by Siim Land
At the end of the 20th century, autophagy had been established as an important function in mammalian cells. However, there wasn’t much proof to autophagy in morphological studies, which is why Dr. Ohsumi’s work was so groundbreaking. His lab gave important insight into the mechanisms of this complex process. By now we don’t know everything about autophagy but we do know something. So, how does it work?
When autophagy gets activated, the organelles of your healthy cells start to hunt out dead or diseased cells and then consume them.
Autophagy is mediated by an organelle called the autophagosome, which combines with other cellular components like the endosome and lysosome to form a double membrane around the cell that’s going to be eaten. The autophagosome then dissolves the cell that are sentenced to death and converts it into energy[212] (See Figure 32).
Figure 32 The Process of Autophagy
Where and how the autophagosome gets formed is currently a mystery for researchers. In yeast, it’s been identified to occur when many ATG proteins converge at a site that’s called ‘pre-autophagosomal structure (PAS)’[213]. Some equivalent structures have been located in mammalian cells as well but detailed information about the PAS is still unknown.
Autophagy gets triggered mostly by nutrient starvation:
In yeast, starvation of nitrogen and other essential factors like carbon, nucleic acid, auxotrophic amino acids, and even sulfate can activate autophagy to some degree[214].
In plant cells, nitrogen and carbon starvation can also trigger autophagy[215]. These were the points of interest for Dr. Ohsumi in yeast as well.
In mammals, autophagy happens in various tissues in different degrees. There’s macroautophagy in the liver, brain, muscle, mitophagy in the mitochondria and Chaperone-Mediated Autophagy (CMA) (See Figure 33). Depletion of amino acids is a strong signal for triggering autophagy but that depends on the type of cell and amino acids because amino acid metabolism differs among tissues.
In vivo, it’s thought that autophagy is regulated mostly by the endocrine system, particularly by insulin. Insulin suppresses liver autophagy by raising blood sugar and signaling the presence of nutrients. Glucagon, which is the counterpart of insulin, releases liver glycogen to be burnt for energy, and that increases autophagy.
Figure 33 Different levels of autophagy
Both amino acids and insulin-like growth factors regulate the nutrient-sensing of mTOR. Suppressing TOR with things like rapamycin and CCI-779 has been shown to induce autophagy in yeast[216] and other animals[217]. However, not all of the autophagy signaling happens through mTOR as some amino acids can suppress autophagy independent of mTOR[218].
Recent reports have shown many other factors to be involved in autophagy regulation, such as Nf-kb[219], reactive oxygen species[220], calcium[221], AMPK[222], and many more. So, what I propose is to not look at autophagy as a binary on-and-off switch but more like a degree dependent state that’s mediated through how depleted and deprived the organism is from certain nutrients.
The main inhibitor of autophagy in muscles is a kinase called Akt. It can regulate autophagy mainly in two ways: (1) a rapid regulation of mTOR activation, and (2) a slower response of gene transcription via FoxO3[223]. FoxO3 controls the transcription of autophagy-related genes, such as LC3 and Bnip3, which mediate the effect of FoxO3 on autophagy. Akt activation blocks FoxO3 and autophagy.
Although there’s still much to learn about autophagy, current research is showing that most of the signaling happens through the pathways of mTOR and AMPK. When under nutrient deprivation, AMPK starts to inhibit cellular growth by suppressing the mTORC1 pathway, which in turn forces the body to catabolize its weakest components.
Autophagy is a catabolic pathway that makes you break down old cells. Although you’re causing protein breakdown, autophagy is needed for muscle homeostasis. With poor autophagy functioning, your body wouldn’t be able to maintain lean tissue. It improves your body’s ability to deal with catabolism and atrophy by promoting protein sparingness. A weakened or inadequate state of autophagy may contribute to aging, and muscle wasting through sarcopenia[224]. That’s why it’s incredibly vital for not only living longer but also to age slower.
However, defective as well as excessive autophagy can lead to substantial muscle disorders and loss of lean tissue[225], which in turn can promote premature aging.
Figure 34 Too much, as well as too little autophagy, is bad for you
You need these anabolic pathways like mTOR and insulin to build new tissue and keep your cells alive. That’s why strength athletes and bodybuilders are so focused on supporting anabolism and preventing muscle catabolism by taking different amino acid supplements and consuming foods that make them grow more. They’re probably over-doing it but that’s a whole nother story with potential consequences on their long-term health.
With that being said, a constant supply of nutrients and access to energy inhibits the body’s ability to induce autophagy and protect against catabolism. In fact, a continuous circulation of both macro- and micronutrients all the time inhibits their usage and uptake by making the cells less responsive. It means that to actually absorb the nutrients you’re feeding yourself, you need to go through periods of mild deprivation as you’ll be more sensitive to those nutrients afterwards again.
Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise[226]. To trigger the activation of autophagy during exercise, the activation of AMPK is also needed[227].
AMPK regulates both protein synthesis and breakdown pathways. AMPK has a vital role in skeletal muscle homeostasis. It’s activated by conditions of energy stress, including nutrient deprivation and vigorous exercise.
Exercise performed in a fasted state shows a higher increase in LC3B-II level compared with a fed state, which suggests exercise done while fasting to have a better autophagic response[228]. This makes sense because you’ll be tapping straight into your body fat for energy instead of burning through the food you ate. When activated, AMPK increases the flux of glycolysis and fatty acid oxidation while at the same time inhibiting gluconeogenesis and fatty acid and cholesterol synthesis.
In addition, AMPK has been recently shown to be a critical regulator of skeletal muscle protein turnover[229]. Protein turnover is the balance between protein build up and protein breakdown over the course of the day.
If your protein synthesis exceeds the amount protein’s being broken down, then you’re in a more anabolic state.
If you’re breaking down more than synthesizing, then you’ll be more catabolic. Or autophagic.
Your body is always in a flux between anabolism and catabolism – growing and degrading. Both of these ends of the spectrum are vital for a healthy life – you want to promote the growth and repair of your vital organs and muscles, but you also want to eliminate and break down the old worn out cells and metabolic debris.
How Long Until Autophagy?
But how much time do you need to starve to activate autophagy? That’s a difficult question because it happens in various degrees almost all the time. It’s not as binary as you’d think but it’s definitely not something you can hack easily either.
In general, suppressing mTOR and insulin will already begin to elevate autophagy in a dose-specific degree. Low blood glucose levels and depleted liver glycogen stores are indicators of energy shortage in the body. As energy depletion continues, the body upregulates its metabolic pathways that are associated with burning stored fat for fuel. Eventually, this leads to a ketotic state with elevated levels of ketones in the blood. Chaperone-mediated autophagy can be maintained with the elevation of endogenous ketones and fatty acids thanks to suppressed insulin and low blood glucose.
Although autophagy is usually accompanied by ketosis, you can still be in ketosis without activating autophagy and you can activate autophagy without being in ketosis. The reason is that of nutrient signaling and mTOR. Fats and exogenous ketones have a negligible effect on insulin but they ma
y still raise mTOR if you consume them in copious amounts and under the wrong circumstances. Likewise, you can see trace amounts of autophagy even while having fasted for 24 hours on a carbohydrate-rich diet if you do things right.
To really gain the benefits of autophagy, you’d have to be fasting for over 48 hours to allow the stem cells and immune system to do their work. That’s why I recommend everyone to fast for at least 3-5 days 2-3 times per year. These extended fasts not only make you burn a lot of body fat very easily but they’ll also recycle the weak cells that are simply dragging you down and giving you potential issues.
Contemporary eating habits like 3 square meals a day, constant snacking, high carb + high fat foods lead to excessive overconsumption and are all anti-autophagic and they inhibit the recycling of old waste material. We are forcing our body to be in chronic hoarding mode with no allowance for spring cleaning.
Even people who eat „clean foods“ but don’t go through nutrient starvation may potentially be walking trash cans. There are many other sources of toxins and inflammation all of us get exposed to starting from air pollution, water, GMOs, plastics, heavy metals and who knows what else. What looks good on the outside doesn’t mean that everything is okay on the inside. Having your autophagy pathways live and active is even more important for living in the modern world.
However, autophagy can do both good as well as harm to the organism. It has a dark side...
The Negative Side Effects of Autophagy
The entire process of autophagy and self-eating is called autophagic flux, which includes (1) the formation of an autophagosome, (2) fusion with lysosomes, and (3) the degradation of the autophagosome.
Autophagy controls inflammation and immunity by eliminating inflammasome activators[230]. Removal of pathogens by autophagy is called xenophagy[231], which has many immune strengthening benefits. However, some bacteria like Brucella use autophagy to replicate themselves[232]. That may cause some bacterial overgrowth or at least prevent its death.
The essential autophagy gene ATG6/BECN1 encoding the Beclin1 protein has been found to suppress tumors in cancer. However, it’s not been found to be that big of a tumor-suppressor as previously thought and sometimes it can even promote cancer due to the self-replicative process[233]. Self-eating can enhance tumor cell fitness against environmental stressors[234], which makes them more resilient against starvation and chemotherapy. It may be that autophagy is better for cancer prevention rather than treatment.
It’s not clear whether autophagy prevents or promotes apoptosis or programmed cell death[235]. The outcome turns out to depend on the stimulus and cell type[236]. Blocking autophagy enhances the pro-apoptotic effect of bufalin on human gastric cancer cells, which is a Chinese medical toxin used for tumor suppression[237], through endoplasmic reticulum stress[238]. In this example, less autophagy led to more cancer cell death because the cancer cells were weaker whereas with autophagy they became stronger.
Essentially, when malignant tumor cells are put under nutritional stress through calorie restriction, autophagy may prevent them from dying by inhibiting apoptosis[239]. So, it’s not all black and white with autophagy – some viruses and pathogens are eliminated by it whereas others hijack its mechanisms and replicate themselves. What’s more, autophagy has various differences in different tissues[240], such as the brain, liver, muscle, and fat. Sometimes it’s good, sometimes it’s not. You want the beneficial autophagy in the liver and brain to clear out plaques but you don’t want to self-eat your lean tissue and muscles.
The point of all this is that cells undergo constant deterioration and slow degradation because of entropy. You can easily end up with suffering from the dysfunctional apoptotic or necrotic processes that may potentially leave the body in a lesser state than before. That’s why it’s important to not only get rid of the bad but to also keep the good, which is how well-functioning autophagy should work.
How to Measure Autophagy
Autophagy can be monitored by two different approaches: (1) direct observation of autophagy-related structures and their fate; and (2) quantification of autophagy-lysosome-dependent degradation of proteins and organelles.
To accurately estimate autophagic activity, it is essential to determine autophagic flux, which is defined as the amount of autophagic degradation. Monitoring autophagic flux is still complicated even in cultured cells and model organisms. and is currently impractical in humans.
At the moment, there is no established method to measure autophagic flux in humans; therefore, it’s very difficult to know exactly how autophagy works in humans. However, studies done in other species have found some similarities and mechanistic principles that regulate autophagy.
As we know by now, autophagy is regulated most by the mTOR and AMPK pathways. To trigger autophagic cell death you need a catabolic catalyst that would increase AMPK and cause cellular stress. Being anabolic and growing will inhibit autophagy by raising mTOR through the insulin/IGF-1 signaling pathway.
To know whether or not you’re more anabolic or catabolic or more mTOR or AMPK activated, you can measure your insulin to glucagon ratio (IGR). Both insulin and glucagon are important for your body’s homeostasis and survival. They will either make you store energy and repair vital tissues (anabolism) or break down backup storage so that you’d survive (catabolism). In general, an increase in IGR is associated with more anabolism – weight gain, muscle growth, fat storage, hyperinsulinemia, and higher risk of hypoglycemia. A reduction in IGR promotes catabolism, fat loss, and prevents hypoglycemia.
Everyone’s average blood glucose levels vary but the consensus is that the normal range for non-diabetics while fasting should be 3.9-6.0 mmol/L (70-100 mg/dL). During the day it would fall around 5.5 mmol/L or 100 mg/dL. However, in my opinion, a healthier person would fall slightly lower than that between 3.0-5.0 mmol/L-s. Deviations of 5-20% will either release insulin to lower blood sugar or increase glucagon to raise blood sugar. It fluctuates throughout the day.
To know your insulin to glucagon ratio you can take blood tests for insulin as well as glucagon from your medical doctor. Here’s what research has found to influence your IGR:
A 1:1 insulin to glucagon ratio: 1.0
While fasting you have lower insulin and more glucagon. Fasting + No Food: ~0.8
While eating the Western Diet with higher carbs there’s more insulin. Carbs + Eating: ~4.0
On a Low Carb Diet, there’s fewer carbs and less insulin. Low Carb + Eating: ~1.3
Consuming protein while fasting causes a drop in insulin by raising glucagon-induced gluconeogenesis. Fasting + Protein: ~0.5
Consuming protein on a low carb diet doesn’t raise insulin and doesn’t significantly affect glucagon. Low Carb + Protein: ~1.3
Consuming protein with high amounts of carbohydrates spikes insulin 20x more than normally because of the anabolic effects. Carbs + Protein: ~70
Amino acids combined with carbohydrates produce a much larger anabolic effect and insulin response than just carbs or protein alone. That’s a significant difference between macronutrients and their anabolic response. Calories in VS calories out just got much more complicated.
The Glucose Ketone Index
You can guestimate your general metabolic health and the insulin-glucagon ratio at home by measuring your blood glucose and ketones with an ordinary ketone meter.
The Glucose Ketone Index (GKI) is a number between the relationship of your ketones and glucose levels. It can help to monitor your general health in relation to your blood glucose levels.
Here’s the Glucose Ketone Index Formula: (Your Glucose Level / 18) / Your Ketones Level = Your Glucose Ketone Index
Measure your blood glucose by pricking your finger and all that. Write down the number you got.
Measure your blood ketones by pricking your finger again (ouch). Write down the number you got.
Divide your blood glucose number by 18. If your device is using mg/dl, then dividing that with 18 converts it over to mmo
l/l
If your device is already showing mmol/l, then you don’t need to divide anything and can skip this step
Divide your result from the previous step by your ketone numbers.
The end result is your GKI.
Figure 35 Glucose Ketone Index Formula
Let’s take an example from my readings during a 5-day water fast:
My blood glucose was at 55 mg/dl which is 3.0 mmol/L (almost hypoglycemic!)
My blood ketones were at 3.4 mmol/l (which is why I didn’t have symptoms of hypoglycemia)
Glucose at 55 mg/dl divided by 18 gives us 3.05
Divide 3.05 by 3.4 and we get 0.9 rounded
In general, having a GKI below 3.0 indicates high levels of ketosis in relation to low levels of glucose; 3-6 shows moderate ketosis, and 6-9 is mild ketosis. Anything above 9 and 10 is no ketosis. Therefore, a lower GKI will reflect an estimated insulin-glucagon ratio by virtue of how glucose and ketones affect that relationship.
In his book, Cancer as a Metabolic Disease, Thomas Seyfried says that the optimal glucose ketone index range for cancer treatment and prevention is between 0.7-2.0, preferably around 1.0. As you saw with my five-day fast glucose ketone index, it’s quite hard to reach and keep your GKI that low. On a daily basis, my own GKI can fall somewhere between 5-13. If you’re metabolically healthy and don’t have any serious disease like cancer, then you don’t have to obsess over your GKI score. It’d be a good thing to dip into the 1.0 range every once in a while during an extended fast but it’s not a necessity.