Monday, March 30, 2020

Look after your lysosomes

I was a little cautious about the efficacy vs toxicity of chloroquine and its derivatives in my last post.

George Henderson just retweeted this snippet;

Sadly the narrow line between the degree of raising lysosomal pH to blunt viral replication and that which might release sufficient cysteine to strip the FeS clusters out from complex I can be crossed quite easily, so it appears.

Worryingly Dr Barman appears to have been one of those people with some degree of metabolic syndrome and who might have been someone most likely to benefit from prophylaxis against coronavirus replication.

My own observation during my very rare trips to our local hospital is that medical professionals are far from immune to metabolic syndrome. Couple that with extreme stress, high viral load exposure, severe sleep deprivation and the sort of food/snacks available in hospitals and you have to worry for the health of these people.

None of them want to have metabolic syndrome, a problem which is built in to our public health guidelines. These people are laying their lives on the line to support the lipid hypothesis. Most of their patients are in hospital secondary to the lipid hypothesis. Those developing ARDS in the ITU do so in a large part as a result of the lipid hypothesis.

Just my rather sad view from the sidelines.


Wednesday, March 25, 2020

From Yeasts to Chloroquine

This paper is from Hughes and Gottschling

An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast

It got a mention in the blog back in 2012 when it was freshly published. The group have gone on to study yeasts, ageing and the lysosome-like vacuole of yeasts. Their core finding is that vacuolar pH controls mitochondrial "health" which controls ageing, at least in their model.

The group has been very busy and earlier this year this paper was published from Hughes' lab:

Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis

The paper describes a very long series (way too many to detail here) of experiments aimed at adjusting vacuolar pH upwards and downwards and observing the effect on the survival of mother yeast cells through repeated cell divisions (replicative age rather than chronological age, there are arguments about which matters most).

Bottom line: Acidifying vacuolar pH extends lifespan, reducing its acidity shortens it.

Why should that be?

Their next series of experiments demonstrated that cysteine toxicity was the driver of early mitochondrial functional decline secondary to loss of vacuolar acidity. Cysteine is normally harmless and essential for life. Your cells love it, just so long as it is within the vacuole (or lysosome in humans), not in the cytoplasm. It's kept there by a vacuolar amino acid transporter driven by the vacuole proton gradient. The pH gradient is generated using a vacuolar vATP-ase to pump protons from the cytoplasm in to the vacuole, using ATP. It's related to the mitochondrial ATP synthase but normally runs in reverse.

If, on a long term basis, vacuolar pH rises (ie the vATP-ase fails), cysteine is released from the vacuole in to the cytoplasm where it auto-oxidises, generating much too much hydrogen peroxide. This reacts with the iron-sulphur clusters of complex I and many other crucial enzymes in the mitochondria. In old age cysteine becomes toxic through vacuolar failure.

I've been interested in this for some time because Barja and Sinclair have both intimated that they are tending to avoid animal proteins in favour of low cysteine/methionine plant proteins. Cysteine is the cellular executioner when vacuole pH rises during the old age of yeasts or lysosomal pH rises in ageing mammalian cells. It's interesting because methionine restriction (which reduces cysteine levels) appears to core to the longevity promotion seen with caloric restriction or protein restriction in mice fed on crapinabag.

You have to wonder whether we are looking at this the wrong way round. What if crapinanbag, based on starch and sucrose, causes early onset lysosomal failure which can be ameliorated by removing the cysteine, which is the cellular execution mechanism?

This would make methionine restriction's longevity extension rather specific to glucose based metabolism. My biases would tend to favour this point of view. There's no data, yet.

As an aside:

Now, I have speculated that both influenza and corona viruses need anabolic processes generated by mTOR activation. This requires acute acidification of the lysosome. Blocking acute lysosomal acidification is one technique currently being investigated for treating the life threatening pneumonia which develops in susceptible individuals during the current COVID-19 pandemic. There are suggestions that chloroquine, a suppressor of lysosomal acidification, might be an effective treatment. My guess is because it blocks anabolism.

There is probably a fine line between suppressing anabolism and releasing a mitochondrial-executing concentration of cysteine.

Neither Hughes nor Gottschling were considering therapeutic inhibition of vacuolar acidification as a stratagem for anything. They were more interested in avoiding long term loss of vacuolar acidity to delay mitochondrial function decline. But blunting anabolism without causing catastrophic cysteine release is a current anti-viral/anti-neoplastic therapeutic target.

You can see that the drug chloroquine a) might work and b) might be very toxic in overdose.

It does currently appear that it might work but we should never forget that "clinical experience is no guarantee of therapeutic efficacy".

However it would be great if it really did work.


Tuesday, March 17, 2020

ARDS and linoleic acid

Adult Respiratory Distress Syndrome is topical at the moment. In the comments to the last post I wondered whether omega six fatty acids, especially linoleic acid, might be a driver of ARDS, which is one of the most intractable ITU problems in response to major infection/trauma/inflammatory insults.

Tucker came up with this abstract

Plasma fatty acid changes and increased lipid peroxidation in patients with adult respiratory distress syndrome

and I peeked at the related papers to find this gem:

An increase in serum C18 unsaturated free fatty acids as a predictor of the development of acute respiratory distress syndrome

Again, only an abstract and mostly describing a pilot study. But here is the critical statement:

"Increases in unsaturated serum acyl chain ratios differentiate between healthy and seriously iII patients, and identify those patients likely to develop ARDS".

That is, the more linoleic (and oleic) acid you have as FFAs in your bloodstream, relative to my beloved palmitic acid, the more likely you are to develop ARDS. Which carries a high risk of death.

That was 1996.  The work will have been done before that, so we have known that linoileic acid is bad news for well over 20 years.

If you are a Standard American on the Standard American Diet, or anyone else in the world poisoned by a cardiologist-promoted PUFA based diet, any weight loss through illness will release significant amounts of linoleic acid from your adipocytes. That might just trigger ARDS in the aftermath of a viral pneumonia.

There's a lot of it about.


BTW Steve Cooksey has a rather nice post up citing a lot of the refs featuring how to maintain an effective innate immune system, so as to avoid the viral pneumonia in the first place. It's a good read.

Saturday, March 07, 2020

Cell surface oxygen consumption (4) Influenza

This press release, from 2013, surfaced on twitter (embarrassingly I have again lost the tweeter due a hat tip for this. Mea culpa. Found him, it was resurfaced/retweeted by Guðmundur Jóhannsson).

Glucose: Potential new target for combating annual seasonal flu

which summarises this paper:

Glycolytic control of vacuolar-type ATPase activity: a mechanism to regulate influenza viral infection.

Over the last few weeks I happen to have been immersed in vacuoles/lysosomes, cysteine toxicity, longevity and yeasts. Oh, and mTORC1, which is deeply associated with lysosomes. So I'm in a mindset of how lysosomes/mTOR control longevity/anabolism.

Anyhoo. Influenza A virus uses lysosomes to maximise its survival. My prediction is that it activates mTOR to induce a marked anabolic state and hijacks that anabolic state to generate lots and lots of influenza A virus particles. It will do that, much as a cancer cell might, by aerobic glycolysis working on the basis that glycolysis, while inefficient, is very, very fast at generating ATP compared to OxPhos. This would suggest that the free availability of glucose secondary to hyperglycaemia (or increased access of glucose to the cytoplasm secondary to hyperinsulinaemia) will increase the success of the influenza virus, as found in Kohio's paper.

Which brings us to anabolism and glycolysis. Not only does aerobic glycolysis supply ATP for anabolism faster than OxPhos can but it also supplies phosphoenolpyruvate for amino acid synthesis, plus other anabolic substrates come from glucose via assorted pathways.

However for every glucose molecule which generates a pair of 1-3 bisphosphoglycerate molecules two NAD+ are consumed. If these glycerate molecules are used for anabolism via phosphoenolpyruvate they will not restore the NAD+ balance by converting to lactate. The basic story is in

Cell surface oxygen consumption (2)


Cell surface oxygen consumption (3)

with an introduction to the concept in

Cell surface oxygen consumption (1)

The glycerophosphate shuttle won't do the job because this too is limited to the speed of OxPhos. Cell surface oxygen consumption does fit the bill for rapid restoration of NAD+.

So. Does influenza virus drive cell surface oxygen consumption to facilitate anabolism at a speed fast enough to keep it one step ahead of the innate immune system?

I don't know.

But another standard (primarily rodent) model RNA virus certainly does.

Oxygen uptake associated with Sendai-virus-stimulated chemiluminescence in rat thymocytes contains a significant non-mitochondrial component

I think this will be a basic feature of rapid anabolism, be that viral or neoplasia related.

Will hyperglycaemia and/or hyperinsulinaemia facilitate viral directed anabolism under infection by another, more topical novel human RNA virus?

Personally, I'm not planning on finding out the hard way when I get around to catching the current bug.


Sunday, February 23, 2020


It came up in conversation with Ally as part of the Paleo Canteen podcast that I like coffee but that it doesn't like me.

Over the years before LC my coffee ingestion had stabilised at around 7 or 8 mugs per day. That's quite a lot. At the time I started on LC I did Atkins induction and cold turkey-ed from all methyl xanthines. The headache was tolerable, especially as I knew exactly why it was there and that it would be gone by about seven days in, which it was. The need for an evening stimulant also disappeared because I no longer fell asleep during the hyperinsulinaemic phase of the post prandial period.

For which I was infamous.

Over the years I have reintroduced coffee a couple of times but  stopped it again due to either minor lower GI upsets or worsening of either low back pain or finger arthritis.

I had done a desultory Pubmed search to see if there was any evidence for clear cut, lectin induced GI damage from coffee which might explain my own signs. When the penny dropped that coffee "beans" were actually seeds rather than legume-like beans I sort of gave up hunting.

So I was avoiding coffee and expected to do so long term. My issue was that I quite like the jittery restlessness which comes from an acute large dose.

In the aftermath of chatting to Ally I received an e-mail for Mason about Dr Paul Mason, his local Dr in Sydney. I have a lot of time for Dr Mason and I really enjoyed his lecture from the 2019 conference.

It turns out that Dr Mason is pretty sure there is a lectin in coffee. Not only that but the lectin is heat labile.

If you boil your coffee for 10 minutes you appear to pretty well destroy the lectin.


I can boil down a double strength cafetiere of coffee to the volume and bitterness of a double espresso in 10 minutes.

The caffeine is still there and absolutely produces the desired pharmacological effect.

For myself, drinking two or three double espressos per day produces tachyphilaxis to the caffeine within a week or two. Withdrawal is mild and sensitivity is pretty well restored within about 4-5 days. I have no interest in using caffeine to blunt caffeine withdrawal, so coffee is probably a weekend treat.

Plant poison, undoubtedly. Contains disgusting antioxidants too, no doubt. At the moment I feel that there is an acceptable trade-off.


For those who enjoy confirmation bias and worm studies:

Lifespan Extension Induced by Caffeine in Caenorhabditis elegans is Partially Dependent on Adenosine Signaling

Lard makes hungry mice live longest

Over the past few weeks I've been looking for papers where Barja's group might have run longevity experiments. This does not seem to have been their forte. They have done lots of observational comparative studies looking at long vs short lived species and lots of interventions to modify mitochondrial membrane lipid composition but no hard-core lifespan measuring studies that I can find.

So Barja threw in the rather off comment about avoiding "excessive intake of animal proteins and fats typical of western diets" in his review without obvious direct testing of these variables on lifespan.

I have to leave the mechanism of calorie restriction, aka protein restriction, aka methionine restriction for another day.

What we can do today is to look at Barja's dreaded animal fats. Like lard.

The data are, sadly, only available from CRON fed mice. This is the study:

The Influence of Dietary Fat Source on Life Span in Calorie Restricted Mice

Diets had their fat source modified thus and also had their calories restricted by 40%:

"The modified AIN-93G diets (% of total kcal) each contained 20.3% protein, 63.8% carbohydrate, and 15.9% fat. Soybean oil was the dietary fat in the control group (standard AIN-93G diet). The dietary fats for the CR groups were soybean oil (high in n-6 fatty acids, 55% linoleic acid, Super Store Industries, Lathrop, CA), lard (high in monounsaturated and saturated fatty acids, ConAgra Foods, Omaha, NE) and fish oil (high in n-3 PUFAs, 18% eicosapentaenoic acid, 12% docosahexaenoic acid, Jedwards International, Inc., Quincy, MA). To meet linoleic acid requirements, the fish oil diet contained 1% (w/w) soybean oil".

Here are the survival curves:

The left hand curve of green circles is from (nearly) ad-lib feeding of crapinabag. The yellow squares showing best survival are from feeding the dreaded animal fats from lard, combined with CRON. The fish oil group, full of EPA and DHA, did worst of the three CRON groups with soy oil being intermediate.

I think beef dripping would have done better than lard and beef suet even better still, but then I would think that.

Peter, saturophile.

Saturday, February 22, 2020

Insulin sensitivity makes you fat: growth hormone receptor deletion

TLDR: Excessive insulin sensitivity sets you up to become obese.

I have to apologise for citing Valter Fastingbar Longo, sometimes you have little choice. This paper

GH Receptor Deficiency in Ecuadorian Adults Is Associated With Obesity and Enhanced Insulin Sensitivity

documents the physiology of humans who are homozygous for a large growth hormone receptor gene defect. They make their GH, lots of it. It does absolutely nothing, having no receptor. GH normally works in opposition to insulin on adipocytes, causing both lipolysis and systemic insulin resistance.

Also, in the absence of GH signalling, these people make no IGF-1 so are of dwarf stature. They are exquisitely insulin sensitive. As in here are the OGTT results. Dark lines are the GHR deficient people:

Plasma glucose is comparable to that of controls throughout, matched for BMI (and lots of other things). But just look at that insulin level, peaking at 25microIU/ml vs 80microIU/ml in controls. The dwarves are very, very insulin sensitive.

And very fat.

Despite having a mean BMI of 27.6 (controls are higher at 29.4) the dwarves have 48% of their weight as fat mass compared to 41% in the controls.

Let's put this in to context: The GHr deficient people are fat because they are insulin sensitive. There is no paradox. We are not thinking that their obesity should have caused insulin resistance, it's that their failure to generate one type of physiological insulin resistance has allowed pathological insulin sensitivity to prevail, hence obesity.

Oh, and leptin:

Leptin in the dwarves with 48% body fat is 7.32ng/ml. Leptin in controls with 41% body fat is 10.36ng/ml, p is just over 0.02 if you are wondering or care.

It looks to me as if these excessively insulin sensitive individuals have yet to reach their "ideal" metabolic level of obesity to counteract their lack of GH signalling. Interesting to wonder what determines the level of adiposity at a given age in the absence of GH signalling. That's not simple.

We have no data on RER under fasting or post prandially. But we can be fairly confident that the fasting RER will be low, reflecting high basal lipolysis from distended adipocytes and post prandial RER will be high as insulin action facilitates glucose metabolism and locks lipids in to adipocytes.

A bit like those insulin sensitive pre-obese humans a couple of posts ago. But these dwarves will have to become very, very obese to behave like normal overweight insulin resistant people.


Addendum, not worth a post in its own right but on-topic:

Does Weight Gain Associated with Thiazolidinedione Use Negatively Affect Cardiometabolic Health?

Epic quote of failed perception:

"This review paper discussed the mechanism of action of TZDs on weight gain and the so-called “glitazone paradox”, the phenomenon that TZD-associated weight gain improves rather than exacerbates insulin resistance".

There is no paradox. Insulin signalling improves with glitazones, this makes you fat.

Tuesday, February 18, 2020

CPT1aL479 resurfaces nicely

Originally from Erik Arnesen, via a retweet by Miki Ben-dor:

Inuit metabolism revisited: what drove the selective sweep of CPT1a L479?

as in

Coconuts and Cornstarch in the Arctic?

The P479L gene for CPT-1a and fatty acid oxidation

The abstract looks very nice, I can't wait to get hold of the full text!


Edit: The paper is long and somewhat repetitive. There is a much neater paper from Amber which people might enjoy:

Evidence on chronic ketosis in traditional Arctic populations

End edit.

Monday, February 17, 2020

Insulin sensitivity makes you fat

TLDR: Excessive insulin sensitivity sets you up to become obese. Becoming obese makes you insulin resistant. Eventually excessive adipocyte size will induce systemic insulin resistance. Further weight gain is still possible given a diet which induces systemic hyperglycaemia combined with a pancreas of steel. Here we go.

I picked this paper up from Pubmed while looking for something else:

Insulin sensitivity is increased and fat oxidation after a high-fat meal is reduced in normal-weight healthy men with strong familial predisposition to overweight

It's very interesting.

Over the years I have collected various models, mostly mouse/rat models, which generate obese, insulin resistant rodents.

These mostly involve damaging the hypothalamus in some way and letting the mice eat ad lib until they reach the desired level of obesity, with the associated insulin resistance. There is the ventromedial hypothalamic injury model

Molecular and metabolic changes in white adipose tissue of the rat during development of ventromedial hypothalamic obesity

The MSG injury model:

Decreased lipolysis and enhanced glycerol and glucose utilization by adipose tissue prior to development of obesity in monosodium glutamate (MSG) treated-rats

Late effects of postnatal administration of monosodium glutamate on insulin action in adult rats

The gold thioglucose injury model:

Adiponectin expression is paradoxically increased in gold-thioglucose-induced obesity

What they all have in common is that the models are always more insulin sensitive in the first weeks after injury compared to the non-injured controls. This excess sensitivity persists until a certain level of obesity is achieved. As obesity increases so does systemic insulin resistance increase (a separate mechanism) until it overwhelms the excess insulin sensitivity and rate of weight gain markedly reduces. The model is now insulin resistant.

Inappropriate insulin sensitivity is what generates the obesity. Insulin resistance limits its progression.

Insulin resistance in adipocytes can, undoubtedly, occur but this is not a feature of the adipocytes in the early stages of obesity. They are insulin sensitive. Insulin acts easily. Adipocytes distend.

Back to the paper. It enrolled young, male, non-obese offspring of obese parents. Let's call them pre-obese. Sadly the paper is from 2004, it's now 2020, I would expect the "pre" prefix might nowadays be redundant. Here are the subject characteristics:

To me it is interesting that the pre-obese chaps were carrying more fat mass than the controls. There is a 1.7kg excess, statistically ns but the trend is there. You have to wonder how close to 0.05 the p value might have been.

Here are the fasting metabolic parameters for both groups:

Notice that the fasting insulin is lower in the group with higher fat mass, provided they have obese parents. It's also interesting that their fasting FFAs are higher than those of the folks with slim parents. This difference is also ns but the numbers after the +/- sign are standard deviations, not standard errors, so my guess these too are close to significance (for what that is worth). I also like the ns elevated trigs, I suspect related to repackaging the elevated fasting FFAs. Which are elevated due to increased adipocyte size allowing increased basal lipolysis. All speculation.

Next we have the insulin response to a quite pleasant sounding, mixed macro, highish fat meal:

The fasting insulin is the one from Table 2, p being 0.007 and for a large percentage of the post-meal eight hour period insulin stays significantly lower in the pre-obese group than in the normal-weight parent group. The pre-obese subjects are consistently more insulin sensitive.

Here is the FFA graph for the same eight hours:

Converting the FFA levels to real money terms it appears that the lean parent group had FFAs of 280micromol/l and the pre-obese people had 390micromol/l. I've already speculated that the elevated FFAs in the pre-obese group are from increased basal lipolysis, not insulin resistance. As soon as insulin is released after the meal FFA levels become identical for eight hours. I've not copied the trigs graph but the trend is for chylomicrons to be the same between groups for 4 hours and then lower in the pre-obese as insulin sequesters fat in adipocytes.

Which group will be metabolising most fat under hypoinsulinaemic, near-basal lipolytic conditions? Pre-obese have elevated fasting FFAs and they're oxidising more fat, 1150 vs 740mg/kg FFM/d,  ns but you can see the trend:

However, as soon as insulin rises fat oxidation drops because insulin sequesters fat in to adipocytes at levels way below those which translocate GLUT4s. It will also divert intracellular FFAs in to intracellular triglycerides. Lipid oxidation under insulin drops to 90mg/kg FFM x 8h compared to 163mg/kg FFM x 8h in the more normal individuals. Giving p less than 0.007.

BTW FFAs stay high in both groups because the meal was around 50% fat. I would predict that a high carbohydrate, low fat meal would have produced a marked drop in FFAs and a rise in RER, both more pronounced in the people with obese parents. No data on that one.

I do not think these pre-obese people have an injury to their hypothalamus. It is more likely the problem is with their adipocytes causing the excess insulin sensitivity.

I think we can ignore discussion comments about the influence of medium chain acyl CoA dehydrogenase variation as a red herring because the pre-obese folks are oxidising more fat under fasting conditions, ie when more lipid is available. The leptin receptor comment is lovely because we know that in mice with a complete leptin receptor deficiency that providing less than 5% of calories from PUFA is highly protective against obesity while providing 15% PUFA in the diet is grossly obesogenic (first link in the blog post). Clearly dietary fatty acid composition trumps even gross leptin signalling deficiency.

What were the diets like in the pre-obese participants? All we know from this study is that the ratio of PUFA:SFA was higher in the pre-obese people:

"The polyunsaturated to saturated (P/S) ratio was 0.34+/-0.06 in the group with overweight parents and 0.31+/-0.09 in the control group".

However you try to reverse engineer the limited data from the results it's hardly 5% vs 15% PUFA, but these people have taken around 25 years of eating a slightly heart-healthier PUFA rich-er diet to gain an excess of 1.7kg of fat mass. My biases are willing to accept this as real.

Maybe it is, maybe not. I'm not exactly a bias free source of opinion.


BTW leptin is consistently lower in the pre-obese group carrying excess fat mass. My suspicion is that their fat cells "feel" empty, so are refusing to signal their true state of fullness. Once the adipocytes become full enough then leptin will increase to give a more accurate representation of the absolute fat mass. This will be associated with the onset  of the more expected insulin resistance of obesity.

Saturday, February 01, 2020

Looking in to the future of Low Energy Diets

I think I picked this up from Jan Vyjidak on Faceache but it's done the rounds on twitter too.

Low-energy total diet replacement intervention in patients with type 2 diabetes mellitus and obesity treated with insulin: a randomized trial

"At randomization, participants commenced a 12-week TDR [total diet replacement] formula LED [low energy diet]... followed by 12 weeks of structured food reintroduction and then ongoing followup in combination with an energy deficit diet at 3-month intervals until 12 months. For the first 12 weeks, all meals were replaced with four formula LED products per day (800–820 kcal/day, 57%
carbohydrate, 14% fat, 26% protein and 3% fiber) in addition to at least 2.25 liters of energy-free beverages. A fiber supplement was recommended, if required, to avoid constipation, a common side effect of using a TDR".

For three months patients were starved on 800kcal per day. At 56% carbohydrate that makes carbs come out at around 100g/d. Oddly enough, restricting carbs to this level allowed a drop in insulin usage. Indeed, there was such a marked drop in insulin usage that some patients coming off insulin all together. I wonder what these starvation subjects would think if you told them that they could have had equal reductions in insulin usage just by restricting the carbohydrate content of their diets to that 100g/d, while still allowing fat and protein to satiety... I suspect  that a) no one has told them this and b) they might not be best pleased to find out retrospectively.

For a second three months a little food was added to their diet, but not much. For the final six months patients were kept a little hungry but not so much as in the first six months of the study.

Here is what the abstract says:

"Results: Mean weight loss at 12 months was 9.8 kg (SD 4.9) in the intervention and 5.6 kg (SD 6.1) in the control group (adjusted mean difference −4.3 kg, 95% CI −6.3 to 2.3, p less than 0.001)".

Here is what the results show for the intervention group:

Here is the same graph but simplified in to three red lines representing the three phases of the study:

You can argue the exact slopes of the lines but overall the pattern is correct. Something like this:

Now it is time to look into the future. Usually this is difficult but I think that in this case the general shape of the graph lets us predict the shape of things to come when related to weight gain. Plus, because it becomes obvious in the later months of the study (from HbA1c values) that insulin is going to have to be added back in, at this time the rate of weight gain might actually increase (dramatically), but we can't know that.

Using a simple maintenance of the status quo (best case scenario) we get this, looking forwards to around about the 24 month mark:

Weight gain, in the aftermath of a year of hunger, might not stop at baseline mass either.

I think it is also possible to look in to the future of glycaemia too, by extending the plot of HbA1c with time, working from the published graph in the results. Taken forwards to 16 months or so, it looks something like this:

Maybe I'm being pessimistic. Maybe sudden tolerance of chronic hunger might kick in and reverse the adverse trends in weight and glycaemia clearly present at the end of the study. Maybe subjects might suddenly become slim and euglycaemic.

Maybe not.


Monday, January 27, 2020

Rory Robertson and Protein Restricted Longevity

I looked at this paper when it did the rounds a fair while ago, saw that the only fat source used was soybean oil and decided that living on soybean oil, sucrose, maltodextrin and wheat starch was not a good idea and so I binned it as the junk it is:

The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice

I missed the embedded problems which have since been brought to light by Rory Robertson, whose slightly over-the-top concerns are voiced here. I would perhaps disagree slightly with some of his opinions but, overall, he makes a rather good case. The first thing to note is that you have to go to the supplementary data to realise that a significant number of groups of mice were lost (and excluded) due to mortality problems. Table S1 describes all thirty of the diets which the study started out with. Five of these diets had to be discontinued because too many mice either died outright or (I suspect) were ordered to be euthanased on the authority of the supervising veterinary surgeon due to concerns about animal welfare. I'm assuming Oz has a Home Office much as the UK does which requires Named Veterinary Surgeons to be employed to supervise animal welfare in all laboratories.

We know this from the legend to supplementary table S1. Here is the last section of that legend (sorry that the small letter superscripting is lost, that's blogger for you):

"a Diets 2 low energy and 6 medium energy were discontinued within 23 weeks. b Diets 3 low energy, 3 medium energy and 6 low energy were discontinued within 10 weeks of treatment. These diets were discontinued due to weight loss (≥ 20%), rectal prolapse or failure to thrive".

Here is table S1 with the discontinued (and removed) groups outlined in red:

So, they started with 30 diets groups but five of them had such high early death rates that they were excluded from the study. This left 25 groups. Other than the legend to supplementary table S1 I am unable to find any reference to the loss of five diet groups anywhere in the main paper, which gives the distinct impression that 25 groups were all that were included from the start. So 17% of the mice died at under 23 weeks in to the study, many of those within less than 10 weeks, and you have to read the supplementary data to find out.

All of the high mortality groups were eating 5% of calories as protein.

Did you pick that up in the abstract? No, you didn't.

Is there any excuse for failing to discuss this crucial finding in the results and discussion sections of the paper? You can decide that. It's not exactly rocket science.

My feeling is that the authors could argue, if they were convinced that protein restriction was key to longevity (amino acids, cysteine, mTOR etc don'tchano), that studying early life mortality has no relevance to late life longevity. Why not leave early mortality to the paediatricians? That is a potentially arguable position and should, as it involved a huge chunk of the study mice, have been reported and been justified (if possible) in the results, discussion and especially in the abstract.

The other slight hiccup is this line from the main paper:

"Median lifespan was greatest for animals whose intakes were low in protein and high in carbohydrate... (Figure 2A)"

Figure 2A is not in a format which lends itself to simple interpretation and, obviously, excludes all of the mice which died or were euthanased at less than 23 weeks of age, all of which were in low protein groups. Anyway, you might want to see a simple table of median lifespan for each of the surviving groups. Like supplementary table S2. I've high-lighted the group which had the longest median lifespan in red:

Looks to me like the longest median lifespan group might have been eating 42% of it's calories as protein... Hmmmm. Worth repeating:

"Median lifespan was greatest for animals whose intakes were low in protein and high in carbohydrate... (Figure 2A)".

vs Table S2 giving 42% protein for longest median lifespan.


Let's make this crystal clear: The data demonstrating the actual outcomes are, absolutely, present in the supplementary data of the paper. It is also absolutely crystal clear that the paper itself, excluding supplementary information, does not accurately represent the the actual findings in the study.

If you had to summarise the paper in human terms you could say that applying severe protein restriction to your kids while topping up their calories with sugar and soyabean oil would hopefully result in them being taken away from you and placed in to care before they died.

Please don't try this at home.


My thanks to Rory Robertson for his attempts to have this paper retracted and more accurately rewritten and to Grant Schofield for tweeting about his efforts.

Saturday, January 25, 2020

Coronary Artery Calcium Score and Scleroderma

Dr Malcolm Kendrik has a very interesting post over on his blog relating to coronary artery calcium scoring. I think it is fair to say that he is not in favour of the test.

My ears pricked up (metaphorically) when he mentioned myositis ossificans, about which he comments "This does not end well".

I have spent some time in the past thinking about pathological arterial calcification, as applied to the aorta of of patients with familial hypercholesterolaemia. Bear in mind that the dietary advice for patients with FH is about the worst you could possibly imagine and, of course, has no evidence base. My thoughts and assorted links are in an old blog post here. At the time I had never heard of Sci-hub so was unable to access this rather neat diagram of the mechanism of action of insulin, Pi and pyrophosphate:

Back to pathological soft tissue calcification. Clearly the obvious question about myositis ossificans has to be to ask whether it is in part driven by hyperinsulinaemia/hyperglycaemia or both.

As far as I am aware this is not a question which had been asked. It is simply genetic and that's it.

However, a similar question has already been answered in relationship to a serious generalised soft tissue mineralisation condition described as "calcinosis and scleroderma", back in a publication from 1932 (apologies to the person who tweeted the link, I didn't note their name to acknowledge. And twitter is ephemeral). That is too long ago to be listed on Pubmed so if you would like to read it you can go and ask Elsevier how much they would like to charge you for a peek in to the past or you can go to that awful place that none of use ever use to download any paper for free.


"Calcinosis and scleroderm" looks to be one of a family of soft tissue calcification diseases. The case report from 1932 describes the complete remission of this extremely unpleasant condition in a child following a period of time on ketogenic diet of the type used at the start of the last century, before dieticians were invented/summoned from Hades.

Did the ketogenic diet resolve this child's pathological calcification by suppressing insulin levels, glucose levels or both? Does it work by lowering alkaline phosphatase production by cells in/around inflammatory lesions? Or by some other mechanism?

Would it do the same for pathological arterial calcification? Given a tool like the ketogenic diet, perhaps there is some logic to CAC testing?

Unless you feel that tissue calcification is an appropriate part of healing until it gets to scleroderma levels...


Tuesday, January 21, 2020

Barja, an aside

I quite enjoyed Barja's review

The Cell Ageing Regulatory System (CARS)

but found this section a little uncomfortable:

Hmmmmmm. Plant based, healthy fruit and vegetables, bad animal fats. Not my sort of outlook really.

In another of his publications here

Highly resistant macromolecular components and low rate of
generation of endogenous damage: Two key traits of longevity

there is this comment

"It was also found that 6–7 weeks of dietary restriction are enough to decrease MitROS production and 8-oxodG in mtDNA and nDNA in rat liver (Gredilla et al., 2001a )".

Gredialla et al (incl Barja) 2001a is

Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source

Here they found, by eyeball, an approximately 50% reduction of in 8-oxodG in mitochondrial DNA after those six weeks of quite severe caloric restriction:

Now let's compare this with the degree of damage reduction (this time using the term 8-OHdG as the marker rather than oxo-8dG, which appears to be the same thing).

Here's the change in mtDNA damage marker in brain mitochondria using F3666, one of the worst ketogenic diets around:

Just by eyeball I make the drop in mtDNA damage out to be greater than 50% by two days and something like 75% by three weeks. On ad-lib food consumption. No hunger.

Considering that F3666 does not extend longevity in mice (it doesn't shorten lifespan either, despite causing liver damage and it does actually improve health during ageing in rodents) this does, for me, slightly knock some of Barjas core ideas.

Sad but true.


Wednesday, January 15, 2020

Stearic acid again

Better post this one while I have a few minutes. I picked it up while looking for refs for Gustavo Barja's epic The Cell Ageing Regulatory System (CARS) in which longevity is tied to the Double Bond Index of the mitochondrial inner membrane (Thanks Bob!). BTW it is possible to modify the DBI but, with current data, it looks like you cannot alter the saturated or MUFA percentages, it is replacing omega 3s with omega 6s which mimics the mitochondria of long lived mammals!

Anyway, here is the cocoa butter paper:

Differential effects of saturated versus unsaturated dietary fatty acids on weight gain and myocellular lipid profiles in mice

Here are the diet compositions:

The line in red is the total percent of calories from linoleic acid in each diet. Here are the body weight changes:

The bottom two lines are the low fat high carbohydrate diet which happens to come in at just 1% linoleic acid and the cocoa butter diet which comes in at 1.4% of calories as linoleic acid. The high palmitic acid gives the most weight gain as it delivers 4.5% of calories as PUFA. Olive oil is a close second, also with 4.5% linoleic acid. The oddity is the safflower oil diet which is very high in PUFA but only gives intermediate obesity. Quite what is going on here is difficult to say but you have to wonder at what level of omega 6 PUFA that "next level up" signalling (lipid peroxide based) kicks in. No data on that, just a guess/excuse from the Protons perspective. There are a number of other studies showing this phenomenon of limited weight gain with safflower oil.

Still, stearic acid as cocoa butter is still looking pretty good. All of the high fat diets were based around different fat sources placed in to the D1245 background so are equally high in sucrose and starch too, comparable amounts across all of the higher fat diets.


Thursday, January 02, 2020

Protons (53) a formula

A couple of things came up in emails recently. First is that I never mention that I had a chat with Ally Houston on the Paleocanteen podcast. It was fun. I think I sound like me. It's here

Second is that karl asked if there was a general formula for working out the F:N ratio for assorted fatty acids.

Edit: cavenewt pointed out that for people unfamiliar with the FADH2:NADH ratio concept there is a reasonable introduction at Protons: FADH2:NADH ratios and MUFA. PubMed-ing Dave Speijer and CoQ makes good reading too. End edit.

There wasn't but given a few minutes and some algebra it works out like this for even-numbered, fully saturated fatty acids of carbon skeleton length n:

F/N   =   (n-1)/(2n-1)

So stearate (C18) is 0.486

Palmitate (C16) is 0.484

Caprylate (C8) is 0.467

For MUFA/PUFA you just subtract one FADH2 per double bond (db). This doesn't affect the NADH term.

F/N  =  (n-1-db)/(2n-1)

Oleate (db = 1) is 0.457

Oleate is the MUFA of stearate. Saturated fats allow us to resist insulin, MUFA allow insulin to act.

Linoleic acid, also C18 but with two double bonds, gives 0.429

This is lower than stearate or oleate. The switch for ROS generation occurs between roughly 0.486 (high physiological ROS) and 0.457 (low physiological ROS). LA is lower than oleic acid.

Glucose has an F/N ratio, from memory, of 0.2 so LA is the "glucose-like" of the common fatty acids, in Mike Eades' terminology, and so will fail to generate fatty acid appropriate ROS. Which will allow continued insulin action when it should be resisted. That will make you fat, and the loss of calories in to adipocytes will make you hungry. The exact opposite of stearic acid...

Happy New Year all.