User:22healpromoter/New sandbox

= Citrate malate shuttle = The citrate-malate shuttle is a biochemical system that transports acetyl-CoA in the mitochondrial matrix across the inner and outer mitochondrial membrane for fatty acid synthesis. As the inner mitochondrial membrane is impermeable to acetyl-CoA, the shuttle system is essential to fatty acid synthesis in the cytosol. It plays an important role in hepatic lipogenesis. The name of the citrate-malate shuttle is derived from the two intermediates citrate and malate that carry the acetyl-CoA molecule across the mitochondrial double membrane.

The citrate-malate shuttle is present in humans and higher eukaryotic organisms, and is closely related to the Krebs cycle. The system is responsible for the transportation of malate into the mitochondrial matrix as an intermediate in the Krebs cycle and the transportation of citrate into the cytosol for secretion in some fungi such as aspergillus niger, that is commercially used to produce citric acid.

= Mechanism = All cells need the energy to survive. Mitochondria is a double-membrane structure in the body cell that generates and transports essential metabolic products. The 3 layers of this structure are the outer membrane, intermembrane space and inner membrane. The space inside the mitochondria is called the mitochondrial matrix, while the region outside is the cytosol. Murrell suggested that the outer membrane allows most small molecules to pass through. In contrast, the inner membrane transports specific molecules only. The shuttle acts as a pump to drive the substances from the inner membrane to the outside. The pump is important for certain metabolic processes, such as fatty acid synthesis and the citric acid cycle.

The citrate malate shuttle system consists of citrate shuttle and malate shuttle, which are carrier proteins. There are 6 major steps in this pathway. Acetyl-CoA is a molecule that is involved in ATP synthesis, protein metabolism, and lipid metabolism. In addition, it is the precursor material in the reaction. As the inner membrane is not permeable to this molecule, a conversion is needed for effective transport.

First, the acetyl group of Acetyl-CoA combines with oxaloacetate to form citrate, releasing the coenzyme group (CoA) in the mitochondrial matrix. In the second step, citrate would bind to citrate transporters. The shuttle delivers citrate from the inner membrane to the intermembrane space. Then, there is a net movement of citrate from intermembrane space to the cytosol across the outer membrane, following the concentration gradient, i.e. from a region of higher citrate concentration to that of lower citrate concentration. After the citrate is transported out, it is broken down into the original reactants. Using ATP as energy, citrate is lyzed into the acetyl group and oxaloacetate. The acetyl group would join the coenzyme in the cytosol, forming acetyl-CoA.

Moreover, oxaloacetate is reduced by NADH (a reducing agent in our body) to malate in the cytosol, releasing free electrons. PubChem put forward the idea that malate would be transported by the malate shuttle, moving from cytosol to matrix. It is a highly efficient transport system. At last, the malate is oxidized by NAD+ (the oxidizing agent) to oxaloacetate again, releasing NADH. The replenishment of oxaloacetate can be achieved. The oxaloacetate can react with the Acetyl-CoA in the first step, completing a cycle.

= Steps = Table 1: Summary of the Reactants and Products involved in 6 Steps of the Citrate-Malate-Shuttle System

The table shows the overall processes and chemical molecules present in the 6 major steps, including their location in cells. A cycle is formed by the system, ensuring that the conversion between Acetyl-CoA, oxaloacetate, citrate and malate can continue itself without the need of foreign molecules addition.

= Significance = Acetyl-CoA is generated in the mitochondrial matrix from two sources: pyruvate decarboxylation in glycolysis and break down of fatty acids through β-oxidation. Pyruvate decarboxylation is the step that connects glycolysis and the Krebs cycle regulated by the pyruvate dehydrogenase complex when blood glucose level is high. Fatty acid β-oxidation occurs when blood glucose level is low and acetyl-CoA is required to generate ATP through the Krebs cycle. The citrate-malate shuttle allows the cell to produce fatty acid with excess acetyl-CoA in mitochondria when blood glucose level is high for storage and use in the future. The principle is similar to that of insulin, which turns excess glucose in the body to glycogen for storage in the liver cells and skeletal muscles, so that the body would still provide itself with glucose by breaking down the glycogen when there is a lack of energy intake. The citrate-malate shuttle enables more compact storage of energy in the form of fatty acid by transporting acetyl-CoA into the cytosol for fatty acid and cholesterol synthesis. The lipids produced can then be stored, so that they can be used in the future.

In a subject with defect in the citrate-malate shuttle, fatty acid synthesis is hindered by the lack of cytosolic acetyl-CoA and the body would not be able to store excess energy in the form of lipids as efficiently as a normal subject.

= Linkage to Krebs cycle = Both citrate and malate involved in the citrate-malate shuttle are necessary intermediates of the Krebs cycle. Usually oxaloacetate in the Krebs cycle is generated from carboxylation of pyruvate in the mitochondrion, however malate generated in the cytosol can also enter the mitochondrion through the transport protein located in the inner mitochondrial membrane to directly join the Krebs cycle.

The mitochondrial transport proteins are encoded by the SLC25 gene in humans and facilitates the transportation of various metabolites, including citrate and malate in the Krebs cycle. These transport proteins control the flow of metabolites in and out of the inner mitochondrial membrane that is impermeable to most molecules. These transport proteins connect carbohydrate metabolism of the Krebs cycle to fatty acid synthesis in lipogenesis by catalysing the transportation of acetyl-CoA out of the mitochondrial matrix into the cytosol, which is done in the form of citrate export from the mitochondria to cytosol. Cytosolic citrate is a key substrate for the generation of energy. It releases acetyl-CoA and provides NADPH for fatty acid synthesis and in subsequent pathways generates NAD+ for glycolysis. Citrate also activates acetyl-CoA carboxylase, an enzyme that is essential in the fatty acid synthesis pathway.

= Associated Diseases =

Alternate metabolic pathway in cancer cell
Recent study proposed that the citrate-malate shuttle may contribute to sustaining cancer cells through a β-oxidation-citrate–malate shuttle metabolic pathway. In normal cells, β-oxidation produces acetyl-CoA which enters the Krebs cycle to produce ATP, and β-oxidation cannot continue if the Krebs cycle is impaired and acetyl-CoA accumulates. However, cancer cells may carry out continuous β-oxidation by connecting it to the citrate-malate shuttle. The new metabolic pathway consists of mitochondrial transport proteins and several enzymes, including ATP-citrate lyase (ACLY) and malate dehydrogenases 1 and 2 (MDH1 and MDH2). The proposed metabolic pathway may explain the Warburg effect and hypoxia in cancer.

The energy efficiency of this pathway is 3.76 times less than the normal β-oxidation Krebs cycle pathway, only producing 26 moles instead of 98 moles of ATP from 1 mole of palmitate.

It is still unsure whether this pathway exists in cancer cells. The lipotoxicity of palmitate and stearate may be a factor preventing this pathway from occurring.

Hepatocarcinogenesis
The liver is a metabolic active tissue in the human body. Any abnormalities in biological processes that affect the liver metabolism might have effects on liver cancer development, i.e. hepatocarcinogenesis. Hepatocellular carcinoma (HCC) is a prevalent type of liver cancer that accounts for over 80% of cases. Lei et al. argued that it is lethal cancer due to the remarkable drug tolerance, potential of metastasis and a high chance of relapse. Scientists have carried out many kinds of research in finding the risk factors of HCC progression. Apart from a hepatic viral infection, metabolic disorders also significantly increase the chance of hepatocarcinogenesis. Several underlying mechanisms might explain the reasons. The responsibilities of the liver include synthesis of cholesterol, fatty acid, triglyceride and distribution of lipid. It is also a major site for detoxification, protein and carbohydrate metabolism. Mitochondria is responsible for oxidation with NAD participation. Step 4 of the shuttle system would produce NAD. In high obesity or insulin resistance patients, their body contains large amounts of fatty acid. The shuttle system might not generate sufficient NAD. As a high NAD level can potentially decrease the chance of liver cancer progression, the patients are more likely to suffer from HCC. Moreover, the mitochondria would overload, leading to an increased reactive oxygen species level in the liver. Those species can induce HCC development due to their ability in DNA damage. Scientists have carried out many kinds of research in finding the risk factors of HCC progression. Apart from a hepatic viral infection, metabolic disorders also significantly increase the chance of hepatocarcinogenesis. Several underlying mechanisms might explain the reasons. The responsibilities of the liver include synthesis of cholesterol, fatty acid, triglyceride and distribution of lipid. It is also a major site for detoxification, protein and carbohydrate metabolism. Mitochondria is responsible for oxidation with NAD participation. Step 4 of the shuttle system would produce NAD. In high obesity or insulin resistance patients, their body contains large amounts of fatty acid. The shuttle system might not generate sufficient NAD. As a high NAD level can potentially decrease the chance of liver cancer progression, the patients are more likely to suffer from HCC. Moreover, the mitochondria would overload, leading to an increased reactive oxygen species level in the liver. Those species can induce HCC development due to their ability in DNA damage.

Scientists have carried out many kinds of research in finding the risk factors of HCC progression. Apart from a hepatic viral infection, metabolic disorders also significantly increase the chance of hepatocarcinogenesis. Several underlying mechanisms might explain the reasons. The responsibilities of the liver include synthesis of cholesterol, fatty acid, triglyceride and distribution of lipid. It is also a major site for detoxification, protein and carbohydrate metabolism. Mitochondria is responsible for oxidation with NAD participation. Step 4 of the shuttle system would produce NAD. In high obesity or insulin resistance patients, their body contains large amounts of fatty acid. The shuttle system might not generate sufficient NAD. As a high NAD level can potentially decrease the chance of liver cancer progression, the patients are more likely to suffer from HCC. Moreover, the mitochondria would overload, leading to an increased reactive oxygen species level in the liver. Those species can induce HCC development due to their ability in DNA damage.

Another risk factor is mutations and overexpressed citrate-malate shuttle. Ras oncogene, which is a high frequency mutated gene in a wide range of cancers, has a significantly close association to HCC. The research of Lei et al., shows that there is a noticeable increase in the HCC patients’ citrate and malate levels, suggesting the possibility of higher activity of citrate-malate shuttle. Those patients carry the Ras gene. The electrons provided by the shuttle play a crucial role in the normal function of mitochondria and ATP generation as an energy source. This mechanism is effective under lowered TCA cycle activity. The shuttle is also involved in the fatty acid synthesis and lactic acid synthesis. In liver cancer cells, the TCA cycle is blocked, causing an excess of pyruvate level. However, the overexpressed citrate-malate shuttle might remove the excessive pyruvate, preventing the natural cell death of the cancer cells. This higher shuttle activity would generate more fatty acid, which is a potential cause of HCC.

= Genetics and Evolution = The replication of mitochondrial DNA follows binary fission, which separates a body into two bodies. The mitochondrial gene of children is inherited from their mother only. If there are any genetic defects or mutations in the mother’s mitochondrial DNA, it would pass to the children. If those changes in genes can cause mitochondrial diseases, the children have a 100% possibility of acquiring the diseases. For the malate-oxaloacetate shuttle, there are 4 major genes involved. They are PMDH1, MDH, PMDH2, mMDH1. PMDH-1 and PMDH-2 encode two different enzymes that provide NAD for the oxidation of malate. In addition, MDH and mMDH1 encode for an enzyme that directly oxidizes malate.

For the malate-oxaloacetate shuttle, there are 4 major genes involved. They are PMDH1, MDH, PMDH2, mMDH1. PMDH-1 and PMDH-2 encode two different enzymes that provide NAD for the oxidation of malate. In addition, MDH and mMDH1 encode for an enzyme that directly oxidizes malate.

SLC25 is a gene that is essential for the synthesis of a wide range of mitochondrial transporters, including citrate shuttle. Mutations in this gene can result in dysfunctional mitochondria, significantly reducing the energy production of our body cells. severe metabolic diseases. It can cause severe symptoms in organs or tissues that have high energy demand. These organs include the liver, brain, heart, kidneys. They require abundant functional mitochondria to function. Mitochondrial disorders caused by defective or reduced SLC25 gene expression can result in diseases, such as CAC deficiency, HHH syndrome, AGC2 deficiency (CTLN2/NICCD), adPEO, Congenital Amish microcephaly, Early epileptic encephalopathy, AAC1 deficiency, PiC (isoform A) deficiency, AGC1 deficiency, Neuropathy with striatal necrosis, and Congenital sideroblastic anaemia.

In addition, the frequency of a gene among organisms indicates how critical the gene is. Genetic variation randomly exists in organisms. The struggle for survival creates a natural selection pressure. Genes that are favourable for the survival of a species in response to the environmental features are preserved and passed along the generation. Nonetheless, the genes that are not favourable would be eliminated out of the gene pool. Palmieir argued that not only is the SLC25 gene found in humans, but also in other animals, or even microorganisms like bacteria and viruses. This might provide evidence for the significance and essentialness of the gene in the survival of organisms. Therefore, SLC25 gene is preserved during evolution.

= References =