This "Devil's Pact" refers to the endosymbiotic event where a free-living bacterium, now known as the mitochondrion, was engulfed by an early eukaryotic cell, leading to a mutually beneficial relationship. This event is crucial for the evolution of eukaryotic cells and their ability to perform aerobic respiration, producing the energy needed for complex cellular processes. Mitochondria, crucial for energy production in eukaryotic cells, can also act as sensors of cellular stress and danger signals, triggering both adaptive and apoptotic responses. While essential for cell survival, mitochondria can release substances that initiate cell death pathways, particularly when damage is severe and irreversible. This dual role makes the relationship between eukaryotic cells and mitochondria a "Devil's Pact" – vital for life but capable of inducing disease and death.  

The liver is also one of the organs richest in mitochondria – the small organelles in cells that convert food into usable energy through a process called metabolism. Consequently, the mitochondria produce high levels of free radicals, as well as antioxidants to keep these free radicals at healthy levels. Both free radicals and antioxidants play a key role in regulating metabolism and are elevated in insulin resistance and fatty liver disease. 

The liver supports mictochondrial health troughout the body.

Mitochondrial dysfunction in the liver can lead to various metabolic disorders, impacting not only liver health but also systemic health.  

One of these antioxidants bilirubin, a yellow-bile substance that is released from the breakdown of biliverdin – its green-bile precursor. Bilirubin is produced at high levels in livers from people with fatty liver disease. Both biliverdin and bilirubin are found naturally in the body and released during the breakdown of heme – the deep red iron-containing molecule in red blood cells. 

Effects of bilirubin on Mitochondrial Function:
Bilirubin can disrupt mitochondrial function in several ways:

Uncoupling Oxidative Phosphorylation: Bilirubin can interfere with the process by which mitochondria generate energy (ATP). 

Mitochondrial Swelling: It can cause mitochondria to swell, potentially leading to damage. 

Cytochrome c Release: Bilirubin can induce the release of cytochrome c, a protein involved in apoptosis (programmed cell death), from the mitochondria. 

Research revealed that increased bilirubin content inside the mitochondria driven by ABCB10 activity is contributing to fatty liver disease. Four mitochondrial ATP-binding cassette (ABC) transporters have been described to date in mammals: ABCB6, ABCB8, ABCB7 and ABCB10. ABCB10 is located in the inner mitochondrial membrane forming homodimers, with the ATP binding domain facing the mitochondrial matrix. 

ABCB10 irregularities, such as deletions or mutations, can lead to mitochondrial damage. This mitochondrial damage can manifest as structural abnormalities, dysfunction, and increased oxidative stress.  

ABCB10 is also involved in iron transport within mitochondria, and its deficiency can impact heme and iron-sulfur cluster biosynthesis, further contributing to mitochondrial dysfunction 

Mitochondrial dysfunction can lead to higher serum bilirubin levels. Disruptions in mitochondrial activity can impair the liver's ability to process and excrete bilirubin, resulting in elevated levels in the blood. When mitochondria are dysfunctional, the liver's ability to conjugate bilirubin is compromised. This leads to an accumulation of unconjugated (water-insoluble) bilirubin in the bloodstream.  

In the brain, bilirubin can be particularly problematic. It can cross the blood-brain barrier and affect neuronal function. Some studies suggest that bilirubin can inhibit mitochondrial respiration and disrupt energy production in brain cells. 

Elevated levels of unconjugated bilirubin, a condition known as hyperbilirubinemia, can be toxic, particularly to the brain. In severe cases, it can lead to neurological damage. While high levels of bilirubin can be harmful, bilirubin itself can also have protective effects, particularly at lower concentrations. It acts as an antioxidant and can help mitigate oxidative stress and inflammation. So, bilirubin, tranported in the body by albumin, is a crucial modulator. 

Hyperbilirubinemia, or high levels of bilirubin in the blood, can precede and contribute to progressive brain disorders, particularly in newborns. This condition, if severe, can lead to bilirubin-induced neurological dysfunction (BIND) and potentially irreversible brain damage, known as kernicterus. 

Serum bilirubin levels, particularly low levels, may serve as a potential marker for certain progressive brain disorders, including ischemic stroke and neurodegenerative diseases like Parkinson's and Alzheimer's. While elevated bilirubin can be a marker of poor outcomes in acute ischemic stroke, low bilirubin levels are increasingly associated with increased risk of neurological issues, including dementia.  

A properly functioning liver is essential for the processing of bilirubin. Bilirubin is transported to the liver where it's conjugated (i.e. combined with glucuronic acid) (made water-soluble). If the liver is damaged or not working properly, it may not be able to efficiently process bilirubin, leading to a buildup in the bloodstream. 

Studies have shown that circulating bilirubin levels tend to increase with age. This increase is observed even when considering factors like red blood cell turnover and other potential confounding variables. While some liver functions may decline with age, the increase in bilirubin is not primarily due to a reduction in bilirubin production within the liver.  

Unconjugated bilirubin serum levels tend to be higher in older adults. This increase is often observed within the normal range of bilirubin levels, but even small increases can be clinically significant. Studies have shown a gradual increase in serum bilirubin levels as people age. Even within the normal range, small increases in bilirubin in older adults can be associated with increased mortality 

Poorly functioning mitochondria can contribute to elevated unconjugated bilirubin levels in the serum.  

Mitochondria are involved in heme degradation, which is a key step in bilirubin production. When mitochondria are not functioning properly, heme breakdown can be impaired, leading to increased production of bilirubin. Mitochondria are also involved in the detoxification of bilirubin, particularly through the process of conjugation. If mitochondria are not working correctly, the liver's ability to conjugate and excrete bilirubin can be compromised, leading to its accumulation in the blood. Bilirubin itself can contribute to mitochondrial dysfunction by inducing oxidative stress, particularly at high concentrations. This creates a cycle where mitochondrial dysfunction leads to increased bilirubin, which then further damages mitochondria, in the brain especially in neuronal and glial cells.   

Mitochondria themselves also have a mechanism for exporting their interneral biliverdin via the ABCB10 transporter. While this export is necessary for regulating mitochondrial redox state and preventing oxidative damage, it can become maladaptive in certain conditions, such as obesity, where it may contribute to insulin resistance.  

Biliverdin Reductase

Biliverdin reductase (BVR) is found in both the cytoplasm and mitochondria. Mitochondrial activity is indirectly involved in the production of biliverdin reductase (BVR), primarily through its role in generating NADPH, a cofactor essential for BVR's enzymatic activity. While BVR itself is not produced within the mitochondria, it relies on mitochondrial function for proper activity. In the mitochondria, BVR can influence cellular energy metabolism and protect against oxidative damage. For example, BVR-A has been shown to transport a protein involved in energy metabolism into the mitochondria, and its loss can impair mitochondrial function and activate the mitochondrial unfolded protein response. The biliverdin reductase (BVR) gene is present in both the mitochondria and the cell nucleus, allowing it to perform diverse functions within the cell. BVR's location and activity are influenced by cellular stress and signaling pathways. It can be found in the membrane, cytoplasm/ER, mitochondria, and nucleus, and its movement between these compartments is regulated.  

BVR acts as a kinase, scaffolding protein, and transcription factor, influencing various cellular processes like glucose metabolism, cell growth, apoptosis, and even synaptic plasticity.  

BVR's movement between these locations is not random. It's regulated by factors like nitrosylation, lipid modification, and phosphorylation, which are often associated with cellular stress and signaling pathways. For example, biliverdin (BV) can induce nitric oxide (NO) production, which in turn can stabilize and facilitate BVR's translocation to the nucleus. Biliverdin reductase, an enzyme crucial for converting biliverdin to bilirubin, relies on several amino acids for its activity. Specifically, cysteine, tyrosine, serine, threonine, lysine, and arginine residues are involved in its catalytic and regulatory functions. At least three cysteine residues (Cys73, Cys280, and Cys291) are present in biliverdin reductase, and modification of these residues can affect enzyme activity. Specifically, the cysteine at position 73 (Cys73) appears to be critical for both NADH- and NADPH-dependent activity. Cysteine residues are essential for the function of biliverdin reductase (BVR), particularly in substrate and cofactor binding, and catalysis. Cysteine can be synthesized via the transsulfuration pathway, using homocysteine and serine as substrates. This pathway, particularly active in the liver, uses vitamin B6-dependent enzymes to convert these precursors into cysteine. Mitochondria don't directly transport cysteine, their dysfunction can create a cascade of effects that indirectly impact cysteine uptake. For example, if mitochondrial dysfunction leads to increased oxidative stress and glutathione depletion, it might trigger an increased demand for cysteine, but the malfunctioning mitochondria may not be able to support the efficient uptake and utilization of cysteine to meet this demand. 

The uptake of amino acids like cysteine into a cell typically requires ATP. Amino acids are polar molecules and don't readily diffuse across the cell membrane, which is made up of a hydrophobic lipid bilayer. Therefore, cells utilize active transport mechanisms, which require energy in the form of ATP, to move amino acids against their concentration gradient into the cell.      

The liver tightly regulates cysteine levels to maintain adequate cysteine for its various metabolic roles, while preventing cysteine toxicity. 

The liver's ability to synthesize cysteine via the transsulfuration pathway (to convert homocysteine (Hcy) into cysteine) is crucial for maintaining cysteine homeostasis, particularly when cysteine intake is limited. The liver also plays a key role in regulating cysteine catabolism through enzymes like cysteine dioxygenase (CDO), which converts cysteine to other metabolites like taurine and sulfate. Cysteine serves as a building block for glutathione and proteins. The liver's ability to regulate cysteine levels is vital for maintaining cellular health and preventing cysteine-related toxicities.  

Mitochondrial dysfunction can interfere with the host cell's production of biliverdin reductase (BVR), potentially impacting its cellular processes. Specifically, reduced BVR-A levels can disrupt insulin signaling and mitochondrial bioenergetics, leading to insulin resistance and potentially neurodegeneration. Furthermore, BVR-A plays a role in transporting a protein (GSK3βS9) into the mitochondria, which is crucial for regulating energy metabolism. When BVR-A is compromised, this transport is hindered, impacting oxidative phosphorylation and activating the mitochondrial unfolded protein response (UPRmt).

Mitochondrial dysfunction can lead to reduced production of biliverdin reductase (BVR). This reduction in BVR activity can impact various cellular processes, including insulin signaling, mitochondrial function, and antioxidant defense. Specifically, BVR plays a role in shuttling phosphorylated GSK3β into mitochondria, influencing oxidative phosphorylation and potentially triggering the mitochondrial unfolded protein response. Additionally, BVR-deficient cells exhibit reduced mitochondrial number, decreased mitochondrial respiration, and increased oxidative stress. 

Biliverdin reductase (BVR) production, particularly the cytosolic enzyme BVR-A, is influenced by mitochondrial ATP levels and cellular energy status. BVR-A plays a crucial role in converting biliverdin to bilirubin, a process that can be affected by mitochondrial function and the availability of ATP. Insulin signaling, which is linked to mitochondrial metabolism, can also impact BVR-A levels and its activity in the cell.     

Vitamin B6 deficiency can lead to lower levels of cysteine and glutathione, although some studies show mixed results. Vitamin B6, in the form of pyridoxal-5'-phosphate (PLP), acts as a coenzyme for enzymes involved in cysteine and glutathione synthesis. Specifically, it's needed for cystathionine β-synthase and cystathionine γ-lyase, which are part of the transsulfuration pathway where cysteine is synthesized from methionine. Additionally, PLP is involved in the synthesis of glycine, a component of glutathione. Vitamin B6 deficiency is prevalent in people with metabolic disorders, with varying degrees of deficiency observed in conditions like type 2 diabetes, renal disease, and rheumatoid arthritis.

There is evidence suggesting that people with type 2 diabetes, renal disease, and rheumatoid arthritis may experience disturbed vitamin B6 uptake or metabolism. This can lead to lower vitamin B6 levels and potentially contribute to disease progression or complications.   

Mitochondria play a crucial role in the metabolism of vitamin B6, specifically by converting absorbed B6 vitamers into the active form, pyridoxal 5'-phosphate (PLP), which acts as a cofactor for various enzymes. While the small intestine absorbs non-phosphorylated B6 vitamers, the conversion to PLP, and its subsequent utilization, occurs within cells, including those within the mitochondria.   

The prevalence of MD (Mitochondrial Disease) is probably underestimated 

Mitochondria are distinct cellular organelles that generate adenosine triphosphate (ATP), the energy carrier in most mammalian cells, from adenosine diphosphate by oxidizing glucose and fatty acids. Acetyl-CoA is a key intermediate generated from the oxidation of glucose and fatty acids that is further metabolized by the tricarboxylic acid (TCA) cycle. The TCA cycle produces reduced flavin adenine dinucleotide and reduced nicotinamide adenine dinucleotide. Reduced nicotinamide adenine dinucleotide and reduced flavin adenine dinucleotide transport energy to the mitochondrial electron transport chain (ETC), a series of reactions known as oxidative phosphorylation. Mitochondria contain two plasma membranes, an inner and an outer membrane. The ETC is located in the inner mitochondrial membrane and consists of five multi-subunit enzyme complexes (complexes I through V) and two electron carriers (ubiquinone, also known as co-enzyme Q10, and cytochrome c).  The ETC is coded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). mtDNA contains 37 genes that code for 13 subunits of complexes I, III, IV and V, as well as the machinery required to translate and transcribe the mtDNA genes into ETC complex subunits. The remainder of the ETC complex subunits are coded by over 850 nDNA genes. nDNA also codes for mitochondrial enzymes that participate in carbohydrate and fatty acid oxidation. Thus, mutations in either genome can impair mitochondrial function and cause ETC complex deficiencies.

Cord dancing; it's all about the 'balance'

MD has a broad phenotypic presentation: children with MD can have normal intelligence, mental retardation or developmental delay. Stressors, such as dehydration, fever and infection can lead to a functional decline and neurodegenerative regression in individuals with MD. 

Mitochondrial dysfunction can be classified as either primary or secondary. Primary mitochondrial dysfunction generally refers to mitochondrial dysfunction caused by a defect in a gene directly involved in the function of mitochondrial systems responsible for producing ATP, whereas secondary mitochondrial dysfunction refers to other metabolic or genetic abnormalities that impair the ability of mitochondria to produce ATP. For example, metabolites produced by toxic substances (for example, environmental toxicants) or by the dysfunction of other metabolic systems that are not specifically involved in producing ATP (for example, increased oxidative stress because of dysfunctional antioxidant pathways) can interfere with the ability of mitochondria to make ATP and lead to secondary mitochondrial dysfunction. Other reported causes of secondary mitochondrial dysfunction include: certain medications; enteric short chain fatty acids, such as propionic acid; elevated concentrations of tumor necrosis factor-α; cerebral folate deficiency; malnutrition; heme, vitamin B6, or iron deficiencies; elevated NO; GSH deficiency; oxidative stress; or exposure to environmental toxicants, such as heavy metals, chemicals, polychlorinated biphenyls or pesticides. Some individuals have findings consistent with MD but do not have an identifiable genetic defect and/or do not meet full criteria for definite or probable MD. It is possible that these individuals have secondary mitochondrial dysfunction or may have an as yet unidentified genetic abnormality. In this review article, we collate evidence of both primary and secondary mitochondrial dysfunction in ASD. 

An imbalance between both the production of free radicals (reactive oxygen species or ROS) and antioxidants by mitochondria can lead to brain damage, a condition known as oxidative stress. The brain, due to its high metabolic rate and oxygen consumption, is particularly vulnerable to this damage, which contributes to various neurological disorders.  

Nuclear genes can influence mitochondria 

Specific genes can interact with the mitochondrial genome to ensure proper mitochondrial function and cellular health. Dysfunctional mutations in these nuclear genes can lead to a variety of mitochondrial diseases.  

Mitochondrial DNA Replication and Maintenance 

DNA Polymerase Gamma (POLG):
This nuclear gene encodes the enzyme responsible for replicating mitochondrial DNA (mtDNA). Mutations in POLG can lead to mtDNA instability and mitochondrial dysfunction. 

OPA1:
This gene encodes a protein involved in mitochondrial fusion and cristae formation. Mutations in OPA1 can cause mitochondrial DNA deletions and are associated with optic atrophy and other mitochondrial diseases. 

Mitochondrial Protein Synthesis 

Nuclear-encoded mitochondrial ribosomal proteins (MRPs):
A large number of nuclear genes encode proteins that are part of the mitochondrial ribosome, responsible for translating mitochondrial mRNA into proteins. Defects in these MRP genes can disrupt mitochondrial protein synthesis and function.

Other nuclear genes involved in mitochondrial translation:
Various other nuclear genes are involved in the intricate process of mitochondrial translation, including those encoding tRNAs, aminoacyl-tRNA synthetases, and factors involved in translation initiation, elongation, and termination. 

Mitochondrial Metabolism 

Nuclear genes encoding mitochondrial enzymes:
Many nuclear genes encode enzymes involved in various metabolic pathways within the mitochondria, such as the citric acid cycle, oxidative phosphorylation, and fatty acid oxidation. Dysfunctional mutations in these genes can disrupt mitochondrial energy production and lead to metabolic disorders.

Genes involved in mitochondrial transport:
Nuclear genes also encode proteins that transport metabolites and other molecules into and out of the mitochondria, ensuring proper substrate delivery for metabolic processes. Nuclear genes play a crucial role in regulating ABCB10 transporter activity and are implicated in disorders related to this transporter. While ABCB10 is a mitochondrial protein, its function is tightly regulated by nuclear genes through various signaling pathways, including nutrient sensing and oxidative stress response. Disruptions in these pathways due to nuclear gene mutations can lead to a range of disorders impacting erythropoiesis, metabolism, and cellular stress responses.    

Mitochondrial-Nuclear Interactions 

Mito-nuclear coevolution:
There is a complex interplay between nuclear and mitochondrial genomes, with nuclear genes evolving to compensate for mitochondrial mutations and vice versa. This coevolution is important for maintaining mitochondrial function and overall cellular health. 

Sex-specific effects:
Nuclear genes can exhibit sex-specific effects on mitochondrial gene expression, with males and females showing different patterns of gene expression in response to mitochondrial variations. 

ATF4 (Activating Transcription Factor 4) plays a complex role in metabolic disorders, with both protective and detrimental effects depending on the specific context and tissue. It's involved in regulating glucose homeostasis, lipid metabolism, and energy expenditure, and its dysfunction can contribute to insulin resistance, obesity, and related metabolic diseases. ATF4 dysfunction, particularly its aberrant activation, is closely linked to mitochondrial dysfunction. ATF4, a transcription factor, responds to cellular stress, including mitochondrial stress. 

When mitochondria are impaired, they can trigger the activation of ATF4, which in turn can exacerbate or contribute to mitochondrial dysfunction through various mechanisms, including the mitochondrial unfolded protein response (UPRmt) and mitophagy. 

In some cases, mitochondrial dysfunction can also impair the ability of ATF4 to be activated or properly carry out its functions, highlighting a complex interplay between these two cellular components.  

ATF4, or Activating Transcription Factor 4, acts as a modulator by regulating the expression of target genes in response to cellular stress. It plays a crucial role in balancing cell survival and death pathways, influencing processes like autophagy, amino acid metabolism, and endoplasmic reticulum stress. Its activity is context-dependent, with potential roles in both promoting and inhibiting cell survival depending on the specific cellular environment and stress conditions.

ATF4 (Activating Transcription Factor 4) plays a complex role in regulating glutathione (GSH) levels, impacting both antioxidant defense and cellular stress responses. ATF4 can influence glutathione metabolism by affecting its synthesis, degradation, and utilization in various cellular processes. Genetic changes in ATF4 (Activating Transcription Factor 4) expression can contribute to metabolic diseases. ATF4 is a transcription factor that plays a crucial role in regulating cellular responses to stress, including metabolic stress. Its dysregulation can lead to disruptions in glucose homeostasis, lipid metabolism, and overall metabolic balance, potentially contributing to conditions like obesity, diabetes, and fatty liver disease.    

In aging individuals, the activity of the transcription factor ATF4 tends to decline. Studies suggest that ATF4 activity can decrease in the liver with age. This decline can impact the liver's ability to respond to stress and maintain metabolic homeostasis. An imbalance between stress and ATF4 (Activating Transcription Factor 4) levels can lead to both metabolic and neuronal diseases.   

ATF4 (Activating Transcription Factor 4) plays a crucial role in collagen synthesis, particularly by regulating the expression of enzymes involved in glycine production. Glycine, the most abundant amino acid in collagen, is synthesized through a pathway that converts the glycolytic intermediate 3-phosphoglycerate into glycine. ATF4 is a key regulator of this serine-glycine synthesis pathway, meaning it controls the production of the enzymes needed to make glycine. A decrease in collagen production, particularly collagen VI, can contribute to brain disorders by impairing neuronal health and potentially accelerating neurodegeneration. Collagen VI, for instance, plays a neuroprotective role by mitigating the harmful effects of amyloid-beta (Aβ) peptides, a hallmark of Alzheimer's disease. Reduced collagen VI levels can exacerbate Aβ toxicity, highlighting its importance in maintaining neuronal integrity. 

Cysteine is crucial for the proper functioning of ATF4, particularly in the context of oxidative stress and amino acid deprivation. ATF4, a transcription factor activated during the Integrated Stress Response (ISR), relies on cysteine for several key functions, including glutathione synthesis, maintaining redox balance, and responding to amino acid imbalances.