Monday, November 29, 2010

I Warned Them This Drug Might Kill People...

by Chris Masterjohn

I was happy to see last night that the two letters to the editor I've written about vitamin E have been cited recently. 

I'm sad that another recent paper did not cite my letter about why their drug might be killing people, which we'll get to below.

The first letter was published in the Journal of the American College of Cardiology.  It was a response to the famous "one meal of saturated fat will kill you" study where the authors found that making a carrot cake and a milk shake with coconut oil rather than safflower oil led to lower anti-inflammatory properties of HDL particles isolated from the subjects' blood.  They attributed the effect to the saturated fat in the coconut oil, and I pointed out that it was probably a result of the vitamin E in the safflower oil.  I developed this argument in much greater detail in my article on this site, "Myth: One Meal of Saturated Fat Can Be Bad."

Little did I know until last night that the widely buzzed-about Krauss paper debunking the association between saturated fat and heart disease in the American Journal of Clinical Nutrition. 

They cited my letter very accurately, apart from the little typo at the end:
In the acute postprandial phase following a meal enriched in saturated or polyunsaturated fat, HDL collected from individuals after a coconut meal compared with a safflower or unsaturated fat meal was associated with a 50-70% increase in intercellular adhesion molecule and vascular cell adhesion molecule (131). Attribution of this effect specifically to the saturated fat of the coconut meal may, however, be confounded by the high [low] concentrations of tocopherol found in coconut oil (132).
Thanks guys!  If they liked my vitamin E letter, I hope I can convince them in the future to change their minds that "substitution of dietary polyunsaturated fat for saturated fat has been shown to lower CVD risk."  But I'm certainly happy that they concluded that "there are few epidemiologic or clinical trial data to support a benefit of replacing saturated fat with carbohydrate" and that "dietary efforts to improve the increasing burden of CVD risk associated with atherogenic dyslipidemia should primarily emphasize the limitation of refined carbohydrate intakes and a reduction in excess adiposity."

On the other hand, a paper about aromatherapy massage oils prepared from virgin coconut oil published in the Journal of the American Oil Chemists' Society used my letter in the most preposterous way imaginable:
Masterjohn [25] demonstrated that coconut oil had anti-inflammatory properties, which could help to reduce the inflammation occurring in muscles.
I did?  I wasn't even a graduate student yet when I wrote that letter.  I'm baffled that I "demonstrated" anything in my bedroom where I typed that letter, except the logical flaws in the paper I was responding to.  The study I commented on was about coconut oil diminishing the anti-inflammatory properties of isolated HDL.  I don't think that study design provided a realistic evaluation of the physiological effects of the coconut oil, but it certainly didn't demonstrate an anti-inflammatory effect of coconut oil!  And what it had to do with muscular function is beyond me.

I imagine they did a pubmed search for "coconut oil anti-inflammatory."  My letter is fourth out of twelve results.  Its title is "The anti-inflammatory properties of safflower oil and coconut oil may be mediated by their respective concentrations of vitamin E."  The authors must have taken "no abstract available" to mean you only have to read the title.  But geez, the whole letter is about one paragraph long.

The second letter was published in The American Heart Journal and argued that safety trials of anacetrapib, a cholesterol ester transfer protein (CETP) blocker, should test the effect of the drug on vitamin E transport.  This was in response to a short, 8-week study of anacetrapib showing no adverse effects.

CETP blockers are the first drugs ever used to test the "reverse cholesterol transport" hypothesis in humans.  This hypothesis says that HDL is beneficial because it takes cholesterol back to the liver and that a high total-to-HDL cholesterol ratio indicates that this process is occuring less efficiently and plays a causal role in atherosclerosis.  The first drug used to test this hypothesis, torcetrapib, killed people left and right.  But they want to believe this is because it increased blood pressure and had other "off-target toxicity," instead of believing that their hypothesis was falsified.  So they invented anacetrapib, another drug that does the same thing without the effects on blood pressure.

My letter cited test tube and cell studies showing that CETP plays an important role in transferring vitamin E to HDL particles, and that HDL particles have a very specific role in delivering vitamin E to the endothelial cells that line the blood vessel wall, where the vitamin E helps suppress the oxidation of LDL and thereby suppresses the development of atherosclerosis.  I concluded as follows:
An 8-week preliminary trial of the CETP inhibitor torcetrapib found no increase in adverse effects [9], but this drug ultimately increased the risk of cardiovascular events and death from all causes [10]. Because part of the protective effect of HDL may be due to its role in vitamin E transport, safety and efficacy trials of CETP inhibitors should take into account their effect on this process.

In other words, the fact that anacetrapib showed no adverse effects after eight weeks should give us little comfort that longer trials will not show it to kill people left and right, because the early safety trials with torcetrapib showed the same thing!

The authors responded that the role of CETP in transporting vitamin E to HDL had not been demonstrated conclusively in live people, and that since large trials using alpha-tocopherol did not prevent heart disease, it is not clear that alterations in vitamin E metabolism would have any effect either.  They came to the following conclusion:
A study to assess long-term efficacy and safety of anacetrapib in patients with coronary heart disease is currently in progress (clinical trials.gov NCT00685776).  The safety information provided by this trial will determine whether or not to initiate a phase III outcome trial with anacetrapib.  Ultimately, it is only by conducting such an outcome trial that the true efficacy and safety of anacetrapib will emerge.

Indeed.  However, it still seems worthwhile to do a very simple measurement of the vitamin E contents of these HDL particles just in case, perhaps, a long-term trial shows that it kills more people.

I was quite happy to see last night that a recent review on vitamin E transport in the journal Molecular Nutrition and Food Research cited this letter.  I'm a bit sad, however, that the people who matter -- the people studying anacetrapib -- continue to ignore it.
As Stan (Heretic) and Peter (Hyperlipid) recently pointed out, the initial results from the anacetrapib trial just came in.  They are published in the New England Journal of Medicine here.
 
Searching the pdf for "tocopherol" or even "vitamin" yields no results.
 
The results are a little disturbing, but almost impossible to interpret. 
 
Among the just over 1600 people who were randomized to receive either anacetrapib or the placebo, forty percent more deaths occurred in the anacetrapib group:
 
The difference, however, was not statistically significant.  This means that we can't be confident it wasn't simply the result of chance.  This is unsurprising, given the small number of people that died.

There were four times as many deaths from cardiovascular causes in the anacetrapib group:

Again, the difference was not statistically significant, and again this is unsurprising given that only five people fell into this category.
We could say, well gee, let's conduct a larger study so this massive increase in cardiovascular deaths can be proven with statistical significance.  But the layers of uninterpretability just keep piling on. 

First, they took 142 out of the 801 people who received anacetrapib off treatment because their LDL-cholesterol fell to less than 25 mg/dL.  All of these people were included in the analysis, but it's unclear whether their cardiovascular risk would better reflect the fact that they initially received the drug or the fact that they were taken off of it.  This appraoch is called "intention to treat analysis" and it's very useful for making policy decisions (if I tell 100 people to take the drug, assuming some stop taking it, how many die?), but it doesn't help us understand whether the drug saves people or kills them.

Second, 3.5 times as many people in the placebo group underwent a revascularization procedure:

This might indicate that anacetrapib reduced the need for revascularization procedures.  On the other hand, the report gives no description of who ordered the revascularization procedure and what criteria they used to order it.  Furthermore, it's unclear whether the revascularization procedures helped or hurt the patients, since even the study protocol provides guidelines for assessing heart attacks and deaths that are caused by revascularization procedures.  They don't tell us whether anyone died as a result of a revascularization procedure.

Thus, this report makes it quite unclear whether anacetrapib is saving people or killing them, and makes it even less clear whether anacetrapib will save people or kill them when it's tested in a much larger trial.

One little experiment that could be done now would be to take the plasma samples from this trial out of the freezer, isolate the HDL, and measure the vitamin E in it.  That would very quickly obviate the authors' concerns expressed in response to my letter last year that "to the best of our knowledge, there are no clinical data indicating a definitive role for CETP in vitamin E exchange," since they could test that hypothesis and either show it true or bury it for good in the course of several days, and would provide some potentially relevant safety data, and would also contribute important knowledge to the body of basic science about vitamin E transport.

But as for saving or killing people, we will have to wait a few years for the final word.

Read more about Chris Masterjohn, PhD, here.

Sunday, November 28, 2010

Does Choline Deficiency Contribute to Fatty Liver in Humans

by Chris Masterjohn

I hope all of those who celebrated had a wonderful Thanksgiving!  Giving thanks is at least as healing as a healthy diet!  For those of you who celebrated it just to eat a lot or for entirely other reasons, I hope you also had a wonderful holiday, and for those of you who did not celebrate anything at all, I hope you had a wonderful weekend.

In my last choline post, I presented my hypothesis that a suboptimal intake of choline, found most abundantly in egg yolks and organ meats like liver, is the preeminent cause of fatty liver disease. This disease may afflict 70-100 million Americans.  If you missed it, you can find the post here:

The Sweet Truth About Liver and Egg Yolks -- Choline Matters More to Fatty Liver Than Sugar, Alcohol, and Fat

After reading the research reviewed in that post, I'm quite convinced that the development of fatty liver is pretty simple: anything that increases the amount of energy that the liver needs to process forms the first part; anything that impairs the ability of the liver to export that energy forms the second part. High intakes of total calories, sugar, fat, or alcohol can all contribute to fatty liver in animals.  But the king of all nutrients needed to export that energy as fat, choline, protects against the disease in all of these animal models.

Of course, a high intake of PUFA contributes to the inflammatory component, and when combined with other toxic factors may also contribute to the fatty component by preventing the liver from exporting its fat.

Does choline play the same role in fatty liver disease in humans?  There are a few reasons to think it does.  The most convincing of these is that it's been demonstrated in folks getting fed intravenously. The most interesting of these is the wide prevalence of a defect in the gene that allows us to synthesize phosphatidylcholine from the amino acid methionine.  Its prevalence is high in the general population but almost complete among those with fatty liver.

The basic mechanisms of fatty liver in humans are the same as they are in animals.  Humans with fatty liver disease have increased lipogenesis (synthesis of fat from carbohydrate) (1), increased circulation of fat released from adipose tissue (2), and decreased secretion of fat from the liver (3).

Although folks with fatty liver release more fatty acids from the fat tissue, the studies cited above show that their livers don't actually synthesize more fat from these fatty acids than the livers of healthy folks do. So those fatty acids may contribute to "lipotoxicity" -- and in fact the creation of triglycerides in the liver is in part probably an effort to protect against the toxicity of excess free fatty acids -- but it seems that the synthesis of fat from carbohydrate is more important than the release of fat from adipose stores.

The increased synthesis of fat almost certainly means that the burning of fat is decreased, because these two phenomena are metabolically linked to each other.  Animal experiments using specific enzyme inhibitors suggest that both need to occur simultaneously to get significant increases in liver fat (4), if you want to induce it without any changes in the diet.

It was until quite recently thought that choline is not an essential nutrient in humans because choline can be synthesized from methionine. In 2001, however, a very small randomized, placebo-controlled trial conducted in 15 patients who developed fatty liver while receiving intravenous total parenteral nutrition (TPN) showed that the addition of two grams of choline chloride to the formula could completely resolve the fatty liver (5).

There are a number of other nutrients that reduce our requirement for choline in addition to methionine.  One is betaine, which occurs most abundantly in wheat, spinach, and beets.  Folate and vitamin B6 are also important.  However, betaine, folate and B6 simply spare choline. We can actually use methionine to methylate a phospholipid called phosphatidylethanolamine to phosphatidylcholine, the principal choline-containing phospholipid.

There's just one problem: doing so requires an enzyme called phosphatidylethanolamine-N-methyltransferase (PEMT).  The presence of at least one defective copy of this gene is extremely prevalent in the population, and it dramatically raises our chances of developing fatty liver on a low-choline diet. 

Here's what one case-control study found when they compared the prevalence of the defective gene among people who had fatty liver and those who didn't (6):



The vast majority of people in this study had at least one defective copy of the gene, but almost everyone who had fatty liver had at least one copy. The difference between the two groups doesn't seem huge, but if we spin the tables and look at who didn't have any copies of the defective gene, which is just another way of looking at the same data, the difference looks pretty big:



And here's the prevalence of people with two copies of the defective gene:


Of course "defective" is a value judgment.  Apparently it's "good enough" to survive at a high prevalence.  But I'll call it "defective" for now for the sake of simplicity.

One recent study suggests that people with defective copies of this gene are much more vulnerable to developing fatty liver on a low-choline diet. 

The study is built on the findings of a previous study published a few years ago, so we'll discuss that one first. In the first study (7), the investigators fed 57 healthy men and women a diet containing roughly the RDA for choline (550 mg choline for every 70 kg bodyweight) for ten days.  Then they switched them to a choline-deficient diet for 42 days.  The diet was adequate (meeting or exceeding the RDA) for the other essential nutrients but contained less than 50 mg choline for every 70 kg bodyweight.  The investigators then increased their choline intake stepwise, replacing 25% of it for ten days, then 50% for another ten days, then 75% for ten days, and then 100% for the final ten days.

38% of young women, 40% of men, and 80% of postmenopausal women developed fatty liver.  81% of these people resolved their fatty liver at some point during the repletion phase, while the others recovered only after returning to a normal diet.

This study is not terribly convincing.  It is essentially an observational study because there was no control group, and rather than giving different people different doses of choline during the repletion period, they all got the same dose at once, increased over time.  The results could be explained in numerous alternative ways.  For example, the low-choline diet could have had some noxious factor in it that the subjects adversely reacted to at first, but to which most of them adapted over time. 

Nevertheless, the study is an interesting pilot study, and it suggests that there may be very wide variability in the choline requirement, and that it may be much higher than the RDA in some people.  And these people were healthy.  As indicated by the genetic data above, people with fatty liver disease -- possibly a third of the population -- might have even higher needs for choline.  And of course, the whole point of my last choline post was that the choline requirement is affected dramatically by what else we eat, not just our genes.

The second study (8) was designed similarly, but they did not monitor variation in the amount of choline needed for repletion.  In this study, they divided postmenopausal women into those who had defective copies of the PEMT gene and those who did not, and they randomly allocated half of them to estrogen therapy.  After all, the fact that twice as many postmenopausal women developed fatty liver in the previous study compared to younger women suggests that estrogen might reduce the choline requirement.

The results are pretty remarkable.  The following results are for "organ dysfunction," which included fatty liver, elevated liver enzymes, or signs of muscle dysfunction, but 90% of the women who developed "organ dysfunction" were diagnosed on the basis of fatty liver.  The PEMT gene had a huge influence on who developed fatty liver:


In the placebo group, only 25% of women with no defects in the PEMT gene developed "organ dysfunction" on the low-choline diet, but a whopping all of them developed organ dysfunction, virtually all of which was fatty liver, if they had at least one copy of the defective gene!

The estrogen had a remarkable protective effect.  Two copies of the defective gene was very rare in this population (since the inclusion criteria required the participants be healthy), and no one was represented in the estrogen group. Nevertheless, even having one copy of the defective gene has a remarkable effect, as we'd expect based on the above graph:

Quite an interaction!  The combination of estrogen and two healthy copies of the PEMT gene abolishes the risk of choline-deficiency associated fatty liver, at least in the short course of a month, on an otherwise adequate diet.  But it seems those with two of the "good" copies are in the minority.

The authors made the following remark about the effect of estrogen:

It makes sense, from an evolutionary perspective, that young women can supply more of their choline requirement from endogenous biosynthesis because pregnancy and lactation are times when the demand for choline is especially high, particularly to support the developing nervous system.
That's a rather odd comment considering the paper made no attempt to unravel the gentic mechanisms by which estrogen is increased during youth or by which estrogen decreases the choline requirement, but it's an interesting tangent.  Certainly, it is biologically more important to provide choline to the brain of a developing fetus and infant than it is to make enough to get the fat out of your liver. 

On the other hand, men were a lot better off than postmenopausal women, and although it's possible for men to lactate, it's very rare. 

Evolutionarily, it makes a lot of sense for men not to lactate, because... ah well, nevermind.  Maybe in another post.

Neither of these studies provide conclusive evidence that suboptimal intakes of choline cause fatty liver in humans, but together the totality of these data make a strong case for testing the hypothesis.  It certainly seems that most of us are vulnerable to needing choline, rather than just methionine and the other nutrients that spare it. The TPN study provides proof of principle that choline does indeed reverse fatty liver in humans. And the universality of the protective effect of choline in diet-induced animal models of fatty liver certainly puts choline at center stage.

PEMT is not the only gene that affects the choline requirement.  I'll be writing much more about the choline requirement and how to obtain it in a future post, and that will cover the effects of other genes as well.

Lets raise another glass to King Choline!  Long live liver and eggs!

Read more about Chris Masterjohn, PhD, here.

Thursday, November 25, 2010

Thanksgiving, A Source of Eternal Joy

by Chris Masterjohn

Sandrine Hahn, a Weston A. Price Foundation chapter leader, created a wonderful Thanksgiving illustration, found here.  It contains a beautiful quote from Konrad von Gesner:
Best of all is it to preserve everything in a pure, still heart, and let there be for every pulse a thanksgiving, and for every breath a song.
Gesner was a sixteenth century Swedish naturalist with many professional pursuits and interests including botany, medicine, mountains, and theology.

Gesner's quote is particularly beautiful because it urges us to be thankful for everything.  That we be thankful every time our heart beats, not simply for our heartbeat, but for whatever we have been given at the very moment our heart beats.

Another quote I very much enjoy is from Alexander Schmemman:
Everyone capable of thanksgiving is capable of salvation and eternal joy.
Schmemman (1921-1983) was an influential Orthodox Christian priest living, teaching, and writing in the United States.

One need not share Gesner's faith or Schmemman's faith to appreciate the liberating and saving power of gratitude.  In a recent conversation with some friends, we ennumerated the many things we were thankful for, but also concluded that we should be thankful for everything, including the things that seem to have caused us harm or seem to have made us suffer, because there are lessons for us hidden deep within each of these things.  This attitude is truly liberating and truly saving, because it frees us from anxiety, greed, anger, and fear.

If we are thankful for everything, we need not worry about what will happen next.  If we are thankful for what we already have, we need not live our lives for the sake of taking more and more.  We will never become angry at what we are thankful for.  If we are thankful for everything, there is nothing to fear.

Of course, we should not be thankful that other people suffer.  Our thankfulness should turn inward, and our compassion should turn outward.  But when we see suffering, we should be thankful for the opportunity to help someone in need.

There are many of you reading this to whom I am very grateful, and I hope to ennumerate all of this as time goes on, and I hope I have appropriately thanked many of you in past posts.  Today is an opportunity to remember what we should remember at every moment our heart beats, with every breath, that the world is marvelous, and we are thankful to be here.

Read more about Chris Masterjohn, PhD, here.

Tuesday, November 23, 2010

The Sweet Truth About Liver and Egg Yolks -- Choline Matters More to Fatty Liver Than Sugar, Alcohol, or Fat

by Chris Masterjohn

In a recent post, I pointed out that perhaps as many as 100 million Americans have some degree of fatty liver disease.  Why?  Alcohol has been blamed since the 1800s, but we currently have an epidemic of nonalcoholic fatty liver.  Some researchers, such as Dr. Robert Lustig, are making the case against fructose.  Naturally, the nutritional establishment and the media will blame anything on saturated fat, and fatty liver is no exception, even when the "saturated fat" is corn oil.

After studying the relevant literature and tracing it much further back in time than anyone else ever bothers to, I've come to the conclusion that neither fat nor sugar nor booze are the master criminals here. Rather, these mischeivous dudes are just the lackeys of the head honcho, choline deficiency.  That's right, folks, it's the disappearance of liver and egg yolks from the American diet that takes most of the blame.

More specifically, I currently believe that dietary fat, whether saturated or unsaturated, and anything that the liver likes to turn into fat, like fructose and ethanol, will promote the accumulation of fat as long as we don't get enough choline. Once that fat accumulates, the critical factor igniting an inflammatory fire to this fat is the consumption of too much PUFA (polyunsaturated fat from vegetable and perhaps fish oils).




Choline (Chloride) -- Is His Deficiency a Villain or Just the Absence of His Superhero-ness? An Irrelevant Question For People Who Philosophize Too Much.  Read On to Find Out Why Choline Is Awesome.


Most reviews about nonalcoholic fatty liver disease (NAFLD) trace the disease back to a 1980 paper published by Jurgen Ludwig and several of his colleagues from the Mayo Clinic (1).  But Ludwig's group never claimed to have discovered NAFLD.  On the contrary, they simply came up with the name nonalocholic steatohepatitis, which they abbreviated "NASH."  They took credit for achieving three things by inventing this new word (2).  First, NASH is really easy to say.  It only takes one syllable.  Second, giving a convenient name for the disease encouraged an organized approach to researching it.  Third, it stopped physicians from assuming their patients were lying about not consuming alcohol just because they had an inflamed liver.

As Ludwig's group acknolwedged, Samuel Zelman had produced the first case series of obesity-associated NAFLD patients back in 1952 (3).  Zelman launched his investigation after observing fatty liver in a hospital aide who drank more than 20 bottles of Coca-Cola every day.  This was before the days of the obesity epidemic, so it took him a full year and a half to find 20 obese people who weren't alcoholics.  All but one of them had some indication of liver damage, and about half of them had pretty substantial NAFLD.

But fatty liver goes back even further than that.  According to one paper I found, fatty liver had been identified in type 1 diabetes at least as far back as 1784 (4).  By the 1930s, some physicians recognized fatty liver as a common occurrence in severe cases of diabetes and it tended to spontaneously resolve when they treated the patients with insulin and a low-carbohydrate diet that included 100 grams of bread and 100 grams of fruits and low-carbohydrate vegetables, with the remainder of energy requirements coming from fat (5).

Severe type 1 diabetes is quite a special case and it is likely that in these instances it was the severe metabolic derangement causing the fatty liver.  This, however, quite clearly cannot explain why as many as 100 million Americans or more currently have the disease.  Nevertheless, it was the study of type 1 diabetes animal models that led to the discovery of the critical role of choline in preventing and curing fatty liver.

Diabetes and Choline

Physiologists first identified the role of insulin deficiency in type 1 diabetes by studying the disease in dogs.  In 1889 they produced diabetes by simply taking out the whole pancreas from these dogs and, after scrambling for a couple decades to identify the active component, they cured the diabetes with insulin in the early 1920s (6).  As cured as their diabetes was, the insulin-treated dogs nevertheless developed severe fatty liver degeneration and ultimately died of liver failure.  Adding raw pancreas to their diet, which was composed of lean meat and sucrose, cured the problem.  As researchers attempted to discover what it was in raw pancreas that cured the disease, they found in the early 1930s that egg yolk lecithin, which is abundant in choline, could cure it (7).  And then they found that choline alone could cure it (8). 

It later turned out that the dogs became deficient in choline and methionine without a pancreas because they weren't producing the digestive enzymes needed to free up those nutrients from the foods they were eating.  Thus, simply providing them with the digestive enzyme trypsin could cure the fatty liver (9).

We now know that choline is necessary to produce a phospholipid called phosphatidylcholine (PC).  This is a critical component of the very low density lipoprotein (VLDL) particle, which we need to make in order to export fats from our livers.  The amino acid methionine can act as a precursor to choline and can also be used to convert a different phospholipid called phosphatidylethanolamine directly into PC.  Thus, the combined deficiency of choline and methionine will severely impair our abilities to package up the fats in our livers and to send them out into the bloodstream (10).

High-Fat Diets and Choline

In 1932 a group of researchers decided to replicate the fatty liver seen in depancreatized dogs in a nondiabetic rat model.  What better way to stuff their livers with fat?  Feed them fat!  Seemed simple enough.  And, in fact, it worked.  Althogh they had trouble reproducing the fatty liver with different colonies of rats or during the summer heat, they produced fatty liver in certain colonies of rats during the winter by replacing 40% of their ordinary cereal-based diet with beef drippings.  Lecithin derived from egg yolk or beef liver (11) or simply choline itself (12) cured the disease.

These experiments were back in the day, before researchers realized that methionine would be able to indirectly cure choline deficiency.  Another group of researchers had the bright idea of trying to replicate this experiment in a group of rats who were consuming sufficient protein to maximize growth.  So they fed them 40% beef drippings but replaced another 20% of their cereal grains with the milk protein casein.

Lo and behold, these researchers discovered a now well known phenomenon that occurs during the process of scientific discovery called the "epic fail" (13).

All of the groups had average levels of liver fat under 7% and could hardly be said to have gotten fatty liver of any sort.

So the researchers had an idea.  Perhaps it was the casein they fed them that was the problem.  And indeed, their hypothesis turned out to be correct: on a choline-free, 40% beef dripping diet, reducing the casein from 20% to 5% doubled the level of fat in the liver (14).

Those of you who have been reading my blog for the past few months may remember that in the wake of Denise Minger's shredding apart of the China Study's epidemiological data, I dug deep down into the buried secrets of Dr. T. Colin Campbell's rat research, and found that he attributed the "protective" effect of 5% casein diets to the massive fatty liver that the rats developed.  As described in "The Curious Case of Campbell's Rats" and the associated addendum, the membrane containing a drug-detoxifying enzyme that he blamed for liver cancer got stuffed with 3-4 times more fat, which may have clogged up the enzyme and stopped it from working.  Well, it turns out that others had shown a similar effect of such a diet on liver fat decades earlier.

Back to the 1930s.  So these researchers repeated their experiment with the different fats, this time using 5% casein.  Ta-da!  The experiment worked much better (13):

The butter produced the worst fat accumulation, where almost a third of the liver was fat!  The animals taking cod liver oil tended to eat a bit less food than the others, which undoubtedly confounded the results at least to some degree, but it still seems from this graph that the more saturated the fat and the longer-chain the fat, the worse the fatty liver was.  In fact, if we blow up the graph, we can see this more easily:


Whoa!  Hold your horses, you might be thinking.  Mr. Masterjohn, haven't you been telling us that saturated fat protects against fatty liver disease?

Well, yes I have, and I stand by that.  But it appears that things are a little more nuanced than they first appeared.  As it turns out, saturated fats increase the choline requirement a bit more than PUFAs do.  Take a look at the results of this 1957 paper that tested the effect of butter and corn oil on the choline requirement (15):

As it turns out, the choline requirement is about 30% higher on a 30% butter diet than on a 30% corn oil diet.  Why would this be?  It's not entirely clear, but I have a good guess.  As I pointed out in my PUFA Report, "How Essential Are the Essential Fatty Acids?", studies in rats, humans, and other primates show that 18-carbon PUFA are burned for energy at an extraordinarily high rate compared to other fats.  In rats, 60% are burned for energy, 20% are broken down into basic building blocks to make more saturated fatty acids, and most of the rest are secreted into the fur.  They do accumulate over time at a slow rate, but the body seems to sense that these fatty acids are an unnecessary oxidative liability and tries to get rid of them however it can.  Thus, perhaps saturated fats require more choline to get them out of the liver because they don't scare the liver into burning them for energy so quickly.

In any case, the clear picture that is emerging is that dietary fat increases the choline requirement, and that high-fat diets promote fatty liver only when the level of choline in the diet is insufficient to meet the extra demand for it caused by the increase in fat.

Fructose, Sucrose, Ethanol, and Choline

The buzz is all about fructose nowadays, but the role of fructose in fatty liver has been known since the 1930s, just like the role of fat.  It also played a critical role in the modification of the AIN-76A standards for purified lab rat diets to the AIN-93 standards in 1993, when sucrose was switched to corn starch, like I explained in "They Did the Same Thing to the Lab Rats That They Did to Us."  Fructose can be provided in the diet as free fructose or as sucrose, which is half fructose and is the type of sugar found in refined, white table sugar.  Fructose is different than glucose in that it goes straight to the liver rather than dispersing throughout the body; thus, the liver converts much of it to fat, sending the fat back into the bloodstream -- providing it has enough choline.  Ethanol, the type of alcohol we get drunk from, is metabolized similarly (16). 

The first studies showing that fructose played a role in fatty liver disease developed out of the discovery of the essential fatty acids.  I've recounted the history of this discovery in my recent article, "Precious Yet Perilous -- Understanding the Essential Fatty Acids."  In 1924, George Burr joined the laboratory of Herbert Evans, where Evans and Katherine Scott Bishop had recently discovered vitamin E.  Evans and Bishop were having trouble reproducing their vitamin E-deficient diet, and Burr helped them develop a highly purified diet based on reprecipitated casein and recrystallized sucrose.  The new diet produced a deficiency that vitamin E couldn't cure, which Burr and his wife Mildred later identified as essential fatty acid (EFA) deficiency

Observing that the annual per capita consumption of sugar in the United States had tripled over the preceding decades from 38 pounds to 115 pounds, Clarence Martin Jackson conducted a comprehensive analysis of the anatomy and tissues of rats fed Burr’s EFA-sufficient, 80% sucrose control diet and compared them to rats fed 45% sucrose or 45%  corn starch (17). Neither the 45% sucrose diet nor the 45% starch diet produced fatty liver, but the 80% sucrose diet produced moderate to severe fatty liver with flattening and displacement of nuclei within the liver cells.  Jackson warned that the liberal provision of cod liver oil, dried yeast and wheat germ satisfied the nutritional needs of the rats in all treatment groups, and that smaller amounts of sucrose may contribute to fatty liver in humans consuming nutritionally deficient diets. 

This is rather remarkable, because much lower concentrations of sucrose started spontaneously producing fatty liver disease in lab rats in the late 1970s and early 1980s once the American Institute of Nutrition set standards for purified rodent diets that relied exclusively on isolated vitamins and minerals rather than whole-food supplements like cod liver oil, yeast, and wheat germ.  We'll never know exactly how much choline was in Jackson's diet, but purified diets with 0.3% methionine and 0.8% choline, though to be adequate in these nutrients, produced moderate fatty liver when the sucrose concentration was increased from 20% to 25-35% (18).

Physicians and researchers had started pinning the blame on alcohol abuse for fatty liver back in the 1800s, so while research was first highlighting the role of sucrose in fatty liver, other research was doing the same for alcohol.  In 1949, however, researchers showed that sucrose and ethanol had equal potential to cause fatty liver and the resulting inflammatory damage, and that increases in dietary protein, extra methionine, and extra choline could all completely protect against this effect (19).

Conversely, much more recent research has shown that sucrose is a requirement for the development of fatty liver disease in a methionine- and choline-deficient (MCD) model.  The MCD model of fatty liver disease is the oldest and most widely used dietary model.  The MCD model produces not only the accumulation of liver fat, but massive inflammation similar to the worst forms of fatty liver disease seen in humans.  What no one ever mentions about this diet is that it is primarily composed of sucrose and it's fat is composed entirely of corn oil!

As you can see here, the MCD diet resulted in only the most miniscule increase in liver fat when it was composed of starch (column 4 vs column 2), but when it was combined with sugar (green bar) it produced a massive increase in liver fat. The researchers also found that the MCD-sugar diet led to full-fledged inflammation, while the MCD-starch diet had no such effect (20). They found that on the sucrose diet, the fructose component led to the synthesis of fat in the liver, and because the choline level was deficient, the liver could not export that fat.

The picture that is clearly emerging from all of these studies is that fat, or anything from which fat is made in the liver, such as fructose and ethanol, are required for the development of fatty liver.  But in addition to this some factor -- overwhelmingly, it appears to be choline deficiency -- must deprive the liver of its ability to export that fat.

Oxidative Stress May Impair Export of Liver Fat

An interesting test tube study using isolated cells found that incubating liver cells with PUFAs would suppress export of fats while incubating the liver cells with saturated fats, vitamin E, or a chelator of free iron would improve the export of fats.  The results indicated that lipid peroxidation, that is, the oxidative destruction of PUFAs, causes liver cells to degrade their VLDL particles before they send them forth into what would be the bloodstream if the experiments were conducted in a live animal.  They also provided preliminary evidence that this occurs in live animals by directly infusing their bloodstreams with DHA (21). 

While it seems possible that excess PUFA might interact with iron overload, ethanol, or some other type of toxicity in order to impair the export of liver fat in the absence of choline deficiency, it nevertheless seems unlikely that excess PUFA in and of itself causes enough oxidative stress to make this happen, because even rats consuming 30% corn oil mentioned above fared fine if they were given enough choline.  However, as we'll see below, excess PUFA are required for the inflammatory part of NAFLD.  And, moreover, in humans there may be many interacting factors that "activate" the ability of excess dietary PUFA to suppress the export of fat from the liver.

MCD-Sugar Sets the Kindling, Corn Oil Lights the Fire

MCD formulas containing 20% fat as either lard or olive oil produce equivalent levels of NAFLD, suggesting that changes in the ratio of saturated to monounsaturated fats is not important (22).  Substitution of carbohydrate, coconut oil, or beef tallow for corn oil similarly offers no protection against accumulation of fat in the liver nor on the injury to liver cells that causes increases in liver enzymes; all these substitutions, however, dramatically decrease lipid peroxidation and and the resulting inflammation (23).  Corn oil probably promotes inflammation both by increasing vulnerability to lipid peroxidation because of its total PUFA content and by decreasing tissue levels of DHA because of its high omega-6-to-omega-3 ratio.

Choline is King!

I have to conclude from all these studies that choline deficiency plays a role in virtually every type of diet-induced fatty liver model.  The fat has to be provided to the liver through either dietary fat or dietary lipogenic substrates like ethanol and fructose, and the fat has to be trapped by impaired export of fats from the liver.  And choline deficiency seems to be the preeminent cause of this.

So does fructose cause fatty liver?  Kind of.  I'm not suggesting fructose is harmless or that you should go out an eat a bowl of fruit loops with your liver and eggs, but the loss of cholesterol-rich foods like egg yolks and organ meats as a result of cholesterol paranoia seems to be at the bottom NAFLD thus far.

There's just one question.  Leptin deficiency and leptin resistance both cause fatty liver.  And obese people are leptin resistant and the majority of obese people have fatty liver.  Can fatty liver or choline deficiency cause leptin resistance?  Or does leptin resistance cause cravings for choline-poor, fatty, and fructose-rich foods?  Here's a web I'll try to untangle in future posts!

Read more about Chris Masterjohn, PhD, here.

Saturday, November 20, 2010

Is the "Receptor for AGEs (RAGE)" Really a Receptor for AGEs?

by Chris Masterjohn

RAGE.  It sounds pretty mad.  Angry.  And it's out to get you.  How?  By punishing you for eating overcooked food, igniting your tissues with the hellfire of inflammation and oxidative stress.

Or not.  Maybe it's just there to make your brain grow.  Welcome to the controversies of science department snack rooms and rarely published critical reviews.

In this blog post I'm going to present some evidence that the Receptor for AGEs (RAGE) is not a receptor for AGEs.  And that RAGE's job isn't to punish you with a hellfire of inflammation and oxidative stress, regardless of whatever you did wrong, cooked food or no.

Thursday, November 18, 2010

Is Insulin Resistance Really Making Us Fat?

by Chris Masterjohn

Disclaimer: I love and respect many low-carbers and low-carb researchers, and think low-carb diets are very appropriate and perhaps necessary for many people.

Many in the low-carb field seem to think that insulin resistance is what is making us fat and even that insulin resistance is caused by... insulin.  Too much carb makes too much insulin and the insulin receptors get scared and run away.  Intriguing theory, but very simplistic.  During the Q&A session of my Wise Traditions talk this weekend, I made the following quip:


Saying that insulin causes insulin resistance is like saying that childhood mortality is caused by children.

Melissa McEwen caught this quote on her live twitter feed.  Melissa also live tweeted many other excellent talks from the conference, including Stephan Guyenet's masterpiece on the traditional diet of the Pacific islands.  He uncovered islands where the traditional diet was over 90% carbohydrate and other islands where the traditional diet was mostly fat, including a whopping 50% of calories just from saturated fat alone.  Neither of the populations had insulin resistance, diabetes, or cardiovascular disease, and none of either island's inhabitants were fat.  In came refined foods, and they became vulnerable to all of these diseases.

Lots of other people live tweeted talks and other events from the conference, and you can see a hodge podge of these tweets here

In this blog post, I'd like to review a couple animal models that strongly suggest that between insulin resistance and leptin resistance, leptin resistance is much more critical to the development of obesity.


The mainstream often cries "eat less and exercise more" whenever someone has trouble losing weight.  Well in fact there's a little molecule called leptin that our fat tissue makes, which slips into our brain, acts on the hypothalamus, and makes us... *drumroll*... eat less and exercise more. Well, at a minimum it makes us eat less and expend more energy when we exercise.

Even though leptin appears to decrease food intake and increase energy expenditure, obese people have very high levels of leptin compared to lean people (1).  When they lose weight, leptin levels go down, but stay way above what you'd find in a lean person. The drop in leptin is accompanied by a drop in thyroid hormone (2, 3), muscular utilization of glucose (3), sympathetic nervous system activity (3), and adrenaline (3), and by changes in the pattern of brain responses to food (4), all of which are reversed by injections of leptin.  These studies have been very small and preliminary, but they support the widely held belief that obese people are leptin resistant.

Insulin has a century or so more research behind it than leptin has, so insulin resistance is a much more well defined phenomenon than leptin resistance.  Recent data suggest that about 39% of overweight Americans have either diabetes, impaired fasting glucose, or elevated fasting insulin (5), which are the clinical manifestations of insulin resistance. 

These numbers are pretty similar to what Zelman reported in 1952 (6).  Zelman wrote long before the obesity epidemic emerged and it took him 18 months, a full year and a half, to find 20 obese people who weren't alcoholics.  He reported that about half of these people were glucose intolerant, meaning that when they were given a load of glucose, their insulin couldn't work fast enough to prevent an abnormal spike in blood sugar. 

Zelman's finding was similar to what had already been reported in larger groups.  His new contribution was to show that upon liver biopsy, all patient but one -- a full 95% -- showed signs of at least mild to moderate liver damage.  The longer the people had been obese, the more damaged their liver was.  Long before the discovery of leptin, a hormone that acts on the hypothalamus, Zelman hypothesized that damage to the hypothalamus caused obesity and cravings for nutrient-poor sweets and fats, that the consumption of too much sugar and fat without sufficient choline, protein, and other nutrients led to liver damage, and he cited another researcher's suggestion that liver damage led to glucose intolerance. 

Why would liver damage lead to glucose intolerance?  The liver not only contributes to clearance of glucose from blood, but, more importantly, the liver produces glucose from protein in a process called gluconeogenesis.  Ordinarily, the liver stops making glucose in response to insulin.  However, if liver damage prevents this response, the liver will keep making glucose even when we don't need any more of it.  More glucose in the blood will cause the pancreas to make more insulin, but the insulin will fail to stop the liver from making more glucose, and a vicious circle will ensue.  With increasing levels of glucose and insulin in the blood, many other tissues such as skeletal muscle and fat may deliberately stop responding to insulin themselves in order to prevent glucose overload in their own cells.

The fact that insulin resistance is not found in all obese people does not mean it plays no causal role, because obesity does not necessarily have the same cause in every person. 

Nevertheless, a look at the genetic animal models of leptin and insulin resistance would suggest that leptin resistance has a much more prominent role in causing obesity and that insulin resistance without leptin resistance may not cause obesity at all.

The agouti yellow mouse was the first genetic animal model of obesity, systematically described as far back as the 1920s, and it later turned out to be leptin resistant (7).  Another early model of obesity was neither dietary nor genetic, but rather involved the removal of the hypothalamus, the principal (but not only) site of leptin action (8).  The common modern genetic models of obesity include the ob/ob mouse (9), the db/db mouse (10), and the fa/fa rat (10).  The ob/ob mouse doesn't produce leptin at all, while the db/db mouse and the fa/fa rat have defects in the leptin receptor.  All three types of animals become insulin resistant and fat.

It's a little bit harder to study the effects of genetic insulin resistance, because mice with no insulin receptors die 2-3 days after birth (11).  Nevertheless, mice can be developed with deletions of the insulin receptor in specific tissues. Deletion of the insulin receptor from liver tissue results in whole-body (systemic) insulin resistance (12).  As predicted above, the insulin resistance in the liver can cause systemic insulin resistance by causing the liver to continue making glucose and sending it out into the blood stream even when it isn't needed. 

But, surprise surprise!  These mice become neither leptin resistant nor fat (13).  In fact, while the effect is not statistically significant, they are slightly more sensitive to leptin and slightly more lean.

Although glucose metabolism tends to normalize after six months, fasting glucose is initially higher.  Feeding them glucose produces much higher blood glucose peaks and dosing them with insulin produces much less effective declines in blood glucose.  What is particularly remarkable, however, is their high insulin levels.  These dramatic changes persist even through adulthood.

In the fasting state, insulin levels are over 7-fold higher:


In the fed state, insulin levels are 23-fold higher:

Their insulin receptors are only knocked out in their liver.  So if high insulin levels are what act on our adipose tissue to make us fat, these mice should be really, really, really fat.

On the contrary, they are quite lean:



Why aren't they fat?  This study showed that they were just as sensitive to leptin, perhaps slightly more sensitive, than controls. Thus, while leptin resistance and insulin resistance often go together, it seems that leptin resistance is a much more important contributor to obesity.

These data should not be considered evidence that insulin resistance can never lead to leptin resistance in humans.  In fact, human hepatic insulin resistance (insulin resistance of the liver) looks nothing like what happens in the hepatic insulin receptor knockout mice.  The livers of these mice don't respond to insulin at all.  In humans, this insulin resistance is "selective."  The livers of "insulin resistant" humans continue to manufacture fat and send it out into the blood as triglycerides in response to insulin, but fail to suppress the production and export of glucose in response to insulin.

In humans, insulin resistance of the liver leads to increased triglycerides in the blood.  One theory that has some experimental support, but is still questioned by some experts, is that increased blood triglycerides decrease the transport of leptin into the brain.  For this and perhaps other reasons, insulin resistance as it occurs in humans could, perhaps, cause leptin resistance.  However, if it does not cause leptin resistance, it is very unlikely to make people fat.

In describing this selectivity, Dr. Robert Lustig recently made the following remark (14):
The reason for this uncoupling of insulin's two main hepatic signaling pathways remains unclear.
I propose that the explanation may be rather simple.  Rather than a result of gluocose toxicity or fat toxicity or fructose toxicity, the development of insulin resistance may be a natural, protective, homeostatic response to energy overload.  It certainly has adverse consequences down the line, but the selectivity does seem to suggest a deliberate attempt of the liver to alter its energy metabolism.  Moreover, the liver is clearly modifying its energy metabolism in a consistent way that exports energy.  In this case, it is exporting both glucose and fat at the same time, rather than suppressing one whenever it engages in the other.

I'll expand on these ideas in an upcoming post arguing that the main culprit preventing the liver from correctly handling its energy is suboptimal intakes of choline, and perhaps lipid peroxidation, which prevent the liver from being able to export the fat that it obtains from dietary fat or that in manufactures anew from things like fructose and ethanol.  Nutrient deficiencies and other issues that compromise the liver's ability to burn energy are also likely involved. 

First, expect a brief review debunking some of the conventional ideas about the so-called "Receptor for AGEs (RAGE)."

Then we can go back to fruit and honey.

Read more about the author, Chris Masterjohn, PhD, here.

Tuesday, November 16, 2010

In Favor of Neolithic Role Playing and Reenactment

by Chris Masterjohn

This post is a tribute to John Durant, Melissa McEwen, and Stephan Guyenet, each of whom brought a whopping pile of curiosity, friendship, and comradery to the 2010 Wise Traditions conference.  Stephan also brought a whopping pile of genius, which he injected into his excellent presentation on traditional diets in the Pacific islands, showing that neither carbs, fat, nor saturated fat make you fat, but rather that modern, refined, industrial foods bring in the diseases of modern civilization.

John Durant has declared his official opposition to caveman role playing, found here.  Apparently loin cloths will not sell the public on paleo.  Ho humph.  So much for comfy clothing.

How about a tall straw hat to sell the public on the 1830s New England digestion-deranging traditional diet?  It was full of sugar, but probably better than what's found on American's plates today.

I used to work at Old Sturbridge Village (OSV), a living history museum set in the 1830s.  Here's me in my work clothes:


At OSV I learned to operate carding, grist, and saw mills, to bottom shoes by hand, and to make pottery, like this guy is doing:


I also learned to put a leash on a calf to take her for a walk (not so easy as it sounds!), to mow grass with a scythe (walking a calf is a lot easier!), and to fire a black powder musket, like this one:


The 1830s were a time of bustling change.  Andrew Jackson, who ran on the campaign "Jackson and No Bank," became president in 1828, and achieved the destruction of America's second central bank by 1836.  1830s America had a number of competing coin currencies:



The progression of capitalism enhanced the ability of women to play an important role in society (in addition to their direct contributions to their family).  The advent of carding mills and other semi-automated processes freed up time that women could use to engage in social and reform movements:


The political scene looked very different.  90% of taxes went to the municipality, and those funds were allocated by direct democracy.  The separation of church and state was taken as literally stated in the first amendment and had no application to municipalities, so often each town had an official church.  It wasn't so terrible, because it's a lot easier to move to a different town than a different country, if you want your taxes to go to your own religion.  Poverty was confronted rather than swept under the rug.  Anyone who couldn't work was taken care of by a family, paid for by the town taxes.  Whichever family volunteered to take care of the indigent for the lowest price got the contract. 

Here are a few things I learned about the New England Diet while I was there:

  • They ate an awful lot of meat, and considered a "good" diet to be one that was high in meat and flour.
  • Vegetables and fruits were considered "garnish" unless they could be elevated in status by mixing with flour and sugar, as in an apple pie.
  • Their "white meat" was cheese, fish, and butter, and its status was above sugars but below meats and grains.
  • They drank very little milk, but made lots of cheese and butter.
  • Wheat didn't grow in the region, but the rich people could buy it, as it was beginning to be imported from Rochester and Baltimore.  Most folks made bread from corn and rye.  The bread was so tough that they used it as a utensil.  For example, they would cut off a more or less spoon-shaped piece and use it to eat their soup.
  • Most people ate with their knife; they used their fork to get the food onto the knife.
  • Their foods were preserved in vinegar, not by lacto-fermentation.
  • They ate a lot.  When Alexis de Tocqueville visited America he remarked that Americans were noted for the huge portions of food they consume.
  • Their number one health complaint was digestive problems.
  • The best thing about this diet?  They ate tons of organ meats.
I suppose to my credit, John might consider this "leaving it to the professionals," since I did in fact get a paycheck.  But I'd encourage anyone who swings by central Massachusetts to take a stroll through OSV.

If you're in for a laugh, our sister living history museum, Pioneer Village, was featured on an episode of South Park:


Enjoy!

Read more about the author, Chris Masterjohn, PhD, here.