How We Handle Hypoxia

Where are we now? We are in a shallow sea. The time is seven hundred fifty million years ago. Ish. (750MYAish) There is no coffee, don’t bother looking. Land plants don’t exist yet.

We have started to wriggle.

Wriggling requires a left and right hand side. You need to be a bilaterian. Your FACE is bilateral. We are in the clade of bilaterians. We are NOT in the clade of sponges, which was the example last time. Sponges are literally made of sponges and just sit there on the bottom filtering seawater. That’s not us. We get around.

Quick recap of everything before now: Earth is formed, life appears, photosynthesizing cyanobacteria appear, then algae. The waste product of the photosynthesizers (oxygen) nearly kills everything else on the planet. The oxygen catastrophe. Oxygen is burny.

But the oxygen is an opportunity. A terminal electron acceptor. It opens up a new niche and multicellular organisms begin to appear. Niches get fishes. End recap.

Now we are beginning to wriggle. There have been a lot of advances in cell type and body organization since sponges. But what stays the same is the stem cells are kept in relatively hypoxic enviroments.

How Do The Cells Know What Oxygen Levels Are?

An inherent fact of multicellularity is that there is an oxygen gradient. Cells further from the surface get less oxygen. This protects them from oxidative damage but also maintains their stemness. But how does each cell know what to be?

Bilaterians have an ingenious system for sensing oxygen and oxidative stress and responding. It is controlled by 3 transcription factors (turns on other genes): HIF-1a, NF-kB and Nrf2. The system is:

1. Mind-bending
2. Uniquely Biological
3. Quirky as all getout

No human would design a system like this. I am writing this from the perspective of a molecular biologist. If you choose to interpret this as existence of a higher power I’m not going to argue. We are inclusive.

Here’s what happens in hypoxia. Keep in mind that we are a worm in a shallow sea that has less oxygen than today and that different currents likely have different oxygen levels. We need to be constantly navigating this.

By Oxygenation-atm.svg: Heinrich D. Hollandderivative work: Loudubewe (talk) – Oxygenation-atm.svg, CC BY-SA 3.0, Link

Skip this if you want.

HIF-1a mRNA is produced continuously under normal oxygen levels, but it is hydroxylated on specific proline residues by proline hydroxylase domain enzymes (PHDs). These are bound by von Hippel-Lindau protein(pVHL), marking them for ubiquination and degradation. The reaction of PHDs requires oxygen, so when oxygen is missing the HIF-1a fails to become hydroxylated and then degraded. It begins to build up. It migrates into the nucleus, dimerizes with HIF-1b (AKA aryl hydrocarbon receptor nuclear translocator), then binds to and turns on (or off) genes that have a hypoxia response element.

Since oxygen is the terminal electron acceptor, when hypoxia strikes the electrons in the mitochondria rapidly build up and spew out, creating a wave of ROS. The ROS oxidize specific cysteine residues in the IKKb complex, which would ordinarily keep NF-kB in the cytoplasm. This triggers K63-linked poly-ubiquination of TRAF6/TAF2, TAK1 autophosphorylation, IKKb phosphorylation, IkBa phosphorylation and degradation, which allows the nuclear entry of NF-kB. Then NF-kB binds to the HIF-1a promoter to turn it on. The now-stabilized HIF-1a protein binds the promotor of TNF-a and turns it on, which in turn binds the promoter of NF-kB and turns it on creating a double-positive feedback loop.

Nrf2 is cytoplasm-bound by its repressor KEAP1. The ROS oxidize cysteine residues on KEAP1, causing it to release the Nrf2. Dropping oxygen levels prevent disulfide bond formation which activates PERK which phosphorylates Nrf2, preventing the re-binding of KEAP1. The slowing down of mitochondrial respiration causes an increase in fumarate, which succinylates KEAP1, preventing its rebinding to Nrf2. Nrf2 is now free to translocate to the nucleus and trun on target genes.

End Skip.

This may seem overly complicated, but the bottom line is that all three transcription factors are simultaneously activated in the event of hypoxia. This is true in every bilaterian. Even if we just count up the copepods, there have been quite a few. They are tiny and the ocean is full of them. They can reproduce in about a week in tropical water. Each female can produce 300-800 eggs in her lifespan. Their clade is 400 million years old. The system works every time.

HIF-1a, NF-kB, Nrf2!!

Copepods have them, so do you!

What They Do

In the last post I pointed out that glycolysis does not use oxygen. Mitochondrial OxPhos does. HIF-1a and NF-kB turn on the key enzymes in glycolysis, allowing more glucose into the cell and allowing the worm to continue making some ATP for wriggling in the low oxygen conditions. NF-kB goes a step further and activates Pyruvate Dehydrogenase Kinase 1, preventing carbohydrate-derived fuel from entering the mitochondria.

Nrf2 activates the antioxidant defense system to eliminate the excess ROS. This includes enzymes in glutathione (the “master antioxidant”) production and ROS detoxification. It ALSO includes the pentose phosphate pathway (PPP). The PPP is an alternative pathway to glycolysis. It is primordial (4 BYAish). The activation of PPP results in the production of mitochondrial NADPH rather than NADH from glycolysis. The NADPH fuels the reduction of glutathione, the reducing power (electrons) of which is used to eliminate the ROS.

Now the lowered flow of pyruvate (electrons) into the mitochondria results in lowered ROS production. The increase in Nrf2 mops up the new level of ROS. Once ROS are under control, newly produced KEAP1 can begin to sequester newly produced Nrf2 and the system reaches a new equilibrium level.

Pretty neat.

It’s Important How We Talk About Things

In biology things are often associated with the way they were discovered. It’s hard to even talk about NF-kB without someone screaming INFLAMMATION but the process I’ve just described has nothing to do with inflammation. NF-kB’s basic job is a metabolic switch from OxPhos to glycolysis. This gets used in many immune functions because glycolysis is anabolic and an immune response is a very anabolic thing.

On the other hand, people are always praising Nrf2 for it’s antioxidant functions. If you want to criticize Nrf2, you have to say, “Nrf2, although hugely beneficial for its roles in antioxidant defense and redox balance, …”.

If your memory was deleted and you had to rewrite the textbooks knowing everything we know now, I think you might write things with a different slant. I would say that HIF-1a, NF-kB and Nrf2 all support hypoxia response in a coordinated fashion, control fuel switching and effect stemness.

As I’ve said, the adipose cells of obese humans are glycolytic. All THREE of them are implicated in obesity.

But only Nrf2 – the good guy – is implicated in increasing expression of G6PD, the controlling enzyme of the PPP. G6PD was already known to be a diet-inducible lipogenic enzyme back in 1959. Plot twist.

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Gene Symbol	Full Name	Pathway	Key Role	NF-κB	HIF-1α	NRF2
SLC2A1	Glucose transporter 1	Glycolysis	Increases glucose uptake	✔️	✔️	❌
HK2	Hexokinase 2	Glycolysis/PPP	Phosphorylates glucose	✔️	✔️	❌
PFKFB3	6-Phosphofructo-2-kinase 3	Glycolysis	Boosts glycolytic flux	✔️	✔️	❌
PKM2	Pyruvate kinase M2	Glycolysis	Pyruvate production & gene regulation	✔️	✔️	❌
LDHA	Lactate dehydrogenase A	Glycolysis	Converts pyruvate to lactate	✔️	✔️	❌
PDK1	Pyruvate dehydrogenase kinase 1	Glycolysis	Inhibits pyruvate entry into TCA	❌	✔️	❌
G6PD	Glucose-6-phosphate dehydrogenase	PPP	Produces NADPH, rate-limiting	❌	❌	✔️
PGD	6-Phosphogluconate dehydrogenase	PPP	Continues NADPH generation	❌	❌	✔️
TKT	Transketolase	PPP (non-ox)	Sugar shuffling, glycolysis link	❌	❌	✔️

Fitch, Walter M., et al. “The Effect of Fructose Feeding on Glycolytic Enzyme Activities of the Normal Rat Liver.” Journal of Biological Chemistry, vol. 234, no. 5, 1959, pp. 1048–51, https://doi.org/10.1016/S0021-9258(18)98127-5.

Kietzmann, Thomas. “Liver Zonation in Health and Disease: Hypoxia and Hypoxia-Inducible Transcription Factors as Concert Masters.” International Journal of Molecular Sciences, vol. 20, no. 9, May 2019, p. 2347, https://doi.org/10.3390/ijms20092347.

Li, Shiri, et al. “The Role of the Nrf2 Signaling in Obesity and Insulin Resistance.” International Journal of Molecular Sciences, vol. 21, no. 18, Sept. 2020, p. 6973, https://doi.org/10.3390/ijms21186973.

Mángano, M. Gabriela, and Luis A. Buatois. “The Rise and Early Evolution of Animals: Where Do We Stand from a Trace-Fossil Perspective?” Interface Focus, vol. 10, no. 4, Aug. 2020, p. 20190103, https://doi.org/10.1098/rsfs.2019.0103.

Vannier, Jean, et al. “Priapulid Worms: Pioneer Horizontal Burrowers at the Precambrian-Cambrian Boundary.” Geology, vol. 38, no. 8, 2010, pp. 711–14, https://doi.org/10.1130/G30829.1.