Increased glucose metabolism and alpha-glucosidase inhibition in Cordyceps militaris water extract-treated HepG2 cells

BACKGROUND/OBJECTIVES: Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism. MATERIALS/METHODS: Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement. RESULTS: CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose. CONCLUSION: CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment.

A study of the lipoprotein lipase inhibitory mechanism of Poncirus trifoliata water extracts / 한국영양학회지

PURPOSE: Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W. METHODS: We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein (C/EBPbeta) RESULTS: The total polyphenol and flavonoid content of PF-W was 52.15 +/- 4.02 and 6.56 +/- 0.47 mg/g, respectively. PF-W treatment decreased LPL content in media to 58 +/- 5% of that in control adipocyte media, and increased LPL content to 117 +/- 3.5% of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and time-dependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of C/EBPbeta, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. CONCLUSION: The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through C/EBPbeta-mediated induction of SorLA expression.

Antiobesity effects of the water-soluble fraction of the ethanol extract of Smilax china L. leaf in 3T3-L1 adipocytes

BACKGROUND/OBJECTIVES: Several medicinal properties of Smilax china L. have been studied including antioxidant, anti-inflammatory, and anti-cancer effects. However, the antiobesity activity and mechanism by which the water-soluble fraction of this plant mediates its effects are not clear. In the present study, we investigated the lipolytic actions of the water-soluble fraction of Smilax china L. leaf ethanol extract (wsSCLE) in 3T3-L1 adipocytes. MATERIALS/METHODS: The wsSCLE was identified by measuring the total polyphenol and flavonoid content. The wsSCLE was evaluated for its effects on cell viability, lipid accumulation, glycerol, and cyclic adenosine monophosphate (cAMP) contents. In addition, western blot analysis was used to evaluate the effects on protein kinase A (PKA), PKA substrates (PKAs), and hormone-sensitive lipase (HSL). For the lipid accumulation assay, 3T3-L1 adipocytes were treated with different doses of wsSCLE for 9 days starting 2 days post-confluence. In other cell experiments, mature 3T3-L1 adipocytes were treated for 24 h with wsSCLE. RESULTS: Results showed that treatment with wsSCLE at 0.05, 0.1, and 0.25 mg/mL had no effect on cell morphology and viability. Without evidence of toxicity, wsSCLE treatment decreased lipid accumulation compared with the untreated adipocyte controls as shown by the lower absorbance of Oil Red O stain. The wsSCLE significantly induced glycerol release and cAMP production in mature 3T3-L1 cells. Furthermore, protein levels of phosphorylated PKA, PKAs, and HSL significantly increased following wsSCLE treatment. CONCLUSION: These results demonstrate that the potential antiobesity activity of wsSCLE is at least in part due to the stimulation of cAMP-PKA-HSL signaling. In addition, the wsSCLE-stimulated lipolysis induced by the signaling is mediated via activation of the beta-adrenergic receptor.

Fatty acid analysis and regulatory effects of citron (Citrus junos Sieb. ex TANAKA) seed oil on nitric oxide production, lipid accumulation, and leptin secretion / 한국영양학회지

PURPOSE: Citron seed oil (CSO) has been reported to have high antioxidant activity. However, the composition and other biologically activities of CSO have not been reported. In this study, we confirmed the fatty acid composition of CSO, which may be beneficial to vascular disease and obesity. METHODS: We investigated the oil composition of CSO using gas chromatography coupled with mass spectrometry (GC-MS) analysis, and cytotoxicity was confirmed by Cell Counting Kit-8 (CCK-8) assay. Nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) was measured using Griess reagent, and lipid accumulation and leptin secretion in 3T3-L1 cells were measured by Oil-Red O staining and commercial ELISA kit, respectively. RESULTS: GC-MS analysis indicated that CSO contains several components, including linoleic acid, oleic acid, palmitic acid, stearic acid, linolenic acid, palmitoleic acid, and arachidic acid. In physiological activity analysis, CSO did not induce cytotoxic effects in HUVECs and 3T3-L1 cells. Further, CSO significantly induced nitric oxide and leptin secretion as well as inhibited lipid accumulation. CONCLUSION: CSO increased NO release, inhibited lipid accumulation, and induced leptin secretion, suggesting it may be useful for the management of vessels and weight gain. Although further studies are required to investigate the safety and mechanism of action of CSO, our results show that the composition and physiological activity of CSO are sufficient for its use as functional edible oil.

Study of the mechanisms underlying increased glucose absorption in Smilax china L. leaf extract-treated HepG2 cells / 한국영양학회지

PURPOSE: Previous studies have shown that treatment with Smilax china L. leaf extract (SCLE) produces antidiabetic effects due to alpha-glucosidase inhibition. In this study, we examined the mechanism underlying these antidiabetic effects by examining glucose uptake in HepG2 cells cultured with SCLE. METHODS: Glucose uptake and glucokinase activity were examined using an assay kit. Expression of glucose transporter (GLUT)-2, GLUT-4, and HNF-1alpha was measured by RT-PCR or western blot. RESULTS: Treatment with SCLE resulted in enhanced glucose uptake in HepG2 cells, and this effect was especially pronounced when cells were cultured in an insulin-free medium. SCLE induced an increase in expression of GLUT-2 but not GLUT-4. The increase in the levels of HNF-1alpha, a GLUT-2 transcription factor, in total protein extract and nuclear fraction suggest that the effects of SCLE may occur at the level of GLUT-2 transcription. In addition, by measuring the change in glucokinase activity following SCLE treatment, we confirmed that SCLE stimulates glucose utilization by direct activation of this enzyme. CONCLUSION: These results demonstrate that the potential antidiabetic activity of SCLE is due at least in part to stimulation of glucose uptake and an increase in glucokinase activity, and that SCLE-stimulated glucose uptake is mediated through enhancement of GLUT-2 expression by inducing expression of its transcription factor, HNF-1alpha.

Exploration of optimum conditions for production of saccharogenic mixed grain beverages and assessment of anti-diabetic activity / 한국영양학회지

PURPOSE: This study was conducted to establish the production conditions through optimization of the production process of beverages using Aspergillus oryzae CF1001, and to analyze volatile compounds and antidiabetic activity. METHODS: The optimum condition was selected using the response surface methodology (RSM), through a regression analysis with the following independent variables gelatinization temperature (GT, X1), saccharogenic time (ST, X2), and dependent variable; DeltaE value (y). The condition with the lowest DeltaE value occurred with combined 45 min ST and 50degrees C GT. The volatile compounds were analyzed quantitatively by GC-MS. RESULTS: Assessment of antidiabetic activity of saccharogenic mixed grain beverage (SMGB) was determined by measurement of alpha-glucosidase inhibition activity, and glucose uptake activity and glucose metabolic protein expression by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. Results of volatile compounds analysis, 62 kinds of volatile compounds were detected in SMGB. Palmitic acid (9.534% ratio), benzaldehyde (8.948% ratio), benzyl ethyl ether (8.792% ratio), ethyl alcohol (8.35% ratio), and 2-amyl furan (4.826% ratio) were abundant in SMGB. We confirmed that alpha-glucosidase inhibition activity, glucose uptake activity, and glucose-metabolic proteins were upregulated by SMGB treatment with concentration dependent manner. CONCLUSION: Saccharogenic mixed grain beverage (SMGB) showed potential antidiabetic activity. Further studies will be needed in order to improve the taste and functionality of SMGB.

Erratum: Funding Acknowledgment

The funding acknowledgment in this article was omitted as published.

Effects of medicinal herb water extracts on expression of hepatic glucokinase, pyruvate dehydrogenase and acetyl-CoA carboxylase mRNA

We studied the anti-diabetic effects of medicinal herb water extracts on expression of hepatic glucokinase (GCK), pyruvate dehydrogenase (PDH), and acetyl-CoA carboxylase (ACC) mRNA. The medicinal herbs used for experiments were Cornus officinalis (CO), Paeonia suffruticosa Andrews (PSA), Discorea japonica Thunb. (DJ), Rehmannia glutinosa (RG), Lycium chinense (LC), and Pyrus pyrifolia (PP). For GCK mRNA expression, CO, RG, and LC water extracts exhibited a more effective activity than other extracts. Cells treated with RG and LC water extracts showed an increase in expression of PDH mRNA to 191% and 124%, respectively, compared to control. Expression of ACC mRNA was significantly higher in LC water extract. These data indicate that CO, RG, and LC water extracts stimulates expression of hepatic GCK, PDH, and ACC mRNA.

Study on antioxidative, antidiabetic and antiobesity activity of solvent fractions of smilax china L. leaf extract / 한국영양학회지

Smilax china L., a native plant found in Asian countries, has several medicinal properties including antioxidant, anti-inflammatory, and anti-cancer effects. Although the root of the plant is commonly used as traditional herbal medicine in Korea and China, the medicinal properties of the leaves have not gained the same attention. In this study, we analyzed the antioxidant activity, alpha-glucosidase inhibitory effect and lipid accumulation inhibition effect of Smilax china L. leaf water extract (SCLE) and its solvent fractions. SCLE was fractionated by using a series of organic solvents, including ethylacetate (EA) and n-butanol (BuOH). The EA fraction had the highest total polyphenol content (440.20 +/- 12.67 mg GAE/g) and total flavonoid content (215.14 +/- 24.83 mg QE/g). The radical scavenging activity IC50 values of the EA fraction for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) were 0.022 mg/mL and 0.13 mg/mL, respectively. Further, SOD-like activity and reducing power values of the EA fraction were higher than those of the other fractions. However, both the alpha-glucosidase and lipid accumulation inhibition assays showed that the BuOH fraction (83.35 +/- 4.18% at 1 mg/mL) and water extract (11.27 +/- 2.67%) were more effective than the EA fraction (64.13 +/- 6.35%, and 45.66 +/- 7.20%). These results provide new insights into the potential anti-diabetic and anti-obesity effects of Smilax china L. leaf.

Inhibitory effects of Capsicum annuum L. water extracts on lipoprotein lipase activity in 3T3-L1 cells

Obesity, an intractable metabolic disease, currently has no medical treatment without side effects, so studies have been actively carried out to find natural compounds that have anti-obesity activity with minimum side effects. In this study, the anti-obesity effects of water extracts of seven Capsicum annuum L. varieties being Putgochu (Pca), Oyee gochu (Oca), Kwari putgochu (Kca), Green pepper (Gca), Yellow paprika (Yca), Red paprika (Rca) and Cheongyang gochu (Cca), were examined through the evaluation of lipoprotein lipase (LPL) mRNA expression level in 3T3-L1 cells (mouse pre-adipocytes). After capsaicin elimination by chloroform defatting, freeze-dried powder of Cca was treated to 3T3-L1 cells and anti-obesity effects were examined by determining the LPL mRNA level using the RT-PCR method. Of the primary fractions, only proven fractions underwent secondary and tertiary refractionating to determine anti-obesity effects. From seven different Capsicum annuum L., there was a significant decrease of the LPL mRNA expression level of 50.9% in Cca treatment compared to the control group. A significant decrease of the LPL mRNA expression level was shown in primary fractions (Fr) 5 (36.2% decrease) and 6 (30.5% decrease) of the Cca water extracts. Due to the impurities checked by UPLC chromatography, Fr 5 and 6 were refractionated to determine the LPL mRNA expression level. Treatment of Fr 6-6 (35.8% decrease) and Fr 5-6 (35.3% decrease) showed a significant decrease in the LPL mRNA expression level. When analyzed using UPLC, major compounds of Fr 6-6 and Fr 5-6 were very similar. Subsequently, we refractionated Fr 6-6 and Fr 5-6 to isolate the major peak for structure elucidation. Treatment of Fr 5-6-1 (26.6% decrease) and Fr 6-6-1 (29.7% decrease) showed a significant decrease in the LPL mRNA expression level. Consequently, the fractions may have a possibility to ameliorate obesity through the decrease of the LPL mRNA expression level.

Comparison of antioxidant, alpha-glucosidase inhibition and anti-inflammatory activities of the leaf and root extracts of Smilax china L / 한국영양학회지

This study was conducted in order to compare the biological activities of leaf and root water extracts of Smilax china L. (SC) by measuring the total polyphenol and flavonoid contents, anti-oxidant activity, inhibitory effect on alpha-glucosidase, and anti-inflammatory gene expression. The total polyphenol and flavonoid contents of SC leaf (SCLE) and root (SCRE) water extracts were 127.93 mg GAE/g and 39.50 mg GAE/g and 41.99 mg QE/g and 1.25 mg QE/g, respectively. The anti-oxidative activities of SCLE and SCRE were measured using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging activity assay and reducing power assay. Both SCLE and SCRE scavenged radicals in a concentration-dependent manner, and SCLE showed stronger radical scavenging activity and reducing power than SCRE; however, both SCLE and SCRE exhibited lower activities than ascorbic acid. Compared to the anti-diabetic drug acarbose, which was used as a positive control, SCLE and SCRE exhibited low alpha-glucosidase inhibition activities; nevertheless, the activity of SCLE was 3.7 fold higher than that of SCRE. Finally, SCLE caused significantly decreased expression of the LPS-induced cytokines, iNOS, and COX-2 mRNA in RAW264.7 cells, indicating anti-inflammatory activity. These results indicate that SCLE might be a potential candidate as an anti-oxidant, anti-diabetic, and anti-inflammatory agent.

Suppressive effects of Schizandra chinensis Baillon water extract on allergy-related cytokine generation and degranulation in IgE-antigen complex-stimulated RBL-2H3 cells

Schizandra chinensis Baillon is a traditional folk medicine plant that is used to treat and prevent several inflammatory diseases and cancer in Korea, but the underlying mechanisms involved in its anti-allergic activity are not fully understood. This study was designed to investigate mechanisms of anti-allergic activity of a Schizandra chinensis Baillon water extract (SCWE) in immunoglobulin E (IgE)-antigen complex-stimulated RBL2H3 cells and to assess whether gastric and intestinal digestion affects the anti-allergic properties of SCWE. Oxidative stress is an important consequence of the allergic inflammatory response. The antioxidant activities of SCWE increased in a concentration-dependent manner. RBL-2H3 cells were sensitized with monoclonal anti-dinitrophenol (DNP) specific IgE, treated with SCWE, and challenged with the antigen DNP-human serum albumin. SCWE inhibited beta-hexosaminidase release and expression of interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein in IgE-antigen complex-stimulated RBL2H3 cells. We found that digested SCWE fully maintained its antioxidant activity and anti-allergic activity against the IgE-antigen complex-induced activation of RBL-2H3 cells. SCWE may be useful for preventing allergic diseases, such as asthma. Thus, SCWE could be used as a natural functional ingredient for allergic diseases in the food and/or pharmaceutical industries.

Erratum: Funding Acknowledgment

The funding acknowledgment in this article was omitted as published.

Anti-Oxidative and Anti-Inflammatory Effect of Water Extract from Perillae semen in RBL-2H3 Cells

The present study was to investigate anti-oxidative and anti-inflammatory activity of Perillae semen in RBL-2H3 basophilic leukemia cells. Inhibitory effect of Perillae semen onto free radical generation was determined by measuring DPPH and hydroxyl radical scavenging activities in vitro. Anti-inflammatory actions of Perillae semen extracts (100, 250, 500 microgram/mL) were assessed by testing their effects on the degranulation of mast cells. For this, beta-hexosaminidase released from RBL-2H3 cells was used and proinflammatory cytokines were measured by an ELISA kit. Our results indicated that Perillae semen water extracts effectively inhibited free radical generation. At the concentration of 500 microgram/mL of water extract, the degranulation of RBL-2H3 cells were inhibited by 42.1%. The IgE-antigen complex increased the accumulation of IL-4 and TNF-alpha secretion in RBL-2H3 cells and treatments with 250 and 500 microgram/mL of Perillae semen extracts suppressed the IgE induced secretion of IL-4 and TNF-alpha protein by 20.5, 26.9% and 14.5, 16.5% respectively. We observed that Perillae semen water extract reduced beta-hexosaminidase, IL-4, and TNF-alpha secretion in RBL-2H3 cells. These results provide that Perillae semen may be beneficial in the treatment of allergic inflammatory disease.

Cyclooxygenase-2 promotes cell proliferation, migration and invasion in U2OS human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor, but the pathogenesis is not well understood. While cyclooxygeanse-2 (COX-2) is known to be closely associated with tumor growth and metastasis in several kinds of human tumors, the function of COX-2 in osteosarcoma is unclear. Therefore, to investigate the function of COX-2 in osteosarcoma, we established stable cell lines overexpressing COX-2 in U2OS human osteosarcoma cells. COX-2 overexpression as well as prostaglandin E(2) treatment promoted proliferation of U2OS cells. In addition, COX-2 overexpression enhanced mobility and invasiveness of U2OS cells, which was accompanied by increases of matrix metalloproteinase-2 and -9 (MMP-2 and -9) activities. Selective COX-2 inhibitors, NS-398 and celecoxib, inhibited cell proliferation and abrogated the enhanced mobility, invasiveness and MMP activities induced by COX-2 overexpression. These results suggest that COX-2 is directly associated with cell proliferation, migration and invasion in human osteosarcoma cells, and the therapeutic value of COX-2 inhibitors should be evaluated continuously.

Inhibitory Effects of an Aqueous Extract of Gynura procumbens on Human Mesangial Cell Proliferation

Gynura procumbens (Lour.) Merr. has been used in some parts of Southeast Asia as a folk medicine to treat kidney diseases, diabetes mellitus, and hyperlipidemia. The present work was undertaken to prove the mechanisms of G. procumbens in the management of glomerular diseases. We investigated the effect of an aqueous extract of G. procumbens on cell proliferation, DNA synthesis, and the expressions of TGF-beta1, PDGF-BB, CDK1, CDK2, and CDK4 in fetal bovine serum-activated human mesangial cells (MCs). The G. procumbens extract inhibited proliferation, DNA synthesis, expressions of PDGF-BB, CDK1, and CDK2 mRNA, and expression of TGF-beta1 protein in MCs. The inhibitory effect of G. procumbens on MC proliferation may be mediated by suppression of PDGF-BB and TGF-beta1 expressions and the modulation of CDK1 and CDK2 expression. Therefore, G. procumbens shows promise as an adjunct therapy in preventing progressive renal diseases.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.