Sunday, May 08, 2022

Protons (70) Uncoupling does suppress insulin signalling

The premise of the last post is that mild mitochondrial uncoupling is protective against fatty liver because it disables insulin signalling and generates heat.

There is a considerable literature looking at "energy stress" in cells induced by marked uncoupling, usually using dinitrophenol (DNP) to profoundly reduce ATP generation from oxidative phosphorylation. The DNP concentration in cell culture to achieve this would usually be 1mM. Oral dosing can transiently achieve plasma levels in mice of around 0.5mM and is probably higher in the liver because it receives the portal blood flow from the site of absorption in the gut, so maybe this has some application to real life. Maybe. These studies are peripherally interesting as dropping ATP this aggressively triggers AMPK activation which translocates GLUT4 to the cell surface to maintain cell viability using ATP from glycolysis. The translocation is independent of any markers of insulin signalling.

An example from 1988

Evidence for two independent pathways of insulin-receptor internalization in hepatocytes and hepatoma cells

The basic premise is that ATP depletion is the activator of AMPK mediated translocation of GLT4s to the plasma membrane. However AMPK is also activated by both fasting and ketogenic diets, neither of which produces an acute ATP deficit. Years ago I suggested that a major activator of AMPK is acute loss of insulin exposure and/or its signalling, independent of ATP status.

So DNP at 1mM (1000μM) in cell culture does indeed deplete ATP and AMPK does indeed translocate GLUT4 under these circumstances. Is the mechanism of action the acute suppression of insulin signalling (secondary to the loss of mitochondrial membrane potential) rather than, or in addition to, the fall in ATP generation per se?

Happily it is quite easy to measure insulin signalling nowadays. It's also possible to use either live mice taking non-lethal doses of DNP or cell culture using therapeutic concentrations of DNP. 

This next paper used DNP in live mice at non lethal dose rates and in cortical neuronal cell culture at 10-40μM concentration as opposed to 1000μM.

The Mitochondrial Uncoupler DNP Triggers Brain Cell mTOR Signaling Network Reprogramming and CREB Pathway Upregulation

Bottom line: Mild uncoupling using a therapeutic concentration of DNP suppresses insulin signalling. In this case they are looking at whole cerebral cortex in their live (until euthanasia for brain removal) mouse model or cortical neuronal cell culture.

"The protein levels of AKT, p-AKT (Thr308), ERK, and p-ERK were examined by immunoblotting which showed that the activated (phosphorylated) forms of these kinases (p-AKT and p-ERK 42/44) were reduced in the cerebral cortex at 24 and 72 h after DNP treatment (Fig. 3c–e). Collectively, these results suggest that insulin receptor signaling is suppressed in cerebral cortical cells in response to mild mitochondrial uncoupling."

That seriously confirms my biases.

I'm perfectly willing to extrapolate from mouse neuronal cells to mouse hepatocytes because this is a basic principle of how I view energy physiology working. I might be wrong, or not.

Low dose DNP and BAM15 will both treat metabolic syndrome in humans.

Conceptually what is happening in metabolic syndrome at the most basic level is that linoleic acid is allowing too many calories in to a cell and this leads to both storage and pathological ROS generation to side-step and/or limit the process. It is a coping mechanism for the failure of LA to signal physiological insulin resistance cf that provided by palmitate.

Uncoupling suppresses insulin signalling. If there are stored calories within the cell they are then made accessible. These calories are used for ox-phos to make up the deficit caused by the uncoupling. At normal weight (ie without excess insulin signalling due to diet) the suppression of insulin signalling will be accommodated by AMPK activation.

Stuff makes sense.



Paul M Hart said...

Slightly different emphasis to Tucker Goodrich then: that LA destroys cardiolipin leading to cellular inability to process glucose, hence insulin resistance: or is this an extension of the same biochemical processes?

Peter said...

Yes. Put six people around a table, all at the same conclusion (LA is bad), and we'll each have a different take. It's not a consensus thing re mechanism.

However failure of electron transfer via damaged cytochrome C *cannot* be ameliorated by uncoupling. There is a blocked electron transport chain so there should be a build up of electrons upstream and a large generation of ROS because there is nowhere for the electrons to go to past complex III. Delta psi should be low because electrons need to actually flow to pump protons. Uncoupling won't help, in fact it would worsen matters. A fully blocked ETC can be tolerated so long as there is ATP from glycolysis to drive ATP synthase in reverse to maintain a functional delta psi. Uncoupling would eliminate this "routine" fix.

I note that uncoupling *does* ameliorate metabolic syndrome, it wouldn't if it was driven by impaired cytochome C function, but it would work if delta psi was too high... It works.


karl said...

OT a bit :
First - to be very clear intracelular vs systemic melotonin are quite different topics.

But it appears there is a photo connection and intracellular melotonin is produced via light.

This paper has a number of references to follow about mitochondria..

What would the effect of high PUFA diet have on intracelular melotonin?

Looks like people are finally starting to follow the photo exposure threads.. much we don't know.

Peter said...


Interesting. I can see why it appeals, but it also look like a very deep rabbit hole!


Jonathan Croxton said...

Hey Peter, I just watched your interview with Megan Hall. You mentioned that there are some unknowns for what makes someone quit getting fat and turn into having diabetes.

You've said many times before that people prevent the metabolic disaster of hyperglycemia + high ffas at the cost of getting fat. But it's not just stuffing existing adipocytes. The body can differentiate new adipocytes as an adaptive response to mop of ffas that are spewed out of existing distended adipocytes.

I suspect the ability to differentiate new adipocytes has a large genetic component. Have you ever masochistically watched one of those documentary shows about extremely obese people? It always seems to "run in the family," so to speak. Of course people in a family also eat the same stuff, including piling on the mayo and ranch dressing, but the same could be said about the entire US, and most people who get seriously obese don't break 300 pounds. (Sorry, 136 kilos)

Another potential genetic factor: in the US there is a trope of people who are hot (read: young and fairly lean) but have terrible gut problems. I'm beginning to think there is variability in people's ability to digest and absorb dietry fat. Not soaking all that soybean oil from the french fries they eat keeps them skinny, but for whatever reason gives them a load of intestinal inflammation. (I honestly think this explains the rise in popularity of carnivore for treating gut problems better than anything else.)

It just makes me think about how variance in genetics has such a profound impact on our health. Failing to absorb dietary fat should be a terrible evolutionary disadvantage. But in this maladaptive envioronment, those people get a stomach ache and others end up on My 272 Kilo Life.

The other grim truth is genetic variance makes demonstrating Protons in the real world terribly difficult. There are always going to be exceptions, like the !Kung and their nuts.

One last thing, you mentiones the curiosity of wild cats eating very oily mice, ues obviously not get fat per Proton's predictions. I think any species could evolve to live on a high-PUFA diet, given enough time. Just as an example, maybe cat mitochondria don't make as much Complex 1, which would be like a human permanently on metformin. Or maybe they make more of that enzyme that Brad has been talking about recently that oxidizes NADH and creates a superoxide (NNT? I can't remember). There are a dozen changes in just gene expression that would make a high-PUFA diet not a big deal. But clearly, humans are not evolved to eat a high-PUFA diet, we are specifically adapted to get our fat from ruminants. Except the !Kung, maybe.

Malcolm said...

"I honestly think this explains the rise in popularity of carnivore for treating gut problems better than anything else."

I can believe this. I know of someone with one of these problems, and their consultant actually advised them to eat more meat! The word is out! :-)

cavenewt said...

karl and Peter re light: I simply cannot remember his last name, but in the past a Dr. Jack Somebody would occasionally comment, and his focus was on the effect of light on cellular activity. Is that the kind of thing you refer to?

Peter said...

Hi Jonathan,

Re cats: They get fat enough on PUFA in modern life with carbohydrate diets. My suspicion is that PUFA do not matter if insulin is low. Pax glucagon and protein...

Re genetics: Yes, there must be a range of responses to the current novel food environment. Genes which were harmless/beneficial might not be so good on the SAD or equivalent. The genetic variations are there long term, don't affect us on carnivore but allow us to demonstrate pathology once eating PUFA +/- sucrose.

cave, yes, he was also very concerned about non ionising radiation altering electron pathways in respiratory complexes. He tended to lose me on many occasions.


Monica said...

Cave: Jack Kruse?

cavenewt said...

Monica: yes, thank you. It was driving me crazy that I could not remember his name. I did check out his website at one point. I never could decide what to think about it all.

Passthecream said...

CN there has been talk of 'phoyotgerapy" for wound and burn healing for quite some time. This usually involves low red to near infrared wavelengths and many of the papers talk about the wavelengths from sunlight which can easily be transmitted through human flesh. These can also usually travel through standard glass and through water so you could experience them in a solarium on a sunny winters day. Or possibly even get some of them from a cast iron heat source. These seem like time tested human pursuits a little less risky than getting lots of full sun UV exposure in summer in a desert region. (Some of that is good too, imo, just not too much.)

Anyway, the focus for this is now shifting onto NIR stimulated melatonin production in most cell types with melatonin being an important cellular antioxidant outside of it's it's role as a hormone.

This fellah sums up the current thinking about vitD versus melatonin versus sunlight. Usual caveats apply, batteries not included.

cavenewt said...

Hey Pass, did you invent a whole new area of science? Phoyotgerapy!

And then there's phototherapy which sounds very similar ;)
I have run across references to this in the past. I know someone who had a treatment for chronic Lyme that involved treating the blood with UV light via an IV. With no discernible effect. And no insurance coverage.

"less risky than getting lots of full sun UV exposure in summer in a desert region"

That barb was aimed straight at me, right? I live in a hot desert region, and in the six years I've been here, haven't used sunscreen and haven't had a sunburn. Working outdoors quite a bit.

Knock on wood!

Passthecream said...

Oh I also live in a dry region with massive summer UV levels. I don't get burned nowadays. I've never used sunscreen, ever.

Passthecream said...

There's a g/h thing going on with my spellcheck. Or with my fingers. Cumulative error + machine learning.

Passthecream said...

But you reminded me of a UV problem for stock which proves that what you eat affects what the sun can do to you, and that vegetables are dangerous. We have St John's Wort as a feral weed pest - hypericum perforatum. Hypericine and other chemicals in it are photoactive such that if stock eat the plant they will get skin lesions out in the sun. It was being investigated as a cancer therapy ( phototherapy aka phoyotogerapy) because certain cancer cells take it up more than healthy cells do and exposing those cells to UV destroys them.

People also take SJW as a sedative and sleeping potion. Bit risky!!!

Peter said...

Yes, and I even have a fairly large bush of it outside my kitchen window. The flowers are quite pretty!


Passthecream said...

To extract the ' therapeutics' all you need is to macerate it in vegetable oil until the oil takes on a deep reddish colour. No harm in that ...

Don't let the goats chew on it.

Peter said...

Happily I have no need of the therapeutics and enjoy the sun too much to risk it! Will keep the goats away tho'...