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Metformin is known to stimulate glucose uptake, but the mechanism for this action is not fully understood. In this study, AMPK activators (AICAR and metformin) increased the expression of T-lymphoma invasion and metastasis-inducing protein-1 (Tiam-1), a Rac1 specific guanine nucleotide exchange factor (GEF), mRNA and protein in skeletal muscle C2C12 cells. Metformin increases the serine-phosphorylation of Tiam-1 by AMPK and induces interaction between Tiam-1 and 14-3-3. Pharmacologic inhibition of AMPK blocks this interaction, indicating that 14-3-3 may be required for induction of Tiam-1 by AMPK. Metformin also increases the phosphorylation of p21-activated kinase 1 (PAK1), a direct downstream target of Rac1, dependent on AMPK. Tiam-1 is down-regulated at high glucose concentrations in cultured cells and in the db/db mouse model of hyperglycemia. Furthermore, Tiam-1 knock-down blocked metformin-induced increase in glucose uptake. These findings suggest that metformin promotes cellular glucose uptake in part through Tiam-1 induction.

It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.

Coenzyme Q10(CoQ10) is a known anti-adipogenic factor. However, the mechanism by which CoQ10 acts is unclear. In this study, we found that CoQ10 increased the phosphorylation of AMP-activated protein kinase (AMPK) in 3T3-L1preadipocytes. CoQ10 induced an increase in cytoplasmic calcium concentrations, which is reflected by increased Fluo-3 intensity under confocal microscopy recording. Either inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) or knock-down CaMKK blocked CoQ10-induced AMPK phosphorylation, suggesting the involvement of calcium in CoQ10-mediated AMPK signaling. CoQ10 also increased the expression of peroxisome proliferator-activated receptor alpha (PPARα) at both the mRNA and protein levels. Knock down of AMPK with siRNA or inhibition of AMPK using an AMPK inhibitor compound C blocked CoQ10-induced expression of PPARα, indicating that AMPK plays a critical role in PPARα induction. In addition, CoQ10 increased fatty acid oxidation in 3T3-L1preadipocytes. The promoter activity of PPARα was increased by CoQ10 in an AMPK-dependent fashion. Moreover, the induction of acyl-CoA oxidase (ACO), a target gene of PPARα, was blocked under the PPARα knock down condition. Furthermore, treatment with CoQ10 blocked differentiation-induced adipogenesis. This blockade was not observed under the PPARα knock-down condition. Collectively, these results demonstrate that CoQ10 induces PPARα expression via the calcium-mediated AMPK signal pathway and suppresses differentiation-induced adipogenesis.

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.

Celastrol, an anti-oxidant flavonoid that is widely distributed in the plant kingdom, has been suggested to have chemopreventive effects on cancer cells: however, the mechanism of this process is not completely understood. In this study, we found that celastrol suppressed the viability of breast cancer MCF-7 cells in an AMP-activated protein kinase (AMPK)-dependent fashion. Celastrol also induced an increase in reactive oxygen species (ROS) levels, leading to AMPK phosphorylation. Protein kinase C (PKC) zeta was also shown to play a role in celastrol-induced ROS generation. In addition, celastrol increased phosphorylation of the pro-apoptotic effector, p53. Inhibition of AMPK blocked celastrol-mediated p53 phosphorylation. Moreover, celastrol increased the expression of tumor suppressor polo like kinase-2 (PLK-2) in a p53-dependent manner. Neither celastrol-induced PLK-2 induction nor celastrol-mediated apoptosis inducing factor poly(ADP-ribose) polymerase-2 (PARP-2) induction was observed in p53 knock-out cells. Furthermore, add-back of PLK-2 resulted in an increase in both celastrol-mediated PARP-2 induction and celastrol-induced apoptotic index sub G1 population. Together, these results suggest that celastrol may have anti-tumor effects on MCF-7 cells via AMPK-induced p53 and PLK-2 pathways.

Quercetin, an anti-oxidant flavonoid that is widely distributed in the plant kingdom, has been suggested to have chemopreventive effects on cancer cells, although the mechanism is not completely understood. In this study, we found that quercetin increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed the viability of HeLa cells. AICAR, an AMPK activator, and quercetin down-regulated heat shock protein (HSP)70 and increased the activity of the pro-apoptotic effector, caspase 3. Knock-down of AMPK blocked quercetin-mediated HSP70 down-regulation. Moreover, knock-down of HSP70 enhanced quercetin-mediated caspase 3 activation. Furthermore, quercetin sustained epidermal growth factor receptor (EGFR) activation by suppressing the phosphatases, PP2a and SHP-2. Finally, quercetin increased the interaction between EGFR and Cbl, and also induced the tyrosine phosphorylation of Cbl. Together, these results suggest that quercetin may have anti-tumor effects on HeLa cells via AMPK-induced HSP70 and down-regulation of EGFR.

Aims Clozapine has previously been implicated in the dysregulation of energy balance and glucose metabolism in the central nervous system, but its effects in the periphery have yet to be thoroughly elucidated. The objective of this study was to characterize the effects of clozapine on AMP-activated protein kinase (AMPK) activity in the skeletal muscles. Main methods Myotube C2C12 cells were incubated under control conditions, or with clozapine. Expression levels of phosphorylation status of AMPK and its direct downstream Acetyl-CoA carboxylase (ACC) were analyzed by Western blot. Intracellular calcium concentration was measured with calcium indicator dye, fluo-3 AM. 2-deoxyglucose uptake was assessed via the scintillation count. Key findings We reported that clozapine activated AMPK in mouse C2C12 myotubes and also stimulated glucose uptake. Clozapine also increased intracellular calcium concentrations of C2C12 cells, and pretreatment with either ethylenediaminetetraacetic acid (EDTA), an extracellular calcium chelator, or 1.8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), a Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, blocked clozapine-induced AMPK activation. Significance These results demonstrate that clozapine increases glucose uptake through CaMKK-AMPK pathway in myotube C2C12 cells.

Curcumin has been shown to exert a variety of beneficial human health effects. However, mechanisms by which curcumin acts are poorly understood. In this study, we report that curcumin activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. In addition, curcumin activated the mitogen-activated protein kinase kinase (MEK)3/6-p38 mitogen-activated protein kinase (MAPK) signaling pathways in the downstream of the AMPK cascade. Moreover, inhibition of either AMPK or p38 MAPK resulted in blockage of curcumin-induced glucose uptake. Furthermore, the administration of curcumin to mice increased AMPK phosphorylation in the skeletal muscles. Taken together, these results indicate that the beneficial health effect of curcumin can be explained by its ability to activate AMPK-p38 MAPK pathways in skeletal muscles.

Aims C-peptide is a product of pro-insulin cleavage. Numerous studies have demonstrated that C-peptide, although not influencing blood glucose control, may play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. The aim of this paper was to present a novel function of C-peptide, focusing on its role in nitric oxide (NO) generation. Main methods Murine macrophage Raw264.7 cells and primary peritoneal macrophages were incubated under control conditions, or with C-peptide. Expression level of iNOS and phosphorylation status of JAK2/STAT1 were analyzed by Western blot. Fluorometric NO assay kit was used to assess the concentration of nitrite in culture medium. Intracellular calcium concentration was measured with calcium indicator dyes, such as Fura-2 and Fluo-3 AM. Key findings C-peptide increased the level of nitrites in murine macrophage Raw264.7 cells. The nitrites production induced by lipopolysaccharide (LPS) was further enhanced by co-treatment of C-peptide. This up-regulation of nitrites generation also correlated with the induction of inducible nitric oxide synthase (iNOS), a prominent marker of macrophage activation. In addition, C-peptide increased the intracellular concentration of calcium levels. Moreover, C-peptide-induced nitrites generation and increase in calcium was observed in freshly isolated primary peritoneal macrophages. In addition, C-peptide specifically affected the Janus activated kinase (JAK)/signal transducer and activated transcription (STAT) pathway. Finally, C-peptide-mediated nitrites generation and JAK2/STAT1 phosphorylation were not detected in the presence of the intracellular calcium chelator, BAPTA-AM. Significance These results suggest that C-peptide may elicit immune modulatory function via modulation of the calcium/JAK–STAT pathway.

Gaegurin-6, an antimicrobial peptide that belongs to the alpha-helix family, was isolated from the skin of Rana rugosa. Gaegurin-6 contains a hydrophobic motif at the N-terminus and a helical region at the C-terminus. Although gaegurin-6 has been implicated in cell signaling, the precise role in insulin secretion is currently unknown. We have attempted to determine whether gaegurin-6 affects insulin secretion and tried to elucidate the relationship between the structural motifs and biological activity. In this study, we have shown that gaegurin-6 stimulates insulin secretion and also increases the intracellular calcium concentration in pancreatic beta Rin5mf cells. Moreover, a corollary study revealed that both the hydrophobicity of the N-terminus and the disulfide bridge of the C-terminus of gaegurin-6 are critical for its effects on insulin secretion. Membrane pore-forming ability is also observed in gaegurin-6, but not in the linear form or the N-terminus hydrophobic amino acid-deleted form. We further showed that these regions of gaegurin-6 are responsible for calcium influx in pancreatic beta Rin5mf cells. Taken together, these results indicate that gaegurin-6 can affect insulin secretion in pancreatic beta cells through the modulation of calcium influx.