[IEEE 2013 Annual International Conference on Emerging Research Areas (AICERA) - 2013 International...
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International Conference on Microelectronics, Communication and Renewable Energy (ICMiCR-2013) Incorporation of Flux Balance Analysis in Pre-β- oxidation Network to Detect Carnitine Translocase in the Cardiac Cell in Homo sapiens 1 Tarika Vijayaragahavan Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, CBD Belapur 400611, Navi Mumbai, India, E-Mail: [email protected] 2 Somnath Tagore Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, CBD Belapur 400611, Navi Mumbai, India, E-Mail: [email protected] Abstract-Fats are immensely important for the survival of the human body. These are stored in the body in the form of fatty acids and reused whenever the body is unable to consume essential fats(at the time of exercise, fasting etc).In the human heart cell, the transport of certain fatty acids into the mitochondria is required to produce energy. This is done via a certain enzyme called Carnitine Translocase .This paper emphasizes on the simulation of the presence of Carnitine Translocase. Metabolic flux balance analysis was done to detect the presence of this enzyme, thus by preventing the accumulation of excess Fatty Acyl Carnitine that is known to give rise to various Cardiovascular diseases such as hypoglycemia, arrhythmia etc. Keywords - Carnitine Translocase, Flux Balance Analysis, Fatty acids, Carnitine shuttle, CPT, Malonyl-CoA. INTRODUCTION The heart requires a high supply to energy in order to function and nourish the human body. Adenosine triphosphate (ATP) an energy rich compound supplies energy to the human heart. The heart has a comparatively low level of ATP content as compared to the process of ATP hydrolysis. Therefore, in order to generate enough ATP, the heart uses various metabolites present in the cell. Most of these metabolites are present in the form of glucose, free fatty acids, essential fatty acids, co-factors, co-enzymes etc. Beta-oxidation of fatty acids, a catabolic process which takes place in the mitochondrial membrane, generates maximum number of ATP molecules required for processing the human heart. The fatty acids with chain lengths of twelve or fewer carbons enter mitochondria without the help of membrane transporters. However, those with fourteen or more carbons which constitute the majority of free fatty acids obtained in the diet cannot pass through the mitochondrial membrane. They pass through the Carnitine shuttle which is composed of various enzymes. One such enzyme is Carnitine Translocase (CT), which is responsible for the transportation of the fatty acyl Carnitine from the outer mitochondrial membrane to the matrix. In this paper we have used Flux Balance Analysis methods to maximize the objective of finding the CT enzyme in the inter-membrane space of mitochondria [1]. I. CARNITINE SHUTTLE The Carnitine shuttle present in the cell consists of three reactions. The first reaction involves the formation of fatty acyl CoA from Fatty acids that are present in the cytosol. The enzymes include fatty-acyl-CoA synthetase and inorganic pyrophosphatase. The fatty acyl CoA esters formed at the cytosolic sites either enter the mitochondrial matrix or are used in the cytosol to synthesize lipids. The second reaction includes the binding of the fatty acyl group to the hydroxyl group of Carnitine to form Fatty acyl Carnitine. The third reaction involves the transport of fatty acyl Carnitine into the mitochondrial membrane by a series of enzymes called isozymes – CPT-1 (Carnitine palmitoyltransferase-1), Carnitine Translocase and CPT-2.The isozyme located at the inner surface of the mitochondrial membrane regenerates fatty acyl CoA and releases it, along with free Carnitine into the matrix. Carnitine re-enters the inter membrane space with Carnitine Translocase. This Carnitine mediated entry process is the rate limiting step for oxidation of fatty acids in mitochondria and hence is the regulating point [2]. II. CARNITINE TRANSLOCASE Carnitine Translocase also known as the Carnitine acylcarnitine Translocase is an enzyme present in the inter-membrane space of mitochondria. This enzyme is the coded by an SLC gene (solute carrier gene, family 25, member 20). The main function of this enzyme is to transport the fatty acyl Carnitine from the outer membrane to the inner membrane, where it is free from its bound form and the free Carnitine moves back to the outer mitochondrial membrane. Fig. 1 denotes the placement of Carnitine translocase. 978-1-4673-5149-2/13/$31.00 ©2013 IEEE
Transcript of [IEEE 2013 Annual International Conference on Emerging Research Areas (AICERA) - 2013 International...
International Conference on Microelectronics, Communication and Renewable Energy (ICMiCR-2013)
Incorporation of Flux Balance Analysis in Pre-β-oxidation Network to Detect Carnitine Translocase in
the Cardiac Cell in Homo sapiens 1Tarika Vijayaragahavan
Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, CBD Belapur
400611, Navi Mumbai, India,E-Mail: [email protected]
2Somnath Tagore Department of Biotechnology and Bioinformatics,
Padmashree Dr. D.Y. Patil University, CBD Belapur 400611, Navi Mumbai, India,
E-Mail: [email protected]
Abstract-Fats are immensely important for the survival of the human body. These are stored in the body in the form of fatty acids and reused whenever the body is unable to consume essential fats(at the time of exercise, fasting etc).In the human heart cell, the transport of certain fatty acids into the mitochondria is required to produce energy. This is done via a certain enzyme called Carnitine Translocase .This paper emphasizes on the simulation of the presence of Carnitine Translocase. Metabolic flux balance analysis was done to detect the presence of this enzyme, thus by preventing the accumulation of excess Fatty Acyl Carnitine that is known to give rise to various Cardiovascular diseases such as hypoglycemia, arrhythmia etc.
Keywords - Carnitine Translocase, Flux Balance Analysis, Fatty acids, Carnitine shuttle, CPT, Malonyl-CoA.
INTRODUCTION
The heart requires a high supply to energy in order to functionand nourish the human body. Adenosine triphosphate (ATP) an energy rich compound supplies energy to the human heart. The heart has a comparatively low level of ATP content as compared to the process of ATP hydrolysis. Therefore, in order to generate enough ATP, the heart uses various metabolites present in the cell. Most of these metabolites are present in the form of glucose, free fatty acids, essential fatty acids, co-factors, co-enzymes etc. Beta-oxidation of fatty acids, a catabolic process which takes place in the mitochondrial membrane, generates maximum number of ATP molecules required for processing the human heart. The fatty acids with chain lengths of twelve or fewer carbons enter mitochondria without the help of membrane transporters. However, those with fourteen or more carbons which constitute the majority of free fatty acids obtained in the diet cannot pass through the mitochondrial membrane. They pass through the Carnitine shuttle which is composed of various enzymes. One such enzyme is Carnitine Translocase (CT), which is responsible for the transportation of the fatty acyl Carnitine from the outer mitochondrial membrane to the matrix. In this paper we have used Flux Balance Analysis methods to maximize
the objective of finding the CT enzyme in the inter-membrane space of mitochondria [1].
I. CARNITINE SHUTTLE
The Carnitine shuttle present in the cell consists of three reactions. The first reaction involves the formation of fatty acyl CoA from Fatty acids that are present in the cytosol. The enzymes include fatty-acyl-CoA synthetase and inorganic pyrophosphatase. The fatty acyl CoA esters formed at the cytosolic sites either enter the mitochondrial matrix or are used in the cytosol to synthesize lipids. The second reaction includes the binding of the fatty acyl group to the hydroxyl group of Carnitine to form Fatty acyl Carnitine. The third reaction involves the transport of fatty acyl Carnitine into the mitochondrial membrane by a series of enzymes called isozymes – CPT-1 (Carnitine palmitoyltransferase-1), Carnitine Translocase and CPT-2.The isozyme located at the inner surface of the mitochondrial membrane regenerates fatty acyl CoA and releases it, along with free Carnitine into the matrix. Carnitine re-enters the inter membrane space with Carnitine Translocase. This Carnitine mediated entry process is the rate limiting step for oxidation of fatty acids in mitochondria and hence is the regulating point [2].
II. CARNITINE TRANSLOCASECarnitine Translocase also known as the Carnitine acylcarnitine Translocase is an enzyme present in the inter-membrane space of mitochondria. This enzyme is the coded by an SLC gene (solute carrier gene, family 25, member 20). The main function of this enzyme is to transport the fatty acyl Carnitine from the outer membrane to the inner membrane, where it is free from its bound form and the free Carnitine moves back to the outer mitochondrial membrane. Fig. 1 denotes the placement of Carnitine translocase.
978-1-4673-5149-2/13/$31.00 ©2013 IEEE
International Conference on Microelectronics, Communication and Renewable Energy (ICMiCR-2013)
Fig. 1 The transport of fatty acid into the mitochondrial membrane.
III. FLUX BALANCE ANALYSIS
Flux balance analysis (FBA) calculates the flow of metabolites through a given metabolic network. This analysis is used to calculate the growth rate of certain metabolites in that network. Metabolic reactions are represented as a stoichiometric matrix of the equation 0. =vS . The values ineach column are the stoichiometric numbers present in the reaction. Negative values are assigned for every flux leaving a metabolite, positive values are assigned to every metabolite that receives a flux. Zero values are given to the metabolites that do not participate in the reaction. The flux through the network is represented by a vector. At quasi- steady state, the mass balance equation is considered at an equation resulting to zero. Any vector that satisfies the equation is said to be the null space of S. We are interested in maximizing the possibility of CT in the inter-membrane space of mitochondrial membrane in the heart cell in humans.
A. THE OBJECTIVE FUNCTION The objective function of our interest is to maximize the occurrence of Carnitine Translocase(CT) enzyme. The simulation was carried using the linprog function that is a pre-defined function present in the MATLAB program. An optimization toolbox was used to carry out the same. A stoichiometric matrix was created on the basis of the network reconstructed. Initial concentration values for the metabolties were given and considered at ‘lower bound’ limit, whereas the ‘upper bound’ limit was kept constant throughout for all metabolites.
B. INHIBITION OF CPT-1 BY MALONYL COA Malonyl- CoA is produced from Acetyl CoA for fatty acid synthesis. However, when produced in excess (due to the consumption of excess carbohydrates), Malonyl CoA is known to inhibit the activity of CPT-1 enzyme. Our first
module consists of the inhibition of malonyl CoA on CPT-1 enzyme. Stoichiometric matrix was constructed accordingly and the flux was calculated. Here, the initial concentration of Acetyl CoA and Malonyl CoA was kept higher than that of CPT-1
C. TRANSPORT OF FATTY ACYL CARNITINE THROUGH CPT-1
Under normal conditions, the fatty acyl CoA is converted to Fatty acyl Carnitine in the presence of the enzyme CPT-1. When Malonyl CoA is not produced in excess, it does not inhibit CPT-1. Hence, in our second module we ruled out the inhibition of Malonyl CoA and included only those reactions that lead to the transport of fatty acyl Carnitine to Carnitine Translocase. Here, the initial concentration of CPT-1 enzyme was kept higher than other metabolites and the then objective function was studied.
IV. RESULTS AND DISCUSSIONSIn our first module, the objective function showed a zero value meaning that due to the inhibition by another metabolite, the fatty acyl CoA was unable to pass through the memebrane and hence it got deposited in the cytosol itself (Table I).
TABLE I. Inhibition of CPT1 by Malonyl CoA
Metabolites Concentration (mM)
Objective Function
Final Concentration (mM)
Acetyl CoA 0.05 0.0 0.05
Malonyl CoA 0.05 0.0 0.05 CPT1 0.005 0.0 0.05Fatty acid 0.0 0.0 0.05 Fatty acyl CoA 0.0 0.0 0.05 Fatty acyl carnitine
0.0 0.0 0.05
Carnitine translocase
0.0 0.0 0.05
CPT2 0.0 0.0 0.05
TABLE II. Transport of Fatty-acyl CoA to carnitine translocase
Metabolites Concentration (mM)
Objective Function
Final Concentration (mM)
Acetyl CoA 0.00 0.0 0.05
Malonyl CoA 0.00 0.0 0.05 CPT1 0.05 0.05 0.05Fatty acid 0.00 0.0 0.05 Fatty acyl CoA 0.00 0.0 0.05 Fatty acyl carnitine
0.00 0.0 0.05
Carnitine translocase
0.00 0.0 0.05
CPT2 0.00 0.0 0.05
International Conference on Microelectronics, Communication and Renewable Energy (ICMiCR-2013)
In our second module, the objective function showed a positive value which meant that the fatty acyl CoA was easily able to pass through the mitochondrial membrane. In this case there was no inhibition by another metabolite (Table II).
V. REFERENCES [1] Gary L, John U, Clifford F, Jagdip J, William, S, “Myocardial Fatty Acid Metabolism in Health and Disease", Physiol Rev., Vol. 90, No. 1, pp. 207-58, 2010. [2] Nelson DL, Cox, MM, Lehninger Principles of Biochemistry, 2004. [3] Jeffrey O, Ines T, Bernhard O, "What is flux balance analysis?" Nat Biotechnol., Vol. 2, No.3, pp. 245-8, 2010. [4] Feist AM, Palsson BO, “The growing scope of applications of genome-scale metabolic reconstructions using Escherichia coli”, Nat Biotech., Vol. 26, pp. 659–667, 2008.
