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Saturday, 23 February 2019

Why the High-Fat Hep C Diet? Rationale and n=1 results.

[pinned post hence the unusual date]

I originally started this blog to publicise the hypothesis that a diet low in carbohydrate and linoleic acid, but high in saturated fat and long-chain PUFA, will inhibit HCV replication.

The blog header with the pig above is the abstract for this hypothesis.

I first worked this out in 2010 after reading Dr Atkins New Diet Revolution while studying HCV replication. The lipid patterns in low-carb dieters - low TG and VLDL, high HDL, normal or high LDL - are those associated with lower viral load and improved response to treatment in HCV cases.
The mechanics of HCV replication and infection support this link.


HCV inhibits PPAR-a, a ketogenic diet reverses this inhibition

I wrote a fairly comprehensive version of the hypothesis in 2012:
http://hopefulgeranium.blogspot.co.nz/2012/02/do-high-carbohydrate-diets-and-pufa.html

Recently I was sent a link to an article that cited this paper:
http://www.journal-of-hepatology.eu/article/S0168-8278(11)00492-2/pdfHCV and the hepatic lipid pathway as a potential treatment target. Bassendine MF, Sheridan DA , Felmlee DJ, et al. Journal of Hepatology 2011 vol. 55 j 1428–1440

This review compiles a great deal of supporting evidence regarding the interaction between HCV and lipids, and between lipids and HCV. The only thing missing is the role of carbohydrate. It mentions multiple lipid synthetic pathways as targets for indirect-acting antiviral drugs (IDAA), pathways which are also well documented as targets of low carbohydrate ketogenic diets, or of saturated fat in the diet (in the case of the LDL-receptor complex).

From 2012:
A little n=1 experimental data; 4 years ago (2008) my viral load was 400,000 units, now after 2 years of low carb dieting and intermittent mild ketosis (2012) it is 26,000.

Later in 2012:
Total Cholesterol:  6.7  H     
Triglyceride:          0.8         
HDL:                     1.63              (63.57)
LDL (calc.)            4.7   H    
Chol/HDL ratio:     4.1          

HCV viral load on this day (21st May 2012): 60,690 IU/mL (4.78 log)



Lipid panel from 07 Feb 2012, during ketogenic diet phase (non-fasting)

Total Cholesterol: 8.9   HH  (347.1)
Triglyceride:         1.3          (115.7)
HDL:                    1.65         (64.35)
LDL (calc):           6.7    H    (261.3)
Chol/HDL ratio:     5.4   H

HCV viral load on this day: 25,704 IU/mL (4.41 log)

From 2014:
On a personal note, I have started an 8-week trial of Sofosbuvir and GS-5816 (Vulcan). It is day 11 and it seems tolerable so far.
A pre-trial blood test on 22nd October was normal except for these counts:
AST 74
ALT 174

and viral load was 600,419 (log 5.78), counts consistent with the tests I've had done this last year.

But the day the trial started, 18th November, before my first dose, things were different:
AST 21

ALT 32
Viral load 27,167 (log 4.43)

The low viral load is easy to explain; I get a consistent 1 log drop (to 14,000-60,000*) when I try to eat very low carb (50g/day or lower) and an elevation to 400-600,000 when my carbohydrate intake is over 50g/day. When I ate very high carb (but took antioxidant supps) it was as high as it was on 22nd October. So for me the tipping point seems to be where ketosis begins, and other variations don't have much effect; it's an on/off switch, not a dial (and the name of that switch is PPAR-alpha).
[edit: though the very low scores are at ketogenic, or nearly so, carb intakes, the exact increase in carbohydrate needed to cause a significant increase in viral load seemed to vary]
(I do however, according to CAPSCAN elastography, have zero excess fat in my liver, which is an effect of low carb in general, as well as avoiding vegetable seed oils).

My belief is that my viral load was much higher than any of these counts previous to 2003. This was the year I started taking antioxidant supplements, eating a bit better (in a normal, confused "healthy eating" pattern), and using herbal antivirals like silybin. Prior to that I was seriously ill, and I believe that my viral load would have reflected my extra autoimmune symptoms, signs of liver failure, and elevated enzymes. Unfortunately in those days one didn't get a PCR unless one was considering donating one's body to interferon, which I was not.

* I don't seem to have a record of the date of the 14,000 VL reading, but will include it when I find it.

Summary:
A very low carbohydrate ketogenic diet, without enough PUFA to lower LDL artificially, had a significant inhibitory effect on HCV viraemia in my case.
Effective DAA drugs for HCV infection are now available. There is a ~98% SVR rate at present. These drugs are expensive, they sometimes have side effects (though much less so than interferon + ribavirin), and interferon + ribavirin is still being used.
If my results are more generally applicable, VLCKD diet offers an adjunct therapy for patients with a high viral load, steatosis that relates to diet and lifestyle as well as HCV infection, or a need to postpone treatment. In people who oppose or cannot complete or afford treatment, it offers a way to manage the disease, and in particular to reverse the autoimmune syndromes caused by immune complexes when viraemia is excessive.


Sunday, 30 September 2018

Egregious - the Richard M. Fleming Story

An RCT paper claiming to show harmful effects of a low-carb diet passed briefly over the internet on the weekend before being shot down in flames.
The title is:

Long‐term health effects of the three major diets under self‐management with advice, yields high adherence and equal weight loss, but very different long‐term cardiovascular health effects as measured by myocardial perfusion imaging and specific markers of inflammatory coronary artery disease.
The paper is published in Clinical Cardiology [edit: not Preventive Cardiology], and is free to access.
https://onlinelibrary.wiley.com/doi/abs/10.1002/clc.23047

Three of the authors work at Fleming's medical imaging company in California, one is a deceased psychologist from Iowa, another is a pediatric nutritionist from New York and one is a Kellogg's employee from Illinois.
How this group was able to run a 12-month diet trial in 120 subjects is something of a mystery.

The conclusions:

One‐year body mass changes did not differ by diet (P .999). Effect sizes (R, R2) were statistically significant for all indices. Coronary blood flow, R (CI 95%) = .48 to .69, improved with low‐to‐moderate‐fat and declined with lowered carbohydrate diets. Inflammatory factor Interleukin‐6 (R = .51 to .71) increased with lowered carbohydrate and decreased with low‐to‐moderate‐fat diets.
Conclusions
One‐year lowered‐carbohydrate diet significantly increases cardiovascular risks, while a low‐to‐moderate‐fat diet significantly reduces cardiovascular risk factors. Vegan diets were intermediate.

First I'll consider all the good reasons to reject this study, but after that I'll do something we should always do, even for the worst study - take it at face value.

1) Lead author Dr Richard M. Fleming is a self-confessed and convicted felony fraudster who has admitted falsifying data in another RCT.

Fleming admitted to knowingly executing and attempting to execute a scheme to defraud Medicare and Medicaid healthcare benefit programs in connection with the delivery of and payment for healthcare benefits, items, and services, namely by submitting payment claims for tomographic myocardial perfusion imaging studies that he did not actually perform. Fleming also pled guilty to one count of felony mail fraud in violation of 18 U.S.C. 1341 and 2 for conduct relating to money paid him to conduct a clinical study of a soy chip food product for the purpose of evaluating health benefits. As Fleming admitted during his guilty plea, he received approximately $35,000 for conducting a clinical trial, but he fabricated data for certain subjects.
https://www.federalregister.gov/documents/2018/09/28/2018-21210/richard-m-fleming-denial-of-hearing-final-debarment-order

2) Fleming obtained Robert Atkins' medical records by deception and shared them with Neal Barnard of the vegan activist group PCRM in 2004, another unethical behaviour and one demonstrating that Fleming has a long-standing animus against Atkins and his diet.

https://theskepticalcardiologist.com/2016/12/18/what-does-the-death-of-robert-atkins-tell-us-about-the-atkins-diet/

Now, vegans can do research into this exact question with a high standard of rigour, see Chris Gardner's studies - one can certainly dispute the interpretation of some results, but not the results themselves. And I have reviewed a vegan diet study favourably here (dealing with another Fleming paper in passing) - good results are good results; I don't doubt these diets can have also cardiovascular benefits over the short-to-medium term, but question their long-term effects on mental health, reproductive health, dental health, joint health etc.

3) The trial protocol number on the paper links to a study that was completed in 2002. This explains how a convicted felony fraudster was able to conduct a study. The protocol was posted in 2006, 4 years after the study concluded, which seems unusual on the Clinical Trials website.


https://clinicaltrials.gov/ct2/show/NCT00324545

4) The study has 35 citations - 15 of these are to Fleming's own papers. This self-spamming, which helps boost an author's citation rate, is frowned upon by reputable journals. One of these references has the word "quantum" in the title. Other references are to news articles and book chapters. The low carbohydrate diet references are more than 15 years old. Ref 16 is curious as an anonymous reviewer is given credit for the wording of a paragraph of interpretation, surely a run-of-the-mill interaction with a reviewer.

5) The novel aspect of this paper may lie in the reference to that novel vegan touchstone, Neu5Gc. Pro-tip - if the vegan diet had a magic mechanism, you ought to know it already; some major low-carb mechanisms have been understood for generations. At this rate, if there is a magic mechanism for vegan health benefit, it will be discovered by a low-carb scientist.

We  now  know  that  these  food  choices  and  their  impact  are  at  least partially precipitated by the inflammatory effect of our diets based given our inability to convert Neu5Ac to Neu5Gc and our bodies immune response to the Neu5Gc present in animal protein. 

At this point, let's take the study at face value. the vegan diet avoided animal protein and Neu5Gc, the low carb diet probably included twice as much protein as the other diets (based on reference 6), but the low fat diet included more animal protein and Neu5Gc than the vegan diet.

One‐year lowered‐carbohydrate diet significantly increases cardiovascular risks, while a low‐to‐moderate‐fat diet significantly reduces cardiovascular risk factors. Vegan diets were intermediate.

So Fleming's own study, taken at face value, doesn't support the Neu5Gc hypothesis. In fact, it's unusual for the vegan diet to be inferior to the low-fat diet in any vegan study, and it's unusual for the low carb diet to be inferior to the low-fat diet in any low-carb study.

6) adherence to diets over 12 months was 100%. Of course, this is unheard of and entirely implausible; if honestly reported, it seems to show considerable gullibility or self-deception in the study team.


 That 100% of participants continued on their respective diet plans through a full year of dieting contrasts  sharply  with  much  of  diet  research  experience  with  drop outs  and  with  common experience with difficulties of dieting and remaining on diets. This success can be attributed to attention to well-established psychological principles of habit acquisition and extinction and of behaviour modification through Bandura [17] counseling.

Bandura's ethos seems sensible enough and appropriate for such a project, except perhaps when the people using it for counselling already believe that one approach is preferable to another.

7) the original report of the 2002 study (ref 6), if it is the same study, reports diet groups differently.





8) Implausible randomisation was the red flag that saw the PREDIMED study and many others retracted. Here randomisation of n=120 into 6 groups produced this result:


The  58  female  and  62  male  participants  were  randomly  assigned  to  equal  dietary  groups  by casting  a  die.  There  were  no  statistical  demographic  differences  between  group  assignments. There  were  no  statistically  significant  differences,  or  even  trends,  between  diet  groups  at  the initiation of the study. Since the groups were unequivocally randomized for all fifteen-baseline indices, statistical inference to the initial population, described by Table 1, is appropriate. 


9) Fleming et al state "A  four-month  post-intervention  analysis was obtained to determine post-intervention treatment, which has not previously been reported in the literature."

Post intervention status was in fact reported at 4 years by the Shai et al DIRECT study group.



10) the sponsor is listed as the Camelot Foundation. A search turned up this mention - Dr Fleming is the editor of a predatory journal, and the Camelot Foundation has little other existence online, it seems to be a 501(c)(3) legal tax-avoidance scheme within Fleming's own business.
https://www.omicsonline.org/editor-profile/richard-maximus-fleming/

11) Cardiovascular improvement by Fleming's medical imaging method correlates with improvement in the TG/HDL ratio. Taken at face value, although TG/HDL doesn't improve in Fleming's "low carb" arm, it does in most of the people reading this who have tried a low carb approach, so if Fleming's diagnostics are accurate this is not bad news. Interleukin 6 also improves during fasting but not a ketogenic diet in a 6-day study, but improves in a low carb diet vs a low fat diet in a 6 month study here (as there was at least one previous study in the literature that came to different conclusions from Fleming et al with regard to an outcome they highlighted, this should really have been cited).





Both LFD and LCD led to similar reductions in body weight, while beneficial effects on glycaemic control were observed in the LCD group only. After 6 months, the levels of IL-1Ra and IL-6 were significantly lower in the LCD group than in the LFD group, 978 (664–1385) versus 1216 (974–1822) pg/mL and 2.15 (1.65–4.27) versus 3.39 (2.25–4.79) pg/mL, both P < 0.05.

Taken at face value, Fleming's possibly fraudulent paper predicts cardiovascular benefit from a low carb diet if people get different results from the ones he claims to have produced, which is usually the case in other studies and in real life...


The question is, how did this paper pass peer review with all the red flags above? [edit]



Credit to @MacroFour and Ivor Cummins @FatEmperor for the links regarding Dr Fleming's colourful past.


Tuesday, 18 September 2018

A Grand Unified Theory of Polyunsaturated Fatty Acid Misbehaviour in Inflammatory Disease

One of the great mysteries of nutrition is the behaviour of polyunsaturated fatty acids (PUFAs). They often look good in the kind of sloppy epidemiology used to drive or latterly protect dietary guidelines*, are more ambiguous in RCTs, and can easily be shown to have deleterious effects in a number of specific medical and experimental conditions that might be expected to have a "canary in the coalmine" validity as warnings when it comes to the longer-term effects of consuming more, sometimes much more, that the essential nutrient requirement for these functional molecules (which is, at a rough consensus, around 3% of energy, with 1% coming from omega-3 PUFAs).

However, higher intakes are sometimes tolerated well; any fairly liberal ketogenic diet including pork or olive oil or nuts or avocado will almost certainly exceed 3%, and even though PUFA over 3% is almost a requirement for the induction of NAFLD, Browning et al reversed NAFLD quickly with a ketogenic diet supplying 15%E as PUFA.[1]

So what gives? What is the nature of the interaction between PUFAs and other dietary components or metabolic states that produces inflammation?

In an earlier blog post I identified the enzyme systems upregulated in NAFLD as those of the microsomal ethanol oxidase system (MEOS) and also showed that the evolutionary function of the MEOS is to degrade PUFA, rather than alcohol which is a latecomer to our diets.
But what activates the MEOS when alcohol does not? How, for example, does fructose send PUFAs down this pathway, and how does this promote inflammation?

I found a clue in this hepatitis C editorial by Jenny Heathcote on a study in which weight loss improved liver function.[1] This is some quite brilliant speculation.

Here is the description of fatty liver due to insulin resistance (HCV causes IR by a pharmacological action of its core protein):

In peripheral tissues, insulin normally downregulates the hormone sensitive lipase (HSL) enzyme responsible for hydrolysis of stored triglycerides from free fatty acids within adipocytes. In patients who are insulin resistant, this enzyme is no longer suppressed. In addition, counterregulatory hormones such as catecholamines, glucagon, and growth hormone are increased in response to increased circulating insulin levels. These counterregulatory hormones stimulate HSL to hydrolyse more triglycerides into free fatty acids, the end result being an increased flux of dietary and stored free fatty acids away from the adipose tissues and towards the liver. Unfortunately, Hickman et al did not measure free fatty acid levels before or after the weight reduction programme. Within the liver, insulin upregulates esterification of free fatty acids to triglycerides. Once the triglycerides are formed, insulin downregulates the secretory pathways, thus favouring increased storage of triglycerides in the cytosolic pool. Furthermore, free fatty acids can themselves upregulate the esterification pathway. The net result is a positive feedback cycle contributing to an ever increasing amount of free fatty acids and triglycerides in the liver. Thus portal hyperinsulinaemia leads to hepatic steatosis.

And here is the description of how hepatic steatosis influences PUFA disposal:

 These studies have suggested that the presence of fat in patients with hepatitis C is associated with markers of progressive liver disease in that fat was associated with increased stellate cell activation, but the mechanism by which this takes place is uncertain. It is possible that this occurs secondary to saturation of beta oxidation pathways within mitochondria which then leads to free fatty acids becoming more available to intracellular microsomes where they undergo lipid peroxidation. There are three main products of microsomal lipid peroxidation: malondialdehyde, 4-hydroxynonenal, and hydrogen peroxide. Malondialdehyde has been shown to activate stellate cells to produce fibrin, and may be responsible at least in part for liver fibrosis in patients with non-alcoholic steatohepatitis.

Malondialdehyde (MDA) and 4-HNE are unsaturated products of PUFA, and H2O2 is also a step in the MEOS disposal of PUFA, requiring catalase for its reduction to H2O + O.




We can see how this relates to the "essential" role that PUFA plays in the development of alcoholic liver disease; not only can the liver become fatty from the conversion of alcohol to triglycerides, but also the disposal of excess ethanol through the MEOS has upregulated this enzyme system (hepatic CYP2E1 is upregulated 10-fold by ethanol); to add insult to injury, the liver's ability to dispose of excess fat via beta oxidation is impaired by the depletion of NAD+ during the conversion of ethanol to fat.

But another clue was supplied by Tucker Goodrich, the PUFA ninja, who found a rodent study showing that 4-HNE and 9-ONE could themselves be cleared if beta-oxidation pathways were upregulated enough, that is, by a ketogenic diet.[3]

Our results showed that livers from rats fed ketogenic diet or high fat mix diet had high ω-6 polyunsaturated fatty acid concentrations and markers of oxidative stress. However, high concentrations of HNE (1.6 ± 0.5 nmol/g) and ONE (0.9 ± 0.2 nmol/g) were only found in livers from rats fed the high fat mix diet. Livers from rats fed the ketogenic diet had low HNE (0.8 ± 0.1 nmol/g) and ONE (0.4 ± 0.07 nmol/g), similar to rats fed the standard diet. A possible explanation is that the predominant pathway of HNE catabolism (i.e. beta oxidation) is activated in the liver by the ketogenic diet. This is consistent with a 10 fold decrease in malonyl-CoA in livers from rats fed a ketogenic diet compared to a standard diet. The accelerated catabolism of HNE lowers HNE and HNE analog concentrations in livers from rats fed the ketogenic diet. On the other hand, rats fed the high fat mix diet had high rates of lipid synthesis and low rates of fatty acid oxidation, resulting in the slowing down of the catabolic disposal of HNE and HNE analogs. Thus, decreased HNE catabolism by a high fat mix diet induces high concentrations of HNE and HNE analogs. The results of the present work suggested a potential causal relationship to metabolic syndrome induced by western diets (i.e. high fat mix), as well as the effects of the ketogenic diet on the catabolism of lipid peroxidation products in liver.

So - any state in which beta-oxidation is inhibited, but fat is present, will see PUFA shunted into the microsome - essentially the MEOS - and a high production of damaging peroxides and aldehydes. This also happens when mice are fed a ketogenic diet, but the aldehydes can be disposed of by beta-oxidation.
Note that the high fat (non-keto) diet in the mouse study was the Surwit diet relatively low in PUFA and MUFA (coconut and soy oil), overloading beta-oxidation with a mixture of ~50% saturated fat and 22.5% sucrose. Don't try this at home, kids. 


For reasons of time I haven't gone into every possible ramification such as the role of peroxisomal oxidation in PUFA disposal, the proper function of the MEOS (making and disposing of eicosanoids), the hormetic effect on antioxidant systems of low level HNE production, and the difference between liver and other fat-burning tissues (i.e. is this relevant to heart disease if the same thing happens in muscle, macrophages, or endothelial cells? Magic 8 ball says very probably).

However, here's a model that allows us to predict and explain the likely role of PUFA in inflammatory diseases at a metabolic level. Especially, for now, liver diseases.




* FFQ epidemiology studies are notoriously inaccurate at capturing intakes of calories (and protein, which often looks wonky in epidemiology). They really can't tell you in what context PUFA is being consumed, and in any case it's hard to see how deep frying oil in food can really be measured - do you even know what your chips (fries) are cooked in and in what part of the FFQ would you put this information?


[1] Browning JD, Baker JA, Rogers T et al. Short-term weight loss and hepatic triglyceride reduction: evidence of a metabolic advantage with dietary carbohydrate restriction. Am J Clin Nutr. 2011 May; 93(5): 1048–1052.

[2] Heathcote J. Weighty issues in hepatitis C. Gut. 2002;51(1):7-8.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1773268/

[3] 
Li Q, Tomcik K, Zhang S, Puchowicz MA, Zhang G-F. Dietary-regulation of catabolic disposal of 4-hydroxynonenal analogs in rat liver. Free radical biology & medicine. 2012;52(6):1043-1053. doi:10.1016/j.freeradbiomed.2011.12.022
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289253/





Monday, 18 June 2018

Never attribute to pathology what can be adequately explained by adaptive physiology

When this paper stumbled across my desk the other day my first thought was of course "Aha! Linoleic acid not so hot, this explains lack of benefit in RCTs as analysed by Steve Hamley".[1]

Unsaturated Fatty Acids Inhibit Cholesterol Efflux from Macrophages by Increasing Degradation of ATP-binding Cassette Transporter A1.[2]

http://www.jbc.org/content/277/7/5692.full


I still think, maybe it does - once there's a pathology in the house, which was often the case in those RCTs - but I can think of an alternative explanation, connecting this paper with my previous post.

It's not obvious why MUFA (oleic acid is the only MUFA ever worth considering, which is odd given the diversity of saturated fats and their effects) should be bad for reverse cholesterol transport. It's in everything that supplies SFA in, overall, comparable amounts, and is 60% of your adipose fat.

Unsaturated Fatty Acids Reduce Cellular ABCA1—Because
ABCA1 controls the rate of apoA-I-mediated lipid efflux, we
assayed the effects of fatty acids on the cell membrane content
of ABCA1. Incubating cells with unsaturated fatty acids caused
a significant decrease in membrane ABCA1 (Fig. 4A). In contrast,
saturated fatty acids had no or little effect on ABCA1
levels. As with lipid efflux, oleate and linoleate reduced ABCA1
membrane protein in a concentration-dependent manner (Fig.
4B). We compared the effects of stearate and linoleate on the
plasma membrane content of ABCA1 by treating cells for 6 h
with fatty acids, biotinylating cell-surface proteins, isolating
ABCA1 by immunoprecipitation, and assaying for biotinylated
(cell-surface) ABCA1 with a streptavidin probe. Results
showed that linoleate, but not stearate, reduced both the total
and plasma membrane content of ABCA1.

Nutritional studies have shown that different fatty acids
have diverse effects on lipoprotein metabolism. It is believed
that substituting dietary saturated fatty acids with cis-unsaturated
fatty acids protects against cardiovascular disease by
lowering plasma LDL levels (reference is Hu et al the way to Willett, 1997).
Our results suggest that,
although reducing atherogenic particles, these dietary manipulations
may suppress cholesterol efflux from macrophages.
This may partially explain why a meta-analysis of clinical
trials showed only a small cardiovascular risk benefit with
modified dietary fat intake.(Ref is Hooper et al, 2001)

The culture medium DMEM supplies 1000mg/L glucose, that's 100 mg/dL or 5.5 mmol/L.





But think - what is the use of this? Remember the last post - UFAs promote fat storage, SFAs do not.
If the macrophage is storing fat, it needs to retain some cholesterol; if it cannot store fat, because all the fat is SFA and mammalian cells cannot synthesise a TG from 3 SFAs, it might as well release cholesterol normally.
That's what I think is going on. Of course, if the macrophage is always storing fat and cholesterol because glucose and insulin are always high, that's part of the pathology (excess lipid droplet formation leading to creation of foam cells), and functionality will be impaired by UFAs in the manner described in this paper as regulation fails to keep pace with overactivation, but we also have to think of every cell in the body as not only performing a function but also as an obligate consumer of the body's different fuels. Even macrophages need something to eat, and even macrophages might want to put a little aside for later when there's a lot on the plate.
An additional question is whether macrophages synthesise cholesterol from LA, as hepatocytes do.

I like the way these authors understand CVD as a disease of cholesterol retention, rather than excess LDL per se. This is a view often kicked to the kerb by those who only have LDL-lowering meds to sell. The European Heart Journal, for example, seems to publish an editorial bashing HDL every other week. We get it, HDL is not a drug target, and not much of a genetic lottery either, but it is still a part of the system nature supplied to regulate the accumulation of cholesterol in cells, and worth making friends with.




[1] Hamley, S. The effect of replacing saturated fat with mostly n-6 polyunsaturated fat on coronary heart disease: a meta-analysis of randomised controlled trials. Nutr J. 2017; 16: 30

[2] Wang, Y, Oram, JF. Unsaturated Fatty Acids Inhibit Cholesterol Efflux from Macrophages by Increasing Degradation of ATP-binding Cassette Transporter A1. February 15, 2002

The Journal of Biological Chemistry. 277, 5692-5697.

Tuesday, 3 April 2018

Uncoupling - Saturated fatty acids and glucose are preferred muscle fuels, but unsaturated fats act as buffers

An intriguing new study looked at 2 different types of enteral feeding in 60 critically ill patients for 7 days. The fat-based formula was 37%E glucose, so this was not a test of a low carb diet, and predictably the differences in glucose and insulin AUC, though trending in the right direction, were not significant.[1]
The significant finding was higher resting energy expenditure (REE) in the higher-fat group.
In my opinion this was not an effect of higher fat feeding but an effect of a high intake of a particular type of fat – no-one in the real world would ever eat 45% of energy as fat from rapeseed and sunflower oil exclusively (if nothing else, natural protein foods would supply other fats not found in the protein isolate used here).

“Fat-based EN formulas contain 45% fat, 37% carbohydrates, 18% protein, and 2.3 g of fiber per 100 ml, whereas glucose-based EN formulas are comprised of 30% fat, 55% carbohydrates, 15% protein, and contain 1.5 g of fiber per 100 ml. Both formulas have a caloric density of 1 kcal/ml and contain rape seed oil and sunflower oil. Initial assessment of resting energy expenditure (REE) was performed for each patient using the technique of indirect calorimetry. Target energy was 25% above the measured REE [13]. Both study groups received early EN that was initiated with the target dosage and continuously administered at a constant rate for 7 days via a nasogastric tube.”

The diet was very high in monounsaturated and polyunsaturated fat, and very low in saturated fat.
Unsaturated fats are well-known to activate uncoupling proteins in the mitochondria of muscle and adipose cells (in brown adipose tissue, there is good evidence that saturated fats can drive uncoupling; brown adipose is a highly functional cell type that exists for this sort of thing rather than storage, so I’m going to ignore it for now).[2,3]




I’m really interested in fuel use by muscle. The big, novel question in physiology today bar none is the lean mass hyper-responder lipid profile discovered by Dave Feldman (@DaveKeto). Because this relates to muscle mass/fat mass (and activity) ratio, and because different fatty acids in people eating normal diets have differential effects on lipid profiles, it’s necessary to know how muscles use fats before we investigate whether this can influence a lipid profile.

Effect of fatty acids on D-[U-14C]glucose oxidation in 1h incubated rat soleus (A) and extensor digitorium longus (EDL) (B) muscles. Muscles were incubated for 1 h in the absence or presence of 10 mU/mL insulin and/or 100 μM of different fatty acids.

Here’s a study on two types of muscle cell isolated from rats which shows a different effect of saturated vs unsaturated fats in extensor digitorium muscle (the soleus muscle, A, is less clear but I'm going with B for now; however the faster oxidising (medium chain) SFAs in A behave like palmitate in B).[4] To summarise the findings, unsaturated fats activate uncoupling; that is, a proportion of the potential energy released by their beta-oxidation is wasted, instead of generating ATP (the more double bonds, the more uncoupling). And this wastage – which will produce extra heat - allows the cell to burn extra glucose at the same time to make up the shortfall in ATP.
This is what is meant by unsaturated fats improving insulin sensitivity. Glucose and saturated fatty acids are the two preferred fuels of muscle cells, but they exist in competition. At times of energy excess, they would be at loggerheads if both were available together without other “softer” fuels. The effect of unsaturated (uncoupling) fats is to buffer the potentially harmful effect of this competition, by occupying the beta-oxidation mechanism (carnitine etc) yet leaving some ADP free for both glucose and SFA catabolism to convert to ATP. When glucose is restricted, the saturated fat level of the blood falls, despite a higher intake, because the competing effect of glucose and its insulin-driven uptake is removed. At this extreme, the buffering effect of unsaturated fat is unnecessary. At fat intakes below 37%, on the other hand, a differential effect on insulin sensitivity can more easily be detected, because glucose is the primary fuel, and insulin is driving SFA synthesis and retention.

(Thus the low fat diet, especially with refined carbs at current availability, was the very thing that painted us into the corner where there might be some reason to worry about the types of fat we use! Who the hell wants to be in that shithole.)

Strictly speaking we don’t need to consume unsaturated fats (beyond tiny EFA amounts of PUFA) because we can make oleic acid MUFA from SFA by DNL elongation and desaturation, although there is genetic variation in the ability to do this. Realistically speaking, this separation of SFA from MUFA in the diet is never going to happen anyway. Humans eat fat of all types, not SFA, MUFA or PUFA.

Although linoleic acid was an uncoupling fat in muscle in vitro, muscle in vivo may not be using much of this fuel. LA has a high rate of conversion to other lipids (cholesterol and SFA) in liver, is used to make eicosanoids and is otherwise peroxidizable, and is still stored in adipose in amounts that seem excessive in proportion to dietary intakes. In practical terms, oleic acid (C18:1) is probably always the dominant uncoupling fatty acid in muscle, and the more unsaturated fats (which would uncouple more) are lousy fuels. This might(?) help to explain the prevalence of CPT1A mutations, which suppress fatty acid oxidation on high fat diets, in populations with a high take of fat from oily fish (Inuit, Faroe Islands, Northern Japan).[5]

There’s another pathway by which UFA protects against SFA-glucose competition in muscle – in humans, triglyceride synthesis always requires at least one UFA.[6] So you can’t store any excess of SFA that turns up in a cell without some UFA; this is also a form of buffering that clears the track for whichever of the preferred fuels is dominant. Without it, incoming glucose would drive SFA elongation into excess ceramide, and the cell would be stuffed.

So in our critically ill population, an enteral diet very high in unsaturated fats produced a higher REE through uncoupling, and improved insulin sensitivity non-significantly (the control diet was relatively high in UFA by normal standards anyway). I’m not sure what the split of muscle vs adipose and other tissue fuel use would have been for bed-rest REE (I'm only using that study for a kicking-off point here). I’m told that elevated REE isn’t desirable in the critically ill. Maybe one day a more sensible enteral formula including, say, beef fat will be tested.

If you are overeating on a higher carb diet, the various energetically futile aspects of UFAs could be protective of your metabolism (but the eicosanoids and peroxidation products of PUFAs, especially LA, could well catch up with you eventually if you rely on those rather than MUFA). If you are restricting carbs, working hard, undereating or IF, or otherwise burning fat, do you really want to generate a lot of heat for less available energy? I can’t see a high degree of uncoupling being of benefit in endurance activities where heat loss is maxed out. I can’t see it being anything but exhausting. Even the Inuit may(?) have evolved to side-step it to some extent.
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references


[1] Wewalka M, Drolz A, Seeland B, et al. Different enteral nutrition formulas have no effect on glucose homeostasis but on diet-induced thermogenesis in critically ill medical patients: a randomized controlled trial. European Journal of Clinical Nutrition. 2018;72,496–503
doi:10.1038/s41430-018-0111-4

[2] Graier WF, Trenker M, Malli R. Mitochondrial Ca2+, the secret behind the function of uncoupling proteins 2 and 3? Cell calcium. 2008;44(1):36-50. doi:10.1016/j.ceca.2008.01.001.

[3] Romestaing C, Piquet M-A, Bedu E, et al. Long term highly saturated fat diet does not induce NASH in Wistar rats. Nutrition & Metabolism. 2007;4:4. doi:10.1186/1743-7075-4-4. LINK

[4] 
Hirabaraa SM, Silveiraa LR, Alberic LC, et al. Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle. Biochimica et Biophysica Acta (BBA) - Bioenergetics
Volume 1757, Issue 1, January 2006, Pages 57-66. LINK

[5] https://yk-health.org/images/3/36/Arctic-Variant-CPT-1.pdf

[6] Henique C, Mansouri A, Fumey G, et al. Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis. J Biol Chem. 2010;
285, 36818-36827. LINK


Tuesday, 30 January 2018

My first podcast interview, over at Break Nutrition

Raphi Sirt of Break Nutrition interviewed me last week for a podcast. This was a far-ranging interview that took me to some unexpected places - but was a chance to expand on the big-picture stuff and the unanswered questions around the situational hypercholesterolaemia that marks the lean mass hyper-responder phenotype and the lessons about insulin resistance hidden in statin trials.

http://breaknutrition.com/episode-37-george-henderson-takes-us-tour-public-health-research/

"But who really knows what this all means?"


For (much) more background on the LMHR question, please also visit Dave Feldman's blog. If you don't know his work, and you're interested in lipids (whether yours or other peoples') I promise your mind will be blown by the Inversion Pattern amongst other matters.

http://cholesterolcode.com/are-you-a-lean-mass-hyper-responder/

http://cholesterolcode.com/impact-of-endurance-running-on-cholesterol/

Also look into the other Break Nutrition podcasts - I know Tucker Goodrich and Gabor Erdosi recorded a very interesting discussion recently, and I'm going to see what else there is.




Tuesday, 5 December 2017

Fibre and the risk of Type 2 Diabetes - the InterAct meta-analysis

Recently the Australian government publicised claims generated by Nutrition Australia, in an opinion paper funded by Kellogg's, that Australians increasing their cereal fibre intake could reduce the cost of CVD and diabetes to the Australian economy:

This research demonstrates that if Australian adults use grain fibre to increase their intake of dietary fibre to target intake levels for chronic disease risk reduction (28g for women, 38g for men):
• The potential healthcare expenditure savings would be approximately $1 billion for CVD and over $285 million for T2D in 2015–16. The savings for CVD would represent approximately 0.6% of total Australian health expenditure and savings for T2D would be around 0.2% of health expenditure.
• The potential productivity cost savings were estimated to be approximately $600 million for CVD and $1.4 billion for T2D. The savings for CVD represent approximately 0.04% of gross domestic product (GDP) and for T2D, approximately 0.08% of GDP.
The total combined economic savings could potentially reach $3.3 billion.


Zoe Harcombe looks into the evidence base here and finds it lacking; however as her article requires a subscription and she asked me to look at the evidence to try to make sense of some convoluted manipulations, I'm going to discuss some extra aspects of the main type 2 diabetes study used, the EPIC-InterAct study and its meta-analysis.[1]

This paper presents the results from a large multicentre epidemiological study from the EPIC cohorts, followed by what is supposed to be a meta-analysis of prospective epidemiological studies (more on that later).The EPIC-InterAct data is the "news" here, and it doesn't support the hypothesis that cereal fibre prevents type 2 diabetes when adjusted for age and sex, or for "lifestyle, diet and BMI" but does in the purely lifestyle and diet adjusted models.
However, if we look at the forest plot, we can see that the associations are all over the place, and there are many countries without a protective association, including France with a non-significant HR of 1.72. Another outlier is the UK with an HR of 0.74 (NS).

What are the differences in these populations? The French cohorts, for some reason, are all 100% female, which isn't the case for any other country. And the largest of the two UK cohorts is EPIC-Oxford.[2]
"The majority of participants recruited by the EPIC Oxford (UK) centre consisted of vegetarian and “health conscious” volunteers from England, Wales, Scotland, and Northern Ireland"
So these health conscious volunteers are probably being compared with people with lower fibre intakes in the less health-conscious UK cohort.
Sweden always interests me because one of their two cohorts is the Malmo Diet and Cancer Study, which uses a 7-day food diary for all subjects, and in Sweden the HR is a more reasonable 0.96. So less confounding by conscientiousness and more accurate diet recall tends to minimize the cereal fibre and type 2 diabetes association. If cereal fibre prevented type 2 diabetes in any important way, it should show up in most of these populations, not just a few.

The InterAct authors then follow up this rather inconsistent evidence by including it in a meta-analysis of all other prospective cohort studies on the question. This is an example of advocacy epidemiology - our study didn't convincingly support our belief, so we'll incorporate it into the totality of less relevant evidence that does. Presumably an intention of InterAct is to inform European dietary recommendations, for which the European evidence is most relevant, and for which it seems to say that the effect of cereal fibre on type 2 diabetes risk depends a great deal on what country you're a citizen of, so cannot be guaranteed either way.

Lo and behold the larger meta produces the strongly supportive associations that informed the Australian Kellogg's paper.
But hang on.
At least one of the studies included, the Finnish Diabetes Prevention Study, isn't a prospective cohort study at all - it's a mixed intervention of diet and exercise advice vs no intervention.
This simply should not have been included. It's a small study so shouldn't have biased the result too much if at all, but it doesn't inspire confidence that the selection of studies for inclusion was as rigorous as it should have been. (It's discussed and excluded from an analysis in a supplementary paper, so this isn't an error).
There are 3 Australian studies, two small ones with protective associations and a large one, Hodge 2004, with none.[3] However Hodge 2004 finds that white bread is associated with type 2 diabetes, and that lower GI carbs, including sugar, aren't. White bread in Australia is so bad that even sugar looks good by comparison. Does it follow that putting a few grams of bran in white bread will improve it? Why not just say "avoid white bread"? Anyway, if fibre isn't associated with type 2 diabetes in a fairly large sample of Australians, who as Zoe Harcombe pointed out tend to have fairly high fibre intakes anyway by OECD standards, why do Aussies need to take their lead from the USA?
Because most of the weighty studies in favour of fibre for type 2 diabetes prevention in the meta-analysis come from the USA. There are two important facts about the USA - low fibre intakes are lower than they are anywhere else (so basically a high intake of deep fried food, white breads, and soda in these groups), and conscientiousness is an identifiable confounding factor in many populations. For example, the Nurses' Health Study and Health Professionals' Follow-up Study; here we have the populations not only given the most advice about fibre being healthy, but also given the job of passing it on to the other US populations. In other words, grain fibre - like red meat - is one of the signifiers separating conscientious Americans from other Americans (it's harder to eat fruit and vege without their fibre, and vege in the US includes fried potatoes). It's almost a class distinction.


Look at this subgroup analysis of the "dose response" (ESM Table 3) - it's ALL about the USA. Zoe and I couldn't get our heads around how this "dose-response" was calculated from such diverse studies that all had differing groupings and cut-offs, but even taken at face value, why would an Australian government claim it showed any health savings for Australians?
If we compare diabetes prevalence between countries with mean fibre intakes, the USA with its abnormally low fibre intake for a high-cereal society fits, but other countries don't.
"The mean±SD fibre intake in the subcohort was 22.9± 6.2 g/day (ranging from 19.9 g/day in Sweden to 25.2 g/day in Denmark; data not shown)."
Germany (7.40%) and Denmark (7.20%) have higher diabetes prevalence rates than Sweden (4.70%).  USA's mean total fibre intake is 16.1 g/day and diabetes prevalence is 10.80%.
Germany and Denmark were the other populations in EPIC-InterAct where fibre had some protective association with type 2 diabetes. I'm not sure about T2D, but the amount of wholegrain associated with a modest degree of ischaemic CVD protection in Malmo was only 2.5 servings a day, and there was a protective association between fibre (mostly cereal) and saturated fat (a high SFA, high fibre diet was the most protective combination).[4,5]
We are probably seeing - in all these studies - a protective effect of eating high quality food with a minimal human interference factor (dairy and ryebread in Scandinavia) and belonging to the social class that is more likely to do this. With regard to iCVD bran may be a "failsafe" source of silicon, needed for vascular resilience and repair (you can get silicon from other foods and water, but bran in a staple food would guarantee it was present in the diet), but it is hard to see how this applies to T2DM. If a microbiotal mechanism was strongly involved, presumably other types of fibre would be more protective than they are. I come back to what whole grains are not. They are not white bread, and what is white bread? It used to be made with alloxan, a chemical used to produce experimental diabetes in animals. Today it contains

INGREDIENTS:  White Bread (Enriched Wheat Flour (Flour, Malted Barley Flour, Niacin, Iron(Ferrous Sulfate, Reduced Iron), Thiamine Mononitrate, Riboflavin, Folic Acid), Water, Yeast, Salt, Soybean Oil, Sugar, Malt, Dough Conditioners(Ascorbic Acid, Calcium Sulfate, Sodium Stearoyl Lactylate),Calcium Propionate(To Retard Mold Growth))
ALLERGENS:  Wheat, Soybeans, Gluten

This Harvard-supplied list is incomplete - NZ white bread contains soy protein; Tip Top's omega 3 loaf supplies;

Wheat Flour, Water, Soy Fibre, Baker's Yeast, Wheat Gluten, Vinegar, Iodised Salt, Vegetable Gum (412), Soy Flour, Canola Oil, Tuna Oil (0.05%) (Contains Fish, Soy), Milk Protein (Sodium Caseinate), Emulsifiers (481, 471, 472e), Vitamins (Thiamin, Folate, Vitamin E, Niacin, Vitamin B6), Minerals (Iron, Zinc).

Woah - the fibre isn't even grain fibre, and there are 3 emulsifiers. Emulsifiers are experimentally linked to obesity and diabetes.
So it's hard to rule out that whole grains just tend to replace a cause of type 2 diabetes in some societies.
So, if you're an American eating a lot of starch from fibre-free foods, replacing these with whole grain foods (which for one thing won't be cooked in oil) should decrease your risk of type 2 diabetes. Replacing them with anything closer to nature will likely have the same effect.
If you're a European, Brexit will be bad news, because you won't be able to move to the one place where fibre is really protective, but you can still go to Sweden and enjoy the best of both worlds.
If you're an Australian, ask yourself, do I eat like the average American who isn't health conscious? If the answer is yes, then sprinkling a bit of bran on your food won't save you. But if you avoid bread or cereals altogether, are you at extra risk of type 2 diabetes? That's what we really want to know, and what none of the epidemiology can tell us at all, unless it's the risk marker epidemiology, which as far as I know says low TG/HDL, high LDL, low HbA1c = lowest T2DM risk.

References

[1] The InterAct Consortium. Dietary fibre and incidence of type 2 diabetes in eight European countries: the EPIC-InterAct Study and a meta-analysis of prospective studies. Diabetologia. 2015;58(7):1394-1408. doi:10.1007/s00125-015-3585-9.ghbhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472947/

[2] The InterAct Consortium, Langenberg C, Sharp S, et al. The InterAct Project: An Examination of the Interaction of Genetic and Lifestyle Factors on the Incidence of Type 2 Diabetes in the EPIC Study. Diabetologia. 2011;54(9):2272-2282. doi:10.1007/s00125-011-2182-9.

[3] Hodge AM, English DR, O'Dea K, Giles GG. Glycemic index and dietary fiber and the risk of type 2 diabetes. Diabetes Care. 2004;27:2701–2706. doi: 10.2337/diacare.27.11.2701
[4] Wallström P, Sonestedt E, Hlebowicz J, et al. Dietary Fiber and Saturated Fat Intake Associations with Cardiovascular Disease Differ by Sex in the Malmö Diet and Cancer Cohort: A Prospective Study. Obukhov AG, ed. PLoS ONE. 2012;7(2):e31637. doi:10.1371/journal.pone.0031637.

[5] Sonestedt E, Hellstrand S, Schulz C-A, et al. The Association between Carbohydrate-Rich Foods and Risk of Cardiovascular Disease Is Not Modified by Genetic Susceptibility to Dyslipidemia as Determined by 80 Validated Variants. Müller M, ed. PLoS ONE. 2015;10(4):e0126104. doi:10.1371/journal.pone.0126104.