Unravelling the molecular mechanisms underlying mitochondrial dysfunction in metabolic
diseases
Mposhi, Archibold
DOI:10.33612/diss.146092791
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Mposhi, A. (2020). Unravelling the molecular mechanisms underlying mitochondrial dysfunction in metabolic diseases. University of Groningen. https://doi.org/10.33612/diss.146092791
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1.1. Mitochondria, more than an energy factory!
Mitochondria are primarily considered as the powerhouses of the cell. They produce energy in the form of adenosine triphosphate (ATP) through the process of oxidative phosphorylation. The number of mitochondria in each cell is largely dependent on the metabolic requirements of the cell1. Tissues or organs such as liver, muscle, brain and heart contain relatively more mitochondria due to their high energy demand1-3. However, besides their role in energy metabolism, mitochondria also play a key role in promoting cell survival by mediating adaptive responses to metabolic and cellular stress. This is a function that hinges on their ability to mediate cell signaling processes through the generation of reactive oxygen species (ROS)4,5.
Mitochondria contain their own DNA (mtDNA) (also see Chapter 2) that codes for 13
subunits of the electron transport chain complex (ETC), which is involved in oxidative phosphorylation. Similar to prokaryotic chromosomes of bacteria, mtDNA molecules are supercoiled and packaged together into nucleoprotein complexes containing variable numbers of mtDNA molecules. These complexes are called nucleoids and they allow mtDNA to freely diffuse through the mitochondrial network6,7. Several hundred nucleoids are present in each cell and 1-10 copies of mtDNA are found in each mitochondrion7. The core component of the nucleoid is the mitochondrial transcription factor A (TFAM) that binds to the mtDNA in a non-sequence selective manner. This induces negative supercoiling of mtDNA and greatly compacts the mitochondrial genome6-8.
The mtDNA is comprised of a “heavy” (H) strand and a “light” (L) strand, differentiated by the higher proportion of guanine and adenine nucleotides in the H strand. Whereas there is at least one promoter region per gene in the nuclear DNA (nDNA), the mtDNA contains only three promoter regions directing expression of all encoded genes. These three promoter regions reside in a non-coding region known as the “displacement loop” (D-loop). DNA transcription is initiated in the D-loop to produce polycistronic transcripts for the simultaneous expression of multiple genes9. Transcription of H-strand genes depends on the H-H-strand promoters 1 and 2 (HSP1, HSP2), while L-strand transcription depends on the L-L-strand promotor (LSP), the latter only directing expression of the ND6 gene encoding NADH-ubiquinone oxidoreductase chain 6. The D-loop is a stable genetic structure in human mtDNA, which comprises 1,124 bps
(approximately 7% of the total mitochondrial size)10,11. The D-loop forms a triple helix structure consisting of the H- and L-strand and a 7S DNA primer12 (also see in Chapter
2). The 7S DNA primer forms a third nascent DNA strand which forms the D-loop’s
triple helix structure13,14. This triple helix structure facilitates mtDNA replication by maintaining accessibility of the DNA to the transcription machinery. Apart from the three promoter regions, the D-loop also contains three conserved sequence blocks (CSBs) and the origin of replication of the H-strand (OH). The CSBs are sequences in the mtDNA molecule that have remained highly conserved throughout evolution. Of the three CSBs in the D-loop, CBSII is particularly important for transcription termination and 7S DNA primer formation15.Given the importance of the D-loop in mtDNA replication and transcription, mutations in this structure can result in dysfunctional mitochondria. Mutations in the mtDNA can be deleterious, resulting in mitochondrial dysfunction, impaired energy metabolism and, as a consequence, the development of metabolic diseases.
Mitochondria divide constantly by fusion and fission processes and form a dynamic interconnecting network depending on the cell’s energy state16,17. Wild type and mutated mtDNA molecules coexist, a phenomenon called heteroplasmy16. Importantly, the detection of mutant mtDNA does not necessarily imply mitochondrial dysfunction, but there is a generally accepted notion that mutational load must exceed 60% of all mtDNA molecules within a given tissue to cause a significant (pathological) phenotype18. Therefore, an increase in mutant mtDNA above this threshold results in dysfunctional mitochondria and this gives rise to mitochondrial diseases, a group of disorders that primarily affect the ETC complex19,20. However, due to heteroplasmy, different clinical presentations are induced, which complicates the characterization and diagnosis of mitochondrial diseases16.
There is need to develop diagnostic tools that can use mtDNA as an indicator of different mitochondrial disease states. In this regard, mtDNA acts as a cell signaling molecule in response to cell damage, which makes it a potent damage-associated molecular pattern (DAMP)21. For instance, mtDNA is a high-affinity substrate for toll-like receptor 9 (TLR9), which promotes inflammation in metabolic diseases, such as in non-alcoholic steatohepatitis (NASH)21. Thus, mitochondria are multipurpose
1.1. Mitochondria, more than an energy factory!
Mitochondria are primarily considered as the powerhouses of the cell. They produce energy in the form of adenosine triphosphate (ATP) through the process of oxidative phosphorylation. The number of mitochondria in each cell is largely dependent on the metabolic requirements of the cell1. Tissues or organs such as liver, muscle, brain and heart contain relatively more mitochondria due to their high energy demand1-3. However, besides their role in energy metabolism, mitochondria also play a key role in promoting cell survival by mediating adaptive responses to metabolic and cellular stress. This is a function that hinges on their ability to mediate cell signaling processes through the generation of reactive oxygen species (ROS)4,5.
Mitochondria contain their own DNA (mtDNA) (also see Chapter 2) that codes for 13
subunits of the electron transport chain complex (ETC), which is involved in oxidative phosphorylation. Similar to prokaryotic chromosomes of bacteria, mtDNA molecules are supercoiled and packaged together into nucleoprotein complexes containing variable numbers of mtDNA molecules. These complexes are called nucleoids and they allow mtDNA to freely diffuse through the mitochondrial network6,7. Several hundred nucleoids are present in each cell and 1-10 copies of mtDNA are found in each mitochondrion7. The core component of the nucleoid is the mitochondrial transcription factor A (TFAM) that binds to the mtDNA in a non-sequence selective manner. This induces negative supercoiling of mtDNA and greatly compacts the mitochondrial genome6-8.
The mtDNA is comprised of a “heavy” (H) strand and a “light” (L) strand, differentiated by the higher proportion of guanine and adenine nucleotides in the H strand. Whereas there is at least one promoter region per gene in the nuclear DNA (nDNA), the mtDNA contains only three promoter regions directing expression of all encoded genes. These three promoter regions reside in a non-coding region known as the “displacement loop” (D-loop). DNA transcription is initiated in the D-loop to produce polycistronic transcripts for the simultaneous expression of multiple genes9. Transcription of H-strand genes depends on the H-H-strand promoters 1 and 2 (HSP1, HSP2), while L-strand transcription depends on the L-L-strand promotor (LSP), the latter only directing expression of the ND6 gene encoding NADH-ubiquinone oxidoreductase chain 6. The D-loop is a stable genetic structure in human mtDNA, which comprises 1,124 bps
(approximately 7% of the total mitochondrial size)10,11. The D-loop forms a triple helix structure consisting of the H- and L-strand and a 7S DNA primer12 (also see in Chapter
2). The 7S DNA primer forms a third nascent DNA strand which forms the D-loop’s
triple helix structure13,14. This triple helix structure facilitates mtDNA replication by maintaining accessibility of the DNA to the transcription machinery. Apart from the three promoter regions, the D-loop also contains three conserved sequence blocks (CSBs) and the origin of replication of the H-strand (OH). The CSBs are sequences in the mtDNA molecule that have remained highly conserved throughout evolution. Of the three CSBs in the D-loop, CBSII is particularly important for transcription termination and 7S DNA primer formation15.Given the importance of the D-loop in mtDNA replication and transcription, mutations in this structure can result in dysfunctional mitochondria. Mutations in the mtDNA can be deleterious, resulting in mitochondrial dysfunction, impaired energy metabolism and, as a consequence, the development of metabolic diseases.
Mitochondria divide constantly by fusion and fission processes and form a dynamic interconnecting network depending on the cell’s energy state16,17. Wild type and mutated mtDNA molecules coexist, a phenomenon called heteroplasmy16. Importantly, the detection of mutant mtDNA does not necessarily imply mitochondrial dysfunction, but there is a generally accepted notion that mutational load must exceed 60% of all mtDNA molecules within a given tissue to cause a significant (pathological) phenotype18. Therefore, an increase in mutant mtDNA above this threshold results in dysfunctional mitochondria and this gives rise to mitochondrial diseases, a group of disorders that primarily affect the ETC complex19,20. However, due to heteroplasmy, different clinical presentations are induced, which complicates the characterization and diagnosis of mitochondrial diseases16.
There is need to develop diagnostic tools that can use mtDNA as an indicator of different mitochondrial disease states. In this regard, mtDNA acts as a cell signaling molecule in response to cell damage, which makes it a potent damage-associated molecular pattern (DAMP)21. For instance, mtDNA is a high-affinity substrate for toll-like receptor 9 (TLR9), which promotes inflammation in metabolic diseases, such as in non-alcoholic steatohepatitis (NASH)21. Thus, mitochondria are multipurpose
organelles whose role is not confined to energy production alone but encompasses a diverse range of metabolic and cell signaling functions.
1.2. Mitochondrial dysfunction in metabolic diseases
Mitochondrial dysfunction has been implicated in ageing, myopathy, cancer, diabetes, obesity, non-alcoholic fatty liver disease (NAFLD) and various other metabolic diseases (Figure 1). It is therefore evident that dysfunctional mitochondria pose
detrimental effects on the overall functioning of the cell and impair organ and tissue function.
1.2.1. Myopathy
Myopathy is a disease that affects the muscle tissue and generally manifests as chronic muscle weakness, fatigue, low ATP generation, exercise intolerance, proximal and elevated levels of serum creatine kinase25. In most mitochondrial diseases, impaired ETC functioning results in a reduced capacity to produce ATP. The various forms of myopathy are grouped in two categories, namely inherited (primary) and acquired (secondary) myopathies. Primary myopathies are a result of genetic (nDNA and mtDNA) defects, while secondary myopathies are associated with external factors, such as drugs and toxic agents that induce mitochondrial defects. Mitochondrial defects in skeletal muscle tissue result in the development of mitochondrial myopathy, and patients suffer from myalgia and fatigue.Mitochondrial myopathies occur as a result of mutations in either mitochondrial or nuclear DNA. With respect to mtDNA-encoded genes, mutations in genes encoding cytochrome b (CYTB)25-27 and cytochrome c oxidase 1 (COX1)28,29 have been associated with some forms of mitochondrial myopathy. A depletion of mtDNA in skeletal muscle can also result in mitochondrial myopathy, while in other cases this disease is characterized by an accumulation of mitochondria in muscle fibers. It has been suggested that these elevated levels of mitochondrial content may be a mechanism to compensate for the impaired energy production.
1.2.2 Non-alcoholic fatty liver disease (NAFLD)
The liver is a vital metabolic organ responsible for the body’s energy metabolism through the metabolism of fats, carbohydrates and proteins, a function supported by a high mitochondrial density (approximately 800 mitochondria per cell)1. Primary functions of the liver include storage of glycogen, vitamins and minerals, detoxification, bile production and synthesis of plasma proteins, such as albumin. NAFLD is a progressive metabolic disorder characterized by the accumulation of lipids in the liver, primarily as a result of dietary habits in the Western lifestyle. NAFLD covers a spectrum of hepatic pathologies, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which is typically accompanied by the development of liver fibrosis that may progress to cirrhosis and predisposes for hepatocellular carcinoma (HCC). NASH is the more progressive subtype of NAFLD, which marks the transition from simple steatosis to steatohepatitis and, as such, is characterized by inflammation of the liver. Prime risk factors for NASH are obesity and type 2 diabetes 22-24. NAFLD is a rapidly increasing health problem and contributes to a third of liver-related deaths in developed countries. The global prevalence of NAFLD is on the rise as many regions of the world become industrialized. As there are no drugs yet available to treat NAFLD, only liver transplantation remains as a last life-saving option for patients with end-stage NAFLD. However, limited availability of donor livers and transplantation-associated complications are major challenges to overcome. Treatments that can reverse mitochondrial dysfunction and improve mitochondrial fitness and cell survival may therefore be potential therapeutic interventions for NAFLD and other chronic liver diseases.
organelles whose role is not confined to energy production alone but encompasses a diverse range of metabolic and cell signaling functions.
1.2. Mitochondrial dysfunction in metabolic diseases
Mitochondrial dysfunction has been implicated in ageing, myopathy, cancer, diabetes, obesity, non-alcoholic fatty liver disease (NAFLD) and various other metabolic diseases (Figure 1). It is therefore evident that dysfunctional mitochondria pose
detrimental effects on the overall functioning of the cell and impair organ and tissue function.
1.2.1. Myopathy
Myopathy is a disease that affects the muscle tissue and generally manifests as chronic muscle weakness, fatigue, low ATP generation, exercise intolerance, proximal and elevated levels of serum creatine kinase25. In most mitochondrial diseases, impaired ETC functioning results in a reduced capacity to produce ATP. The various forms of myopathy are grouped in two categories, namely inherited (primary) and acquired (secondary) myopathies. Primary myopathies are a result of genetic (nDNA and mtDNA) defects, while secondary myopathies are associated with external factors, such as drugs and toxic agents that induce mitochondrial defects. Mitochondrial defects in skeletal muscle tissue result in the development of mitochondrial myopathy, and patients suffer from myalgia and fatigue.Mitochondrial myopathies occur as a result of mutations in either mitochondrial or nuclear DNA. With respect to mtDNA-encoded genes, mutations in genes encoding cytochrome b (CYTB)25-27 and cytochrome c oxidase 1 (COX1)28,29 have been associated with some forms of mitochondrial myopathy. A depletion of mtDNA in skeletal muscle can also result in mitochondrial myopathy, while in other cases this disease is characterized by an accumulation of mitochondria in muscle fibers. It has been suggested that these elevated levels of mitochondrial content may be a mechanism to compensate for the impaired energy production.
1.2.2 Non-alcoholic fatty liver disease (NAFLD)
The liver is a vital metabolic organ responsible for the body’s energy metabolism through the metabolism of fats, carbohydrates and proteins, a function supported by a high mitochondrial density (approximately 800 mitochondria per cell)1. Primary functions of the liver include storage of glycogen, vitamins and minerals, detoxification, bile production and synthesis of plasma proteins, such as albumin. NAFLD is a progressive metabolic disorder characterized by the accumulation of lipids in the liver, primarily as a result of dietary habits in the Western lifestyle. NAFLD covers a spectrum of hepatic pathologies, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which is typically accompanied by the development of liver fibrosis that may progress to cirrhosis and predisposes for hepatocellular carcinoma (HCC). NASH is the more progressive subtype of NAFLD, which marks the transition from simple steatosis to steatohepatitis and, as such, is characterized by inflammation of the liver. Prime risk factors for NASH are obesity and type 2 diabetes 22-24. NAFLD is a rapidly increasing health problem and contributes to a third of liver-related deaths in developed countries. The global prevalence of NAFLD is on the rise as many regions of the world become industrialized. As there are no drugs yet available to treat NAFLD, only liver transplantation remains as a last life-saving option for patients with end-stage NAFLD. However, limited availability of donor livers and transplantation-associated complications are major challenges to overcome. Treatments that can reverse mitochondrial dysfunction and improve mitochondrial fitness and cell survival may therefore be potential therapeutic interventions for NAFLD and other chronic liver diseases.
Figure 1: Illustration of diseases associated with mitochondrial dysfunction, in particular metabolic and
neurodegenerative diseases.
1.2.3. Striking the balance, mitochondrial biogenesis versus mitophagy
In healthy cells, mitochondria appear dispersed throughout the cytoplasm as an interconnected network, which changes in response to the cell’s energy demands or due to stress conditions17. In most metabolic diseases associated with mitochondrial dysfunction, mitochondrial structure changes and the number of dysfunctional mitochondria increases. Mitochondria are essential regulators of caspase activation and apoptotic cell death through a process known as mitochondrial outer membrane permeabilization (MOMP). MOMP leads to the release of various proteins from the mitochondrial intermembrane space, such as cytochrome c, which activates cytosolic caspases and results in apoptosis. Cytochrome c leakage from mitochondria is a key process that initiates apoptotic cell death, and this is an indicator for dysfunctional mitochondria. Cells can prevent caspase-mediated cell death by inhibiting mitochondrial membrane permeabilization and also by maintaining a delicate balance between mitophagy to selectively remove dysfunctional mitochondria and mitochondrial biogenesis to replenish mitochondria1,30-34 (Figure 2). These are important adaptations to cellular stress, which ensure that cells have healthy functional mitochondria30,35. In Caenorhabditis elegans it has been shown that mitophagy
complements mitochondrial biogenesis by preventing the accumulation of dysfunctional mitochondria resulting in a prolonged lifespan34.
Figure 2: The delicate balance between mitophagy and mitochondrial biogenesis. Under stress
conditions, mitochondrial reactive oxygen species (ROS) mediate signaling. The production of ROS following exposure to stress induces the expression of antioxidant genes such as HO-1 which is involved in the endogenous production of carbon monoxide (CO). CO may provide a therapeutic avenue in the maintenance of a healthy mitochondrial population through the induction of mitochondrial biogenesis. The action of CO on mitochondria creates ROS which facilitates signal transduction to enable mitochondrial biogenesis. The balance between mitochondrial biogenesis and mitophagy maintains mitochondrial content during cellular stress conditions. A) High levels of mitophagy result in depleted mitochondrial content which may result in altered mitochondrial metabolism and impaired ATP generating capacity; B) an equilibrium between mitochondrial biogenesis and mitophagy maintains the mitochondrial content at optimal levels; C) Induction of mitochondrial biogenesis results in an increase in mitochondrial content in response to a high energy demand.
1.3. Modelling metabolic diseases 1.3.1. Modelling NASH in vivo
NASH is a complex disease, which requires comprehensive in vitro and in vivo modelling to help understand the various mechanisms that underly its development and progression36. Currently, various in vivo models, such as genetic mouse, rat and
Figure 1: Illustration of diseases associated with mitochondrial dysfunction, in particular metabolic and
neurodegenerative diseases.
1.2.3. Striking the balance, mitochondrial biogenesis versus mitophagy
In healthy cells, mitochondria appear dispersed throughout the cytoplasm as an interconnected network, which changes in response to the cell’s energy demands or due to stress conditions17. In most metabolic diseases associated with mitochondrial dysfunction, mitochondrial structure changes and the number of dysfunctional mitochondria increases. Mitochondria are essential regulators of caspase activation and apoptotic cell death through a process known as mitochondrial outer membrane permeabilization (MOMP). MOMP leads to the release of various proteins from the mitochondrial intermembrane space, such as cytochrome c, which activates cytosolic caspases and results in apoptosis. Cytochrome c leakage from mitochondria is a key process that initiates apoptotic cell death, and this is an indicator for dysfunctional mitochondria. Cells can prevent caspase-mediated cell death by inhibiting mitochondrial membrane permeabilization and also by maintaining a delicate balance between mitophagy to selectively remove dysfunctional mitochondria and mitochondrial biogenesis to replenish mitochondria1,30-34 (Figure 2). These are important adaptations to cellular stress, which ensure that cells have healthy functional mitochondria30,35. In Caenorhabditis elegans it has been shown that mitophagy
complements mitochondrial biogenesis by preventing the accumulation of dysfunctional mitochondria resulting in a prolonged lifespan34.
Figure 2: The delicate balance between mitophagy and mitochondrial biogenesis. Under stress
conditions, mitochondrial reactive oxygen species (ROS) mediate signaling. The production of ROS following exposure to stress induces the expression of antioxidant genes such as HO-1 which is involved in the endogenous production of carbon monoxide (CO). CO may provide a therapeutic avenue in the maintenance of a healthy mitochondrial population through the induction of mitochondrial biogenesis. The action of CO on mitochondria creates ROS which facilitates signal transduction to enable mitochondrial biogenesis. The balance between mitochondrial biogenesis and mitophagy maintains mitochondrial content during cellular stress conditions. A) High levels of mitophagy result in depleted mitochondrial content which may result in altered mitochondrial metabolism and impaired ATP generating capacity; B) an equilibrium between mitochondrial biogenesis and mitophagy maintains the mitochondrial content at optimal levels; C) Induction of mitochondrial biogenesis results in an increase in mitochondrial content in response to a high energy demand.
1.3. Modelling metabolic diseases 1.3.1. Modelling NASH in vivo
NASH is a complex disease, which requires comprehensive in vitro and in vivo modelling to help understand the various mechanisms that underly its development and progression36. Currently, various in vivo models, such as genetic mouse, rat and
zebrafish models37 and diet-induced mouse models, are in use to dissect NASH (see
Chapter 6). However, such experiments do not allow rapid screening of experimental
therapeutic drugs. Moreover, translation from mouse to human often fails. On the other hand, in vitro approaches using human cells often take only the lipid or only the inflammatory component into consideration, while NAFLD progression (e.g. NASH) is characterized by the coexistence of lipid accumulation and inflammation. Several studies have shown that inflammation is an underlying factor that exacerbates lipid accumulation in NASH38,39. Furthermore, it is known that the development of NASH is orchestrated by factors such as insulin resistance, genetic and epigenetic changes. The effect of inflammatory cytokines on hepatic lipid metabolism, including the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, has not been extensively studied. Individuals with the PNPLA3 I148M genotype (rs738409) are susceptible to develop NASH40,41. In addition, increased PNPLA3 expression is associated with NASH and fatty acid loading is known to induce the expression of PNPLA3 in in vitro conditions 42. Due to the multifactorial nature of NASH, it is imperative that the “ideal model” should consider the myriad of factors that are associated with the disease. It remains unclear whether inflammatory cytokines regulate PNPLA3 expression in NASH and whether modulating PNPLA3 expression can attenuate progression of NASH.
1.3.2. Modelling NASH in vitro (two-hit hypothesis vs multifactorial hypothesis)
In vitro modelling of NASH under controlled mono-culture conditions consisting of
either primary hepatocytes or liver-derived cell lines is used as an approach to elucidate molecular mechanisms that underlie its etiology. In NASH models, the “double hit” hypothesis proposes that hepatic lipid accumulation renders the first hit while inflammation, mitochondrial dysfunction, oxidative stress and/or endoplasmic reticulum stress provide the second hit. In vitro NASH models that are currently in use involve loading lipids into cells and then introducing secondary insults, such as cytokines (TNFα, IL-1β and IL-6)38,43. The “multiple hit” hypothesis considers the multifactorial nature of NASH and proposes the inclusion of all factors when studying the disease. However, this approach is poised with the challenge of determining the order in which these factors occur leading the development of NASH.
1.4. Mitochondrial DNA methylation, a role in energy metabolism
The term mitochondrial epigenetics is a broad term that describes the layer of information on top of the mtDNA sequence that governs how the mitochondria function. This remains a hotly debated topic given the controversies reported about the actual existence of methylation of mtDNA methylation (reviewed in Chapter 2).
Interestingly, increased levels of mitochondrial DNA methyltransferase (mt-DNMT-1) and mtDNA methylation have been recently reported in NAFLD patients and this strongly correlated with progression of the disease. In particular, the ND6 gene was highly methylated in NAFLD patients 44. However, it remains elusive whether indeed such epigenetic changes in the mtDNA contribute to mitochondrial dysfunction in NAFLD patients.
1.5. Carbon monoxide as a modulator of hepatic metabolism
Mitochondrial dysfunction in the liver presents dire consequences, which include impaired hepatic energy metabolism. Various studies have identified modulators of mitochondrial metabolism, which include a broad array of molecules ranging from microRNAs to complex chemical compounds45-47. Carbon monoxide (CO) is a simple molecule, known to the general public as a toxic gas. Interestingly, this gaseous compound is endogenously produced by cells during the degradation of haem by haem oxygenases (HO)48 and low (non-toxic) doses of gaseous CO show health promoting effects and harbor therapeutic potential in the treatment of various pathophysiological conditions. CO exposure protects vital organs in mice, such as the brain, heart, lung and liver, during sepsis, hypoxia and organ transplantation48,49. Though several mechanisms have been proposed to explain the cytoprotective action of CO, the exact mechanism remains elusive48,50-53. Various pro-survival pathways are activated when a cell is exposed to oxidative stress, including the HO-1/CO pathway. HO-1 and CO promote cell survival in liver, heart, brain and muscle tissue54-57 and this is, in part, due to the modulation of mitochondrial metabolic activity and mitochondrial biogenesis58-61. HO-1/CO-induced mitochondrial biogenesis is dependent on the activation of the master regulator of cellular oxidative stress, NF-E2-related factor-2 (NRF2), which regulates HMOX-1 expression, the gene encoding HO-1 62-64.
CO as a potential modulator of mitochondrial metabolism could provide a possible therapeutic benefit for patients with metabolic diseases, such as NAFLD. However,
zebrafish models37 and diet-induced mouse models, are in use to dissect NASH (see
Chapter 6). However, such experiments do not allow rapid screening of experimental
therapeutic drugs. Moreover, translation from mouse to human often fails. On the other hand, in vitro approaches using human cells often take only the lipid or only the inflammatory component into consideration, while NAFLD progression (e.g. NASH) is characterized by the coexistence of lipid accumulation and inflammation. Several studies have shown that inflammation is an underlying factor that exacerbates lipid accumulation in NASH38,39. Furthermore, it is known that the development of NASH is orchestrated by factors such as insulin resistance, genetic and epigenetic changes. The effect of inflammatory cytokines on hepatic lipid metabolism, including the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, has not been extensively studied. Individuals with the PNPLA3 I148M genotype (rs738409) are susceptible to develop NASH40,41. In addition, increased PNPLA3 expression is associated with NASH and fatty acid loading is known to induce the expression of PNPLA3 in in vitro conditions 42. Due to the multifactorial nature of NASH, it is imperative that the “ideal model” should consider the myriad of factors that are associated with the disease. It remains unclear whether inflammatory cytokines regulate PNPLA3 expression in NASH and whether modulating PNPLA3 expression can attenuate progression of NASH.
1.3.2. Modelling NASH in vitro (two-hit hypothesis vs multifactorial hypothesis)
In vitro modelling of NASH under controlled mono-culture conditions consisting of
either primary hepatocytes or liver-derived cell lines is used as an approach to elucidate molecular mechanisms that underlie its etiology. In NASH models, the “double hit” hypothesis proposes that hepatic lipid accumulation renders the first hit while inflammation, mitochondrial dysfunction, oxidative stress and/or endoplasmic reticulum stress provide the second hit. In vitro NASH models that are currently in use involve loading lipids into cells and then introducing secondary insults, such as cytokines (TNFα, IL-1β and IL-6)38,43. The “multiple hit” hypothesis considers the multifactorial nature of NASH and proposes the inclusion of all factors when studying the disease. However, this approach is poised with the challenge of determining the order in which these factors occur leading the development of NASH.
1.4. Mitochondrial DNA methylation, a role in energy metabolism
The term mitochondrial epigenetics is a broad term that describes the layer of information on top of the mtDNA sequence that governs how the mitochondria function. This remains a hotly debated topic given the controversies reported about the actual existence of methylation of mtDNA methylation (reviewed in Chapter 2).
Interestingly, increased levels of mitochondrial DNA methyltransferase (mt-DNMT-1) and mtDNA methylation have been recently reported in NAFLD patients and this strongly correlated with progression of the disease. In particular, the ND6 gene was highly methylated in NAFLD patients 44. However, it remains elusive whether indeed such epigenetic changes in the mtDNA contribute to mitochondrial dysfunction in NAFLD patients.
1.5. Carbon monoxide as a modulator of hepatic metabolism
Mitochondrial dysfunction in the liver presents dire consequences, which include impaired hepatic energy metabolism. Various studies have identified modulators of mitochondrial metabolism, which include a broad array of molecules ranging from microRNAs to complex chemical compounds45-47. Carbon monoxide (CO) is a simple molecule, known to the general public as a toxic gas. Interestingly, this gaseous compound is endogenously produced by cells during the degradation of haem by haem oxygenases (HO)48 and low (non-toxic) doses of gaseous CO show health promoting effects and harbor therapeutic potential in the treatment of various pathophysiological conditions. CO exposure protects vital organs in mice, such as the brain, heart, lung and liver, during sepsis, hypoxia and organ transplantation48,49. Though several mechanisms have been proposed to explain the cytoprotective action of CO, the exact mechanism remains elusive48,50-53. Various pro-survival pathways are activated when a cell is exposed to oxidative stress, including the HO-1/CO pathway. HO-1 and CO promote cell survival in liver, heart, brain and muscle tissue54-57 and this is, in part, due to the modulation of mitochondrial metabolic activity and mitochondrial biogenesis58-61. HO-1/CO-induced mitochondrial biogenesis is dependent on the activation of the master regulator of cellular oxidative stress, NF-E2-related factor-2 (NRF2), which regulates HMOX-1 expression, the gene encoding HO-1 62-64.
CO as a potential modulator of mitochondrial metabolism could provide a possible therapeutic benefit for patients with metabolic diseases, such as NAFLD. However,
there are major challenges associated with the use of CO as a therapeutic drug and these include selective targeting of the diseased tissue and controlled site-specific CO release to prevent potential systemic CO toxicity. To prevent toxicity, CO release should occur at a constant rate that maintains non-toxic CO levels below 10% COHb48,65 in the body whilst ensuring that the CO concentration is sufficient to induce the intended therapeutic effect in the target tissue. To establish this, CO-releasing molecules (CORMs) that enable the delivery of CO in a more practical, controllable and accurate way to the target site have been developed48,66,67.
1.6. Aims and Outline of this thesis
NASH and myopathy are complex metabolic diseases, both characterized by mitochondrial dysfunction although the underlying causes are not well understood. Though controversial, the relevance of methylation-derived changes in mtDNA in relation to mitochondrial dysfunction may hold answers to some of the unexplained causes of metabolic diseases. This thesis investigates the impact mtDNA methylation on mitochondrial health and metabolism. Furthermore, mtDNA methylation is posed as a cause of mitochondrial dysfunction and a pro-cell survival mechanism. This thesis also investigates the effects of CO on promoting hepatocyte proliferation and maintaining mitochondrial fitness in in vitro NAFLD models.
1.6.1. Part 1: Investigating the role of mtDNA methylation in metabolic diseases Chapter 2 of this thesis provides a concise overview of diseases that have been
previously associated with differential mtDNA methylation and proposes epigenetic mechanisms that may govern the expression of mitochondrial genes. In this review, we sought to understand the role of mtDNA methylation in causing mitochondrial dysfunction based on previous studies, which have suggested that methylation may have an effect mtDNA transcription and replication. The ensuing debate on the existence of mtDNA methylation is also brought into the scope of this chapter to give insight into the advances that have been made in the field of mitochondrial epigenetics. Differential DNA methylation has been previously reported in NASH patients and in
Chapter 3, we investigate whether mtDNA methylation may have a role in promoting
mitochondrial dysfunction. By artificially inducing mtDNA methylation, we set out to
analyze whether methylation impairs mitochondrial respiration, affects expression of mitochondrial genes and also whether it disturbs lipid metabolism. One of the pertinent questions was whether mtDNA methylation is a cause or consequence of lipid accumulation in hepatocytes during the progression of NASH. Furthermore, we wanted to gain insights in how mitochondrial gene expression changes during the progression of NAFLD, from simple steatosis to NASH. Understanding the intracellular mitochondrial dynamics could shed light on mitochondrial dysfunction in NASH and this information may be vital in determining the therapeutic windows within which even mild interventions may have the greatest chances of success.
Chapter 4 investigates whether mitochondrial dysfunction and impaired ATP
generating capacity in patients with myopathy may be explained by changes in mtDNA methylation. In this study, we investigate whether mtDNA methylation associates with myopathy in patients without the known disease-associated genetic mutations in either mtDNA or nDNA. This chapter further addresses the functional relevance of mtDNA methylation in muscle tissue.
1.6.2. Part 2: Therapeutic prospects of CO in NASH models
Chapter 5 presents CO-releasing molecule 3 (CORM3) as a treatment option that may
promote liver regeneration through the modulation of mitochondrial metabolism. Earlier studies suggested that CO promotes hepatic regeneration in mice by stimulating hepatic stellate cells to release the hepatocyte growth factor (HGF). In this chapter, we investigated the direct effects CORM3 on hepatocytes proliferation.
Chapter 6 takes a step further to investigate the effects of CO in in vitro models of
simple steatosis and NASH. In this chapter, the effects of CORM3 on lipid metabolism, anti-inflammation and the antioxidant response are investigated.
The main findings presented in this thesis are summarized in Chapter 7 and provides
an integrated discussion on how mitochondrial dysfunction may promote the development of metabolic diseases, such as NASH and myopathy. The discussion also covers the therapeutic potential of CO as an enhancer of mitochondrial metabolism and liver regeneration.
there are major challenges associated with the use of CO as a therapeutic drug and these include selective targeting of the diseased tissue and controlled site-specific CO release to prevent potential systemic CO toxicity. To prevent toxicity, CO release should occur at a constant rate that maintains non-toxic CO levels below 10% COHb48,65 in the body whilst ensuring that the CO concentration is sufficient to induce the intended therapeutic effect in the target tissue. To establish this, CO-releasing molecules (CORMs) that enable the delivery of CO in a more practical, controllable and accurate way to the target site have been developed48,66,67.
1.6. Aims and Outline of this thesis
NASH and myopathy are complex metabolic diseases, both characterized by mitochondrial dysfunction although the underlying causes are not well understood. Though controversial, the relevance of methylation-derived changes in mtDNA in relation to mitochondrial dysfunction may hold answers to some of the unexplained causes of metabolic diseases. This thesis investigates the impact mtDNA methylation on mitochondrial health and metabolism. Furthermore, mtDNA methylation is posed as a cause of mitochondrial dysfunction and a pro-cell survival mechanism. This thesis also investigates the effects of CO on promoting hepatocyte proliferation and maintaining mitochondrial fitness in in vitro NAFLD models.
1.6.1. Part 1: Investigating the role of mtDNA methylation in metabolic diseases Chapter 2 of this thesis provides a concise overview of diseases that have been
previously associated with differential mtDNA methylation and proposes epigenetic mechanisms that may govern the expression of mitochondrial genes. In this review, we sought to understand the role of mtDNA methylation in causing mitochondrial dysfunction based on previous studies, which have suggested that methylation may have an effect mtDNA transcription and replication. The ensuing debate on the existence of mtDNA methylation is also brought into the scope of this chapter to give insight into the advances that have been made in the field of mitochondrial epigenetics. Differential DNA methylation has been previously reported in NASH patients and in
Chapter 3, we investigate whether mtDNA methylation may have a role in promoting
mitochondrial dysfunction. By artificially inducing mtDNA methylation, we set out to
analyze whether methylation impairs mitochondrial respiration, affects expression of mitochondrial genes and also whether it disturbs lipid metabolism. One of the pertinent questions was whether mtDNA methylation is a cause or consequence of lipid accumulation in hepatocytes during the progression of NASH. Furthermore, we wanted to gain insights in how mitochondrial gene expression changes during the progression of NAFLD, from simple steatosis to NASH. Understanding the intracellular mitochondrial dynamics could shed light on mitochondrial dysfunction in NASH and this information may be vital in determining the therapeutic windows within which even mild interventions may have the greatest chances of success.
Chapter 4 investigates whether mitochondrial dysfunction and impaired ATP
generating capacity in patients with myopathy may be explained by changes in mtDNA methylation. In this study, we investigate whether mtDNA methylation associates with myopathy in patients without the known disease-associated genetic mutations in either mtDNA or nDNA. This chapter further addresses the functional relevance of mtDNA methylation in muscle tissue.
1.6.2. Part 2: Therapeutic prospects of CO in NASH models
Chapter 5 presents CO-releasing molecule 3 (CORM3) as a treatment option that may
promote liver regeneration through the modulation of mitochondrial metabolism. Earlier studies suggested that CO promotes hepatic regeneration in mice by stimulating hepatic stellate cells to release the hepatocyte growth factor (HGF). In this chapter, we investigated the direct effects CORM3 on hepatocytes proliferation.
Chapter 6 takes a step further to investigate the effects of CO in in vitro models of
simple steatosis and NASH. In this chapter, the effects of CORM3 on lipid metabolism, anti-inflammation and the antioxidant response are investigated.
The main findings presented in this thesis are summarized in Chapter 7 and provides
an integrated discussion on how mitochondrial dysfunction may promote the development of metabolic diseases, such as NASH and myopathy. The discussion also covers the therapeutic potential of CO as an enhancer of mitochondrial metabolism and liver regeneration.
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3. Shay JW, Pierce DJ, Werbin H. Mitochondrial DNA copy number is proportional to total cell DNA under a variety of growth conditions. J Biol Chem.
1990;265(25):14802-14807.
4. Chin BY, Jiang G, Wegiel B, et al. Hypoxia-inducible factor 1alpha stabilization by carbon monoxide results in cytoprotective preconditioning. Proc Natl Acad Sci U S A. 2007;104(12):5109-5114.
5. Shadel GS, Horvath TL. Mitochondrial ROS signaling in organismal homeostasis.
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17. Ferree A, Shirihai O. Mitochondrial dynamics: The intersection of form and function. Adv Exp Med Biol. 2012;748:13-40.
18. Rossignol R, Faustin B, Rocher C, Malgat M, Mazat JP, Letellier T. Mitochondrial threshold effects. Biochem J. 2003;370(Pt 3):751-762.
19. Nunnari J, Suomalainen A. Mitochondria: In sickness and in health. Cell. 2012;148(6):1145-1159.
20. Niyazov DM, Kahler SG, Frye RE. Primary mitochondrial disease and secondary mitochondrial dysfunction: Importance of distinction for diagnosis and treatment. Mol
Syndromol. 2016;7(3):122-137.
21. Garcia-Martinez I, Santoro N, Chen Y, et al. Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9. J Clin Invest.
2016;126(3):859-864.
22. Rector RS, Thyfault JP, Uptergrove GM, et al. Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model. J Hepatol. 2010;52(5):727-736.
23. Nassir F, Ibdah JA. Role of mitochondria in nonalcoholic fatty liver disease. Int J
Mol Sci. 2014;15(5):8713-8742.
24. Dyson JK, Anstee QM, McPherson S. Non-alcoholic fatty liver disease: A practical approach to treatment. Frontline Gastroenterol. 2014;5(4):277-286.
25. Massie R, Wong LJ, Milone M. Exercise intolerance due to cytochrome b mutation. Muscle Nerve. 2010;42(1):136-140.
26. Emmanuele V, Sotiriou E, Rios PG, et al. A novel mutation in the mitochondrial DNA cytochrome b gene (MTCYB) in a patient with mitochondrial
encephalomyopathy, lactic acidosis, and strokelike episodes syndrome. J Child
Neurol. 2013;28(2):236-242.
27. Legros F, Chatzoglou E, Frachon P, et al. Functional characterization of novel mutations in the human cytochrome b gene. Eur J Hum Genet. 2001;9(7):510-518. 28. Horvath R, Schoser BG, Muller-Hocker J, Volpel M, Jaksch M, Lochmuller H. Mutations in mtDNA-encoded cytochrome c oxidase subunit genes causing isolated myopathy or severe encephalomyopathy. Neuromuscul Disord. 2005;15(12):851-857.
29. Massie R, Wang J, Chen LC, et al. Mitochondrial myopathy due to novel missense mutation in the cytochrome c oxidase 1 gene. J Neurol Sci. 2012;319(1-2):158-163.
30. Dam AD, Mitchell AS, Quadrilatero J. Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts. Biochim Biophys Acta. 2013;1833(12):3426-3435.
31. Cherry AD, Suliman HB, Bartz RR, Piantadosi CA. Peroxisome proliferator-activated receptor gamma co-activator 1-alpha as a critical co-activator of the murine hepatic oxidative stress response and mitochondrial biogenesis in staphylococcus aureus sepsis. J Biol Chem. 2014;289(1):41-52.
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34. Palikaras K, Lionaki E, Tavernarakis N. Coordination of mitophagy and
mitochondrial biogenesis during ageing in C. elegans. Nature. 2015;521(7553):525-528.
35. Jornayvaz FR, Shulman GI. Regulation of mitochondrial biogenesis. Essays
Biochem. 2010;47:69-84.
36. Kanuri G, Bergheim I. In vitro and in vivo models of non-alcoholic fatty liver disease (NAFLD). Int J Mol Sci. 2013;14(6):11963-11980.
37. Willebrords J, Pereira IV, Maes M, et al. Strategies, models and biomarkers in experimental non-alcoholic fatty liver disease research. Prog Lipid Res.
2015;59:106-125.
38. Ma KL, Ruan XZ, Powis SH, Chen Y, Moorhead JF, Varghese Z. Inflammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of
apolipoprotein E knockout mice. Hepatology. 2008;48(3):770-781.
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41. Linden D, Ahnmark A, Pingitore P, et al. Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice. Mol Metab. 2019;22:49-61.
42. Bruschi FV, Tardelli M, Herac M, Claudel T, Trauner M. Metabolic regulation of hepatic PNPLA3 expression and severity of liver fibrosis in patients with NASH. Liver
Int. 2020;40(5):1098-1110.
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45. Chen Y, Verfaillie CM. MicroRNAs: The fine modulators of liver development and function. Liver Int. 2014;34(7):976-990.
46. Hochreuter MY, Altıntaş A, Garde C, et al. Identification of two microRNA nodes as potential cooperative modulators of liver metabolism. Hepatol Res. 2019;49(12):1451-1465.
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48. Babu D, Motterlini R, Lefebvre RA. CO and CO-releasing molecules (CO-RMs) in acute gastrointestinal inflammation. Br J Pharmacol. 2014.
49. Liang F, Cao J, Qin WT, Wang X, Qiu XF, Sun BW. Regulatory effect and mechanisms of carbon monoxide-releasing molecule II on hepatic energy metabolism in septic mice. World J Gastroenterol. 2014;20(12):3301-3311. 50. Queiroga CS, Almeida AS, Alves PM, Brenner C, Vieira HL. Carbon monoxide prevents hepatic mitochondrial membrane permeabilization. BMC Cell Biol. 2011;12:10-2121-12-10.
51. Queiroga CS, Almeida AS, Vieira HL. Carbon monoxide targeting mitochondria.
Biochem Res Int. 2012;2012:749845.
52. Almeida AS, Figueiredo-Pereira C, Vieira HL. Carbon monoxide and
mitochondria-modulation of cell metabolism, redox response and cell death. Front
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53. Sharma VS, Magde D. Activation of soluble guanylate cyclase by carbon monoxide and nitric oxide: A mechanistic model. Methods. 1999;19(4):494-505. 54. Hinds TD,Jr, Sodhi K, Meadows C, et al. Increased HO-1 levels ameliorate fatty liver development through a reduction of heme and recruitment of FGF21. Obesity
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58. Suliman HB, Carraway MS, Tatro LG, Piantadosi CA. A new activating role for CO in cardiac mitochondrial biogenesis. J Cell Sci. 2007;120(Pt 2):299-308. 59. Almeida AS, Queiroga CS, Sousa MF, Alves PM, Vieira HL. Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in astrocytes: Role of bcl-2.
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Regulation of mitochondrial gene expression, the
epigenetic enigma
A Mposhi1,2, MGP van der Wijst1, KN Faber2, MG Rots1.
1. Epigenetic Editing, Department of Medical Biology and Pathology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The
Netherlands.
2. Department of Hepatology and Gastroenterology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
