ndrom Sy es tic & e G n Asadi et al., J Genet Syndr Gene Ther 2016, 7:6 e e n G e f T o Journal of Genetic Syndromes & DOI: 10.4172/2157-7412.1000314 h l e a r n a r p u y o J ISSN: 2157-7412 Gene Therapy Research Article Open Access Assessment of Pep, Fndc5, C2c12, Pgc1α Pattern of Genes Expression during Differentiation of Human Embryonic Stem Cells into Heart Cells Shahin Asadi1*, Zahra Gholizadeh1, Mahsa Sadat Mir Jamali2 and Mina Niknia2 1Research Center for Stem Cell and Drug Applied Research Center, Tabriz University of Medical Sciences in modern biology, Iran 2Young Researchers and Elite Club, Tabriz Branch, Islamic Azad University, Tabriz, Iran Abstract FNDC5 gene with another protein called Xuemei Proxy (PEP) is a 209 amino acid protein coding. These genes mainly in heart tissue, skeletal muscle and brain expressed. This study aimed to clarify the pattern of expression of this gene in mouse embryonic cells Heart cells taken. The mouse embryonic stem cells as a model for cardiac differentiation induced by ascorbic acid used and the pattern of expression of PEP at certain stages of differentiation were analyzed by Real-Time PCR technique. The results show a dramatic increase in PEP gene expression in the adult cardiomyocytes. PEP increased expression of genes may have a role in later stages cardiogenesis is possible that further studies are needed to identify it. Keywords: Protein proxy xuemei; Cardiac differentiation; Embryonic The ectodomain was proposed to be cleaved to give a soluble stem cells and mice; Real-time PCR peptide hormone named irisin. Separately it was proposed that irisin is secreted from muscle in response to exercise, and may mediate some Introduction beneficial effects of exercise in humans and the potential for generating Fibronectin type III domain-containing protein 5, the precursor of weight loss and blocking diabetes has been suggested [2,5-11]. Others irisin, is a protein that is encoded by the FNDC5 gene [1]. Irisin is a questioned these findings [1,12-14] (Figure 3). cleaved version of FNDC5, named after the Greek messenger goddess The FNDC5 gene encodes a pro hormone, a single-pass type I Iris [2] (Figures 1 and 2). membrane protein (human, 212 amino acids; mouse and rat, 209 amino acids) that is upregulated by muscular exercise and undergoes post- Fibronectin domain-containing protein 5 is a membrane protein translational processing to generate irisin. The sequence of the protein comprising a short cytoplasmic domain, a transmembrane segment, includes a signal peptide, a single fibronectin type III domain and a and an ectodomain consisting of a ~100 kDa fibronectin type III (FNIII) C-terminal hydrophobic domain that is anchored in the cell membrane. domain. FNDC5 was discovered during a genome search for fibronectin type III domains [3] and independently in a search for peroxisomal proteins [1,4]. Figure 1: The amino acid sequence encoded by the gene FNDC5. Figure 3: How FNDC5 gene and PGC1a neuron in the human brain functions. *Corresponding author: Shahin Asadi, Research Center for Stem Cell and Drug Ap- plied Research Center, Tabriz University of Medical Sciences in modern biology, Iran, Tel: +989379923364; E-mail: [email protected] Received October 25, 2016; Accepted November 21, 2016; Published November 28, 2016 Citation: Asadi S, Gholizadeh Z, Jamali MSM, Niknia M (2016) Assessment of Pep, Fndc5, C2c12, Pgc1α Pattern of Genes Expression during Differentiation of Human Embryonic Stem Cells into Heart Cells. J Genet Syndr Gene Ther 7: 314. doi: 10.4172/2157-7412.1000314 Copyright: © 2016 Asadi S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted Figure 2: Structure of N-terminal and C-terminal gene FNDC5 the region. use, distribution, and reproduction in any medium, provided the original author and source are credited. J Genet Syndr Gene Ther, an open access journal ISSN: 2157-7412 Volume 7 • Issue 6 • 1000314 Citation: Asadi S, Gholizadeh Z, Jamali MSM, Niknia M (2016) Assessment of Pep, Fndc5, C2c12, Pgc1α Pattern of Genes Expression during Differentiation of Human Embryonic Stem Cells into Heart Cells. J Genet Syndr Gene Ther 7: 314. doi: 10.4172/2157-7412.1000314 Page 2 of 10 The production of irisin is similar to the shedding and release of it a health promoting hormone [5,6]. However this proposal has been other hormones and hormone-like polypeptides, such as epidermal challenged [17] because FNDC5 is upregulated only in highly active growth factor and TGF alpha, from transmembrane precursors. After elderly humans [12]. the N-terminal signal peptide is removed, the peptide is proteolytically A 2016 in vitro study of white and brown fat cell tissue found cleaved from the C-terminal moiety, glycosylated and released as a dose-related upregulation of a protein called UCP1 that contributes hormone of 112 amino acids (in human, amino acids 32-143 of the full- to the browning of white fat and found other markers that would length protein; in mouse and rat, amino acids 29-140) that comprises indicate that the white cells were browning and that fat cells were more most of the FNIII repeat region. metabolically active. Many of the stem cells became a type of cell that The sequence of irisin, the cleaved and secreted portion of FNDC5, matures into bone. The tissue treated with irisin produced about 40 is highly conserved in mammals; the human and murine sequences are percent fewer mature fat cells [18]. In mice, irisin released from skeletal identical [2]. However, the start codon of human FNDC5 is mutated muscle during exercise acts directly on bone by increasing cortical to ATA, which causes it to be expressed at only 1% the level of other bone mineral density, bone perimeter and polar moment of inertia. animals with the normal ATG start - though its circulation levels are still on par with other key human hormones, such as insulin [15]. Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme in the lyase family used in the metabolic pathway of gluconeogenesis. It A difference in the nucleotide sequence of human FNDC5 from converts oxaloacetate into phosphoenolpyruvate and carbon dioxide that of mouse Fndc5 creates a different initiation codon, potentially [1,19,20]. It is found in two forms, cytosolic and mitochondrial. generating a protein that begins at methionine-76 (Met-76). A protein initiated at Met-76 would be missing the signal peptide and would be In humans there are two isoforms of PEPCK: a cytosolic form trapped in the cytoplasm. Via mass spectrometry, irisin has been found (SwissProt P35558) and a mitochondrial isoform (SwissProt Q16822) to circulate in humans in levels similar to other key hormones, such as which have 63.4% sequence identity. The cytosolic form is important insulin [15] (Figure 4). in gluconeogenesis. However, there is a known transport mechanism to move PEP from the mitochondria to the cytosol, using specific Exercise causes increased expression in muscle of peroxisome membrane transport proteins. proliferator-activated receptor gamma co-activator 1 alpha (PGC- 1alpha), which is involved in adaptation to exercise. In mice, this X-ray structures of PEPCK provide insight into the structure and the causes production of the FNDC5 protein which is cleaved to give a mechanism of PEPCK enzymatic activity. The mitochondrial isoform of new product irisin [2,7]. Due to its production through a mechanism chicken liver PEPCK complexed with Mn2+, Mn2+-phosphoenolpyruvate initiated by muscular contraction, irisin has been classified as a (PEP), and Mn2+-GDP provides information about its structure and how myokine [16]. this enzyme catalyzes reactions [2]. Delbaere et al. resolved PEPCK in E. Based on the findings that FNDC5 induces thermogenin expression coli and found the active site sitting between a C-terminal domain and in fat cells, overexpression of FNDC5 in the liver of mice prevents diet- an N-terminal domain. The active site was observed to be closed upon induced weight gain, and FNDC5 mRNA levels are elevated in human rotation of these domains [3]. muscle samples after exercise, it has been proposed that irisin promotes Phosphoryl groups are transferred during PEPCK action, which is the conversion of white fat to brown fat in humans which would make likely facilitated by the eclipsed conformation of the phosphoryl groups when ATP is bound to PEPCK [3]. Since the eclipsed formation is one that is high in energy, phosphoryl group transfer has a decreased energy of activation, meaning that the groups will transfer more readily. This transfer likely happens via a mechanism similar to SN2 displacement [3]. PEPCK gene transcription occurs in many species, and the amino acid sequence of PEPCK is distinct for each species. For example, its structure and its specificity differ in humans, Escherichia coli (E. coli) and the parasite Trypanosoma cruzi [4] (Figure 5). PEPCase converts oxaloacetate into phosphoenolpyruvate and carbon dioxide. As PEPCK acts at the junction between glycolysis and the Krebs cycle, it causes decarboxylation of a C4 molecule, creating a C3 molecule. As the first committed step in gluconeogenesis, PEPCK decarboxylates and phosphorylates oxaloacetate (OAA) for its conversion to PEP, when GTP is present. As a phosphate is transferred, the reaction results in a GDP molecule. When pyruvate kinase-the enzyme that normally catalyzes the reaction that converts PEP to pyruvate-is knocked out in mutants of Bacillus subtilis, PEPCK participates in one of the replacement anaplerotic reactions, working in the reverse direction of its normal function, converting PEP to OAA Figure 4: How FNDC5 gene and PGC1a gene function interacts with Irisin in [5]. Although this reaction is possible, the kinetics is so unfavorable human brain memory a cell.