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The fat storage system

Perhaps not a very sexy name but why name it something else than what it is?

After studying the functioning of our lipoproteins for a good 2 years (and still not finished learning!), it has finally become clear what those lipoprotein are meant for. As complex as it may be, the end goal is really simple:

  • To facilitate the storage of fat
  • To prepare for the storage of fat
  • To recuperate storage structure when it is no longer needed
  • Transfer fat storage from one location to another

Compare it with the complexity of a swiss watch. All that mind-boggling complexity of the internal components, in the end to simply show you the hour, minutes and seconds.

I want to show you how these lipids relate to the storage objective. Not only that, by understanding how it functions I also want to provide a perspective on how the system can be protective of atherosclerosis, indeed even reverse plaque formation (!)

A very bold claim to make indeed. Just consider it speculative from my side. I present you the info and you make up your own mind. It will never be substantial until research is done to elucidate this further.

What I’m going to present you now, although elaborate, is still a simplified picture. Elaborate enough to show how the system works and simplified enough to show the most important components.

The basics

This picture, for most people who have already looked into the lipids, will be fairly familiar.

  • Dietary fat gets stored in adipose tissue
  • 1st phase delivery is done by chylomicrons
  • 2nd phase delivery is done by the interplay of various lipoprotein (VLDL, IDL, LDL, HDL)

The role of liver-produced lipids and cholesterol

The fat needs to be pushed in a lipid droplet which resides inside the fat cell. So both the lipid droplet and the fat cell need to expand. They both have a membrane so the membrane needs to expand as well and that requires phospholipids and cholesterol.

Those phospholipids and cholesterol come from the liver-produced lipoprotein (VLDL, IDL, LDL). Keep those phospholipids in mind when you see growing or shrinking lipid storage because I won’t mention them any further.

Not important for the system although interesting, the type of fatty acids used to make up the phospholipids will determine how much stability is required and thus how much cholesterol will be absorbed in the membrane. This is why cholesterol values can vary depending on the type of fat you eat.

When we get into the fasted state, the fat cells will start to release the fat. Now the reverse happens. Both the lipid droplet and the fat cell will shrink so they need to reduce their membranes and thus also release the cholesterol that is in it.

This means the cell will have too much cholesterol and will get rid of it via HDL.

The fat goes out of the fat cells and will be used throughout the body. In the muscle cells specifically, the cells will store the lipids that may have been reduced from previous activity. So here the lipid droplets will grow and, you guessed it, also here the membrane of the droplet requires cholesterol.

At the bottom left you see the shrinking lipid content represented of the fat cells while on the bottom right you have the growing lipid content of the muscle cells.

As we proceed longer into the fasted state, the muscle reaches an equilibrium where the size of the lipid droplet is maintained at a roughly equal size. Keep in mind though, there is a high turnover in the lipid droplet. So there is a continuous breakdown and buildup of triglycerides but the end result is essentially zero change in size. There is no need for additional cholesterol or the removal of cholesterol.

Before the lipoprotein can support this delivery of membrane components, they must have the right composition. This change in composition is facilitated by cholesterol ester transfer protein (CETP). The liver produces CETP under stimulation of insulin so the level of insulin drives the level of CETP activity.

With CETP:

  • HDL-C exchanges cholesterol for triglycerides (TG) with VLDL, IDL and LDL (ApoB100 containing lipoprotein)
  • HDL is dedicated to remodeling ApoB100 lipoprotein
  • This means that VLDL, IDL and LDL lose their TG and in return gain cholesterol. They become cholesterol-enriched.  
  • This also means that HDL loses its cholesterol content and increases its TG content.
  • By being TG-rich, more HDL will be taken up by the liver creating lower circulating HDL particles in the blood
  • By being cholesterol-enriched, ApoB100 lipoprotein can be taken up by the cells

Without CETP:

  • HDL cannot offload its cholesterol to the ApoB100 lipoprotein and instead stays in circulation longer and accumulates more cholesterol
  • HDL is dedicated to cholesterol collection from cells
  • ApoE and LCAT drive HDL cholesterol accumulation so that it becomes big enough for uptake by the liver
  • The ApoB100 lipoprotein remain rich in TG and cannot be taken up by the cells

This is already becoming complex but you get a picture of how important CETP is. CETP will come back when we look at atherosclerosis..

Also note in the illustrations above, the free fatty acids aka non-esterified fatty acids (NEFA) (meaning not bound their usual glycerol backbone) are transported via albumin. This is an abundant available protein responsible for transporting the energy itself that will be used directly in the cells and also will be esterified (binding to glycerol) as triglycerides and stored in the lipid droplets inside the cells.

Getting everything across the cell membrane

There are 2 important factors to absorb the fatty acids and membrane structures. Note again, we focus primarily on cholesterol although the phospholipids are part of it.

These are not just gates that are open 100%, accepting anything at all times. How are they regulated? How do we get the NEFA and cholesterol across?

LPL

  • ApoC-II reduces on a high fat diet. This means that more NEFA is imported via albumin
  • Sterol regulatory element-binding protein 1 (SREBP1) is activated under lower cellular cholesterol content and reduces LPL expression
    • => This is a negative feedback which reduces NEFA import. When there is a shortage of cholesterol to build storage then you need to lower NEFA to avoid accumulation in the cell
  • The fat cell LPL is activated by insulin.  Muscle cell LPL is activated by low insulin
    • => Insulin prioritizes fat storage in fat cells. => When insulin lowers, the energy is distributed and taken up by the other cells
  • LPL is increased during and after exercise. During those moments, there is an increased demand for energy

LDLr

  • Requires cholesterol-rich lipoprotein (VLDL, IDL, LDL)
  • Endocytosis of the lipoprotein (complete uptake of the lipoprotein)
  • Support expansion of the lipid droplet membrane via cholesterol import
  • Insulin activates SREBP1 and SREBP1 activates LDLr. As expected because SREBP1 shows there is a need for cholesterol and when driven by insulin it means we are focusing on storing fat.

LDLr in the muscle

  • exercise, fasting activates SREBP1 via MAPK. Similar to insulin, we also want to store fat simply to respond to a higher fat metabolism need. When fat metabolism increases, there is need for a bigger local buffer, a bigger lipid droplet.

In summary so far, we can say that a diet existing out of high fat quantities in combination with a high fat metabolism causes a continuous growing and shrinking of the fat cells. If that is combined with exercise then also the muscles are faced with this growing and shrinking. The timeframe and the volume of fat handled causes those cells and lipid droplets to grow to larger sizes and to shrink to smaller sizes, compared to a diet and lifestyle where glucose is a major energy source. This leads to a lower flux of the fat storage.

Such an intensified remodeling on high fat/high activity requires more support from the circulating lipoprotein!

This has to be supported by the lipoproteins that we are familiar with. VLDL, LDL and HDL. Moreover, in order to be able to receive the next bolus of incoming fat it is good to have the right types and level of lipoprotein ready to support this.

What is the best way to accommodate a high fat meal so that we can swiftly provide the necessary membrane components for expansion?

  • TG-rich (large buoyant) LDL
  • High cholesterol levels on HDL

When the meal comes in, insulin comes up and will stimulate CETP. With those 2 listed above, CETP can immediately remodel LDL to become enriched with cholesterol so that it can be taken up by the fat cells. Both lipoproteins need to be sufficiently high to support a faster clearance of the fat into storage.

It’s my engineer thinking but would it be just a coincidence that LDL goes up under such high fat intake and consumption? This high flux signals the need for a greater storage capacity so doesn’t it make sense to have more of the membrane components ready to maximize the storage capacity? Could that be the reason why fasting LDL particles and HDL cholesterol are high? As soon as CETP kicks into action you’ll now have a lot of LDL particles ready to accept cholesterol and support the membrane expansion of fat cells and their lipid droplets.

You NEED high LDL and HDL when you have a high dietary fat intake

High fasting LDL, HDL and low TG for lean individuals on a ketogenic diet

With the above information you understand the need for it. But how do we arrive at that situation? The following are general mechanisms that apply to everybody but the extend to which they get applied is determined by many factors such as the amount of fat you eat, exercise level, your level of insulin etc… There are other factors but insulin is a very big player in all of this.

  • Under fasted conditions, insulin is sufficiently low to keep cholesterol production very low. When fasted we are not interested in storing fat. The liver preserves most fatty acids for ketogenesis, bile production and its own fat metabolism. Both fatty acids and cholesterol production is low in such a way that it has shifted from VLDL production towards large buoyant LDL
  • Large buoyant LDL stays in circulation longer because it is cholesterol poor, ApoE rich and thus has a lower ability to be taken up by LDLr for endocytosis. That ApoE enrichment is also the reason why small dense LDL (sdLDL) has a lower affinity for LDLr
  • Low insulin keeps CETP low, thus VLDL and LDL cannot acquire sufficient cholesterol from HDL for endocytosis by LDLr
  • Due to low CETP, the cholesterol load on HDL increases and minimizes exchange with VLDL, IDL, LDL.
  • With very low to no production of VLDL sized lipoprotein, there are almost no lipoproteins that can acquire ApoC-II for lipolysis by LPL thus almost all skeletal muscle NEFA are derived from albumin

So on the production side we have lower production but more of the lipoprotein in the LDL size, in circulation there is no remodeling taking place and due to this the uptake by the skeletal muscle is much lower.

When the skeletal muscles are full and reduce their uptake of fatty acids, this will lead to a higher return of those fatty acids towards the liver. So the release of fatty acids from the fat cells is temporarily higher than the uptake by the other cells. This accumulation leads to a larger availability of fatty acids in the liver so that it can augment its production of TG-rich LDL.

Now it will depend on how big this discrepancy is. This higher level of fatty acids in the liver also causes more ketones (BHB) to be produced. The body may respond to this by slightly increasing insulin. This will cap BHB production, reduce the rate of fatty acid release from fat cells and slightly increase cholesterol production allowing the liver to temporarily produce more VLDL-sized lipoprotein. It will also slightly elevate CETP so that storage can be facilitated to get rid of the excess fat in circulation.

You may notice this as a slight elevation in your fasted triglycerides. It is only temporarily as it is part of an exercise to balance out demand and supply.

Buffering for energy usage

and issues with it

Still following so far? OK. Now lets have a look at issues with storage.

Just like with glucose, the fat is stored in large quantity in a central place and from there distributed to local storage in the muscle. Central is put in quotes, mainly referring to the adipocytes. It is supposed to sit under our skin and well.. our skin is completely wrapped around our body so it is a bit strange to say central.

I’ve illustrated glycogen here just for comparison. Just note that I’m continuing only talking about the fat.

Storage limitation -> muscle insulin resistance

At some point, all of these buffers have to signal when they are full and (temporarily) cannot take in any more fat. This signaling is provided by 1,2DAG. An intermediate fatty acid that is formed when synthesizing triglycerides for storage.

  • TG synthesis generates 1,2DAG
  • Accumulation leads to translocation of 1,2DAG into the cell membrane
  • 1,2DAG attracts Protein Kinase C (PKC) to the membrane
  • PKC internalizes LDLr -> No uptake of cholesterol-rich lipoprotein
  • PKC inhibits Insulin Receptor Signaling (IRS) -> insulin resistance!
  • 1,2DAG is preferentially hydrolyzed (broken down) for fat metabolism
  • Exercise, fasting, high fat diets increase fat metabolism
    • Increased clearance of 1,2DAG via DGAT1 expression (exercise)
    • Restores insulin sensitivity
    • Increases SREBP1 expression -> increases LDLr expression -> increases lipid storage capacity

It does create a conflict though because the accumulation also indicates the need for more cholesterol but 1,2DAG prevents this by blocking LDLr. You cannot signal stop and store more at the same time so how do we end up in this situation?

  • Increased storage in lipid droplet reduces cellular cholesterol
  • lower cellular cholesterol -> SREBP1 activation -> increase LDLr expression
  • 1,2DAG accumulation threshold blocks LDLr via PKC
    • Prioritize cellular cholesterol production instead of via extracellular uptake
    • Reduce cellular fatty acid accumulation

In and of itself this is a normal mechanism. By not taking up both lipids and membrane components, other cells can manage to top up their reserves so you get an even distribution. The problem however is when every cell is topped up and you still have too much in circulation.

It will lead to higher insulin levels because too much energy in circulation drives up insulin to try and store that excess. That will lead to more VLDL-bound TG, higher CETP thus higher cholesterol bound to LDL (higher cholesterol/particle ratio) and lower HDL.

But with cells that have enough fat accumulated, they say no to insulin so insulin does what it is supposed to do, form the lipoprotein particles that are required for storage but all the cells keep their doors closed.

Exactly the pathologic lipid profile that we know and should fear.

  • Increased VLDL triglycerides
  • Increased cholesterol-rich LDL
  • Decreased HDL (lower cholesterol/particle ratio)

Why should we fear it? Because it has a good proxy value towards atherosclerosis.

Atherosclerosis

saved by enhanced fat metabolism?

As mentioned in the introduction, it may actually mean reversal of plaque! The very low insulin obtained through being lean, very low carbohydrate diet with high fat intake creates cholesterol-enriched – ApoE-enriched HDL which drives up their LCAT activity. LCAT stimulates cholesterol release from macrophages.

Macrophages in atherosclerosis aren’t only gobbling up cholesterol, they also accumulate triglycerides. It turns out though that cholesterol efflux helps to switch gears into fat metabolism so the macrophage can reduce its fat storage. If you cannot store the fat, you have to start using it for energy.

This whole deep dive into research led me to the following, to my view important, observation. These M1 macrophages actually maintain import and storage of fatty acids (through LDLr). Only by ‘forcing’ cholesterol efflux are we able to reverse the situation, pushing them to metabolize the fat. PPARy is known to do this and PPARy is activated by BHB, thus a ketogenic diet. PPARy is also activated under low cellular cholesterol, via SREBP1. This has been evidenced in liver and fat cells but likely also applies to macrophages. And a deactivation of PPARy was accomplished by re-addition of cholesterol.

PPARy expression hence seems subject to a tight and fast control by alterations in intracellular cholesterol levels, and this effect is mediated by the SREBP family of transcription factors.

source: https://pubmed.ncbi.nlm.nih.gov/10409739/ “Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: implications for adipocyte differentiation and metabolism”

A paper showed me that macrophages are able to restore insulin resistance in skeletal muscle of diabetics. Insulin resistance that is caused by 1,2DAG as we have seen. So if macrophages carry the capacity to somehow neutralize 1,2DAG from the membrane then maybe they apply this capacity also to their own membrane.

This is still an area I need to find research on but we already see that macrophages do not exhibit a stop mechanism like other cells.

That would explain why they get so big with fat and cholesterol. The only way out of this situation is to enhance the efflux of cholesterol from the macrophages. And the only way to do that is by creating big cholesterol-rich HDL so that it can acquire ApoE and drive up LCAT. And the only way to do that is by getting your insulin very low. And the only way to do that sustainably(!) is by adopting a very low carb diet and getting lean.

Get those cells to push out cholesterol like it’s nobodies business!

—– T H E – E N D —–

Sources

My previous writings on this topic with references that over time have contributed to my understanding.

Further resources, specifically for this article

LXR

https://pubmed.ncbi.nlm.nih.gov/12697904/ “Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433546/ “Liver X receptors in lipid signaling and membrane homeostasis”

Cholesterol efflux

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159082/ “Cholesterol efflux and atheroprotection: Advancing the concept of reverse cholesterol transport”

https://pubmed.ncbi.nlm.nih.gov/17390217/ “Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes”

Lipids on a ketogenic diet

https://academic.oup.com/jn/article/135/6/1339/4663837 “Modification of Lipoproteins by Very Low-Carbohydrate Diets”

LPL receptor

https://www.ncbi.nlm.nih.gov/books/NBK537040/ “Biochemistry, Lipoprotein Lipase”

https://www.sciencedirect.com/science/article/pii/S0022227520380901 “Effects of dietary carbohydrate and fat on plasma lipoproteins and apolipoproteins C-II and C-III in healthy men”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705268/ “Apolipoprotein C-II: New findings related to genetics, biochemistry, and role in triglyceride metabolism”

https://www.ahajournals.org/doi/full/10.1161/01.ATV.19.3.472 “Role of ApoCs in Lipoprotein Metabolism”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705268/ “Apolipoprotein C-II: New findings related to genetics, biochemistry, and role in triglyceride metabolism”

https://pubmed.ncbi.nlm.nih.gov/11090277/ “Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes”

LDL in skeletal muscle

https://pubmed.ncbi.nlm.nih.gov/9696990/ “Skeletal muscle lipoprotein lipase: molecular regulation and physiological effects in relation to exercise”

https://www.sciencedirect.com/science/article/pii/S0925443902001692 “Expression and regulation by insulin of low-density lipoprotein receptor-related protein mRNA in human skeletal muscle”

https://diabetes.diabetesjournals.org/content/50/11/2585 “In Muscle-Specific Lipoprotein Lipase−Overexpressing Mice, Muscle Triglyceride Content Is Increased Without Inhibition of Insulin-Stimulated Whole-Body and Muscle-Specific Glucose Uptake”

https://pubmed.ncbi.nlm.nih.gov/17046550/ “Fasting decreases free fatty acid turnover in mice overexpressing skeletal muscle lipoprotein lipase”

in liver

https://pubmed.ncbi.nlm.nih.gov/12951168/ “Mechanisms of HDL lowering in insulin resistant, hypertriglyceridemic states: the combined effect of HDL triglyceride enrichment and elevated hepatic lipase activity”

LDL receptor

https://www.ahajournals.org/doi/10.1161/circresaha.114.301621 “PCSK9 – A key modulator of cardiovascular health”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3071588/ “Activation of LDL Receptor (LDLR) Expression by Small RNAs Complementary to a Noncoding Transcript that Overlaps the LDLR Promoter”

https://pubmed.ncbi.nlm.nih.gov/16449296/ “Exercise training and calorie restriction increase SREBP-1 expression and intramuscular triglyceride in skeletal muscle”

https://diabetes.diabetesjournals.org/content/69/5/848 “Exercise and Muscle Lipid Content, Composition, and Localization: Influence on Muscle Insulin Sensitivity”

https://pubmed.ncbi.nlm.nih.gov/9010277/ “Low density lipoprotein receptor expression and function in human polymorphonuclear leucocytes”

https://pubmed.ncbi.nlm.nih.gov/3237238/ “The binding of lipoproteins to human muscle cells: binding and uptake of LDL, HDL, and alpha-tocopherol”

https://journals.physiology.org/doi/full/10.1152/ajpendo.00543.2005 “Exercise training and calorie restriction increase SREBP-1 expression and intramuscular triglyceride in skeletal muscle”

https://www.cell.com/fulltext/S0092-8674(00)80213-5 “The SREBP Pathway: Regulation of Cholesterol Metabolism by Proteolysis of a Membrane-Bound Transcription Factor”

https://www.ahajournals.org/doi/full/10.1161/01.ATV.18.3.466 “Influence of ApoE Content on Receptor Binding of Large, Buoyant LDL in Subjects With Different LDL Subclass Phenotypes”

https://pubmed.ncbi.nlm.nih.gov/8254047/ -> https://dm5migu4zj3pb.cloudfront.net/manuscripts/116000/116915/cache/116915.1-20201218131521-covered-253bed37ca4c1ab43d105aefdf7b5536.pdf “Accumulation of “small dense” low density lipoproteins (LDL) in a homozygous patients with familial defective apolipoprotein B-100 results from heterogenous interaction of LDL subfractions with the LDL receptor”

https://pubmed.ncbi.nlm.nih.gov/16670767/ “Putting cholesterol in its place: apoE and reverse cholesterol transport”

Lipids

https://www.sciencedirect.com/science/article/pii/S0022227520380901 “Effects of dietary carbohydrate and fat on plasma lipoproteins and apolipoproteins C-II and C-III in healthy men”

https://www.britannica.com/science/lipid/Classification-and-formation “Classification and formation”

Macrophages

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3767414/ “Lipid Droplets And Cellular Lipid Metabolism”

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0056601 “Skeletal Muscle Insulin Resistance Associated with Cholesterol-Induced Activation of Macrophages Is Prevented by High Density Lipoprotein”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868788/ “Macrophage PPARγ is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957345/ “High-Density Lipoprotein Maintains Skeletal Muscle Function by Modulating Cellular Respiration in Mice”

https://pubmed.ncbi.nlm.nih.gov/29217413/ “Apolipoprotein E-containing high-density lipoprotein (HDL) modifies the impact of cholesterol-overloaded HDL on incident coronary heart disease risk: A community-based cohort study”

https://pubmed.ncbi.nlm.nih.gov/16670775/ “HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoE- and ABCG1-dependent pathway”

https://www.nature.com/articles/aps201093 “A novel model of cholesterol efflux from lipid-loaded cells”

https://www.cell.com/cell-metabolism/references/S1550-4131(07)00166-0 “PPARγ Activation Primes Human Monocytes into Alternative M2 Macrophages with Anti-inflammatory Properties”

https://pubmed.ncbi.nlm.nih.gov/22207731/ “Regulation of lipid droplet cholesterol efflux from macrophage foam cells”

https://www.nature.com/articles/s41598-018-34305-x “A reduced M1-like/M2-like ratio of macrophages in healthy adipose tissue expansion during SGLT2 inhibition”

Lipid droplet

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575688/ “DAG tales: the multiple faces of diacylglycerol—stereochemistry, metabolism, and signaling”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526153/ “The Biophysics and Cell Biology of Lipid Droplets”

Muscle cell

https://www.sciencedirect.com/science/article/pii/S0005273615003867 “Alteration of lipid membrane structure and dynamics by diacylglycerols with unsaturated chains”

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0056601 “Skeletal Muscle Insulin Resistance Associated with Cholesterol-Induced Activation of Macrophages Is Prevented by High Density Lipoprotein”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1665384/ “High triacylglycerol turnover rate in human skeletal muscle”

https://journals.physiology.org/doi/full/10.1152/ajpendo.00543.2005 “Exercise training and calorie restriction increase SREBP-1 expression and intramuscular triglyceride in skeletal muscle”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866250/ “Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510842/ “Studies on the Substrate and Stereo/Regioselectivity of Adipose Triglyceride Lipase, Hormone-sensitive Lipase, and Diacylglycerol-O-acyltransferases”

Dawn phenomenon

https://joe.bioscientifica.com/view/journals/joe/231/3/235.xml “Melatonin modifies basal and stimulated insulin secretion via NADPH oxidase”

https://pubmed.ncbi.nlm.nih.gov/6368151/  “Fasting early morning rise in peripheral insulin: evidence of the dawn phenomenon in nondiabetes”

PPARy

https://pubmed.ncbi.nlm.nih.gov/10409739/ “Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: implications for adipocyte differentiation and metabolism”

Metabolism on a ketogenic diet

Let me first clarify that this article is about a true ketogenic diet, meaning you actually produce higher levels of BHB. Why am I saying this? Because I notice quite a number of people who leave out most carbs but they don’t necessarily reach the ketogenic state due to various reasons. I’ve always said and will keep on repeating: Measure your blood BHB level to validate if you are in a ketogenic state.

That said, what is this article about? I had this idea for a while where I suspect that there is a reduction of metabolism on a ketogenic diet. This is noted by a drop in the thyroid hormone T3 and a rise in reverse T3 (inactive T3). Yet we notice a higher energy consumption which tells us that metabolism is higher. We also notice a higher heat production on a ketogenic diet. So the question is if this increase in heat production is of such magnitude that it covers both the increase versus a standard diet and the reduction in metabolism versus a standard diet.

This is not proportional, just for illustration purposes. Note though that scientifically it will be argued that heat production is part of the metabolism. I would argue it is part of energy loss but it is nor part of cell metabolism in the sense that it doesn’t contribute to metabolism. Rather it is a result of metabolism just like we produce water and CO2 which gets dissipated from our body.

The efficiency of the metabolism will determine how much heat is produced versus how much workable energy for the cell is produced in the form of ATP.

What I want to do next is look at evidence that shows if there is a lower metabolism yet a higher heat production but also at the numbers to get an estimation of how much more heat could be produced.

Before getting into the details, here is an article from Stephen Phinney, PhD (a highly respected low carb researcher) who argues for increased sensitivity to T3 because metabolism is maintained while recognizing the drop in T3. However I do not agree to this sensitivity hypothesis because as stated, I believe metabolism does go down yet the heat production goes up so that total energy consumption is maintained or even elevated.

So to recap…

  • Energy consumption is equal or increases
  • Energy production (metabolism; ATP production) decreases
  • Heat production (energy loss) increases

Lower T3

Phinney referred to unpublished data from Volek showing in a calorie restricted setting (estimated weight maintenance – 500 kcal) a drop from 4.2pmol/L to 3.5pmol/L, low-fat to KD for 14 overweight men. That is 17% lower.

“Comparison of energy-restricted very low-carbohydrate and low-fat diets on weight loss and body composition in overweight men and women” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC538279/

A second reference that he provided shows an iso-caloric comparison of 85%, 44% and 2% carbohydrates for 6 men during 11 days. The T3 levels measured were 1.78, 1.71 and 1.33 nmol/L. The 2% carb is 23% lower in T3 than the 44% carbs.

“Isocaloric carbohydrate deprivation induces protein catabolism despite a low T3-syndrome in healthy men” https://pubmed.ncbi.nlm.nih.gov/11167929/

1nmol = 1000 pmol so I think there is a mistake in units used. In any case, this second study already gives us an important indication. It seems that the level of carbohydrates in the diet determines the level of T3. Still, it could also be depending on the protein. Despite that this is kept equal in the diet, the drop in carbohydrates allows glucagon to be more active resulting in more gluconeogenesis which also catabolizes amino acids into glucose thus resulting in a lowering of the plasma amino acids.

The study noted an increase in urinary nitrogen excretion (10.91; 12.79; 15.89 g/24h). A 24% increase in protein catabolism. Please do note that switching to a ketogenic diet takes roughly 11 days for the body to adapt in order to reduce the protein catabolism. Search for “transition” on that page.

Insulin stimulates muscle protein synthesis (under sufficient leucine). By drastically lowering insulin this would make (muscle) protein more prone to catabolism unless there are counter measures. Lowering metabolism via T3 reduction, besides protection from BHB and growth hormone, is an essential element to reduce amino acid requirements in a cell that wants to build protein.

“Thyroid hormone stimulates protein synthesis in the cardiomyocyte by activating the Akt-mTOR and p70S6K pathways” https://pubmed.ncbi.nlm.nih.gov/16717100/

“Effect of T3-induced hyperthyroidism on mitochondrial and cytoplasmic protein synthesis rates in oxidative and glycolytic tissues in rats” https://journals.physiology.org/doi/full/10.1152/ajpendo.00397.2006

“T3 increases mitochondrial ATP production in oxidative muscle despite increased expression of UCP2 and -3” https://journals.physiology.org/doi/full/10.1152/ajpendo.2001.280.5.E761

We see that hypothyroidism results in a reduction of amino acid efflux from skeletal muscle and a reduction in protein synthesis in various tissues.

Amino acid release (alanine, glycine, tyrosine, glutamine) is increased in hyperthyroid skeletal muscle [9] while it is decreased in hypothyroidism [9, 15, 22].

…

in hypothyroidism hepatic synthesis of intracellular or secretory proteins is reduced by 20% and 50%, respectively [18, 40].

…

Total cellular protein toss during a starvation period is reduced by 50% in the hypothyroid state, mainly due to a decrease in the cytosolic compartment, while in hyperthyroidism there is no change [44]. In starvation, mobilization of hepatic proteins is decreased in the thyroid deficient state, providing a prolonged conservation [44]

source: “Thyroid hormone action on intermediary metabolism. Part III. Protein metabolism in hyper- and hypothyroidism” https://pubmed.ncbi.nlm.nih.gov/6231411/

What we have seen here is that T3 modulates (increase/decrease) the basal level of ATP production and mTORC1 stimulation affecting amino acid utilization for protein synthesis and replacement.

Circulating amino acids are measured and accordingly the body responds. Stimulate synthesis when there is abundance, conserve when there is shortage.

The ketone molecule BHB reduces protein catabolism but before we get to this point we first have to generate BHB and for that dietary protein has to be low enough in the first place.

Increased UCP

UCP allows for the loss of electrons that dissipate as heat during metabolism. Ahigher level of UCP will mean that more heat will be generated and less ATP.

In the following rat study they gave a control diet, control+sucrose drink, low protein-high carb or low protein-high fat diet. The authors concluded the following:

Brown adipose tissue protein content and thermogenic capacity (assessed from purine nucleotide binding to isolated mitochondria) were greater than control values in sucrose-fed and protein-deficient animals, and the greatest levels of activity were seen in low protein–fed rats with a high fat intake.

Why greater in the high fat versus the high carb? Because dietary carbs result in a direct feed of glucose to the brain so there is more protection from catabolism due to the higher insulin stimulation. Dietary fat doesn’t stimulate insulin as much and in contrast requires the dissociation of the fatty acids from the glycerol backbone so that the glycerol can serve as a gluconeogenic source. But what to do with that abundance of free fatty acids? Get rid of them via thermogenesis. With a low protein-high fat diet you need a higher level of fat metabolism to get the same level of protection from amino acid catabolism.

The thermic effect seems to be double that from the control group.

The acute thermogenic response (% rise in oxygen consumption) to a standard balanced-nutrient meal was higher (12%) in sucrose-supplemented and in low protein groups (15-16%) than in control rats (8%).

“Influence of Carbohydrate and Fat Intake on Diet-Induced Thermogenesis and Brown Fat Activity in Rats Fed Low Protein Diets” https://academic.oup.com/jn/article-abstract/117/10/1721/4780321?redirectedFrom=fulltext

The next animal study gave the rats a ketogenic diet with 9% protein. Versus the control group they ate the same amount of calories, had reduced weight gain and generated 11% more heat. The 66% calorie restricted group had obtained a similar fat mass and lean mass composition but without the heat production.

Fig. 1.
Fig. 2.

“A high-fat, ketogenic diet induces a unique metabolic state in mice” https://journals.physiology.org/doi/full/10.1152/ajpendo.00717.2006

I do want to warn about animal studies though and caution with interpretation. 2 main issues could influence the results.

  • 1) isocaloric feeding: if a low protein diet requires more fat burning then the ad lib feeding could result in a higher dietary intake. By giving the same calories as the control diet you reduce the protein catabolism protection. This may lead to a lower thermogenesis capacity than they would do naturally and at the same time have a reduction in growth.
source: “Isoenergetic Feeding of Low Carbohydrate-High Fat Diets Does Not Increase Brown Adipose Tissue Thermogenic Capacity in Rats” https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038997
  • 2) Too low protein: when feeding animals very low protein amounts or even absent protein, they will reduce protein assembly. In order to increase heat production, fat needs to be metabolized via an increase in UCP expression and at the same time the machinery for fat metabolism needs to be enhanced. This means proteins need to be assembled to form the necessary enzymes, hormones etc. If the level of circulating amino acids go too low then those protein assemblies could be under pressure as well.

When evaluating this in humans we see a similar result. Energy expenditure goes up when carbohydrates are exchanged for fat while keeping protein equal. The subjects on low carb had a TEE of 2713 kcal/d and this changed at the end with an increase of 270 kcal/d so roughly 10% which is in line with the animal study.

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“Effects of a low carbohydrate diet on energy expenditure during weight loss maintenance: randomized trial” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233655/

Finally

So we see from the reduction in T3 that cell metabolism in general slows down while we see in skeletal muscle and brown adipose tissue that heat production goes up. Lean mass growth is slowed down.

What this essentially means is that the heat production increase is not just the additional energy expenditure that is observed, it also covers whatever the reduction is in metabolism. Keep in mind that 17~23% T3 reduction which by itself results in a lower heat production. The calorie-restricted mice had a 6% reduction in heat versus the control while the KD had an almost 8% increase in heat production versus control.

It all depends on the level of dietary protein and how it is combined with carbs or fat.

Longevity is associated by a low IGF-1 and T3 in those who live well above 90 years. As long as we can maintain or even build muscle strength, reducing protein and carb while increasing fat intake may be a good strategy to reduce overall tissue metabolism.

“Familial longevity is associated with decreased thyroid function” https://pubmed.ncbi.nlm.nih.gov/20739380/

The level of protein has a major role in signaling ideal reproduction times. Lowered levels indicate a less than optimal time with a need to be conservative.

On the menu you can choose:

  • with increased heat production (high fat)
  • without heat production (high carb)

The choice is yours.

———————————— T H E – E N D ————————————

A ketogenic diet increases the brain glucose uptake mechanism

When looking into acetoacetone uptake by the brain, I bumped against the following article. I don’t have full access but the abstract showed me the following:

Similar trends were observed for (18)FDG uptake with a 1.9-2.6 times increase on the KD and F(asting), respectively (P < 0.05).

FDG -> radiolabeled glucose such as used to trace cancer. But more importantly, a good doubling of the uptake of glucose! So I started thinking, is it the case that our brain is sucking up so much glucose? Or is this just a side effect of being ketotic and getting a big bolus of glucose administered?

“Mild experimental ketosis increases brain uptake of 11C-acetoacetate and 18F-fluorodeoxyglucose: a dual-tracer PET imaging study in rats” https://pubmed.ncbi.nlm.nih.gov/21605500/

———————–

I find it interesting because astrocytes produce more lactate on starvation and I assume on keto as well. Why? To increase the expression of MCT1 transporters on the endothelial cells in the blood-brain-barrier which allows a higher uptake of BHB.

Pyruvate can otherwise be reduced to lactate by lactate dehydrogenase (LDH). This lactate can be released in the extracellular space through monocarboxylate transporters (MCTs).

“Brain Energy Metabolism: Focus on Astrocyte-Neuron Metabolic Cooperation” https://www.sciencedirect.com/science/article/pii/S1550413111004207

Astrocytes can store small amounts of glycogen which they, if necessary, break down to glucose and metabolize to lactate (Falkowska et al. 2015). To fulfill this functional characteristic, astrocytes are highly metabolically flexible and can rapidly upregulate glycolysis. In the event of an undersupply, astrocytes thus ensure the survival and function of neurons by providing lactate (Kasischke et al. 2004; Pellerin and Magistretti 1994).

“Long-Term Glucose Starvation Induces Inflammatory Responses and Phenotype Switch in Primary Cortical Rat Astrocytes” https://link.springer.com/article/10.1007/s12031-021-01800-2

———————–

So it seems that astrocytes start to increase lactate production when glucose is running low and they use their little glycogen buffers for that. Cool. So that coincides nicely with an increase in ketones and takes care of the BHB uptake, balancing out low glucose with increased BHB.

I guess it would make sense for the brain to increase its ability to take up glucose which is then indicated by the first link I provided. So I don’t think the brain is actually continuously taking up so much glucose, it is just that it has opened the gates to maximally receive glucose.

Further a reference that shows increased glucose uptake under chronic hypoglycemia

“Chronic hypoglycemia increases brain glucose transport” https://pubmed.ncbi.nlm.nih.gov/3532819/

A first candidate to look at what could cause that increase in uptake is the GLUT1 transporter. And indeed, the following article looked at GLUT1 expression in the BBB under glucose deprivation, hypoxia and the two combined.

r/ketoscience - Keto increases the ability to receive glucose in the brain?

“Glucose deprivation and hypoxia increase the expression of the GLUT1 glucose transporter via a specific mRNA cis-acting regulatory element” https://onlinelibrary.wiley.com/doi/pdfdirect/10.1046/j.0022-3042.2001.00756.x

Summary

  • astrocytes sense low glucose availability
  • astrocytes increase lactate production
  • lactate increases MCT1 expression
  • increased MCT1 enables more influx of BHB into the brain
  • low glucose increases GLUT1 expression in the BBB to maximally take up glucose

So this shows a whole balancing mechanism, it allows a shift from purely glucose to glucose and BHB.

———————–

For people who interpret this as ketones being a backup and think this is showing the brain needs glucose… I’d say the truth is somewhere in the middle 😉 Consider the following quote. There may be a point that can be crossed when too much glucose is available and can be considered toxic when that downregulates GLUT1 in the BBB to the point that the brain doesn’t get enough glucose. Also here the astrocytes may start to produce lactate but it won’t do any good because under hyperglycemia, there won’t be any BHB produced.

Glucose transport into the brain is depressed in chronically hyperglycemic (diabetic) rats.

“Chronic hypoglycemia increases brain glucose transport” https://pubmed.ncbi.nlm.nih.gov/3532819/

Compared with normal control rats, the GLUT(1) mRNA was reduced by 46.08%, 29.80%, 19.22% (P < 0.01) in DM1, DM2, and DM3 group, respectively; and the GLUT(3) mRNA was reduced by 75.00%, 46.75%, and 17.89% (P < 0.01) in DM1, DM2, and DM3 group, respectively.

“Influence of blood glucose on the expression of glucose trans-porter proteins 1 and 3 in the brain of diabetic rats” https://pubmed.ncbi.nlm.nih.gov/17935675/

But I find contrasting evidence. In the following article they noted no effect on GLUT1 expression. Different rat models so who knows what the case is for humans..

“Blood-brain barrier glucose transporter: effects of hypo- and hyperglycemia revisited” https://pubmed.ncbi.nlm.nih.gov/9886075/

Why is this important? We see reduced expression of GLUT1 in Alzheimer’s. Is it a genetic issue or not and can it be partially prevented or even reverted when going on a low carb diet? Food for thought…

“GLUT1 reductions exacerbate Alzheimer’s disease vasculoneuronal dysfunction and degeneration” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734893/

– – – T H E – E N D – – –

Insight into evolution

Where do we come from and where is it taking us?

Lately I’ve been exposed to different, seemingly unrelated pieces of information. However putting them all on the table, a pattern started to emerge. Join me in a philosophical trip making sense out of evolution and what it could possibly have in storage for us. To put a bit of structure in it, there’s the history, our present and the future.

The beginning

When we talk about evolution we talk about life. But what is life? The only element that is an absolute necessity is reproduction. If something cannot reproduce there is no lineage, there is no evolution. We just have static elements.

Life itself cannot exist at random. You cannot throw a bunch of molecules together and have reproduction. Reproduction is the result of an organization of molecules in such a way that they together not only produce something but actually replicate itself. Such an organization doesn’t come easy. In fact, it has taken a good 1,5 billion years before we saw the rise of the first single-cell life before the right match was found.

And that wasn’t the end of the story naturally. Reproduction requires resources, raw material. Putting things together requires energy. Reproduction thus depends on raw material and energy. As such, life is confronted with another problem. If you just allow unrestricted reproduction, how are you going to obtain that material and energy?

Maybe the material and energy is floating around you but a little bit to far and you miss it so life had to develop the ability to move. If you look at such examples today, you’ll see single-cell life with moving parts. Movement costs energy.

But where do we move to? Just move around? It is going to be hit or miss to find material and energy. We certainly don’t want to be moving out of an environment that is ideal for reproduction. So evolutionary pressure requires the development of environmental sensors, a sense of direction so that the single cell life form can position itself in a more ideal location for reproduction.

The elements that are required for reproduction are not always abundant everywhere. There’s also the problem of competition. As a cell is able to reproduce, more and more start to exist. They all want to reproduce so they compete with each other for the resources. This will create scarcity of resources and life will cease to exist.

Again, evolution comes up with a solution. Cells start to lower their needs when there is a lack of resources. They start to differentiate in state. Grow when there is abundance and wait for more ideal times by reducing requirements.

We are now again millions away from our first reproduction moment and still are primarily focusing on successful reproduction. It doesn’t stop here and will actually never stop.

One more last step before we’ll do a fast forward. As a single cell we have developed a good set of capabilities to succeed in reproduction. Curiously though, cells evolved further. It seems to be beneficial to work together rather than compete against each other. When cells group together and in agreement with each other allow for specialization, the group as a whole develops capabilities that are not possible for an individual cell.

That is interesting, just as the molecules had to come together in the right formation to create the capability to reproduce, now cells do it at their level. Just as the many molecules are different from each other providing a piece of the functionality, so do the cells when they start to work together. They start to differentiate and contribute specific functionality of which the group will benefit. Benefit in what way? Again, improve their ability to reproduce.

A brain, skeleton and muscle are the pinnacle that allows movement, detection, observation etc.. to get the cooperation of cells, that now have formed a body, in a more liberal way to the resources that it so desperately needs to reproduce.

Our more recent history

Fast forwarding now. Evolution has developed many different life forms. They all specialize to ensure reproduction in their living environment.

We’re in the era where Neanderthals are roaming around the world. They managed to survive for a good 400K. Much longer than we as modern humans have been around yet look at what we have done in a much shorter time span.

If you think brain size reflects intelligence, you’ll be disappointed. They had a similar size, if not even bigger. Our own brains have in fact reduced in size since the introduction of agriculture. Never mind brain size, let’s look at what is being done with that brain.

We see that modern humans developed art work and evolved in tools, hunting techniques. Neanderthals did not. We suspect they were able to imitate and have some evidence of that at the point where Neanderthals encountered modern humans. Modern humans did not exterminate the Neanderthals as the interbreeding seems to suggest.

The different elements I’ve read about seem to suggest to me that Neanderthals were missing a creative element. Creativity is an important requirement to resolve problems. It also allows you to explore, discover, run through scenarios, imagine things that do not exist.

There are theories that indicate modern humans have hunted the large mammals to extinction. How would Neanderthals survive in such a situation? Switch over to hunting smaller game? They would have had to adapt their tools, strategies, update their learnings, techniques etc.. and we see no evidence of that.

The fractures of their bones indicate their hunting technique made use of close encounters with their prey. When we look at wild life, the predators are using their own body as weapons. Modern humans, thanks to that creativity I’m guessing, have moved beyond that. Another success story for increasing the chance of reproduction.

Just as cells were at some point in competition with each other for resources, so may Neanderthals and modern humans have been. Again a natural selection of a feature that improves the ability to reproduce has been selected for.

Creativity is not something that emerged from our encounter with Neanderthals though. We already had it when we came out of Africa. Our evolution suggests that we evolved alongside large mammals in Africa. Hunter and prey co-evolved in a way that sparked the development of creativity to overcome problems in obtaining prey.

Modern days

Another fast forward and we are today. What evolution did to molecules to for a cell, what evolution did to cells to form a body, so does evolution continue and created groups out of those bodies. As humans we have formed groups in many different forms but the same rules continue to apply. The group is the next level of cooperation.

Throughout our history, evolution has experimented with many different groups in many different ways and locations. Societies have risen and fallen. These groups were not able to sustain themselves long enough. Communities emerged and disappeared. The group was not able to secure food and energy to continue reproduction.

What we can see around us today is the latest most successful form.

The bodies (us) specialize to support the existence of the group. Some are specialized at growing food. Some specialize at generating energy. This specialization is freeing up time for others to specialize in their respective area, all for the benefit of the group existence. We have scientists who use their creativity and intelligence to discover how things are working. We have engineers who take those discoveries and combine it with their own creativity to come up with new applications. The group has enabled bodies to specialize in repairing damaged bodies etc.. All these bodies depend on the specialization of the others.

As we’ve seen, layer after layer new capabilities start to emerge. What are those group capabilities. As a group we first of all protect the survival of the group because now we have moved reproduction up to the level of the group. We are currently worrying about climate change. The group has advanced enough to overcome this problem. Either that or the group will disappear.

Currently our focus is to secure our energy need as a group. We started out with burning wood, then charcoal. Then we invented electricity and use charcoal, wind, water etc for it but our energy need continues to grow and we know the resources to produce that energy are finite.

Thanks to the group, that problem is being addressed by bringing solar fusion within the reach of the group. There are several initiatives to create a sustained hydrogen fusion reactor which will deliver more energy than the amount that is needed to keep it running.

More incredible group stuff is happening. We are actually preparing a trip to Mars. We are upscaling our knowledge of everything at an immense rate. What we can discover, know, build… as a group far exceeds what can be done by an individual body.

The capabilities of the group is however determined by the capabilities of the layer beneath. What the group is doing is going through another level of selective pressure to favor intelligence in the bodies that make up the group.

Some people may not like this from a moral or ethical point of view but intelligence is being rewarded more than anything else in our current groups. It is not a matter of fairness, it is a necessity. The group needs to survive. Just as a single cell was floating around and had to adapt, so does the group. Raw material and energy remain limited.

The group needs to develop methods now to become more efficient to reduce the need for raw material. And we are doing that by continuously improving the scale of our devices and tools. I’ve already pointed out to securing energy via the fusion reactor. More and more is the group in need of bodies who can come up with solutions that enhance the survivability of the group and when possible allow it to reproduce.

Intelligence is selected for in favor of raw muscle power. Intelligence is more important and can overcome the lack of muscle power by inventing robots which will replace the shortcomings of diminished muscle power.

Future

This group level existence allows me to have a positive outlook. As each next level in evolution shows us that cooperation rather than competition is the best way forward, we can expect war, tribalism, religion, country borders.. whatever we have done in the past to separate us from others will disappear as it doesn’t help the group to advance.

Our current group configuration may not be the final last form but it may evolve, through trial and error, to the most optimal form to sustain reproduction because that is what life is about.

The group will have specialized in preservation, in creating a sustainable eco-system for the group. It will have specialized in mastering the vast amount of energy that is available in the universe. The group will know intrinsically that cooperation is the way forward.

With such lessons learned we will move out into outer space.

How will we look like when we evolved our intelligence to a high enough level? In order to have a brain that can improve the way we think we actually may need more volume. I’ve discussed before with the Neanderthals that brain size is not necessarily related to intelligence but that is as an absolute parameter.

There is however a rule in life that requires a balance within the organs. In our own evolution, our gut size had to shrink to allow a bigger brain. The brain is very energy hungry so growing a bigger brain means that something else has to reduce consumption. This may lead us to optimize food intake further, shrinking more of the gut or some other solution like reducing our muscle mass which we can only afford if our brain can compensate the lack of muscle. We can afford a reduction in muscle if we have a smaller skeleton.

Does that sound familiar? A big head and skinny body? Maybe you are laughing now but realize that I’ve not stated anything unreasonable 😉

Keeping in mind those lessons learned and understanding how we evolved, how would we travel through space? As hungry pirates completely stripping every planet of the resources that we can use or do you think it is more closely to something like Star Trek where we just observe.

Putting the science fiction aside for a minute. I see us supporting development of other life forms with a minimal interference. Self-development with a little kick in the right direction if you will and from time to time come back and see how it is going. Just like we do lab research. Probably we’ll make some errors at this level as wel but over time will improve. We will inspect other planets other galaxies out of curiosity just to see what is out there and if we can further improve our understanding.

As a group we will seek cooperation with other groups if we find any so that we can get to the next level. From molecules to cells to bodies to groups to whatever comes next.

Does that sound like a plausible future? Could it then be feasible that life on an other planet already got to this point and kicked us in the right direction?

Back to the present

I’m fascinated by Göbekli Tepe. This place has been dated around 11 500 before present. It is an enormous structure of which only a small portion has been excavated. What makes it so special is that, following our modern day thinking, there are some elements that don’t really seem to fit. For example, given the size there must have been a huge organization to get everything in place. This requires a society of workers, others who provide food for the workers. You need local storage facilities, some form of containers for water and so on. No signs of any of that.

We can see an evolution of tools and buildings throughout history, yet for this location none of that. It just seems to have popped into existence.

So I let my imagination mix in with the future outlook I just described earlier. What if there was life on another planet that gave us that kick in the right direction? What if that kick was to put a monument? How would we respond to it with our creativity, curiosity and intelligence?

We would gather around it, not understanding what it is. Inspect it and start using our creativity. These blocks of stone are huge. A small person like ourselves cannot lift these stones so whoever did it must have been huge and strong, very powerful. Who are they? Maybe this has led us to the concept of gods. Where are they? Maybe they’ll come back so we’ll come back as well to meet them.

It becomes a meeting place where people exchange their ideas. Because it is a unique reference point, unique like we need them to navigate the landscape and remember our routes, routes establish themselves to and from the monument. Trading and exchange of goods increases. A few individuals start to settle locally to support the trade and local needs. Whoever travelled from far away may be hungry when they get to this point so we feed them.

Some of those grains collected by the gatherers falls on the ground unnoticed but due to increased return they notice the same plants as those that they got the grains from elsewhere. And the rest is history..

Finally

Reproduction is at the core of life, evolution is the result of a cooperation to improve the outcome of a common goal. With every new layer of cooperation, capabilities emerge that were not possible before, further securing reproduction and thereby sustaining life.

How will life be for us in a few thousand years from now? I bet we’ll still play by the same rules 😉

—– T H E – E N D —–

CETP

Read it carefully as it may help you assess your risk for cardiovascular disease!

In my previous article comparing pathological versus physiological high LDL-C levels, one of the marked differences is that HDL-C goes down under the pathological situation versus up on the physiological case.

This difference comes down to cholesterol ester transfer protein or CETP. That has triggered my curiosity so naturally I wanted to understand more of it.

What does it do? Where does it come from? And since we see our HDL-C levels modulated by diet due to this CETP activity.. how does diet modulate CETP?

Exchanger

First, what does it do? CETP is responsible for the exchange of triglycerides for cholesterol between the lipid particles. HDL-particles will give up a cholesterol ester in exchange for a triglyceride from ApoB-containing particles such as VLDL, IDL or LDL.

What normally happens is that the HDL-particle gets loaded up with triglycerides. The liver will take up triglyceride rich HDL-particles via hepatic lipase. Stripped from its triglycerides, the remnant HDL is put back into circulation but risks higher clearance via the kidneys.

“New insights into the mechanism of low high-density lipoprotein cholesterol in obesity – Changes in HDL component” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207906/#__sec6title

This explains why we see HDL-C trend upwards for athletes and lean individuals on a ketogenic diet. It is due to their CETP activity being reduced.

But it raises a question. It could explain why LDL particles become large buoyant in size as the particle is not unloading its triglycerides to the HDL particle. But the HDL particle normally also exchanges with VLDL particles so how come the lipid profile results in low triglyceride levels under reduced CETP activity?

This may be difficult to answer but could be driven by a reduction in VLDL-sized ApoB particles. Under fasted conditions, the liver may be producing LDL-sized ApoB particles directly which is true under prolonged fasted conditions where the liver is more tuned towards fat metabolism.

Macrophages

Low CETP activity induces higher cholesterol efflux from macrophages to HDL-particles. When we look at subjects with homozygous CETP deficiency we see a 2~3-fold increase in efflux.

“HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoE- and ABCG1-dependent pathway” https://pubmed.ncbi.nlm.nih.gov/16670775/

But we are not all genetically deficient in CETP. When using a drug that inhibits CETP activity, we also not a higher efflux capacity.

“Cholesterol Efflux Potential and Anti-inflammatory Properties of HDL following Treatment with Niacin or Anacetrapib” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917780/

Metabolic syndrome

Given that lower CETP activity is beneficial as it increases HDL-C and causes a greater efflux of cholesterol from macrophages, we want to know what the level is for ourselves.

HDL-C by itself is already a good proxy but the following study shows that CETP activity is also associated with increased BMI, fasting glucose and c-peptide. This explains why obese people tend to have lower HDL-C

“Effect of adiposity on plasma lipid transfer protein activities: a possible link between insulin resistance and high density lipoprotein metabolism” https://pubmed.ncbi.nlm.nih.gov/8033953/

When reviewing patients with metabolic syndrome, they found increased CETP activity (and PCSK9).

“Circulating PCSK9 levels and CETP plasma activity are independently associated in patients with metabolic diseases” https://cardiab.biomedcentral.com/articles/10.1186/s12933-016-0428-z

Small dense LDL

In type 2 diabetes patients it has been demonstrated that CETP contributes significantly to the increased levels of small dense LDL by preferential CE transfer from HDL to small dense LDL, as well as through an indirect mechanism involving enhanced CE transfer from HDL to VLDL-1(114). … Recent evidence has indicated that a primary acceptor for CETP-mediated HDL cholesteryl ester transfer in normolipidemic subjects is a large, buoyant, triglyceride-enriched LDL subclass (116).

source: “Metabolic origins and clinical significance of LDL heterogeneity” https://www.sciencedirect.com/science/article/pii/S0022227520328005

CETP targets the large buoyant LDL particles and turns them into small dense LDL particles. Small dense LDL is a biomarker for atherosclerosis.

Here are some numbers from a study looking at events across 2 years in people with metabolic syndrome. In those with events: LDL size was lower (P < 0.0001), due to reduced larger subclasses and increased small, dense LDL (all P < 0.0001). After multivariate analysis for independent risk factors: elevated small, dense LDL (OR 11.7, P = 0.0004). Now that is impressive.

“Small Dense Low-Density Lipoprotein as Biomarker for Atherosclerotic Diseases” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441126/

“Small, dense low-density lipoproteins (LDL) are predictors of cardio- and cerebro-vascular events in subjects with the metabolic syndrome” https://pubmed.ncbi.nlm.nih.gov/18771560/

Insight

If you paid close attention to the above then you may have noticed something.

HDL-particles have 2 jobs. Doing one makes it bad at the other and this is regulated by CETP activity.

  • Recycling of triglycerides from LDL-particles to the liver
  • Taking up cholesterol from macrophages

When CETP activity is high, HDL volume reduces from circulation because it is taken up by the liver (and also by the kidneys). With less of them available, there is less availability to take up cholesterol from macrophages.

When CETP is low, there is greater capacity to take up cholesterol from macrophages. As a result LDL particles will have to hold on to their triglycerides more.

CETP activator

So what causes CETP to be activated or turned down?

CETP is secreted by the liver and this is regulated by liver x protein (LXP). Higher stimulation of LXP causes higher levels of CETP.

Interestingly higher LXP also causes a higher stimulation of GLUT5 expression in the duodenum and adipose tissue.

“Identification of the fructose transporter GLUT5 (SLC2A5) as a novel target of nuclear receptor LXR” https://www.nature.com/articles/s41598-019-45803-x

The duodenum is particularly interesting because it is thought that most of the fructose is processed by the bacteria in the small intestine. This idea is based on the following study which, to my view, people incorrectly extrapolate to humans. The study was done in mice and I doubt they have a similar gut microbiome. Secondly fructose that would be absorbed by the liver and converted to fat would not immediately show up in the circulation.

“The Small Intestine Converts Dietary Fructose into Glucose and Organic Acids” https://www.sciencedirect.com/science/article/pii/S1550413117307295

It is important to find out what causes higher expression of LXP. Causing more fructose to be absorbed into the body is really not a good thing. It already creates a bad lipid profile for cardiovascular disease.

LXP activity

I figured it had something to do with fat metabolism since we see evidence of very low CETP activity in very lean individuals on a ketogenic diet. The diet features things such as PPAR-alpha increase, PGC-1alpha increase, AMPK increase. So a quick look around and indeed, it seems that AMPK activations directly inhibits liver x receptor (LXR). The receptor is a protein and sometimes referred to as LXP.

These results indicate that AMPK directly inhibits ligand-induced LXR activity in addition to blocking production of endogenous LXR ligands.

source: “Mechanism of AMPK suppression of LXR-dependent Srebp-1c transcription” https://pubmed.ncbi.nlm.nih.gov/21647332/

This indicates that lean ketogenic individuals have remarkably elevated AMPK levels. It shouldn’t be a surprise really. This has already been studied very well. An example here shows that AMPK levels double in the liver when the animals are put on a ketogenic diet. 2-fold higher in the liver and 2 to 3-fold higher in muscle.

“A high-fat, ketogenic diet induces a unique metabolic state in mice” https://journals.physiology.org/doi/full/10.1152/ajpendo.00717.2006

Pharma Failure?

It is tempting that given all of the above you can simply develop a drug to inhibit CETP. So they did and had to stop the trial early because it caused more deaths. I couldn’t find much input about due to what the patients died from but there was a follow-up study that indicates it was not caused by the inhibition of CETP but due to side-toxicity.

The study said the following about the CETP inhibition:

Recent analysis suggests that failure may have been caused by off-target toxicity and that HDL is functional and promotes regression of atherosclerosis. New studies highlight the central importance of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 in reducing macrophage foam cell formation, inflammation, and atherosclerosis. A variety of approaches to increasing HDL may eventually be successful in treating atherosclerosis.

source: “HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis” https://pubmed.ncbi.nlm.nih.gov/18460328/

Finally

It seems clear to me that you need to get into a state where your body lowers CETP activity naturally and thereby increases your HDL-C. In my previous article on pathological high LDL we clearly see that a low HDL-C is a risk indicator along with obesity etc.

My advice: get onto the ketogenic diet, get lean and smile when you see your HDL-C rising because you know it is working.

I wish you Good Luck and enjoy the rest of your day!

—- T H E – E N D —-

Pathologic LDL cholesterol

Throughout all my investigations on the ins and outs of cholesterol, energy metabolism, diseases such as T2 diabetes, cancer, Alzheimer’s, ageing etc.. I learned a lot about the environment of which the circulating lipids can be seen as a proxy for it.

In the low carb community, as people get leaner there is a tendency for their LDL cholesterol (LDL-C) to rise over time. Naturally there is a concern because we are all very familiar with the message of increased risk for heart disease, atherosclerosis specifically.

What I want to do with this article is convince you of the possibility that not all high levels of LDL-C are equally bad.

Let’s assume for a minute that it does exist, a situation in which high LDL-C is healthy, except that situation is rarely seen. You have 10 of these people. Now you mix them in with a group of 990 people who have high LDL-C but these 990 people have a situation in which their high LDL-C is a proxy for high risk of atherosclerosis. Now you run a study on them, not knowing about these 2 different situations. You will compare this group against another group of 1000 people with low LDL-C and you follow them up for a very long and see which group has the most heart attacks. The group with high LDL-C has more heart attacks so naturally your conclusion is that high LDL-C is indicative of a higher risk. On average that is indeed true. The problem is that this is averaged out.

Those 10 people are swamped by the 990 and will not be noticed. This will lead research to think that all high LDL-C is bad.

What I will do now is first of all assume that there is such a split where high LDL-C is a proxy for ill health and where it is not a problem. I will call one group pathological LDL-C (patLDL) and the other group physiological LDL-C (phyLDL). This is analogous to pathological insulin resistance and physiological insulin resistance.

Quite a number of people have looked into this so I’m leaning heavily on their insights and will be mixing in my own. I hope to contribute in this topic of “high LDL-C always being bad” by making a side by side comparison.

For the record: this is analysis done for myself. I am just sharing this information with you and it is completely up to yourself to first of all validate what I’m saying is correct and it is your own responsibility for the actions you will take or not. In no way am I advising what you should do.

That said, lets look at patLDL first and then see if we can come up with research that shows there is indeed a phyLDL profile and check if the risk factors of patLDL are also present under phyLDL. If they are present, then it would be fair to conclude phyLDL is not really a thing isn’t it?

patLDL

First of all what are the official risk factors according to the NHS from the UK? I’ve highlighted the ones which are lifestyle dependent, where you can take action to change them.

https://www.nhs.uk/conditions/atherosclerosis/

In the US, their NIH department of National Heart, Blood and Lung institute is a bit more elaborate. I’ve highlighted a number of interesting focus points for later on.

Major Risk Factors

  • Unhealthy blood cholesterol levels. This includes high LDL cholesterol and low HDL cholesterol.
  • High blood pressure. At or above 140/90 mmHg over time. If you have diabetes or chronic kidney disease, high blood pressure is defined as 130/80 mmHg or higher.
  • Smoking. Smoking can damage and tighten blood vessels, raise cholesterol levels, and raise blood pressure. Smoking also doesn’t allow enough oxygen to reach the body’s tissues.
  • Insulin resistance. Insulin resistance may lead to diabetes.
  • Diabetes. The body’s blood sugar level is too high because the body doesn’t make enough insulin or doesn’t use its insulin properly.
  • Overweight or obesity.
  • Lack of physical activity. A lack of physical activity can worsen other risk factors for atherosclerosis, such as unhealthy blood cholesterol levels, high blood pressure, diabetes, and overweight and obesity.
  • Unhealthy diet. Foods that are high in saturated and trans fats, cholesterol, sodium (salt), and sugar can worsen other atherosclerosis risk factors.
  • Older age.
  • Family history of early heart disease.

“Studies show that an increasing number of children and youth are at risk for atherosclerosis. This is due to a number of causes, including rising childhood obesity rates.“

Emerging Risk Factors

  • High levels of a protein called C-reactive protein (CRP) in the blood may raise the risk for atherosclerosis and heart attack. High levels of CRP are a sign of inflammation in the body.
  • Inflammation is the body’s response to injury or infection. Damage to the arteries’ inner walls seems to trigger inflammation and help plaque grow.
  • People who have low CRP levels may develop atherosclerosis at a slower rate than people who have high CRP levels. Research is under way to find out whether reducing inflammation and lowering CRP levels also can reduce the risk for atherosclerosis.
  • High levels of triglycerides (tri-GLIH-seh-rides) in the blood also may raise the risk for atherosclerosis, especially in women. Triglycerides are a type of fat.
  • Studies are under way to find out whether genetics may play a role in atherosclerosis risk.

Other Factors

  • Sleep apnea. Sleep apnea is a disorder that causes one or more pauses in breathing or shallow breaths while you sleep. Untreated sleep apnea can raise your risk for high blood pressure, diabetes, and even a heart attack or stroke.
  • Stress. Research shows that the most commonly reported “trigger” for a heart attack is an emotionally upsetting event, especially one involving anger.
  • Alcohol. Heavy drinking can damage the heart muscle and worsen other risk factors for atherosclerosis.

https://www.nhlbi.nih.gov/health-topics/atherosclerosis

These are all well established risk factors. If the list is a bit overwhelming and hard to grasp at once, let me break down these factors in the light of the lipid profile that shows increased risk.

Lipid profile:

  • High LDL-C
  • Low HDL-C
  • High Triglycerides

Along with:

  • High blood pressure
  • Overweight/obesity
  • T2 diabetes
  • Raised inflammation
  • High levels of CRP
  • Insulin resistance
  • Smoking
  • Chronic Kidney Disease

Apart from smoking, all the other risk markers are all linked to insulin resistance.

Is it feasible that the lipid profile reflects insulin resistance?

The 3 major components of the dyslipidemia of insulin resistance are increased triglyceride levels, decreased high-density lipoprotein (HDL) cholesterol, and changes in the composition of low-density lipoprotein (LDL) cholesterol.

source: “Insulin resistance and lipid metabolism” https://pubmed.ncbi.nlm.nih.gov/10418856/

The LDL-C/HDL-C ratio in men and LDL-C/HDL-C, TG/HDL-C, and non-HDL-C/HDL-C ratios in women might be clinically significant predictors of IR in healthy Korean adults.

source: “What is the most appropriate lipid profile ratio predictor for insulin resistance in each sex? A cross-sectional study in Korean populations (The Fifth Korea National Health and Nutrition Examination Survey)” https://dmsjournal.biomedcentral.com/articles/10.1186/s13098-015-0051-2

What you see in these ratios is that the risk goes up as LDL or triglycerides goes up and the risk goes down as HDL-C goes up.

Insulin resistance, as manifested by a high triglyceride/HDL-c ratio, was associated with adverse cardiovascular outcomes more than other lipid metrics, including LDL-c, which had little concordance.

source: “Study of the Use of Lipid Panels as a Marker of Insulin Resistance to Determine Cardiovascular Risk” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625988/

We see here the same ratio back and they even found no use in looking at LDL-C.

It seems to support the case to state that the lipid profile is pathological if your LDL-C goes up, HDL-C goes down and triglycerides go up. Perhaps even more important is that your lipids are evolving into this scenario over time so that you see which way you are going.

If you keep all conditions the same, smoking or not and your activity level then only food is left to have a serious impact on your lipid profile.

phyLDL

You may or may not know this but food can have a very dramatic impact on your lipids.

Having read the previous section where you saw almost all risk factors, including the lipid profile, linked to insulin resistance…

Would you consider to be at risk for CVD under the following conditions?:

  • Lean (instead of obese)
  • Active (instead of sedentary)
  • Insulin sensitive (instead of insulin resistant)
  • Low hsCRP (instead of high hsCRP)
  • Low blood pressure (instead of high blood pressure)
  • No chronic kidney disease (instead of having it)
  • Not smoking (instead of smoking)
  • High HDL-C (instead of low HDL-C)
  • Low triglycerides (instead of high triglycerides)
  • Yet high LDL-C

So out of all the risk factors, only your LDL-C matches while we have seen that the ratios are better predictors, not the isolated LDL-C value. And the LDL-C value was seen in the light of insulin resistance.

Would you truly believe you are at risk?

Such a profile does exist among humans but is rarely seen. Rarely because it takes a specific diet and lean humans. Could they be the 10 people in our group of 1000 where 990 really are at an increased risk?

But before we look at the diet, lets have a look at endurance athletes who we can view as an example of fit and healthy individuals. I want to see what is happening to their lipid profile and then see how the lipid profile of the diet matches with it.

Endurance Athletes

Let’s just list up a few studies and see what they have to say about the lipid profile.

  1. Low LDL-C/HDL-C and low triglycerides

“Long distance runners and body-builders exhibit elevated plasma levels of lipoprotein(a)” https://pubmed.ncbi.nlm.nih.gov/8187216/

2. Higher HDL-C and lower TC/HDL-C (TC = total cholesterol)

“Elevated high-density lipoprotein cholesterol levels in older endurance athletes” https://pubmed.ncbi.nlm.nih.gov/6465022/

3. Higher HDL-C and lower triglycerides

“High density lipoprotein metabolism in endurance athletes and sedentary men” https://pubmed.ncbi.nlm.nih.gov/2060090/

4. Higher HDL-C and lower triglycerides. This study looked at the clearance rate of triglycerides and found a strong negative relation with fasting triglycerides and a strong positive relation with HDL-C levels. So the lower your triglycerides and the higher your HDL-C, the faster you are able to clear triglycerides from the circulation.

“Elevated high-density lipoprotein cholesterol in endurance athletes is related to enhanced plasma triglyceride clearance” https://pubmed.ncbi.nlm.nih.gov/3374323/

5. Increase in HDL-C and lower triglycerides, if triglycerides started out high.

“The influence of exercise on the concentrations of triglyceride and cholesterol in human plasma” https://pubmed.ncbi.nlm.nih.gov/6376133/

I think you get the picture by now. They all report increase in HDL-C and a reduction in triglycerides. Yet none of them report on LDL-C except for the last one saying there is hardly any change.

If LDL-C would be so important for health, then why don’t we see it noticeably reduced in endurance athletes?

We have a bit of a baseline now so let’s look at how diet could be matching with the changes that we see in endurance athletes.

Diet

People who go on a ketogenic diet which is high in dietary fat intake and very low in carbohydrate intake (to stimulate easier ketone production) often tend to do it for weight loss. As they get leaner they may experience a rise in LDL-C. The most lean subjects, who may have gotten onto the diet while they were already lean, see their levels go sky high. Often they hear from their doctor that they have never seen this before.

I collected a sample from such people’s self-reported figures to see the correlation with ApoE version but that is not important. The graph shows you averages of in total 52 people. You can ignore the 2/3 and 4/4 groups because they have only 4 and 3 samples.

They have low triglycerides and very high HDL-C levels. The athletes usually have HDL-C levels of around 60 mg/dL from what I’ve seen in the papers.

Most of the people behind these numbers report an active lifestyle although that is not the case for all of them but they all have low body fat in common with athletes. They are generally health conscious and generally fit the phyLDL conditions.

Diet and Endurance

Just a trivia, how about mixing the diet in with endurance athletes? This study has been done and here are the results.

You see here that the HDL-C has gone up significantly higher. This has positively affected the triglyceride/HDL-C ratio. Triglycerides were already low in both groups. Also here we see a higher LDL-C in the low carb group.

“Paradox of hypercholesterolaemia in highly trained, keto-adapted athletes” https://pubmed.ncbi.nlm.nih.gov/30305928/

patLDL versus phyLDL

But back to the diet itself. Let’s review that list again. How does it compare to the pathological risk factors?

Lipid profile:

  • Very high LDL-C
  • Very high HDL-C
  • Low triglycerides

Taking everything into account, this profile is far from resembling that of the patLDL.

Finally

As you can see in this article, the profile of the lipids and risk factors associated with CVD do not fit with athletes nor with lean people on a ketogenic diet, nor with athletes on a ketogenic diet.

This comparison shows us that there is clearly a difference. There is indeed a case to create when elevated LDL-C is a bad sign but it should be seen in light with all the other factors. For the majority of people who are not on a ketogenic diet (the 990 people) you need to be worried. But for the small group of others who are on a ketogenic diet and fit the physiological high LDL-C, I’m not worried about CVD risk.

All the risk factors are not present and the lipid profile is different. LDL-C should not be looked at in isolation.

—- T H E – E N D —-

The key to enhance performance on a high-fat diet

There is a professor (Louise Burke) who looked at athlete performance extensively in elite race-walkers. Shamefully the high-fat community has criticized her and attacked her because the results that she came up with showed that when it comes to world class athletes, there is no performance benefit and more likely there is a slight decrease.

That critique has been met with more research and the results remained the same.

Rather than criticizing and recognizing the facts for the given circumstances, I started to wonder why there could be an issue with performance. We’ve seen great benefits from fat adapted athletes meaning improved performance and markers but not at the high intensity that needs to be sustained during a race.

Why?

We know that under high intensity the contribution of fat to energy diminishes. It is not a great amount but still, it reduces.

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source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2278845/
the same data represented as a graph

The figure above comes from people who were not fat adapted. They have their peak fat oxidation at around 55% of VO2Max. When we look at a study from Jeff Volek then we see that maximum fat oxidation can shifts towards a higher intensity. The high carb group shows the same results as before, max fat oxidation at 55% while the LC group (very low carb) reached the peak at 70%.

source: https://pubmed.ncbi.nlm.nih.gov/26892521/

That is an impressive result so why doesn’t it result in improved performance at the highest intensities?

Carnitine

Without going into too much details, carnitine is needed to bring the long chain fatty acids (LCFA) into the mitochondria so that the fat can be used to produce ATP.

The following presentation helps greatly to understand why carnitine availability is an issue.

In short, at higher intensities there is not enough available carnitine to support the import of LCFA so that there is even a negative effect on its ability to import those fats.

That is a major blocking point if you are primarily fueled by fat.

In high carb athletes we see a strong reduction after 65% of VO2Max intensities. My guess is that we’ll see a similar effect at around 70~75% for the fat-adapted athletes.

source: https://www.youtube.com/watch?v=M-9hS_dPdrI

This is supported by another study looking at free carnitine. Subjects started at an average of 15.9 mmol per kg of dry weight. Exercise at 70% and 100% VO2Max resulted in a drop to 5.9 and 4.6. Less than a 3rd remains.

“Muscle carnitine metabolism during incremental dynamic exercise in humans” https://pubmed.ncbi.nlm.nih.gov/2327259/

Increase carnitine content

So naturally people will have the reflex of supplementing carnitine and indeed, when they succeed we see a number of changes. It translates into a greater reduction in lactate which shows that more fat is used for energy production.

source: https://www.youtube.com/watch?v=M-9hS_dPdrI

When testing the results of a 20% increase in carnitine content in a time trial effort of 30 minutes at 80% VO2Max we see that all participants improved and on average had an 11% increase in performance. The study subjects were recreational athletes so the results may not equal in a similar performance gain for top-level athletes but it shows promise and 11% is a very big deal if this could be achieved in such athletes.

“Chronic oral ingestion of L-carnitine and carbohydrate increases muscle carnitine content and alters muscle fuel metabolism during exercise in humans” https://pubmed.ncbi.nlm.nih.gov/21224234/

How?

Naturally if it offers a performance enhancement we’re interested. It turns out that it is not as simple as taking a carnitine supplement. Bacteria in the gut seem to love it and not even intravenous supplementation made any change.

What researchers did find out is that it requires insulin and works via a sodium transporter. So they recommend to take carbohydrates along with the supplement.

But research comes with varying results. For example 3 months of supplementation shows no increase while 6 months do.

Three months of dietary supplementation with a combination of carnitine and CHO had no effect, but after 6 months muscle carnitine content increased by 21%. The necessity of using a very long supplementation period demonstrates the difficulties involved and explains the failure of previous studies with shorter intervention periods.

source: “Boosting fat burning with carnitine: an old friend comes out from the shadow” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099008/

If you however want to stay on a ketogenic diet and be as fat adapted as possible, I think there are alternative ways to increment muscle carnitine levels.

I know we are not chicken but when looking at what they did, they were able to increase the body mass while reducing the fat content. How they achieved this was by varying protein content in the diet and supplementing with carnitine. It looks like the combination is a great way to increase the absorption and usage.

“Effects of dietary L-carnitine supplementation and protein level on performance and degree of meatness and fatness of broilers” https://pubmed.ncbi.nlm.nih.gov/9404545/

From the youtube presentation it was clear that insulin is required. They experimented on one hand with 80gr carbs and later on also had experiments with 40gr carbs mixed with protein and had similar results.

Purely a guess but I think that the insulin response to a meal is already sufficient for the absorption so low carb athletes could make sure that they take a supplement during meals.

But also here the results are conflicting. The inclusion of carbs and protein seem to blunt the uptake in an acute phase. Conflicting because from the youtube presentation there was successful results using a mix of 40gr carbs and 13gr protein in 24 weeks.

“Protein ingestion acutely inhibits insulin-stimulated muscle carnitine uptake in healthy young men” https://pubmed.ncbi.nlm.nih.gov/26675771/

The following study, although a very specific case seems to hint at a redistribution mechanism. This may indicate why it could take some time before muscle carnitine increases. It is possible that there is a lot of tissue in the body that takes up carnitine so regular and prolonged higher intake of carnitine may be necessary to create a saturation effect before muscle carnitine increases.

“Muscle and plasma carnitine levels and urinary carnitine excretion in multiply injured patients on total parenteral nutrition” https://pubmed.ncbi.nlm.nih.gov/16829425/

There is also a trend towards carnivore eating, it would be interesting to compare carnitine levels in these people/athletes to see if natural sources are sufficient to increase levels.

So clearly more research is required and hopefully we can see another step up in athlete performance.

  • What are the best tolerable ways to increase carnitine in the shortest period of time?
  • How high can carnitine levels be pushed in athletes?
  • Does increased carnitine provide a performance benefit for high-fat athletes and does that benefit still hold if we also increase carnitine levels in high-carb athletes?
  • Do we have any non-invasive proxies so that we can avoid muscle biopsies to check carnitine levels?
  • Where lays the true limit of oxygen availability?

A final word on the fat part

One other way to overcome the limitations that carnitine levels impose is to enhance the availability of short and medium chain fatty acids in the body. However, levels must be sustained for the whole duration of the race. It is likely that the absorption of these fatty acids into the mitochondria goes faster and will be depleted or reduced quicker. Fueling with fats during the race is not that straightforward.

A study looking at fat oxidation noted a quite clear difference. This was achieved through infusion so not a practical approach during racing but it shows the potential.

Furthermore, the percentage of oleate uptake oxidized decreased from 67.7 +/- 2.8% (40% VO2peak) to 51.8 +/- 4.6% (80% VO2peak, P < 0.05), whereas the percentage of octanoate oxidized was similar during exercise at 40 and 80% VO2peak (84.8 +/- 2.7 vs. 89.3 +/- 2.7%, respectively)

“Regulation of plasma fatty acid oxidation during low- and high-intensity exercise” https://pubmed.ncbi.nlm.nih.gov/9227453/

When looking at high performance and circulating fat we see that there is also a reduction in circulating fatty acids which also support the idea that in case of highest performance you need to fuel fatty acids.

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“The Effects of a Ketogenic Diet on Exercise Metabolism and Physical Performance in Off-Road Cyclists” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113752/

—– E N D —–

Cold on the outside – Warm on the inside

Introduction

We are now in the year 2020.  A lot of research has been performed regarding different diets yet still so much controversy exists around whether or not we should eat a lot of carbohydrates or fat or protein.  Those are the 3 macronutrients that are played around with in research.  Diets with different compositions compete with each other for being the best at weight loss, health and longevity.

So will I be able to provide a definite answer?  I cannot claim that I do but I will present you the material I have been able to gather to provide a picture that will hopefully bring more clarity (and probably as always a lot of questions too).

I have always been interested in health and in lifespan.  Healthspan means to stay as healthy as possible for as long as possible and minimize the time in which health deteriorates followed by death.  It is my suspicion that if we can live optimally by increasing our healthspan then we may also be pushing our lifespan to what we are naturally capable of.

This has brought me to the point where it is important to know how all of the cells within the human body work together and what makes each cell survive individually.

In order to get a good picture of things, there are a few individual concepts that we need to go through before we can talk about a potential way to affect our health- and lifespan positively.

What are we?

It may come as a surprise to some but “we”, “you” and “I” don’t really exist.  Our body is a cooperation of cells.  Throughout evolution individual cells have started to form bodies in order to increase their chances at survival and reproduction.  In its most rudimentary form these bodies have helped to avoid being destroyed, being eaten or damaged by environmental elements and also helped in waiting for more ideal times to replicate. 

Replicating is the essence of life.

It feels strange to think that everything we do in our lives, the complexity of our society, is driven by the desire to survive and reproduce by the billions of cells that we are made up of.

But it is true, the cells that we are made out of are the actual life forms.

Reproduction

Via the book “Lifespan: Why We Age–And Why We Don’t Have to” of author David Sinclair, PhD. it became clear that cells have 2 distinct states in which they operate.  They are either running in a mode of repair and maintenance or, when the times are right, they turn to proliferation.  When they proliferate, the cells spend much less of their energy on repair and maintenance.  All energy goes to creating the building blocks for new versions of themselves.

The Hayflick limitation

The cells in our body are continuously proliferating.  All of our organs are undergoing cell renewal to some degree although brain cells practically don’t as everything we’ve learned and remember depends on the connections between the cells.  If such a cell would die then that memory connection is lost.

With each cell division, the telomeres that protect the unraveling of the DNA in our cells, gets shorter and shorter.  When it is too short, the cell fails to replicate.  This effectively puts a limit to how many generations can exist.

When that limit is reached the cell will become senescent over time.  It will gradually lose its identity.  Although all cells have the same DNA in their nucleus, they all differentiate into a specific cell type (a heart cell, lung cell, muscle cell etc).  Throughout time, the cell will accumulate DNA damage causing it to behave differently and lose that identity.

“Hayflick Limit” https://en.wikipedia.org/wiki/Hayflick_limit

There are possibilities for cells to overcome this limit because we have an enzyme called telomerase which repairs the telomere ending of our DNA.  In humans we primarily see this in cancer and stem cells.  Both types are undifferentiated to some degree which suggest that it is important to limit growth when the cell is part of a community such as an organ.

Healthspan

So what does this tell us with respect to health span?  It is important to spare ourselves from DNA damage and mutations to stay healthy and when we are grown up, if we can slow down the cell replication over time, then we are adding time to our existence so prolonging our lives.

How can we achieve this?  By reducing cell replication we automatically drive up the potential for cell maintenance and DNA repair.

Cell metabolism

One of the ways that DNA is repaired is by a group of enzymes called sirtuins.  The enzyme SIRT1 performs repair at the nucleic DNA.  The activity of SIRT1 is regulated by the ratio NAD/NADH.  A higher NAD availability is required for SIRT1.

A cell that is stimulated in metabolism has a lower SIRT1 activity.  This metabolism is modulated by our thyroid hormone free T3 (fT3) so a potential suggestion is to reduce this stimulation.  fT3 stimulates cell proliferation as shown in different cell lines, which is opposite of what we want to achieve.

“3-Iodothyronamine and 3,5,3′-triiodo-L-thyronine reduce SIRT1 protein expression in the HepG2 cell line” https://www.degruyter.com/view/journals/hmbci/41/1/article-20190045.xml

“3,5,3′-triiodothyronine (T3) stimulates cell proliferation through the activation of the PI3K/Akt pathway and reactive oxygen species (ROS) production in chick embryo hepatocytes” https://pubmed.ncbi.nlm.nih.gov/22366194/

Caloric intake

One of the ways to reduce fT3 is to reduce caloric intake.  This has been shown successful in many different species to extend lifespan.

“ENDOCRINE ALTERATIONS IN RESPONSE TO CALORIE RESTRICTION IN HUMANS” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856718/

“Diversion of peripheral thyroxine metabolism from activating to inactivating pathways during complete fasting” https://pubmed.ncbi.nlm.nih.gov/1150863/

Caloric restriction definitely does its job by increasing SIRT1 activity and NAD availability.  This is not the case in all tissue according to this study.  They noted a lower ratio in the liver while the muscle and white adipose tissue both had increased ratios.

“Tissue-specific regulation of SIRT1 by calorie restriction” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2492662/

But is it achievable to subject ourselves to a lifelong caloric restriction?  It comes with a whole host of side effects such as fatigue, feeling cold, feeling hungry and if pushed too far then it is also detrimental for your psychological well being.

“Caloric Restriction in Humans: Impact on Physiological, Psychological, and Behavioral Outcomes” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014770/

“Long-term calorie restriction, but not endurance exercise, lowers core body temperature in humans” https://pubmed.ncbi.nlm.nih.gov/21483032/

https://en.wikipedia.org/wiki/Minnesota_Starvation_Experiment#Results

In an 11-year study on rhesus monkeys they found that the resting energy expenditure was down for about 13%.  It did come with the desired effect of health and lifespan.

“Energy expenditure of rhesus monkeys subjected to 11 years of dietary restriction” https://pubmed.ncbi.nlm.nih.gov/12519821/

It could be manageable to live in a warm climate so the reduction in body temperature is less of an issue but still the lack of energy, possible mental impact and hunger feeling will remain a challenge.

Growth stimulating hormones

There are 2 other important hormones affected by caloric intake and those are insulin and IGF-1.

“Effects of insulin on cellular growth and proliferation” https://www.sciencedirect.com/science/article/abs/pii/0024320581904823

“Insulin-Like Growth Factor-1 Promotes G1/S Cell Cycle Progression through Bidirectional Regulation of Cyclins and Cyclin-Dependent Kinase Inhibitors via the Phosphatidylinositol 3-Kinase/Akt Pathway in Developing Rat Cerebral Cortex” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256126/

It is not a coincidence that food intake drives up metabolism via these hormones and T3.  When nutrients become available, it is an ideal moment to proliferate, to replace malfunctioning cells with new ones.  The cells that make up our body see this as a signal to grow.

Food

Carbohydrates and protein are responsible for insulin and IGF-1 stimulation for the biggest part.

Our current diet causes chronic inflammation which requires renewal of cells but also tends to build up insulin resistance over time.  The muscle and liver are the highest recipients of insulin-driven glucose uptake.  Over time they become resistant leading to prolonged elevated levels of insulin which affects all cells in the body.

Protein itself stimulates IGF-1.  Dietary protein delivers the cells the necessary building blocks to function.  The amino acids that make up the protein can even stimulate the thyroid to produce more fT3.

“Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336265/

“Low Protein Intake is Associated with a Major Reduction in IGF-1, Cancer, and Overall Mortality in the 65 and Younger but Not Older Population” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988204/

“Effect of excitatory amino acids on serum TSH and thyroid hormone levels in freely moving rats” https://pubmed.ncbi.nlm.nih.gov/11251371/

It looks like we have no choice!  We can’t live without food and when we take in food we stimulate the growth mechanism.  Although it is correct that we can’t live without food, the composition of our food can be changed in such a way that we benefit the most from it in terms of health span.

Can we somehow reduce metabolism while remaining warm, energetic and mentally focused?

Climate

In order to keep warm, we can look at what animals do in colder more northern climates.

When we compare animal life in a warm climate versus a cold climate then we notice a change in survival strategy.  In cold climates, surviving the next day does not depend on food but on heat production and protection from cooling down and freezing to death.

The answer that nature has provided to us is fat.  By developing a subcutaneous layer we have created a bit of insulation.  But insulation is nothing if both sides of the layer are equal in temperature so we have our own heat production (thermogenesis) as a second important component.

That heat production is not a static element.  It can be intensified via cold exposure but also by consuming high amounts of fat.  The traditional Inuit diet consists of high amounts of fat.  One of the side effects of consuming a diet low on fat is that they feel colder due to it.

“We were really getting three-quarters of our calories from fat.” 

“The Inuit Paradox” https://www.discovermagazine.com/health/the-inuit-paradox

“When one eats seal, you are full all day. When you eat packaged foods, two hours later you get cold. If [you] eat Inuit food, you stay warm.”

“The Changing Landscape of Arctic Traditional Food” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944111/

Given the above info regarding metabolism, having a higher production of heat thanks to consuming fat would give us a way around the colder body temperature that is normally part of a lower metabolism.  That is, if this fat consumption does not increase our cell metabolism.

Into a bit of science

Thermogenesis is currently being researched for longevity.  This is for other reasons though because the researchers suspect benefits from it by reducing diseases.  Although that by itself would also have the potential to help you to extend healthspan, which is certainly what we are after, it may not necessarily indicate an extension of lifespan.

Longevity research into metabolic diseases is heating up

One interesting bit from the article is the following:

thermogenic adipose tissue activation in response to cold exposure induces the release of eosinophil-activating cytokines by type 2 innate lymphoid cells.

Type 2 innate lymphoid cells (ILC2) will be present in the lungs to surveil potential viral infections.  When they pick up the relevant signal, they’ll stimulate mucus production and also increase other effects such as memory b-cell recruitment.

Stimulated by IL-33, these ILC2’s are also required to convert white adipose tissue into beige (due to the increase in mitochondria).

“IL-33-driven ILC2/eosinophil Axis in Fat Is Induced by Sympathetic Tone and Suppressed by Obesity” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003423/

Already by keeping insulin low, studies in mice show that the WAT becomes more active and, depending on the location, starts to express more UCP1 for heat production.

“Adipose depot-specific upregulation of Ucp1 or mitochondrial oxidative complex proteins are early consequences of genetic insulin reduction in mice” https://journals.physiology.org/doi/abs/10.1152/ajpendo.00128.2020?journalCode=ajpendo

When we further investigate, we see that there is an increase in uptake of T4 into the brown and beige adipose tissue under stimulation of the sympathetic nervous system.  This T4 is locally converted into T3 to drive up the metabolism of the fat cell itself and thereby its heat production.

“A-FABP mediates adaptive thermogenesis by promoting intracellular activation of thyroid hormones in brown adipocytes” https://www.nature.com/articles/ncomms14147

This is very interesting as it gives us a clue on how this higher metabolism is achieved.  Not by stimulating metabolism overall in the body but very much focused in fat cells themselves.

I can only speculate about it but by taking up more T4, there is less conversion towards T3.  This could be counteracted by increased T4 production.  What it could mean is that at least we don’t have a systemic increase in metabolism in order to increase warmth production.

Interestingly all these effects also take place during cold exposure.

What does it all mean?

If longevity depends on reducing growth and health depends on the time spent in maintenance/repair then a very high fat diet could be a possible solution.  

I’ve only covered the metabolic part in this article but the consequences reach further than just metabolism.  By making the switch from primarily glucose to primarily ketones and fat we can modulate what happens inside our cells.

As indicated under the section “Cell Metabolism”, nuclear DNA repair is mediated by SIRT1.  Intrinsically linked, a very high fat diet raises SIRT1.  We can see this already in the brain of mice but there is other research indicating the same.

“Mechanism of Action of Ketogenic Diet Treatment: Impact of Decanoic Acid and Beta—Hydroxybutyrate on Sirtuins and Energy Metabolism in Hippocampal Murine Neurons” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468807/

“Ketogenic Diet Modulates NAD+-Dependent Enzymes and Reduces DNA Damage in Hippocampus” https://www.frontiersin.org/articles/10.3389/fncel.2018.00263/full

I’m saying intrinsically because a reliance on fat and higher dietary fat intake seems to signal to the overall organism that our body is that times are not right for reproduction, that we are living in a tougher climate so that time is better spent on repair and maintenance.

My speculation

Rather than mimicking starvation, to me the overall picture looks like a high fat diet is meant to sustain life through cold climates.  Fat is THE energy source in winter.  

  • The activation of the ILC2’s represent a higher surveillance against viral infections.  Respiratory infections is something we see typically return every winter.
  • Increased heat production is required to survive the cold through the night and through the day.  We don’t need this anymore today but it was necessary in our past.
  • Winter typically is harder to hunt and find food.  The extra fat mass in our body helps to endure sporadic tough days.
  • Sporadic because in our past there were large mammals available who delivered a big volume of fat.  Our food during winter likely was always high in fat availability.

Keep in mind though that a very high diet does mean consuming a lot of fat.  Not just avoiding carbohydrates.  Frequently people are afraid of calories and think they will gain from it.

If you keep carbohydrates out of your diet and keep protein intake to a minimum then you fill up the rest with fat to satiety.  If you don’t eat enough fat, you will feel cold.

Keep in mind the rhesus monkeys, they had a 13% lower metabolism.  Estimations of increased energy metabolism on a high fat diet are indicating +/- 12% extra metabolism on top of a normal diet.  

As a wild guess, we can compensate for the reduction in metabolism by letting thermogenesis cover an extra 29% of calories.

2000kcal – 13% = 1740kcal (on reduced metabolism)

2000kcal + 12% = 2240kcal (on a very high fat diet)

The experiment in mice showed a state that mimics caloric restriction yet showed an 11% increase in metabolism, primarily due to heat production.

“A high-fat, ketogenic diet induces a unique metabolic state in mice” https://journals.physiology.org/doi/full/10.1152/ajpendo.00717.2006

Studies have also been conducted in humans where a similar pattern of higher energy consumption is visible.

“Energy Requirement Is Higher During Weight-Loss Maintenance in Adults Consuming a Low- Compared with High-Carbohydrate Diet” https://pubmed.ncbi.nlm.nih.gov/32470981/

If you want to stay warm during winter.. don’t pass on the butter 😉

—- END —-

Cancer.. where to look for clues

I’ve been interested in cancer for quite a while. Even before I got affected, the complexity and the link with metabolism caught my attention. Despite digging up all the details of how cancer develops and works, I somehow never could find a distinct feature of a cancer cell. I could not find a single feature that I could not find back in other normal cells.

They do what is normal for growing cells, they do what is normal for cells under hypoxic conditions. They behave similar to embryonic cell proliferation, to immune cell proliferation. Except… they don’t differentiate. Why don’t they differentiate?

“But isn’t it clear” you may ask? “It is a genetic disease right? So mutated genes of course!” Really? Thomas Seyfried already showed that by taking the nucleus (where the cell genes are located) and putting them in another cell doesn’t create a tumor cell. However, putting the mitochondria from a cancer cell into a normal cell does cause the cell to become cancerous.

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source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3941741/

Mutated nuclear DNA doesn’t seem to cause a cell to be cancerous. The mitochondria however contain mitochondrial DNA. Perhaps mutations there cause cancer? That doesn’t stride well with the way mitochondria work.

They are highly susceptible to damage indeed but through evolution they developed a build-in mechanism to eliminate malfunction parts. Through fission and fusion they continuously break up and digest malfunctioning mitochondria via a process called mitophagy to then build up again towards bigger properly functioning mitochondria.

Then along comes my investigation on the root cause of atherosclerosis and it drives my attention to the role macrophages play in the pathology. There I get to learn about how monocytes get stuck at locations of inflammation via signaling molecules (cytokines). Locally they start to perform their job where they stimulate satellite cells to become active and start proliferating. Again this is happening through signaling via cytokines. The macrophages also change profile in this process. This causes their metabolism to switch from glycolysis towards fat oxidation. And once more this is triggered by external signaling.

After all this comes along a video on my feed in youtube. PhD Mina Bissell from UC Berkeley explains about her life work. She shows how the extracellular matrix (ECM) is driving behavior.

Her work supports Thomas Seyfried. When implanting tumor cells into a chicken wing, it develops like a cancer. Those cells in a petri dish develop like a cancer. Inserted into the wing of an embryo, they behave like normal cells despite having mutated nuclear genes! The context, the surrounding is different, not the cells!

Proliferate or differentiate under influence of the ECM. This is very important. It shows why cell cultures fail to provide similar results in vivo. They are missing the context.

ECM

What are the components of the ECM? Here’s a good introductory video.

Notice here lamina which has been central in the work of Bissell.

  • Collagen
  • Proteoglycans
  • Lamina
  • Fibronectin

If you watch the video then notice at some point he says how the structure gives cells some resistance to migrate, they ‘feel’ there is no room to proliferate! The cells ‘feel’ this through interaction with the ECM.

When he discusses proteoglycans, he explains about hyaluronic acid (HA or also known as hyaluronan) and how it attracts water so that together it forms a gel-like structure.

I found the following article a true eye-opener. They did research towards the effect of a breakdown of the HA. When this structure is lost around cells, they change metabolism towards glycolysis and take up an accelerated migration pattern.

When HA is lost, the inhibitory effect on GLUT1 translocation to the membrane is lost. This allows the cells to increase glucose uptake in support of glycolysis. This switch in metabolism is what cells do to proliferate. This is what enables them to turn on the right genes for building copies of themselves and construct the raw material for building these copies. This is not a special feature of cancer and you certainly don’t need a mutated gene.

As you can see in the next picture, every single cell line they tested this with responds in a similar way, starting to increase glucose uptake and increase lactic acid production. They used primary, immortalized, murine, human cells, as well as cancer cells. A very diverse array of nuclear genetic material yet they are all responding.

Loss of HA also allows cells to migrate more which reminds us of metastasis in cancer.

“Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization” https://www.sciencedirect.com/science/article/pii/S009286741831033X

When we look at how muscle repair works then we note a similar behavior for satellite cells (which get activated and proliferate thanks to macrophage signaling). What we note is that amongst others, the ECM factors (collagen, fibronectin) are listed as regulators of their proliferating state. Including beta1 integrin which was used by Bissell.

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How much research is done on cells without providing an ECM? Wouldn’t it invalidate their applicability? By not providing an ECM during testing, we know what cells do without it. They proliferate. You don’t need a cancer cell, you don’t need genetic mutations. Trying to develop drugs that interfere with the growth will likely interfere with the growth of all cells. It is not specific enough.

Unless loss of ECM is part of cancer but that is not the case.

But it begs the question, is it possible that the root cause of cancer has to be found in a disturbance in the cell environment? In its ECM?

Hypoxia

I suspect a situation of chronic hypoxia in the case of atherosclerosis because the cause of hypoxia is outside of the region that is affected by hypoxia. As a result the region itself tries to recover from it but is unable to.

We have to start somewhere so why not start with the hypothesis that in a similar way there is a disturbance in blood supply to a region in the body. This triggers inflammation and leads to a response to heal. But what if the response is not sufficient to fix the hypoxia? We go from acute to chronic.

Hypoxia certainly affects the ECM. It activates breakdown of the basement membrane while at the same time building up collagen.

“Hypoxia and the extracellular matrix: drivers of tumour metastasis” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4283800/

What I understand from the basement membrane is that it gives the group of cells their purpose. It helps cells to differentiate, provide structure for organ development and so on. The basement membrane is stabilized by type IV collagen but hypoxia also upregulates type IV collagen-breakdown enzymes (MMP2, MMP9).

“Basement Membranes: Cell Scaffoldings and Signaling Platforms” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039528/

Tumor blood vessels

One thing I keep reading is how the neovascularization fails. The whole idea is to improve blood supply so that oxygen can be delivered to resolve the hypoxia. But it fails to do this properly.

An external file that holds a picture, illustration, etc. Object name is cshperspectmed-ANG-006536_F1.jpg
source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282494/

Although there are manifold VEGF signals sent out for vascularisation, it could be that the proliferation of cells had a chance to build up in volume to interfere with proper development, interfere with the proper structure formation.

“Tumor Endothelial Cells” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282494/

Such defect in proper vascularisation could signal the entry into a more chronic state of hypoxia and exposes the inability to resolve the hypoxic situation.

Hypothesis

Something goes wrong at the very end of the microvessels, a malfunction of some sort. Because of this, oxygen delivery by the blood fails to reach a very small area which causes that area to become hypoxic.

Similar to atherosclerosis, the cause for the hypoxia is nearby but not in the area itself. This starts to set the normal hypoxia reactions in motion: macrophage attraction, cell growth, ECM remodeling, neovascularisation etc. all the steps needed to resolve the situation and all hallmarks that we are familiar with looking at cancer.

What should stop the growth however is proper vascularisation. But this fails because the cause of the initial damage to the blood vessel is still there. That damaged area is closest to the hypoxic region and from that damaged area it would normally start to grow new blood vessels.

But because the point to start new blood vessels from is also the point that is damaged, the formation of new blood vessels is impaired and is unable to rescue the hypoxic region in time.

Support for the hypothesis

A first detailed look at the situation shows us that loss of fatty acid synthase (FAS) enzyme increases malonyl-coa which acts as an inhibitor for mTORC1. This impairs the ability to grow new vessels.

“Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation” https://pubmed.ncbi.nlm.nih.gov/30146486/

insulin resistance

I know diabetes and hypertension are risk factors for cancer. I’m also aware that diabetes patients risk amputation due to capillary damage.

In the following article they explain the mechanism. FAS binds to Nitric Oxide Synthase (NOS). What they did was knock out FAS in endothelial cells. This caused the blood vessels to become leaky and “unable to generate new blood vessel growth“. This aligns well with the impairment of mTORC1.

“Root cause of blood vessel damage in diabetes discovered” https://www.sciencedaily.com/releases/2011/01/110129081530.htm

Looking at hypertension we see that the insulin-triggered vasodilation is impaired in the capillaries.

“Capillary recruitment is impaired in essential hypertension and relates to insulin’s metabolic and vascular actions” https://academic.oup.com/cardiovascres/article/49/1/161/292764

Vasodilation is dependent on NOS so what this all seems to suggest is that the endothelial cells have become insulin-resistant. They don’t respond to insulin signaling anymore.

“Role of Insulin Resistance in Endothelial Dysfunction” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594115

I’m not fully clear why but we see higher circulating levels of FAS in diabetes. One possibility that we see is that FAS is mainly produced in the liver and possibly insulin resistance in the liver may cause FAS to be secreted into circulation.

“Extracellular Fatty Acid Synthase: A Possible Surrogate Biomarker of Insulin Resistance” https://diabetes.diabetesjournals.org/content/59/6/1506

“Circulating serum fatty acid synthase is elevated in patients with diabetes and carotid artery stenosis and is LDL-associated” https://www.sciencedirect.com/science/article/pii/S0021915019304411

Smoking

We know smoking is a risk factor for both atherosclerosis and for cancer. Although the following article is about smoking and atherosclerosis, I bring it up because it shows how smoking causes damage to the endothelial cells. Smoke isn’t selective to your heart. It is also increases the risk factor for amputation.

“Cigarette smoking, endothelial injury and cardiovascular disease” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517732/#__sec4title

“A comparison of diabetic smokers and non-smokers who undergo lower extremity amputation: a retrospective review of 112 patients” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3474996/

We would expect this in the hypothesis if cancer originates from damaged blood vessels.

Fatty acid types

The following paper found that palmitic acid and linoleic acid cause a similar impairment. Both types of fatty acids are more prevalent in our diet (linoleic acid, palmitic acid) and from endogenous production (palmitic acid) due to a high carbohydrate diet combined with insulin resistance.

“Free Fatty Acids Inhibit Insulin Signaling–Stimulated Endothelial Nitric Oxide Synthase Activation Through Upregulating PTEN or Inhibiting Akt Kinase” https://diabetes.diabetesjournals.org/content/55/8/2301

Thomas Seyfried

The experiment that Seyfried showed us is somewhat a result after the facts. Proliferation is driven by the state of metabolism (glycolysis or oxphos). But the state of metabolism is switched (not driven) by the signals from the environments. In response to this signal, the structure of the mitochondria change to support this mode.

Glioblastoma cells in different ECM mediums (added on 2021.04.07)

The following paper shows nicely how the environment of the cells influences their behavior and morphology. They compared a typical collagen versus a GBM-patient tissue derived ECM. With the picture you can already see how the same implantation of cells proceed differently whereby only the ECM is different.

figure3

“The mode and dynamics of glioblastoma cell invasion into a decellularized tissue-derived extracellular matrix-based three-dimensional tumor model” https://www.nature.com/articles/s41598-018-22681-3

————————————-

I do not think cancer nor atherosclerosis can be defined as a metabolic disease because I do not see any impairment in metabolism. It is a problem with oxygen delivery driven by endothelial dysfunction. The nearby affected region is then inflicted with hypoxia which starts to drive the remodeling of the ECM. The remodeling drives the cells to their dedifferentiated embryonic state. The lactate production is to implement attachment as is seen under true embryonic development. Unfortunately, with impaired endothelial cells from which the vascularization should take place, this step of the process fails to be done properly so that the hypoxic situation remains unresolved.

What needs to be cured is endothelial dysfunction.

Current cancer standards of care

If this hypothesis turns out true, the current treatment options for cancer may actually cause cancer through the same mechanism, damage to the blood vessels.

Radiation therapy causes endothelial dysfunction.

“Radiation therapy impairs endothelium-dependent vasodilation in humans” https://www.sciencedirect.com/science/article/pii/S0735109700011906

But also chemotherapy results in impaired endothelial function. They try to deliver anti-angiogenesis resulting in an increased cardiovascular risk, trombosis, hypertension etc.

“Vascular Complications of Cancer Chemotherapy” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4989034/

Second cancers, following treatment of the first cancer are recognized to be related to the treatment. Do note this is correlation, not necessarily causation.

“Second Cancers Related to Treatment” https://www.cancer.org/treatment/treatments-and-side-effects/physical-side-effects/second-cancers-in-adults/treatment-risks.html

The curious case of macrophages and LDL cholesterol uptake in the pathology of atherosclerosis

Recently I wrote an article exploring what could be at the root cause of atherosclerosis. After the publication a comment pointed to the macrophage and cholesterol not being discussed. I didn’t do this because the root cause brought me to a point before macrophage and LDL-cholesterol comes into the picture.

But it got me thinking… what about those macrophages? Why do they take up that cholesterol? What are they supposed to do with it? Why aren’t they doing anything with it? Is it normal for them to just sit there and engulf the LDL cholesterol? Do they take up the LDL by accident and this disables them to function?

Nobody seems to talk about the role of macrophages, why is that? Do they do everything as is supposed to in the development of atherosclerosis? How is the LDL causing an issue here? Is it the fault of cholesterol itself that it gets stuck there? Or is there more to it?

They do correlate well with the lipids and severity of the atherosclerotic plaque. Could there be a problem with the macrophages?

“Relation of Plaque Lipid Composition and Morphology to the Stability of Human Aortic Plaques” https://www.ahajournals.org/doi/10.1161/01.ATV.17.7.1337

I have read my fair share of research in the last 3 years and this gets remarkably little attention but there is no lack of research. As you can see there are a lot of questions to raise and clear up so it is time for another dive into an area where I know nothing about, exciting!

My very basic understanding about macrophages started with them being cells that can kill pathogens and malfunctioning cells by taking them up and dismantling them, releasing the pieces as debris for recycling or elimination by the liver.

Result

I want to start with the result first because there is a lot of ground to cover. The full article is quite detailed so for those who cannot spare the time, here is the result in brief.

Hypoxia causes macrophages to become immobilized. As a first step they become a pro-inflammatory glycolytic M1 type in order to stimulate growth. These M1’s prefer the uptake of oxidized LDL and accumulate their lipid. After a while they should differentiate into the anti-inflammatory M2 type which consumes the accumulated lipids through fatty acid oxidation for energy. This has nothing to do with creating disease but are all part of resolving the inflammatory situation. It is the healing process.

However, although the elements are there to perform this change from M1 towards M2, this process becomes only partially successful resulting in macrophages that fall in between showing properties of both M1 and M2. As a result they maintain M1 features including the uptake of oxidized LDL. The environment signals them in both directions.

My own interpretation of the research lead me to believe that the inflammation as a consequence of the hypoxia is not resolved by the actions of the macrophages although this is what they are supposed to do (as you can see in muscle repair). They normally resolve the hypxia-induced inflammation by resolving the hypoxia through cell regeneration and vascularisation.

If you have read my article on the root cause then you see that the cause is outside of the reach for macrophages to resolve it.

Total Picture

Due to the existence of a hypoxic region at the bifurcation as a result from contracted or hardened cell wall due to factors such as smoking, high insulinemia, fructose etc… You get a region in the blood flow that does not deliver sufficient oxygen to the deepest layer of the intima.

The furthest away from oxygen delivery becomes the most hypoxic and starts to send out inflammatory markers.

Monocytes infiltrate and are immobilized by hypoxia and differentiate into M1 macrophages by the environmental stimulus. As M1 macrophages they take up oxidized LDL so that they can collect lipids to support their proliferation. Normally the further progression of actions from M1 macrophages result in a morphology towards M2 macrophages.

These M2 macrophages use the accumulated fatty acids for fuel resulting in a clearance of the fatty acid content. We see that there is vascularisation penetrating from the vasa vasorum so the initial hypoxic region closest to the media is rescued from that side.

But at the same time the original M1 macrophages also stimulated growth of the intima creating more distance between the hypoxic region and the endothelial wall. On one hand the issue is resolved but on the other hand the problematic area now just shifted further away from the media and still the problem of the reduced blood flow isn’t solved.

Because this issue with the blood flow remains, you get a continuous growth stimulation leading to a further narrowing of the blood passage.

And now for the research that led me to this conclusion.

Where I started

A first thing I did was get me some better background info on macrophages. I know they are part of the immune system so a refresher is always in place. An excellent video to start with for a basic overview is the following.

Important to understand is that, as you can see from the video above, these cells develop and morph into different types. Macrophages circulate as monocytes and when triggered they develop into different types of macrophages.

Next up is a visit to wikipedia on macrophages itself. It has a section on macrophage subtypes but underneath I noticed a section on muscle regeneration. That is interesting. In my root cause research, vascular smooth muscle cells are affected. I know these cells are not exactly the same as skeletal muscle cells but still.. why not have a look at the interplay between skeletal muscle and macrophages. Maybe we can learn something from it.

The next video shows how muscle damage and repair is supported by monocytes-turned-macrophages.

What we can learn here is that macrophages play an active role in the environment and again note how macrophages of the M1 type transition into the M2 type. These are the 2 main categories that I’ll look into but note that there are many sub-categories and versions in between depending on the tissue they are located at etc…

By taking up malfunctioning cells macrophages have learned to extract the lipids for further usage. Related to this, a distinct feature is that M1 types are more prone to fatty acid synthesis while the M2 favor fatty acid oxidation. They operate in a hypoxic area so they revert to glycolysis, something we recognize from cancers cells that also live in hypoxic conditions.

“Macrophages and lipid metabolism” https://www.sciencedirect.com/science/article/pii/S0008874918300327

This is offering a first clue as to why M1’s take up lipids. Proliferating cells require lipids to build these new cells. Proliferating cells require lipids for membrane construction and other organelles that also have membranes.

M1

When the monocytes migrate into the inflammed area, hypoxia is one of the drivers to change their phenotype towards M1 macrophages.

“Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis” https://pubmed.ncbi.nlm.nih.gov/27444197

These type of macrophages stimulate inflammation in order to trigger proliferation of satellite cells which then further become differentiated and form newly repaired tissue.

These macrophages are also know to stimulate proteoglycans (PGs) to stimulate the buildup of the extracellular matrix which will hold the new tissue. These proteoglycans are the earliest evidence seen of atherosclerosis before lipids accumulate.

Walton [8] showed this mucoid thickening of the intima occurs before lipid infiltration and is composed primarily of collagen, PGs, and ECM. Thus, although lipid accumulation in the artery wall is considered an early event in atherosclerosis, lipid retention is not the initiating event, and the fatty streak is not the first sign of atherosclerotic injury [11,12].

source: https://www.ncbi.nlm.nih.gov/books/NBK2029/

“Monocyte to Macrophage Differentiation: Synthesis and Secretion of a Complex Extracellular Matrix” https://pubmed.ncbi.nlm.nih.gov/22351750/

It tells us that these macrophages arrive to the site of injury and then start to accumulate lipids. This order of sequence is important as some believe LDL gets stuck, oxidizes (or not) and cause inflammation and then macrophages arrive to resolve this.

The correct order is that inflammation attracts macrophages and then they start to accumulate lipids.

This alone is a reason to question if lowering LDL is a meaningful thing to do.

M2

These macrophages are the ones who help the proliferated satellite cells to differentiate and form restored tissue.

One of the contributing factors to make the switch from M1 to M2 is the activation through PPAR-γ and IL-4. PPAR-γ is activated by fatty acids so to no surprise, the M1 needs to accumulate fatty acids in order to transition.

“New Insights on the Role of Lipid Metabolism in the Metabolic Reprogramming of Macrophages” https://www.frontiersin.org/articles/10.3389/fimmu.2019.02993/full

It is a complex world, the IL-4 cytokines come from Th2, activating PPAR-γ so these T-cells also have a role to play in the process.

“PPARγ Activation Primes Human Monocytes into Alternative M2 Macrophages with Anti-inflammatory Properties” https://www.cell.com/cell-metabolism/references/S1550-4131(07)00166-0

“The Hunt for the Source of Primary Interleukin-4: How We Discovered That Natural Killer T Cells and Basophils Determine T Helper Type 2 Cell Differentiation In Vivo” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924770/

Uptake of LDL

There are different scavenger receptors on the cell surface that take up fatty acids from circulation. LOX-1 and CD36 are 2 types of such scavenger receptors. CD36s are particularly fond of oxidized LDL (oxLDL). Just as they learned to extract fatty acids from dying cells, they also extract the fatty acids from oxLDL. Under normal circumstances, the cholesterol is expelled from the cell and picked up by HDL particles via a process called reverse cholesterol transport.

source: https://www.sciencedirect.com/science/article/pii/S0008874918300327

With such an important function for HDL it should not be a surprise that low HDL is stated as being a strong and independent risk factor for atherosclerosis! But does increasing HDL lower your risk? Attempts have been made and failed to show benefit. Is that a surprise though? Do you expect a fix if the root cause is not addressed?

“HDL-cholesterol and cardiovascular disease: rethinking our approach” https://pubmed.ncbi.nlm.nih.gov/26192490/

Another issue is the hypoxia itself, this needs to be resolved for the cholesterol export to function properly.

“Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis” https://pubmed.ncbi.nlm.nih.gov/27444197/

Interesting to note is that the availability of cholesterol within the cell triggers the transcription to export the cholesterol.

Accumulation of cellular cholesterol leads to activation of several transcription factors, including PPARγ, LXRs and RXRs which subsequently regulate expression of their target genes including transporters such as ABCA1 and ABCG1 which regulate the efflux of free cholesterol and scavenger receptors. Alternatively, passive efflux of free cholesterol can also occur.

source: https://www.sciencedirect.com/science/article/pii/S0008874918300327

So the idea is to export cholesterol yet we do find cholesterol back in the plaque so it seems that yet again the unresolved hypoxia is playing a disturbing factor.

But back to the oxLDL…

Oxidative stress

In areas of low oxygen you get an increase in ROS production which interacts with the LDL to oxidize it. Being in an environment with high risk of oxLDL, macrophages prefer oxLDL. That is not a coincidence.

oxidized LDL has also been reported to induce the expression of the M2 macrophage phenotypic marker arginase 1 via activation of peroxisome proliferator activated receptor-γ (PPARγ)79, and oxidized phospholipids present in oxidized LDL induce a macrophage phenotype distinct from M1 or M2 that has been termed Mox, which is characterized by increased expression of nuclear respiratory factor 2 (NRF2)-dependent genes and reactive oxygen species80. It is likely that T helper 1 (Th1) and Th2 cells in plaques secrete macrophage-polarizing factors81 that also contribute to the balance of M1 and M2 macrophages. Nonetheless, the factors in the plaque microenvironment that promote the polarization of these cells in vivo remain incompletely defined.

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357520/

This is a difficult area to conclude from because of its complexity but we can already see that oxLDL is a factor that contributes to the change away from the pro-inflammatory M1 type. Rather than considering oxLDL an issue, it may even be a necessary factor in the path towards tissue repair and resolution of the inflammation. But why aren’t they moving towards M2?

Why don’t they change to M2?

In order to have a possible answer I first looked at some info again on how this is handled in muscle tissue.

We see here that BACH1 is an inhibiting component for the transcription to move towards the M2 (repair macrophage) type. BACH1 gets inhibited by binding to heme.

Heme itself gets degraded by Heme Oxygenase 1. This enzyme gets upregulated by oxLDL but also by hypoxia and we see this in the plaque.

HO-1 is transcriptionally upregulated as a sensitive antiinflammatory protein by various types of oxidative stress, such as oxidized LDL,14 ultra-violet radiation,15 thiol scavengers,16 and hypoxia,17,18 as well as substrate heme19 in the cardiovascular system.

source: https://www.ahajournals.org/doi/10.1161/01.atv.0000178169.95781.49

There are likely other factors but this again makes an important link with hypoxia. If the status of hypoxia cannot be resolved then this creates a chronic situation of more prevalent M1 macrophage phenotypical lipid uptake.

But the factor of oxidized LDL is also intriguing so I took a closer look at the reference. It seems there are specific molecules required in the oxLDL to trigger HO-1. This may actually represent an issue in the research as there are many ways to create oxLDL. Preferrably the test is done with oxLDL extracted from human sera.

Lysophosphatidylcholine, one of the major components present in oxidized LDL, was ineffective to induce the gene expression, suggesting that other lipophilic substances derived from LDL oxidation are responsible for the induction of HO-1

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1858397/

Why don’t macrophages emigrate away?

A reason why macrophages stay where they are is because of hypoxia but also cholesterol accumulation induces expression of netrin 1, a molecule that prevents them to migrate. This is very important because as I showed in my previous article, there may be a chronic situation of hypoxia that initiates the rest of the pathology.

Macrophage expression of these migration inhibitory molecules are also induced during hypoxia, which is intimately linked to atherosclerosis26,95

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357520/

In the process of resolving the situation there is vascularization, small micro vessels that are formed to feed the affected area so that it can receive nutrients and oxygen. This is how muscle develop and become stronger.

What would happen if that situation of hypoxia cannot get resolved?

LMHR

Some people on a ketogenic diet see their LDL cholesterol increase dramatically (typified Lean Mass Hyper Responder or LMHR). Levels above 300mg/dL is not unusual but typically they also see their HDL double compared to the days they were on a high carb diet.

Naturally such people are severely encouraged by their doctor to either quit the diet or use statins to lower the cholesterol because current assumption is that it causes atherosclerosis.

One of the elements in the physiology is that this CD36 receptor is enhanced by skeletal muscle. This means that if oxidized LDL is part of the issue, more of it is taken up by the skeletal muscle for energy usage.

oxLDL

OxLDL is no stranger to the body so is it really just bad? With my current understanding I suspect that the body has foreseen a way to make it part of the overall functioning. The oxidized form may no longer be able to perform the same functionality as LDL so rather than throwing it away, cells have developed a way to use the fatty acids that it contains.

This would be ideal and could explain the preference of the CD36 receptor for oxLDL. Recuperate what is possible. They contain energy which helps survival. Being on a ketogenic diet you especially depend on fatty acids for energy production so our LMHR’s could be clearing more of these oxLDL from the circulation leading to an overall lower level.

But we can only claim this if there is no proportional increase in oxLDL as a percentage of total LDL.

“Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin” https://pubmed.ncbi.nlm.nih.gov/10480880/

And again, such affinity for oxLDL by the CD36 scavenger has a reason so oxLDL must be part of the design. It cannot be bad by itself but perhaps in the quantities that it is present, it may overwhelm the system.

expression of the fatty acid translocase Cd36 gene was higher in the LCHF group (mouse study)

source: https://link.springer.com/article/10.1186/s12986-016-0132-8

Previously, we reported that FoxO1 activation in C2C12 muscle cells recruits the fatty acid translocase CD36 to the plasma membrane and increases fatty acid uptake and oxidation. This, together with FoxO1 induction of lipoprotein lipase, would promote the reliance on fatty acid utilization characteristic of the fasted muscle.

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386936/

It makes me suspect that absolute levels of oxLDL are more important than your LDL-c level.

The following article even shows that the LDL receptor is downregulated (!) in macrophages so that non-oxidized LDL have a more difficult time being absorbed while the scavenger receptors are not downregulated.

Fig. 1
source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627698/

among 425 patients with acute coronary syndrome (ACS), the HR of AMI recurrence or ACS-related death at the 5-year follow-up was 2.88 (95% CI: 1.93–4.32) for 1 mmol/L increase in circulating ox-LDL

“Association between circulating oxidized low-density lipoprotein and atherosclerotic cardiovascular disease” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627698/

When a hazard ratio goes above 2 then it gets me interested for a possible link. The article discusses other studies that showed no meaningful HR so we have to be careful here and look at each study to see what differs between them for reaching different conclusions. It could also show that oxLDL is just one factor of many that make up the pathology of atherosclerosis.

The article continues to show that different statins are able to lower the volume of oxLDL. So if statins exert an effect, is it because they lower LDL cholesterol overall or because they lower oxLDL?

A different paper reports on the ability of Atorvastatin to suppress the uptake of oxLDL by macrophages and at the same time lowers their CD36 expression.

“Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes” https://pubmed.ncbi.nlm.nih.gov/12119208/

Whatever the answer, it doesn’t change the observed mechanism of reduced uptake of normal LDL. And with how statins exert their effect, should we consider our total LDL-c level as an issue?

I’m more inclined to focus on oxLDL. Not to eliminate it but to reduce quantity.

HDL

Also when we see the role that HDL has to play, we can conclude it is not necessarily the cholesterol itself that can form an issue. LMHR’s more often than not have very high HDL cholesterol to assist in this uptake from cells that do not need the cholesterol such as the macrophages. But since all cells that have increased their dependency on fat metabolism have increased uptake of oxLDL, there could simple be more HDL required.

Is the level in function of how much is needed? We can’t be too quick to conclude. They don’t necessarily have a higher availability for cholesterol uptake by HDL from macrophages.

This is a very difficult area of research I think because it will all come down to the rate at which this happens and the amount of oxLDL that is in circulation.

“Macrophage‐mediated cholesterol handling in atherosclerosis – Formation of cholesterol esters” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717859/#jcmm12689-sec-0007title

Furthermore the cholesterol efflux speed depends on the ABC1 transporter which is down regulated by unsaturated fat, not saturated fat. The ketogenic diet favors more on the side of saturated or mono-unsaturated fat and certainly promotes avoiding poly-unsaturated fat.

“Unsaturated fatty acids repress expression of ATP binding cassette transporter A1 and G1 in RAW 264.7 macrophages” https://pubmed.ncbi.nlm.nih.gov/22209005/

In addition, do not forget that HDL also has an anti-oxidant function for LDL.

Finally

What would I recommend knowing what I know so far? Handle the root cause and don’t worry about cholesterol but do look into what causes LDL to oxidize. Check out my article on oxidized LDL for some of the protective factors. You can find them more towards the end.

I hope this article has given you again some more background on how things work and help you in your search to be in control of your health.

– – – THE END – – –

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