Metformin (14) Normals

One of the flakiest aspects of Protons is the concept that the glycerophosphate shuttle transforms cytoplasmic NADH in to a mitochondrial FADH2 input, altering the FADH2:NADH ratio in a direction which favours reverse electron transport through complex I, given a reduced CoQ couple, and this leads to a) continued ROS generation to maintain insulin signalling and b) easy achievement of high ROS once a cell is replete with calories, ie when it is then necessary to resist further insulin mediated calorie ingress.

So the glycerophosphate shuttle should mediate both insulin signalling and insulin resistance, essentially controlled by the redox state of the CoQ couple. If CoQH2:CoQ is moderate ROS are still adequate to activate insulin signalling, if CoQH2:CoQ ratio is high ROS will also be high and insulin signalling will be curtailed.

Metformin blunts insulin signalling, in addition to reducing hepatic glucose output, by blockade of the glycerophosphate shuttle at mtG3Pdh.

Finding evidence to confirm this bias is not easy. I hope we all recall the poor Polish girl with SHORT syndrome who fell in to the hands of the endocrinologists who "treated" her insulin "resistance" with metformin. That ended well...

Metformin (11) a SHORT paradox

However, I tripped over this next paper while looking for something else. Oddly enough when I saved it it turned out I had already got a copy and had put up a one liner post about it

Metformin (12) You don't need to be SHORT

but didn't explain its significance. Here's the paper:

Effects of short-term metformin treatment on insulin sensitivity of blood glucose and free fatty acids

and here we go as to why I like it.

These are the results of treating either DMT2 people or people with a family history of DMT2 using metformin. There is an almost infinite supply of such responses in Pubmed, metformin is clearly insulin sensitising. If you wanted to you could say that metformin reduces insulin resistance. And then blanche at the thought of trying to define exactly what you mean by "insulin resistance".

I guess the first small fly in the ointment is the fasting FFAs,  as I've highlighted here

It looks like the rise in FFAs was not statistically significant but it must have come damned close. Increased insulin sensitivity should suppress FFAs.

So if we consider the insulin and glucose responses to be a direct result of suppressed gluconeogenesis in the liver and subsequent decreased hepatic glucose output then the rise in FFAs becomes as simple as being a direct consequence of metformin decreasing insulin signalling in adipocytes via blockade of mtG3Pdh plus a decreased absolute level insulin per se due to reduced hepatic glucose output. From my point of view all the adipocytes "see" is less  absolute insulin plus less ROS generated via the glycerophosphate shuttle, so less insulin signalling. They release FFAs.

If we then look at an obese person who is still insulin sensitive they have no fasting hyperglycaemia and no fasting hyperinsulinaemia.

Giving metformin for 10 days has essentially no effect on fasting insulin or glucose. Gluconeogenesis will still be reduced by metformin but the accompanying blunting of hepatic insulin signalling (via blockade of mtG3Pdh) allows more glucose release, so the two roughly balance out. This may not be the case under peak metformin effect, diabetic rats do drop their blood glucose on an IV dose of metformin at a therapeutic level of hepatic glucose output suppression. This may well be dependent of how much metformin you give, by which route and at which time you measure. Fed vs fasted would matter as well.

On feeding 75g of glucose to normal people for an OGTT insulin signalling becomes important and the decreased insulin signalling intrinsic to metformin shows as mildly impaired glucose tolerance (statistically ns but clinically 130mg/dl vs 170mg/dl might be undesirable) but because of blunted insulin signalling a significant compensatory hyperinsulinaemia is needed to get even this rather impaired glucose response.

So. In insulin sensitive people metformin's easily comprehensible action to obtund insulin signalling shows, under high glucose and high insulin conditions, as an impaired glucose tolerance with compensatory hyperinsulinaemia.

With supra-maximal dietary glucose (the specific intention in an OGTT is maximal activation of the insulin system) suppression of hepatic glucose output becomes irrelevant and all we see is mtG3Pdh inhibition manifest as poor insulin signalling. As it did for the poor girl with SHORT syndrome.

Nice.

I'm now wondering if there is any logical way of working out why people with DMT2 almost always (and clearly unexpectedly) benefit from metformin, while people with simple obesity behave exactly as you would expect them to do under an insulin signal blunting agent, especially during an OGTT.

It might be possible. I'm thinking about it.

Peter

Metformin (13) PowerPoint doodles

Sorry if this is a bit repetitive, I have a paper on metformin I'd like to discuss and just to make sure I understand how metformin works I've re-read this paper from many moons ago, just to be sure I'm clear 

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

So here we go with PowerPoint once more:

This the route for glucose production starting from glycerol

which is very straight forward. Let's flesh it out with some enzymes. For gluconeogensis we need to convert glycerol-3-P in to DHAP, this is performed by mitochondrial glycerophosphate dehydrogenase thus:

Of course this is half of the glycerophosphate shuttle, the other half being thus:

and we can put the two together to show the full shuttle

and then we can inhibit mtG3Pdh using metformin

and gluconeogensis grinds to a halt. Glycerol-3-P and NADH accumulate in the cytoplasm. If cytG3Pdh tries to run in reverse (which it can) it simply forces the accumulation of NADH in the cytoplasm, to which there are limitations. There's also a secondary change as regards lactate.

Here's how lactate is used, as it usually is, for gluconeogenesis. 

using lactate dehydrogenase and NAD+ to generate the pyruvate

so of course an high NADH level under metformin will make this process impossible, even if the pyruvate could later get past the inhibited glycerophosphate shuttle. Rising NADH in cytoplasm stops the conversion of lactate to pyruvate. Lactate accumulates.

That's it. Metformin suppresses hepatic glucose output by inhibiting gluconeogensis at the mtG3Pdh step. It acts it within minutes of an IV bolus in an alive rat at therapeutic plasma concentrations. Lactate accumulation is secondary to the redox changes in the cytoplasm.

Let's rephrase that: metformin blockades mtG3Pdh to produce an effect which imitates the suppression of hepatic glucose output by insulin.

This has absolutely nothing to do with enhanced insulin signalling. At all.

Hold on to that.

Peter

Endocannabinoids in brain and adipocytes

Well, these are interesting:

Adipocyte cannabinoid receptor CB1 regulates energy homeostasis and alternatively activated macrophages

Adipocyte cannabinoid CB1 receptor deficiency alleviates high fat diet induced memory deficit, depressive-like behavior, neuroinflammation and impairment in adult neurogenesis

This is very clever stuff. It is possible to engineer in to mice the ability to induce permanent knock-out for the CB1 receptor gene, only in adipocytes and only after the whole mouse is treated with tamoxifen for a few days. So you can grow mice which are phenotypically normal on either chow or an high fat diet (circa 6% LA plus unspecified carbohydrate source) until they are 16 weeks into the study then permanently delete, using tamoxifen exposure, all CB1 receptors on all adipocytes.

Okay, so what happens to food intake if you delete the adipocyte CB1 receptor? It's interesting because some of us (myself included) feel that pathological insulin sensitivity in adipocytes is dominant over brain physiology in terms of weight gain/loss. This paper sums up isolated adipocytes and CB1 activation: 

The CB1 endocannabinoid system modulates adipocyte insulin sensitivity

Without endocannabinoid facilitation of insulin sensitivity you should stop losing calories in to adipocyte fat stores, so you should stop being hungry...

Nothing happens.

Upper line is mice fed on an high fat diet for 16 weeks and then tamoxifen treated to eliminate the adipocyte CB1 receptors in those with the tamoxifen sensitive knockout mechanism. This graph is an amalgamation of sections B and D of Figure 1. I've pulled and stretched both vertical and horizontal scales so they now match for calories and for weeks to make clear what happened. Tamoxifen exposure is circled:

I think we can conclusively say that when adipocytes can no longer listen to the insulin sensitising effects of CB1 receptor activation there is no fall in food intake. Which supports the idea that

CB1 activation -> brain -> eat

so you could say:

↑ dietary LA -> ↑ endocannabinoids -> brain -> eat -> get fat

Nice and simple. It's almost as if appetite regulation by the endocannabinoid system might be brain-centric and adipocytes might be unimportant.

Except. Here's what happens to the weights above baseline. Pulled and stretched and tidied from sections C and E of Figure 1, tamoxifen exposure circled again, as above:

So, from week 17 to week 26 the high fat fed adipocyte CB1 receptor knockout mice continued to eat as much as the high fat fed wild type mice but dropped their weight to match that of the control group.

Without dropping their food intake or linoleic acid exposure. They eat as much junk food as they like and lose weight...

Obviously they uncouple. Not just in brown adipose tissue, white adipose tissue beiges too.

Now: You could simply conclude that brain CB1 receptor activation makes you continue to eat extra and adipose tissue, now without CB1 receptor activation, becomes a calorie sink via uncoupling to dispose of those excess calories eaten under brain CB1 activation. Metabolism is hypercaloric, adipocytes off-load those excess calories. Brain first.

Or: You could conclude that when adipocytes lose their CB1 receptor with its insulin sensitising effect they become less able to store calories and, on an individual cell basis, decide to oxidise their suddenly available stored lipid. If adipocyte hypertrophy includes a lot of linoleic acid then this LA is released and it acts as the best bulk facilitator (along with some 4-HNE) of uncoupling protein activation available. So loss of calories occurs within adipocytes through uncoupling protein activation. Basal lipolysis falls with decreasing lipid droplet size and the brain senses a loss of systemically available calories so maintains food intake to maintain energy homeostasis, ie the adipocytes still control energy availability which controls the brain's action. Metabolism is hypocaloric necessitating food intake. Adipocytes first.

I was going to stop at this point and I probably should have but here's some more rambling anyway.

It has just occurred to me that we can make this comparison:

Mixed diet + LA -> ↑ insulin signal in adipocytes -> loss of calories in to fat droplets -> obesity -> obesity being due to calorie loss in to physical triglyceride storage in adipocytes -> need to eat more (brain sensing ↓ systemic available calories). Getting fat makes you hungry. Fundamental.

This is my standard view of obesity, first pointed out (in my case) via Gary Taubes, although the LA component comes from Protons.

Now we can view the situation under adipocyte CB1 receptor knockout as:

↓ insulin action in adipocytes via ↓ CB1 receptor -> ↑ lipolysis -> ↑ UCP activation -> ↑ loss of fat, but still within adipocytes, only now as CO2 and H2O eventually excreted by lungs/kidneys rather than being released as "calorie carrying FFAs" -> ↓ energy delivery to circulation -> need to eat more (brain sensing ↓ systemic available calories).

Calorie loss in to storage and calorie loss in to uncoupling look the same to the brain.

One makes you fat, the other makes you hot.

Obviously today we already have research drugs to block peripheral CB1 receptors which don't cross to the brain so don't cause the suicidal ideation that central CB1 receptor blockade produces. This would allow you to eat a diet of utter crap without developing excess adipocyte size. Which might be a good thing long term, or might not. Or you could just take an uncoupler such as BAM15 or low dose/slow release DNP. Or not.

Or maybe there are other ways of reducing insulin/LA mediated "loss" of calories in to physical storage within adipocyte lipid droplets and instead activate physiological uncoupling proteins and lose fat directly as CO2 and H2O via lungs and kidneys. We all know this image of what happens when you profoundly drop systemic insulin (and associated adipocyte insulin signalling) in mice, even with continued LA exposure:

taken from here

A high-fat, ketogenic diet induces a unique metabolic state in mice

with a little support from here 

The ketogenic diet increases mitochondrial uncoupling protein levels and activity

You can't 100% take the brain out of the framework but the brain is, fundamentally, looking at available energy, largely controlled by adipocytes and their insulin signalling.

Peter

Adipose triglyceride lipase knockout mice

People may have noticed that I'm quite interested in basal lipolysis, adipocyte size and metabolic syndrome. That is correct.

What happens if you delete adipose triglyceride lipase (ATGL) so you can't have basal lipolysis? This paper gives some answers as to what happens to mice with ATGL knockout

Hypophagia and metabolic adaptations in mice with defective ATGL-mediated lipolysis cause resistance to HFD-induced obesity

Well, they die. Not unexpected. Here are the survival curves, one group of ATGL knockouts fed chow and another fed an "high fat diet" based on a modified D12492 (mostly extra sucrose with the lard)

If you consider which organ runs (in health) almost exclusively on fatty acid oxidation it will come as no surprise that the mice die of dilated cardiomyopathy secondary to lipid accumulation and mitochondrial failure. Sooner and more rapidly on the high fat diet.

You can get round this problem by engineering the ATGL gene just in to cardiac myocytes. Then the animals live long enough to allow you to study the effects of ATGL deficiency in the absence of a dead myocardium. The whole of the paper, other than Figure S1, uses mice with this protected myocardium (denoted cTg). WT/cTg denotes normal ATGL throughout their body plus extra myocardial ATGL (phenotypically normal) or AKO/cTg without ATGL everywhere other than their myocardium. So ignore the cTg label part, its WT vs AKO re adipocytes throughout the paper

So here's the paradox.

On chow WT mice carry approximately 2g of fat and AKO mice carry 5g of fat, much as the histology suggests and as you might expect. Figure 1 summarises the top left and top right groups of mice:

Things get more interesting when we compare the high fat fed WT mice with the high fat fed AKO mice, thats the bottom left and bottom right. Both groups have increased fat mass but the AKO mice are less obese than the WT mice. Like this

Note that all of the vertical scales are different. But there we have it, knocking out ATGL long term blunts the obesity induced by D12492, somewhat. The effect kicks in slowly but is well established by the end of the study at 22 weeks (solid black squares are AKO)

The paper goes in to some detail about PPAR-γ2 suppression in AKO mice which can be reverse by the diabetes PPAR agonist rosiglitazone.

Which ultimately translates as the adipocytes adapt to being unable to offload triglycerides by suppressing every aspect of lipid uptake and storage that they can.

"... the expression of genes involved in lipogenesis and fat storage such as PPAR-γ2 (−95%), C/EBPα (−30%), and SREBP1c (−78%) were significantly lower in gWAT from HFD-fed AKO/cTg mice than from WT/cTg."

The AKO mouse adipocytes, which cannot off-load lipid, compensate by progressively rejecting lipid ingress.

Does this make the adipocytes insulin resistant? Or the mice insulin resistant?

They didn't look at this at the adipocyte level and the interactions are too complex to guess how adipocytes might respond to physiological or pharmacological exposure to insulin.

What we do know is that the AKO mice fed a high fat diet are still very insulin sensitive at the whole body level despite their adipocytes eventually down regulating all aspects of lipid accumulation. Here's the  intra-peritoneal glucose tolerance test result. All of the following results are high fat diet based.

If you can read Table S1 in the original paper (too faint to reproduce here) you can see that fasting insulin in the AKO mice is 0.1ng/ml vs WT at 1.0ng/ml. Fasting glucose is also low at 164mg/dl in the AKO mice vs 212mg/dl in WT. Sorry for all the Noddy units. HOMA-IR score would be very, very low for AKO mice.

It doesn't matter what size the adipocytes of an AKO mouse are, they are not going to perform basal lipolysis. Under fasting conditions in normal mice FFAs go up as augmented lipolysis frees FFAs and there is little insulin to limit further lipolysis and FFA release. We need elevated FFAs under fasting.

In Table S1 again the WT mice have a fed FFA level of 0.72mM which rises to 0.93mM on a 4h fast, as it should do. In the AKO mice fed FFAs are 0.65mM and drop to 0.45mM on a 4h fast. They drop on fasting, so we can assume that the initial 0.65mM fed value is largely diet derived and so, with no food and no basal lipolysis, FFA levels have to fall.

Which they do.

My premise from Protons is that insulin resistance is an adaptive response to the delivery of FFAs. Under fasting this is ideal. In the presence of elevated glucose and insulin then the elevated FFAs from distended adipocytes cause caloric oversupply to the whole body and insulin resistance has to kick in to adapt. It is an antioxidant defence mechanism to limit ROS generation to physiological levels.

No ATGL -> perennialy low FFAs -> no need to resist insulin -> insulin sensitive

If we look at the hyperinsulinaemic clamp data we can see that both skeletal muscle (SM) and heart in AKO mice are really good at taking up 2-deoxyglucose. The liver, on a diet of 28% sucrose by weight, is also *very* insulin sensitive, with near total suppression of hepatic glucose production (HGP) during the clamp:

Of course the interesting bar chart is the right hand end one. The basal FFA levels are the ones cited above after a 4h fast. Hyperinsulinaemia  with normoglycaemia lowers fasting FFAs a little, but without statistical significance, in WT mice obese from D12492. Doing the same in AKO mice produces a marked fall from low levels to even lower levels, probably somewhere around 0.2mM.

Clearly the excess of plasma free fatty acids, derived from elevated basal lipolysis and which necessitate insulin resistance ie trigger metabolic syndrome, is not present in the AKO mice. Whatever the size of their adipocyte lipid droplets there is no fatty acid release. My guess for the fall in FFAs is that residual post prandial FFAs are being allowed in to muscle and liver cells using CD36 translocated to the cell surface in parallel to GLUT4s in response to the clamp.

Asides before I finish: What does the term "hypophagia" in the title of the paper actually mean? It means that the mice are NOT HUNGRY. They eat ad lib until they are satiated. Because they have down regulated their ability to "sequester" calories in to adipocytes, they sense adequate calories are available earlier so stop eating earlier. They are not "hypophagic", their lack of hunger is manifest as eating less. They're not under-eating. They're eating exactly the correct amount of food to supply their metabolic needs. It doesn't matter that the food appears to be hedonistic, rewarding or addictive (stop sniggering) as it appears to be in the WT mice. Under exactly the same hedonistic/rewarding/addictive food environment (you really must stop sniggering, and so must I) as the WT mice the AKO mice are simply NOT HUNGRY. The brain is such a secondary organ compared to the adipocyte.

Another aside: Where does hepatic insulin resistance in fructose fed mice (like the WT here) come from? Look here

Metabolic fate of fructose in human adipocytes: a targeted 13C tracer fate association study

Yes. Fructose, if it gets as far as adipocytes, forces FFA release. This will use ATGL. These FFAs will end up in the liver and have to be repackaged as VLDLs to be returned to the adipocytes. If insulin sensitivity is pathologically high (ie linoleic acid exposure) those FFAs in the liver will be stored there giving fatty liver, NAFLD etc. The AKO mice will obviously catabolise fructose without any problem but will be incapable of fatty liver because they cannot transfer fatty acids out of adipocytes to get to hepatocytes. Hence the incredible ability to suppress hepatic glucose production during the clamp in AKO mice. See HGP in the above figure. Adipocyte AGTL is essential for NAFLD on an high fructose diet.

Yet another aside: Ethanol does exactly the same as fructose, same mechanism:

Chronic alcohol exposure stimulates adipose tissue lipolysis in mice: role of reverse triglyceride transport in the pathogenesis of alcoholic steatosis

Okay, I'll shut up now. The role of ATGL in converting linoleic acid induced insulin sensitisation in to whole body insulin resistance and metabolic syndrome is central. This extends to NAFLD and ALD.

Physiology is comprehensible.

Peter

I was going to hit post but two more asides have presented themselves to my brain. Rosiglitazone more than eliminates the down regulation of PPAR-γ2 in AKO mice to give a slightly more obese mouse than the WT high fat fed mice. Does this restore insulin resistance too? Of course not, those bigger adipocytes still can't do lipolysis. The group must know this so they simply didn't run IPGTTs on the rosi-fat mice.

Also re adipocytes under clamp conditions. They looked at behaviour of skeletal muscle cells, heart cells and liver cells under hyperinsulinaemia. But not adipocytes. They know these adipocytes are resistant to insulin, so they didn't check. In a paper on AGTL and adipocyte function. They know this. I love glaring holes from carefully crafted methods sections where you can see the not-investigated leverages.

Oh, and at the time of this study they were clearly looking to find a drug to recreated the benefits of AGTL knockout on weight gain (for high PUFA, high sucrose fed humans). If they had found one I guess they would just have crossed their fingers that it wouldn't trigger dilated cardiomyopathy.

Oh, and another. High PUFA, high sucrose diets clearly do not trigger insulin resistance in the absence of pathologically distended adipocytes forcing elevated basal lipolysis and caloric overload induced high delta psi. The high linoleic acid only generates the 4-HNE to augment insulin resistance and ultimately shut down ETC function when caloric supply and delta psi is excessive. These AKO mice have access to obesogenic levels of PUFA and 21% atmospheric oxygen, yet essentially zero insulin resistance. 

Now I really will stop.

How can insulin resistance cause weight gain? (5) MCTs

The problems posed to the Protons hypothesis by the failure of fully hydrogenated coconut oil to suppress obesity in rodent models continues to draw me. In the recent past I've gone to Pubmed and searched on "octanoate + ROS" and "coconut oil + ROS".

This paper is pure confirmation bias for saturates resisting insulin signalling via ROS:

Modulation of adipocyte lipogenesis by octanoate: involvement of reactive oxygen species

This is from 2006 and the software for identifying and quantifying changes in cell appearance was not available (and looks like colour imaging wasn't either!). Now, these are 3T3-L1 "adipocytes" which are the "sort of" fat cells that many people work with in obesity research. 

The white signifies ROS generation. Upper left is control and upper right is with added 1000μM pure octanoate. So a model, using fasting levels of FFA of a composition never seen in vivo. But it generates lots of ROS which can be normalised by adding N-acetyl cysteine. Not only that but it inhibits lipogenesis too:

Bear in mind that you can show almost anything you like by adjusting the conditions during the development of 3T3-L1 adipocyte-like cells, but at face value octanoate is a potential weight loss drug. They didn't look at insulin signalling per se but reduced adipogenesis combined with high ROS generation implies ROS mediated insulin resistance. To me anyway.

The exact opposite is encapsulated in this paper:

Coconut oil protects cortical neurons from amyloid beta toxicity by enhancing signaling of cell survival pathways

Here they used rat brain neurons in culture and used a mix of fatty acids from coconut oil giving in the region of 100μM of mixed, mostly medium chain length fatty acids. So slightly different. We can ignore all of the data relating to Aβ because it's irrelevant and I think that's the wrong paradigm for Alzheimers disease anyway. What I want are the data relating to ROS generation from medium chain triglycerides. This is what they found:

Okay, 24h of incubation with mixed, mostly MCT fatty acids has no affect on ROS. If there is any trend it is downwards. From the Protons perspective reduced ROS lead to preservation of insulin signalling when it should be limited, ie reduced ROS allow excess caloric ingress. 

So look at this. Amongst the many things they measured they looked at the phosphorylation of AKT and it was increased. More insulin signalling will facilitate more fat storage, under fixed conditions:

So what are the differences between the two studies?

In this study these cells are being grown in Neurobasal™ Medium  which comes with the usual 25mM of glucose, enough to support the growth and replication of cells in the complete absence of fatty acids. Some degree of insulin signalling is on-going because it's routine to add supplement B-27 which specifically contains insulin. How much insulin is a trade secret but it is clearly a reasonable amount to ensure effective utilisation of glucose for growth but is unlikely to provide pharmacological levels of insulin exposure which would generate insulin resistance per se. This gives a certain level of phosphorylation of AKT as an effect of that insulin. That's the control cells. Simply adding 100μM of mostly MCTs increases insulin signalling at the pAKT level.

We are talking physiological insulin levels with high glucose and a little coconut oil, compatible with normal conditions after a carbohydrate based meal.

If we go back to the initial octanoate paper we are now talking about the same 25mM of glucose, ie higher than recently fed levels, plus a "fasting" exposure to 1000μM of octanoate. Oh, and 170mM of insulin (that's that 170 times peak post prandial level from previous discussions). This mix is the equivalent of the inability to suppress FFAs under high glucose/insulin, aka loss of metabolic flexibility, aka metabolic syndrome.

Here these adipocytes are awash in a sea of calories and an absolutely supra maximal level of insulin. I think it is reasonable to suggest their electron transport chains are screaming and their need to resist insulin is marked. Using lots of ROS. Almost certainly generated by high delta psi.

What might this tell us?

Under semi-physiological conditions adding even relatively small amounts of mostly medium chain fatty acids increases the phosphorylation of AKT, at a fixed level of insulin. Obviously, to anyone other than myself, that increase in pAKT would be a Good Thing. Insulin sensitising is good, yes? Recall this one:

Small amounts of dietary medium-chain fatty acids protect against insulin resistance during caloric excess in humans

with the effect of eliminating the correct physiological resistance to insulin signalling under forced overfeeding of humans:

Both solid columns are the control state, left hand hatched brown column is the increased insulin resistance of overfeeding fat and the green hatched column on the right is overfeeding very similar fat but with partial replacement by MCT lipids. You can avoid insulin resistance from overfeeding mostly saturated fat using MCT lipids. "Stopping" this insulin resistance is considered a Good Thing.

Except enhancing insulin signalling enhances fat storage.

Summary: The closer you get to physiological conditions the more the suggestion is that medium chain triglycerides facilitate insulin mediated fat storage.

Random thought: You have to wonder if they also stimulate overall growth via facilitated insulin signalling. As in milk fatty acids might be designed, along with milk proteins, to turn baby cows in to grown up cows. Ditto breast fed baby humans.

"How" is a much harder question. Right from near the start of Protons I have accepted at Dr Speijer's idea that very long chain fatty acids, especially saturated VLCFAs go to peroxisomes because mitochondrial oxidation would generate toxic levels of ROS. Later it dawned on me that VLCFA PUFAs probably go to peroxisomes too as they would generate too little ROS for healthy signalling. Now it looks like MCTs carry a different message and are processed differently to longer saturates to provide the specific advantage of maximal growth pre-weaning. Generally adult ROS signalling seems to come from 16 and 18 carbon fatty acids, saturates to resist insulin and MUFA to work in cooperation with insulin. And linoleic acid to muck up the whole system once you get above a certain level of consumption.

There are, if you set your model correctly, parallels between MCTs and linoleic acid of the ROS front. Which leaves me thinking about coconut based cultures with excellent insulin sensitivity but no obesity.

Interesting things, MCTs!

Peter

How can insulin resistance cause weight gain? (4) amplification

Okay. Here are the basics of insulin facilitated glucose oxidation:

Insulin -> Insulin Receptor (IR) -> G coupled protein -> NADPH oxidase 4 (NOX4) -> ROS

These ROS are the spark which triggers insulin signalling.

They act to inhibit assorted protein tyrosine phosphatases (PTPs). With PTPs disabled the normally suppressed auto-phosphorylation of the IR activates and its self activation allows calories to flood in to the cell. So NOX4 starts the process. It's a eukaryote system, there is nothing fundamental about it, just as insulin signalling is an eukaryote system. But it uses ROS because they are fundamental.

Once glucose calories are entering the cell they are supplied to the mitochondria which generate ROS by the rise in F:N ratio, triggered by the glycerophosphate shuttle (my opinion) converting glucose derived cytoplasmic NADH into mitochondrially targeted FADH2. My expectation is that this degree of ROS generation will keep the PTPs suppressed and so the insulin receptor phosphorylated/active while ever the cell is using glucose and so still wants glucose calories to enter.

These ROS are physiological and represent the normal control and maintenance of insulin signalling at peak levels. The next step is the use of ROS to disable insulin signalling.

There are times when it is necessary to actively suppress insulin signalling. This is most easily visualised by considering a cell which has received more than enough insulin mediated calories to meet its needs. Under these circumstances there is a surfeit of ATP which activates a negative feedback acting on ATP synthase. The inhibition of ATP synthase means that delta psi is no longer being dissipated so it will increase. At values above 140mV the rate of ROS generation increases exponentially and reaches levels that will directly act on the proteins of the insulin receptor and signalling cascade to de-activate them.

This is beautifully illustrated in this paper from 1974, previously discussed here

Evidence for Electron Transfer Reactions Involved in the Cu2+-dependent Thiol Activation of Fat Cell Glucose Utilization

It's tracking the evolution of CO2 from radiolabelled glucose and using H2O2 as a direct replacement for insulin to facilitate glucose uptake/oxidation by adipocytes.

It is very clear that 0.01mM of H2O2 initiates glucose uptake/oxidation, ie with absolutely no insulin present then 0.01 mM of H2O2 has the signalling action of a low dose of insulin. I think of this as equivalent to the NOX4 action. There is a level of H2O2 at around 0.3mM which performs the function of peak insulin exposure. When H2O2 exposure is further increased to 4mM or 5mM then the H2O2 disables its own ability to replace the function of insulin.

Let's make this absolutely clear, insulin is in no way fundamental. It can be completely replaced by H2O2 which, in common with superoxide, is the core signalling system and is ubiquitous as a growth/reproductive signal going right back to bacteria, preserved in their behaviour today. H2O2 is not an insulin mimetic, it is the core signal. Suppressing this ROS signal in modern bacteria, which never use insulin, inhibits their growth and reproduction.

Aside: No one has yet worked out how bacteria generate superoxide or H2O2 today. It doesn't seem to be via NOX enzymes which are, sadly, purely eukaryotic. I say sadly because they are extremely simple enzymes with a six helix tube through the cell membrane containing two Fe moieties as a "wire" to carry electrons to extracellular O2 from a bolted-on NADPH oxidase within the cell. If we ignore the multiple control systems which can also be added then it's very simple. The core of NOX just looks primordial. But it isn't. Sigh. End aside.

I hope I have established that there is a primordial signalling system which uses low concentrations of ROS to initiate nutrient uptake and usage to a certain maximal effect, above which a negative feedback using the same ROS disables further nutrient uptake/utilisation.

I've left fatty acid oxidation out of this narrative to keep it simple.

All of which leads us back to 4-HNE.

We can accept that the NOX4 minor contribution and the mitochondrial major contribution to ROS are generated in absolute proximity to the lipid membranes. All lipid membranes have functional needs for PUFA for structural purposes and if these are directly abutted to the electron transport chain complexes which are producing the above ROS signals then we can expect a proportion of the ROS to interact with those PUFA. The best studied end product is 4-HNE.

Superoxide is a poorly mobile signalling molecule ideal for short distance signalling but with limited  ability to cross lipid membranes. H2O2 is more stable and better able to cross lipid membranes so makes a good intermediate distance signal within a cell. 4-HNE carries the same information about the state of the ROS generation from the electron transport chain (pax NOX4) but is stable enough to be transported through the blood stream in measurable and modifiable concentrations. Because it carries the same information as superoxide/H2O2 it should come as no surprise it elicits the same response. In fact I view it as an amplifier of the ROS signal. It seems that 0.1μM 4-HNE can give a similar effect on adipocyte lipid accumulation as 0.3mM (ie 300μM) H2O2. Roughly. And anything over 5μM 4-HNE disables insulin signalling, equivalent to 5mM H2O2.

Obviously Protons makes this simple story a little more complex as you add in fatty acids and the influence of linoleic acid which both reduces and increases ROS generation as well as being the core substrate for generating 4-HNE per se, but I think this will do for today.

The above description makes the whole system look static. It's not. All of it oscillates, delta psi and ROS generation. You can see why.

Peter

Throw away thoughts:

If ROS are a growth signal, so too should be 4-HNE. Anyone can Pubmed "cancer + 4-HNE".

Also H2O2 is a biological warfare molecule. Macrophages "throw" H2O2 at bacteria to kill them. Any PUFA in the area of the battleground will generate 4-HNE. This is a medium distance signal to recruit more macrophages to join the fight. These are not directly related to metabolism but clearly important. Actually, you could ask whether high levels of both H2O2 and 4-HNE kill bacteria metabolically. There's a thought.

Then you can get in to plasma 4-HNE and it's effects on lipoprotein PUFA components. 4-HNE is a direct generator of ROS in its own right (it's function is as an amplifier of the ROS signal after all) so filling lipoproteins with linoleic acid while elevating plasma 4-HNE will attract macrophages to the extra 4-HNE wherever those lipoproteins stick. Like arterial/arteriolar walls. Stroke? Heart attack?

Hmmm. All fun stuff. Blame your cardiologist.

How can insulin resistance cause weight gain? (3) AD-9308

This is a random, throw away post. A one-liner, don't laugh. I happened on this paper following a link in a link to a link during my 4-HNE reading:

 Disruption of the mGsta4 Gene Increases Life Span of C57BL Mice

This sort of comment makes me sit up and take notice

"Surprisingly, the opposite was true."

as in

"We expected that disruption of mGsta4, a murine gene encoding a major antielectrophilic enzyme, will parallel the effect of a similar intervention in C. elegans and curtail the life span of the knockout mice. Surprisingly, the opposite was true. In the present article, we report this observation and provide a possible explanation for this unexpected effect."

The enzyme from the gene mGsta4 is a membrane associated glutathione-S transferase which is important (like ALDH2 in previous posts) for the detoxification of ROS-derived substances such as 4-HNE (amongst others). The best explanation for the findings in the study is that 4-HNE accumulates in tissues of mGsta4 null mice which activates extra defence mechanisms against electrophilic molecules. The end result is near-normalisation of tissue 4-HNE combined with an extended lifespan. At least in Bl/6 mice. But not in C elegans, here it does the opposite. Choose your model wisely before tinkering with your metabolism.


Apart from the somewhat amusing concept that reducing the detoxification of 4-HNE might be a longevity ploy, it made me wonder about the ALDH2 mutations in the East Asian human populations. Full introduction of this mutated gene in mice exposed to an high linoleic acid plus high sucrose diet leads to supplementary obesity. This is unlikely to be quite so problematic on a more species appropriate diet for either mice or humans. The ALDH2 modified mice were normal weight on chow.

For a mutation to persist there has to be an advantage to the individual carrying that mutation which increases their probability of reproductive success. Null mGsta4 mice show no change to their maximum life span but have a 10% increase in median life span and have a marked increase of 36% in lifespan at the 10th percentile, ie during the peak reproductive period of life.

Does the same happen with the reduced efficacy gene for ALDH2 in people as happens in the null mGsta4 gene in Bl/6 mice? Would this show as an increased healthspan but without increased peak longevity? Provided you don't live on fudge made from sucrose and corn oil of course. I tend to think median lifespan extension probably correlates with increased healthspan but that peak longevity might be more of a luck-based failure to die. Hmmmm...

So the law of unintended consequences makes me think long and hard about ALDH2 activation using a drug such as AD-9308. You could use it to reduce 4-HNE from an obesogenic 1.0~ishμM to almost nothing and so reverse whatever benefit has been preserved by the ALDH2 down regulatory mutation in humans. After all 560 million East Asians carry the gene which appears to have been designed to make us fat and insulin resistant when fed a high linoleic acid, high sucrose diet. But not if eating real food. So lets develop a drug for all East Asians to be able to eat junk food without getting fat! Might this simply result in looking good in your coffin?

Evolution is not stupid. It leaves that to drug developers.

Peter

How can insulin resistance cause weight gain? (2) 0.1 μM

When you type adipocyte + 4-HNE to Pubmed, this is your first hit

4-hydroxynonenal causes impairment of human subcutaneous adipogenesis and induction of adipocyte insulin resistance

and it is, of course, just a reinforcement of my biases which suggest insulin resistance should limit lipid accumulation in adipocytes

"Exposure to multiple doses of 4-HNE led to reduction in growth and differentiation of preadipocytes in both groups (Figure 4A), marked by upregulation of anti-adipogenic FABP4 and down regulation of the adipogenic FASN and SREBF1 genes (Figure 4B). "

Here is the rather pretty part A of Figure 4. Look carefully and you will see two stains have been used. The hard-to-see dark blue stain is DAPI and stains nuclei, ie it gives you a cell count so you can compare equal numbers of cells in a given view (or count cells in a proliferation assay). The fluorescent green staining shows lipid and can be used to assess how much lipid a pre-adipocyte has accumulated during the differentiation process in to an adipocyte. All by AI assisted automated counting systems.

The top row are differentiated adipocytes from insulin sensitive obese ladies. On the left of the top row we have pre-adipocytes after "normal" differentiation, with lots of lipid. On the right of the top row we can see that differentiation under 10μM 4-HNE pretty well completely stops lipid accumulation.

The bottom row features adipocytes from ladies who are already insulin resistant. Their pre-adipocytes accumulate very little lipid even after "normal" differentiation and this is made a bit worse by exposure to 4-HNE. Basically these cells are already f*cked and adding 10μM 4-HNE only makes things a little bit worse.

Okay. Biases confirmed. But why did they use 10μM 4-HNE? They did a dose response experiment looking at pre-adipocyte proliferation during differentiation using that pretty blue DAPI nucleus stain. Their benchmark was cell growth without 4-HNE exposure and they then used increasing 4-HNE exposure to see what the dose response was like:

I'd guess they used 10μM because it had some effect but was not as catastrophic as 40μM. Seems fair. It looks like 2.5μM was harmless and 5μM was borderline in terms suppression of proliferation. So 10μM got used for most of the rest of the paper because they were primarily interested in insulin resistance.

None of which helps me explain this

or the other gem from Tucker in here

Acute toxicity oftrans-5-hydroxy-2-nonenal in fisher 344 rats

which is a toxicological study injecting rats with exogenous 4-HNE:

This gives a clue as to what is going on. At 10mg/kg of injected 4-HNE the rats gain weight, at higher doses they don't.

There are a wealth of studies showing 4-HNE causes insulin resistance and a fair few which show that this is demonstrable by decreased phosphorylation of AKT, usually using a (quite high) dose around 100μM. To see what happens at lower doses we need some more digging and it's not too hard to find this review:

Electrophilic Aldehyde 4-Hydroxy-2-Nonenal Mediated Signaling and Mitochondrial Dysfunction

From section 5.2 we have this snippet:

"Depending on the concentration, 4-HNE can be beneficial or detrimental to cells. Many studies in the literature suggest that a 4-HNE concentration below 2 μM leads to cell survival and proliferation. However, a concentration above 10 μM is detrimental to the cell, leading to genotoxicity and cell death"

and further down the same section is

"A study carried out investigating vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells showed that a low concentration of 4-HNE (0.1 μM) leads to increased secretion of VEGF, while its expression is blocked when the 4-HNE concentration is greater than 5 μM"

Following ref [96] to

The expression and function of Vascular Endothelial Growth Factor in Retinal Pigment Epithelial (RPE) cells is regulated by 4-Hydroxynonenal (HNE) and Glutathione s-transferase4-4

does confirm that 4-HNE is a proliferation agonist at low concentrations (0.1 μM) and becomes borderline at 5μM, so all of the work on growing adipocytes at 10μM in the first study I mentioned are way above the levels which might be expected to promote adipogenesis and lipid accumulation in adipocytes.

So.

I would suggest that reducing the function of ALDH2 in mice as in

A common East Asian-specific ALDH2 mutationcauses obesity and insulin resistance: therapeuticeffect of reducing toxic aldehydes by ALDH2activation

does not achieve sustained 4-HNE at or above 5μM, more likely the figure will be below 1μM, simply based on looking at the phenotype. I'd bet AKT phosphorylation would be enhanced.

Equally in

Acute toxicity of trans-5-hydroxy-2-nonenal in fisher 344 rats

I would expect 10mg/kg exogenous 4-HNE to provide less than 5μM 4-HNE to adipocytes and anything over 30mg/kg to provide in excess of 5μM to adipocytes.

That would make sense.

In the next post I'll consider why this might be the case.

Peter

How can insulin resistance cause weight gain? (1) ALDH2

Mea culpa. Sorry all for neglected comments else where on the blog and IRL. Blame Tucker for this link in here... Here's the next post.

There are three studies which have profoundly shaped my thinking about weight gain. First is this one

Insulin Resistance Associated with Lower Rates of Weight Gain in Pima Indians

Insulin resistance is protective against future weight gain (but not metabolic syndrome!)

Second is this one

Fat metabolism in formerly obese women

in which formerly obese women (ie at high risk of weight regain) are much more insulin sensitive than closely matched control women:

And finally this one:

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

in which normal weight, young people with a marked predisposition to obesity are more insulin sensitive than a matched control group:

So, when Tucker presents us with this paper looking at mice engineered to mimic people with reduced ability to detoxify the known generator of insulin resistance, 4-HNE (amongst other molecules)

A common East Asian-specific ALDH2 mutation causes obesity and insulin resistance: therapeutic effect of reducing toxic aldehydes by ALDH2 activation

then I have to think about this graph:

which is very exciting from the Protons perspective. 4-HNE augmentation causes insulin resistance *and* causes obesity. Yet obesity is the result of excessive insulin *sensitivity*, and is clearly visible (in this study) between weeks four and 16, as weights diverge:

Obviously by week 28 both groups of mice are going to be solidly insulin resistant so will gain minimal extra weight. It's weeks 4-16 we're interested in. You don't get fat without storing fat. We use insulin signalling to store fat. Resisting insulin signalling resists fat storage. A paradox, sent to provide insight.  That's worth a post in its own right.

Fascinating stuff. Lots to dig in to.

Peter

Delta psi and ROS

I'll post this now as I've gone off down a related but more concise rabbit hole for the next posts.

Okay. Here are a couple more building blocks for the future, snippets from the slightly dubious 

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

Here is the pattern of ROS generation from isolated mitochondria fed with increasing concentrations of palmitoyl carnitine:

Clearly there is a rise in ROS with increasing PCarn exposure, peaking at 18microM and decreasing to 72microM. There were too few functional preparations to include the 36microM value in statistical analysis. There is also evidence of uncoupling at 72microM. So 18microM gives maximum ROS generation. All measurements were taken under oligomycin to mimic state 4 respiration, ie fed with PCarn but conversion of ADP to ATP was not allowed.

And here is the delta psi developed by PCarn (plus extra carnitine) at increasing concentrations of PCarn. Note that there is a consistent delta psi estimated using between 3microM and 18microM PCarn with no suggestion of an upward trend:

Other places in the paper suggest that this delta psi is around 140mV (see below) and that exposure to PCarn at lower or higher concentrations will produce less ROS than the 18microM exposure does. To get more information about ROS generation they used oligomycin to establish a high delta psi then lowered it a little with 15pM FCCP (right hand end of the graph below) or a lot with 100pM FCCP (left hand end of the graph. As 18microM PCarn can only support 140mV I'm guessing that both voltages are below this value.

Here PCarn, the triangles, shows significant ROS generation at low delta psi with a modest rise to high delta psi, the latter being a little under 140mV. Glutamate shows a delta psi responsive rate of ROS generation.

In this final graph, ignoring the O2 consumption, we can look at the end derived delta psi values from three substrates:

The (expected) delta psi for 18microM PCarn is at 140mV (this is the max which PCarn at any concentration will support in this model), for saturating glutamate with blocked complex II it's 170mV and for succinate (driving through complex II) it's somewhere around 190mV.

What I am driving at is that palmitate, whilst generating ROS, does this without the requirement for, and not as a result of, high delta psi. For glutamate the higher the delta psi, the higher the ROS.

There is no suggestion that PCarn at 18microM is uncoupling yet delta psi is relatively low. What interests me is whether this is being achieved through the inhibition of complex I. In the last post I talked about the role of glutathiolation of proteins as a consequence of localised ROS generation and the role which that glutathiolation might have in the avoidance of situations where unbridled supply of metabolic substrate would lead to very high delta psi and an unacceptable rise in ROS generation. Recall Nick Lane's concept that a cell will sacrifice ATP production from ox-phos rather than generate excess ROS.

How much glutathione was available and what state complex I's glutathiolation might be were not in the remit of this study. So a lot of unknowns.

These are isolated mitochondria. There are, in real life, many controls to the supply of calories to mitochondria, as near by as the carnitine palmitoyltransferases, further away at the cell surface (GLUT4 and CD36) and as far away as adipocytes and hepatocytes. 

Again I have further ideas about ROS and it has to be made very clear that I am making huge extrapolations from isolated mitochondria towards what might happen in an intact cell with insulin exposure, insulin receptors, phosphatases, a controllable supply of Ca2+, superoxide dismutase, catalase etc etc, all in the correct locations to do what is impossible for an isolated mitochondrion to do.

So caution. But ultimately ROS from fatty acid oxidation generates ROS without the need for a high delta psi. Primarily NADH supplying substrates produce ROS in association with (and probably caused by) high delta psi.

Peter