67-kDa laminin receptor mediates oolonghomobisflavan B-induced cell growth inhibition in melanoma.

BACKGROUND: Oolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (-)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PURPOSE: OHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. METHODS: The effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. RESULTS: Cell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. CONCLUSIONS: Taken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.

Mitochondrial protease ClpP supplementation ameliorates diet-induced NASH in mice.

BACKGROUND & AIMS: Mitochondrial dysfunction is considered a pathogenic linker in the development of non-alcoholic steatohepatitis (NASH). Inappropriate mitochondrial protein-quality control, possibly induced by insufficiency of the mitochondrial matrix caseinolytic protease P (ClpP), can potentially cause mitochondrial dysfunction. Herein, we aimed to investigate hepatic ClpP levels in a diet-induced model of NASH and determine whether supplementation of ClpP can ameliorate diet-induced NASH. METHODS: NASH was induced by a high-fat/high-fructose (HF/HFr) diet in C57BL/6J mice. Stress/inflammatory signals were induced in mouse primary hepatocytes (MPHs) by treatment with palmitate/oleate (PA/OA). ClpP levels in hepatocytes were reduced using the RNAi-mediated gene knockdown technique but increased through the viral transduction of ClpP. ClpP activation was induced by administering a chemical activator of ClpP. RESULTS: Hepatic ClpP protein levels in C57BL/6J mice fed a HF/HFr diet were lower than the levels in those fed a normal chow diet. PA/OA treatment also decreased the ClpP protein levels in MPHs. Overexpression or activation of ClpP reversed PA/OA-induced mitochondrial dysfunction and stress/inflammatory signal activation in MPHs, whereas ClpP knockdown induced mitochondrial dysfunction and stress/inflammatory signals in these cells. On the other hand, ClpP overexpression or activation improved HF/HFr-induced NASH characteristics such as hepatic steatosis, inflammation, fibrosis, and injury in the C57BL/6J mice, whereas ClpP knockdown further augmented steatohepatitis in mice fed a HF/HFr diet. CONCLUSIONS: Reduced ClpP expression and subsequent mitochondrial dysfunction are key to the development of diet-induced NASH. ClpP supplementation through viral transduction or chemical activation represents a potential therapeutic strategy to prevent diet-induced NASH. LAY SUMMARY: Western diets, containing high fat and high fructose, often induce non-alcoholic steatohepatitis (NASH). Mitochondrial dysfunction is considered pathogenically linked to diet-induced NASH. We observed that the mitochondrial protease ClpP decreased in the livers of mice fed a western diet and supplementation of ClpP ameliorated western diet-induced NASH.

Glucosyl-hesperidin enhances the cyclic guanosine monophosphate-inducing effect of a green tea polyphenol EGCG.

Animal and clinical studies have revealed that (-)-epigallocatechin-3-O-gallate (EGCG), one of the major bioactive polyphenols in green tea, showed several pharmacological effects including anti-obesity effect and anti-inflammatory effect. We previously reported that the second messenger cyclic guanosine monophosphate (cGMP) mediates its anti-inflammatory and anti-cancer properties. Here we demonstrated that glucosyl-hesperidin, enhances the cGMP-inducing effects of green tea extract in vivo. Moreover, glucosyl-hesperidin intake potentiated the green tea-elicited upregulation of the anti-inflammatory factor, toll-interacting protein.

Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via regulation of VEGFR2 signaling pathway in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys japonicus A. Berger (O. japonicus), so-called Wa-song in Korea, a traditional food and medicine that grows on mountain rocks and roof tiles. Wa-song containing various phenolic compounds have been reported as a medicinal plant for prevention of fibrosis, cancer, inflammation, and oxidative damage. AIM OF THE STUDY: The present study was designed to examine the anti-angiogenic effects of cultivated Orostachys japonicus 70% ethanol extract (CE) in vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: CE was prepared with 70% ethanol. HUVECs, rat aortic rings, and matrigel plug in mice were treated with CE (10-20 µg/mL) and VEGF (20-50 ng/mL), and the anti-angiogenic activities of CE were analyzed by SRB, wound healing, trans-well invasion, capillary-like tubule formation, rat aortas, Western blot, and matrigel plug assay. Phenolic compounds in CE were analyzed using a high-performance liquid chromatography (HPLC)-PDA system. RESULTS: Treatment of CE (10-20 µg/mL) markedly suppressed proliferation of HUVECs in the presence (from 136.5% to 112.2%) or absence of VEGF (from 100.0% to 92.1%). The proliferation inhibitory effect of CE was caused by G0/G1 cell cycle arrest, and the decrease of CDK-2, CDK-4, Cyclin D1 and Cyclin E1. Furthermore, CE treatment showed significant angiogenesis inhibitory effects on motility, invasion and micro-vessel formation of HUVECs, rat aortic rings and subcutaneous matrigels under VEGF-stimulation condition. In HUVECs, CE-induced anti-angiogenic effect was regulated by inhibition of the PI3K/AKT/mTOR, MAPK/p38, MAPK/ERK, FAK-Src, and VEGF-VEGFR2 signaling pathways. CONCLUSION: This study demonstrated that CE might be used as a potential natural substance, multi-targeted angiogenesis inhibitor, functional food material.

Studies on the Anti-Angiogenic Activities of Wild and Cultivated Orostachys japonicus Extracts in Human Umbilical Vein Endothelial Cells.

Orostachys japonicus has traditionally been used as a food product and a fork medicine in Asia to treat various diseases. Angiogenesis is a critical process that contributes to various chronic diseases via excessive delivery of oxygen and nutrients. Common anti-angiogenic drugs have serious problems related to high costs and side effects; thus, natural products with low costs and no cytotoxicity have garnered increasing interest. In this study, we evaluated and compared the anti-angiogenic effects and phenolic compound contents between wild (WOEs) and cultivated O. japonicus extracts (COEs) prepared under various extract conditions. WOEs and COEs suppressed cell proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited vascular endothelial growth factor-induced chemotactic migration, invasion, and capillary-like tube formation in HUVECs. Among COEs, that prepared by 70% EtOH (70% CE) showed the most effective anti-angiogenic activity in HUVECs. When compared to WOEs, total polyphenol and total flavonoid contents were 1.28 to 4.38 times higher in COEs, and 70% CE contained the greatest flavonoid contents (28.28 ± 0.93 mg%), as well as the highest levels of major phenolic compounds including gallic acid (21.84 µg/mL), epicatechin-gallate (6.58 µg/mL), kaempferol (6.32 µg/mL), and quercetin (8.55 µg/mL). Although further studies are required to identify the molecular mechanisms behind these anti-angiogenic effects, 70% CE could be used as an herbal medicine, functional food ingredient, and potent angiogenesis inhibitor. PRACTICAL APPLICATION: Environmental factors such as altitude, nutrients, exposure to sunlight, and temperature can influence the type and quantity of bioactive components in plants. The advantage of cultivated plants is that the above-mentioned factors can be artificially adjusted compared to wild plants. Based on economic efficiency, productivity, and consistent quality, anti-angiogenesis activity of cultivated O. japonicus is of greater commercial value as a functional food than wild O. japonicus.

Efficacy and Safety of Voglibose Plus Metformin in Patients with Type 2 Diabetes Mellitus: A Randomized Controlled Trial.

BACKGROUND: Combination of metformin to reduce the fasting plasma glucose level and an α-glucosidase inhibitor to decrease the postprandial glucose level is expected to generate a complementary effect. We compared the efficacy and safety of a fixed-dose combination of voglibose plus metformin (vogmet) with metformin monotherapy in drug-naïve newly-diagnosed type 2 diabetes mellitus. METHODS: A total of 187 eligible patients aged 20 to 70 years, with a glycosylated hemoglobin (HbA1c) level of 7.0% to 11.0%, were randomized into either vogmet or metformin treatments for 24 weeks. A change in the HbA1c level from baseline was measured at week 24. RESULTS: The reduction in the levels of HbA1c was -1.62%±0.07% in the vogmet group and -1.31%±0.07% in the metformin group (P=0.003), and significantly more vogmet-treated patients achieved the target HbA1c levels of <6.5% (P=0.002) or <7% (P=0.039). Glycemic variability was also significantly improved with vogmet treatment, estimated by M-values (P=0.004). Gastrointestinal adverse events and hypoglycemia (%) were numerically lower in the vogmet-treated group. Moreover, a significant weight loss was observed with vogmet treatment compared with metformin (-1.63 kg vs. -0.86 kg, P=0.039). CONCLUSION: Vogmet is a safe antihyperglycemic agent that controls blood glucose level effectively, yields weight loss, and is superior to metformin in terms of various key glycemic parameters without increasing the risk of hypoglycemia.

Involvement of iron depletion in palmitate-induced lipotoxicity of beta cells.

High levels of plasma free fatty acid are thought to contribute to the loss of pancreatic beta-cells in type 2 diabetes. In particular, saturated fatty acid such as palmitate or stearate can induce apoptosis in cultured beta cells (lipotoxicity). Endoplasmic reticulum stress is a critical mediator of free fatty acid-induced lipotoxicity. Recently, disorders in mitochondrial respiratory metabolism have been linked to lipotoxicity. Since iron is a critical component of respiratory metabolism, this study is initiated to determine whether abnormal iron metabolism is involved in palmitate-induced beta cell death. Immunoblotting analysis showed that treatment of INS-1 beta cells with palmitate reduced the level of transferrin receptor 1 (TfR1), but increased the level of heavy chain ferritin (FTH). In addition, palmitate reduced intracellular labile iron pool. Whereas iron depletion through treatment with iron-chelators deferoxamine or deferasirox augmented palmitate-induced cell death, iron supplementation with ferric chloride, ferrous sulfate, or holo-transferrin significantly protected cells against palmitate-induced death. Furthermore, overexpression of TfR1 reduced palmitate-induced cell death, whereas knockdown of TfR1 augmented cell death. In particular, treatment with deferoxamine increased the level of endoplasmic reticulum (ER) stress markers phospho-PERK, phospho-eIF2α, CHOP and phospho-c-Jun N-terminal kinase. Treatment with chemical chaperone significantly protected cells against deferoxamine-induced apoptosis. Iron supplementation also protected cells against palmitate-induced primary islet death. These data suggest that iron depletion plays an important role in palmitate-induced beta cell death through inducing ER stress. Therefore, attempts to block iron depletion might be able to prevent beta cell loss in type 2 diabetes.

Protective effect of nicotinamide on high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells is attributed to its inhibitory activity to sirtuins.

This study was initiated to determine whether the protective effect of nicotinamide (NAM) on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death was due to its role as an anti-oxidant, nicotinamide dinucleotide (NAD+) precursor, or inhibitor of NAD+-consuming enzymes such as poly (ADP-ribose) polymerase (PARP) or sirtuins. All anti-oxidants tested were not protective against HG/PA-induced INS-1 cell death. Direct supplementation of NAD+ or indirect supplementation through NAD+ salvage or de novo pathway did not protect the death. Knockdown of the NAD+ salvage pathway enzymes such as nicotinamide phosphoribosyl transferase (NAMPT) or nicotinamide mononucleotide adenyltransferase (NMNAT) did not augment death. On the other hand, pharmacological inhibition or knockdown of PARP did not affect death. However, sirtinol as an inhibitor of NAD-dependant deacetylase or knockdown of SIRT3 or SIRT4 significantly reduced the HG/PA-induced death. These data suggest that protective effect of NAM on beta cell glucolipotoxicity is attributed to its inhibitory activity on sirtuins.

Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes.

Elevated fatty acid levels have been thought to contribute to insulin resistance. Repression of the glucose transporter 4 (GLUT4) gene as well as impaired GLUT4 translocation may be a mediator for fatty acid-induced insulin resistance. This study was initiated to determine whether palmitate treatment repressed GLUT4 expression, whether glucose/fatty acid metabolism influenced palmitate-induced GLUT4 gene repression (PIGR), and whether attempts to prevent PIGR restored palmitate-induced impairment of glucose uptake (PIIGU) in C2 myotubes. Not only stimulators of fatty acid oxidation, such as bezafibrate, AICAR, and TOFA, but also TCA cycle substrates, such as pyruvate, leucine/glutamine, and α-ketoisocaproate/monomethyl succinate, significantly prevented PIGR. In particular, supplementing with pyruvate through methyl pyruvate resulted in nearly complete prevention of PIIGU, whereas palmitate treatment reduced the intracellular pyruvate level. These results suggest that pyruvate depletion plays a critical role in PIGR and PIIGU; thus, pyruvate supplementation may help prevent obesity-induced insulin resistance in muscle cells.

Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells.

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the ß-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved ß-oxidation activity.

Utilization patterns and cost of complementary and alternative medicine compared to conventional medicine in patients with type 2 diabetes mellitus.

AIMS: We evaluated the use and annual cost of complementary and alternative medicine (CAM) compared to conventional medicine in type 2 diabetes mellitus (DM) in the Korean population. METHODS: We analyzed the database of 2752 DM patients obtained from the Korean National Diabetes Program (KNDP). The cost data of conventional medicine starting 1-year before enrolment of the KNDP were obtained from the hospital electronic database. The cost data of CAM over the same period were obtained from questionnaires. RESULTS: Among the 2752 subjects, 677 patients (24.6%) used CAM, with the most common type being red ginseng and herbal medicine. Patients with a higher income, neuropathy, and self-monitoring of blood glucose (SMBG) were more likely to use CAM. Men, those with a higher education level and income, no cerebrovascular accident (CVA) history, and SMBG showed a relatively higher cost of CAM of total medical cost. The independent predictors for CAM were a higher income, the existence of diabetic neuropathy, no CVA history, and SMBG. CONCLUSIONS: Use and cost of CAM varied depending on income, accompanying complications and SMBG. To evaluate the total medical costs in DM patients, a comprehensive approach considering not only conventional cost but also CAM is required.

Intake of brown rice lees reduces waist circumference and improves metabolic parameters in type 2 diabetes.

Intake of whole grains has been associated with lower risks of type 2 diabetes and cardiovascular disease. Brown rice is unrefined whole grain and is produced by removing the outermost layers containing the germ and bran, which are rich in nutrients including dietary fiber, vitamins, minerals, and other unmeasured dietary constituents. The lees of brown rice (LB) are by-products of its fermentation in the process of manufacturing takju, a Korean turbid rice wine. In this study, we hypothesized that intake of LB would reduce waist circumference, a strong risk factor for cardiovascular disease in type 2 diabetic patients. A randomized, double-blind, placebo-controlled study was scheduled for 12 weeks. Thirty subjects were randomly assigned to receive a supplement prepared from the LB or from a mixed-grain dietary product (MG). Body weight, waist circumference, body composition, lipid profiles, and other laboratory parameters were measured. The LB group showed greater reduction in waist circumference (LB: 87.9 ± 8.8 to 85.1 ± 9.0 cm; MG: 86.9 ± 8.8 to 86.0 ± 9.3 cm; P = .032). In addition, the consumption of LB resulted in a significantly greater decrease in the level of aspartate transaminase (LB: 25.4 ± 8.5 to 21.0 ± 5.1 IU/mL; MG: 22.5 ± 5.3 to 22.4 ± 5.7 IU/mL; P = .044) and alanine transaminase (LB: 28.6 ± 11.3 to 21.9 ± 8.2 IU/mL; MG: 24.4 ± 7.5 to 24.5 ± 9.9 IU/mL; P = .038). Consumption of the LB was associated with a decreased waist circumference in type 2 diabetic patients. Further study is required to evaluate the metabolic effect of the extract of the LB in type 2 diabetes.

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death.

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.

Korean red ginseng (Panax ginseng) improves insulin sensitivity and attenuates the development of diabetes in Otsuka Long-Evans Tokushima fatty rats.

Ginseng has been reported to ameliorate hyperglycemia in experimental and clinical studies; however, its mechanism of action remains unclear. In this study, we investigated the metabolic effects and putative molecular mechanisms of Korean red ginseng (KRG, Panax ginseng) in animal models for type 2 diabetes mellitus (T2DM) and peripheral insulin-responsive cell lines. Korean red ginseng was administered orally at a dose of 200 mg/(kg d) to Otsuka Long-Evans Tokushima fatty rats for 40 weeks. Initially, chronic administration of KRG reduced weight gain and visceral fat mass in the early period without altering food intake. The KRG-treated Otsuka Long-Evans Tokushima fatty rats showed improved insulin sensitivity and significantly preserved glucose tolerance compared with untreated control animals up to 50 weeks of age, implying that KRG attenuated the development of overt diabetes. KRG promoted fatty acid oxidation by the activation of adenosine monophosphate-activated protein kinase (AMPK) and phosphorylation of acetyl-coenzyme A carboxylase in skeletal muscle and cultured C2C12 muscle cells. Increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, nuclear respiratory factor-1, cytochrome c, cytochrome c oxidase-4, and glucose transporter 4 by KRG treatment indicates that activated AMPK also enhanced mitochondrial biogenesis and glucose utilization in skeletal muscle. Although these findings suggest that KRG is likely to have beneficial effects on the amelioration of insulin resistance and the prevention of T2DM through the activation of AMPK, further clinical studies are required to evaluate the use of KRG as a supplementary agent for T2DM.

Involvement of Ca2+, CaMK II and PKA in EGb 761-induced insulin secretion in INS-1 cells.

EGb 761, a standardized form of Ginkgo biloba L. (Ginkgoaceae) leaf extract, was recently reported to increase pancreatic beta-cell function. To determine whether EGb 761 elicits insulin secretion directly, we treated INS-1 rat beta cells with EGb 761 and then measured insulin release. Treatment of EGb 761 (50 microg/ml) significantly stimulated insulin secretion in INS-1 cells, compared with untreated control (p<0.05) and the stimulatory effect of EGb 761 on insulin secretion was dose-dependent. To elucidate the mechanism of EGb 761-induced insulin secretion, we investigated the involvement of calcium. The treatment with nifedipine, an L-type calcium channel blocker, prevented EGb 761-induced insulin secretion and furthermore, EGb 761 itself elevated [Ca(2+)](i), suggesting the involvement of calcium in this process. To identity the protein kinases involved in EGb 761-induced insulin secretion, INS-1 cells were treated with different kinase inhibitors and their effects on EGb 761-induced secretion were investigated. KN62 and H89, calium/calmodulin kinase (CaMK) II and protein kinase A (PKA) inhibitor, respectively, significantly reduced EGb 761-induced insulin secretion. Immunoblotting studies showed an increase in the phosphorylated-forms of CaMK II and of PKA substrates after EGb 761 treatment. Our data suggest that EGb 761-induced insulin secretion is mediated by [Ca(2+)](i) elevation and subsequent activation of CaMK II and PKA.