PGC-1-related Coactivator Research Articles

Adult skeletal muscle peroxisome proliferator-activated receptor γ -related coactivator 1 is involved in maintaining mitochondrial content.

The peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family of transcriptional coactivators are regulators of mitochondrial oxidative capacity and content in skeletal muscle. Many of these conclusions are based primarily on gain-of-function studies using muscle-specific overexpression of PGC1s. We have previously reported that genetic deletion of both PGC-1α and PGC-1β in adult skeletal muscle resulted in a significant reduction in oxidative capacity with no effect on mitochondrial content. However, the contribution of PGC-1-related coactivator (PRC), the third PGC-1 family member, in regulating skeletal muscle mitochondria is unknown. Therefore, we generated an inducible skeletal muscle-specific PRC knockout mouse (iMS-PRC-KO) to assess the contribution of PRC in skeletal muscle mitochondrial function. We measured mRNA expression of electron transport chain (ETC) subunits as well as markers of mitochondrial content in the iMS-PRC-KO animals and observed an increase in ETC gene expression and mitochondrial content. Furthermore, the increase in ETC gene expression and mitochondrial content was associated with increased expression of PGC-1α and PGC-1β. We therefore generated an adult-inducible PGC-1 knockout mouse in which all PGC-1 family members are deleted (iMS-PGC-1TKO). The iMS-PGC-1TKO animals exhibited a reduction in ETC mRNA expression and mitochondrial content. These data suggest that in the absence of PRC alone, compensation occurs by increasing PGC-1α and PGC-1β to maintain mitochondrial content. Moreover, the removal of all three PGC-1s in skeletal muscle results in a reduction in both ETC mRNA expression and mitochondrial content. Taken together, these results suggest that PRC plays a role in maintaining baseline mitochondrial content in skeletal muscle.

Concerted Action of PGC-1-related Coactivator (PRC) and c-MYC in the Stress Response to Mitochondrial Dysfunction

PGC-1-related coactivator (PRC) has a dual function in growth-regulated mitochondrial biogenesis and as a sensor of metabolic stress. PRC induction by mitochondrial inhibitors, intracellular ROS, or topoisomerase I inhibition orchestrates an inflammatory program associated with the adaptation to cellular stress. Activation of this program is accompanied by the coordinate expression of c-MYC, which is linked kinetically to that of PRC in response to multiple stress inducers. Here, we show that the c-MYC inhibitor 10058-F4 blocks the induction of c-MYC, PRC, and representative PRC-dependent stress genes by the respiratory chain uncoupler, carbonyl cyanide m-chlorophenyl hydrazine (CCCP). This result, confirmed by the suppression of PRC induction by c-MYC siRNA silencing, demonstrates a requirement for c-MYC in orchestrating the stress program. PRC steady-state expression was markedly increased upon mutation of two GSK-3 serine phosphorylation sites within the carboxyl-terminal domain. The negative control of PRC expression by GSK-3 was consistent with the phosphor-inactivation of GSK-3β by CCCP and by the induction of PRC by the GSK-3 inhibitor AZD2858. Unlike PRC, which was induced post-translationally through increased protein half-life, c-MYC was induced predominantly at the mRNA level. Moreover, suppression of Akt activation by the Akt inhibitor MK-2206 blocked the CCCP induction of PRC, c-MYC, and representative PRC stress genes, demonstrating a requirement for Akt signaling. MK-2206 also inhibited the phosphor-inactivation of GSK-3β by CCCP, a result consistent with the ability of Akt to phosphorylate, and thereby suppress GSK-3 activity. Thus, PRC and c-MYC can act in concert through Akt-GSK-3 signaling to reprogram gene expression in response to mitochondrial stress.

Oxaloacetate enhances neuronal cell bioenergetic fluxes and infrastructure.

We tested how the addition of oxaloacetate (OAA) to SH-SY5Y cells affected bioenergetic fluxes and infrastructure, and compared the effects of OAA to malate, pyruvate, and glucose deprivation. OAA displayed pro-glycolysis and pro-respiration effects. OAA pro-glycolysis effects were not a consequence of decarboxylation to pyruvate because unlike OAA, pyruvate lowered the glycolysis flux. Malate did not alter glycolysis flux and reduced mitochondrial respiration. Glucose deprivation essentially eliminated glycolysis and increased mitochondrial respiration. OAA increased, while malate decreased, the cell NAD+/NADH ratio. Cytosolic malate dehydrogenase 1 protein increased with OAA treatment, but not with malate or glucose deprivation. Glucose deprivation increased protein levels of ATP citrate lyase, an enzyme which produces cytosolic OAA, whereas OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased cytochrome oxidase subunit 2, PGC1α, PGC1β, and PGC1 related co-activator protein levels. OAA increased total and phosphorylated SIRT1 protein. We conclude that adding OAA to SH-SY5Y cells can support or enhance both glycolysis and respiration fluxes. These effects appear to depend, at least partly, on OAA causing a shift in the cell redox balance to a more oxidized state, that it is not a glycolysis pathway intermediate, and possibly its ability to act in an anaplerotic fashion. We examined how oxaloacetate (OAA) affects bioenergetic fluxes. To advance the understanding of how OAA mediates these changes, we compared the effects of OAA to malate, pyruvate, and glucose deprivation. We further examined how OAA affects levels of enzymes that facilitate its cytosolic metabolism, and found OAA increased the expression of malate dehydrogenase 1 (MDH1-cytosolic). We propose the following: OAA supports both glycolysis and respiration fluxes, shifts the cell redox balance toward a more oxidized state, and acts in an anaplerotic fashion. Abbreviations not defined in the text: MDH2, malate dehydrogenase 2 (mitochondrial).

Genome-Wide Association Study to Identify Genes Related to Renal Mercury Concentrations in Mice.

Background:Following human mercury (Hg) exposure, the metal accumulates in considerable concentrations in kidney, liver, and brain. Although the toxicokinetics of Hg have been studied extensively, factors responsible for interindividual variation in humans are largely unknown. Differences in accumulation of renal Hg between inbred mouse strains suggest a genetic interstrain variation regulating retention or/and excretion of Hg. A.SW, DBA/2 and BALB/C mouse strains accumulate higher amounts of Hg than B10.S.Objectives:We aimed to find candidate genes associated with regulation of renal Hg concentrations.Methods:A.SW, B10.S and their F1 and F2 offspring were exposed for 6 weeks to 2.0 mg Hg/L drinking water. Genotyping with microsatellites was conducted on 84 F2 mice for genome-wide scanning with ion pair reverse-phase high-performance liquid chromatography (IP RP HPLC). Quantitative trait loci (QTL) were established. Denaturing HPLC was used to detect single nucleotide polymorphisms for haplotyping and fine mapping in 184 and 32 F2 mice, respectively. Candidate genes (Pprc1, Btrc and Nfkb2) verified by fine mapping and QTL were further investigated by real-time polymerase chain reaction. Genes enhanced by Pprc1 (Nrf1 and Nrf2) were included for gene expression analysis.Results:Renal Hg concentrations differed significantly between A.SW and B10.S mice and between males and females within each strain. QTL analysis showed a peak logarithm of odds ratio score 5.78 on chromosome 19 (p = 0.002). Haplotype and fine mapping associated the Hg accumulation with Pprc1, which encodes PGC-1-related coactivator (PRC), a coactivator for proteins involved in detoxification. Pprc1 and two genes coactivated by Pprc1 (Nrf1 and Nrf2) had significantly lower gene expression in the A.SW strain than in the B10.S strain.Conclusions:This study supports Pprc1 as a key regulator for renal Hg excretion.Citation:Alkaissi H, Ekstrand J, Jawad A, Nielsen JB, Havarinasab S, Soderkvist P, Hultman P. 2016. Genome-wide association study to identify genes related to renal mercury concentrations in mice. Environ Health Perspect 124:920–926; http://dx.doi.org/10.1289/ehp.1409284

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

PGC-1-Related Coactivator (PRC) Is an Important Regulator of Microglia M2 Polarization

Microglia can be driven to adopt M1 and M2 phenotypes. While the distinct functions of M1 and M2 microglia have been intensively studied, the former are considered proinflammatory and the latter anti-inflammatory. PGC-1-related coactivator (PRC), an important member in the peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family, plays an essential role in metabolic regulation and cell proliferation. The effects of PRC on microglia M2 polarization are still unknown. In this study, we found that PRC expression was robustly induced in M2 microglia. Overexpression of PRC promotes microglia M2 polarization by increasing the expression of arginase-1 (Arg1), resistin-like α (Retnla, Fizz1), and chitinase 3-like 3 (Chi3l3, Ym1). In contrast, the silence of PRC attenuates microglia M2 polarization. Mechanistically, PRC was found to cooperate with signal transducer and activator of transcription 6 (STAT6) to induce an M2 genetic program. Overall, our findings identify PRC as what we believe to be a novel regulator of microglia M2 polarization.

Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection

We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17β-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) but increased levels of the other PGC-1 isoforms: PGC-1β and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1α via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1α downregulation. In contrast to the decrease in PGC-1α, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1β and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/nuclear DNA ratio were increased. We examined a downstream target of PGC-1β, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1β and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection.

Effects of Roux-en-Y gastric bypass and ileal transposition surgeries on glucose and lipid metabolism in skeletal muscle and liver

BackgroundRoux-en Y gastric bypass (RYGB) and ileal transposition (IT) surgeries produce weight loss and improve diabetic control; however, the mechanisms of glycemic improvements are largely unknown. Because skeletal muscle and liver play a key role in glucose homeostasis, we compared the effects of RYGB and IT surgeries on key molecules of glucose and lipid metabolism in muscle and liver. MethodsSprague-Dawley rats were subjected to RYGB, IT, or sham surgeries; sham-animals were ad-lib fed or pair-fed to RYGB rats (n = 7-9/group). At 8 weeks postoperatively, blood samples were collected for glucagon-like peptide-1 (GLP-1) and insulin analyses by ELISA. Leg muscle and liver tissues were analyzed for mRNA (RT-qPCR) and/or protein abundance (immuno blotting) of important molecules of glucose and lipid metabolism [glucose transporter-4 (GLUT-4), hexokinase, phosphofructokinase (PFK), adenosine monophosphate activated protein kinase-α (AMPKα), cytochrome C oxidase-IV (COX-IV), citrate synthase, carnitine palmitoyl transferase-1 (CPT-1), medium-chain acyl-CoA dehydrogenase (MCAD), peroxisome proliferator-activated receptor gamma co-activator 1 α (PGC-1 α), PGC-1-related coactivator (PRC), uncoupling protein-3 (UCP-3)]. ResultsPlasma GLP-1 concentrations were increased comparably with RYGB and IT. RYGB and IT increased muscle GLUT-4 protein content, muscle hexokinase mRNA, and liver PFK mRNA. IT increased muscle AMPKα and COX-IV protein content and liver citrate synthase activity. IT increased muscle CPT-1, MCAD and PRC mRNA, whereas RYGB increased UCP-3 mRNA in muscle and liver, and PGC-1 α mRNA in liver. ConclusionThe data suggest that RYGB and IT surgeries lead to enhanced GLP-1 secretion and produce similar stimulatory effects on important molecules of glucose metabolism but differential effects on key molecules of lipid oxidation in muscle and liver.

PGC-1-related coactivator (PRC) negatively regulates endothelial adhesion of monocytes via inhibition of NF κB activity

PGC-1-related coactivator (PRC) is a growth-regulated transcriptional cofactor known to activate many of the nuclear genes specifying mitochondrial respiratory function. Endothelial dysfunction is a prominent feature found in many inflammatory diseases. Adhesion molecules, such as VCAM-1, mediate the attachment of monocytes to endothelial cells, thereby playing an important role in endothelial inflammation. The effects of PRC in regards to endothelial inflammation remain unknown. In this study, our findings show that PRC can be inhibited by the inflammatory cytokine LPS in cultured human umbilical vein endothelial cells (HUVECs). In the presence of LPS, the expression of endothelial cell adhesion molecular, such as VCAM1 and E-selectin, is found to be increased. These effects can be negated by overexpression of PRC. Importantly, monocyte adhesion to endothelial cells caused by LPS is significantly attenuated by PRC. In addition, overexpression of PRC protects mitochondrial metabolic function and suppresses the rate of glycolysis against LPS. It is also found that overexpression of PRC decreases the transcriptional activity of NF-κB. These findings suggest that PRC is a negative regulator of endothelial inflammation.

Activation of a PGC-1-related Coactivator (PRC)-dependent Inflammatory Stress Program Linked to Apoptosis and Premature Senescence

PGC-1-related coactivator (PRC), a growth-regulated member of the PGC-1 coactivator family, contributes to the expression of the mitochondrial respiratory apparatus. PRC also orchestrates a robust response to metabolic stress by promoting the expression of multiple genes specifying inflammation, proliferation, and metabolic reprogramming. Here, we demonstrate that this PRC-dependent stress program is activated during apoptosis and senescence, two major protective mechanisms against cellular dysfunction. Both PRC and its targets (IL1α, SPRR2D, and SPRR2F) were rapidly induced by menadione, an agent that promotes apoptosis through the generation of intracellular oxidants. Menadione-induced apoptosis and the PRC stress program were blocked by the antioxidant N-acetylcysteine. The PRC stress response was also activated by the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN-38), an inducer of premature senescence in tumor cells. Cells treated with SN-38 displayed morphological characteristics of senescence and express senescence-associated β-galactosidase activity. In contrast to menadione, the SN-38 induction of the PRC program occurred over an extended time course and was antioxidant-insensitive. The potential adaptive function of the PRC stress response was investigated by treating cells with meclizine, a drug that promotes glycolytic energy metabolism and has been linked to cardio- and neuroprotection against ischemia-reperfusion injury. Meclizine increased lactate production and was a potent inducer of the PRC stress program, suggesting that PRC may contribute to the protective effects of meclizine. Finally, c-MYC and PRC were coordinately induced under all conditions tested, implicating c-MYC in the biological response to metabolic stress. The results suggest a general role for PRC in the adaptive response to cellular dysfunction.

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

PGC-1-related Coactivator (PRC), a Sensor of Metabolic Stress, Orchestrates a Redox-sensitive Program of Inflammatory Gene Expression

PGC-1-related coactivator (PRC) is a growth-regulated transcriptional cofactor that activates many nuclear genes specifying mitochondrial respiratory function. Stable PRC silencing in U2OS cells results in a complex phenotype typical of mitochondrial dysfunction including abundant abnormal mitochondria, reduced respiratory subunit expression, diminished respiratory enzymes and ATP levels, and elevated lactate production. The PRC response to metabolic stress was investigated by subjecting cells to metabolic insults including treatment with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), expression of a dominant negative allele of nuclear respiratory factor 1 (NRF-1), and glucose deprivation. These treatments led to constitutively elevated PRC protein levels, a departure from its normal transient expression upon the initiation of cell growth. A microarray screen identified 45 genes that require PRC for their induction by CCCP. A subset of these genes specific to inflammation and cell stress was also induced by dominant negative NRF-1 and by glucose deprivation, suggesting that diverse metabolic insults converge on the same PRC-dependent inflammatory program. The PRC-dependent inflammatory response was inhibited by N-acetylcysteine, suggesting that PRC may contribute to the inflammatory microenvironment linked to oxidant signaling. The induction of this PRC-dependent program may be an early event in adaptations linked to cancer and degenerative diseases.

Resistance exercise enhances the molecular signaling of mitochondrial biogenesis induced by endurance exercise in human skeletal muscle

Combining endurance and strength training (concurrent training) may change the adaptation compared with single mode training. However, the site of interaction and the mechanisms are unclear. We have investigated the hypothesis that molecular signaling of mitochondrial biogenesis after endurance exercise is impaired by resistance exercise. Ten healthy subjects performed either only endurance exercise (E; 1-h cycling at ∼65% of maximal oxygen uptake), or endurance exercise followed by resistance exercise (ER; 1-h cycling + 6 sets of leg press at 70-80% of 1 repetition maximum) in a randomized cross-over design. Muscle biopsies were obtained before and after exercise (1 and 3 h postcycling). The mRNA of genes related to mitochondrial biogenesis [(peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1)α, PGC-1-related coactivator (PRC)] related coactivator) and substrate regulation (pyruvate dehydrogenase kinase-4) increased after both E and ER, but the mRNA levels were about twofold higher after ER (P < 0.01). Phosphorylation of proteins involved in the signaling cascade of protein synthesis [mammalian target of rapamycin (mTOR), ribosomal S6 kinase 1, and eukaryotic elongation factor 2] was altered after ER but not after E. Moreover, ER induced a larger increase in mRNA of genes associated with positive mTOR signaling (cMyc and Rheb). Phosphorylation of AMP-activated protein kinase, acetyl-CoA carboxylase, and Akt increased similarly at 1 h postcycling (P < 0.01) after both types of exercise. Contrary to our hypothesis, the results demonstrate that ER, performed after E, amplifies the adaptive signaling response of mitochondrial biogenesis compared with single-mode endurance exercise. The mechanism may relate to a cross talk between signaling pathways mediated by mTOR. The results suggest that concurrent training may be beneficial for the adaptation of muscle oxidative capacity.

Nitric Oxide and Calcium Participate in the Fine Regulation of Mitochondrial Biogenesis in Follicular Thyroid Carcinoma Cells

Members of the peroxisome proliferator-activated receptor γ coactivator-1 family (i.e. PGC-1α, PGC-1β, and the PGC-1-related coactivator (PRC)) are key regulators of mitochondrial biogenesis and function. These regulators serve as mediators between environmental or endogenous signals and the transcriptional machinery governing mitochondrial biogenesis. The FTC-133 and RO82 W-1 follicular thyroid carcinoma cell lines, which present significantly different numbers of mitochondria, metabolic mechanisms, and expression levels of PRC and PGC-1α, may employ retrograde signaling in response to respiratory dysfunction. Nitric oxide (NO) and calcium have been hypothesized to participate in this activity. We investigated the effects of the S-nitroso-N-acetyl-DL-penicillamine-NO donor, on the expression of genes involved in mitochondrial biogenesis and cellular metabolic functions in FTC-133 and RO82 W-1 cells by measuring lactate dehydrogenase and cytochrome c oxidase (COX) activities. We studied the action of ionomycin and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (i.e. a calcium ionophore and a cytosolic calcium chelator) on whole genome expression and mitochondrial biogenesis in RO82 W-1 cells. COX activity and the dynamics of endoplasmic reticulum and mitochondrial networks were analyzed in regard to calcium-modulating treatments. In the FTC-133 and RO82 W-1 cells, the mitochondrial biogenesis induced by NO was mainly related to PRC expression as a retrograde mitochondrial signaling. Ionomycin diminished COX activity and negatively regulated PRC-mediated mitochondrial biogenesis in RO82 W-1 cells, whereas BAPTA/AM produced the opposite effects with a reorganization of the mitochondrial network. This is the first demonstration that NO and calcium regulate mitochondrial biogenesis through the PRC pathway in thyroid cell lines.

Interactions between the consumption of a high-fat diet and fasting in the regulation of fatty acid oxidation enzyme gene expression: an evaluation of potential mechanisms

The consumption of high-fat diets (HFDs) and fasting are known to increase the expression of enzymes involved in fatty acid oxidation (FAO). However, it has been reported that the ability of physiological stressors to induce enzymes of FAO in skeletal muscle is blunted with obesity. In this regard, we sought to explore the effects and potential mechanisms of an HFD on the expression of FAO enzymes in the fed and fasted state. The consumption of an HFD increased the mRNA expression or protein content of medium-chain acyl-CoA dehydrogenase (MCAD), uncoupling protein-3 (UCP3), and pyruvate dehydrogenase kinase 4 (PDK4) in the fed state. Fasting increased the mRNA expression of PDK4, MCAD, and UCP-3, and the protein content of UCP-3 in chow but not HFD rats. HFDs did not increase carnitine palmitoyl transfer-1 (CPT-1) mRNA levels in the fed state and the effects of fasting were markedly reduced compared with chow-fed rats. The expression of peroxisome-proliferator-activated receptor-γ coactivator-1β (PGC-1β) was increased in muscle from HFD rats in the fed state, while PGC-1-related coactivator (PRC) was increased with fasting in chow-fed but not HFD rats. Plasma fatty acid levels were elevated in the fed state from HFD rats but not increased further with fasting, whereas fasting increased plasma fatty acids in chow-fed animals. Fasting-mediated increases in plasma epinephrine, and the activation of PKA and AMPK in skeletal muscle were similar between chow and HFD rats. p38 MAPK phosphorylation was increased with fasting in chow-fed but not HFD rats. Our findings suggest that a blunted effect of fasting on the induction of PDK4, MCAD, and UCP3 in skeletal muscle from HFD rats is likely a result of already elevated levels of these enzymes, the induction of which is associated with increases in plasma fatty acid and PGC-1β. On the other hand, a blunted induction of PRC and CPT-1 mRNA may be explained by decreases in p38 MAPK signaling.

Defective DNA Replication Impairs Mitochondrial Biogenesis In Human Failing Hearts

Mitochondrial dysfunction plays a pivotal role in the development of heart failure. Animal studies suggest that impaired mitochondrial biogenesis attributable to downregulation of the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 transcriptional pathway is integral of mitochondrial dysfunction in heart failure. The study sought to define mechanisms underlying the impaired mitochondrial biogenesis and function in human heart failure. We collected left ventricular tissue from end-stage heart failure patients and from nonfailing hearts (n=23, and 19, respectively). The mitochondrial DNA (mtDNA) content was decreased by >40% in the failing hearts, after normalization for a moderate decrease in citrate synthase activity (P<0.05). This was accompanied by reductions in mtDNA-encoded proteins (by 25% to 80%) at both mRNA and protein level (P<0.05). The mRNA levels of PGC-1alpha/beta and PRC (PGC-1-related coactivator) were unchanged, whereas PGC-1alpha protein increased by 58% in the failing hearts. Among the PGC-1 coactivating targets, the expression of estrogen-related receptor alpha and its downstream genes decreased by up to 50% (P<0.05), whereas peroxisome proliferator-activated receptor alpha and its downstream gene expression were unchanged in the failing hearts. The formation of D-loop in the mtDNA was normal but D-loop extension, which dictates the replication process of mtDNA, was decreased by 75% in the failing hearts. Furthermore, DNA oxidative damage was increased by 50% in the failing hearts. Mitochondrial biogenesis is severely impaired as evidenced by reduced mtDNA replication and depletion of mtDNA in the human failing heart. These defects are independent of the downregulation of the PGC-1 expression suggesting novel mechanisms for mitochondrial dysfunction in heart failure.

Estrogen‐related receptor α and PGC‐1‐related coactivator constitute a novel complex mediating the biogenesis of functional mitochondria

Mitochondrial biogenesis, which depends on nuclear as well as mitochondrial genes, occurs in response to increased cellular ATP demand. The nuclear transcriptional factors, estrogen-related receptor alpha (ERRalpha) and nuclear respiratory factors 1 and 2, are associated with the coordination of the transcriptional machinery governing mitochondrial biogenesis, whereas coactivators of the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family serve as mediators between the environment and this machinery. In the context of proliferating cells, PGC-1-related coactivator (PRC) is a member of the PGC-1 family, which is known to act in partnership with nuclear respiratory factors, but no functional interference between PRC and ERRalpha has been described so far. We explored three thyroid cell lines, FTC-133, XTC.UC1 and RO 82 W-1, each characterized by a different mitochondrial content, and studied their behavior towards PRC and ERRalpha in terms of respiratory efficiency. Overexpression of PRC and ERRalpha led to increased respiratory chain capacity and mitochondrial mass. The inhibition of ERRalpha decreased cell growth and respiratory chain capacity in all three cell lines. However, the inhibition of PRC and ERRalpha produced a greater effect in the oxidative cell model, decreasing the mitochondrial mass and the phosphorylating respiration, whereas the nonphosphorylating respiration remained unchanged. We therefore hypothesize that the ERRalpha-PRC complex plays a role in arresting the cell cycle through the regulation of oxidative phosphorylation in oxidative cells, and through some other pathway in glycolytic cells.

PGC-1-Related Coactivator Modulates Mitochondrial-Nuclear Crosstalk through Endogenous Nitric Oxide in a Cellular Model of Oncocytic Thyroid Tumours

BackgroundThe PGC-1 related coactivator (PRC), which shares structural and functional features with PGC-1α, is believed to regulate several metabolic pathways as well as mitochondrial biogenesis. Its involvement in the early programming of cell proliferation suggests the existence of finely regulated crosstalk between mitochondrial functions and the cell cycle status.Methodology/Principal FindingsPRC-regulated pathways were explored in a cell-line model derived from mitochondrial-rich tumours with an essentially oxidative metabolism and specifically high PRC expression. The functional status of mitochondria was compared to the results of microarray analysis under conditions of temporal PRC inhibition. To specify the fine PRC regulation, the expression levels of the genes and proteins involved in the oxidative phosphorylation process were studied by real time quantitative PCR and western blotting. As in earlier studies on PGC-1α, we investigated the role of nitric oxide in PRC-regulated mitochondrial biogenesis and determined its action in the control of the phosphorylation status of the mitogen-activated protein kinase pathway.Conclusion/SignificanceWe found that nitric oxide rapidly influences PRC expression at the transcriptional level. Focusing on mitochondrial energetic metabolism, we observed that PRC differentially controls respiratory chain complexes and coupling efficiency in a time-dependent manner to maintain mitochondrial homeostasis. Our results highlight the key role of PRC in the rapid modulation of metabolic functions in response to the status of the cell cycle.

A Role for the Transcriptional Coactivator PRC1 in Mitochondrial Biogenesis During Erythroid Development.

Abstract 3642Poster Board III-578Red cell development requires unparalleled mitochondrial metabolic and biosynthetic capacities to support hemoglobin formation. In addition to meeting the energetic requirements of the erythroblast and reticulocyte, mitochondria are the site of multiple steps in heme biosynthesis and are essential for iron utilization. As such, perturbation of mitochondrial function in hematopoietic cells appears to differentially affect red cell development. For instance, we show that the drug chloramphenicol (an inhibitor of mitochondrial protein synthesis) preferentially inhibits erythroid colony formation with relatively little effect on other types of myeloid progenitors. Similarly, we show that knockout of the mitochondrial superoxide dismutase (Sod2) preferentially inhibits BFU-E, CFU-E and GEMM colonies. Clinically, several mutations in mitochondrial DNA or in nuclear encoded mitochondria-localized proteins preferentially disrupt erythroid development resulting in sideroblastic anemia. In order to better understand regulation of mitochondrial activity during red cell development/differentiation, we have investigated expression of transcription factors and coactivators implicated as regulators of mitochondrial biogenesis in other tissues including PGC-1 α and β, ERR proteins, NRF 1 and NRF 2 in normal and Sod2 deficient murine erythroblasts. Microarray and qPCR data suggest that neither of the classic transcriptional coactivators PGC-1 α or β are expressed at appreciable levels in erythroblasts. However, a PGC-1 related coactivator, PRC1 is detectable at both the transcript and protein level in developing erythroid cells. In the Sod2 deficiency model, one of the most prominent transcriptional changes we have observed in erythroblasts is a broad down regulation of expression of nuclear encoded mitochondria localized protein transcripts including multiple components of the electron transport chain, TCA cycle enzymes and mitochondrial ribosomal proteins. We show that PRC1 expression is down regulated in Sod2 deficient cells—suggesting that this coactivator plays a key role in mitochondrial biogenesis during erythroid development. Further, using the MEL cell line, we show that PRC1 expression is turned on following induction of differentiation with DMSO. These results identify PRC1 as a novel, and perhaps exclusive regulator of mitochondrial biogenesis during erythroid development. A better understanding of regulation of PRC1 expression and identification of transcriptional targets of PRC1 during erythroid development will improve our understanding of how perturbations in mitochondrial physiology affect the red cell compartment. Disclosures:No relevant conflicts of interest to declare.

The effects of apelin treatment on skeletal muscle mitochondrial content

Adipose tissue is recognized as a key player in the regulation of whole body metabolism. Apelin, is a recently identified adipokine that when given to mice results in increases in skeletal muscle uncoupling protein 3 (UCP3) content. Similarly, acute apelin treatment has been shown to increase the activity of 5'-AMP-activated protein kinase (AMPK), a reputed mediator of skeletal muscle mitochondrial biogenesis. Given these findings, we sought to determine the effects of apelin on skeletal muscle mitochondrial content. Male Wistar rats were given daily intraperitoneal injections of apelin-13 (100 nmol/kg) for 2 wk. We made the novel observation that the activities of citrate synthase, cytochrome c oxidase, and beta-hydroxyacyl coA dehydrogenase (betaHAD) were increased in triceps but not heart and soleus muscles from apelin-treated rats. When confirming these results we found that both nuclear and mitochondrial-encoded subunits of the respiratory chain were increased in triceps from apelin-treated rats. Similarly, apelin treatment increased the protein content of components of the mitochondrial import and assembly pathway. The increases in mitochondrial marker proteins were associated with increases in proliferator-activated receptor-gamma coactivator-1 (PGC-1beta) but not PGC-1alpha or Pgc-1-related co-activator (PRC) mRNA expression. Chronic and acute apelin treatment did not increase the protein content and/or phosphorylation status of AMPK and its downstream substrate acetyl-CoA carboxylase. These findings are the first to demonstrate that apelin treatment can induce skeletal muscle mitochondrial content. Given the lack of an effect of apelin on AMPK signaling and PGC-1alpha mRNA expression, these results suggest that apelin increases skeletal muscle mitochondrial content through a mechanism that is distinct from that of more robust physiological stressors.