Sunday, May 20, 2018

Guddling in the dark for a respiratory quotient (2)

OK. I've been bugged by the RQ values which come out of CLAMS equipment. For months. Multiple papers have values that don't make sense, which is an issue making me doubt my sanity and is damaging to my personal extensive set of confirmation biases. They also produce basic contradictions of the physiology of the oxidation of glucose vs the oxidation of fatty acids. But now I think I can go back and explain much of the peculiarity in this image from Kahn's group, the one which triggered this previous post. Two sets of mice, on the same chow, having sustained differences in RQ, in a way I couldn't understand:

Well now I think I can. The insight needed comes from a non related paper on the effect of knocking out the sweet taste receptor in mice.

So, let's have a look at the RQ data from the CLAMS apparatus in this paper:

Disruption of the sugar-sensing receptor T1R2 attenuates metabolic derangements associated with diet-induced obesity

And in particular from this next set of graphs. They are interesting because they cover two complete days in the CLAMS equipment.

Look at the grey and the black lines first, these are control and sweet receptor KO mice, being fed pretty standard low fat crapinabag. We can see that between day one dark period and day two dark period in the CLAMS equipment that a) the peak height of the RQ rises, b) carbohydrate oxidation rises and c) lipid oxidation falls. No one did stats to compare these two days with each other so I don't know what the p value might be, but the trend certainly looks very real to me. No one altered the food macros between day one and day two.

I think mice, like humans, cut their calories when you put them in to a respiratory chamber or in to a CLAMS apparatus. Certainly for the first two or three days of mice in CLAMS.

In this T1R2 KO study you can see evidence for the the improving intake of (carbohydrate based) food between day one and day two which is inseparably linked to the rising RQ. There had been a day of acclimatisation before the two study days which will have minimised this trend but it's very much still visible. The change is less obvious (but still present) for the fat fed mice (red coloured traces) but it's not so clear cut in these mice because they are largely either oxidising fat from their diet or fat from their adipocytes, both of which look pretty much the same from the RQ point of view.

Core insight. You can alter RQ away from the macros of a (high carbohydrate) diet by simply eating less. Adipocyte fat makes up the difference in calories and this automatically drops the RQ. Let's take this idea and look at the RQ figure from Kahn's paper.

In a normal mouse, on normal low fat chow, the RQ during the early feeding period rises to above 1.0 due to de novo lipogenesis combined with fat repletion/storage. Kahn's control mice (SHAM) started with a RQ at around 0.88 and they slept through the light period so dropped their RQ to around 0.75. When they started to feed with the arrival of the dark period (on their high carbohydrate chow) in the CLAMS apparatus the RQ only rises to 0.88. In real mice eating this sort of chow the RQ should rise to 1.2 for a few hours.

The RQ will not rise to 1.0 if the mice fail to eat enough carbohydrate to meet or exceed their energy needs. If they don't eat enough they will continue to utilise body fat and this will lower their RQ below that of a mouse eating to weight stability. These mice did not eat enough carbohydrate to suppress fat oxidation. The RQ says so. The absolute food intakes are below but these are fairly irrelevant compared to the RQ data.

They were put in to the CLAMS apparatus with no suggestion (in the methods) of an acclimatisation period. I think the mice were frightened and dropped their food intake. We can see from the RQ of both groups that at no point does this even approach, let alone exceed, 1.0 (in the acclimatised mice in the T1R2 KO paper figure the RQ reaches 1.1 at the peak of feeding on day 2, ie CLAMS day 3).

Summary: Both of Kahn's groups are oxidising fat to give a low RQ on an high carbohydrate diet and the only way they can be doing this is if the fat is coming from their adipocytes. They are not eating enough of their carbohydrate based diet to maintain fat storage or to raise the RQ.

That just leaves us to ask why the control group are oxidising more fat than the intervention group. Is this a magical preference of the intervention group to oxidise glucose? Is that why they are so slim? I know that sounds strange but that's the conclusion in the paper. Logically, oxidising FAT makes you slim. Storing fat makes you fat. But that's under ad-lib conditions. At the time of the hypocaloric 24 hours for Kahn's mice in the CLAMS apparatus we have these data available from which we can estimate fat stores at a given time, week ten in particular:

The white diamonds are still the controls/sham operated. They carry about 10g of fat at week ten. The black squares are the intervention group, they carry about 5g of body fat at this time. If both groups cut calories acutely during their day of anorexia in the CLAMS apparatus, I would expect the animals with the most adipose tissue to release the most FFAs. And oxidise the most FFAs. That has to be the control group, because they're a lot fatter to start with. The intervention group has less stored fat available, so oxidises less fat. Interestingly the fatter control group ate 3.9g of carbohydrate based food in the CLAMS vs 4.2g for the slimmer intervention group (ns), which just might reflect less hunger in the fatter control mice because they are accessing their bigger fat stores, ie the control mice appear to run on fat (because they are doing so on this one single day). A lack of stored fat in the intervention group is why they ate more (carbohydrate based) food and utilised less adipose derived fat. So this group appears to run on carbs, judging by their RQ. But the RQ over the rest of their lives, in either group, while eating normally, to satiety, outside the CLAMS apparatus, is a mystery.

But yay! The CLAMS equipment is almost certainly working correctly. The RQ graph is very explicable. It probably tells us NOTHING about the substrate oxidation under non-CLAMS conditions, but we can speculate about that in another post.

Trying to make inferences about overall post-intervention metabolism, based on a single day in a very novel (and possibly frightening) environment under acute hypocaloric conditions will set you up for a hiding to nothing as far as comprehension and understanding of your intervention are concerned. But given enough thought the results at least become comprehensible.

Now I'm happy.


Now that I'm happy I might have a think about why the slim, subcutaneous to visceral fat-transplanted mice in Kahn's study might have come to remain slim. Clearly, from the fat mass graph, the transplantation of subcutaneous fat in to the location of visceral fat does do something...


cavenewt said...

Two questions.

1. "If both groups cut calories acutely during their day of anorexia in the CLAMS apparatus, I would expect the animals with the most adipose tissue to release the most FFAs. And oxidise the most FFAs. That has to be the control group, because they're a lot fatter to start with. The intervention group has less stored fat available, so oxidises less fat."

The rate at which stored fat is released depends on the total amount of fat in adipocytes? Sort of like a fully-inflated balloon releasing air faster than a limp balloon? Is this a known behavior? The alternative would be fat cells releasing fat at a static rate; in that case the slimmer cells would run out of fuel sooner (starve).

2. "...will set you up for a hiding to nothing as far as comprehension and understanding of your intervention are concerned."

This is either a typo or a Britishism that I'm unfamiliar with. Translation appreciated.

annlee said...

Yeah, I'd be stressed in there. Or the human equivalent.

Peter said...

Cave, I hadn't realise it was Britspeak. I guess it has flavours of damned if you do, damned if you don't. Or not a hope in hell.

These poor folks are trying to piece together why adding SC fat to the omentum might keep you slim and metabolically healthy and they're starting from incomprehensible data because they didn't acclimatise their mice to the CLAMS apparatus. That does not bode too well...

Re the fat loss, maybe I should have been more upfront about how speculative that aspect is. The drop in fed RQ for both groups is unarguable, the differential between groups must be due to differential lipolysis but whether that is due to differential fat mass vs some other cause for limited lipolysis in the intervention group is unclear. We know it happened, I'm guessing about how, feels like using Occam's Razor.


raphi said...

If i understand correctly, your Occam's Razor is

1) scared rats
2) dropped their calories low enough such that
3) lipolysis increased sufficiently to increase fat ox
4) which increased their RQ
5) especially in the fatter rats since lipolysis is more permissive in the fat than in the slimmer rats

i follow you until point (5).

although fatter rats have more fat to burn they're by definition relatively better at *not* burning fat. so although a sufficient restriction in carbs might be enough to get them burning fat, shouldn't this increased lipolysis happen more readily in the leaner rats? (they are by definition better at burning fat, are they not?).

But since we're dealing with T1R2 double-knockouts (a big carbohydrate sensor), maybe that changes my assumptions about fat accessibility and may in fact support your contention that lipolysis is more responsive in the fatter rats, explaining their higher RQ.

as you said, who knows given the acclimation question and discrepancy between caloric intake and RQs...

Peter said...


That sums it up nicely except that at 4) I'm pretty certain you meant decreases RQ... 5) applies to the adipose transplanted rat study. The T1R2-KO study is a separate thought train. I only looked at it because a FB post was put up suggesting that Sweet Taste Receptors = high "reward" = overeating = getting fat. It was a quick glance at sweet taste receptors to confirm the lack of physiological perception involved in being a Reward-itard that found me the paper, but sitting there was this 2 day graph in a CLAMS apparatus showing interesting (and probably important) trends.


Peter said...

BTW point 5) might clarify as I work through the fat transplant paper, now freed from the incomprehensible influence of the RQ data... But that might be an even more speculative post, so no guarantees!


raphi said...


Whoops yes it should be DECREASE for point (4)

"Sweet Taste Receptors = high "reward" = overeating = getting fat." ==> as always, describing things as rewarding/palatable does nothing but...describe it. nothing is explained and time is wasted. i podcasted about sweet taste receptors T1R2, T1R3 and TRPM5 with Gabor Erdosi

the taste receptors essentially integrate higher level signals to produce an appropriate insulin response (beta cells have those receptors). doesn't appear to be incretin mediated interestingly. it's also apparent that small doses of circulating fructose must reach a (low) threshold to potentiate GSIS. in any case, nothing to do with 'reward'...

cavenewt said...

"Reward-itard". This coinage should be rewarded with an award of some sort.

Peter said...

Hee hee. Perhaps a kilo of adipose tissue. Preferably omental from a ruminant??????

karl said...

Looking at pictures of CLAMS equipment one can see that there is no place to hide.. So I'm guessing it spikes the adrenaline response.. doing this long term - 24 hours - not so sure what happens. Clearly short term it diverts blood from the intestines to muscles..

Not thinking about controlling for this reminds me of what Richard Feynman wrote about mice maze studies.

Thinking about adrenaline and stress also reminds me of what Dr. Kendrick has written about stress and CAD - it is funny - they do want to run cholesterol tests - but don't measure cortisol or do anything to evaluate chronic stress which effects a list of steroidal hormones.

Eric said...

I apologize forthis unrelated post. NYT ran an article yesterday on continued use of antibiotics in feedstock that linked on this older article on antibiotics and weight gain:

Focusses on gut biome, not mitichondriae.

Peter said...

Hi Eric, yes, ab's are certainly growth promoters, I can remember farmers being not too displeased when I'd have to prescribe in-food oral abs for a pig unit back in the '80s, when I did that sort of thing. Likely works on several levels but I can't help but think that FIAF and bacterial failure to grow might be related as well as microbiome and mitochondrial targets. You'd have to look at all sorts of things to try to pick it apart,,,