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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]

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.


[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

[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

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


[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.

"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.

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.