BNIP3-Dependent Mitophagy via PGC1α Promotes Cartilage Degradation.

Since mitochondria are suggested to be important regulators in maintaining cartilage homeostasis, turnover of mitochondria through mitochondrial biogenesis and mitochondrial degradation may play an important role in the pathogenesis of osteoarthritis (OA). Here, we found that mitochondrial dysfunction is closely associated with OA pathogenesis and identified the peroxisome proliferator-activated receptor-gamma co-activator 1-alpha (PGC1α) as a potent regulator. The expression level of PGC1α was significantly decreased under OA conditions, and knockdown of PGC1α dramatically elevated the cartilage degradation by upregulating cartilage degrading enzymes and apoptotic cell death. Interestingly, the knockdown of PGC1α activated the parkin RBR E3 ubiquitin protein ligase (PRKN)-independent selective mitochondria autophagy (mitophagy) pathway through the upregulation of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3). The overexpression of BNIP3 stimulated mitophagy and cartilage degradation by upregulating cartilage-degrading enzymes and chondrocyte death. We identified microRNA (miR)-126-5p as an upstream regulator for PGC1α and confirmed the direct binding between miR-126-5p and 3' untranslated region (UTR) of PGC1α. An in vivo OA mouse model induced by the destabilization of medial meniscus (DMM) surgery, and the delivery of antago-miR-126 via intra-articular injection significantly decreased cartilage degradation. In sum, the loss of PGC1α in chondrocytes due to upregulation of miR-126-5p during OA pathogenesis resulted in the activation of PRKN-independent mitophagy through the upregulation of BNIP3 and stimulated cartilage degradation and apoptotic death of chondrocytes. Therefore, the regulation of PGC1α:BNIP3 mitophagy axis could be of therapeutic benefit to cartilage-degrading diseases.

Suppression of PGC-1α Drives Metabolic Dysfunction in TGFß2-Induced EMT of Retinal Pigment Epithelial Cells.

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFß2) suppressed PGC-1α expression. Correspondingly, TGFß2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFß2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFß2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFß2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFß2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.

Andrographolide modulates HNF4α activity imparting on hepatic metabolism.

Hepatic nuclear factor 4 alpha (HNF4α) drives the expression of apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B), governing hepatic very-low-density lipoprotein (VLDL) production and secretion. Andrographolide (AP) is a major constituent isolated from Andrographis paniculata. We found that AP can disrupt the interaction between HNF4α and its coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Virtual docking and mutational analysis indicated that arginine 235 of HNF4α is essential for binding to AP. As a consequence of antagonizing the activity of HNF4α, AP suppresses the expression of ApoB, MTP and PLA2G12B and reduces the rate of hepatic VLDL secretion in vivo. AP additionally reduced gluconeogenesis via down-regulating the expression of HNF4α target genes phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pc). Collectively, our results suggest that AP affects liver function via modulating the transcriptional activity of HNF4α.

Peroxisome proliferator-activated receptor-γ coactivator 1α-mediated pathway as a possible therapeutic target in endometriosis.

STUDY QUESTION: Is a peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)-mediated pathway involved in the development of endometriosis? SUMMARY ANSWER: PGC-1α plays critical roles in inflammation and cell proliferation of endometriotic tissues and may be involved in the development of endometriosis. WHAT IS KNOWN ALREADY: Expression levels of PGC-1α are higher in ovarian endometrioma (OE) than normal endometrium (NE). PGC-1α also stimulates aromatase activity and promotes local estrogen biosynthesis in OE. STUDY DESIGN, SIZE, DURATION: This is a case-controlled biological study using endometrial cells and tissues derived from 23 women with, and 10 women without, OE. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ectopic endometriotic and eutopic endometrial stromal cells (SCs) were isolated and maintained in culture. PGC-1α was either overexpressed in the cells or knocked down using siRNA. The expression of PGC-1α and other factors during endometriosis was examined using real-time PCR and western blotting, cell proliferation was measured using Cell Counting Kit-8 (WST-8) assays and transcriptional activity was assessed using luciferase reporter assays. MAIN RESULTS AND THE ROLE OF CHANCE: PGC-1α overexpression promoted the proliferation of OESCs in a time-dependent manner (P < 0.01 versus control) but not NESCs. PGC-1α stimulated aromatase (P < 0.01 versus control) and interleukin (IL)-6/IL-8 mRNA expression levels (P < 0.05 versus control for each) and led to inhibitor kappa B phosphorylation protein expression and upregulation of the apoptosis inhibitors X-linked inhibitor of apoptosis protein and survivin at mRNA level (P < 0.05 versus control for each). HX531, a selective retinoid-X receptor-α (RXRα) antagonist, suppressed the PGC-1α-induced cell proliferation (P < 0.05 versus control), aromatase/IL-6/IL-8/survivin mRNA expression (P < 0.05 versus control for each) and transcription reporter activity of PGC-1α in a dose-dependent manner (P < 0.01 versus control). Moreover, HX531 downregulated PGC-1α-induced aromatase-promoter PI.3-II transcripts in OESCs, and PGC-1α knockdown reduced aromatase, IL-6/IL-8 and antiapoptotic factors mRNA expression (P < 0.05 versus control for each). Notably, the Histogram score, which was used for quantifying RXRα status, was markedly higher in OE than in NE tissue (P < 0.01). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Only OE tissues were included in this study, while peritoneal and deep infiltrating endometriotic tissues were not. Therefore, these findings might not be generalized to other types of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: In OESC, PGC-1α stimulated cell proliferation and was involved in local estrogen biosynthesis, inflammation and apoptosis, and these effects of PGC-1α were inhibited by HX531. The suppression of PGC-1α-induced proliferation by HX531 in OESCs but not NESCs suggests that the PGC-1α-RXRα axis could play critical roles in promoting endometriosis. This is the first report of a relationship between PGC-1α and inhibitor of apoptosis proteins in endometriosis. Based on these findings, the PGC-1α-mediated pathway could represent a potential target in molecular therapy of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): The study is supported in part by Grants-in-Aid for Scientific Research (15 K10681 and 15 K10726) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan). The authors have no conflicts of interest to disclose.

Shikonin exerts antitumor activity by causing mitochondrial dysfunction in hepatocellular carcinoma through PKM2-AMPK-PGC1α signaling pathway.

Shikonin, a naphthoquinone derivative isolated from the root of Lithospermum erythrorhizon, exhibits broad-spectrum antitumor activity via different molecular mechanisms. In this study, we investigated the effect of shikonin on mitochondrial dysfunction in hepatocellular carcinoma (HCC). Our results showed that shikonin inhibited the proliferation, migration, and invasiveness of HCCLM3 cells, and promoted cell apoptosis in a dose-dependent manner. More importantly, shikonin affected mitochondrial function by disrupting mitochondrial membrane potential and oxidative stress (OS) status. Furthermore, shikonin decreased the oxygen consumption rate of HCCLM3 cells, as well as the levels of ATP and metabolites involved in the tricarboxylic acid cycle (TCA cycle). We also investigated the molecular mechanisms underlying the regulation of mitochondrial function by shikonin as an inhibitor of PKM2. Shikonin decreased the expression of PKM2 in the mitochondria and affected other metabolic pathways (AMPK and PGC1α pathways), which aggravated the oxidative stress and nutrient deficiency. Our results indicate a novel role of shikonin in triggering mitochondria dysfunction via the PKM2-AMPK-PGC1α signaling pathway and provide a promising therapeutic approach for the treatment of HCC.

Mutual Antagonism of PINK1/Parkin and PGC-1α Contributes to Maintenance of Mitochondrial Homeostasis in Rotenone-Induced Neurotoxicity.

Parkinson's disease (PD) is a progressive, selective, and age-related neurodegenerative disease. The pathogenic focus of PD is mitochondrial dysfunction. When mitochondrial homeostasis was damaged, it can lead to reactive oxygen species formation to further accelerate the accumulation of dysfunctional mitochondria, resulting in a vicious cycle harmful to the neuron. PINK1 and Parkin, two proteins that are linked to PD, play vital roles in mitophagy, which was very important in maintaining mitochondrial homeostasis. Thus, at present, we explored mitochondrial biogenesis, mitophagy, and fission/fusion in rotenone-induced dopamine neurotoxicity. In particular, we focused on interactions between the PINK1/Parkin pathway and PGC-1α in the regulation of mitochondrial homeostasis impairment. The results indicated that both the autophagy and mitophagy levels increased significantly and were accompanied by altered levels of PINK1/Parkin proteins in rotenone-induced neurotoxicity. PINK1 influenced mitochondrial biogenesis by inhibiting PGC-1α and mtTFA protein expression as well as the mtDNA copy number. PGC-1α, in turn, inhibited PINK1/Parkin protein expression and the mitophagy levels. Furthermore, the results demonstrated that PINK1 influenced mitochondrial fission/fusion by regulating MFN2 and phosphorylating Drp1. In summary, mutual antagonism of the PINK1/Parkin pathway and PGC-1α formed a balance that regulated mitochondrial biogenesis, fission/fusion, and mitophagy. These effects contributed to the maintenance of mitochondrial homeostasis in rotenone-induced neurotoxicity.

PGC-1α Participates in the Protective Effect of Chronic Intermittent Hypobaric Hypoxia on Cardiomyocytes.

BACKGROUND/AIMS: Myocardial ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injury is always characterized by Ca2+ overload, energy metabolism disorder and necrocytosis of cardiomyocytes. We showed previously that chronic intermittent hypobaric hypoxia (CIHH) improves cardiac function during I/R through improving cardiac glucose metabolism. However, the underlying cellular and molecular mechanisms of CIHH treatment improving energy metabolism in cardiomyocytes are still unclear. In this study, we determined whether and how CIHH protects cardiomyocytes from Ca2+ overload and necrocytosis through energy regulating pathway. METHODS: Adult male Sprague-Dawley rats were randomly divided into two groups: control (CON) and CIHH group. CIHH rats received a hypobaric hypoxia simulating 5,000-m altitude for 28 days, 6 hours each day, in hypobaric chamber. Rat ventricular myocytes were obtained by enzymatic dissociation. The intracellular calcium concentration ([Ca2+]i) and cTnI protein expression were used to evaluate the degree of cardiomyocytes injury during and after H/R. The mRNA and protein expressions involved in cardiac energy metabolism were determined using quantitative PCR and Western blot techniques. PGC-1α siRNA adenovirus transfection was used to knock down PGC-1α gene expression of cardiomyocytes to determine the effect of PGC-1α in the energy regulating pathway. RESULTS: H/R increased [Ca2+]i and cTnI protein expression in cardiomyocytes. CIHH treatment decreased [Ca2+]i (p< 0.01) and cTnI protein expression (p< 0.01) in cardiomyocytes after H/R. Both mRNA and protein expression of PGC-1α increased after CIHH treatment, which was reversed by PGC-1α siRNA adenovirus transfection. Furthermore, CIHH treatment increased the expression of HIF-1α, AMPK and p-AMPK in cardiomyocytes, and pretreatment with AMPK inhibitor dorsomorphin abolished the enhancement of PGC-1α protein expression in cardiomyocytes by CIHH (p< 0.01). In addition, PGC-1α knock down also abolished the increased protein level of GLUT4 (p< 0.01) and decreased the protein level of CPT-1b (p< 0.05) in cardiomyocytes by CIHH treatment. CONCLUSION: CIHH treatment could reduce the calcium overload and H/R injury in cardiomyocytes by up-regulating the expression of PGC-1α and regulating the energy metabolism of glucose and lipid. The HIF-1α-AMPK signaling pathway might be involved in the process.

Resistin destroys mitochondrial biogenesis by inhibiting the PGC-1α/ NRF1/TFAM signaling pathway.

Mitochondrial biogenesis deficits in neuronal cells are associated with the pathological progression of neurodegenerative diseases. Resistin, a secretory adipocytokine, possesses multiple physiological functions in diverse cells and tissues. However, the effects of resistin on mitochondrial biogenesis in neuronal cells are still elusive. In the current study, we found that resistin caused a sustainable decrease in mitochondrial contents, including mitochondrial DNA/nuclear DNA ratio (mtDNA/nDNA), mitochondrial mass, cytochrome b protein expression, and cytochrome c oxidase activity, which were correlated with "loss of mitochondrial function" including reduced mitochondrial respiration rate and ATP production in human SH-SY5Y neuronal cells. Indeed, resistin treatment destroyed the expression of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis, as well as its downstream target genes including nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM). Notably, overexpression of PGC-1α could completely rescue mitochondrial biogenesis and mitochondrial deficits induced by resistin. Mechanistically, inhibition of 5'-adenosine monophosphate-activated protein kinase (AMPK) was shown to mediate the inhibitory effects of resistin on mitochondrial biogenesis.

AMPK/PGC1α activation by melatonin attenuates acute doxorubicin cardiotoxicity via alleviating mitochondrial oxidative damage and apoptosis.

Doxorubicin (DOX) is a highly effective anticancer anthracycline drug, but its side effects at the level of the heart has limited its widespread clinical application. Melatonin is a documented potent antioxidant, nontoxic and cardioprotective agent, and it is involved in maintaining mitochondrial homeostasis and function. The present study established acute DOX-induced cardiotoxicity models in both H9c2 cells incubated with 1 µM DOX and C57BL/6 mice treated with DOX (20 mg/kg cumulative dose). Melatonin markedly alleviated the DOX-induced acute cardiac dysfunction and myocardial injury. Both in vivo and in vitro studies verified that melatonin inhibited DOX-induced mitochondrial dysfunction and morphological disorders, apoptosis, and oxidative stress via the activation of AMPK and upregulation of PGC1α with its downstream signaling (NRF1, TFAM and UCP2). These effects were reversed by the use of AMPK siRNA or PGC1α siRNA in H9c2 cells, and were also negated by the cotreatment with AMPK inhibitor Compound C in vivo. Moreover, PGC1α knockdown was without effect on the AMPK phosphorylation induced by melatonin in the DOX treated H9c2 cells. Therefore, AMPK/PGC1α pathway activation may represent a new mechanism for melatonin exerted protection against acute DOX cardiotoxicity through preservation of mitochondrial homeostasis and alleviation of oxidative stress and apoptosis.

Swainsonine induces apoptosis of rat cardiomyocytes via mitochondria­mediated pathway.

Swainsonine is an Astragalus membranaceus extract. It is indole, alkaloid, and soluble in water. Its effect on rat cardiomyocytes apoptosis, and the mechanisms underlying that effect, were investigated by inducing apoptosis in H9c2 cells. This was detected by MTT assay, Annexin V-FITC/propidium iodide double staining and western blotting. Flow cytometry and fluorescence microscopy were used to confirm swainsonine's effect on mitochondrial membrane potential and levels of reactive oxygen species, while an ATP-dependent bioluminescence assay kit served to find the ATP contents. Assessment was also carried out for peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) expression levels as well as those of such apoptosis-associated proteins as Cytochrome c, Caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). Overall, indications were that swainsonine may have the potential to inhibit viability of cells, decrease expression of PGC-1α, induce mitochondrial dysfunction, upregulate Cytochrome c, Bax and Caspase-3, and downregulate Bcl-2. The suggestion would be that apoptosis may be induced through signalling pathways in H9c2 cells mediated by mitochondria.

Mutant p53 controls tumor metabolism and metastasis by regulating PGC-1α.

Mutant forms of p53 protein often possess protumorigenic functions, conferring increased survival and migration to tumor cells via their "gain-of-function" activity. Whether and how a common polymorphism in TP53 at amino acid 72 (Pro72Arg; referred to here as P72 and R72) impacts this gain of function has not been determined. We show that mutant p53 enhances migration and metastasis of tumors through the ability to bind and regulate PGC-1α and that this regulation is markedly impacted by the codon 72 polymorphism. Tumor cells with the R72 variant of mutant p53 show increased PGC-1α function along with greatly increased mitochondrial function and metastatic capability. Breast cancers containing mutant p53 and the R72 variant show poorer prognosis compared with P72. The combined results reveal PGC-1α as a novel "gain-of-function" partner of mutant p53 and indicate that the codon 72 polymorphism influences the impact of mutant p53 on metabolism and metastasis.

MicroRNA-148a deficiency promotes hepatic lipid metabolism and hepatocarcinogenesis in mice.

miRNAs are involved in many physiologic and disease processes by virtue of degrading specific mRNAs or inhibiting their translation. miR-148a has been implicated in the control of tumor growth and cholesterol and triglyceride homeostasis using in vitro or in vivo gene expression- and silencing-based approaches. Here miR-148a knockout (KO) mice were used to investigate the intrinsic role of miR-148a in liver physiology and hepatocarcinogenesis in mice. miR-148a downregulation was found to be correlated with poor clinical outcomes in hepatocellular carcinoma (HCC) patients. Under regular chow diet (RCD) or high fat diet (HFD), miR-148a deletion significantly accelerated DEN-induced hepatocarcinogenesis in mice. Mechanistically, miR-148a deletion promotes lipid metabolic disorders in mice. Moreover, restoration of miR-148a reversed these defects. Finally, miR-148a was found to directly inhibit several key regulators of hepatocarcinogenesis and lipid metabolism. These findings reveal crucial roles for miR-148a in the hepatic lipid metabolism and hepatocarcinogenesis. They further identify miR-148a as a potential therapeutic target for certain liver diseases, including cancer.

Endoplasmic Reticulum Stress-Induced CHOP Inhibits PGC-1α and Causes Mitochondrial Dysfunction in Diabetic Embryopathy.

Endoplasmic reticulum (ER) stress has been implicated in the development of maternal diabetes-induced neural tube defects (NTDs). ER stress-induced C/EBP homologous protein (CHOP) plays an important role in the pro-apoptotic execution pathways. However, the molecular mechanism underlying ER stress- and CHOP-induced neuroepithelium cell apoptosis in diabetic embryopathy is still unclear. Deletion of the Chop gene significantly reduced maternal diabetes-induced NTDs. CHOP deficiency abrogated maternal diabetes-induced mitochondrial dysfunction and neuroepithelium cell apoptosis. Further analysis demonstrated that CHOP repressed the expression of peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α), an essential regulator for mitochondrial biogenesis and function. Both CHOP deficiency in vivo and knockdown in vitro restore high glucose-suppressed PGC-1α expression. In contrast, CHOP overexpression mimicked inhibition of PGC-1α by high glucose. In response to the ER stress inducer tunicamycin, PGC-1α expression was decreased, whereas the ER stress inhibitor 4-phenylbutyric acid blocked high glucose-suppressed PGC-1α expression. Moreover, maternal diabetes in vivo and high glucose in vitro promoted the interaction between CHOP and the PGC-1α transcriptional regulator CCAAT/enhancer binding protein-ß (C/EBPß), and reduced C/EBPß binding to the PGC-1α promoter leading to markedly decrease in PGC-1α expression. Together, our findings support the hypothesis that maternal diabetes-induced ER stress increases CHOP expression which represses PGC-1α through suppressing the C/EBPß transcriptional activity, subsequently induces mitochondrial dysfunction and ultimately results in NTDs.

Supranutritional selenium intake from enriched milk casein impairs hepatic insulin sensitivity via attenuated IRS/PI3K/AKT signaling and decreased PGC-1α expression in male Sprague-Dawley rats.

Selenium (Se)-enriched milk provides antioxidant benefits and has therapeutic potential against cancer. However, both antidiabetic and prodiabetic effects have been attributed to Se. Our objective was to evaluate the effect of Se-enriched milk casein on insulin sensitivity in rats when given at the requirement of 0.25 ppm Se and supranutritionally on both low- and high-fat diets. Two hundred sixteen male Sprague-Dawley rats were fed low- or high-fat diets containing one, two or eight times the Se requirement in a randomized block design. After 7 weeks, 72 rats were subjected to the hyperinsulinemic-euglycemic clamp with [3-3H]glucose infusion to estimate glucose fluxes. Tissues were collected from the remaining 144 rats 8 min after ip saline or insulin injection. During hyperinsulinemic-euglycemic clamps, glucose infusion rate was 22% lower (P=.058), and endogenous glucose production was 76% higher (P=.054) when Se content increased from one to eight times the requirement on low-fat diets, indicating impaired hepatic insulin sensitivity. Se also decreased the ability for insulin to stimulate Akt phosphorylation at Thr308. Hepatic oxidation state and expression of selenoprotein P and glutathione peroxidase-1 were unaffected while expression of insulin receptor substrate (IRS)-1 and-2 and PPARγ coactivator-1α (PGC-1α) decreased with supranutritional Se and high-fat intake. In addition, hepatic expression of regulatory and catalytic subunits of phosphatidylinositol 3-kinase (PI3K) decreased with supranutritional intake of Se. Se intake from enriched casein up to eight times the requirement impairs hepatic insulin sensitivity in a mechanism similar to fat feeding, via attenuated IRS/PI3K/Akt signaling and decreased PGC-1α expression.

Leucine supplementation at the onset of high-fat feeding does not prevent weight gain or improve glycemic regulation in male Sprague-Dawley rats.

Obesity is a major public health concern and it is essential to identify effective treatments and preventative strategies to stop continued increases in obesity rates. The potential functional roles of the branched chain amino acid leucine make this amino acid an attractive candidate for the treatment and/or prevention of obesity. The objective of this study was to determine if long-term leucine supplementation could prevent the development of obesity and reduce the risk factors for chronic disease in rats fed a high-fat (60 % fat) diet. Male Sprague-Dawley rats (n = 30 per dietary treatment) were meal-fed (3 meals/day) either a control, low-fat diet (LF), control + leucine (LFL), high-fat (HF), or high-fat + leucine (HFL) for 42 days. On day 42, rats were sacrificed at 0, 30, or 90 min postprandial. Animals fed the HF and HFL diets had higher (P < 0.05) final body weights and weight gain compared to animals fed the LF and LFL diets. Leucine supplementation increased epididymal fat mass (P < 0.05) and decreased muscle mass (P < 0.05). There was no effect of leucine supplementation on postprandial glucose or insulin response. However, there was a significant effect (P < 0.05) of diet and time on free fatty acid concentrations. There was no effect of leucine on muscle markers of protein synthesis (4E-BP1, p70S6K) or energy metabolism (Akt, AMPK). Leucine supplementation decreased (P < 0.05) PGC1α expression and increased (P < 0.05) PPARγ expression in skeletal muscle. In conclusion, long-term leucine supplementation does not prevent weight gain, improve body composition, or improve glycemic control in rats fed a high-fat diet.

PPAR-α Agonist WY-14643 Inhibits LPS-Induced Inflammation in Synovial Fibroblasts via NF-kB Pathway.

Osteoarthritis (OA), the most prevalent form of arthritis that results from breakdown of joint cartilage and underlying bone, has been viewed as a chronic condition manifested by persistence of inflammatory responses and infiltration of lymphocytes. Regulation of the inflammatory responses in synovial fibroblasts might be useful to prevent the development and deterioration of osteoarthritis. WY-14643, a potent peroxisome proliferator activator receptor-α (PPAR-α) agonist, has been described to beneficially regulate inflammation in many mammalian cells. Here, we investigate the potential anti-inflammatory role of WY-14643 in lipopolysaccharide (LPS)-induced synovial fibroblasts. WY-14643 greatly inhibited the production of NO and PGE2 induced by LPS. In addition, the mRNA expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1), and tissue factor (TF) was significantly suppressed by WY-14643, as well as the secretion of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1). Furthermore, the transcription activity and nuclear translocation of NF-kB were found to be markedly decreased by WY-14643, while the phosphorylation of IkB was enhanced, indicating that the anti-inflammatory role of WY-14643 was meditated by NF-kB-dependent pathway. The application of WY-14643 failed to carry out its anti-inflammatory function in PPAR-α silenced cells, suggesting the role of PPAR-α. These findings may facilitate further studies investigating the translation of pharmacological PPAR-α activation into clinical therapy of OA.

Protective effects of PGC-1α via the mitochondrial pathway in rat brains after intracerebral hemorrhage.

UNLABELLED: Peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) is a transcriptional co-activator that coordinately regulates genes required for mitochondrial biogenesis, which stimulates mitochondrial activity. It is also a major factor in the up-regulation of antioxidant activities that are a response to oxidative stress. However, the role of PGC-1α after intracerebral hemorrhage (ICH) has not been studied. The purpose of the present work was to investigate the effects and mechanism of PGC-1α after ICH in the brain. Brain injury was induced by injecting autologous arterial blood (50µL) into the rat brain. PGC-1α siRNAs were injected into rat brains 24h prior to ICH. Then, 72h after ICH, brains were collected for investigation. Post-assessment included western blot analysis, RT-PCR assay, neurobehavioral function testing, measurement of brain water content, high-performance liquid chromatography (HPLC), and projection electron microscopy on ICH rat models. The concentration of PGC-1α was higher in the ipsilateral striatum after ICH, peaking around 72h after ICH. The expression of NRF-1, TFAM, SOD2, UCP2, mitochondrial DNA, ATP concentration, mitochondrial quantity, and brain water content were increased 72h after ICH. However, the neurological score was decreased 72h after ICH. Treatment with PGC-1α siRNAs significantly decreased the neurological score, ATP concentration, number of mitochondria, expression of NRF-1, TFAM, SOD2, UCP2, and mitochondrial DNA, and increased brain water content and formation of mitochondrial myelin layer structures. In conclusion, our data suggest that PGC-1α protects rat brains via a mitochondrial pathway following ICH. KEY WORDS: PGC-1α intracerebral hemorrhage(ICH); mitochondrial; neuroprotection.

MiR-485-3p and miR-485-5p suppress breast cancer cell metastasis by inhibiting PGC-1α expression.

Breast cancer is the worldwide leading cause of cancer mortality in women. The majority of deaths from breast cancer arise from metastasis of local tumors. Cancer cells support their rapid proliferation by diverting metabolites into anabolic pathways, but during cancer metastasis, the proliferative program of invasive cancer cells is suspended for a migratory phenotype. In this study, we demonstrated that both mature forms of miRNA-485, miR-485-3p and miR-485-5p were involved in regulating mitochondrial respiration, cell migration and cell invasion in breast cancer cells by directly targeting and inhibiting the expression of PGC-1α. Specifically, the expression levels of both miR-485-3p and miR-485-5p were decreased in breast cancer tissues. Overexpression of miR-485-3p and miR-485-5p suppressed mitochondrial respiration and potential for cell migration and invasion in vitro, and also inhibited spontaneous metastasis of breast cancer cells in vivo. The suppression of mitochondrial respiration and cell invasion could be partially relieved by restoration of PGC-1α expression.