Blood "energy" content

"One-liner" post. This is exciting (picked up on twitter from Mike Eades):

Effects of dietary carbohydrate content on circulating metabolic fuel availability in the postprandial state

It's Ludwig's group. I've skim read but not looked at the detail. I like what I see.

Over the last year or so I've ventured in to the morass of older papers about the CNS response to infusion of various metabolic substrates where you get bogged down in the various neural groups which respond in various ways to high vs low glucose etc. It's messy and it's rare for people to have asked the questions in quite the way I might have phrased them.

Eventually I simply started adding up the energy content of "blood" in various states, especially under extended fasting when hunger becomes blunted. Being me I tended to add them up in terms of how much NADH and FADH2 might be available. I kept getting pushed towards the idea that hunger might be a simple matter of the energy content of the blood supplying the hypothalamus. Clearly that is one core thing that the CNS monitors (using ROS of course).

Could hunger be this simple?

Okay, there is also clearly a neural input (think hepatic FFA infusion via the portal vein suppressing food intake) but ultimately if the brain is being perfused with too few calories, it is going to do anything it can to make you eat. The classic is reactive hypoglycaemia or insulin induced hunger where I suspect the problem is (in myself in pre low carb days) not absolute hypoglycaemia (I could get this at BG around 4.5mmol/l) but the accompanying low FFA availability giving low brain stem energy availability. But of course measuring FFAs is not as simple as measuring glucose...

Anyway, it's fantastic to see some serious researches looking at the concept of blood energy content. They will have to add the Protons concept eventually, to explain why things happen as they do, but they're on an exciting trajectory.

Peter

Fancy some serology? (3) In Japan

I notice that the COVID-19 state of emergency has been lifted in the last remaining areas of Japan as of last Monday.

I think they lost about 800 people in the pandemic. The seroprevalence in Tokyo is at least 6% in the populace attending a community clinic or two and at least 10% in healthcare workers. Exposure has widespread.

All countries have had their individual approaches to managing the pandemic, some sensible, others less so. What worked and what didn't will probably be lost in the avalanche of lies used to cover the arses of incompetent politicians, certainly here in the UK.

I found this ancient (2014) snippet by accident somewhere on t'internet:

"But one country has managed to keep obesity down with the help of a controversial government policy that probably wouldn't fly in the U.S. That country is Japan, where only about 3.5% of the population is classified as obese, compared to rates as high as 30% or greater in countries like the U.S. And it's not just a generally healthier diet and lifestyle that's kept the Japanese trim.

Citizens must adhere to government-mandated waistline limits or face consequences. The government has established waistline limits for adults ages 40 to 74. Men must maintain a waistline at or below 33.5 inches; for women, the limit is 35.4 inches. The "metabo law" went into effect in 2008, with the goal of reducing the country's overweight population by 25% by 2015. The government's anti-obesity campaign aims to keep "metabolic syndrome" — a number of factors that heighten the risk of developing diabetes and vascular diseases, such as obesity and high blood pressure, glucose and cholesterol levels — in check, thus minimizing the ballooning health care costs of Japan's massive ageing population.

Those who stray beyond the state-mandated waistlines are required to attend counseling and support sessions. Local governments and companies that don't meet specific targets are fined, sometimes quite heavily".


From Snopes (FWIW) it seems this is basically true, assuming the numbers for waistlines are real:

"Japan requires citizens between the ages of 45 and 74 to have their waistlines measured once a year and potentially seek medical attention.

Unlike individuals, however, companies and local governments can be assessed financial penalties if the citizens in their charge do not meet government standards".

I guess that having a national policy to limit metabolic syndrome might or might not have any influence of the course of a pandemic which targets people with metabolic syndrome.

We'll never know...

While the obvious initial advice for mitigating infection with SARS-CoV-2 was to try not to be elderly and to try not to be diabetic it now looks like simply trying not to be diabetic might have been all that mattered.

Peter

Fancy some serology? (2)

I thought I would just take a break from trying to simplify the Protons electron transport chain as regards ultra low fat diets and talk about sensitivity and specificity of serology tests for a break.

People may have noticed I'm quite keen on serology and am rather less than enthusiastic about PCR for test, track and trace in a situation where the SARS-CoV-2 virus is present throughout the country, as it is here in the UK. Stupid is as stupid does.

However, serology is not quite as straight forward as I might like either.

There are a number of serology tests coming on to the market, and many have a 100% sensitivity and 98% specificity. It difficult to express how phenomenally accurate these test are. If I submit a blood sample for some routine analysis I accept that 95% for these sorts of accuracy assessments is pretty good, we're dealing with biological systems, there is room for grey zones.

So presently serology has a 100% sensitivity. That means it will always pick up seropositive people. If you have antibodies, this test will find them. Always. Getting a negative needs some thought.

This is addressed by specificity. A 98% specificity means that a negative on the test will be correct 98 times out of 100. If the test says you don't have antibodies, it is also most likely correct, a one in fifty error rate there.

It is difficult to over emphasise quite how good these values for sensitivity and specificity are for a lab test. They are very, very good.

At detecting antibodies.

If antibodies are found in a healthy person it is, with a test this good, pretty well certain that they have been exposed to the disease and, in the absence of illness, that they are immune. Or at least they were at the time of exposure.

Sadly human immune systems can be recalcitrant in cooperating with serology.

The Royal College of Pathologists short presentation on serology is now up on Youtube

The COVID 19 pandemic: testing – serological diagnostics for COVID 19

and here is a screenshot from just after 18 minutes in:

The dotted red line is the lower limit of the serology assay used. All of the patients have had known, absolutely certain, clinical disease. If you use a serology test which is 100% sensitive and 98% specific, you will pick up everyone over the red dashed line. A negative result will be correct 98% of the time.  That is what a highly sensitive, highly specific test does. To put that in a more visually clear image here is another screenshot:

Again, below the red dotted line you will be classified as seronegative, you are seronegative. That does not mean that you have not been exposed. It doesn't matter how good your test is. The test cannot see below the red line, above the red line the test is phenomenal. This not a problem with the test, the test is not for exposure/recovery from the disease. It is just for antibodies above a certain level. This is the limit of serology testing, it is undermined by the ability to recover from this infection without seroconverting. It happens, it's on the graphs. It's not the test's fault.

To a large extent recovery without seroconversion suggest that the innate immune system is at work (or you are simply unable to become infected) and that would fit nicely with the reports suggesting that hyperglycaemia over 10mmol/l is bad news and hyperglycaemia below 10mmol/l gives a slightly better outlook in severely ill patients. Hyperglycaemia is a good way of suppressing the innate immune system.

Passthecream put an interesting link in the comments of the last serology post which suggests the innate immune system is also adaptive, it remembers, no antibodies needed...

Adaptation in the Innate Immune System and Heterologous Innate Immunity

Sadly, at our rudimentary level of understanding of the immune system, we are in no position to assess whether a given person might be seronegative but still immune.

Having said all of this, it's worth remembering that being seropositive without having being ill suggests you are immune. It's interesting to see the WHO position on this. The WHO currently suggests that there is no evidence that having antibodies confers immunity.

That is interesting and absolutely, currently, technically correct. Thus far seropositive people have never been challenge-tested with virulent virus, so there can be no evidence that antibodies are protective (try getting that one through ethics committee review!). It is theoretically possible that a person could have been exposed to virulent virus, have never been ill, have developed antibodies, and yet is still be susceptible to the virus. You can imagine that this might be the case.

Well, actually, I can't.

So, if you are the head of an ITU in a UK district general hospital in London and you find you are one of those lucky people who are solidly IgG seropositive without ever having been ill, what would you do as regards PPE for yourself?

As the WHO says, there is no evidence that being seropositive is protective, as yet. But for antibodies produced in vivo, by someone who was never unwell, for these antibodies not to be protective would have to be a first of a kind as regards immunology (vaccine induced seropositivity is a whole different ball game).

I love this guy:

COVID-19: ICU care, long-term effects and immunity with Dr Richard Breeze

(Hat tip to Unknown for the link and no, Breeze didn't use any PPE while treating the large wave of COVID-19 patients which passed through Lewisham District General Hospital's upgraded ITU)

Knowledge over protocol. He also strikes me as the sort of person who might look at a patient on a ventilator who was developing barotrauma because "protocol" suggest "Xml/kg" as the "correct" tidal volume setting and who might reach over and reduce (gasp) the tidal volume setting. Just my guess. Or avoid intubated ventilation if at all possible (which was what they did).

You have to contrast this with the hospital managers who discharged SARS-CoV-2 positive patients in to unprotected nursing homes because "it's protocol".

I get the impression that good medics (and there are some excellent ones out there) don't seem to be the sort of people that become the politico-medics who guide the government...

Peter

Aside: I just can't get over Dr Breeze working without PPE. Sort of thing I might have done under the circumstances. I can't believe it was allowed nowadays!

Low fat vs low carb again (2)

For your enjoyment I have simplified this graph from

Hyperinsulinemia Drives Diet-Induced Obesity Independently of Brain Insulin Production

out of Jim Johnson's lab:

down to this graph (at great effort) to show only the mice on standard chow:

These are the insulin responses to an IP glucose tolerance test at a year of age in mice which have been fed on good quality chow all of their lives. The mice in the top curve are phenotypically normal in their insulin response to glucose, the mice in the lower curve have had three out of four of their insulin genes knocked out. They weigh the same.

We all know from the Surwit posts that the normal insulin exposure mice have an 11% decrease in median lifespan compared to low insulin exposed mice, Jim Johnson's lab again.

None of us is in a position to have our insulin genes partially silenced from before birth, but we do have a choice as to how much insulin we expose ourselves to, based on our dietary choices.

What we need to know is what the insulin response to a given meal might be if we were to try to imitate the partial insulin gene knockout mice. Very few studies have provided this sort of information but the current pre print from Hall et al does just this. Here is the graph

The red curve is from a group of people fed a single meal of a mildly ketogenic diet. With insulin peaking between 20 and 30micoU/ml this is quite similar to the value in mice with reduced insulin gene load, those pan out at around the 22microU/ml mark (don't you wish everyone just used picomoles all the time? Well, I do). Or you can eat low fat, plant based and choose to expose yourself to over 100microU/ml of insulin. Doing this you might still lose a little weight (another post, eventually), you might lose a little fat but you also might lose a few years of lifespan as the insulin drives ageing with its associated chronic diseases.

How many years? If the median lifespan for humans is around 70-80 years and we are talking about an 11% reduction that gives us a ballpark of just under a decade lost. As a thought experiment.

PBLF, plant based low fat. ABLC, animal based low carbohydrate.

There is no choice.

Peter

Total aside: I really hope that Hall keeps the title of this paper unchanged in the version which eventually gets published. It's a single sentence of prose which encapsulates what is wrong with nutrition research. It is absolutely, totally factually accurate, while being completely selective in its choice of factual content to give an absolutely misleading impression. As a declaration of bias it is unbeatable. I love it.

Low fat vs low carb again

I guess everyone knows about this pre-print

A plant-based, low-fat diet decreases ad libitum energy intake compared to an animal-based, ketogenic diet: An inpatient randomized controlled trial

There is a wealth of data to enjoy and a lot to say from the Protons and insulin point of view but just a brief look gives us equal weight loss, equal fat loss and the sort of changes in fat free mass you would expect from likely shifts in glycogen and its associated water:



Clearly the extra 600kcal eaten under ketogenic conditions did nothing to blunt fat loss, much as we would expect from the low carb perspective. The extra calories did not evaporate, they were lost through increased energy expenditure, especially during sleep and while sedentary:

These people have uncoupled metabolism during the period of eating the ketogenic diet, they generate heat. As measured within the limits of indirect calorimetry. You could argue about a greater faecal, urinary or breath mediated loss of calories too but that's less important than a measured equivalent weight loss despite higher, extremely accurately measured caloric intake.

That's all pretty boring.

What is really, really interesting is the equivalent spontaneous weight loss under the period of high carbohydrate intake. Over the years I've looked at the carbosis vs ketosis for potential mechanisms and this study may go some way to clarifying what is going on. The very low fat eating certainly does not limit the penetration of either glucose nor insulin past the liver. Both spike systemically after every meal. But still there is spontaneous weight loss due to a suppressed appetite.

Under low fat eating less "waste" heat is generated, metabolism is coupled. Tightly coupled metabolism means people needed less calories. The subjects, under very low fat eating, lost weight without any biochemical markers of inadequate calories. Just as they did under ketogenic eating.

That's really interesting. With data, lots of it including important things like the effect of a typical meal on blood glucose, insulin and lactate. Plenty to work with. Needs a lot of thinking about.

Peter

BTW does this sound like a metabolic advantage to ketogenic eating? Rhetorical question, 24h energy expenditure combined with utterly accurate food intake measurements tells us something...

Fancy some serology?

Just a one liner-ish type post.

I had the privilege of listening-in to one of the weekly Royal College of Pathologist webinars on the SARS-CoV-2 virus, this one on serology testing. These webinars are really fantastic, they are given as a 15 minute presentation by a scientist at the top of their field, in the complete absence of political interference or the sort of financial pressures applied to produce a 100% specific, 98% sensitive serology test to make billions of dollars for a commercial company. They have spent their careers as coronavirus "enthusiasts". The presentations are by pathologists, for pathologists. They are technical and utterly honest (as far as I can tell).

So. There are three types of people in the world. If you have had SARS-CoV-2, confirmed by PCR, have been seriously unwell, hospitalised, needed supplementary oxygen and been considered for a respirator/ITU admission then the chances are good that you will be solidly seropositive for SARS-CoV-2 on a blood sample in recovery. I would suggest that your medical history might be quite a big hint in this direction, which might render the use of the serology test under these circumstances somewhat superfluous.

The second type of person has also had SARS-CoV-2, confirmed by PCR test, been clearly unwell but not so unwell as to need any hospital admission for management. With the best possible testing using multiple different antibodies and different test techniques these people are very, very difficult to detect on a serology basis. Many will be negative on serology within the limits of what we have available now and what will be developed commercially. That is worth thinking about.

The third type of person has never been ill, has never been PCR tested, has no idea whether they have been exposed to SARS-CoV-2 or not. These are the apparently healthy population, the sort of people John Ioannidis sampled in Santa Clara County.

Of the 3300 people Ioannidis tested, 2.5-4.2% turned out to be sero-positive. Listening to the RCPath webinar on the problems of serology testing in mildly unwell people (let alone those apparently never unwell) this implies that the values from Ioannidis might well be the absolute, rock bottom, tip of the iceberg minimum. Exposure has probably been much, much higher in this still healthy population.

I find that rather hopeful.

It is difficult to describe how badly I feel that the COVID-19 pandemic has been managed here in the UK. I don't make political posts on the blog (or anywhere else) but the level of utter incompetence of our current government is breathtaking. I suppose a different administration could have done worse, but that's hard to imagine.

Peter

Surwit diet and derivatives (3) 5LJ5 vs D12330: Chow vs Surwit

TLDR: A "healthy", complex carbohydrate, low glycaemic index diet appears to markedly shorten the median lifespan of mice when compared to a diet of maltodextrin/sucrose with hydrogenated coconut oil, irrespective of obesity or insulin gene dose.

This is the second excellent paper from Jim Johnson's lab:

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

It is slightly different from the 2012 paper as these mice are full knockout for the Ins1 gene and this time it is the Ins2 gene that is present as a full complement or at half knockout, to adjust the insulin gene dosage.

The study was never intended to compare the two diets, the diets were simply intended to provide a fairly normal insulin environment using a rodent chow against an high insulin environment generated by a Surwit type diet. It was the insulin exposure which was the focus of the study.

But, ultimately, the study did compare the two diets and in some detail.

Just to summarise the diets. Both had 4% of calories as PUFA, primarily linoleic acid. The 5LJ5 chow used a slow release carbohydrate (as uncooked ground wheat) combined with a little extra protein from soybean meal. The D12330 (Surwit type) diet was the usual hydrogenated coconut oil with maltodextrin/sucrose plus casein as the sole protein source.

Maximum individual longevities came out as expected, with the 5LJ5 coming out best and the low insulin gene dose conferring benefit to both diets.

These are the mean lifespans of the four longest lived mice in each group (top decile) as shown by the open circles/squares on the bar chart, taken from the end stage of the survival curves as shown here:

That's relatively unexciting and no one would be surprised by it.

What surprised me was the longevity advantage to the Surwit diet groups when assessed at median life span. Not only did the Surwit groups both do a great deal better than the chow groups at median lifespan but there was only a very small improvement (about 3%) obtained by reducing insulin exposure. In fact the normal gene-dose, obese, high insulin-exposure Surwit diet group (purple) had a longer median lifespan compared to the reduced insulin-exposure group that was on chow. Which was better again than that of the ordinary mice fed on chow.

If we simply ignore the reduced insulin exposure groups we can also suggest, based on these data, that the unmodified Surwit diet produces a median longevity gain in the order of 16% over a top-of-the-range excellently formulated lab animal breeding chow.

If the Surwit diet was a drug it would knock spots off of metformin, rapamycin, ethanol, caffeine or glucosamine for median lifespan extension. These mostly gain around 10% in median life span extension.

I accept that, for the four mice which made it in to extreme old age, there is a small disadvantage to the Surwit diet, but this only becomes apparent at those lifespans at over 750 days of age, out of a max of just over 900 days.

A quick look round the literature shows us that feeding a 60% fat diet where the PUFA content comes out at around 15% of total calories (high PUFA lard as the fat source), combined with Surwit-like levels of maltodextrin/sucrose, is a catastrophe. As in this one using TD.06414.

At the risk of speculating; there may be a host of problems triggered by a wheat/soybean based diet which do not appear to occur with a casein/saturated fat based diet, certainly until extreme old age is achieved. Or there could be some specific advantage to a highly saturated fat based diet which over rides the problems provided by maltodextrin/sucrose. Lots of possibilities, no obvious answers!

Fascinating study.

Peter

Surwit diet and derivatives (2) It's the insulin

I've been spending some time re-reading a couple of papers out of Jim Johnson's lab and I'll start with this one because it has a core message which is absolutely crucial and possibly under appreciated. Sorry if the text is a bit repetitive but the idea is not completely intuitive.

Hyperinsulinemia Drives Diet-Induced Obesity Independently of Brain Insulin Production

The mice in this study were full knockouts for the Ins2 gene and also had either no knockout or half knockout of the Ins1 gene. Reduced insulin gene dose reduces insulin secretion which completely protected them from the obesogenic effect of the Surwit (D12330) diet. You cannot become hyperinsulinaemic in response to Surwit's maltodextrin/sucrose if you have only one out of 4 insulin genes functioning (with two out of four you can). Lack of hyperinsulinaemia normalises fat storage as one effect. Lack of hyperinsulinaemia also eliminates long term insulin-induced insulin resistance as a second, non related effect. Both effects are independently the direct result of reduced insulin exposure. The mice stay slim because they are eu-insulinaemic on a Surwit diet. The mice stay insulin sensitive because they are eu-insulinaemic on a Surwit diet. One cause, two responses. Shared causality tends to give correlated effects. But we all know about correleations and causality...

Despite being insulin sensitive the low Ins2 mice do not become obese because their knockouts stop them making enough insulin to achieve this. They are insulin sensitive but they are genetically unable use their insulin sensitivity. They are beautiful, to me at least. In an abstract sense.

Okay, have some graphs:

Top line in pink, obesogenic diet with normalish insulin phenotype, they get fat. Red line is the obesogenic diet with blunted insulin secretion. They don't get fat.

And insulin resistance: just consider the 52 week values here, these mice are a bit hit and miss re glucose/insulin function very early in life. By a year they show their true phenotype.

Just to reiterate: On the obesogenic diet fasting insulin is high because there has been a year of exposure to high insulin from the maltodextrin/sucrose of the Surwit diet when combined with a fairly normal pancreas, pink circles. The red triangles are the same diet but with blunted insulin exposure due to their Ins1 partial gene knockout. Ergo, low insulin exposure is causative of low insulin resistance at a year of age, even on the Surwit diet.

So. Insulin sensitivity, a surrogate for low insulin exposure, is a Good Thing. Using that insulin sensitivity by increasing insulin exposure will make you fat and insulin resistant as two separate effects from the same change.

I'll take a brief pause here for that to sink in before looking at the next paper from Jim Johnson's lab which translates these findings in to longevity studies. Which are really weird.

Peter

Surwit diet and derivatives

As a counterbalance to the papers documenting the obesogenic effect of PUFA containing diets I'd like to have a brief aside about the Surwit diet. There's a nice list of the ingredients here.

The first thing to say is that it is based around hydrogenated coconut oil. I would guess this is fully hydrogenated coconut oil, though the manufacturers do not specify. To supply a small amount of PUFA some soybean oil is added to give just under 7% of fat as PUFA which, at 60% of calories as fat, translates to 4% of total calories as PUFA. That is low yet the diet is one of the most obesogenic on the market.

There is more to obesity than PUFA (gasp).

The whole drive of the Surwit diet appears to be to supply glucose in a form which will be rapidly absorbed and so allow penetration beyond the liver and in to the systemic circulation. Maltodextrin is used for this. The diet uses a medium chain length polymer of glucose which has a glycaemic index in the same ball park as pure glucose.

Systemic hyperglycaemia is combined with sucrose at 17% of calories to induce pathological insulin resistance and a little extra hyperglycaemia. This is combined with the normal physiological insulin resistance associated with oxidising saturated fats. The need for extremely high levels of insulin to maintain normoglycaemia appears to be adequate to force lipid storage in the face of adipocyte insulin resistance. It certainly works, even with minimal PUFA.

Then I got side tracked in to Surwit's early work. A great paper is this one using F1850 for a publication in 1988:

Diet-Induced Type II Diabetes in C57BL/6J Mice

F1850 is still interesting despite having a bit more linoleic acid (around 7% of calories). This still feels like quite a low dose for a mouse diet. The paper has some great graphs. Let's have a look and ask some questions.

Take two mouse strains (Bl/6J and A/J mice), feed both on sucrose, maltodextrin and modest linoleic acid lard supplying about 7% of calories as PUFA. Both strains get fat, like this:

The bottom two lines are the two strains of mice on standard crapinabag and we can ignore these. Today's quiz is to identify the top two lines based on the fasting blood glucose/insulin data.

Here are the fasting insulin and glucose levels after 24 weeks of exposure to either control or obesogenic diet:

Grey columns represents the crapinabag fed mice, black columns the obesogenic F1850 diet fed mice. It is quite clear that while the A/J mice became modestly insulin resistant it was the Bl/6J mice which became severely insulin resistant, with marked increases in fasting insulin and glucose. On the same diet as the A/J mice.

Now, match the mouse strain to the upper traces on the weight gain graphs. We can ignore the crapinabag fed lower lines. Of the two upper lines one represents a set of mice which is fatter than the other. Are B/J or Bl/6J fattest? From the insulin data...

If you carry the idea in your head that insulin causes obesity you might expect the hyperinsulinaemic Bl6/J mice with a fasting insulin of 150microU/ml to be fattest. If you consider that the mice on sugar become fat because they find the sugary food more "rewarding" (it's the same food, idiot!) you might come to the idea that obesity is caused by overeating and that obesity secondarily causes insulin resistance (no sniggering at the back there). And both are incorrect.

The fattest mice are the A/J mice. They are fattest because they are the most insulin sensitive.

Insulin signalling, not insulin per se, causes fat storage.

The Bl/6 mice, on the same diet as the B/J mice, are more insulin resistant so resist insulin induced weight gain. Significant weight gain still happens because gross hyperinsulinaemia can overcome insulin resistance. But the greater weight gain for the A/J mice is much easier by using less insulin combined with lower insulin resistance.

The A/J mice become "fat but fit" based on glucose/insulin levels. Given long enough they will, undoubtedly, join the Bl/6J mice but they have a longer way to go to become adequately obese to resist insulin. But I have no doubt this will happen on F1850.

I think the difference between the strains is genetic and that Bl/6 based mice generate enough ROS to generate significant insulin resistance at much lower levels of insulin signalling than B/J mice do. If I had to guess at a gene I would go for their truncated supercomplex assembly protein causing failure of supercomplex formation and so allowing electron leakage to molecular oxygen from the ETC at times when no such thing should happen, at least not until the CoQ couple is a great deal more reduced.

But that's just speculation, which is fun.

Humans look more like B/J mice (normal ETC) than Bl/6J mice to me.

Bottom line: Insulin signalling is what makes you fat. Insulin resistance resists this. It's all in the data.

Peter

Addendum. Insulin sensitivity is a Good Thing. But it makes you fat, which is a Bad Thing. Let's rephrase that: Insulin sensitivity is a Good Thing provided you don't use it! Perhaps that concept is worth a post in it's own right.

The miracle of safflower oil (4) Soybean oil is just as good

I picked this paper up from George Henderson via twitter

Effects of dietary fat on gut microbiota and faecal metabolites, and their relationship with cardiometabolic risk factors: a 6-month randomised controlled-feeding trial

which is part of the same study as this one

Effects of Macronutrient Distribution on Weight and Related Cardiometabolic Profile in Healthy Non-Obese Chinese: A 6-month, Randomized Controlled-Feeding Trial

The study is amazing. The researchers provided all of the food to all of the participants for six months. The only fat source was soybean oil. It could almost be a rodent study. This is what the study says:

"The three diets were isocaloric, the primary distinguishing feature being their fat and carbohydrate content (Table 1). By replacing a proportion of energy derived from carbohydrates (white rice and wheat flour, the most consumed carbohydrate sources in China contributing to 70% and 17% total carbohydrate respectively) with fats (soybean oil, the most consumed edible oil in China rich in unsaturated fatty acids)..."

Just a few things to point out: The diets were isocaloric. No one was allowed to select their calorie intake. Blokes got just over 2000kcal/day, women got 1700kcal/day. Some supplementary fruit was allowed, to be recorded as and when eaten.

Here are the baseline diets for each of the intervention groups:

Here is what the intervention diets looked like:

Particularly note the exact match of calories normally eaten at baseline compared with that supplied by the study intervention, which was utterly accurately measured out in the study kitchen.

What happened to the weights during the intervention? This did:

The yellow line is 24% PUFA, red is 18% PUFA and blue is 11% PUFA, all by energy intake.

The first thing to say is that all participants lost weight. I guess that has something deep to say about the accuracy of three day dietary records!

However the weight loss was far from uniform and was clearly inversely related to the level of PUFA in the diet. Less PUFA, greater weight loss, 2kg in eight weeks for the lowest PUFA group. This cannot be explained by reduced food intake because all food calories were provided, in exact amounts, by the study kitchens. Diets were isocaloric...

Protons would suggest that the higher the PUFA content the more dietary fat was "lost" in to adipocytes then not subsequently released. From the cico-tard point of view just over a kilo of fat was "not released" from adipocytes over eight weeks of weight loss in the high PUFA vs the low PUFA group. That's 1000g over 56 days or just under 20g/day. This would have to be countered by a decrease in metabolic rate, in NEAT or specific activity. Or more cheating, recorded or not recorded. The study authors assure us there was no cheating.

After eight to 12 weeks all subjects started to regain weight. The regain slope suggests about a kilo per year on a rigidly fixed calorie, weight reducing food intake.

These people were 23 years old and weighed 60kg. If they stuck with this diet over 30 years they might end up at 90kg. Of course this wouldn't happen if they really stuck with the diet. As weight gain tried to continued on a fixed calorie intake, hunger would increase.

You cannot argue with hunger for 30 years. The subjects would break the diet and eat more. This manifest lack of "willpower" or gross "gluttony" would take the blame for the increased weight gain, which would be blamed on the increased calorie intake.

But the weight regain was already there on a rigidly fixed calorie intake. A calorie intake which gave eight weeks of initial weight loss. This still gave a progressive weight gain with no declared increase in caloric intake.

The beauty of PUFA and the Protons concept.

Peter

NB It is difficult to emphasise how good this study is. Just ignore anything the authors have to say.

The miracle of safflower oil (3)

TLDR: PUFA in a mixed diet are obesogenic. PUFA under hypoinsulinaemic conditions are not. I doubt they get a free pass long term.



There have been some interesting snippets on Twitter recently, triggered by Diet Doctor's discussion about vegetable oils here. Perhaps the most controversial quote is this one:

"Disclaimer: Vegetable oils are routinely recommended as “heart healthy.” There is high-quality evidence demonstrating that replacing saturated fat with vegetable oils reduces LDL cholesterol levels. But at this point, there is inconsistent evidence whether this translates into fewer heart events or lower rates of cardiovascular mortality".

This is absolutely incorrect for people with pre existing cardiovascular disease as it was found, in a randomised control trial using safflower oil, that increasing vegetable oil for bulk calories will increase all cause mortality (p = 0.05), cardiovascular disease mortality (p = 0.04) and coronary heart disease mortality (p = 0.04). Mortality is an utterly hard end point and particularly the all cause mortality is an end point which cannot be argued with.

Let's rephrase that: in the context of a mixed diet in people with established heart disease vegetable oil (from safflower seeds) is going to increase you risk of death, especially from cardiovascular disease.

The main issue is to ask whether this still applies under low carbohydrate eating conditions. Given the role of insulin in CVD this is far from certain. But context will be crucial here and who would like to be the guinea pig?

The interesting twitter conversation goes like this:

Dr Westman: "In 20 yrs of clinical research and practice using LCHF/keto, I’ve never even mentioned reducing omega 6s, and it works wonderfully. Just cutting carbs gets the job done!"

Tucker: "I disagree, but @drericwestman is an excellent physician who does great work. This is more about determining ultimate causation so we can address people who can't just go low-carb, which is most of the planet".

I think both people are correct. I came to LC because it works. Over decades I've read studies where it works pretty much invariably on a group basis and studies from the mainstream usually advise progressively increasing carbs if they want to knock low carb and secure future funding. You have to pay the mortgage.

I am perfectly willing to accept that consuming carbohydrate in a rapidly absorbable form will overwhelm the liver's ability to protect the systemic circulation from hyperglycaemia so will require systemic hyperinsulinaemia to control that systemic hyperglycaemia. In particular hyperinsulinaemia comes with its attendant problems (ie most of medicine) but obesity only occurs when hyperinsulaemia is marked enough to overcome insulin-induced insulin resistance. I have no doubt this can occur without PUFA but I think it is massively easier in the presence of PUFA, which delay normal insulin-induced insulin resistance in the immediate post prandial period.

The role of polyunsaturated fatty acids is to stop adipocytes developing insulin resistance by limiting ROS generation. Combining hyperinsulinaemia with hypersensitive somatic cells is a recipe for maximising lipid storage in adipocytes and simultaneous packing lipid in to muscles, pancreas and anywhere else you care to imagine that sprouts an insulin receptor (most brain cells excepted).

Eating a low carbohydrate diet side-steps the problem by reducing absolute levels of systemic insulin. Down a set of unrelated rabbit holes I'm looking at what might control hunger under LC eating and PUFA may have some influence on this, but it is clearly a small effect when compared to the same dose of PUFA combined with an insulogenic diet.

Ultimately at low levels of insulin it doesn't matter how well or badly adipocytes respond to/resist insulin. There is so little insulin about that FFAs and ketones are able supply the body's energy needs, given some excess fat (especially visceral fat) available to be utilised.

Back to long term speculation: Do PUFA matter for non-insulin reasons on a low carb diet? Recall that López-Domínguez et al used a low calorie semi-starvation model (which is a partial mimic of low carb eating) to look at longevity in rodents (post is here). It certainly matters under their study conditions but the effect is small enough that I doubt it would show in any way for someone at 40 years of age under a year or two's exposure to a high PUFA but low carbohydrate diet. For those of us in this for the long haul it's much easier not to be the test case and PUFA avoidance seems prudent to me.

And I am undoubtedly still a low carb eater.

Peter

The miracle of safflower oil (2)

Just a brief mention of this one:

Use of dietary linoleic acid for secondary prevention of coronary heart disease and death: evaluation of recovered data from the Sydney Diet Heart Study and updated meta-analysis

merely because they used the same miraculous safflower oil as featured in the last post. This was a secondary prevention trial and increased safflower oil derived linoleic acid to around 15% of calories, again with no attempt to remove the ubiquitous industrial trans fatty acids from the control diet.

Here is the all cause mortality over 5 years

It's interesting that the increased death rate kicked in almost immediately, ie there is a case to be made for direct toxicity rather than the rather abstract concept of accelerated ageing that I've speculated about previously.

Ramsden published these recovered data in 2013. I guess seven years might be a little too soon for it to have filtered down to the World Health Organisation or Public Health England.

Talk about blood on their hands.

Peter

The miracle of safflower oil

TLDR: Increasing insulin sensitivity makes you fat.

This study is a bit of a mess because there are no control groups. People either got the safflower intervention or the conjugated linoleic acid intervention, then they were crossed over:

Comparison of dietary conjugated linoleic acid with safflower oil on body composition in obese postmenopausal women with type 2 diabetes mellitus

I'm going to ignore all of the CLA/post CLA data and look at the subjects who got just safflower oil, a total of eight capsules per day, two with each meal plus two at bedtime, eight grams a day of the oil for the first 16 weeks of the study. The safflower oil was 78% linoleic acid, regularly checked by gas chromatography.

Looking at Table 3 there was no change in total fat mass (and subjects didn't gain any weight on the scales) by DEXA scan while there was a loss of 1.2kg of "truncal" adipose tissue. With a PUFA supplement. It appears that DEXA scanning cannot differentiate between visceral and subcutaneous fat in the trunk area. The authors can't quite claim that there was selective loss of visceral fat but I think it is very likely that this did happen.

Throw in a fall in fasting glucose and a downward trend in fasting insulin levels coupled with a rise in adiponectin, some muscle gain and well, that's pretty impressive. You can, absolutely, see why people might have the idea that PUFA could be very positive for metabolic health.


How might one view this from a Protons perspective, other than reaching for a bottle of safflower oil?

I think the first thing to consider is the (probable) loss of visceral fat. Visceral fat, in my opinion, is utterly harmless. It contains the most insulin sensitive adipocytes in the body. If you are chronically hyperinsulinaemic, especially overnight, your insulin may never drop low enough to release any significant lipid from your visceral fat. So visceral fat is a surrogate for nocturnal hyperinsulinaemia, which is what is actually bad for you.

We have values for 10h fasting insulin; at enrolment it was 19.9microU/ml and this dropped to 18.2microU/ml over the first 16 weeks of the study. I would not expect 19.9microU/ml to maintain visceral fat and 18.2microU/ml to melt it away. I think it is much more likely that the gross hyperinsulinaemia induced by the sort of evening meal recommended by the ADA for diabetic people might well have resolved faster with safflower oil supplementation than it did without safflower oil, ie the duration of the period of gross hyperinsulinaemia through the night was reduced. Fasting levels were unchanged but the time spent above this ought to have been reduced.

We just have to revisit the Spanish study to see why:














This graph is over eight hours, 10 hours would be similar. These are healthy volunteers, the hyperinsulinaemia would be worse in DMT2 patients eating a high carbohydrate meal. Black squares are butter, white triangles are a high PUFA seed oil. The higher the PUFA content of the meal, the faster insulin level drops. Adding PUFA a mixed meal should allow insulin to drop faster and sooner than saturated fats. This happens because PUFA fail to generate the ROS needed to maintain the physiological insulin resistance which ought to occur post prandially to limit calorie ingress in to cells, adipocytes included. This leaves glucose and fatty acids available to signal satiety to the brain. Also noted in the Spanish study was that PUFA induced more rapid clearance of chylomicrons and more rapid drop in FFAs compared to saturated fats. As I wondered at the time, where do the FFAs and chylomicrons go to?

They go in to adipocytes, because the adipocytes cannot say "no" if PUFA generate too little ROS.

So this drug (safflower oil) allows increased insulin sensitivity (reminiscent of the "glitazones") or, rather, it fails to generate the ROS needed to limit the over expansion of adipocytes, which shows as increased insulin sensitivity during peak insulin exposure. This increased insulin sensitivity puts calories in to adipocytes rapidly so reduces the need for sustained hyperinsulinaemia. All adipocytes gain fat, but the faster fall in insulin allows an increase in the time window where visceral fat can actually release at least some FFAs to the systemic circulation via the portal vein and liver. Visceral fat shrinks, non-visceral fat expands.

The "benefit" of reducing visceral fat in this way during fasting is paid for by increasing the non-visceral fat depots in the immediate post prandial period. The extra fat in non-visceral adipose tissue will come primarily from the diet and the lost fat from visceral adipocytes will be used to provide fasting calories. In this particular study, the amount gained by non-visceral adipocytes was roughly equal to that lost by visceral adipocytes, it's probably random chance that the numbers balanced. And DEXA seems a pretty crude technology to use to work in small numbers of grams of adipose tissue, just looking at the non-balancing cited changes in fat and lean tissue mass in the results.

These processes can continue until non-visceral fat mass eventually become high enough that the loss of FFAs due to adipocyte distension over rides the insulin sensitising effect of the safflower oil. At this point overall insulin exposure will increase and visceral fat will return, on top of a higher mass of non-visceral adipose tissue. It will take longer than 16 weeks.

If you are an obese diabetic taking part in a study like this you should see a prompt but transient improvement in insulin sensitivity. This enhanced sensitivity should allow more non-visceral fat gain until you convert to being a somewhat more obese diabetic. It nicely illustrates that extra PUFA convert you from being established "obese" to being "pre-more-obese". Time is all that is needed to convert you from being "pre-more-obese" to simply"more-obese".

But your lab numbers will improve transiently in the first part of the intervention.

Peter

Double Bond Index and longevity in humans

Preamble: I've had this post written for some time (there are a fair few in this category) but this tweet from the World Health Organisation has prompted me to hit the publish button. In particular this piece of advice begs the question of incompetence vs malicious intent (I doubt the latter):

Here's the post:

I thought I would revisit the idea of trans fatty acids because the late Fred Kummerow got an honourable mention on twitter recently. He is largely responsible for the removal of industrial trans fatty acids from the food chain. No one would argue that that was not a Good Thing.

Back in the 1970s a study was completed which applied a diet from which saturated fats were largely removed and linoleic acid, mostly from corn oil, was increased to about 13% of calories. In a "control" diet saturated fats were as unchanged as practical and linoleic acid limited to just under 5% of calories. That should be a pretty good test of the miraculous benefits of dietary PUFA for blood cholesterol lowering.

However the "control" diet just happened to be specifically increased in industrial trans fatty acids from commercial margarine (1960s style USA margarine), though no one knows by how much, ie there was no genuine "control" diet.

This is what the diets looked like

Cholesterol lowering diet:

"Liquid corn oil was used in place of the usual hospital cooking fats (including hydrogenated oils) and was also added to numerous food items (for example, salad dressings, filled beef (lean ground beef with added oil), filled milk, and filled cheeses). Soft corn oil polyunsaturated margarine was used in place of butter. This intervention produced a mean reduction in dietary saturated fat by about 50% (from 18.5% to 9.2% of calories) and increased linoleic acid intake by more than 280% (from about 3.4% to 13.2% of calories)".

"Control" diet:

"It was designed to appear similar to the experimental diet. Notably, free surplus USDA food commodities including common margarines and shortenings were key components of the control diet, making the daily per participant allocation from the state of Minnesota adequate to cover the full costs. As common margarines and shortenings of this period were rich sources of industrially produced trans fatty acids, the control diet contained substantial quantities of trans fat. Compared with the pre-randomization hospital diet, the control diet did not change saturated fat intake but did substantially increase linoleic acid intake (by about 38%, from 3.4% to 4.7% of calories)".

You have to wonder about the inclusion of trans fats in the control diet. Did Ancel Keys (co-principal non-author) realise, even as long ago as the mid 1960s, that trans fats were bad? A little stacking of the deck has never been been considered an issue when it might help support the lipid hypothesis.

The experiment, designed to confirm the benefits of PUFA, failed completely. There was zero benefit from cholesterol lowering using dietary linoleic acid. Keys never published the results, hence my use of the term "co-principal non-author" because non published study results cannot have any authors. Happily enough data were excavated by Ramsden et al 40 years later to be published in 2016 as

Re-evaluation of the traditional diet-heart hypothesis: analysis of recovered data from Minnesota Coronary Experiment (1968-73)

There was, overall, no effect on total mortality when comparing the two interventions. To rephrase that: Increasing dietary linoleic acid was no worse than increasing trans fats, overall.

OK. So we could stop there with nothing more insightful than an observation of the moral and scientific bankruptcy of the architects of the lipid hypothesis. Nothing new there.




But what we actually have here is a study comparing two diets, one with a marked increase in the double bond index (DBI) of the lipids vs one with a modest increase in DBI, if we ignore the problems of trans fats.

We have something resembling the CRON mouse study in which lard as the lipid source gave a greater longevity benefit compared to fish oil or soya oil. We can view the present study as an intervention which altered mitochondrial membrane lipid composition in a direction of enhanced ageing based on increased DBI of those membrane lipids. But this time in humans, and with no calorie restriction.

Rather than looking at specific diseases we can ask whether increasing the DBI of your mitochondrial lipids might simply make you biologically older than your chronological age. This should show in the all cause mortality data, irrespective of the cause of death (ignoring "One flew over the cuckoo's nest" scenarios, even though this was a mental hospital study). However this would be hard to isolate in younger people because they are far enough, chronologically, away from death that ageing them by 10 years (a totally fictitious value, merely used for illustrative purposes) wouldn't show up much in all cause mortality. Assuming the risk of death at 40 years of age is similar to that at 50 years of age, nothing will show.

But, if you are 65 years of age and eating 13% of your calories as corn oil derived polyunsaturated fats makes you behave biologically as if you are 75 years of age, this just might show as increased all cause mortality.

Ramsden provides us with these graphs. In people under 65 years of age nothing shows:

but in people over 65 years of age there is visibly increased all cause mortality in the corn oil subjects:

Because the raw data for these graphs could not be recovered it is impossible to perform any sort of statistical analysis but it looks to me like there might be some indication that basing your diet around corn oil PUFA might be worse than eating trans fats, late in life. Given the raw data I suspect it might be possible to calculate how much linoleic acid might shorten your lifespan and by implication I would expect it might also shorten your healthspan, which could actually be worse.

Trans fats come out unexpectedly well. You have to wonder how much more benefit removing linoleic acid might provide, especially if you are an elderly person trying to avoid ARDS in the ITU.

Peter

ARDS isoprostanes and isofurans

This study from 2012 uses a model and it's based on mice. No one, ever, develops ARDS by inhaling reagent grade lipopolysaccharide following an intra tracheal injection of said LPS. So some caution.

Low levels of tissue factor lead to alveolar hemorrhage, potentiating murine acute lung injury and oxidative stress

I was interested, not because of the tissue factor knockout, but in the control group because I wanted quantification of how much haemorrhage occurred in to the alveoli during the progression of ARDS. I was modestly interested in the concept that pulmonary haemorrhage might reduce systemic haemoglobin value, elevate bilirubin, elevate ferritin and induce oxidative stress due to free haemoglobin or its derivatives. It can do some of these things but what caught my eye was the section on inflammatory markers within the small "human" arm of the study.  This is what they found in real people with real ARDS:

"Patients with diffuse alveolar hemorrhage had progressively increasing BAL isoprostanes and isofurans as their sequential BAL aliquots become more bloody. These findings suggest that liberation of free hemoglobin into the airspace and intraalveolar lipid peroxidation may be important mechanisms of clinical acute lung injury".

I would just comment that while both isoprostanes and isofurans measured in the study are arachidonic acid non-enzymic derived peroxidation products I can see no reason why the toxic derivatives of linoleic acid peroxidation would not also be formed, though these weren't measured in the study.

This would fit well with the likelihood of who is most likely to develop ARDS being predicted by the proportion of polyunsaturated fatty acids in their plasma free fatty acid pool on admission to the ITU.

The role in ARDS of pulmonary haemorrhage, intra-alveolar red cell lysis, free haemoglobin and severe oxidative stress in the airspaces is fascinating and is likely to be a routine feature of all types of ARDS. I got here by trying to decide whether the widely discussed concept that SARS-CoV-2 might be doing something special based on viral protein modelling suggesting the displacement of the Fe atoms from haemoglobin could be causal of whole body damage via systemic hypoxia. Looking at the clinical data coming out of Wuhan and what we have known about ARDS for decades makes me doubtful that we need the "iron hypothesis". I'd not say that it is incorrect, just that an awful lot of the clinical data fit with a severe viral infection induced ARDS including pulmonary haemorrhage in patients loaded with polyunsaturated fatty acids.

Peter



I wrote this post some time ago and felt it wasn't really interesting enough to put up, but the iron/haemoglobin idea keeps resurfacing. Loss of Fe from Hb will undoubtedly impair oxygen delivery but it would not cause arterial oxygen desaturation without concomitant lung dysfunction. With normal lung function whatever undamaged haemoglobin remains would saturate perfectly well. A pulse oximeter would still render a reading of 100% saturated even if little functional haemoglobin remained and tissue oxygen delivery was very low, assuming the colour of the haemoglobin derivatives did not interfere with pulse oximeter function. This is the situation in "normal" profound anaemia. In one of the Wuhan reports clinical anaemia on presentation was undoubtedly associated with decreased survival (though this was not related to mean haemoglobin levels) but equally so were decreased platelet count and decreased albumin levels. These would be compatible with serious problems from multiple tissue haemorrhage, lungs included, as part of the advanced stages of SIRS (systemic inflammatory response syndrome) where DIC (disseminated intravascular coagulation, better known as Death Is Coming) becomes one of the terminal features.

The discussion of mechanical ventilation techniques in causing/avoiding pressure injury to the lungs is almost as old as the eternal discussion of colloid vs crystalloid for volume resuscitation. Personally I prefer crystalloids and would favour IPPV techniques which avoid barotrauma to the lungs if at all possible. But that's just me.

Look after your lysosomes: Hydroxychloroquine

This fascinating:

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

Forty two patients were enrolled. Sixteen were not given or refused hydroxychloroquine and none of these were admitted to the ITU, none walked out of the hospital and none were nauseated enough to withdraw from the trial.

Of the 26 patients given hydroxychloroquine three were subsequently excluded from the study because they were admitted to the ITU. Oops. One died (not one of those admitted to the ITU). One walked out of the hospital and never came back. Another was too nauseated on the drug to continue in the study.

Exclude these six patients and in the remaining 20 patients hydroxychloroquine was reported as successful at clearing the virus on nasopharyngeal swabbing.

You have to wonder if the poor patient who actually died while taking hydroxychloroquine ended up with high pH lysosomes which leaked enough cysteine to extract the FeS clusters from the nearby complex I FeS chains. Or simply died of pneumonia before he/she could be admitted to the ITU. We'll never know. I hope the patients "lost" in to the ITU made a full recovery.

Perhaps the patients who accepted hydroxychloroquine were simply iller in the first place and were so more willing to accept an experimental drug. I do hope so but I feel that the initial data on hydroxychloroquine are not looking too promising.

Peter

Edit: On the plus side there might be an effect if given early. However only time and trials will tell if there really is an effect or whether Turkey happens to have done several other things correctly in addition to using hydroxychloroquine. At least they are not reporting toxicity. End edit.

Coronavirus is possibly everywhere

This tweet

https://twitter.com/Yascha_Mounk/status/1246240181440540672

links to this news report

https://www.lastampa.it/topnews/primo-piano/2020/04/02/news/coronavirus-castiglione-d-adda-e-un-caso-di-studio-il-70-dei-donatori-di-sangue-e-positivo-1.38666481

which should be headline news everywhere.

In Lombardy 40 out of 60 blood donations from healthy donors with no history of coronavirus illness are seropositive. They have been exposed, infected but were never ill.

In comments to a pervious post I suggested that the best advice re coronavirus was a) try not to be elderly and b) try not to be diabetic.

Obviously assessment a) was incorrect.

Being elderly is probably only a problem if you drop in to category b) as well. ie age is a surrogate for risk of metabolic syndrome or as Kraft would have described it "diabetes in-situ". There is a simple solution to diabetes in-situ.

Testing of UK blood donations, if it duplicates Italy, should allow some return to normality with continued or increased protection for those most at risk while we get their metabolic syndrome under control. That should take a few weeks of LC eating and might have to be maintained long term. Damn, bacon and eggs for breakfast every day and steak with broccoli and 'shrooms for supper. Cheese and olives for lunch if you're hungry. Sounds awful I know but sacrifices will have to be made.

Peter

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;

https://twitter.com/UnitedRda/status/1244299325963829248

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.

Peter

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 are 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.

Peter

ARDS and linoleic acid

Acute 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.

Peter

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.

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)

and

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.

Peter

Coffee

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 Carnivory.com 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.

So....

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.

Peter

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.