Metformin Induces Lipogenesis and Apoptosis in H4IIE Hepatocellular Carcinoma Cells.

Metformin is the most widely used anti-diabetic drug that helps maintain normal blood glucose levels primarily by suppressing hepatic gluconeogenesis in type II diabetic patients. We previously found that metformin induces apoptotic death in H4IIE rat hepatocellular carcinoma cells. Despite its anti-diabetic roles, the effect of metformin on hepatic de novo lipogenesis (DNL) remains unclear. We investigated the effect of metformin on hepatic DNL and apoptotic cell death in H4IIE cells. Metformin treatment stimulated glucose consumption, lactate production, intracellular fat accumulation, and the expressions of lipogenic proteins. It also stimulated apoptosis but reduced autophagic responses. These metformin-induced changes were clearly reversed by compound C, an inhibitor of AMP-activated protein kinase (AMPK). Interestingly, metformin massively increased the production of reactive oxygen species (ROS), which was completely blocked by compound C. Metformin also stimulated the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). Finally, inhibition of p38MAPK mimicked the effects of compound C, and suppressed the metformin-induced fat accumulation and apoptosis. Taken together, metformin stimulates dysregulated glucose metabolism, intracellular fat accumulation, and apoptosis. Our findings suggest that metformin induces excessive glucose-induced DNL, oxidative stress by ROS generation, activation of AMPK and p38MAPK, suppression of autophagy, and ultimately apoptosis.

Anti-cancer effects of fenbendazole on 5-fluorouracil-resistant colorectal cancer cells.

Benzimidazole anthelmintic agents have been recently repurposed to overcome cancers resistant to conventional therapies. To evaluate the anti-cancer effects of benzimidazole on resistant cells, various cell death pathways were investigated in 5-fluorouracil-resistant colorectal cancer cells. The viability of wild-type and 5-fluorouracil-resistant SNU-C5 colorectal cancer cells was assayed, followed by Western blotting. Flow cytometry assays for cell death and cell cycle was also performed to analyze the anti-cancer effects of benzimidazole. When compared with albendazole, fenbendazole showed higher susceptibility to 5-fluorouracil-resistant SNU-C5 cells and was used in subsequent experiments. Flow cytometry revealed that fenbendazole significantly induces apoptosis as well as cell cycle arrest at G2/M phase on both cells. When compared with wild-type SNU-C5 cells, 5-fluorouracil-resistant SNU-C5 cells showed reduced autophagy, increased ferroptosis and ferroptosis-augmented apoptosis, and less activation of caspase-8 and p53. These results suggest that fenbendazole may be a potential alternative treatment in 5-fluorouracil-resistant cancer cells, and the anticancer activity of fenbendazole does not require p53 in 5-fluorouracil-resistant SNU-C5 cells.

Fenbendazole Suppresses Growth and Induces Apoptosis of Actively Growing H4IIE Hepatocellular Carcinoma Cells via p21-Mediated Cell-Cycle Arrest.

Bendimidazole anthelmintics (BAs) have gained interest for their anticancer activity. The anticancer activity is mediated via multiple intracellular changes, which are not consistent under different conditions even in the same cells. We investigated the anticancer activity of fenbendazole (FZ, one of BAs) under two different growth conditions. The growth rate of H4IIE cells was dose-dependently decreased by FZ only in actively growing cells but not in fully confluent quiescent cells. Apoptosis-associated changes were also induced by FZ in actively growing cells. Markers of autophagy were not changed by FZ. The number of cells was markedly increased in sub-G1 phase but decreased in S- and G2/M phases by FZ. FZ up-regulated p21 (an inhibitor of cyclin-CDK) but suppressed the expression of cell cycle-promoting proteins (cyclin D1 and cyclin B1). FZ did not affect integrin αV or n-cadherin expression as well as cell migration. Glycolytic changes (glucose consumption and lactate production) and the generation of reactive oxygen species (ROS) were not affected by FZ. Although the activity of mitogen-activated protein kinases (MAPKs) was altered by FZ, the inhibition of MAPKs did not affect the pro-apoptotic activity of FZ. Taken together, FZ selectively suppressed the growth of cells via p21-mediated cell cycle arrest at G1/S and G2/M, and resulted in apoptosis only in actively growing cells but not in quiescent cells. Glucose metabolism, ROS generation, and MAPKs are unlikely targets of FZ at least in H4IIE rat hepatocellular carcinoma cells used in this study.

Antitumor Effect of Metformin in Combination with Binimetinib on Melanoma Cells.

Cutaneous melanoma is a fatal disease for patients with distant metastasis. Metformin is the most widely used anti-diabetic drug, and proved to suppress cell proliferation and metastasis in diverse cancers including melanoma. We previously reported that MEK inhibitor trametinib increases the expression of epithelial-mesenchymal transition (EMT) regulators and melanoma cell motility, which are suppressed by addition of metformin in A375 melanoma cells. To confirm our findings further, we first evaluated the effect of metformin in combination with another MEK inhibitor binimetinib on cell viability in G361 melanoma cells. We then investigated whether binimetinib affects the expression of EMT regulators and cell motility. We finally monitored the effect of metformin on binimetinib-induced cell migration. Cell viability assay showed that combination index (CI) value at ED50 is 0.80, suggesting synergy for the combination of metformin with binimetinib. Our results also revealed that binimetinib increased the expression of EMT regulators such as integrin αV, fibronectin and slug, which correlate well with the enhanced cell migration in wound healing assay. Metformin, on the contrary, suppressed the expression of sparc, integrin αV, fibronectin and N-cadherin with the reduced cell motility. The combination treatment showed that metformin counteracts the binimetinib-induced increase of cell motility. Overall, these results suggest that metformin with binimetinib might be useful as a potential therapeutic adjuvant against cell survival and metastatic activity in melanoma patients.

Effect of Metformin in Combination With Trametinib and Paclitaxel on Cell Survival and Metastasis in Melanoma Cells.

BACKGROUND/AIM: Despite clinical benefit from treatment with dabrafenib and trametinib in melanoma patients with BRAF mutations, half relapse within months and one-third are unresponsive to treatment. We evaluated the anticancer potential of metformin in combination with trametinib plus paclitaxel, against four melanoma cell lines. MATERIALS AND METHODS: Metformin with trametinib and paclitaxel was tested for effects on cell viability, signaling molecules in MAPK and mTOR pathways, factors involved in epithelial-mesenchymal transition (EMT), and cell motility. RESULTS: The combination of metformin with trametinib and paclitaxel showed differential growth inhibitory effects; synergistic effects were observed in a cell line in which metformin suppresses ERK activity, whereas the combination showed antagonistic effects in a cell line with metformin-induced ERK activation. Trametinib or paclitaxel increased the expression of EMT regulators and melanoma cell motility, which were suppressed by combining metformin with trametinib and paclitaxel. CONCLUSION: The combined treatment of metformin with trametinib and paclitaxel showed divergent effects on melanoma cell viability. Metformin might be useful as a potential adjuvant against cell proliferation and metastatic activity in melanoma patients.

Antitumor Activity of Combination Therapy with Metformin and Trametinib in Non-Small Cell Lung Cancer Cells.

Metformin has been widely used as an antidiabetic drug, and reported to inhibit cell proliferation in many cancers including non-small cell lung cancer (NSCLC). In NSCLC cells, metformin suppresses PI3K/AKT/mTOR signaling pathway, but effect of metformin on RAS/ RAF/MEK/ERK signaling pathway is controversial; several studies showed the inhibition of ERK activity, while others demonstrated the activation of ERK in response to metformin exposure. Metformin-induced activation of ERK is therapeutically important, since metformin could enhance cell proliferation through RAS/RAF/MEK/ERK pathway and lead to impairment of its anticancer activity suppressing PI3K/AKT/mTOR pathway, requiring blockade of both signaling pathways for more efficient antitumor effect. The present study tested the combination therapy of metformin and trametinib by monitoring the alterations of regulatory effector proteins of cell signaling pathways and the effect of the combination on cell viability in NCI-H2087 NSCLC cells with NRAS and BRAF mutations. We show that metformin alone blocks PI3K/AKT/mTOR signaling pathway but induces the activation and phosphorylation of ERK. The combination therapy synergistically decreased cell viability in treatment with low doses of two drugs, while it gave antagonistic effect with high doses. These findings suggest that the efficacy of metformin and trametinib combination therapy may depend on the alteration of ERK activity induced by metformin and specific cellular context of cancer cells.

Metformin Induces Oxidative Stress-Mediated Apoptosis without the Blockade of Glycolysis in H4IIE Hepatocellular Carcinoma Cells.

Metformin, a widely prescribed anti-diabetic drug, also exerts anti-cancer effects in different types of cancers. Although a number of molecular mechanisms have been suggested, the metabolic features underlying metformin's anti-cancer activity is not fully understood enough. Because cancer cells have been known to prefer inefficient aerobic glycolysis to support their proliferation, it is important to clarify by which metformin affects metabolism to suppress the proliferation of cancer cells. Here, we report the metabolic changes induced by metformin and its relevance to the induction of apoptosis in H4II rat hepatocellular carcinoma cells. H4IIE cells were treated with metformin and other reagents in culture media with various nutritional compositions. Glutamine as well as pyruvate enhanced the viability of H4IIE cells in glucose-deprived conditions. Protective effects of glucose and pyruvate were comparable at same concentrations (5 mM). Metformin induced apoptosis irrespective of any nutritional conditions. Glucose consumption and lactate production were stimulated by metformin. Inhibition of glycolysis by 2-deoxyglucose suppressed the metformin-induced lactate production but additively enhanced metformin's pro-apoptotic effect. These results indicate that metformin does not interfere but accelerate glycolysis. Unexpectedly, the production of reactive oxygen species (ROS) was markedly stimulated by metformin. A potent antioxidant, N-acetylcysteine (NAC) suppressed all pro-apoptotic changes as well as ROS generation induced by metformin. Taken together, metformin does not interfere with glycolysis but promotes apoptosis by enhancing oxidative stress.

Synergistic Enhancement of Paclitaxel-induced Inhibition of Cell Growth by Metformin in Melanoma Cells.

Melanoma is one of the most aggressive and treatment-resistant malignancies. Antidiabetic drug metformin has been reported to inhibit cell proliferation and metastasis in many cancers, including melanoma. Metformin suppresses the mammalian target of rapamycin (mTOR) and our previous study showed that it also inhibits the activity of extracellular signal-regulated kinase (ERK). Paclitaxel is currently prescribed for treatment of melanoma. However, paclitaxel induced the activation of ERK/mitogen-activated protein kinase (MAPK) pathway, a cell signaling pathway implicated in cell survival and proliferation. Therefore, we reasoned that combined treatment of paclitaxel with metformin could be more effective in the suppression of cell proliferation than treatment of paclitaxel alone. Here, we investigated the combinatory effect of paclitaxel and metformin on the cell survival in SK-MEL-28 melanoma cell line. Our study shows that the combination of paclitaxel and metformin has synergistic effect on cell survival and suppresses the expression of proteins involved in cancer metastasis. These findings suggest that the combination of paclitaxel and metformin can be a possible therapeutic option for treatment of melanoma.

Combination of BEZ235 and Metformin Has Synergistic Effect on Cell Viability in Colorectal Cancer Cells.

Patients with type II diabetes mellitus are more susceptible to colorectal cancer (CRC) incidence than non-diabetics. The anti-diabetic drug metformin is most commonly prescribed for the treatment of this disease and has recently shown antitumor effect in preclinical studies. The aberrant mutational activation in the components of RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathway is very frequently observed in CRC. We previously reported that metformin inhibits the phosphorylation of ERK and BEZ235, a dual inhibitor of PI3K and mTOR, has anti-tumor activity against HCT15 CRC cells harboring mutations of KRAS and PIK3CA. Therefore, we hypothesized that simultaneous inhibition of two pathways by combining metformin with BEZ235 could be more effective in the suppression of proliferation than single agent treatment in HCT15 CRC cells. Here, we investigated the combinatory effect of metformin and BEZ235 on the cell survival in HCT15 CRC cells. Our study shows that both of the two signaling pathways can be blocked by this combinational strategy: metformin suppressed both pathways by inhibiting the phosphorylation of ERK, 4E-BP1 and S6, and BEZ235 suppressed PI3K/AKT/ mTOR pathway by reducing the phosphorylation of 4E-BP1 and S6. This combination treatment synergistically reduced cell viability. The combination index (CI) values ranged from 0.44 to 0.88, indicating synergism for the combination. These results offer a preclinical rationale for the potential therapeutic option for the treatment of CRC.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm across mitochondrial inner membrane.

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨm). Therefore, pharmacological manipulation of ΔΨm can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨm against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 µM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca2+ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨm were completely abolished in K+-free medium on pure isolated mitochondria. Taken together, results demonstrate that K+ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K+ influx is probably mediated, at least in part, by activation of mitochondrial K+ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Metformin Synergistically Potentiates the Antitumor Effects of Imatinib in Colorectal Cancer Cells.

Metformin is the most commonly prescribed anti-diabetic drug with relatively minor side effect. Substantial evidence has suggested that metformin is associated with decreased cancer risk and anticancer activity against diverse cancer cells. The tyrosine kinase inhibitor imatinib has shown powerful activity for treatment of chronic myeloid leukemia and also induces growth arrest and apoptosis in colorectal cancer cells. In this study, we tested the combination of imatinib and metformin against HCT15 colorectal cancer cells for effects on cell viability, cell cycle and autophagy. Our data show that metformin synergistically enhances the imatinib cytotoxicity in HCT15 cells as indicated by combination and drug reduction indices. We also demonstrate that the combination causes synergistic down-regulation of pERK, cell cycle arrest in S and G2/M phases via reduction of cyclin B1 level. Moreover, the combination resulted in autophagy induction as revealed by increased acidic vesicular organelles and cleaved form of LC3-II. Inhibition of autophagic process by chloroquine led to decreased cell viability, suggesting that induction of autophagy seems to play a cell protective role that may act against anticancer effects. In conclusion, our present data suggest that metformin in combination with imatinib might be a promising therapeutic option in colorectal cancer.

Tangeretin Improves Glucose Uptake in a Coculture of Hypertrophic Adipocytes and Macrophages by Attenuating Inflammatory Changes.

Obesity is characterized by a state of chronic low-grade inflammation and insulin resistance, which are aggravated by the interaction between hypertrophic adipocytes and macrophages. In this study, we investigated the effects of tangeretin on inflammatory changes and glucose uptake in a coculture of hypertrophic adipocytes and macrophages. Tangeretin decreased nitric oxide production and the expression of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, inducible nitric oxide synthase, and cyclooxygenase-2 in a coculture of 3T3-L1 adipocytes and RAW 264.7 cells. Tangeretin also increased glucose uptake in the coculture system, but did not affect the phosphorylation of insulin receptor substrate (IRS) and Akt. These results suggest that tangeretin improves insulin resistance by attenuating obesity-induced inflammation in adipose tissue.

Blockage of Autophagy Rescues the Dual PI3K/mTOR Inhibitor BEZ235-induced Growth Inhibition of Colorectal Cancer Cells.

Molecular targeting for the altered signaling pathways has been proven to be effective for the treatment ofmany types of human cancer, including colorectal cancer (CRC). The dual phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor BEZ235 has shown to exhibit potent antitumor activity against solid tumors. Autophagy is a cellular lysosomal catabolic process to maintain metabolic homeostasis, which has been known to be induced in response to many therapeutic agents in cancer cells. This process is negatively regulated by mTOR and often acts as prosurvival or prodeath mechanism following cancer therapeutics. The current study was designed to investigate the antiproliferation activity of BEZ235 and to evaluate the role of autophagy induced by BEZ235 using HCT15 CRC cells bearing ras oncogene mutation. We found that BEZ235 decreases cell viability, which was mostly dependent on G1 arrest of cell cycle via suppression of cyclin A expression. BEZ235 affects PI3K/Akt/mTOR signaling pathway by increasing the phosphorylation of AKT at Ser(473) and RAS/RAF/MEK/ERK pathway by decreasing the phosphorylation of ERK at Tyr(204). BEZ235 also stimulated autophagy induction as evidenced by the increased expression of LC3-II and abundant acidic vesicular organelles (AVOs) in the cytoplasm. In addition, the combination of BEZ235 with autophagy inhibitor chloroquine, a known antagonist of autophagy, counteracted the antiproliferation effect of BEZ235. Thus, our study indicates that autophagy induced in response to BEZ235 treatment appears to act as cell death mechanism in HCT15 CRC cells.

Effects of dihydrotestosterone on rat dermal papilla cells in vitro.

Androgenetic alopecia involves the action of dihydrotestosterone (DHT) on dermal papilla cells (DPCs) that line the base of the hair follicle. However, the mechanism of DHT action is not completely understood. The effects of DHT on DPCs, regulatory cells that function in follicle growth and the hair cycle, were examined in immortalized cells derived from rat vibrissa follicles. DHT did not affect the proliferation of immortalized DPCs. However, flow cytometry analysis revealed that DHT increased cell-cycle arrest in these cells, which was accompanied by an increase in the p27(kip1) level and by decreases in cyclin E, cyclin D1, and cyclin-dependent kinase 2 levels. DHT treatment resulted in the phosphorylation and nuclear translocation of Smad2/3, a mediator of the transforming growth factor-ß (TGF-ß) signaling pathway, which leads to the catagen phase of the hair cycle. DHT also induced the phosphorylation and nuclear translocation of heat shock protein 27 (HSP27). Moreover, DHT decreased the levels of total and nuclear ß-catenin, an important regulator of hair growth and proliferation, while lithium chloride, a glycogen synthase kinase-3ß inhibitor, attenuated the DHT-induced downregulation of the ß-catenin level. On the other hand, DHT increased the phosphorylation of mammalian target of rapamycin (mTOR), a regulator of proliferation, in immortalized DPCs. These results illustrate that DHT could shorten the duration of the hair growth cycle by initiating cell-cycle arrest, downregulating the ß-catenin level, and upregulating the TGF-ß/Smad and HSP27 level, whereas activation of mTOR by DHT could attenuate the inhibition of hair growth cycle in immortalized DPCs.

p-Synephrine suppresses glucose production but not lipid accumulation in H4IIE liver cells.

p-Synephrine, the primary protoalkaloid in the extract of bitter orange and other citrus species, has gained interest due to its lipolytic activity in adipose tissues. We previously found that p-synephrine stimulates glucose consumption via AMP-activated protein kinase (AMPK) in L6 skeletal muscle cells. This study investigated the effect of p-synephrine on glucose production and lipid accumulation in H4IIE rat liver cells. Glucose production was increased in H4llE cells that were incubated in glucose-free medium but decreased dose dependently (1-100 µM) with p-synephrine treatment. Protein levels of glucose-6-phosphatase (G6Pase) and phosphoenol pyruvate carboxykinase (PEPCK) were also decreased by treatment (4 h) with p-synephrine. Antagonists against α- and ß-adrenergic receptors (phentolamine and propranolol) and other inhibitors against signaling molecules did not interrupt p-synephrine-induced suppression in glucose production. However, H7 (an inhibitor of serine/threonine kinases PKA, PKC, and PKG) significantly blocked p-synephrine-induced suppression of glucose production and further increased basal glucose production. Unlike the suppressive effect on glucose production, p-synephrine failed to affect palmitic acid-induced cytoplasmic lipid accumulation. Protein levels of fatty acid synthase (FAS) and phosphorylation levels of AMPK and ACC were not changed by p-synephrine. Altogether, p-synephrine can suppress glucose production but does not affect lipid accumulation in H4IIE liver cells.

Biphasic activity of chloroquine in human colorectal cancer cells.

Autophagy is a homeostatic degradation process that is involved in tumor development and normal development. Autophagy is induced in cancer cells in response to chemotherapeutic agents, and inhibition of autophagy results in enhanced cancer cell death or survival. Chloroquine (CQ), an anti-malarial devrepug, is a lysosomotropic agent and is currently used as a potential anticancer agent as well as an autophagy inhibitor. Here, we evaluate the characteristics of these dual activities of CQ using human colorectal cancer cell line HCT15. The results show that CQ inhibited cell viability in dose-and time-dependent manner in the range between 20 to 80 uM, while CQ did not show any antiproliferative activity at 5 and 10 uM. Cotreatment of CQ with antitumor agent NVP-BEZ235, a dual inhibitor of PI3K/mTOR, rescued the cell viability at low concentrations meaning that CQ acted as an autophagy inhibitor, but CQ induced the lethal effect at high concentrations. Acridine orange staining revealed that CQ at high doses induced lysosomal membrane permeabilization (LMP). High doses of CQ produced cellular reactive oxygen species (ROS) and cotreatment of antioxidants, such as NAC and trolox, with high doses of CQ rescued the cell viability. These results suggest that CQ may exert its dual activities, as autophagy inhibitor or LMP inducer, in concentration-dependent manner.

Mild mitochondrial depolarization is involved in a neuroprotective mechanism of Citrus sunki peel extract.

Mitochondrial membrane potential (∆Ψm ) contributes to determining a driving force for calcium to enter the mitochondria. It has been demonstrated that even a small mitochondrial depolarization is sufficient to prevent mitochondrial calcium overload and the subsequent apoptosis. Therefore, mild mitochondrial depolarization has been recently evaluated as a novel mechanism of neuroprotection via inhibiting neurotoxic mitochondrial calcium overload during neuronal insults. In the present study, using both real-time recording and flow cytometric analyses of ∆Ψm , we demonstrated that ethanolic peel extract of Citrus sunki Hort. ex Tanaka (CPE) and its active compounds are capable of inducing a mild mitochondrial depolarization. Polymethoxylated flavones such as nobiletin and tangeretin were found as the active compounds responsible for CPE effects on ∆Ψm . Neuronal viability was significantly increased in a dose-dependent manner by CPE treatment in H2 O2 -stimulated HT-22 cells as an in vitro neuronal insult model. CPE treatment significantly inhibited H2 O2 -induced apoptotic processes such as chromatin condensation, caspase 3 activation and anti-poly (ADP-ribose) polymerase (PARP) cleavage. CPE treatment significantly blocked mitochondrial calcium overload in H2 O2 -stimulated HT-22 neurons as indicated by rhod-2 acetoxymethyl ester. Taken together, our findings suggest that CPE and its active compounds may be considered as promising neuroprotective agents via inducing a mild mitochondrial depolarization.

p-Synephrine stimulates glucose consumption via AMPK in L6 skeletal muscle cells.

Interest in p-synephrine, the primary protoalkaloid in the extract of bitter orange and other citrus species, has increased due to its various pharmacological effects and related adverse effects. The lipolytic activity of p-synephrine has been repeatedly revealed by in vitro and in vivo studies and p-synephrine is currently marketed as a dietary supplement for weight loss. The present study investigated the effect of p-synephrine on glucose consumption and its action mechanism in L6 skeletal muscle cells. Treatment of L6 skeletal muscle cells with p-synephrine (0-100µM) did not affect cell viability and increased basal glucose consumption up to 50% over the control in a dose-dependent manner. The basal- or insulin-stimulated lactic acid production as well as glucose consumption was significantly increased by the addition of p-synephrine. p-Synephrine stimulated the phosphorylation of AMPK but not of Akt. p-Synephrine-induced glucose consumption was sensitive to the inhibition of AMPK but not to the inhibition of PI3 kinase. p-Synephrine also stimulated the translocation of Glut4 from the cytoplasm to the plasma membrane; this stimulation was suppressed by the inhibition of AMPK, but not of PI3 kinase. Taken together, p-synephrine can stimulate glucose consumption (Glut4-dependent glucose uptake) by stimulating AMPK activity, regardless of insulin-stimulated PI3 kinase-Akt activity in L6 skeletal muscle cells.

Effects of brown alga, Ecklonia cava on glucose and lipid metabolism in C57BL/KsJ-db/db mice, a model of type 2 diabetes mellitus.

Recently, there has been a growing interest in alternative therapies of marine algae for diabetes. Therefore, the anti-diabetic effects of brown alga, Ecklonia cava was investigated in type 2 diabetic animal. Male C57BL/KsJ-db/db (db/db) mice were divided into control, dieckol rich extract of E. cava (AG-dieckol), or rosiglitazone (RG) groups. The blood glucose, blood glycosylated hemoglobin levels, and plasma insulin levels were significantly lower in the AG-dieckol and RG groups than in the control db/db mice group, while glucose tolerance was significantly improved in the AG-dieckol group. AG-dieckol markedly lowered plasma and hepatic lipids concentration compared to the control db/db mice group. The antioxidant enzyme activities were significantly higher in the AG-dieckol group than in the control db/db mice group, yet its TBARS level was markedly lower compared to the RG group. With regard to hepatic glucose regulating enzyme activities, glucokinase activity was enhanced in the AG-dieckol group mice, while glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities in the AG-dieckol group mice were significantly lowered than those in the control db/db mice group. These results suggest that AG-dieckol exert an anti-diabetic effect in type 2 diabetic mice by improving the glucose and lipid metabolism and antioxidant enzymes.

6-Hydroxydopamine-induced PC12 cell death is mediated by MEF2D down-regulation.

Recently, it was reported that in a 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model, neuronal cell death is associated with the cdk5-mediated hyperphosphorylation of myocyte enhancer factor 2 (MEF2), a transcription factor that is critically required for neuronal survival. In the present study, we investigated the possible involvement of cdk5-mediated MEF2D down-regulation on 6-hydroxydopamine (6-OHDA)-induced PC12 cell death. 6-OHDA was found to significantly increase nitric oxide (NO) production and to induce apoptosis in a time-dependent manner in PC12 cells. Furthermore, 6-OHDA was found to markedly reduce MEF2D levels under conditions that could induce PC12 cell apoptosis. In addition, PC12 cell death and MEF2D degradation by 6-OHDA were prevented by the cdk5 inhibitor roscovitine, but roscovitine could not restore the 6-OHDA-induced inactivation of Akt. These results suggest that the cell death and MEF2D degradation caused by 6-OHDA are dependent on cdk5 activity. On the other hand, roscovitine enhanced the 6-OHDA-induced activations of ERK1/2 and JNK, but reduced the 6-OHDA-induced activation of p38. These results suggest that PC12 cell death by 6-OHDA appears to be regulated by the down-regulation of MEF2D via some interaction between cdk5 and MAP kinase.