High-resolution respirometry for evaluation of mitochondrial function on brain and heart homogenates in a rat model of cardiac arrest and cardiopulmonary resuscitation.

The physiology and physiopathology process of mitochondrial function following cardiac arrest remains poorly understood. We aimed to assess mitochondrial respiratory function on the heart and brain homogenates from cardiac arrest rats. The expression level of SIRT1/PGC-1α pathway was measured by immunoblotting. 30 rats were assigned to the CA group and the sham group. Rats of CA were subjected to 6 min of untreated ventricular fibrillation (VF) followed by 8 min of cardiopulmonary resuscitation (CPR). Mitochondrial respiratory function was compromised following CA and I/R injury, as indicated by CIL (451.46 ± 71.48 vs. 909.91 ± 5.51 pmol/min*mg for the heart and 464.14 ± 8.22 vs. 570.53 ± 56.33 pmol/min*mg for the brain), CI (564.04 ± 64.34 vs. 2729.52 ± 347.39 pmol/min*mg for the heart and 726.07 ± 85.78 vs. 1762.82 ± 262.04 pmol/min*mg for the brain), RCR (1.88 ± 0.46 vs. 3.57 ± 0.38 for the heart and 2.05 ± 0.19 vs. 3.49 ± 0.19, for the brain) and OXPHOS coupling efficiency (0.45 ± 0.11 vs. 0.72 ± 0.03 for the heart and 0.52 ± 0.05 vs. 0.71 ± 0.01 for the brain). However, routine respiration was lower in the heart and comparable in the brain after CA. CIV did not change in the heart but was enhanced in the brain. Furthermore, both SIRT1 and PGC-1α were downregulated concurrently in the heart and brain. The mitochondrial respiratory function was compromised following CA and I/R injury, and the major affected respiratory state is complex I-linked respiration. Furthermore, the heart and the brain respond differently to the global I/R injury after CA in mitochondrial respiratory function. Inhibition of the SIRT1/PGC-1α pathway may be a major contributor to the impaired mitochondrial respiratory function.

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.

Cardiac hypertrophy drives PGC-1α suppression associated with enhanced O-glycosylation.

The peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolism and is essential for normal cardiac function. Its activity is suppressed during pressure overload induced cardiac hypertrophy and such suppression at least partially contributes to the associated morbidity. The O-linked ß-N-acetylglucosamine post-translational modification (O-GlcNAc) of proteins is a glucose-derived metabolic signal. The relationship between O-GlcNAc, and PGC-1α activity in cardiac hypertrophy is unknown. We hypothesized that hypertrophy-induced suppression of PGC-1α was at least partially regulated by O-GlcNAc signaling. Treatment of neonatal rat cardiac myocytes with phenylephrine (an inducer of cardiomyocyte hypertrophy) significantly enhanced global O-GlcNAc signaling. Quantitative real-time PCR analysis revealed a downregulation of PGC-1α with concomitant suppression of fatty acid oxidation/mitochondrial genes. Transverse aortic constriction in mice decreased the basal expression of PGC-1α and its downstream genes. Reduction of O-GlcNAc signaling alleviated suppression of PGC-1α and most of its downstream genes. Interestingly, augmentation of O-GlcNAc signaling with glucosamine or PUGNAC (a O-GlcNAcase inhibitor) reduced glucose starvation-induced PGC-1α upregulation even in the absence of hypertrophy. Finally, we found that PGC-1α itself is O-GlcNAcylated. Together, these results reveal the recruitment of O-GlcNAc signaling as a potentially novel regulator of PGC-1α activity during cardiac hypertrophy. Furthermore, O-GlcNAc signaling may mediate constitutive suppression of PGC-1α activity in the heart. Such findings illuminate new possibilities regarding the inter-regulation of O-GlcNAc signaling and also may have some implications for metabolic dysregulation during cardiac diseases.

ß1-Adrenoceptor antibodies induce PPCM via inhibition of PGC-1α related pathway.

OBJECTIVES: Peripartum cardiomyopathy (PPCM) is a pregnancy-associated and life-threatening cardiac disease. However, the causes and pathogenesis are not fully understood. Accumulating studies show that cardiomyopathy often appears to be associated with elevated levels of ß1-adrenoceptor (ß1AR) antibodies, indicating a possible involvement of ß1AR antibodies in the development of PPCM. DESIGN: We injected the antigen peptide segment of the ß1AR into the postpartum Wistar rats to make the immune models and their cardiac function was detected by echocardiography. Also, the concentration of ß1AR antibodies and apoptosis rate of left ventricular myocytes was tested by SA-ELISA, TUNEL, HE staining, qRT-PCR and western blot methods. Finally, the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its related proteins were examined by qRT-PCR and western blot methods. RESULTS: We found that the level of ß1AR antibodies in the serum was significantly increased and the postpartum rats exhibited symptoms of PPCM after autoimmunity. Moreover, the expression of peroxisome PGC-1α, which was a master regulator of mitochondrial metabolism, and its downstream transcript vascular endothelial growth factor (VEGF), was decreased in autoimmune perinatal rats. In addition, the expression of the apoptosis factor caspase 3 as well as the apoptosis rate of left ventricular myocytes was significantly increased. CONCLUSIONS: The results suggested that the symptoms of PPCM that appeared in autoimmune perinatal rats may be due to the increase of ß1AR antibodies, which inhibited the pathway associated with peroxisome PGC-1α.

Inhibition of the Peroxisome Proliferator-Activated Receptor gamma Coactivator 1-alpha (PGC-1α)/Sirtuin 3 (SIRT3) Pathway Aggravates Oxidative Stress After Experimental Subarachnoid Hemorrhage.

BACKGROUND Emerging evidence shows that Sirtuin 3 (SIRT3) can exert an antioxidative effect in various neurodegenerative diseases, but whether and how SIRT3 modulates neuronal death after subarachnoid hemorrhage (SAH) remains to be elucidated. MATERIAL AND METHODS Experimental SAH was induced in adult mice by prechiasmatic cistern injection and primary neurons by OxyHb incubation. The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) and SIRT3 protein levels were examined at different time points after SAH induction. The PGC-1alpha protein gene knockdown in vivo and in vitro was achieved by transfection of lentivirus (LV) vectors expressing shPGC-1alpha or negative control (NC). Western blot, oxidative stress index, histopathology, neurological function, and cell viability analysis was performed. RESULTS Results showed that the PGC-1alpha/SIRT3 pathway was remarkably activated in vivo and in vitro after SAH. LV-shPGC-1alpha treatment significantly inhibited the activation of this pathway after SAH, accompanied by deteriorated neurologic function, aggravated oxidative stress, increased neuronal apoptosis, and enhanced cytotoxicity compared with the mice or primary neurons treated with LV-NC only. CONCLUSIONS The present results highlight the detrimental PGC-1alpha/SIRT3 pathway, involving regulation of the endogenous antioxidant activity against neuronal damage, which may provide a potential therapeutic target in SAH.

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.

Baicalin ameliorates hepatic insulin resistance and gluconeogenic activity through inhibition of p38 MAPK/PGC-1α pathway.

BACKGROUND: Although the results of our and other studies show that baicalin can enhance glucose uptake and insulin sensitivity in skeletal muscle and adipocytes of mice, the specific metabolic contribution of baicalin on hepatic insulin resistance and gluconeogenic activity is still unclear. PURPOSE: The aim of this study is to investigate whether baicalin is involved in regulation of hepatic insulin resistance and gluconeogenic activity and its underlying mechanisms. STUDY DESIGN/METHODS: In the present study, high-fat diet-induced obese mice were given 50 mg/kg baicalin intraperitoneally (i.p.) once a day for 21 consecutive days, and hepatocytes were treated with baicalin (100 µM) or metformin (100 µM) in the presence of glucagon (200 nM) for 12 h. Then insulin resistance indexes and genes related to gluconeogenesis were examined in liver tissues. RESULTS: The present findings showed that baicalin decreased body weight, HOMA-IR, and alleviated high fat diet-induced glucose intolerance, hyperglycemia and insulin resistance in diet-induced obese mice. Furthermore, baicalin markedly suppressed p-p38 MAPK, p-CREB, FoxO1, PGC-1α, PEPCK and G6Pase expression in liver of obese mice and hepatocytes. Moreover, inhibition of gluconeogenic genes by baicalin was also strengthened by p38MAPK inhibitor in hepatocytes. CONCLUSION: Baicalin suppressed expression of PGC-1α and gluconeogenic genes, and reduced glucose production in high-fat diet-induced obese mice. Baicalin ameliorated hepatic insulin resistance and gluconeogenic activity mainly through inhibition of p38 MAPK/PGC-1α signal pathway. This study provides a possibility of using baicalin to treat hyperglycemia and hepatic insulin resistance in clinic.

PGC-1α, a potential therapeutic target against kidney aging.

Aging is defined as changes in an organism over time. The proportion of the aged population is markedly increasing worldwide. The kidney, as an essential organ with a high energy requirement, is one of the most susceptible organs to aging. It is involved in glucose metabolism via gluconeogenesis, glucose filtration and reabsorption, and glucose utilization. Proximal tubular epithelial cells (PTECs) depend on lipid metabolism to meet the high demand for ATP. Recent studies have shown that aging-related kidney dysfunction is highly associated with metabolic changes in the kidney. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a transcriptional coactivator, plays a major role in the regulation of mitochondrial biogenesis, peroxisomal biogenesis, and glucose and lipid metabolism. PGC-1α is abundant in tissues, including kidney PTECs, which demand high energy. Many in vitro and in vivo studies have demonstrated that the activation of PGC-1α by genetic or pharmacological intervention prevents telomere shortening and aging-related changes in the skeletal muscle, heart, and brain. The activation of PGC-1α can also prevent kidney dysfunction in various kidney diseases. Therefore, a better understanding of the effect of PGC-1α activation in various organs on aging and kidney diseases may unveil a potential therapeutic strategy against kidney aging.

dl-3n-butylphthalide reduces oxygen-glucose deprivation-induced endothelial cell damage by increasing PGC-1α.

OBJECTIVE: Animal experiments verified that dl-3-n-butylphthalide (NBP) can protect vascular endothelial cells from ischemic damage and promote vascular proliferation in ischemic stroke treatment, but the underlying mechanism has not been fully clarified. This study aimed to investigate the effects of NBP on peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α) expression in endothelial cells exposed to oxygen-glucose deprivation (OGD) and to clarify the related molecular mechanism. MATERIALS AND METHODS: SV40-transformed aortic rat endothelial cell line was cultured and subjected to OGD in the presence or absence of NBP. The cell viability was evaluated by using thiazolyl blue tetrazolium bromide (MTT) method. The cellular endothelial nitric oxide synthase (eNOS) activity was measured by using eNOS activity assay. The nuclear changes were assessed with Hoechst 33342 fluorescent dye. The immunofluorescence analysis and Western blotting assay were conducted to evaluate the protein expression. RESULTS: We found that NBP could significantly prevent endothelial cells from OGD-induced injuries, in terms of cell morphology and cell viability. Both immunofluorescence analysis and Western blot findings confirmed that the NBP treatment further enhanced PGC-1α expression during OGD, which was prevented in the presence of selective endothelial nitric oxide synthetase (eNOS) inhibitor N5-(1-Iminoethyl)-L-ornithine-HCL (L-NIO). Furthermore, we found that NBP could protect the eNOS activity about by 40% during OGD and did not influence the eNOS protein level in the spectrophotometric-based analysis. CONCLUSIONS: NBP maintained the endothelial PGC-1α expression via regulating eNOS activity during the exposure to OGD; therefore, it presented its protective function to cell viability and vascular proliferation.

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.

Dihydromyricetin Attenuates Dexamethasone-Induced Muscle Atrophy by Improving Mitochondrial Function via the PGC-1α Pathway.

BACKGROUND/AIMS: Skeletal muscle atrophy is an important health issue and can impose tremendous economic burdens on healthcare systems. Glucocorticoids (GCs) are well-known factors that result in muscle atrophy observed in numerous pathological conditions. Therefore, the development of effective and safe therapeutic strategies for GC-induced muscle atrophy has significant clinical implications. Some natural compounds have been shown to effectively prevent muscle atrophy under several wasting conditions. Dihydromyricetin (DM), the most abundant flavonoid in Ampelopsis grossedentata, has a broad range of health benefits, but its effects on muscle atrophy are unclear. The purpose of this study was to evaluate the effects and underlying mechanisms of DM on muscle atrophy induced by the synthetic GC dexamethasone (Dex). METHODS: The effects of DM on Dex-induced muscle atrophy were assessed in Sprague-Dawley rats and L6 myotubes. Muscle mass and myofiber cross-sectional areas were analyzed in gastrocnemius muscles. Muscle function was evaluated by a grip strength test. Myosin heavy chain (MHC) content and myotube diameter were measured in myotubes. Mitochondrial morphology was observed by transmission electron microscopy and confocal laser scanning microscopy. Mitochondrial DNA (mtDNA) was quantified by real-time PCR. Mitochondrial respiratory chain complex activities were examined using the MitoProfile Rapid Microplate Assay Kit, and mitochondrial membrane potential was assessed by JC-1 staining. Protein levels of mitochondrial biogenesis and dynamics markers were detected by western blotting. Myotubes were transfected with siRNAs targeting peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), mitochondrial transcription factor A (TFAM) and mitofusin-2 (mfn2) to determine the underlying mechanisms. RESULTS: In vivo, DM preserved muscles from weight and average fiber cross-sectional area losses and improved grip strength. In vitro, DM prevented the decrease in MHC content and myotube diameter. Moreover, DM stimulated mitochondrial biogenesis and promoted mitochondrial fusion, rescued the reduced mtDNA content, improved mitochondrial morphology, prevented the collapse in mitochondrial membrane potential and enhanced mitochondrial respiratory chain complex activities; these changes restored mitochondrial function and improved protein metabolism, contributing to the prevention of Dex-induced muscle atrophy. Furthermore, the protective effects of DM on mitochondrial function and muscle atrophy were alleviated by PGC-1α siRNA, TFAM siRNA and mfn2 siRNA transfection in vitro. CONCLUSION: DM attenuated Dex-induced muscle atrophy by reversing mitochondrial dysfunction, which was partially mediated by the PGC-1α/TFAM and PGC-1α/mfn2 signaling pathways. Our findings may open new avenues for identifying natural compounds that improve mitochondrial function as promising candidates for the management of muscle atrophy.

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.