Mfn2 Promoter Research Articles

DNA Methylation-Mediated Mfn2 Gene Regulation in the Brain: A Role in Brain Trauma-Induced Mitochondrial Dysfunction and Memory Deficits.

Repeated mild traumatic brain injuries (rMTBI) affect mitochondrial homeostasis in the brain. However, mechanisms of long-lasting neurobehavioral effects of rMTBI are largely unknown. Mitofusin 2 (Mfn2) is a critical component of tethering complexes in mitochondria-associated membranes (MAMs) and thereby plays a pivotal role in mitochondrial functions. Herein, we investigated the implications of DNA methylation in the Mfn2 gene regulation, and its consequences on mitochondrial dysfunction in the hippocampus after rMTBI. rMTBI dramatically reduced the mitochondrial mass, which was concomitant with decrease in Mfn2 mRNA and protein levels. DNA hypermethylation at the Mfn2 gene promoter was observed post 30days of rMTBI. The treatment of 5-Azacytidine, a pan DNA methyltransferase inhibitor, normalized DNA methylation levels at Mfn2 promoter, which further resulted into restoration of Mfn2 function. The normalization of Mfn2 function was well correlated with recovery in memory deficits in rMTBI-exposed rats. Since, glutamate excitotoxicity serves as a primary insult after TBI, we employed in vitro model of glutamate excitotoxicity in human neuronal cell line SH-SY5Y to investigate the causal epigenetic mechanisms of Mfn2 gene regulation. The glutamate excitotoxicity reduced Mfn2 levels via DNA hypermethylation at Mfn2 promoter. Loss of Mfn2 caused significant surge in cellular and mitochondrial ROS levels with lowered mitochondrial membrane potential in cultured SH-SY5Y cells. Like rMTBI, these consequences of glutamate excitotoxicity were also prevented by 5-AzaC pre-treatment. Therefore, DNA methylation serves as a vital epigenetic mechanism involved in Mfn2 expression in the brain; and this Mfn2 gene regulation may play a pivotal role in rMTBI-induced persistent cognitive deficits. Closed head weight drop injury method was employed to induce repeated mild traumatic brain (rMTBI) in jury in adult, male Wistar rats. rMTBI causes hyper DNA methylation at the Mfn2 promoter and lowers the Mfn2 expression triggering mitochondrial dysfunction. However, the treatment of 5-azacytidine normalizes DNA methylation at the Mfn2 promoter and restores mitochondrial function.

Paeonol protects against doxorubicin-induced cardiotoxicity by promoting Mfn2-mediated mitochondrial fusion through activating the PKCε-Stat3 pathway

IntroductionThe anti-cancer medication doxorubicin (Dox) is largely restricted in clinical usage due to its significant cardiotoxicity. The only medication approved by the FDA for Dox-induced cardiotoxicity is dexrazoxane, while it may reduce the sensitivity of cancer cells to chemotherapy and is restricted for use. There is an urgent need for the development of safe and effective medicines to alleviate Dox-induced cardiotoxicity. ObjectivesThe objective of this study was to determine whether Paeonol (Pae) has the ability to protect against Dox-induced cardiotoxicity and if so, what are the underlying mechanisms involved. MethodsSprague-Dawley rats and primary cardiomyocytes were used to create Dox-induced cardiotoxicity models. Pae's effects on myocardial damage, mitochondrial function, mitochondrial dynamics and signaling pathways were studied using a range of experimental methods. ResultsPae enhanced Mfn2-mediated mitochondrial fusion, restored mitochondrial function and cardiac performance both in vivo and in vitro under the Dox conditions. The protective properties of Pae were blunted when Mfn2 was knocked down or knocked out in Dox-induced cardiomyocytes and hearts respectively. Mechanistically, Pae promoted Mfn2-mediated mitochondria fusion by activating the transcription factor Stat3, which bound to the Mfn2 promoter in a direct manner and up-regulated its transcriptional expression. Furthermore, molecular docking, surface plasmon resonance and co-immunoprecipitation studies showed that Pae’s direct target was PKCε, which interacted with Stat3 and enabled its phosphorylation and activation. Pae-induced Stat3 phosphorylation and Mfn2-mediated mitochondrial fusion were inhibited when PKCε was knocked down. Furthermore, Pae did not interfere with Dox's antitumor efficacy in several tumor cells. ConclusionPae protects the heart against Dox-induced damage by stimulating mitochondrial fusion via the PKCε-Stat3-Mfn2 pathway, indicating that Pae might be a promising therapeutic therapy for Dox-induced cardiotoxicity while maintaining Dox's anticancer activity.

LARS2 Regulates Apoptosis via ROS-Mediated Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Ovarian Granulosa Cells.

Several studies have indicated that mutations of LARS2 are associated with premature ovarian insufficiency (POI). However, the pathogenic mechanism of LARS2 in POI has not been reported yet. In the present study, the expression levels of LARS2 and E2F1 in granulosa cells (GCs) of POI patients were examined. CCK-8 and Edu assay were performed to determine the effect of LARS2 on cell proliferation. Apoptosis rate, mitochondrial membrane potential, reactive oxygen species (ROS), and cytoplasm Ca2+ levels were analyzed by flow cytometry. Western blot was conducted to evaluate the expression level of genes affected by LARS2. Transmission electron microscopy (TEM) was used to observe mitochondrial structure in GCs. Chromatin immunoprecipitation (ChIP) was used to evaluate the regulatory effect of E2F1 on Mfn-2 expression. Our results showed that LARS2 expression was downregulated in GCs of POI patients. Silencing of LARS2 inhibited cell proliferation and promoted the apoptosis of GCs. Meanwhile, LARS2 knockdown could induce mitochondrial dysfunction and accumulation of ROS levels. Moreover, ROS was found to be involved in the antiproliferation, proapoptotic, and endoplasmic reticulum (ER) stress effects of LARS2 knockdown. Furthermore, we also found that the expression level of E2F1 was positively correlated with LARS2. In addition, E2F1 could bind at the -61/-46 region of Mfn-2 promoter and regulated MFN-2 transcription. These findings demonstrated that LARS2 could promote the expression of E2F1. E2F1 mediated the effect of LARS2 on Mfn-2 expression via targeting the promoter region of Mfn-2, in which subsequently regulated cell proliferation and apoptosis, which resulted in the etiology of POI. This study will provide useful information for further investigations on the LARS2 in the occurrence of POI.

Mitochondrial fusion and maintenance of mitochondrial homeostasis in diabetic retinopathy

Mitochondria are dynamic in structure, and undergo continuous fusion-fission to maintain their homeostasis. In diabetes, retinal mitochondria are swollen, their membrane is damaged and mitochondrial fusion protein, mitofusin 2 (Mfn2), is decreased. DNA methylation machinery is also activated and methylation status of genes implicated in mitochondrial damage and biogenesis is altered. This study aims to investigate the role of mitochondrial fusion in the development of diabetic retinopathy, and to illustrate the molecular mechanism responsible for Mfn2 suppression. Using human retinal endothelial cells, manipulated for Mfn2, we investigated the role of fusion in mitochondrial structural and functional damage in diabetes. The molecular mechanism of its suppression in diabetic milieu was determined by investigating Mfn2 promoter DNA methylation, and confirmed using molecular and pharmacological inhibitors of DNA methylation. Similar studies were performed in the retinal microvasculature (prepared by hypotonic shock method) of diabetic rats, and human donors with documented diabetic retinopathy. Overexpression of Mfn2 prevented glucose-induced increase in mitochondrial fragmentation, decrease in complex III activity and increase in membrane permeability, mtDNA damage and apoptosis. High glucose hypermethylated Mfn2 promoter and decreased transcription factor (SP1) binding, and Dnmt inhibition protected Mfn2 promoter from these changes. In streptozotocin-induced diabetic rats, intravitreal administration of Dnmt1-siRNA attenuated Mfn2 promoter hypermethylation and restored its expression. Human donors with diabetic retinopathy confirmed Mfn2 promoter DNA hypermethylation. Thus, regulating Mfn2 and its epigenetic modifications by molecular/pharmacological means will protect mitochondrial homeostasis in diabetes, and could attenuate the development of retinopathy in diabetic patients.

335 - Modulation of dynamic mitochondria by epigenetics in diabetic retinopathy

Introduction Mitochondria are highly dynamic, and they continually fuse and divide. Fusion mixes the contents of two mitochondria, and dilutes injured mitochondrial proteins and DNA. Outer membrane fusion is mediated by fusion proteins, mitofusins (Mfn), and Mfn2 itself is sufficient to modulate mitochondrial metabolism. In the pathogenesis of diabetic retinopathy, mitochondrial structure and DNA are damaged, oxygen consumption is decreased and Mfn2 levels are decreased, and damaged mitochondria accelerates capillary cell apoptosis. Gene expression is also regulated by epigenetic modifications, and these modifications are implicated in the development of diabetic retinopathy. Aim of this study is to investigate the role of epigenetic modifications in the regulation of Mfn2 in diabetic retinopathy. Methods In human retinal endothelial cells, incubated in normal (5mM) or high glucose (20mM) for four days, in the presence/absence of DNA methyltransferase (Dnmt) inhibitors (5-Azacytidine or Dnmt1-siRNA), methylated cytosine (5mC) levels at Mfn2 promoter were quantified by immunecapturing method. Mitochondrial stability was evaluated by measuring membrane permeability, complex-III activity and transcription of mtDNA (cytochrome b, Cytb), and cell apoptosis was quantified by ELISA method using monoclonal antibodies against DNA and histones. Key parameters were evaluated in the retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreal administration of Dnmt1-siRNA. Results Cells exposed to high glucose had increased 5mC levels at Mfn2 promoter. Dnmt inhibition ameliorated increase in 5mC levels, decrease in Mfn2 expression, and protected mitochondrial stability. In the same cells, glucose-induced increase in apoptosis was also ameliorated. Similarly, Dnmt1-siRNA prevented diabetes-induced increase in 5mC at Mfn2 promoter and decrease in its expression. Conclusions DNA methylation of Mfn2 promoter in diabetes, impairs mitochondrial stability, leading to accelerated apoptosis. Thus, targeting Mfn2 DNA methylation could help maintain mitochondrial homeostasis, and halt/retard the development of diabetic retinopathy.

Epigallocatechin-3-gallate regulates mitofusin 2 expression through the peroxisome proliferator-activated receptor-γ coactivator-1α and estrogen-related receptor-α pathway.

Our previous study showed that epigallocatechin-3-gallate (EGCG) inhibition of human aortic smooth muscle cell (HASMC) proliferation might be mediated via upregulation of mitofusin 2 (Mfn-2). Studies on the mechanism of Mfn-2 inhibition of cell proliferation have mainly focused on downstream signaling. However, it is still not clear how upstream signaling molecules regulate Mfn-2. The promoter region of the Mfn-2 gene contains cis-acting elements of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and estrogen-related receptor-α (ERR-α), suggesting a possible link between EGCG, Mfn-2, and PGC-1α/ERR-α. However, the effect of EGCG on PGC-1α/ERR-α remains unknown. In this study, we investigated the role of PGC-1α/ERR-α in the regulation of Mfn-2 induced by EGCG and assessed the underlying mechanisms. The effects of EGCG on cell proliferation of cultured HASMCs were observed by a cell counting kit-8 (CCK8) and 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. Mfn-2, PGC-1α, and ERR-α gene and protein levels were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis. PGC-1α gene-silencing (PGC-1α small interfering RNA [siRNA]) was achieved by RNA interference and Mfn-2 promoter and peroxisome proliferator response element (PPRE) functional activity was achieved by a luciferase transfection assay. The results showed that the ERR-α-specific antagonist XCT-790 and PGC-1α siRNA decreased the expression of Mfn-2, thus antagonizing the inhibition of HASMC proliferation induced by EGCG. EGCG enhanced Mfn-2 promoter (-352 to 459) activity, while XCT-790 and PGC-1α siRNA abrogated this effect. PGC-1α stimulating Mfn-2 expression was dependent on intact ERR-α binding in the Mfn-2 promoter. The transcriptional effect of PGC-1α on the Mfn-2 promoter required the integrity of the -432 to 459 region and supported that Mfn-2 was a key target gene of PGC-1α. These results imply that PGC-1α/ERR-α played important physiological roles in inhibiting the proliferation of HASMCs by modulating Mfn-2 gene expression. Hence, EGCG regulated Mfn-2 expression likely through the PGC-1α/ERR-α pathway.

Epigenetics and Mitochondrial Stability in Diabetic Retinopathy

Mitochondria constantly fuse and divide to form a cellular network, and this dynamic fusion-fission controls their function and genome stability. Although both, mitofusin 1 and 2 (Mfn1 and 2) regulate outer mitochondrial membrane fusion, Mfn2 itself is sufficient to modulate mitochondrial metabolism. In diabetes, retinal mitochondria are swollen, respiration is impaired, Mfn2 is decreased and mtDNA is damaged. Diabetes also facilitates epigenetic modifications, altering gene expressions without changing their DNA sequences. This study aims to investigate the role of DNA methylation of Mfn2 in the development of diabetic retinopathy. Methods: Human retinal endothelial cells, incubated in normal (5mmols/L) or high glucose (20mmols/L) for four days, in the presence/absence of DNA methyltransferase (Dnmt) inhibitors (5-Azacytidine or Dnmt1-siRNA), were analyzed for methylated cytosine (5mC) at Mfn2 promoter by immunocapturing method. Mitochondrial integrity was evaluated by membrane permeability, coenzyme Q: cytochrome-c reductase (complex-III) activity, and transcripts of mtDNA-encoded cytochrome b (Cytb). Similar measurements were made in the retinal microvessels from streptozotocin-induced diabetic mice receiving 5-Aza (2.5mg/kg, i.p.) or Dnmt1-siRNA (2µg/2µl, intravitreal). Results: High glucose increased 5mC levels at Mfn2 promoter by over 2 fold, and decreased Mfn2 expression. Dnmt inhibition ameliorated glucose-induced increase in 5mC at Mfn2 and mitochondrial membrane permeability, and decrease in Mfn2 expression, complex III activity and Cytb transcripts. Similarly, in mouse retinal microvessels, inhibition of Dnmt prevented diabetes-induced increase in 5mC at Mfn2 promoter and decrease in Mfn2 expression. Conclusions: In diabetes, epigenetic modifications of Mfn2 promoter impair mitochondrial functional and genomic stability. Thus, targeting DNA methylation could help maintain mitochondrial homeostasis, and halt/retard the development of diabetic retinopathy. Disclosure R. Kowluru: None. M. Mishra: None.

Histone Decacetylase Inhibitors Prevent Mitochondrial Fragmentation and Elicit Early Neuroprotection against MPP+

Parkinson's disease (PD) is a common neurodegenerative disease, characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra. Recent investigations have shown that mitochondrial fragmentation, an early event during apoptosis, is implicated in the degeneration of DA neurons in PD, and more importantly, preventing mitochondrial fragmentation could rescue cell death in several PD models. Therefore, mitochondrial dynamics may be a therapeutic target for early intervention in PD. However, much remains unknown about the mechanism underlying mitochondrial fragmentation in PD. The alterations in mitochondrial morphology, cell apoptosis, and mitochondrial shaping protein levels were detected after SH-SY5Y cells were treated with various doses of MPP+ or rotenone. Mitochondrial fragmentation is an early event during apoptosis caused by MPP+ but not rotenone, and Trichostatin A (TSA), a commonly used histone deacetylase (HDAC) inhibitor, selectively rescues mitochondrial fragmentation and cell death induced by lower doses of MPP+. Mitochondrial fragmentation triggered by lower doses of MPP+ may be a result of Mfn2 down-regulation, which could be completely reversed by TSA. Further investigation suggests that TSA prevents MPP+-induced Mfn2 down-regulation via inhibiting histone deacetylation over Mfn2 promoter and alleviating its transcriptional dysfunction. Histone deacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection in PD cell model induced by MPP+. Hence, HDAC inhibitors may be a potential early treatment for PD.

Hepatitis B virus X protein inhibits p53-mediated upregulation of mitofusin-2 in hepatocellular carcinoma cells

The hepatitis B virus X (HBx) protein has many significant roles in hepatocellular carcinoma (HCC). Our previous research demonstrated that mitofusion-2 (Mfn2), a potential tumor suppressor gene in HCC, is a novel direct target of p53 that exerts apoptotic effects via the mitochondrial apoptotic pathway. However, the relationship between HBx and Mfn2 expression in the development of HCC is unknown. We found that HBx had little direct effect on the expression of Mfn2 or p53 in HCC cells not treated with doxorubicin. However, HBx inhibited the upregulation of Mfn2 in HBx-transfected HCC cells simultaneously treated with doxorubicin or cotransfected with p53 plasmid, as evidenced by Western Blot and real-time PCR. Through electrophoretic mobility shift analysis, we confirmed that HBx interfered with the binding event of the p53 protein and the p53 binding site-oligo of the Mfn2 promoter. Moreover, luciferase assays revealed that the activity of the Mfn2 promoter did not increase when transfected with HBx plasmid in doxorubicin-treated HepG2 cells. These results indicate that HBx impacts p53-mediated transcription of Mfn2, providing insight into the negative effect of HBx against p53-dependent chemotherapeutic agents, such as doxorubicin, used in the treatment of HCC.

The promoter activity of human Mfn2 depends on Sp1 in vascular smooth muscle cells

Mitofusin-2 (Mfn2) expression is dysregulated in vascular proliferative disorders and its overexpression attenuates the proliferation of vascular smooth muscle cells (VSMCs) and neointimal lesion development after balloon angioplasty. We sought to gain insight into the mechanisms that control Mfn2 expression in VSMCs. We cloned and characterized 2 kb of the 5'-flanking region of the human Mfn2 gene. Its TATA-less promoter contains a CpG island. In keeping with this, 5'-rapid amplification of cDNA ends revealed six transcriptional start sites (TSSs), of which TSS2 and TSS5 were the most frequently used. The strong CpG island was found to be non-methylated under conditions characterized by large differences in Mfn2 gene expression. The proximal Mfn2 promoter contains six putative Sp1 motifs. Sp1 binds to the Mfn2 promoter and its overexpression activates the Mfn2 promoter in VSMCs. Chemical inhibition of Sp1 reduced Mfn2 expression, and Sp1 silencing reduced transcriptional activity of the Mfn2 promoter. In keeping with this view, Sp1 and Mfn2 mRNA levels were down-regulated in the aorta early after an atherogenic diet in apolipoprotein E-knockout mice or in VSMCs cultured in the presence of low serum. Sp1 is a key factor in maintaining basal Mfn2 transcription in VSMCs. Given the anti-proliferative actions of Mfn2, Sp1-induced Mfn2 transcription may represent a mechanism for prevention of VSMC proliferation and neointimal lesion and development.

Mitofusin-2 is a novel direct target of p53

The tumor suppressor p53 modulates transcription of a number of target genes involved in cell cycle arrest, apoptosis, DNA repair, and other important cellular responses. Mitofusin-2 (Mfn2) is a novel suppressor of cell proliferation that may also exert apoptotic effects via the mitochondrial apoptotic pathway. Through bioinformatics analysis, we identified a p53 binding site in the Mfn2 promoter. Consistent with this, we showed that the p53 protein binds the Mfn2 promoter directly both in vitro and in vivo. Additionally, we found that Mfn2 mRNA and protein levels are up-regulated in a p53-dependent manner. Furthermore, luciferase assays revealed that the activity of the wild-type Mfn2 promoter, but not a mutated version of the promoter, was up-regulated by p53. These results indicate that Mfn2 is a novel p53-inducible target gene, which provides insight into the regulation of Mfn2 and its associated activities in the inhibition of cell proliferation, promotion of apoptosis, and modulation of tumor suppression.

Krüppel-like factor 4 interacts with p300 to activate mitofusin 2 gene expression induced by all-trans retinoic acid in VSMCs

To elucidate how krüppel-like factor 4 (KLF4) activates mitofusin 2 (mfn-2) expression in all-trans retinoic acid (ATRA)-induced vascular smooth muscle cell (VSMC) differentiation. The mfn-2 promoter-reporter constructs and the KLF4 acetylation-deficient or phosphorylation-deficient mutants were constructed. Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression. The luciferase assay and chromatin immunoprecipitation were used to detect the transactivation of KLF4 on mfn-2 gene expression. Co-immunoprecipitation and GST pull-down assays were used to determine the modification of KLF4 and interaction of KLF4 with p300 in VSMCs. KLF4 mediated ATRA-induced mfn-2 expression in VSMCs. KLF4 bound directly to the mfn-2 promoter and activated its transcription. ATRA increased the interaction of KLF4 with p300 by inducing KLF4 phosphorylation via activation of JNK and p38 MAPK signaling. KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter. ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.

Peroxisome proliferator-activated receptor δ regulates mitofusin 2 expression in the heart

Mitofusin 2 (Mfn2) has been proposed as an important mitochondrial protein in maintaining mitochondrial network and bioenergetics. Mfn2 is highly expressed in the heart, but is downregulated in response to hypertrophic stimuli. However, little is known about how Mfn2's expression is regulated in cardiomyocytes. Here, we have investigated how Mfn2 expression in the heart responds to fasting condition and determined if Mfn2 is one of those PPARδ-selective target genes that are involved in myocardial energy metabolism. Fasting for 48 h in mice led to a robust increase of Mfn2 expression in the heart. On the other hand, cardiomyocyte-restricted PPARδ deficiency in mice led to substantially diminished cardiac expression of Mfn2 transcript and protein compared to that of controls. Fasting induced cardiac expression of Mfn2 was blunted in cardiomyocyte-restricted PPARδ deficient hearts. Moreover, PPARδ-selective ligand treatment in cultured cardiomyocytes induced elevated Mfn2 expression. A functional PPRE consensus sequence located at − 837 to − 817 bp upstream of the mouse Mfn2 promoter was identified and confirmed by Electrophoretic Mobility Shift Assays and Luciferase Promoter Reporter Assays. We conclude that Mfn2 is a PPARδ-selective target, which may play an important role in regulating myocardial energy homeostasis.

Mitochondrial Fusion Is Increased by the Nuclear Coactivator PGC-1β

BackgroundThere is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression.Methodology/Principal FindingsHere, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1β is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1β increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1β-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1β increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor α (ERRα).Conclusions/SignificanceTaken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1β in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.

Evidence for a Mitochondrial Regulatory Pathway Defined by Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α, Estrogen-Related Receptor-α, and Mitofusin 2

Mitofusin 2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion and regulates mitochondrial metabolism in mammalian cells. Here, we show that Mfn2 gene expression is induced in skeletal muscle and brown adipose tissue by conditions associated with enhanced energy expenditure, such as cold exposure or beta(3)-adrenergic agonist treatment. In keeping with the role of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1 alpha on energy expenditure, we demonstrate a stimulatory effect of PGC-1 alpha on Mfn2 mRNA and protein expression in muscle cells. PGC-1 alpha also stimulated the activity of the Mfn2 promoter, which required the integrity of estrogen-related receptor-alpha (ERR alpha)-binding elements located at -413/-398. ERR alpha also activated the transcriptional activity of the Mfn2 promoter, and the effects were synergic with those of PGC-1 alpha. Mfn2 loss of function reduced the stimulatory effect of PGC-1 alpha on mitochondrial membrane potential. Exposure to cold substantially increased Mfn2 gene expression in skeletal muscle from heterozygous Mfn2 knock-out mice, which occurred in the presence of higher levels of PGC-1 alpha mRNA compared with control mice. Our results indicate the existence of a regulatory pathway involving PGC-1 alpha, ERR alpha, and Mfn2. Alterations in this regulatory pathway may participate in the pathophysiology of insulin-resistant conditions and type 2 diabetes.