Aging and what causes it. Does calorie restriction help?

Aging affects everyone, whether they care or not. We might not pay much attention to it, but it’s a fascinating phenomenon. Right now, it’s still unclear how to slow it down, stop it, or even reverse it. Some animal experiments suggest possible directions, but first, we should clarify what aging actually is and how to measure it. That’s not a simple problem. The easiest way is to count the time from birth until now—but that’s probably not what interests us. What we really want to know is how we compare to others in a similar population. Are we more worn out or less? Is there a chance we’ll live longer than others, or not? Let’s explore this using studies and logical reasoning.

First, we need to define how we measure aging. Health might be a good metric. We don’t just want to live long if it means being bedridden, unaware of our surroundings, with a failing body and mind. That’s not a good vision of longevity. We want to stay active and healthy. We want all our organs fully functional, all necessary cells working properly. How do we measure that? Maybe by muscle strength? Or walking speed? I’ve discussed this before in relation to supplementing glycine and NAC—components of glutathione (GSH), the main antioxidant that removes H₂O₂ produced during oxidative phosphorylation in energy production. It worked remarkably well, with 75-year-olds showing significant rejuvenation. That’s a promising lead, and we’ll follow it.

Another important clue is cellular senescence, which can be identified by proteins like p21. We even have some idea how to trigger it—oxidative stress, such as from H₂O₂. The mechanism likely involves the enzyme phospholipase A2 (PLA2), which cleaves free fatty acids from membrane phospholipids. These act as signaling molecules for other enzymes, including those regulating mitochondrial uncoupling (UCP).

If, instead of oleic acid, the products of PUFA auto-oxidation (like 4-hydroxy-2-nonenal, HNE) are released, cellular senescence is triggered. So, what happens if we deactivate PLA2? No oxylipins are released, and senescence doesn’t occur. PLA2 is activated by hydrogen peroxide. What happens if we add the antioxidants? Could it also suppresses senescence? Let’s look at a study: It clearly shows that disabling PLA2 (Pla2r1 KO) reduces age-related metabolic changes. Antioxidant protection remains intact, and insulin sensitivity is preserved.

PLA2 knockdown (KO) limits the onset of p21 cellular senescence under both control (CD) and high-fat (WD) diets.

Turning off PLA2 (KO) prevents age-related changes in glucose metabolism (Old).

We will now leave PLA2 and look at another study on the mechanism of aging. The authors logically explain the mechanism of aging and support their reasoning experimentally. It concerns DNA damage related to the slowing of repair mechanisms. Generally, we have the idea that our DNA is unchanging, stored in the cell nucleus. Our environment is and always has been not entirely friendly. For example, we are, and in the past were likely much more, exposed to ultraviolet and radioactive radiation. We must also account for radiation from common minerals, remnants of cosmic radiation, etc. Our DNA is thus damaged daily in every cell of our body. Fortunately, we have a whole range of repair mechanisms in every cell. These take such good care of our DNA that we don't actually perceive these effects at all. It's different in cases of congenital genetic disorders of any of the repair mechanisms. These people, if their condition even allows them to survive, must protect themselves from UV radiation, radiation, etc. It can be fatal for them.

Long RNA sequences with defects block RNA polymerase, thus changing the ratios between proteins and resulting in insufficient transcription of long genes into proteins.

The genetic code is long, but for enzyme production, only a relatively short segment is always used - it unwinds, is copied into RNA, which leaves the nucleus and serves as a punch tape for the protein-making machine, RNA polymerase. If the DNA information is correct, the correct sequence is formed, which when folded functions as a new enzyme, a new nano-machine. Over time, despite repair mechanisms, errors appear in DNA. The longer the code, the greater the probability of faulty sequences. An error in the sequence causes the protein assembly machine to jam. It stops producing new proteins. Gradually, more RNA polymerases arrive and a traffic jam forms, like on a highway. Shorter sequences have fewer defects, longer ones have more. This causes changes in enzyme presence ratios. This causes changes in gene expression. Some of these changes are relatively well studied and are used as a measure of aging.

What does this imply? If we want to slow aging, we should protect our DNA in all cells from invaders that damage our DNA. We probably can't influence background radiation much, but UV radiation we can. But why can aging be influenced by turning off PLA2? By turning off PLA2, we don't remove the root cause, i.e., H2O2 from oxidative phosphorylation. The most likely mechanism I see is that even if phospholipids in membranes are oxidized, the problem only arises when they are released. This is also related to the fact that if we can maintain fat burning, oxylipins will also be burned.

The question is whether DNA damage is caused by limited repair mechanisms or by oxidative stress directly. I would lean more toward the first option, that oxylipins slow the activity of repair enzymes. We know that HNE attaches very actively to various enzymes, slowing or stopping their function. So if it attaches to repair enzymes, they won't be able to repair DNA and the damage will remain unresolved. Wait, what does this remind me of? Yes, it's the principle of cellular senescence! Cells stop dividing and wait for repair of damaged DNA, because unwound DNA can no longer be repaired - information from the neighboring strand would be lost.

So let's recap. Our membranes gradually fill with polyunsaturated phospholipids that replace the monounsaturated ones. From these phospholipids, PLA2 releases free fatty acids as needed (by higher H2O2 levels). But their composition changes with age, gradually increasing arachidonic acid, which oxidizes into e.g. HNE, which then suppresses the function of many enzymes, including those that repair DNA and burn fats. If we leave PUFAs in membranes, the function isn't suppressed. Aging slows down. If PUFAs are released from membranes, it's necessary to have a functional antioxidant system, i.e., enough GSH and NADPH. If we can prevent cellular senescence, overall aging will also slow down. Not all organs age at the same rate - lifespan is limited by the organ that ages fastest.

Now let's look at a study that examined the effect of calorie restriction on lifespan. The conclusion is that calorie restriction can indeed lead to lifespan extension, most notably in animals that gained the most weight on unlimited calorie intake. If we think about it, the whole study is essentially about the harm of overeating. Calorie restriction just normalizes intake to levels of natural diet. Artificially created food, fast carbs processed for maximum digestibility, lead to overeating, lead to insufficient filling of liver glycogen stores, which causes this overeating. Excess fuel in the cell causes hydrogen peroxide buildup and the cell should in this situation activate PLA2, release monounsaturated oleic acid from phospholipids, and this should trigger mitochondrial uncoupling, i.e., purposefully waste energy as heat. But if membranes contain excess arachidonic acid, we already know this triggers cellular senescence, i.e., accelerated aging. It's just another type of protection.

The greater the overeating, the shorter the life and the greater the effect of caloric restriction. Bad food must be intentionally limited so as not to harm. The right food is taken at the right speed without overeating.

Calorie restriction preserves the antioxidant system that relies on GSH and NADPH for longer. Older mice (22 months, black bar) have sufficient GSH recovery if they do not overeat.

However, overeating doesn't always have to be a means of shortening life. As an example, we can use a study that Petro Dobromylskyj brought into the discussion on network X. If we overfeed mice with coconut oil without omega-6 linoleic acid and without sugar, cellular senescence isn't triggered and the mice live on average longer (median) than on standard low-fat diet. I assume this is again about cellular membrane composition. Coconut oil doesn't produce oxylipins (HNE), similarly linoleic acid doesn't either if it's enriched with antioxidant protecting fats, vitamin E, it doesn't produce oxylipins (HNE). The result is an obese mouse that isn't sick and reaches relatively old age (for a mouse).

The conclusion might be that if we want to reach old age, we must prevent cellular senescence. There are multiple ways to trigger cellular senescence. The most common mechanism is ultraviolet radiation, radiation, loss of antioxidant protection and poisoning by products of autooxidation of omega-6 linoleic acid.

Addition

I have included references to studies on the correlation between PLA2 and PARP-1. The proposed mechanism appears to be possible; activation of PLA2R1 via H2O2 suppresses DNA repair by inhibiting the enzyme PARP-1. Suppression of PARP-1 increases senescence and reduces the amount of ROS. This seems to be an effective method for reducing the peroxidation of polyunsaturated phospholipids and is associated with a decrease in HNE levels. This has shown positive effects in an autism model in mice. However, the main culprit might be the excessively high content of polyunsaturated phospholipids in the membranes, which triggers senescence. If stable monounsaturated phospholipids were predominant in the membranes, there would not be excessive ROS elevation, and cellular senescence might not need to be triggered to reduce it.

I'll save some thoughts in the pictures from the studies so I don't forget them.

A mouse model of autism (VPA) produces too much ROS and increases the level of oxylipin HNE derived from omega-6, i.e. vegetable oils. This activates DNA repair by PARP-1 but does not reduce ROS. 5-AIQ inhibits PARP-1 and inhibits DNA repair, two different concentrations, but also activates MKP-1, which tries to switch the metabolism to fermentative, i.e. not using oxygen because the enzymes produce too much ROS. This reduces the amount of ROS produced and reduces the concentration of HNE, the cells survive but senescence is triggered.

Turning off DNA repair (siPARP-1) to reduce ROS can be problematic, senescent cells interfere and disrupt metabolism. Senescence is triggered by MKP-1, a signal to switch to fermentation and reduce ROS by removing phosphate groups placed on enzymes by kinases. If we turn off this signal (siMKP-1), caspase (caspase-3) is activated, which causes so much ROS that the dysfunctional cell is disassembled in a controlled manner into its individual components. This can sometimes be useful, as it allows metabolism to be repaired.

Another study, intestinal epithelial cells, what they do by poisoning them with oxylipin molecules HNE, causes apoptosis, so in this particular case. I estimate that small amounts of 4-hydroxy-2-nonenal (4-HNE, HNE) cause senescence rather than higher concentrations of apoptosis. This is because at higher concentrations, in addition to suppressing PARP-1, MKP-1 is also suppressed. The antioxidant N-acetyl cysteine (NAC) reduces the concentration of ROS so that cell poisoning does not occur.

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References:

Caloric restriction and the aging process: a critique

Loss of Pla2r1 decreases cellular senescence and age-related alterations caused by aging and Western diets

Genome-wide RNA polymerase stalling shapes the transcriptome during aging

Reduced Circulating Insulin Enhances Insulin Sensitivity in Old Mice and Extends Lifespan

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

H₂O₂-Activated Mitochondrial Phospholipase iPLA₂γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging

PARP-1 Inhibition Ameliorates Neuronal Damage in Valproic Acid-Induced Models of Autism Spectrum Disorder through the MKP-1/p38 Pathway

4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells