Saturday, December 11, 2021

Protons (67) a formula revised for butter oil

A couple of months ago Tucker emailed me this study

Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome

The study itself, while interesting, is not the point. The point is that the diet used contained the infamous 42% of calories from fat and was obesogenic. In common with a number of other studies I have been mulling over, the fat was butter oil. This is the butter oil composition

which has 2.3% linoleic acid in 42% of calories giving just over 1% of calories as LA. All I am interested in here is the comparison between 14 weeks on control low fat diet (CD) vs 14 weeks on a low linoleic acid Western Diet (WD). Here are the fat mass data (bodyweight mirrored this)

and here are the IPGTT results. We're just comparing the black control (CON) with the red western diet (WD) lines

So I think we can say that 14 weeks on butter oil is obesogenic and has the appropriate insulin resistance as we expect from any obesity with its increased basal lipolysis of large adipocytes coupled with Protons effects on the non adipose tissues.


The diet looks like this:

If anyone thinks this looks like fudge, here is the recipe for a kilo of the stuff

Take 340g of table sugar, add 200g of anhydrous butter fat, bulk it up with 150g of corn starch to help it set and throw in some casein if you feel like it. Mix and extrude (you could cut in in to cubes for human consumption).


Looks yummy. Rewarding. Don't snigger!

End aside.

Butter oil is one of those features of study designs which produce obesity without linoleic acid.

Again, prompted by Tucker, I re-drafted the F:N ratios of MUFA and PUFA in the last post to account for the consumption of one NADH to provide an NADPH for rearranging each double bond during beta oxidation and we can add these revised ratios and some for selected saturate values to the composition table of butter oil like this:

In red are linoleic acid and the short chain fatty acids of an equal or lower F:N ratio cf LA. I threw in oleic acid and C8 caprylic (blue numbers) too to point out that caprylic acid, though higher than LA, is now lower than oleic (I view oleic acid as the mammalian default for "normal" insulin sensitivity) and so might be obesogenic.

So, from the F:N ratio Protons perspective, we have a modest supply of short chain fatty acids of potentially greater insulin sensitising ability (hence obesity promoting) than linoleic acid itself.

The effect of butyrate as a dietary supplement on obesity is controversial and reviewed here

Butyrate: A Double-Edged Sword for Health?

Conclusions totally depend on how you set your study up, what you consider good and what you consider bad. Bear in mind that butyrate is the darling of fibre-philes so consider publication bias too. Conversely it is to a large extent consumed by the colonic epithelium, so not a lot gets through to the systemic circulation. But some clearly does. The snippet of Figure 2 which caught my eye was this section:

I would just point out that anything which decreases lipolysis and increases adipocyte glucose uptake is NOT going to make you skinny. It might make you insulin sensitive (bravo), at least until you get fat enough to leak excess FFAs via augmented basal lipolysis.

You could of course just say butter oil fudge is highly Rewarding, so makes rats and mice fat. How can you tell it's Rewarding? Because it makes rats and mice fat. Except corn oil is also very Rewarding but ad-lib preferential consumption fails to induce obesity. For obesity there is the absolute necessity of calories to enter adipocytes and then stay there. Long term. Dopamine release in the brain might make you choose to eat something over something else but without pathological energy storage... Shrug.

My own concept of how butter oil/sucrose causes obesity is limited by the clear fact that there is no way of simply saying a given F:N ratio will always produce obesity. Too many variables for this to be set in concrete, which allows the hypothesis to side step conflicting evidence. You have been warned.

Random thought: Sucrose (when it doesn't produce a slim insulin sensitive phenotype) usually produces hyperinsulinaemia and insulin resistance (often "skinny fat"). The higher the insulin levels the more effective insulin sensitising dietary components (linoleic acid and now possibly SCFAs) are at allowing that high level of insulin to generate obesity. Probably why the cornstarch is added to the fudge, to augment the insulin response.

As always, alternative explanations welcome.

My thanks to Basti in the comments after the last post which crystalised a lot of this current post. And to Tucker twice over.


Wednesday, December 08, 2021

Protons (53) a formula revised

Back in Protons (53) a formula I wrote down how to work out the F:N ratio of (even chain) fatty acids with varying double bonds:

F/N = (n-1-db)/(2n-1)

where n is the length of the carbon chain and db is the number of double bonds.

Oleic C18 is 18-1-1 divided by 36-1, ie 16/35 = 0.457
Linoleic 18-1-2 divided by 36-1, ie 15/35 = 0.423

This is fine up to C18 but C20 and above are targeted to peroxisomes rather than mitochondria so the need for an F:N ratio fades. Peroxisomes have their own signalling systems but research on them is in its infancy.

Anyhoo, Tucker mentioned off blog that during the multistep processing of double bonds there is a step which consumes NADPH. This will have to be re-reduced from the resultant NADP+ by the Krebs Cycle where NADH producing steps have iso enzymes capable of generating NADPH instead of NADH. That reduces the NADH supply to the electron transport chain by 1 NADH per double bond requiring NADPH, so complicates the formula.

The formula ends up as:

F/N = (n-1-db)/(2n-1-db)

It makes a relatively small change to the ratio as the denominator is a much larger number than the numerator.

Oleic acid, originally 0.457 becomes

18-1-1 divided by 36-1-1, ie 16/34 = 0.471

and linoleic acid, originally 0.423 becomes

18-1-2 divided by 36-1-2, ie 15/33 = 0.455

The latter is interesting as it moves linoleic acid upwards towards MUFA and the saturates because the denominator drops.

The value for caprylic (shortest saturate in common consumption) is 0.467 and with the new LA now at 0.455, they are getting closer. Also caprylic is now at lower F:N ratio than oleic. I just wonder if this is part of the explanation of the coconut based diets used by Surwit to induce obesity with LA still limited to 4% of calories...

Thanks to Tucker for the NADPH requirement insight.


Tuesday, December 07, 2021

!Kung Bushmen and mongongo nuts yet again

Well, I got that wrong about conjugated linoleic acid (CLA) from mongongo nuts.

The !Kung people eat their mongongo nuts and the large amount of alpha-eleostearic acid converts to 9cis, 11trans CLA:

Alpha-eleostearic acid (9Z11E13E-18:3) is quickly converted to conjugated linoleic acid (9Z11E-18:2) in rats

This 9c, 11t CLA is exactly the same isomer as rumenic acid, the primary CLA of ruminant meat/dairy fats.

It's not a lipolytic agent. Not from monongo nuts, not from ruminants.

For lipolysis you want 10trans, 12cis CLA.

Manufacturing a bulk supply of CLA for marketing as a fat loss drug uses a process of treating ordinary linoleic acid with a catalytic industrial process which isomerises the LA in to roughly a 50:50 mix of 9c, 11t CLA and 10t, 12c CLA plus some odds and sods

The 10t, 12c isomer is a lipolytic agent of some potency. There's a nice review here:
However it does not appear to be found as a normal component of any biological system as far as I know, though I'm open to someone finding a source. At the moment it looks like it is a drug, manufactured from linoleic acid by an industrial process. The chemical formula might be identical to rumenic acid but on a "shape", charge distribution and metabolism basis (the location and orientation of the double bonds really matters to enzymes) the two have nothing what so ever in common.

In addition to weight loss 10t, 12c CLA can also trigger adipocyte apoptosis. A little apoptosis might be fine if you have adipose tissue hyperplasia (too many adipocytes, rather than too distended adipocytes) but if you have normal levels of overly large adipocytes it will place the burden of accepting excess insulin mediated lipid for storage in to the remaining, already overly large, adipocytes. Or your liver.

This is essentially a lipodystrophy, certainly if taken far enough. As in congenital and acquired lipodystrophies, this will be associated with glucose intolerance, insulin resistance and functional type 2 diabetes. In rodent models you can drive this process somewhat further than you can in human clinical trials. So this study uses mice:

Conjugated Linoleic Acid Supplementation Reduces Adipose Tissue by Apoptosis and Develops Lipodystrophy in Mice

Bear in mind this is a model and has been set up to produce an extreme black/white result and it delivers.

Oral glucose tolerance test and intraperitoneal insulin tolerance tests:

Here are the weights of various organs of interest (check the liver):

Note that the model was set up so there was no weight loss with the 10t, 12c CLA treated group. There is massive adipose tissue loss, adipocyte number loss but no weight loss (it's a model, people are clever). All of the lipid which should be in adipocytes ends up in the liver. What does a 4.44g liver look like in an adipocyte depleted mouse? Like this:

This is the diet:

"The semipurified diet was a low-fat diet and on a calorie basis contained 63% carbohydrate, 11% safflower oil, and 26% protein. Safflower oil was used as a source of fat. Safflower oil (high-oleic type) contained 46% oleic acid (18:1 n-9) and 45% linoleic acid (18:2 n-6) from total fatty acids. CLA was prepared as a free fatty acid at Rinoru Oil Mills (Nagoya, Japan) and stored frozen in plastic bottles blanketed with nitrogen. Linoleic acid was isomerized to CLA with isomers (34% c9, t11/t9, and c11; 36% t10 and c12; 3% c9, c11/c10, and c12; 2% t9, t11/t10, and t12 from total fatty acids). In the CLA-fed group, to keep fat intake constant in the 2 groups, 25% of the safflower oil was replaced with CLA"

That's a quarter of 11% of calories as mixed isomer CLA, the sort you might take as a supplement, ie around 3% of calories. About a third of this is the active 10t, 12c CLA, ie around one percent of calories.

If a human consumes 2000kcal/d then that's 20kcal or 2g of 10t, 12c CLA per day. In this now rather well thumbed study

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

they were using 6.4g/d mixed CLA isomers. That will be around 3g/d 10t, 12c CLA. That's exactly the ball park used to produce lipodystrophy and diabetes in mice. The same phenomenon occurs in pigs where after slaughter back fat can be extracted, weighted and processed to detect apoptosis:

Supplementation with conjugated linoeic acid decreases pig back fat deposition by inducing adipocyte apoptosis

Comparable studies would be difficult in humans but least pigs aren't mice.

Where does this leave the !Kung and their mongongo nuts? Well, they certainly never see any 10t, 12c CLA, our liver only converts alpha-eleostearic acid to rumenic acid (assuming we're like rats). This latter is either a weak or non lipolytic/apoptosis agent. Does that leave the !Kung as inexplicable?


It turns out that alpha-eleostearic acid is a rather potent lipolytic agent in its own right, it also induces apoptosis in fat cells in a similar manner to 10t, 12c CLA. Bitter melon seed oil is another, quite well studied source of alpha-eleostearic acid. This gives the flavour:

Mongongo nuts are lipolytic until their alpha-eleostearic acid content is detoxified to rumenic acid. To me, this suggests that living on mongongo nuts may carry weight control benefits at some risk of generating a degree of lipodystrophy, however small. I doubt anyone has gone studying adipocytes from the !Kung for markers of apoptosis. It looks like there will be a trade off between degree of lipolysis, giving small, low basal lipolysis adipocytes vs lost adipocytes giving larger, more basally lipolytic remaining adipocytes. I suspect the dose makes the poison.

I think it's probably unimportant to go in to detail about how alpha-eleostearic acid and 10t, 12c CLA induce lipolysis/apoptosis but, not surprisingly, it involves the generation of ROS for both.