Identification of Bioactive Components from Ruellia tuberosa L. on Improving Glucose Uptake in TNF-α-Induced Insulin-Resistant Mouse FL83B Hepatocytes.

Ruellia tuberosa L. (RTL) has been used as a folk medicine to cure diabetes in Asia. RTL was previously reported to alleviate hyperglycemia, insulin resistance (IR), abnormal hepatic detoxification, and liver steatosis. However, the potential bioactive compounds of RTL have still not been identified. The aim of this study was to investigate the bioactive compounds in RTL ethyl acetate (EA) fractions by using a glucose uptake assay in TNF-α-treated mouse FL83B hepatocytes to discover a mechanism by which to improve IR. The bioactive compounds were identified by the high-performance liquid chromatography (HPLC) assay. Using the Sephadex LH20 gel packing chromatography column, the EAF5 fraction was isolated from RTL and significantly increased glucose uptake in TNF-α-treated FL83B cells. Moreover, the MCI gel packing chromatography column separated EAF5 into five subfractions and had no significant cytotoxic effect in FL83B cells when treated at the concentration of 25 µg/ml. Among the subfractions, EAF5-5 markedly enhanced glucose uptake in TNF-α-treated FL83B cells. The possible bioactive compounds of the EAF5-5 fraction that were identified by the HPLC assay include syringic acid, p-coumaric acid, and cirsimaritin. The bioactive compound with the best effect of increasing glucose uptake was p-coumaric acid, but its effect alone was not as good as the combined effect of all three compounds of the EAF5-5 fraction. Thus, we speculate that the antidiabetic effect of RTL may be the result of multiple active ingredients.

Alleviative effect of Ruellia tuberosa L. on NAFLD and hepatic lipid accumulation via modulating hepatic de novo lipogenesis in high-fat diet plus streptozotocin-induced diabetic rats.

Ruellia tuberosa L. (RTL) exhibits phytochemical activities and has been used as a folk medicine for curing diabetes mellitus in East Asia for decades. This study investigated the effect of RTL aqueous and ethanolic extracts on nonalcoholic fatty liver disease (NAFLD) and hepatic lipid accumulation in high-fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) rats. Administration of RTL aqueous extract (RTLW) or ethanolic extract (RTLE) at dosage of 100 or 400 mg/kg body weight for 4 weeks was carried out in HFD/STZ-induced T2DM rats. Liver weight, adipose (epididymal and perirenal adipose tissues) weight, hepatic triglyceride level, and de novo lipogenesis (DNL)-associated protein expression were monitored after scarification. The results revealed that RTLW and RTLE reduced relative liver weight and relative fat weights in HFD/STZ-induced T2DM rats. RTLW and RTLE also ameliorated NAFLD and hepatic triglyceride (TG) accumulation in diabetic rats. Moreover, hepatic DNL-regulated enzymes such as sterol regulatory element-binding protein-1 (SREBP1) and fatty acid synthase (FAS) expression were significantly suppressed by RTLE (100 and 400 mg/kg body weight) in diabetic rats. The evidences of this study suggest that RTL possesses potential on alleviating NAFLD and lipid accumulation via regulating DNL in the liver of HFD/STZ-induced T2DM rats.

Protective effect of Ruellia tuberosa L. extracts against abnormal expression of hepatic detoxification enzymes in diabetic rats.

Ruellia tuberosa L. (RTL) has been used as a folk medicine for curing diabetes mellitus in East Asia decades. This study investigated the effect of RTL on hepatic detoxification enzyme expression in diabetic rats. Male Wistar rats were fed a high fat diet (HFD) and intraperitoneally injected with streptozotocin (STZ) to induce diabetes. Subsequently, rats received oral administrations of 100 or 400 mg kg-1 body weight RTL extract, in either water (RTLW) or ethanol (RTLE), once a day for 4 weeks. The real-time PCR analyses showed that abnormality of hepatic phase I and II detoxification enzyme expression was observed in diabetic rats. However, both RTLW and RTLE significantly normalized the expression of hepatic phase I detoxification enzymes such as CYP 2E1, and expression of phase II detoxification enzymes such as UGT 1A7 and GST M1 in diabetic rats. Furthermore, we found that fasting serum glucose, hemoglobin A1C (HbA1C) and the area under the curve of oral glucose tolerance test (AUCOGTT) levels were significantly reduced in both RTLW and RTLE treated diabetic rats. Moreover, both RTLW and RTLE significantly increased the activity of hepatic anti-oxidative enzymes such as superoxide dismutase (SOD) in diabetic rats. The present study suggests that RTL may ameliorate abnormal hepatic detoxification function via alleviating hyperglycemia and enhancing hepatic antioxidant capacity in HFD/STZ-induced diabetic rats.

Vescalagin from Pink Wax Apple [Syzygium samarangense (Blume) Merrill and Perry] Alleviates Hepatic Insulin Resistance and Ameliorates Glycemic Metabolism Abnormality in Rats Fed a High-Fructose Diet.

This study investigates the ameliorative effect of vescalagin (VES) isolated from Pink wax apple fruit on hepatic insulin resistance and abnormal carbohydrate metabolism in high-fructose diet (HFD)-induced hyperglycemic rats. The results show that in HFD rats, VES significantly reduced the values of the area under the curve for glucose in an oral glucose tolerance test and the homeostasis model assessment of insulin resistance index. VES significantly enhanced the activity of hepatic antioxidant enzymes while reducing thiobarbituric acid-reactive substances in HFD rats. Western blot assay revealed that VES reduced hepatic protein expression involved in inflammation pathways while up-regulating expression of hepatic insulin signaling-related proteins. Moreover, VES up-regulated the expression of hepatic glycogen synthase and hepatic glycolysis-related proteins while down-regulating hepatic gluconeogenesis-related proteins in HFD rats. This study suggests some therapeutic potential of VES in preventing the progression of diabetes mellitus.

Gallic acid ameliorates hyperglycemia and improves hepatic carbohydrate metabolism in rats fed a high-fructose diet.

Herein, we investigated the hypoglycemic effect of plant gallic acid (GA) on glucose uptake in an insulin-resistant cell culture model and on hepatic carbohydrate metabolism in rats with a high-fructose diet (HFD)-induced diabetes. Our hypothesis is that GA ameliorates hyperglycemia via alleviating hepatic insulin resistance by suppressing hepatic inflammation and improves abnormal hepatic carbohydrate metabolism by suppressing hepatic gluconeogenesis and enhancing the hepatic glycogenesis and glycolysis pathways in HFD-induced diabetic rats. Gallic acid increased glucose uptake activity by 19.2% at a concentration of 6.25 µg/mL in insulin-resistant FL83B mouse hepatocytes. In HFD-induced diabetic rats, GA significantly alleviated hyperglycemia, reduced the values of the area under the curve for glucose in an oral glucose tolerance test, and reduced the scores of the homeostasis model assessment of insulin resistance index. The levels of serum C-peptide and fructosamine and cardiovascular risk index scores were also significantly decreased in HFD rats treated with GA. Moreover, GA up-regulated the expression of hepatic insulin signal transduction-related proteins, including insulin receptor, insulin receptor substrate 1, phosphatidylinositol-3 kinase, Akt/protein kinase B, and glucose transporter 2, in HFD rats. Gallic acid also down-regulated the expression of hepatic gluconeogenesis-related proteins, such as fructose-1,6-bisphosphatase, and up-regulated expression of hepatic glycogen synthase and glycolysis-related proteins, including hexokinase, phosphofructokinase, and aldolase, in HFD rats. Our findings indicate that GA has potential as a health food ingredient to prevent diabetes mellitus.

CDKN1A-mediated responsiveness of MLL-AF4-positive acute lymphoblastic leukemia to Aurora kinase-A inhibitors.

Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. In this study, we investigated the effects and molecular mechanisms of Aurora kinase inhibitors in acute lymphoblastic leukemia (ALL). Western blot analysis showed that both Aurora-A and Aurora-B are overexpressed in ALL cell lines and primary ALL cells. Both VE-465 and VX-680 effectively inhibited Aurora kinase activities in nine ALL cell lines, which exhibited different susceptibilities to the inhibitors. Cells sensitive to Aurora kinase inhibitors underwent apoptosis at an IC50 of ∼10-30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 µM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility of ALL cell lines was not correlated with the expression level or activation status of Aurora kinases. Interestingly, RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora kinase-A inhibitors treatment. Complementary DNA (cDNA) microarray analysis suggested that CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Most importantly, primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to Aurora kinase inhibitors. Our study suggests CDKN1A could be a potential biomarker in determining the drug responsiveness of Aurora kinase inhibitors in ALL, particularly in MLL-AF4-positive patients.

Graptopetalum paraguayense and resveratrol ameliorates carboxymethyllysine (CML)-induced pancreas dysfunction and hyperglycemia.

Hyperglycemia is associated with advanced glycation end products (AGEs). Recently, AGEs were found to cause pancreatic damage, oxidative stress, and hyperglycemia through the AGE receptor. Carboxymethyllysine (CML) is an AGE but whether it induces pancreatic dysfunction remains unclear. Graptopetalum paraguayense, a vegetable consumed in Taiwan, has been used in folk medicine and is an antioxidant that protects against liver damage. We investigated the protective properties of G. paraguayense 95% ethanol extracts (GPEs) against CML-induced pancreatic damage. The results indicated that resveratrol, GPE, and gallic acid (the active compound of GPE) increased insulin synthesis via upregulation of pancreatic peroxisome proliferator activated-receptor-γ (PPARγ) and pancreatic-duodenal homeobox-1 (PDX-1) but inhibited the expression of CML-mediated CCAAT/enhancer binding protein-ß (C/EBPß), a negative regulator of insulin production. Moreover, resveratrol and GPE also strongly activated nuclear factor-erythroid 2-related factor 2 (Nrf2) to attenuate oxidative stress and improve insulin sensitivity in the liver and muscle of CML-injected C57BL/6 mice and resulted in reduced blood glucose levels. Taken together, these findings suggested that GPE and gallic acid could potentially be used as a food supplement to protect against pancreatic damage and the development of diabetes.

An extract from wax apple (Syzygium samarangense (Blume) Merrill and Perry) effects glycogenesis and glycolysis pathways in tumor necrosis factor-α-treated FL83B mouse hepatocytes.

FL83B mouse hepatocytes were treated with tumor necrosis factor-α (TNF-α) to induce insulin resistance to investigate the effect of a wax apple aqueous extract (WAE) in insulin-resistant mouse hepatocytes. The uptake of 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]-2-deoxyglucose (2 NBDG), a fluorescent D-glucose derivative, was performed, and the metabolism of carbohydrates was evaluated by examining the expression of glycogenesis or glycolysis-related proteins in insulin-resistant hepatocytes. The results show that WAE significantly improves the uptake of glucose and enhances glycogen content in insulin-resistant FL83B mouse hepatocytes. The results from Western blot analysis also reveal that WAE increases the expression of glycogen synthase (GS), hexokinase (HXK), glucose-6-phosphate dehydrogenase (G6PD), phosphofructokinase (PFK) and aldolase in TNF-α treated cells, indicating that WAE may ameliorate glucose metabolism by promoting glycogen synthesis and the glycolysis pathways in insulin-resistant FL83B mouse hepatocytes.

Effect of water extracts from edible Myrtaceae plants on uptake of 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in TNF-α-treated FL83B mouse hepatocytes.

This study investigated the glucose uptake activity of the water extracts from the leaves and fruit of edible Myrtaceae plants, including guava (Psidium guajava Linn.), wax apples [Syzygium samarangense (Blume) Merr. and L.M. Perry], Pu-Tau [Syzygium jambo (L.) Alston], and Kan-Shi Pu-Tau (Syzygium cumini Linn.) in FL83B mouse hepatocytes. The fluorescent dye 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose was used to estimate the uptake ability of the cells. Glucose uptake test showed that pink wax apple fruit extract (PWFE) exhibits the highest glucose uptake activity, at an increment of 21% in the insulin-resistant FL83B mouse hepatocytes as compared with the TNF-α-treated control group. Vescalagin was isolated using column chromatography of PWFE. This compound, at the concentration of 6.25 µg/mL, exhibits the same glucose uptake improvement in insulin-resistant cells as PWFE at a 100-µg/mL dose. We postulate that vescalagin is an active component in PWFE that may alleviate the insulin resistance in mouse hepatocytes.

Hypotriglyceridemic and hypoglycemic effects of vescalagin from Pink wax apple [Syzygium samarangense (Blume) Merrill and Perry cv. Pink] in high-fructose diet-induced diabetic rats.

Vescalagin, an active component from Pink wax apple [Syzygium samarangense (Blume) Merrill and Perry cv. Pink] fruit, with glucose uptake enhancing ability in insulin-resistant FL83B mouse hepatocytes, as shown in our previous study, was further evaluated for its hypotriglyceridemic and hypoglycemic effects in high-fructose diet (HFD)-induced diabetic rats. Wistar rats were fed HFD for 16 weeks and orally administered with vescalagin from Pink wax apple daily during the last 4 weeks. The results of biochemical parameters showed that fasting blood glucose, C-peptide, fructosamine, triglyceride and free fatty acid contents decreased by 44.7%, 46.2%, 4.0%, 42.5%, and 10.8%, respectively, in the HFD-induced diabetic rats administered with vescalagin at 30 mg/kg body weight in comparison with those of control HFD-induced diabetic rats. However, high-density-lipoprotein-cholesterol content increased by 14.4% in the HFD rats treated with vescalagin. The present study reveals that vescalagin could have therapeutic value against diabetic progression via its anti-hypertriglyceridemic and anti-hyperglycemic effects.

Fraction from wax apple [Syzygium samarangense (Blume) Merrill and Perry] fruit extract ameliorates insulin resistance via modulating insulin signaling and inflammation pathway in tumor necrosis factor α-treated FL83B mouse hepatocytes.

Inflammation is associated with the development of insulin resistance in Type 2 diabetes mellitus. In the present study, mouse FL83B cells were treated with tumor necrosis factor-alpha (TNF-α) to induce insulin resistance, and then co-incubated with a fraction from wax apple fruit extract (FWFE). This fraction significantly increased the uptake of the nonradioactive fluorescent indicator 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) in insulin resistant cells. Western blot analysis revealed that, compared with the TNF-α-treated control group, FWFE increased the expression of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), protein kinase B (Akt/PKB), phosphatidylinositol-3 kinase (PI3K), and glucose transporter 2 (GLUT-2), and increased IR tyrosyl phosporylation, in insulin resistant FL83B cells. However, FWFE decreased phosphorylation of c-Jun N-terminal kinases (JNK), but not the expression of the intercellular signal-regulated kinases (ERK), in the same cells. These results suggest that FWFE might alleviate insulin resistance in TNF-α-treated FL83B cells by activating PI3K-Akt/PKB signaling and inhibiting inflammatory response via suppression of JNK, rather than ERK, activation.

Epigenetic silencing of CCAAT/enhancer-binding protein delta activity by YY1/polycomb group/DNA methyltransferase complex.

Human CCAAT/enhancer-binding protein delta (CEBPD) has been reported as a tumor suppressor because it both induces growth arrest involved in differentiation and plays a crucial role as a regulator of pro-apoptotic gene expression. In this study, CEBPD gene expression is down-regulated, and "loss of function" alterations in CEBPD gene expression are observed in cervical cancer and hepatocellular carcinoma. Suppressor of zeste 12 (SUZ12), a component of the polycomb repressive complex 2 (PRC2), silences CEBPD promoter activity, enhancing the methylation of exogenous CEBPD promoter through the proximal CpG islands. Moreover, this molecular approach is consistent with the opposite mRNA expression pattern between SUZ12 and CEBPD in cervical cancer and hepatocellular carcinoma patients. We further demonstrated that Yin-Yang-1 (YY1) physically interacts with SUZ12 and can act as a mediator to recruit the polycomb group proteins and DNA methyltransferases to participate in the CEBPD gene silencing process. Taking these results into consideration, we not only demonstrate the advantage of SUZ12-silenced CEBPD expression in tumor formation but also clarify an in vivo evidence for YY1-mediated silencing paths of SUZ12 and DNA methyltransferases on the CEBPD promoter.