The life and times of cholesterol
Its evolution and crucial importance to our biology
We are told about good cholesterol and bad cholesterol. However, the good cholesterol is only considered good because it is carried by high-density lipoproteins (HDLs) to the liver, which then has the option of sending it to the intestine to be eliminated in faeces. So, it’s only good because we rid ourselves of it. It follows that even the good cholesterol must have been bad, and therefore all cholesterol is bad and that’s all there is to cholesterol. The intent of this post is to give cholesterol a better, balanced and more nuanced press.
The Great Oxidation Event
The emergence of cholesterol in our evolutionary progression can be traced back to the Great Oxidation Event (GOE) that occurred around 2.4 billion years ago and that marked a turning point for life on Earth. Until then, the Earth’s atmosphere (and oceans) did not contain oxygen, and all life (bacteria and archaea) was anoxic. When certain bacteria (cyanobacteria) evolved photosynthesis as a means for generating virtually inexhaustible energy, the molecular oxygen that was released as a byproduct of their metabolism initiated the GOE that lasts until today.
For the first billion years or so, the oxygen released by cyanobacteria was sequestered in the seabed and on land by reacting with abundant metals such as iron, producing iron oxides (hematite — aka rust, magnetite) and forming iron ores such as those laid down in the Precambrian age (over 550 million years ago) in the Pilbara region of Western Australia. Meanwhile, ~800km to the Pilbara’s south, in the shallow sea lagoons of Shark Bay, are to be found mound-like stromatolites formed by the accumulation of calcium secreted by aeons of cyanobacteria. Today, persistently and imperceptibly, they carry on with the terraforming their predecessors initiated.
While sequestration slowed the rate of free oxygen accumulation, ultimately the sinks filled and atmospheric and oceanic oxygen began to rise more rapidly. Many other factors contributed to this period of geo-engineering, both terrestrial (volcanic, tectonic) and biological (oxygenation accelerated with the emergence of algae and lichens, more so land plants). It was a climatically-tumultuous period. For example, the newly introduced atmospheric oxygen degraded methane gas in the primordial atmosphere, nulling its greenhouse-gas effects, cooling the planet and leading to a prolonged era referred to as ‘snowball earth’ (Makganeyene glaciation).
It is powerful and unpredictable climatic forces like these that we recklessly tease today.
In any event, planetary oxygenation took hold, and a new reality was established for life on Earth. The first mass extinctions followed. However, while life had to adapt to oxygen and its damaging effects, evolution thrives under stress and there were new evolutionary niches to be explored that led to profound changes to life.
A new Domain
The Domain is the highest taxonomic rank of organisms. Before the GOE, there were only two Domains to life — Bacteria and Archaea. A consequence of the GOE was the emergence of a third Domain: Eukarya, beginning about 1 billion years ago and likely derived from an Archeal lineage. We belong to this Domain, as does everything else in the Kingdoms of Animalia, Plantae and Fungi.
The distinctive feature of eukaryotes was the development of a central nucleus that held their DNA (in bacteria and archaea, the DNA floats around the cell). This may have been a strategy for protecting DNA from oxidative damage by enclosing it in a separate organelle within the cell with added protections. In addition, eukaryotes developed strategies for controlling oxygen entry into the cell itself, and for neutralising reactive oxygen species (ROS, otherwise known as free radicals).
Evolution selected for a molecule for both these tasks — a sterol. The cholesterol we carry in our bodies today is the sterol that evolved from that ancestoral lineage, as did the equivalent in plants (phytosterols) and fungi (ergosterol). Indeed, all the cells in our body that have a DNA nucleus can synthesise cholesterol (because they are all eukaryotes), and the initial steps in the pathway for the cellular synthesis of cholesterol in animals is exactly the same as that found in plants and fungi — animals, plants and fungi are building on the first eukaryotic blueprint before going their separate ways.
It is for this reason that we should think of sterols as one of the basic building blocks of life.
Cell membrane permeability
In keeping with the early evolution of sterols and eukaryotes, cholesterol makes up a significant part (~30%) of the membranes of all cells in our body. The bulk of the remaining membrane is made up of phospholipids (PLs, two fatty acids (FAs) combined with a water-friendly phosphate group).
How well the PLs can pack together to form the membrane will depend on their FA composition. Typically, a PL might contain one saturated FA and one unsaturated FA. Recall that the carbon chains in saturated FAs are straight, while those of unsaturated FAs contain bends, thereby limiting how well PLs will pack.
Cholesterol helps pack the cell membrane more fully. That is, it ‘fills in gaps’ between the PLs. This restricts membrane permeability and better protects the cell from its environment, particularly from free-radicals damage. As well, cholesterol helps bind the PLs together, strengthening and stabilising the membrane.
As well as being distributed across a membrane, cholesterol also aggregates to form localised ‘rafts’. These are important for holding various proteins and molecular arrangements that enable and regulate the passage of molecules across the cell membrane in both directions. This links the external and internal cellular environments and is a means for transmembrane signalling.
Cholesterol regulates fluidity in a cell membrane while maintaining its integrity. Fluidity is the extent to which PLs or cholesterol can move over the membrane, thereby modulating its shape and other properties. The opposite of fluidity is viscosity, which results in a less adaptable form (such as found in plants). Cholesterol allows membranes to be highly dynamic structures. A single PL can travel over the surface of a small cell (about the size of a bacterium) in ~1 second.
This biological capability, conferred by cholesterol to animal cells, enables the cells to change shape, be distorted, or stretched and compressed, without damaging the membrane. If the membrane is breached, cholesterol will bandaid the damage. This allows for a multitude of functions found in animals, including physical movement (muscle contraction), expanding or stretching (bladder, stomach, skin, lungs) and reshaping (eye lens). Properties that are not found in cholesterol-free plants.
It is thought that membrane fluidity in animal cells heralded the onset of predator/prey behaviour. Early in life, nutrients flowed passively across cellular membranes. However, with the development of more fluid membranes, a cell could engulf nutrients (and eventually other cells) by enclosing around them and internalising the prey. At a cellular level, this is called endocytosis, and it is ubiquitous to our biology (for example, it is how certain immune cells engulf and dispose of pathogens). As multicellular animal organisms evolved, this predator/prey characteristic became steadily more developed and competitive.
The brain and cholesterol
Cholesterol is essential for brain structure and function — the brain contains around ~20% of total body cholesterol, while accounting for only ~2% of bodyweight. However, cholesterol cannot cross the blood-brain barrier (BBB), and all of the brain’s cholesterol requirement is met by synthesis within the brain. The biochemical pathway for synthesis (see later) is the same for the brain as for cells outside the brain.
The main cells in the brain that synthesise cholesterol are two sub-classes of glial cells (oligodendrocytes and astrocytes). Glial cells are more abundant by far than neurons in the brain (although they are smaller) and they have many roles, including immune defence.
Early in brain development, oligodendrocytes use cholesterol to form insulating sheaths (myelin) around large-diameter nerve fibres to speed up neural transmission. About 70%of the brain’s cholesterol is tied up in myelin.
The other 30% is synthesised by astrocytes thoughout life. Functioning adult neurons need cholesterol for repair or reshaping of their membranes, for synaptic transmission, for the genesis of new synaptic connections and for modulating synaptic plasticity (essential for learning and memory).
Cholesterol is trafficked between astrocytes and neurons in lipoproteins similar to the high-density lipoproteins (HDLs) in the peripheral circulation (see previous post). However, as with cholesterol, HDL from the circulation will not cross the BBB (although its ApoA-1 proteins can). Consequently, brain HDL must also be synthesised in situ.
The brain HDL is formed with ApoE, synthesised primarily by astrocytes. Some ApoA-1 that crosses the BBB is also incorporated. Neurons can also synthesise ApoE, but only do so if they are stressed or injured and have an increased need for cholesterol for membrane repair. Like its peripheral counterpart, brain HDL has antioxidant and anti-inflammatory properties. Unlike its peripheral counterpart, brain HDL does not traffic triglycerides because there is virtually no triglyceride in the brain.
There are genetic variants to ApoE, and ApoE-4 is of particular interest in neurodegenerative disorders such as Alzheimer’s disease. ApoE-4 is more readily degraded, and less effective at trafficking cholesterol to neurons. Reduced cholesterol availability can lead to neuronal degeneration and neuronal death. Neurotoxic fragments from E4 degradation may accumulate and do further damage.
As well as neuronal integrity and synaptic transmission, the brain uses cholesterol to synthesise neurosteroids, which can rapidly modulate neuronal excitability and inhibition. In particular, they can up-regulate the brain’s major inhibitory system (the gaba-ergic system) and have anti-seizure properties. As well, neurosteroids can moderate anxiety, stress and depression. Neurosteroids are produced by astrocytes and by excitatory (glutamatergic) neurons. Their precursors can also be synthesised from steroid hormones in peripheral tissue (in turn synthesised from cholesterol). These neurosteroid precursors readily cross over the BBB into the brain and influence brain function.
Finally, the brain is very good at recycling cholesterol, and the daily need for synthesis is kept to a minimum. If the brain needs to eliminate cholesterol to maintain balance, neurons oxidise it to create an oxysterol (24(S)HC), which will cross the BBB and be cleared by the liver.
Oxysterols refer to a diverse class of oxidised cholesterol molecules. These are synthesised under enzymatic control from cholesterol (see ‘synthesis of cholesterol’), or they can arise spontaneously (non-enzymatically) when cholesterol encounters various reactive oxygen species (free radicals). They are small in number (est. 0.002% of cholesterol is oxidised) however, they are increasingly being linked to important biological functions including the regulation of immune function and the response to certain viral and bacterial infections. In addition, they form one of the feedback loops that regulate cholesterol synthesis.
The deliberate enzymatic synthesis of oxysterols implies they confer a functional benefit, although small numbers make them difficult to study. Spontaneous non-enzymatic oxidation of cholesterol has led to the suggestion that cholesterol is part of our antioxidant protection system. This is on the basis of oxidant interception combined with the efficient liver metabolism of oxysterols, as well as the evolutionary relationship between cholesterol and oxygen. Oxysterols can be ‘reverse-metabolised’ in the liver to cholesterol, to other cholesterol products (e.g. bile), or be discharged through the bile duct and eliminated in faeces.
The liver synthesises bile from cholesterol, and sends it to the gallbladder for storage. The bile is released after a meal to aid in the digestion of fats and fat-soluble vitamins. There is no alternative pathway for bile synthesis other than cholesterol.
Cholesterol is necessary for the synthesis of all 20 of our steroid hormones (including testosterone and the oestrogen family of hormones). There is no other mechanism whereby these hormones can be made. Dietary sources for steroid hormones are insignificant.
The close link of sterols with steroids is implicated by their names. A ‘sterol’ is an abbreviated synonym for a ‘steroid alcohol’, that is, a steroid with a OH (hydroxyl group) attached.
The final precursor to cholesterol in the cholesterol-synthesis pathway is used for the synthesis of vitamin D by skin cells in the presence of UV light. Vitamin D maintains and regulates calcium levels in the body and is, therefore, important for growing or maintaining our skeleton. The synthesis of vitamin D is important, because there are few natural dietary sources of significance — mostly fatty fish (salmon, tuna), eggs and liver. Vitamin D deficiency is common in western societies. Consequently, many refined foods are fortified with vitamin D. This is one reason why current dietary guidelines do not entirely discourage consumption of refined grains.
Synthesis of cholesterol
The process of synthesising cholesterol is long — around 30 biochemical steps involving multiple enzymes — complex and energy demanding. Which means that evolution has invested heavily in cholesterol. I have put together this flowchart, which may be helpful to follow as I describe cholesterol synthesis.
The pathway starts with a two-carbon molecule, acetate. Our cells can make acetate out of any macronutrient — glucose (from carbohydrate), amino acids (from proteins), fatty acids (from fats), ketones (from fatty acids) or ethanol (from alcohol). Acetate can be used for energy production, or it can be used to synthesise other molecules such as glucose, fatty acids, triglycerides or, as with the present case, cholesterol.
It takes only a few steps convert acetate to acetyl-CoA and then to HMGCoA (I will just use acronyms). However, the crucial step is the subsequent conversion of HMGCoA to mevalonate, catalysed by the enzyme HMGR (indicated in red). This is the rate-limiting step for cholesterol production because it is irreversible and, once mevalonate is formed, the synthesis of cholesterol is inevitable provided the substrates are available.
Our cells use HMGR to regulate cholesterol synthesis, by raising or lowering the availability of HMGR based on four different feedback loops, the main one being the level of cholesterol already in the cell (from cholesterol synthesis, or uptake from circulating low-density lipoproteins). Statins also act at this level, out-competing HMGR to block downstream cholesterol production and the byproducts of cholesterol synthesis.
Seven more steps lead to the production of squalene. Along the way, a number of biologically-important byproducts are generated, including coenzyme Q10 and haem(a), both of which have crucial roles in the production of cellular energy (by the electron transport chain), and isoprenoids that are involved in protein-protein and lipid-protein binding.
Up to this point, the biochemical reactions have not involved oxygen, and it is likely that these initial steps were present in ancestral eukaryotes and bacteria. While bacteria do not synthesise sterols, they can go on to synthesise another class of compounds (hopanoids) from squalene, which gives bacterial membranes some similar properties.
All eukaryotic cells from animals, plants and fungi have this mevalonate pathway in common, starting with acetate and ending with squalene. This is an ancient pathway. However, the availability of oxygen may have removed squalene as an evolutionary bottleneck, and allowed for the formation of newer molecules with more useful properties. It is at this point that animals, plants and fungi diverge, each evolving their own versions of sterols. The structural molecular diagrams in the top-right indicate how subtle are the differences in these sterols.
The oxidation of squalene to oxidosqualine is the first branch-point that, through a multi-step process (not indicated), leads to plant phytosterols such as sistosterol. It is curious that while animals and fungi have evolved only the one sterol (each), plants have a number.
One further step from oxidosqualine to lanosterol, gives us the earliest common ancestor sterol for animals and fungi that then go their separate ways.
For the animals, there follow an additional ~18 steps after lanosterol, requiring 10 oxygen molecules and 15 enzymes, before one molecule of cholesterol can be formed.
Furthermore, cells do not rely on just one pathway to go from lanosterol to cholesterol, they run two in parallel — the Bloch Pathway and the Kandutsch- Russell Pathway. These pathways share enzymes at various steps that enable them to regulate each other. Cells are taking no chance with the synthesis of cholesterol, ensuring that there is more than one way to get there. A third pathway (not shown) called the Shunt Pathway, seems to further regulate the balance between the two cholesterol pathways.
The last step in the Kandutch-Russell Pathway, before cholesterol is finally formed, branches to the synthesis of vitamin D in skin cells. Finally, cholesterol itself can undergo further transformations to produce oxysterols, bile and steroid hormones, as previously described.
I invite you to contemplate this flow-chart for a moment. It is not only a depiction of cholesterol synthesis, remarkable enough as that process is. It also represents a cross-sectional snapshot of a few billion years of the painstaking evolution of life.