Horng-Mo Lee

N^ε-(carboxymethyl) lysine (CML) plays causal roles in diabetic complications. In the present study, we investigated whether CML-induced HIF-1α accumulation and epithelial-mesenchymal transition (EMT) in HK-2 renal proximal tubular epithelial cells. Treatment with CML-BSA increased reactive oxygen species (ROS) production reduced the mitochondrial membrane potential and induced mitochondrial fragmentation. Pre-treatment of cells with antioxidant, α-lipoic acid, normalised the ROS production and restored the mitochondrial membrane potential. These changes were accompanied with morphological changes of epithelial mesenchymal transition. CML-BSA increased the protein level of hypoxia-inducible factor-1α (HIF-1α), and the EMT-associated transcription factor, TWIST. These effects were reversed by α-lipoic acid. CML-BSA increased the protein levels of mesenchymal-specific markers, including vimentin, α-smooth muscle actin, which were alleviated by pre-treatment with α-lipoic acid. Our data suggest that CML-BSA induces EMT through a ROS/HIF-1α/TWIST-dependent mechanism, and that α-lipoic acid may alleviate the CML-induced EMT in renal tubular cells.

Iron or oxygen regulates the stability of hypoxia inducible factor-1α (HIF-1α). We investigated whether ferrous glycinate would affect HIF-1α accumulation, aerobic glycolysis and mitochondrial energy metabolism in human A549 lung cancer cells. Incubation of A549 cells with ferrous glycinate decreased the protein levels of HIF-1α, which was abrogated by proteosome inhibitor, or prolyl hydroxylase inhibitor. The addition of ferrous glycinate decreased protein levels of glucose transporter-1, hexokinase-2, and lactate dehydrogenase A, and decreased pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase (PDH) phosphorylation in A549 cells. Ferrous glycinate also increased the expression of the mitochondrial transcription factor A (TFAM), and the mitochondrial protein, cytochrome c oxidase (COX-IV). Silencing of HIF-1α expression mimicked the effects of ferrous glycinate on PDK-1, PDH, TFAM and COX-IV in A549 cells. Ferrous glycinate increased mitochondrial membrane potential and ATP production in A549 cells. These results suggest that ferrous glycinate may reverse Warburg effect through down regulating HIF-1α in A549 cells.

Purpose Most of the existing studies on postcoital bleeding (PCB) in Western countries. To date, no study has focused on the various PCB-related comorbidities in Taiwan women. This work aims to analyze and compare the presence or absence of PCB among Taiwanese women with gynecological comorbidity. Methods This study is a population-based retrospective cohort investigation. Outpatients with PCB after the index date were considered. A total of 2377 female patients with PCB (ICD-9 626.7) were identified using a nationwide outpatient sample from 2001 to 2010. For comparison, 7131 cases were randomly matched with the study group in terms of gender and age. Results The PCB incidence rate was 39–59 cases/100,000 Taiwanese women, with mean age (±SD) of 36.74 ± 10.79 years, median age of 36 years, and mode age of 29 years. Women with PCB exhibited 1.47-fold risk of cervical dysplasia and 1.59-fold risk for malignant neoplasm of cervix. Young women with PCB showed high risk of cervical cancer. The most common benign diseases among PCB- related comorbidities were cervical erosion and ectropion (20.66%), followed by vaginitis and vulvovaginitis (19.18%). Comparison between PCB and non-PCB groups indicated several significant high-risk comorbidities including cervical polyps, cervical erosion, leukoplakia of cervix, intrauterine contraceptive device, cervicitis, vaginitis, menopause, dyspareunia, and vulvodynia. Conclusions This study provides evidence that PCB-related comorbidities manifested benign diseases (51.58%), lower genital tract infection (46.11%), and cervical cancer (2.31%). Thus, healthcare providers must ensure that appropriate routine screening tests and counseling are given to women with PCB.

Denervation-mediated skeletal muscle atrophy results from the loss of electric stimulation and leads to protein degradation, which is critically regulated by the well-confirmed transcriptional co-activator peroxisome proliferator co-activator 1 alpha (PGC-1α). No adequate treatments of muscle wasting are available. Pyrroloquinoline quinone (PQQ), a naturally occurring antioxidant component with multiple functions including mitochondrial modulation, demonstrates the ability to protect against muscle dysfunction. However, it remains unclear whether PQQ enhances PGC-1α activation and resists skeletal muscle atrophy in mice subjected to a denervation operation. This work investigates the expression of PGC-1α and mitochondrial function in the skeletal muscle of denervated mice administered PQQ. The C57BL6/J mouse was subjected to a hindlimb sciatic axotomy. A PQQ-containing ALZET® osmotic pump (equivalent to 4.5 mg/day/kg b.w.) was implanted subcutaneously into the right lower abdomen of the mouse. In the time course study, the mouse was sacrificed and the gastrocnemius muscle was prepared for further myopathological staining, energy metabolism analysis, western blotting, and real-time quantitative PCR studies. We observed that PQQ administration abolished the denervation-induced decrease in muscle mass and reduced mitochondrial activities, as evidenced by the reduced fiber size and the decreased expression of cytochrome c oxidase and NADH-tetrazolium reductase. Bioenergetic analysis demonstrated that PQQ reprogrammed the denervation-induced increase in the mitochondrial oxygen consumption rate (OCR) and led to an increase in the extracellular acidification rate (ECAR), a measurement of the glycolytic metabolism. The protein levels of PGC-1α and the electron transport chain (ETC) complexes were also increased by treatment with PQQ. Furthermore, PQQ administration highly enhanced the expression of oxidative fibers and maintained the type II glycolytic fibers. This pre-clinical in vivo study suggests that PQQ may provide a potent therapeutic benefit for the treatment of denervation-induced atrophy by activating PGC-1α and maintaining the mitochondrial ETC complex in skeletal muscles.

N^ε-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic β-cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in the cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, the effects of α-lipoic acid (ALA) on mitochondria in AGE-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 wk increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxide production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells. Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 protein suggest that fragmented mitochondria were associated with increased mitophagic activity, and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic β-cells. The findings from this study suggest that increased concentration of AGEs may damage β-cells and reduce insulin secretion.

A shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis is the biochemical hallmark of malignant cancer cells. In the present study, we demonstrated that Nodal stimulated the expression of glycolytic enzymes and decreased reliance on mitochondrial oxidative phosphorylation in human glioma cancer cells. The shift in glucose metabolism was mediated by induction of the hypoxia-inducible factor (HIF). Nodal protein expression was shown to be correlated with expression levels of glucose transporter (Glut)–1, hexokinase (HK)-II, pyruvate dehydrogenase kinase (PDK)–1, the phosphorylation level of pyruvate dehydrogenase (PDH), glucose uptake, and lactate accumulation in human glioma cells. These effects were inversely correlated with mitochondrial oxygen consumption and ATP production. Knockdown of Nodal expression with specific small hairpin RNA reduced Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation. Nodal knockdown also reduced glucose uptake and lactate generation, which in turn increased mitochondrial membrane potential (Ψ), O₂ utilization, and ATP synthesis. The ectopic expression of Nodal in low-expressing Nodal glioma cells resulted in the opposite results compared with those of Nodal knockdown glioma cells. Treatment of cells with recombinant Nodal increased HIF-1 expression, and this effect was regulated at the transcriptional level. Blockage of the Nodal receptor by a pharmacological inhibitor or Nodal knockdown in U87MG cells decreased HIF-1α expression. Furthermore, HIF-1α knockdown in U87MG cells decreased Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation, which were similar to results in Nodal knockdown cells. Taken together, these results suggest that Nodal affects energy metabolism through HIF-1α.

Monacolin K is the secondary metabolite isolated from Monascus spp. It is the natural form of lovastatin, which is clinically used to reduce the synthesis of cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. In the present study, monacolin K increased protein expression of SIRT1 and phosphorylation level of AMP-activated protein kinase (AMPK) in HepG2 cells. Through activation of SIRT1/AMPK pathway, monacolin K increased phosphorylation of acetyl CoA carboxylase and caused nuclear translocation of forkhead box O1. The western blotting results showed that monacolin K increased expression of adipose triglyceride lipase but decreased abundances of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP1). Monacolin K also decreased the intracellular accumulation of lipids as demonstrated by Oil Red O staining. In addition, the immunostaining showed that monacolin K prevented the nuclear translocation of SREBP1, indicating the association with down-regulation of FAS. All the demonstrated effects of monacolin K were counteracted by nicotinamide or compound C, the inhibitors of SIRT1 or AMPK. In summary, monacolin K reduces the lipid content through SIRT1/AMPK pathway in HepG2 cells, which promotes catabolism and inhibits anabolism of lipid.

Aims/hypothesis Sirtuin 1 (SIRT1) is a longevity-associated protein, which regulates energy metabolism and lifespan in response to nutrient deprivation. It has been proposed to be a therapeutic target for obesity and metabolic syndrome. We investigated whether α-lipoic acid (ALA) exerts a lipid-lowering effect through regulation of SIRT1 activation and production in C₂C₁₂ myotubes. Methods ALA-stimulated AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), adipose triacylglycerol lipase (ATGL) and fatty acid synthase (FAS) production, as well as intracellular triacylglycerol accumulation and fatty acid β-oxidation were analysed in the absence or presence of a SIRT1 inhibitor (nicotinamide), SIRT1 small interfering (si) RNA and an AMPK inhibitor (compound C) in C₂C₁₂ myotubes. Mice with streptozotocin/nicotinamide-induced diabetes and db/db mice fed on a high-fat diet were used to study the ALA-mediated lipid-lowering effects in vivo. Results ALA increased the NAD⁺/NADH ratio to enhance SIRT1 activity and production in C₂C₁₂ myotubes. ALA subsequently increased AMPK and ACC phosphorylation, leading to increased palmitate β-oxidation and decreased intracellular triacylglycerol accumulation in C₂C₁₂ myotubes. In cells treated with nicotinamide or transfected with SIRT1 siRNA, ALA-mediated AMPK/ACC phosphorylation, intracellular triacylglycerol accumulation and palmitate β-oxidation were reduced, suggesting that SIRT1 is an upstream regulator of AMPK. ALA increased ATGL and suppressed FAS protein production in C₂C₁₂ myotubes. Oral administration of ALA in diabetic mice fed on a high-fat diet and db/db mice dramatically reduced the body weight and visceral fat content. Conclusions/interpretation ALA activates both SIRT1 and AMPK, which leads to lipid-lowering effects in vitro and in vivo. These findings suggest that ALA may have beneficial effects in the treatment of dyslipidaemia and obesity.

Background: Invasiveness and metastasis are the most common characteristics of non small cell lung cancer (NSCLC) and causes of tumour-related morbidity and mortality. Mitogen-activated protein kinases (MAPKs) signalling pathways have been shown to play critical roles in tumorigenesis. However, the precise pathological role(s) of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cancers has been controversial such that the up-regulation of MKP-1 in different cancers does not always correlate to a better prognosis. In this study, we showed that the induction of MKP-1 lead to a significant retardation of proliferation and metastasis in NSCLC cells. We also established that rosiglitazone (a PPARγ agonist) elevated MKP-1 expression level in NSCLC cells and inhibited tumour metastasis. Methods: Both wildtype and dominant negative forms of MKP-1 were constitutively expressed in NSCLC cell line H441GL. The migration and invasion abilities of these cells were examined in vitro. MKP-1 modulating agents such as rosiglitazone and triptolide were used to demonstrate MKP-1's role in tumorigenesis. Bioluminescent imaging was utilized to study tumorigenesis of MKP-1 over-expressing H441GL cells and anti-metastatic effect of rosiglitazone. Results: Over-expression of MKP-1 reduced NSCLC cell proliferation rate as well as cell invasive and migratory abilities, evident by the reduced expression levels of MMP-2 and CXCR4. Mice inoculated with MKP-1 over-expressing H441 cells did not develop NSCLC while their control wildtype H441 inoculated littermates developed NSCLC and bone metastasis. Pharmacologically, rosiglitazone, a peroxisome proliferator activated receptor-γ (PPARγ) agonist appeared to induce MKP-1 expression while reduce MMP-2 and CXCR4 expression. H441GL-inoculated mice receiving daily oral rosiglitazone treatment demonstrated a significant inhibition of bone metastasis when compared to mice receiving sham treatment. We found that rosiglitazone treatment impeded the ability of cell migration and invasion in vitro. Cells pre-treated with triptolide (a MKP-1 inhibitor), reversed rosiglitazone-mediated cell invasion and migration. Conclusion: The induction of MKP-1 could significantly suppress the proliferative and metastatic abilities of NSCLC both in vitro and in vivo. Therefore, MKP-1 could be considered as a potential therapeutic target in NSCLC therapy and PPARγ agonists could be explored for combined chemotherapy.

Human malignant glioma cells are characterized by local invasion. In the present study, we investigated the role of osteopontin (OPN) in the invasiveness of human glioma cells isolated from grade IV tumors. We found that the expression levels of OPN in these cell lines paralleled matrix metalloproteinase-2 (MMP-2) expression and cell invasiveness potential. When U87MG glioma cells (with a high-OPN expression level) were stably transformed with specific small hairpin RNA to knock down OPN expression, MMP-2 secretion, cell invasiveness, and tumor growth in implanted brains were dramatically reduced. Conversely, forced expression of OPN in GBM-SKH glioma cells (which expressed OPN at a low level) increased MMP-2 secretion, enhanced cell invasiveness, and increased tumor growth in a rodent xenograft model. Expression of OPN was associated with increased expression of vimentin and decreased expression of glial fibrillary acidic protein. Treatment of glioma cells with 5-aza-2′-deoxycytidine (5-aza-dC) suppressed OPN expression in a concentration-dependent manner. Suppression of OPN expression by 5-aza-dC was associated with reductions in MMP-2 secretion, vimentin expression, cell invasion, intravasation, and tumor growth. These data suggest that OPN may play important roles in regulating cell invasion in glioma cells and that 5-aza-dC may serve as a therapeutic agent for human gliomas.