Hyperlipid by Petro Dobromylskyj

26 November 2022

You need to get calories from somewhere, should it be from carbohydrate or fat?
  • Choose your insulin sensitivity well
    People may or may not realise that I dislike meta-analysis. In the words of the great Malcolm Kendrick "One, two, skip a few, 99, one hundred". Or one of my uni lecturers, "The meta-analysis of dross is still dross". On a practical basis they just provide information overload to give a result determined by the selection criteria from which the individual results threads can be hard to extract.

    So this is not one of my go-to type studies but, because it confirms my biases, I'll cite it here

    which came up on a twitter feed of Tucker's.

    The study looked at isocaloric replacement of 5% of carbohydrate calories with various fats or changing the number of double bonds in 5% of calories using various fatty acid substitutions under controlled conditions.

    The important result of this data trawl is this, extracted here from Table 2

    I think it is quite clear that particularly replacing saturated fat with PUFA (mostly linoleic acid) decreases insulin resistance. The more double bonds added, the greater the insulin resistance reduction.

    That's good, right? We all want to be insulin sensitive, right?

    So if I give you these HOMA scores, from a different study, which of the groups would you like to belong to?

    Before you choose you might want to ask what the difference is between the groups. This is the original table from

    Insulin sensitivity is increased and fat oxidation after a high-fat meal is reduced in normal-weight healthy men with strong familial predisposition to overweight

    which I've mentioned previously.

    OK. Choose your parents wisely.

    Statistically, if both of your parents are obese, you are in a bad place for staying slim. You are very likely to be MORE insulin sensitive (p less than 0.05) than your luckier mates with skinny parents. Personally I think genes have very little to do with obesity. Learning your food habits at your parents' knee has a very much larger influence on your future waistline.

    How much linoleic acid to you have to add to your diet to lower your HOMA-IR score from 1.6 (ie normal) down to 1.1 (going to end up like your overweight parents)?

    I don't know for a human but for a mouse it's generally enough to increase LA from around 4% of calories to something over 6% of calories that gets the job done.

    Any hypothesis of obesity has to be able to account for the above data. EBM or CIM.

  • Brand on 4-HNE facilitated uncoupling
    This is one of those papers with strong confirmation bias reinforcing properties, so care is needed. I do indeed have some difficulties with the paper, some of which I'll point out as we go along.

    Brand again.

    High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

    So. They wanted to look at how mitochondrial membrane potential controls uncoupling. They also wanted to differentiate between "endogenous" uncoupling and 4-HNE facilitated uncoupling. This is not easy. Certain steps need to be taken. Each step moves you away from reality and closer to answering the question you have in mind. Which may or may not reflect reality.

    They used adenine nucleotide translocase (ANT), the enzyme which exchanges ADP for ATP across the inner mitochondrial membrane because a) it uncouples and is closely related to standard UCPs and b) there is a ton of the stuff on all mitochondrial membranes which makes quantitative measurements possible. They also have an inhibitor which allows more information to be extracted.

    Here's the setup:

    They needed to eliminate FFA induced uncoupling (and energy supply) so added BSA to the medium to soak up endogenous FFAs.

    They needed to actively control energisation of the mitochondrial membrane so blocked complex I completely with high dose rotenone to stop oxidation of NADH from endogenous malate or glutamate catabolism.

    When they wanted to activate energy supply they used succinate alone working through complex II, which is still functional.

    Obviously, to look at uncoupling, they had to block ATP synthase with oligomycin.

    Next they equilibrated the proton concentration across the inner membrane using the H+/K+ exchanger nigericin to allow a them to use a voltage only sensitive probe to measure membrane potential.

    The degree of mitochondrial activation from succinate was controlled by either titrating with malonate (complex II inhibitor) or cyanide (complex IV inhibitor) to give a stable measurable membrane voltage.

    Finally, apparently 4-HNE is active at around 1μM but you need higher concentrations to get an easily measurable effect, especially in the presence of de-fatted BSA, so here they used 35μM.

    Having done many (but not all) of the above things they end up with this graph in Figure 1:

    which makes everything clear.

    This is my biggest problem with the paper. From graph A they extract the information in bar chart B and bar chart C which give details of the calculated proton leaks at 2.5 minutes and 5.0 minutes in to activation with succinate. Which demonstrate quite clearly that HNE facilitated uncoupling via ANT has kicked in by 2.5 minutes and is marked by 5 minutes. This is chart B, extracted from the grey lines in graph A at around 137mV, both time points:

    And chart C is from the dark lines in the lower right hand end of graph A at 175mV, only at time 2.5 minutes: 

    My problem is that, after many hours trying to curve fit and calculate in Powerpoint, I can never quite extract the numbers from graph A to fit charts B and C.

    If we assume that the research group have the raw data are getting their calculations correct we can have this as the main summing up:

    "In liver, time at high membrane potential in the presence of HNE results in a striking increase in proton conductance, which may be interpreted in two ways. First, HNE may slowly form covalent adducts with ANT, progressively converting ANT into a form with high proton conductance, or secondly, HNE metabolism may progressively form fatty acids [41], which then activate proton conductance [28]."

    The authors consider option two to be unlikely.

    If this paper really does hold water we have a situation where high membrane voltage, working through 4-HNE, produces long acting uncoupling in proportion to the time spent at high membrane voltage.

    Hence you can run a mitochondrial preparation with supra maximal substrate supply and, rather than exploding/imploding, the correct physiological "decision" is to uncouple in proportion to the excess membrane potential and stabilise it at something close to functionality.

    High delta psi -> ROS -> 4-HNE -> ANT/UCP -> damage limitation.

    That's how things should work.

    Of course that's not always how things pan out.

  • FFA vs 4-HNE for activating uncoupling
    Another basic mitochondrial concept. This is Brand again. The paper features mitochondria extracted from yeast cells which have been transfected with a plasmid for the mammalian UCP-1 gene.

    Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

    UCP-1 is the odd man out of UCPs, its primary function is thermogenesis in adipose tissue but it seems that the control systems are similar across the whole family of proteins. UCP-1 is useful because the degree of proton leak is huge compared to other UCPs, which makes measurements using isolated mitochondrial preparations easier.

    It turns out that, in addition to palmitate (and other fatty acids) many lipid derivatives also activate uncoupling, 4-HNE being one of the best studied.

    They isolated mitochondria from their yeasts and fed them with either 4-HNE, palmitate or a combination of the two and looked at the degree of uncoupling (using O2 consumption under oligomycin as the surrogate, as you do).

    The two red rectangles are the degree of uncoupling induced by either 4-HNE alone or palmitate alone. Both do something. If you simply add the two red rectangles together you get the blue one, which is what you would expect if the two agents were additive. The yellow rectangle is what you actually do get, ie significantly more uncoupling because the combination is synergistic. There are papers which suggest the 4-HNE is essential for palmitate to uncouple but that might be model dependent. The above experiments are using a membrane potential of 87mV, ie quite low. A high membrane potential might have generated enough 4-HNE in situ to mask the effect of exogenous 4-HNE. Or done other things, next post.

    Philosophically I view the 4-HNE in this role as a signal that some degree of ROS related "damage" has occurred to the PUFA components of the mitochondrial membrane. A little too much in the way of ROS produces a degree of lipid damage which facilitates uncoupling, which drops mitochondrial membrane potential and lowers ROS generation and subsequent damage. I have the quotation marks around "damage" because the degree of damage is that at which evolution has decided is acceptable before stepping in with an effective intervention, ie the damage is permissible and non injurious to the cell.

    Bottom line: Fatty acids and lipid oxidative derivatives of PUFA both support uncoupling. Their mechanisms appear to be different and to be synergistic. We can go on to look at some aspects of their regulation in the next post.

  • Insulin increases coupling in mitochondria
    Back in the 1990s Veech's lab noted that supra maximal insulin, combined with glucose at 11mmol/l, markedly improved the ability of an isolated rat heart to pump oxygenated perfusion fluid compared with glucose alone. The mechanism of the effect was not explicable from their model but was very clear cut and the time scale of onset suggested a covalent bonding process.

    Substrate signaling by insulin: a ketone bodies ratio mimics insulin action in heart

    Macroscopically, the amount of work done per mole of oxygen consumed increased. As this was without an increase in glycolysis the implication is that insulin increases the coupling of mitochondria.

    I was left with the idea at the time, reinforced occasionally by other finds, that insulin was has a major effect of increasing coupling within mitochondria.

    Insulin clearly has many, many effects within a cell. It's not possible to examine any of these using isolated mitochondrial preparations because they have no cytoplasm to respond to insulin. You need intact cells.

    I recently came across this rather nice paper:

    Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation

    It looks at an assortment of muscle derived cells in much the same way as mitochondrial preparations examine mitochondrial performance, but here whole cells used, a small step closer to reality than isolated mitochondria. They have intact cytoplasm so can function on "normal" substrates such as glucose or palmitic acid. The cells are not even "permeabilised".

    They used standard mitochondrial techniques such as full uncoupling with FCCP to assess the maximum possible oxygen consumption and oligomycin to assess peak oxygen consumption from proton leak in the absence of a functional ATP synthase. So they can provide standard mitochondrial study parameters like respiratory control ratio and make estimates of the degree of (un)coupling of respiration and of the efficiency of ATP generation.

    All good but even better they then went on to look at the effect of fairly physiological concentrations of insulin on these parameters. And to look at the effect of palmitate alone and palmitate in combination with insulin.

    They are looking at mitochondrial function within intact cells, with functional cell surface receptors and cytoplasmic signalling cascades. Insulin was used at 10nmol/l (10,000pmol/l) which is only just above peak post prandial levels and even their 100nmol/l dose is still way below the millimolar concentrations commonly used to assess the effects of supra maximal insulin stimulation on cell preparations.

    Their palmitate dose rate is hard to assess as they presented it bound to albumin with an estimated free palmitate of 20nmol/l, ie 0.02micromol/l. Almost every other study simply measures/specifies total palmitate in solution so making comparisons is hard. Obviously the 400-2000micromol/l of FFAs which are normal in fasted human plasma are almost completely albumin bound, so it's hard to tell if the estimated 20nmol/l of free palmitate used in the study is high or low. It certainly has an effect.

    These are the graphs of oxygen consumption from the human derived muscle cells/myotubes:

    The graphs are not intuitive. First, everything is normalised to the rate of oxygen consumption under the influence of oligomycin (between times 20 and 40 minutes) ie state 4oligomycin, and are expressed as a percentage of this. So the sections of the graph in the "dip" after the line labelled "OLI" are baseline and labelled 100, ie 100%.

    Under oligomycin there is a complete blockade of ATP synthase so any oxygen consumption has to be facilitated by uncoupling. The absolute values will not be identical with vs without insulin, they are just deliberately aligned at 100. The absolute values will differ based on the activity of uncoupling proteins.

    Once FCCP is added there is complete uncoupling of all respiration (while ATP synthase still remains blocked with oligomycin) and so this represents the maximum possible flow of electrons down the ETC to complex IV, with no buildup of proton gradient to inhibit this. These peak values are probably identical whether insulin has or hadn't been applied because FCCP is supra maximal in its uncoupling so subtleties of UCPs become irrelevant. No one has added palmitoylcarnitine either.

    This shows as a greater percentage *increase* when insulin has been applied earlier, ie the oligomycin phase had different absolute oxygen consumptions with or without insulin. I think it's just convention to set up the graphs as they are.

    So glucose + insulin couples respiration compared to glucose without insulin.

    Which reiterates Veech's findings.

    Okay. So we can calculate the coupling efficiency of mitochondria respiring on glucose with or w/o insulin and express it as a fraction of unity. Insulin always increases the coupling of respiration when oxidising glucose. Black bars with insulin:

    The study didn't look at delta psi or ROS generation so we have no way of knowing exactly what happens to these parameters.

    Adding palmitate completely blocks (and probably (ns) decreases) the increase in respiratory coupling seen when insulin is added to glucose. Right hand columns labelled as added PA:

    The change downward looks to have come very close to statistical significance. This suggest that small (possibly) doses of palmitate negate insulin's coupling effect and trend towards actively reversing it.

    What is also interesting is the left hand pair of bars. The white bar is insulin + glucose and the black bar is insulin, glucose and "empty" bovine serum albumin (BSA). The BSA produces a statistically significant increase in coupling of respiration. This is in a cell prep which has not been treated with exogenous fatty acids. There are enough fatty acids "floating around" to interfere with insulin's coupling action on mitochondria.

    My assumption is that the empty BSA scavenges free fatty acids by supplying a sequestration site for any FFAs in the culture. Reminiscent of the effect of carnitine in a previous post.

    Let's make this completely clear: Mitochondria in cells exposed to insulin are more coupled compared to those without insulin. Adding extra palmitic acid reduces this extra coupling. Removing background levels of free fatty acids enhances insulin's coupling effect.

    Insulin is an enhancer of coupling in the mitochondria of intact cells. It's effect appears to be mediated through changes in free fatty acid availability which are known mediators of activation of uncoupling proteins.

    TLDR: All isolated mitochondrial preparations are devoid of insulin signalling so will automatically be uncoupled to some degree, which goes some way to explaining continued oxygen consumption under oligomycin. Especially using supra maximal NADH generating substrates. But it doesn't help explain the regulation of membrane potential to around 180mV under high substrate supply.

    Other things might.

  • Beta oxidation intermediates control ETC function
    Back to the paper provided by met4health, briefly mentioned previously:

    Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

    It's a very interesting paper. It brings to light some of the problems of using isolated mitochondrial preparations. The basic summary is that if you compare the oxidation of palmitoylcarnitine to either pyruvate/malate or glutamate/malate there is significant ROS generation with palmitate, even at low delta psi, compared to the primarily NADH generating substrates (which produce zero ROS at 180mV delta psi in these preparations).

    Figure 3 is perhaps the most interesting. For section C they blocked the function of ATP synthase with oligomycin to raise delta psi and then titrated delta psi downwards by uncoupling with FCCP to give either a low or high delta psi. Then they fed the preparation with either palmitoylcarnitine (plus extra carnitine) or glutamate/malate.

    The right hand bar graph is derived from the left hand curve and shows that delta psi has some influence but, under both delta psi conditions, there are many more ROS produced under palmitate oxidation than G/M. Delta psi clearly has an effect but substrate also has a marked influence.

    That's the convincing part of the paper. There is no insight as to mechanism but my biases assume it will be F:N ratio related. I might have left it there but I can't.

    The rest of the results are hugely influenced by this statement from the methods:

    "Unless otherwise stated, determinations were made in the presence of oligomycin (3µg/ml) to inhibit ATP synthesis, a condition used in previous studies of the mechanisms of ROS formation in isolated mitochondria and permeabilized muscle fibers (e.g. see Refs. 4, 13, 34, and 38)."

    Translation: We blocked ATP synthase because everyone does it.

    So pretty well all of the results appear to have been produced during a complete blockade of ATP synthase.

    These preparations are always looking at ROS generation when the only dissipation route for membrane potential is some form of uncoupling (UCPs, NNT, various proton assisted co-transporters)

    There is no other way to allow O2 consumption under oligomycin. See last post on Brand's review.

    This begs the question of how is it possible to feed a mitochondrial preparation supra maximal amounts of NADH substrates (pyruvate/malate) consuming relatively large amounts of oxygen in the absence of any way of dissipating the proton gradient across the inner mitochondrial membrane without ATP synthase being active?

    The answer is that there must be some sort of uncoupling going on. The delta psi of 180mV is completely normal but this does not mean that there is no proton leak through the inner mitochondrial membrane. It merely means that the leak (defined by an O2 consumption of 21nmol O/min/mg of mitochondrial protein) is sufficient to avoid raising delta psi to massive levels in the face of a supra maximal supply of pyruvate/malate. The proton leak is also kept low enough not to drop delta psi. This smacks of regulation.

    By comparison palmitoylcarnitine at 18micromol/l has less oxygen consumption and supports a lower delta psi, in the region of 145mV.

    This is clear in section A of Figure 3.

    Both of these values for O2 consumption under oligomycin *have* to be facilitated via uncoupling.

    What is also clear is that, with oxygen consumption lower under palmitoylcarnitine, there is less uncoupling than for P/M. 

    Has anyone noticed that in Figure 3 there is a flick between 18µM palmitoylcarnitine and 18µM palmitoylcarnitine plus 2mM carnitine between various graphs?

    The authors of the paper considered that, with palmitoylcarnitine, the low delta psi combined with low O2 consumption might be due to an inhibitory effect of fatty acid oxidation intermediates on either FAO itself or on ETC function.

    Adding 2mM carnitine appears to remove such intermediates. I've not been in to the chemistry but it looks like the carnitine exports them from the mitochondria. There's something about this in the supplementary data. I'm just accepting it happens for today. About which I'm a little cautious.

    Adding the extra carnitine makes the oxidation of palmitoylcarnitine look just like supra maximal P/M or G/M.

    Here's the oxygen consumption bar chart from supplementary data Figure 3. We're looking at the left hand pair. White bar is palmitoylcarnitine 18µM consuming (as before) 10nmol O/min/mg. Black bar is after the extra carnitine was added. Oxygen consumption is around 25nmol O/min/mg (and delta psi did the same) and is now comparable across the metabolic substrates, black bars:

    This increase in O2 consumption means that, under oligomycin, that uncoupling has markedly increased and is directly equivalent to supra maximal NADH sources.

    To me this implies that normal fatty acid oxidation (ie without extra carnitine) is a self limiting process. 

    If the paper is correct (don't forget they are working with oligomycin blocked preparations) a high delta psi is not a feature of palmitoylcarnitine oxidation.  The oxidation of palmitoylcarnitine suports ROS generation irrespective of delta psi. To make me really happy it would be nice to generate ROS with different fatty acids and look at the effect of the F:N ratio on ROS generation.

    There are certain implications to these thoughts. First is that physiology is very keen to keep delta psi in the region of 180mV or lower and applies some degree of uncoupling to achieve this. This appears to be independent of fatty acid induced uncoupling.

    This is possibly very important. Any supra maximal supply of substrate in a non-phosphorylating mitochondrial prep (state 4oligomycin) has to have a method to stabilise delta psi at around 180mV. How? Another post there.

    Second is that FAO intermediates down regulate the ETC performance directly, limiting delta psi to 145mV from palmitoylcarnitine 18µM unless those FAO intermediates are removed.

    Finally, FAO appears to generate ROS moderately independently of delta psi. Certainly palmitoylcarnitine does.

    Now for a long-time-ago throwback:

    Does everyone recall the Dutch chaps who didn't eat for 60 hours and so rendered their mitochondria "dysfunctional"?

    Prolonged fasting identifies skeletal muscle mitochondrial dysfunction as consequence rather than cause of human insulin resistance

    Permeablised  muscle fibres behave pretty much like mitochondrial preparations.

    "Despite an increase in whole-body fat oxidation, we observed an overall reduction in both coupled state 3 respiration and maximally uncoupled [here using FCCP] respiration in permeabilized skeletal muscle fibers..."

    The RCR using an uncoupler and oligomycin, ie state 3FCCP / state 4oligomycin, fell markedly with fasting, hence the term "skeletal muscle mitochondrial dysfunction" in the title.

    But 60 hours of fasting cannot possibly destroy your mitochondria. People can pushbike hundreds of kilometres over 5 days without eating anything at all. Their mitochondria work.

    I would suspect that this is a fully physiological control system designed to cope, at the mitochondrial level of fatty acid oxidation, with a potentially limitless supply of energy from the fatty acids released from adipocytes under low insulin/insulin signalling conditions.

    In this study using permeablised muscle fibres you could probably have reversed the effect completely by treating with 2mM carnitine.

    But why would you want to? Apart from gaining insight as to what is normal physiology of course.

    Summary: fatty acid oxidation is a self regulating system at the level of beta oxidation rather than at the level of the Krebs Cycle. I suspect that the FAO intermediates will act directly on the electron transport chain.

    If this is correct it will provide insight in to other elevated FAO conditions, ie obesity with insulin resistance, where fatty acids should modify (appropriately) ETC function to avoid energetic overload.

    I've had suspicions that this has to be the case for a long time. Finally I'm getting to see a little progress.