-alisol A 24-acetate, alisol C, alisol C 23-acetate, alisol L, alisol A, alisol A 23-acetate, alisol A 24-acetate, alisol L 23-acetate, alisol B, alisol B 23-acetate, 11-deoxy-alisol B and 11-deoxy-alisol B 23-acetate) in high-fat diet-induced IR mice and plamitate-treated IR C2C12 cells, respectively. A dose of 200 mg/kg of ART was orally administered to IR mice, and different doses (25, 50, and 100 μg/ml) of ARTC groups were treated to IR C2C12 cells. IPGTT, IPITT, body weight, Hb1AC, FFA, TNF-α, MCP-1, and IR-associated gene expression (p-AMPK, p-IRS-1, PI3K, p-AKT, p-JNK, and GLUT4) were measured in IR mice. Glucose uptake, TNF-α, MCP-1, and IR-associated gene expression were also measured in IR C2C12 cells. Results showed that ART alleviated high-fat diet-induced IR in the skeletal muscle of mice, and this finding was further validated by ARTC. This study demonstrated that ART presented a notable IR alleviating effect by regulating IR-associated gene expression, and triterpenes were the material basis for the IR alleviating activity of AR.


Introduction
In recent years, increasing research attention has been paid to novel, effective antidiabetes agents derived from natural sources [1]. Alismatis rhizoma (AR) is the tuber of Alisma orientale (Sam.) Juz. (Alismataceae), which is an aquatic plant that has been cultivated mainly in oriental countries (e.g., China, Japan, and Korea) but is also widely distributed in other areas (e.g., North America and Europe). AR has various activities, such as diuretic, hyperlipidemic, inflammatory, antitumoral, and damp-heat clearing activities [2][3][4][5][6][7][8]. AR is traditional folk medicine that has long been used to promote health and longevity (Sheng Nong's herbal classic) for more than several thousand years. AR is currently included in the Pharmacopoeia of China not only as a diuretic but also as a representative hypoglycemic and hypolipidemic traditional Chinese herbal medicine. AR is an important component of many famous Chinese formulas for hypoglycemic from Febrile and Miscellaneous Disease (Shang Han Lun in Chinese) or Synopsis of Golden Chamber, including Zexie docotion, Ba wei shen qi wan, and Liu wei di huang wan [5,9,10], for usage as hypoglycemic because of their low toxicity, high effectiveness, and minimal side effects. Our previous studies reported the hypoglycemic activity of the ethanol extract of AR [11,12]. Fourteen terpenoids were isolated from the ethanol extract of AR triterpenes (ART), which could promote glucose uptake in 3T3-L1 cells [13]. Meanwhile, the potential effect of ART on glucose metabolism, especially insulin resistance (IR), has not been investigated.
Type 2 diabetes (T2MD) comprises 90% of the total number of diabetes cases around the world. IR is an early metabolic abnormality in the progression of T2DM [14]. IR, which is characterized by low efficacy of insulin-induced glucose uptake [15], is caused by the dysregulated insulinrelated signaling and glucose metabolism defects, such as impaired glucose transportation, phosphorylation, oxidation, and glycogen synthesis [16][17][18]. Apart from such wellrecognized factors, low-grade inflammation is also an important contributing factor to IR [19]. Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and monocyte chemotactic protein-1 (MCP-1), aggravate IR in patients [20,21]. As a result, alleviation and drug intervention of IR are major tasks to lessen the risk of T2DM.

ART Preparation.
According to our previous study [35], the extraction and purification method of Alismatis Rhizoma Triterpenes was as follows. Dried rhizomes of Alismatis Rhizoma were ground into powder (24 mesh) and then twice extracted by a decoction with 80% ethanol for 1 h. e filtrate was concentrated and subjected to chromatography on an HP20 macroreticular resin (Beijing Greenherbs Science and Technology Co., Beijing, China) column by using deionized water, 40% ethanol, and 75% ethanol as eluent. After collecting and concentrating the 75% ethanol fraction, the ART yield was determined to be 0.72%. An ACQUITY UHPLC I-Class system coupled with a Xevo XS quadrupole time of flight mass spectrometer (Waters, Milford, MA, USA) was used, and the LC conditions were based on a previous LC-Q-TOF-MS method [12,36]. Chromatographic separation was carried out at 40°C on a Waters CORTECS C18 column (2.1 mm × 100 mm; 1.6 μm), with 0.1% of formic acid in water as mobile phase A and acetonitrile B as mobile phase B. Gradient elution was performed as follows: 45%-45% B for 0-0.5 min, 46%-65% B for 0.5-2 min, 65%-90% B for 2-7 min, and 90%-100% B at 7-10 min. e flow rate was 0.25 mL/min. e mass-spectrometry conditions were optimized as follows: dissolvent gas temperature, 500°C; capillary voltage, 3.5 kV; source temperature, 150°C; dissolvent gas flow, 800 L/h; and cone gas flow, 50 L/h. e MS scan range was m/z 50-1000, and the collision energy was set at 20 eV. For quantitation of ART, standard and sample solutions were prepared as follows: a series of working standard solutions of fourteen analytes [(1) 16-oxo-alisol A, (2) 16-oxo-alisol A 23-acetate, (3) 16-oxoalisol A 24-acetate, (4) alisol C, (5) alisol C 23-acetate, (6) alisol L, (7) alisol A, (8) alisol A 23-acetate, (9) alisol A 24-acetate, (10) alisol L 23-acetate, (11) alisol B, (12) alisol B 23-acetate, (13) 11-deoxy-alisol B, (14) 11-deoxy-alisol B 23-acetate] were freshly prepared by diluting the mixed standard solutions with acetonitrile at the ratios of 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL (Table S1). e ART samples: 20 mg powders were dissolved with 50 mL acetonitrile, then centrifuged at 12000 rpm for 10 min. e supernatant was diluted 100 times with acetonitrile to obtain a sample solution. Sample and working standard solutions were injected into LC-MS to acquire peak area responses (the detailed quantitative ion channel was shown in Table S2). Chromatogram of the sample (a) and standard solutions (b) were shown in Figure 2. According to the working standard concentration linearity curve (Table S1), fourteen triterpenes in ART were detected. ART containing fourteen triterpenes amounted to 885.9 mg/g ( Table 1). e ingredients of ARTC were obtained according to our previous research [37]. en, ARTC was prepared as follows: fourteen pure triterpenes were accurately weighed and mixed: 16-     (CHOW, D12450B, 10% calories from fat) and HFD (D12492, 60% calories from fat) were purchased from Guangdong Animal Center (Guangzhou, China). After 10 weeks, the mice were fasted for 12 h and then fasting blood glucose (FBG), fasting insulin (FINS), and IPITT were measured. Homeostasis model assessment of IR index (HOMA-IR) was also calculated to determine whether the IR model was established successfully ( Figure S1). e HOMA-IR formula was as follows:

Mouse Grouping and Administration.
After establishing the IR-mouse model, the model group was randomly divided into three groups, namely, a positive control group (HFD + POS), an HFD group, and an HFD + ART group. Each group had 10 mice whose weights were recorded. e CHOW group was given a standard diet, the HFD + POS group was given HFD added with 200 mg/kg/d metformin, and the HFD + ART group was given HFD added with 200 mg/kg/d ART. All drugs were mixed as feed supplements through the equivalent incremental method and fed for 4 weeks. Positive drug metformin was bought from Farmhispania (Farmhispania S.A. Barcelona, Spain). After intervention for 16 h, the culture media (with/ without 100 nM insulin) was changed and incubated for 30 min.

Statistical Analysis.
All data were analyzed using SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA). Results are expressed as the mean ± SEM. All data were analyzed by one-way ANOVA, and Student's t-test was subsequently conducted for multiple comparisons. P < 0.05 was considered as a significant difference between each group.

ART Treatment Improves IR by Regulating Body Weight,
Glucose Tolerance, HbA1C, FFA, and Inflammatory Factors in IR Mice. A high-fat diet-induced IR model (HFD) was established following our previous studies [11,38] to explore the ART therapeutic potential in IR mice. We used ART treatment to intervene with these mice and observe the response. At the time point of 10 weeks, the IR model was established ( Figure S1). After ART (200 mg/kg) treatment for 4 weeks (11-14 weeks), the body weight of IR mice (HFD + ART group) was significantly decreased compared with that of HFD group mice (Figure 3(a)). Efficacy monitoring indicators were considered. Specifically, the level of FBG, FINS, HbA1c, FFA with a close relationship with IR occurrence and promotes the development of IR-type diabetes [39]) in serum was determined, and the HOMA-IR in each group was conducted. After ART treatment, FBG, FIINS, HbA1C, FFA concentration, and HOMA-IR were decreased compared with those of HFD group mice (Figures 3(b)-3(f )). IPGTT and IPIGG were performed after 4 weeks of ART treatment to investigate the ART effect on glucose. As shown in Figure 3(g), the HFD + ART group exhibited a significantly low level of blood glucose concentrations at 0, 15, 30, 60, and 120 min in IPGTT assay compared with HFD group mice. e area under the curve (AUC) of blood glucose concentrations was also significantly decreased in the HFD + ART group than in the HFD group (P < 0.01) (Figure 3(h)). In the IPITT assay, HFD + ART group showed more significant and faster glucose reduction than the HFD group at 0, 15, 30, 60, and 120 min (P < 0.01) (Figure 3(i)). e AUC of blood glucose concentrations at HFD + ART group was lower than that at HFD group (P < 0.05) (Figure 3(j)).
ese results indicated that ART treatment could promote glucose metabolism. Meanwhile, the HFD-induced upregulation of serum tumor TNF-α and MCP-1 expression was also reduced by ART treatment (Figures 3(k) and 3(l)).

ART Treatment Regulates IR-Associated Protein
Expression. We analyzed IR-associated protein expression in the skeletal muscle of mice using Western blot analysis to evaluate the evidence base of ART treatment of IR. As shown in Figure 4, HFD group mice showed significantly downregulated AMPK (phosphorylated, Figure 4(a)), AKT (phosphorylated, Figure 4(b)), PI3K (Figure 4(c)), and GLUT4 (Figure 4(d)) compared with CHOW group. Moreover, HFD group mice showed significantly upregulated relative protein expression of IRS-1 (phosphorylated, Figure 4(e)) and JNK (phosphorylated, Figure 4(f )) compared with CHOW. Interestingly, ART treatment could reverse all IR-associated gene dysregulations (Figure 4). e relative expression levels of p-AMPK, p-AKT, p-IRS-1, and p-JNK protein were quantified by gray analysis and normalized to corresponding nonphosphorylated proteins. e relative expression levels of PI3K protein were quantified by gray analysis and normalized to actin. e relative expression levels of GLUT4 protein were quantified by gray analysis and normalized to Na + − K + -ATPase. All results were represented as the mean ± SEM. ART treatment could regulate IR-associated gene expression in the skeletal muscle of high-fat diet-induced IR mice via AMPK/JNK and IRS-1/ PI3K/AKT/GLUT4 signaling pathway.

ARTC Ameliorates Glucose Consumption and IR-Associated Inflammation in Skeletal Muscle Cells.
Cell viability in response to different PA concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, and 1 mM) and ARTC concentrations (0, 12.5, 25, 50, 100, 200, 300, and 400 μg/ml) was measured after treatment for 24 h (Figures 5(a) and 5(b)) to test the cytotoxicity of PA and ARTC. As a result, PA at 0.5 mM reduced cell viability to approximately 85% of control, and 25-100 μg/ml ARTC doses were chosen to further explore the improvement effect in IR cell. PA-induced IR C2C12 cell model was constructed [40]. After 16 hours of PA intervention on C2C12 cells, the glucose consumption of the cells decreased by 12.95% (P < 0.01) compared with that of the control group. In this model, ARTC treatment could reverse 6 Evidence-Based Complementary and Alternative Medicine

ARTC Treatment Could Regulate IR-Associated Protein
Expression in PA-Induced IR C2C12 Cells. Additional comprehensive protein detection was performed to further confirm the ARTC regulation on IR-associated genes. In PAinduced IR cells, the protein expression levels of p-AMPK, p-JNK PI3K, p-AKT, and GLUT4 were suppressed. By contrast, the level of p-IRIS-1 increased significantly. As shown in Figure 6, the expression levels of p-AMPK ( Figure 6(a)), p-JNK (Figure 6(b)) PI3K (Figure 6(c)), p-AKT (Figure 6(d)), p-IRIS-1 (Figure 6(e)), and GLUT4 ( Figure 6(f )) could be reversed after ARTC intervention. Furthermore, the relationship was dose dependent. e relative expression levels of p-AMPK, p-AKT, p-IRS-1, and p-JNK protein were quantified by gray analysis and normalized to corresponding nonphosphorylated proteins. e relative expression levels of PI3K protein were quantified by gray analysis and normalized to actin. e relative expression levels of GLUT4 protein were quantified by gray analysis and normalized to Na + − K + -ATPase. All results were represented as the mean ± SEM. ese results indicated that ARTC treatment had a significant regulatory effect on the expression of IR-associated genes related to AMPK/JNK and IRS-1/PI3K/AKT/GLUT4 signaling pathway in C2C12 cells induced by PA.

Discussion
IR is a complex metabolic syndrome that may occur with obesity, diabetes, or cardiovascular diseases and lead to many abnormalities [41]. In a mouse model, IR has manifested as hyperglycemia, hyperinsulinemia, increased HOMA-IR, impaired glucose tolerance and insulin tolerance, dysglycemia, and inflammatory factor disorder [42][43][44]. At the cellular level, IR primarily manifests as glucose metabolism disorder caused by insulin stimulation to target cells or tissues, especially skeletal muscle tissue [45]. Glucose metabolism usually refers to the process of glucose absorption and utilization by organisms and includes mostly AMPK/JNK and IRS-1/PI3K/AKT/GLUT4-mediated insulin signaling pathway disorder.
Currently recognized IR-mouse models include transgenic, knockout, genetic inherited (spontaneous), and inducible IR [46]. Owing to their stability and convenience, male C57BL/6J mice fed with HFD for 10 weeks were adopted as the IR-mouse model [11,38]. To determine the actual material foundation of the anti-IR effect of ART from AR, UPLC-Q-TOF-MS was used to identify and determine the major ingredients from ART. To evaluate the anti-IR effect in vitro, we used the ARTC (a mixture of 16-oxo-alisol A, 16-oxo-alisol A 23-acetate, 16-oxo-alisol A 24-acetate, alisol C, alisol C 23-acetate, alisol L, alisol A, alisol A 23acetate, alisol A 24-acetate, alisol L 23-acetate, alisol B, alisol B 23-acetate, 11-deoxy-alisol B, and 11-deoxy-alisol B 23acetate, whose contents were equal to that of the ART extract). In order to prove triterpenes are medicinal ingredients that alleviate high-fat diet-induced insulin resistance in skeletal muscle of mice, both in vivo and in vitro experiments were conducted. In vivo, as for AR triterpenes extract (ART) preparation, the ethanol extraction process and macroporous adsorption resin purify process of AR was used. As a result, the ART containing 14 structures clearly triterpenes amounted to 885.9 mg/g (the purity of triterpenes in ART has reached 88.59%). In vitro, in order to further confirm that triterpenes are the medicinal ingredients, ART component compatibility (ARTC, the composition and quantity of triterpenes equivalent to the ART extract triterpenes) was used for palmitate-treated IR C2C12 cells to exclude other components (such as sesquiterpenoids). So, ARTC preparation: fourteen pure triterpenes were accurately weighed and mixed: 16- So, we investigated the anti-IR therapeutic effect of ART in an IR-mouse model and a C2C12 cell model. First, the IRmouse model fed with HFD for 10 weeks was successfully constructed. rough FBG level measurement, IPGTT assay, and HOMA-IR score calculation, we found that the HFD group showed significant IR characters, indicating that the IR-mouse model was established. en, after ART therapy for 4 weeks, the body weight significantly decreased.
rough IPGTT and IPIGG assays, we found that ART could improve IR. Meanwhile, HbA1c as a standard marker for glycemic excursion in diabetic patients was also identified as an IR-prediction marker [47]. Notably, IR is a disease with complex networks of glucose metabolism and fat metabolism. When the tissue is insulin sensitive, excessive FFA in circulation creates IR [48]. Afterwards, the increase in lipolysis produces and releases more FFA, which forms the inflammatory basis in IR disease [49]. Herein, HbA1c, FFA, TNF-α, and MCP-1 level in the ART treatment group all significantly decreased compared with the HFD group. Moreover, considering the critical role of skeletal muscle in insulin-stimulated systemic glucose metabolism, skeletal muscle glucose metabolism disorders could affect systemic glucose homeostasis and insulin sensitivity [50,51]. Skeletal muscle is an important tissue for the body's glucose uptake and utilization and plays a major role in maintaining the dynamic balance of blood glucose. Accordingly, we detected IR-associated gene expression in mouse skeletal muscle and found that ART treatment regulated the AMPK/JNK and IRS-1/PI3K/AKT/GLUT4 signaling pathway. AMPK also ameliorated inflammation and maintained energy homeostasis by inhibiting NF-κB activity. IR induced by HFD activated JNK, which led to decreased phosphorylation of IRS-1 tyrosine residues and prevented the transfer of GLUT4 from the intracellular storage to the plasma membrane through the IRS-1/PI3K/Akt signaling cascades. IRS-1/ PI3K/Akt signaling pathways are the classical pathways in the regulation of glucose uptake and metabolism [14,[24][25][26][27][28][29]. PI3K becomes the focus on the effects of cell growth and cell proliferation by nutrition, especially glucose uptake and cellcycle regulation. AKT is involved in the signaling pathway of glucose-stimulated insulin secretion in skeletal muscle cells as signaling molecules in insulin secretion, the effects of translocation of GLUT4 in skeletal muscle [52,53]. e above research shows that the AMPK/JNK and IRS-1/PI3K/ AKT/GLUT4 signaling pathway is closely implicated in HFD-induced IR. Previous studies have suggested that in HFD-induced hyperlipidemia mouse, the methanol extract of the tuber of Alisma orientale can lower serum lipid levels and prevents hepatic steatosis pathogenesis by inhibiting the expression of hepatic lipogenic genes [7,54]. Triterpenes isolated from AR have the activity of promoting glucose uptake [55]. Recent studies have also shown that alisol A-24acetate promotes glucose uptake via the activation of AMPK, phosphorylation of JNK and p38 in C2C12 myotubes. [56]. Alisol B 23-acetate protects against nonalcoholic steatohepatitis in mice via farnesoid X receptor activation [57]. Furthermore, the NF-κB signaling pathway is obviously suppressed in liver cells treated with alisol F and 25anhydroalisol F [58]. Alisol A, alisol C, alisol B 23-acetate, alisol A-24-acetate, alisol C-23-acetate, and 16-oxo-alisolA have been described with modulating effects of glucose uptake [13]. So, compared to these triterpene compounds, the ART is characterized by multiple triterpenes components and may show better activity in improving the IR than a single component, different triterpenes in AR have different sets of gene targets [5]. So, the overall effect of all triterpenes may be better than the pure one triterpene.
Although AR compounds have been found to improve genes related to glucose and lipid metabolism, the regulation of the insulin signaling pathway has not yet been systematically reported. To further facilitate evidence-based cellular exploration, the IR model of C2C12 cells induced by PA was accustomed. After the ARTC intervention, we found that ARTC treatment promoted PA-induced glucose consumption in a dose-dependent manner [39]. e intervention of ART reduced the expression of TNF-α and MCP-1 levels in the IR model cells, implying the anti-inflammatory effect of ART. It also led to the activation of AMPK and AKTand the inhibition of IκBα, NF-κB, JNK, and IRS-1, ultimately promoting GLUT4 expression. In summary, ARTC exerted a significant regulatory effect on IR-associated genes related to the AMPK/JNK and IRS-1/PI3K/AKT/ GLUT4 signaling pathway in C2C12 cells induced by PA.

Conclusions
e above results showed that ART attenuated the impairment of AMPK/JNK and IRS-1/PI3K/AKT/GLUT4mediated insulin signaling in skeletal muscles of HFD-fed mice. ART treatment significantly ameliorated HFD-induced weight gain and improved glucose tolerance in mice. e expression levels of proinflammatory cytokines were markedly attenuated by ART in various in vivo models. ART treatment markedly augmented AMPK phosphorylation expression in the skeletal muscle of mice. Furthermore, the intervention of ARTC reduced the expression levels of TNFα and MCP-1 in IR model cells, leading to the activation of AMPK and AKT/PI3K and the inhibition of IκBα, NF-κB, JNK, and IRS-1, ultimately promoting GLUT4 expression.
In conclusion, our results demonstrated that ART markedly ameliorated IR in vivo and in vitro. ART exerted a notable alleviated IR effect by regulating IR-associated gene expression, and triterpenes were the material basis for the alleviation of IR activity by AR.

Data Availability
e data used to support the findings of this study are available within the article and the supplementary materials.

Conflicts of Interest
e authors declare no conflicts of interest. Table S1: Linearity curve, linear rang, and linear correlation coefficient r of the 14 compounds. Table S2: e detailed quantitative ion channel of 14 triterpenes in ART by UPLC-MS, Table S3: e information of all antibodies. Figure S1: Insulin resistant mouse model was constructed.