Hyperlipid by Petro Dobromylskyj

14 June 2021

You need to get calories from somewhere, should it be from carbohydrate or fat?
  • Random musings on uncoupling (4) coconut
    Feeding mice on a high sucrose, low linoleic acid diet activates FGF21 production by the liver which stimulates heat generation in brown adipose tissue, leading to a lean phenotype, marked insulin sensitivity and poor glucose tolerance secondary to down regulated glucokinase in the liver. This latter is not surprising as fructokinase has a much higher rate constant for fructose phosphorylation than glucokinase does. Use it or lose it applies, even if only temporarily, so glucokinase down regulates. A bit like eating a low carb diet also down regulates glucokinase.

    Edit, this one too

    End edit

    My basic feeling was that fructose generated a caloric overload in the liver. Rather than dealing with this issue using hepatocyte mitochondrial uncoupling the task of dealing with the excess was delegated to brown adipose tissue and FGF21 was the messenger. "Higher  level" signalling. BAT uncouples on behalf of the liver. 

    Of course that immediately suggests that other caloric overloads, especially if uncontrolled, might do the same thing. George Henderson tweeted this paper, which I've known about for years but have never gone in to in great detail:

    Long term highly saturated fat diet does not induce NASH in Wistar rats

    I hadn't realised how much uncoupling these rats were doing. They all weighed pretty much the same but caloric intake was way higher in the butter fed rats and even higher still in the coconut fed rats. That's interesting compared to coconut oil used in the Surwit type diets but these current diets are low in PUFA and sucrose free. Here are the caloric intakes:

    The coconut based diet was particularly interesting as the rats were consuming twice the calories of the chow fed rats and weighed exactly the same. You could argue that coconut just tastes better than chow and the rats over ate then uncoupled. Or, more interestingly, you could suggest that medium chain fatty acids enter liver mitochondria in an unregulated manner and generate large amounts of input to the electron transport chain. If hepatocytes are experiencing a caloric overload what else should they do other than generate FGF21 and sub contract a calorie disposal solution to the BAT?

    This arrangement would benefit, as with fructose, from the hepatocytes being the primary cells targeted to receive the unregulated caloric supply and is a good reason for keeping MCTs out of chylomicrons and passing them directly to the liver via the portal vein. Which is what happens.

    So we can look at this study (just ignore everything about stress response and how a few ketones will do horrendous things to you):

    Dietary Manipulations That Induce Ketosis Activate the HPA Axis in Male Rats and Mice: A Potential Role for Fibroblast Growth Factor-21

    Here is what gavaging a chow fed rat with MCT oil does to FGF21 an hour later

    LCT stands for corn oil. The acute effect of a low dose is almost nothing. Corn oil enters the systemic circulation in chylomicrons via the thoracic duct. It will be obesogenic as per ROS/Protons and only very mildly stimulating of FGF21 generation. Long term at high dose rates it will, as we've noted, uncouple enough to offset the metabolic syndrome induced as per ROS/Protons and result in a slim rodent which needs to over eat mildly to compensate for the side effect of uncoupling.

    After coconut oil the uncoupling effect via FGF21 is marked so the compensatory eating has also got to be marked because the primary source of calories floods liver mitochondria with medium chain fatty acids.

    So......... Localised hepatic caloric overload is a stimulus for FGF21 production leading to BAT thermal caloric disposal. As far as the rest of the body is concerned there is just the BAT caloric loss induced deficit to be perceived. There is a hypercaloric state in hepatocytes and a hypocaloric state in other systems, hypothalmus included. Food intake rises to maintain a normal energy supply to avoid weight loss.

    Note the arrow of causality. The rats/mice are not over eating and burning off the excess. They are eating extra using an appropriate appetite to cope with BAT calorie expenditure/loss. They might not want to be hot but they have no choice. They eat to make up for it.


    BTW there is this:

    with alcohol being another hepatocyte caloric overload source which also generates FGF21 to "dispose" of the excess hepatic calories via BAT.

    Using AMPK.

    Which is where things get complicated.
  • Random musings on uncoupling (3) oxygen consumption
    This is an nice little study from Japan looking at the effect of fat composition of diet on oxygen consumption before and after a meal:

    Diet-Induced Thermogenesis Is Lower in Rats Fed a Lard Diet Than in Those Fed a High Oleic Acid Safflower Oil Diet, a Safflower Oil Diet or a Linseed Oil Diet

    We can accept changes in oxygen consumption as a pretty good surrogate for the degree of uncoupling.

    These are the diets used

    The sucrose content is around 5% of calories so no confounder there. Fat is consistently 40% of calories and they measured the composition of the fats used. Like this

    This lard is Japanese lard, produced in the early years of the 1990s. It's 7% linoleic acid. With lipids at 40 % of the calories in the diet this means that overall the LA provides 2.8% of the calories. So this is not an obesogenic diet*. However we get very little information about that because the rats were grown under a time restricted, calorie restricted protocol. An energy intake value was chosen as about the amount of food that a hungry rat would eat in an hour. This amount was fed twice a day. So there is no browsing allowed which suggests that some modest degree of caloric restriction is in place and the absolute supply of calories will be the same for all groups, despite each group have differing overall metabolic needs.

    *[If the old anecdote about pork consumption in Okinawa is true this lipid profile might explain any possible longevity effect.]

    The high oleic safflower oil diet provides 6% of calories as LA, the safflower oil diet provides 30% of calories as LA and the linseed oil diet provides 6% LA but this is combined with 21% as ALA. Alpha linolenic acid, from the ROS/Protons perspective, is an extreme version of LA although hard evidence for this is very thin on the ground. There are many studies using ALA which show that it is marvellous stuff at any dose rate but these studies almost always use "pair fed" or fixed calorie, mildly restricted protocols.

    The protocol here also describes confirming that none of the diets generated lipid peroxides before being fed to the rats. The group is quite meticulous, so refreshing.

    Here are the oxygen consumptions, indicating the degree of uncoupling present in the immediate post-meal period:

    We can see that lard at 2.8 % of calories as LA shows very little uncoupling. Diets with LA between 6% and 30% LA uncouple more, although there is no evidence of a graduated response, and that a mix of 6%LA with 21% ALA uncouples the most, although this latter is only statistically ns greater than for the other PUFA groups, with a group size of six rats.

    So. Given a fixed, mildly restricted calorie intake, we can take the lard fed rats as being very close to metabolically "normal", and eating closest to what they might want if fed ad-lib. Then we have three groups of rats, fed exactly the same number of calories, whose diet has been modified to induced a significant amount of uncoupling. All animals are at the same room temperature so obligatory thermogenic needs should be equal. So higher PUFA groups of animals will waste some of their consumed energy and not be allowed to replace it. What happens to body weights?

    The low PUFA lard fed rats are the heaviest and carry about 5% more carcass fat than the PUFA fed rats. I consider them to be at normal body weight. Visceral fat, a surrogate for metabolic damage, is identical across the groups, all are free from clinical metabolic syndrome.

    I would argue that the heavier rats are the least hungry, the high oleic acid safflower fed rats are a little more hungry and the two high PUFA diet rats are the most hungry. Quite what would have happened if the study had included an ad-lib fed arm we will never know.

    So here's some speculation about "what if" there had been an ad-lib arm to the study:

    The lard fed rats (this time) are our normal group and would weigh only a little more due to relief from calorie restriction. It would be nice if the ad-lib fed 6% LA group might have ended up with modest excess energy storage as fat gain and shown extra food consumption to match the extra weight gain. The high ALA fed group should have consumed even more food with less or similar weight gain because uncoupling is having a significant effect. Finally the group with 30% calories as LA should have come out somewhere between. The group having 30% of LA calories seems to be just on the border between where the ROS calorie storage effect transitions to the uncoupling effect in terms of dominance. From other studies 45% of calories as LA might have had the uncoupling effect absolutely dominating, so a slim phenotype would show, but that would be impossible in a diet where only 40% of calories are from fat in total.

    How does this fit in to cellular "hunger" concept? At levels of linoleic acid where facilitated diversion of calories to storage predominates, excessive insulin signalling is dominant. The cell is dealing with an hypercaloric state.

    Under marked uncoupling conditions the cellular state is the opposite. A separate defence mechanism against caloric excess is being activated, whether there is a caloric excess or not, by dropping the mitochondrial membrane potential because the closer a diet gets to 45% LA diet the more it provides a supra-physiological level of LA for the uncoupling protein function to maximise, combined with copious supplies of oxidative products such as 4-HNE to activate those uncoupling proteins.

    At this point ROS generation is suppressed because, whatever the FADH2:NADH ratio, high mitochondrial membrane potential is still essential for reverse electron transport. There will be a transition from failure to limit ROS generation (forcing a cellular energy surfeit) to a reduction in insulin signalling as the result of a process which also involves the direct loss of calories by uncoupling (an hypocaloric state). The fact that suppressed insulin signalling releases fatty acids is over ridden by their loss through overactive uncoupling.

    So PUFA are able to produce both cellular repletion and cellular hunger depending on the concentration.

    It feels counterintuitive that a metabolite should, by one action, generate an hypercaloric state with excess energy storage and yet, by a separate process, go on to produce the opposite effect via uncoupling at higher levels of exposure.

    But this appears to be the case.

    It becomes much clearer using pharmacological uncoupling, which takes us back to 2,4-dinotrophenol.

  • Random musings on uncoupling (2) revised
    Okay, here is how I ended the last post:

    "It's also worth pointing out that this appears to be an ancient system and that high PUFA exposure might uncouple in anticipation of the cellular caloric influx which PUFA signify. It has become pre emptive and has, certainly in rodents, largely been shifted from "all" cells primarily to the brown adipose tissue. The PUFA signal might also be very central to the browning of white adipose tissue to beige. That's a process you would never want to have to use, being in a situation where generating beige adipose tissue might be helpful is not somewhere you want to go."

    which is wooly thinking, to say the least.

    Uncoupling is triggered by ROS generation using a locally available PUFA derived lipoxide signal combined with whatever fatty acids are available in the immediate vicinity of the mitochonrial inner membrane uncoupling proteins. A supply of PUFA is absolutely needed for the signalling molecule generation (4-HNE and related) and intact PUFA have been selected to uncouple better than saturated fats do. These features might be related.

    PUFA are always present in the inner mitochondrial and have many functions. this function of acting as a safety valve appears to be one of them. It will not need to be specifically linked to bulk PUFA induced cellular caloric excess. I envisage it as a response to any excess caloric ingress, hyperglycaemia or markedly elevated FFAs post prandially (or even elevated levels of systemic fructose) when the law of mass action (ie a large concentration gradient) overwhelms the normal response of insulin resistance when cells are replete.

    I view this aspect as the ancient system. It applies to any caloric overload and happens to use a PUFA/ROS signal to limit excessive mitochondrial membrane potential using uncoupling.

    The fact that this system is functional at levels of PUFA intake far in excess of those that a particular species (humans) might be adapted to is perhaps unexpected but does seem to be the case, but this is more understandable if it is viewed as a generic safety mechanism.

    Whether those slim rodent models consuming 45% of their calories as linoleic acid are dealing with excess caloric ingress by uncoupling or whether they are actually under caloric deficit because the emergency uncoupling system is being activated inappropriately due to oversupply of signalling precursors/uncoupling facilitating fatty acids is not clear.

  • Seasonality
  • Random musings on uncoupling (1)
    I thought I'd just take a break from trying to find any studies where sucrose-in-chow causes obesity in the absence of greater than 8% of calories as linoleic acid. That's becoming rather frustrating but is turning up some interesting studies on uncoupling and PUFA along the way.

    So just for this post I thought I'd get even more speculative than normal about uncoupling.

    Thermogenesis. Thermogenesis makes you hungry. That is not a completely intuitive statement.

    It's easiest if we start with a food source which generates heat without utilising uncoupling because there are far less variables to think about. So think protein. Deconstructing a protein chain, processing amino acids to their core constituent energetic compounds such as pyruvate, glutamate etc requires energy and this energy shows up as heat.

    Let's say 1000kcal of protein generates 300kcal of heat. I've no idea of (or interest in) the exact value, I just know you can warm a hypothermic patient post operatively using an IV amino acid infusion.

    So if you are used to eating 2000kcal of fat a day to run your metabolism, you metabolism requires 2000kcal, tightly controlled. Let's also imagine you live in a thermoneutral environment and so are not muddying the water with (usually necessary) thermogenesis to maintain your body temperature.

    If you swap your 2000kcal of fat for 1000kcal of fat plus 1000kcal of protein things change. The 1000kcal of protein provides 700kcal of usable energy and 300kcal of waste heat, which you don't need as you are in a thermoneutral environment.

    So you get hot and uncomfortable and have a 300kcal deficit. You cannot run your metabolism of 1700kcal, you need 2000kcal. The hypothalamus notices this 300kcal deficit. What would you do? You would feel hungry and eat enough extra food to ensure that you actually get the 2000kcal you need for metabolism, tolerating the excess heat generation as an unwanted side effect. You would stay weight stable, eat a little extra to hunger and be sweaty.

    Forced overfeeding is equally straightforward. You eat too much, uncouple, lose heat and hope you don't really live in a theroneutral environment.

    Next is what happens under spontaneous eating but including more than 8% of calories as linoleic acid in your diet.

    Here my hypothesis is that excess calories are available, the cell fills up and poorly-opposed insulin allows more calories to enter and for those calories to be sequestered out of the way as lipid (and probably glycogen too). From the cellular point of view energy status is fine (not overloaded) so long as the excess calories entering are being sequestered away from metabolism. The hypothalamus might perceive too few calories in the arterial blood in direct proportion to those being lost into storage in the periphery. So you eat more.

    The next step in thinking is 2,4-dinotrophenol. This is a classical uncoupler and probably the most effective weight loss drug, particularly for fat loss, ever marketed. Sadly the therapeutic margin is narrow, unpredictable and can change suddenly.

    High dose rate, rapid weigh loss DNP administration uncouples respiration to the point of ATP reduction and massive heat generation. AMPK is activated by the consequences of the fall in ATP, ensuring effective fat oxidation. With a marked fall in mitochondrial membrane potential there is going to be a cessation of reverse electron transport and the mitochondrial component of the ROS generation essential to maintain insulin signalling will collapse. At this point calories will be entering the cell through AMPK facilitated GLUT4s (and probably CD36s as well) and will not be diverted to storage but used for a combination of running metabolism plus extra calories equal to those lost as heat.

    I understand from reading around a little that DNP does, indeed, make you hungry. The calories pouring though the mitochondria are coming from fat primarily and if the fat supply cannot keep up with the uncoupling-augmented metabolic needs then blood energy content will fall and the hypothalamus will notice. Also interesting is the use of drugs such as caffeine and ephedrine to control that hunger, both of which are reputed to work. They increase basal and sympathomimetic induced lipolysis, supply more fat and so control the hunger. So the loss of fat from adipocytes due to failed insulin signalling cannot quite keep up with the increased metabolic heat production without a little help. Not surprising because the shrinkage of adipocytes is from a failure of insulin signalling to facilitate fatty acid uptake combined with unopposed basal/sympathetic lipolysis. Neither is directly related to the huge loss of calories from unrestrained uncoupling. It surprises me a little that the supply and demand are so closely matched in such a complex system, especially with a major spanner dropped in to the works.

    Which just leaves us with PUFA. These appear to facilitate uncoupling in proportion to the amount present in the diet, even on a meal by meal basis. My mental image for this phenomenon is that, intrinsically, PUFA allow too many calories in to a cell if insulin is the facilitating hormone. The more pronounced this effect, the more the need for uncoupling.

    Modest excess, say over 8% of the diet by calories, works by the standard ROS/Protons concept of sequestration of excess calories. But you can only sequester so many excess calories and very high percentages of PUFA have the potential to overwhelm the system. We are talking 35% or over for uncoupling to predominate, but I think this might be a linear effect which is over-shaddowed by the ROS effect at lower concentrations but comes to dominate at very high concentrations.

    At these very high levels of uncoupling the body is in caloric deficit because it is actually losing the calories as heat. It is metabolically the equivalent of the hunger of a high fat (10-30% PUFA) diet but does not involve the distention of adipocytes to achieve it. The degree of hunger would be in proportion to the deficit between lipolysis and heat loss via uncoupling and would require (not allow) a few extra calories to be eaten.

    EDIT: This last section is poor logic. It might be worth a post to clarify or just a an edit to correct. I'm thinking about it.  I'll take it out and put a more considered discussion up as a follow on post. END EDIT.