Fructose (06) Acipimox tangent

I've glibly stated that I consider insulin resistance to be a simple but imperfect adaptation to the inability of insulin to suppress basal lipolysis, that is the rate of FFA release from isolated adipocytes in an ex vivo situation.

One of the best suppressors of basal lipolysis is the nicotinic acid derivative acipimox.

There's a review of our knowledge in 2007 here:

Nicotinic Acid Receptor Subtypes and Their Ligands

which has a nice diagram like this, slightly edited:

The process is quite simple. Acipimox, a nicotinic acid derivative (NA), interacts with a cell surface receptor (here HM74A but it has many, many names) which is coupled to a signaling G protein (G) which inhibits adenyl cyclase (AC), lowering cellular cyclic AMP so deactivating a cAMP dependent protein kinase (PKA) which does something magical to all lipases to shut down lipolysis.

Despite the fact that acipimox can reverse diabetes overnight it has been a clinical complete flop and is nowadays only used to improved a few lab numbers of people eating a diet of complete crap.

I woke up this morning thinking about why "Magic happens" in the above diagram. Not how, but why.

Niacin is a drug acting on the ketone body receptor of adipocytes. The endogenous activator is shown as beta-hydroxy butyrate:

The obvious explanation for ketone bodies inhibiting lipolysis is that this is a negative feedback loop. The original diagram also had FFAs inhibiting lipolysis too.

There are upper limits to the body's tolerance of elevated FFAs and ketosis is a marker that decreasing the FFA supply to the liver might be a good idea.

The most obvious inhibitor of lipolysis via hormone sensitive lipase is insulin. Under ketosis (ignoring MCT exposure) insulin is at absolutely rock bottom levels and if you want something to limit lipolysis under hypoinsulinaemia you had better develop a system which does so independent of insulin's action.

Ketones do that (pax hyperglucagonaemia and ketoacidosis, there are limits).

So if a cell has elevated basal lipolysis (which cannot be shut down by insulin) choosing an alternative mechanism, insulin independent, for suppressing lipolysis is effective stratagem.

Acipimox activates this process in the absence of the normal physiological ligand, ie ketones.

ATGL is key to mediating lipolysis triggered by increased adipocyte lipid droplet size where as elevated insulin exposure cannot do this. You can shut down this elevated basal lipolysis with a "ketone mimetic".

Which reverses type two diabetes. Until the drug wears off.

Peter

Oh, another thought: Is this how exogenous ketones improve insulin sensitivity? Never mind all those complicated intracellular switches (I told you I was lazy). The "problem" is that adipocytes are leaking FFAs. Acipimox fixes this problem. Acipimox is a ketone mimetic. Do exogenous ketones "mimic" acipimox? Amusing thought for the day!

Fructose (05) Four out of five diabetics

This follow on study looking at fructose in people with type 2 diabetes was a bit of a disappointment. 

TLDR: Fructose works pretty well in four out of five people with diabetes just as it does in "normal" people with a relatively poor OGTT result.

Acute Fructose Administration Improves Oral Glucose Tolerance in Adults With Type 2 Diabetes

This is what the title of the study is describing:

It's clear that adding 7.5g of fructose to a 75g OGTT load improved both glucose and insulin curves. There is nothing exciting about this. What I thought might be interesting was that, in a study of five subjects, two of them were outliers of sorts.

I'd hoped these outliers might give more insight in to the state of ROS signalling within hepatocytes of people with severe diabetes. Not really, much of the data you need is not in the paper. For completeness here are the anomalous results:

Subject 2 failed to drop their systemic glucose in response to added fructose, like this

Not unsurprisingly they also failed to drop their insulin level, clearly insulin level follows exposure of the pancreas to systemic glucose, which didn't change, so neither did insulin.

Can we guess what might have been happening in hepatocytes to nullify the "beneficial" effects of fructose? Let's assume that fructose is being absorbed, entering hepatocytes and generating ROS via a NOX enzyme in proportion to the rate of fructose ingress. So the question becomes

"Under what circumstances does a modest increase in ROS fail to activate the insulin cascade?"

We have no idea what the fasting insulin level was, nor the fasting glucose, so this is a little difficult. We are told that subject 2 had an HbA1c of 9.0 or 10.1, one of the highest in the study, which implies the worst average glucose excursion over the last three months or so. We could choose extreme insulin resistance, failing beta cell function or a combination of both to explain this. The fact that they produced a minimal increase in AUC for insulin when presented with 75g of glucose with or without fructose suggests that beta cell function was limited and underlying hyperglycaemia might both be present. I favour this explanation.

Both hyperinsulinaemia -> NOX4 activation via G protein coupled signalling and hyperglycaemia -> NOX2 activation via calmodulin kinase signalling have the potential to spill ROS over from activating via phosphatase inhibition to being inhibitory via insulin receptor substrate inhibition. Adding a small increase in ROS from fructose to a maximally stimulated system may affect both aspects, producing no net change. That's my guess.

The second unusual result was from subject 5. They produced a perfectly reasonable drop in systemic glucose in response to fructose addition but had an unusual increased insulin response despite reduced systemic glucose.

I struggle to explain this "paradoxical" rise in insulin despite a successful fructose induced fall in pancreatic exposure to systemic glucose. You could argue that the increase in insulin came first and this lowered the systemic glucose level but this would make quite an exception and need either a pancreatic response to a very small systemic fructose rise or a pancreatic effect derived from gut hormone signals, none of which were measured and none of which are needed for an explanation of the fructose effect in all other subjects.

If I had to guess I would suggest the fasting insulin level was very high in this person. We know from this 2011 study that OGTTs are reproducible on repeat testing (in "normal" people) over 3 days unless you have to be hyperinsulinaemic in order to be "normal":

Reproducibility of multiple repeated oral glucose tolerance tests

"However, our cases of individuals who exhibited hyperinsulinaemia in order to maintain glucose homeostasis, suggest that repeated OGTT’s may not produce a reliable estimation of insulin sensitivity for people with pre-diabetes and diabetes."

Subject 5 was also an individual with very high HbA1c (9.0 or 10.1) suggesting poor control and compatible with high insulin levels. So the insulin anomaly may just be random finding in one person secondary to chronic hyperinsulinaemia... We'll never know.

Overall the fructose effect on hepatic glucose output seems to be genuinely maintained in patients with DMT2 unless they are approaching seriously poor levels of control.

Should individuals with more "mild" diabetes add a little fructose to each bowl of porridge they consume in real life?

Rhetorical question.

Peter

Fructose (04) Normal Adults

Now it's time to think about this paper

Acute Fructose Administration Decreases the Glycemic Response to an Oral Glucose Tolerance Test in Normal Adults

These are the results from an OGTT on a set of healthy human volunteers who took 75g of glucose with or without 7.5g of fructose. From the top graph you can see the results are more than a little inconclusive. If you instead plot change from baseline you get the slightly more convincing pair of curves below and if you compare the areas under the curves for the second plot you get p less than 0.05.

So fructose addition"almost" or "just" works. I think it was this set of findings which made Cherrington's group go for the canine study with a continuous duodenal infusion of glucose with or without extremely low dose fructose inclusion. So much more control and stability, over hours, than a 75g OGTT in a human where an awful lot of changes occur in the first 30 minutes.

Never the less some very interesting findings did come out of the human study.

As the authors noted, and published, some "normal" people have better OGTT results than others. And they also noted that, if you have a relatively poor OGTT result, you respond well to fructose. If you have a good OGTT result you don't respond to supplemental fructose at all. There is a correlation. It looks like this:

Okay, along the x axis we have a measure of how "good" an individual's OGTT result was. Low values are best, higher values are poorest. The 

y axis indicates how much improvement in OGTT occurred with adding 10% fructose. There is a clear cut cluster at the left hand end. These individual people have the best normal OGTT results and do not respond to fructose supplementation, at all:

At the right hand area of the plot we have people who are trending towards impaired glucose tolerance by having a poor OGTT result and these clearly have a nice response to supplementary fructose:

We can then take those five people at the bottom left of the correlation plot and make a plot of their OGTT results with and without fructose and it comes out like this (using change from baseline glucose, not the absolute values, of course):

I think this is clear cut, if your OGTT only spikes your BG by 4.0mmol/l there is no benefit from adding fructose.

Next these are the six individuals for whom the straight glucose OGTT spiked their blood glucose by 5.0mmol/l:

Very clearly these six people benefit from added fructose.

If you have poor glucose tolerance you benefit from adding fructose. You become almost normal!

How come?

You could, if you wanted, take a diagram like this

which I found in this comprehensive review:

Fructose metabolism in humans – what isotopic tracer studies tell us

(as a link kindly supplied by Jaromir in previous comments.) and try to work out exactly what nudging 75g of glucose metabolism by the co-adminstration of 7.5g of fructose might do to OGTT results in a real live human being. Sadly this is utterly beyond my abilities so I have to fall back on my own trusty crutch, the very simple ideas of ROS and what is meant by impaired glucose tolerance.

The last post was a summary of what I think the role of ROS from differing sources have on insulin signalling. In particular this concept matters:

Insulin acts (among many other places) far, far away from the liver, on adipocytes to suppress lipolysis when there is a copious supply of glucose/insulin available. My definition of impaired glucose tolerance is the inability of adipocytes to limit their release of FFAs to the circulation under the influence of this insulin. In the above doodle insulin shuts down FFAs to 50micromol/l. That's normal. Now let's repeat this illustration with a residual supply of FFAs at around 200micromol/l under an OGTT.

Insulin still docks with the insulin receptor, NOX4 still produces activating ROS but mitochondrial FFA oxidation is producing some degree of partial blockade of the insulin cascade at the insulin receptor substrate level:

The solution to this problem is simple, hyperinsulinaemia. This will not reduce the FFA levels from high basal lipolysis but will allow a "forced" increase in activating ROS and so increase phosphatase inhibition and so increase the insulin cascade to overcome the partial "obstruction" at the IRS level, like this:

Let's be absolutely clear. The hyperinsulinaemia is a sticking plaster placed on to the excess basal lipolysis secondary to adipocyte distension (linoleic acid derived). It is NOT a cure, it's a bodge.

The above is what I consider the metabolic explanation of people with poor glucose tolerance under an ordinary OGTT. The message is that the hyperinsulinaemia is just a tool to increase NOX4 derived ROS to a high enough level to allow the cell to overcome the inhibitory effect of excess FFA oxidation which should not be there.

The extra ROS are what makes this happens and the cost is hyperinsulinaemia.

Now lets add in some fructose.

The fructose (all in green) enters the cell rapidly and talks to its own NOX enzyme generating its own extracellular superoxide.  This dismutates and re enters the cell at the location where ROS are used to activate insulin signalling by disabling inhibitory phosphatases. So fructose derived ROS accentuate the insulin cascade to restore insulin signalling, much as hyperinsulinaemia would do, but without the hyperinsulinaemia.

Again, the signal is the ROS.

Again, it's not a cure, it's a sticking plaster. We are simply using an alternative source of supplementary ROS to activate the insulin cascade. I've changed this last feature to green as well, just to signify it's being helped by fructose derived ROS. I don't mean to suggest this is fructose catabolism, just fructose-ROS facilitated pAKT activation et sequitur.

Activating the insulin cascade in hepatocytes without any action anywhere else will lower glucose penetration to the systemic circulation and normalise the OGTT result.

Recall there is a specific problem causing the poor OGTT result and we have not fixed this problem. Things look better but the abnormal basal lipolysis derived FFAs are still there.

In people with an excellent OGTT result the insulin cascade pretty well fully activates and adding a few extra ROS seems to make little difference in this model. Of course we know that there are changes in the liver of very normal individuals secondary to low dose fructose because we already know the results of the subsequent canine studies.

However two of the normal people had a worsening of their OGTT area under the curve, one by quite a lot, clearly visible circled in red here on the correlation graph:

These two individuals had an excellent OGTT without fructose so we can assume they are perfectly capable of suppressing adipose lipolysis to levels which don't interfere with insulin signalling. You have to ask yourself if it is possible that the ROS generated by fructose and its NOX were sufficiently numerous that they diffused as far inwards from the cell surface as to start to act on the insulin receptor substrate in a mild version of the marked inhibitory effect of FFA oxidation derived ROS. I've put this speculation in as a dotted green line and reduced the overall insulin cascade curving arrow to show some limitation of downstream signalling:

When we come to think about grossly toxic doses of fructose this generation of impaired insulin signalling is likely to be come a primary feature. Perhaps we have a hint here. Just guessing. 

We are not given enough individual subject level data to see what happened to any parameter other than glucose in this study. We only get the whole group aggregate data for most parameters.

I think the concept outlined makes sense and seems quite simple. Fructose feels like it is starting to yield some of its secrets.

Time to look at fructose and type 2 diabetes next.

Peter

Fructose (03) NOX vs RET

This post is not about any specific study. It just gives the background to understand the post after, which is already written but I need to sit on it for 24h and correct the worst of the typos/logic errors. Okay...

Before we can go on to understand the role of fructose as an insulin sensitiser/mimetic I think a little more explanation of NOX enzymes might be in order. So this is a very simple view of NOX and ROS vs reverse electron transfer through complex I and ROS.

So let's begin here again, don't forget this is the cell surface, mitochondria will reappear further down the page:

I want to start with the red square which we can pull out thus:

The basic story is that insulin "talks" to NOX4, activates it and the resulting ROS deactivate a number of (inhibitory) phosphatases, which allows the insulin signalling cascade to take off. That little red arrow needs some elaboration and is an oversimplification.

I have mentioned before that the basic NOX core looks extremely primordial (thought it appears it is actually an eukaryotic invention) and it is better represented like this, taken from here:

The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology

The NADPH binds to a protein containing an FAD moiety which accepts a pair of electrons to form FADH2, temporarily. This seems to be a "bolt-on" NADPH oxidase which can supply high energy electrons to any process you care to bolt the oxidase on to. Here it feeds electrons to a "wire" to the outside of the cell. The six transmembrane helices form a tunnel containing two haem iron groups which are the "wire". One electron at a time travels from FADH2 "down hill" towards the exterior of the cell. But to fully traverse the cell membrane it is travelling against the cell membrane voltage and can only make it if oxygen is docked on to the outer end of the "wire" as terminal acceptor, like this:

Obviously superoxide, as a charged particle, is not going to re-enter the cell. Some NOX members have another "bolted-on" sub-unit which means the NOX produces H2O2 rather than superoxide, which has a much better chance of entering the cell. There is also extracellular SOD3 which can dismutate superoxide to H2O2 to re-enter the cell.

So we can now look at the activation of insulin signalling again but with a modified NOX doodle:

There might be quite a lot of arrows but the set-up isn't really that complicated. The core message is that the ROS which activate the insulin cascade are of extracellular origin. We will see later that they can influence more distant intracellular sites but I want to stay with simple activating physiology of the insulin cascade here for today. As far as insulin signal activation the triggering ROS are of extracellular origin. Hence the location of the "ROS" labels in the initial image from which the above doodle is derived.

Next we have to think of fasting. Here's a view of ROS signalling under fasting conditions. FFAs can be between 1000 and 3000micromol/l in healthy people under an extended fast. To survive under these conditions it is helpful to reduce both the absolute level of insulin and the level of signalling in the insulin cascade from whatever insulin is present. This is how it works:

The oxidation of FFAs will generate ROS by reverse electron transfer through complex I irrespective of mitochondrial membrane potential (as long as this is within physiological limits) and will be a primary mechanism for resisting glucose usage wherever FFAs can substitute to allow glucose sparing for tissues where glucose is an essential requirement. Of course long chain saturated fats do this best but all mitochondrially targeted fats do it to some degree.

Before I pause to make some more doodles on more interesting subjects we just have to add in how FFA supply is regulated. At the start of an OGTT glucose is rapidly absorbed and penetrates to the systemic circulation and stimulates insulin secretion (pax GLP-1 and related overlays to this system) from the pancreas. In healthy people this insulin might drop FFAs as low as 50micromol/l:

This has nothing to do with an individual cell, it's a distant effect of insulin acting on adipocytes to suppress lipolysis and so suppress FFA supply to the whole body. Which will markedly reduce mitochondrially generated ROS. Any residual mitochondrial FFA derived ROS might even be at insulin facilitating levels:

Ultimately the above doodles describe models, which are simple extremes and only distantly relevant to the integrated performance of the complexities of glucose and fatty acid derived energy production and ROS control. But they form a reasonable framework to go on to explore the findings in Cherrington's human OGTT studies.

TLDR

Insulin -> NOX -> cell surface low ROS -> activating

FFAs -> RET -> mitochondrial high ROS -> inhibiting

So these are next, normal adults first:

Acute Fructose Administration Decreases the Glycemic Response to an Oral Glucose Tolerance Test in Normal Adults

Acute fructose administration improves oral glucose tolerance in adults with type 2 diabetes

Peter

Fructose (02) Obesogen

I guess we could do worse than to being this post here;

Inclusion of low amounts of fructose with an intraduodenal glucose load markedly reduces postprandial hyperglycemia and hyperinsulinemia in the conscious dog 2002

It's a follow on study after an initial dog study from 1998 which demonstrated that, much like glucose, in a tightly controlled somatostatin/insulin/glucagon model, low dose fructose markedly suppresses hepatic glucose output and markedly increases glycogen formation in healthy dogs. On a fixed insulin background, low dose fructose behaves as an insulin mimetic.

This follow on study in 2002 used hormonally intact dogs instrumented for portal vein, hepatic vein, femoral vein and femoral artery access. In addition they were cannulated to allow intra-duodenal infusion of glucose or glucose + fructose. This gets rid of all of the pesky delay in gastric emptying involved in an OGTT and allows time to assess effects under steady state conditions rather than the brief and dynamic changes which occur under an OGTT. Between the first and second canine studies the group had performed a couple of sets of human OGTT studies +/- fructose with somewhat challenging results, which I suspect is what led to this latest canine study, to really control as many variables as practical.

Aside: There is a horrible typo in the plasma glucose graphs throughout the study. For glucose they clearly mean milli moles/l, not micro moles/l. Arghhh. I think the fructose at 100micromoles/l is likely to be correct. I can't face trying to confirm whether their infusion rates are correct re micro vs milli. Ugh. I think their data are fine per se, unfortunate re typos. End aside.

The intra-duodenal glucose infusion was pitched to generate a modest, stable hyperglycaemia at ~10mmol/l. In the intervention section this glucose infusion remained unchanged at 44.4μmol/kg/min but an additional infusion of fructose was added at 2.2μmol/kg/min, ie around 5% fructose in addition to the original glucose. This is what happened to the systemic plasma glucose levels:

If anyone had developed a drug to produce this effect in diabetic patients it would be impressive. But that's not all. Look at what insulin levels were needed to achieve that drop in blood glucose:

Adding in 2.2μmol/kg/min of fructose to an hyperglycaemic glucose infusion profoundly lowers the insulin levels needed to maintain a modestly reduced hyperglycaemia value. In a hormonally intact dog preparation.

That's amazing if correct. Fructose appears to be profoundly insulin "sensitising" or mimicking.

Almost nothing is being "done" with the fructose. It enters the liver, drops though fructolysis and is excreted largely as lactate, well in excess of that produced by the glucose alone. I hope we're all aware that fructolysis bypasses those regulatory steps which make glycolysis a tightly regulated process. Here's the lactate change:

Next we can quickly doodle in the fructolysis pathway taken from here

Fructose Metabolism in Cancer

which has this as a generic cell capable of metabolising fructose. In the liver the various GLUTs will be different but the same principle applies:

We can add in fructolysis with its unregulated, high flow pathway to lactate as typically seen in our current discussion of hepatocytes. Like this:

Equally we can put in the tightly regulated glycolysis route to either ox phos and/or lactate, usually a bit of both. But regulated by pH, citrate, ATP or oxygen availability.

We can put both pathways in together and throw in the insulin receptor too:

We are now in a position to add in the core features to an understanding of what is going on. Like this:

and then we can fade the background to let us concentrate on the generation of ROS:

Here we have the situation under intra-duodenal glucose infusion alone. ROS are being generated by glucose ingress per se using NOX2 and also by insulin docking with the insulin receptor to activate NOX4. Both insulin level and the entrance of glucose in to cells without insulin are tightly controlled.

Neither glucose nor insulin are the signal to activate the insulin signalling cascade. That is purely the prerogative of ROS, at physiologically appropriate levels. The end result is a glucose value of around 10mmol/l and an insulin value at around 250pmol/l.

This is what happens if we add 2.2μmol/kg/min of fructose to the glucose infusion. We achieve a portal vein fructose concentration of 100μmol/l which will generate significant ROS, in direct proportion to the rate of entry of fructose to the cell, which will be high:

The fructose generated ROS will increase the ROS signal. This is the activator of the insulin cascade. The ROS signal will result in the phosphorylation of AKT, the activation of glycogen synthesis and will decrease the penetration of glucose past the liver and in to the systemic circulation. Systemic glucose will fall, less pancreatic secretion of insulin will be needed.

We get what we see in the study.

My personal definition of an obesogenic drug is one which activates "insulin" signalling in the absence of insulin. Fructose generates "unexpected" ROS when entering liver cells at a concentration of 100μmol/l. I think it is perfectly likely that, should one achieve an adipocyte exposure of 100μmol/l of fructose, that a similar effect might occur.

In Laughlin's 2014 review

Normal Roles for Dietary Fructose in Carbohydrate Metabolism

there is the suggestion that peak systemic fructose concentrations healthy men after a high sugar exposure might be about 0.5mmol/l, ie 500μmol/l and a fasting level might be a fraction of a μmol/l.

There are a number of studies showing fasting fructose levels as high as 2.0mmol/l (ie 100 times Laughlin's accepted level) with post fructose loading reaching over 17.0mmol/l. That makes life easy for everyone. If you want to show fructose is harmless or beneficial you chose the low studies. If you want toxicities you choose the high studies.

But are low doses of fructose beneficial at all? Is insulin cascade activation good or bad? Are the benefits of reduced absolute insulin exposure offset by the ROS mediated activation of insulin cascade?

Whichever fructose measurement camp you fall in to, a normal human being can easily maintain systemic fructose levels > 100μmol/l in the aftermath of even a modest fructose load, which in the dog studies I consider generates enough ROS to be markedly facilitating of what we could call an insulin sensitising or mimetic exposure. Perhaps we should apply this to adipocytes.

Does fructose make you insulin sensitive? Of course it does. Does activating insulin signalling make you fat? Of course it does.

Except, of course, when it does the opposite.

Peter

Fructose (01) There's a signal

If we start from this diagram

produced in 2006, well ahead of its time in here:

Role of Insulin-Induced Reactive Oxygen Species in the Insulin Signaling Pathway

we can add in the standard ROS signalling pathway, triggered by insulin, without any hint of speculation thus:

Our next move is to look beyond the insulin signal and concentrate more on the glucose -> NOX (NADPH oxidase) ignoring the mitochondria, ie these components of the diagram

and especially consider the highlighted process in red.

Nowadays we have a little more information, taken from here

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes

As they conclude

"Our results showed that acute Hi-Glu induces cardiac myocyte ROS production via O-GlcNAcylation of CaMKIIδ and consequent activation of NOX2 ROS production in the cytosol, but not in the mitochondria."

which lets us add a little detail to the diagram:

So it's worth noting that severe (30mM) hyperglycaemia can activate NOX2 ROS generation in intact mice or their isolated cardiac myocytes through a now determined mechanism without exogenous insulin. In general this is a Bad Thing. Like this:

That's fine, as far as it goes. But it's mice or cells. And 30mM glucose.

Does this happen in real live people? Well, that depends. It's not easy but if you take some normal people, paralyse their hormonal signalling systems with somatostatin combined with a replacement infusion of basal insulin/glucagon/growth hormone you can certainly use an abrupt switch from plasma glucose at 5mmol/l to 10mmol/l and see what happens. In the control group at least, from the ancient days of 2002, all that happens is that, as assessed by glucose tracer, 10mmol/l of "hyperglycaemia" suppresses hepatic glucose output. Without increased insulin exposure and hence without any change in insulin receptor mediated signalling.

Mild hyperglycaemia -> suppressed hepatic glucose output in normal people.

Important: Mild hyperglycaemia appears to be a "functional insulin mimetic".

Without any increase in insulin exposure I would predict that a small rise in plasma glucose will result in some phosphorylation of AKT, by non-insulin mediated ROS generation.

We can now explain this finding in the control group of this paper, ignoring the (beneficial) role of [redacted] in type 2 diabetes, to which I will return on another day. 

[Redacted] improves the ability of hyperglycemia per se to regulate glucose production in type 2 diabetes

We can posit this:

Now it's time to just peek at fructose [which I redacted previously] to see if we can shed some light on to its function in health/disease and ask:

Does fructose cause obesity? Yes and no.

Does it cause insulin resistance without obesity? Yes and no.

Obviously the correct answer is that we are asking the wrong questions.

To answer any questions about insulin "sensitivity/resistance" we have to be asking about ROS. Exactly the same questions about fructose and ROS generation as we already have the answers to about glucose 10mmol/l vs 30mmol/l and ROS generation.

First off let's make a sweeping assumption that fructose exposure is like glucose exposure. This is almost certainly correct. Something like this:

No one has a mechanism in terms of which intermediate signal, which kinase or which of the NOX(s) are involved. Yet. That will come. At least one NOX is where the ROS originate from.

If you are a rat/mouse on dry chow and your only access to any sort of fluid contains either 10g or 20g of fructose in every 100ml of water this is what is going to happen. There is an essentially infinite supply of such studies, just search Pubmed for "Fructose" and "ROS":

What are much, much harder to find are the studies which suggest that this is a Real Thing:

There is quite a literature supporting this latter image too but none of it shows under "ROS" searching. I gained access to it through this review, to which I am very grateful:

Normal Roles for Dietary Fructose in Carbohydrate Metabolism

It's from 2014 and the author has no concept of insulin being a superficial over-laid veneer on the underlying ROS signalling system, or even that there is an ROS signal involved. And the literature cited is from pre-pAKT days, so we have to reverse engineer the gross rodent/human studies to think about them in terms of ROS as the core signal. Even today we seem have essentially no idea of how fructose signals to NOX to generate ROS.

But it does.

Peter

Metformin (15) ROS

Time to talk about insulin signalling, metformin and the phosphorylation of AKT.

I borrowed this image

from this paper

An Intimate Relationship between ROS and Insulin Signalling: Implications for Antioxidant Treatment of Fatty Liver Disease

because, unlike most images you will pull out by searching "insulin cascade", this one actually features a role for ROS and so forms a good basis for discussing metformin. So once agin it's PowerPoint doodle time. I'm going to miss out everything from the paper about anti-oxidants and PGC-1𝛼 etc.

First of all let's simply reduce the diagram to the canonical insulin signalling pathway by using a few blottings out:

In the simplistic world of insulin signalling, insulin arrives and this happens:

As part of the process AKT is phosphorylated and this is one of the core signals to activate the translocation of GLT4 and CD36 to the cell surface, facilitating caloric ingress:

There are several AKTs and they can be phosphorylated in various places but the simple message is that

Insulin -> insulin cascade -> pAKT -> caloric ingress

It is now very well accepted that the trigger for this cascade is the generation of ROS by NADPH oxidase 4 (NOX4) in response to the docking of insulin with its receptor. Like this:

Of course the glucose and fatty acids which enter the cell have to go somewhere so we can add in a mitochondrion:

which produces its own ROS

There is also a signalling cascade which carries information about these ROS. My supposition is that low levels of mitochondrial ROS act, as do those from NOX4, to facilitate the activation of insulin signalling.

However once the cell is calorie replete ROS generation rises markedly and the resulting generated high level of ROS acts to shut down insulin signalling at the insulin receptor substrate 1 (IRS1 on the diagram) point.

At this stage the whole insulin cascade stops functioning, as it should, because the cell is calorie replete. As part of this shutting down process the level of phosphorylation of AKT cannot be increased, no matter how much insulin is applied:

Think about what is happening. There is nothing wrong with the system. The failure to further phosphorylate AKT is not a fault for some drug developer to "correct". It is the direct result of evolution happening on to the ideal system for monitoring and controlling calorie ingress.

The substance which is the best for a cell to monitor, for maximal survival, is mitochondrial superoxide (+/-H2O2).

It's not insulin, it's not pAKT, it's not ATP, it's not NADH.

It is ROS.

So we can observe that phosphorylation of AKT, as a core part of the activation system for insulin signalling, is only allowed to occur provided the resultant ROS generation is within evolutionarily acceptable limits.

Now let's revisit

Insulin Resistance Induced by Hyperinsulinemia Coincides with a Persistent Alteration at the Insulin Receptor Tyrosine Kinase Domain

and add some detail to this graph:

I have pointed out in the past that the three concentrations of insulin used to generate this graph are five times, seventeen times and one hundred and seventy times the approximate upper limit of physiological exposure. All three levels produce exactly the same level of AKT phosphorylation because all three concentrations produce the maximal ROS tolerable to the cell. These ROS disable signalling at the level of IRS1.

I'm now going to modify the above diagram to include just the red box and stretch it to make it easier to see:

and add in some imaginary, more physiological, insulin concentrations:

I've assumed insulin in cell culture acts within the same five minutes as the supra-maximal doses do and the amount of pAKT formation stays near constant once set, as it does for supra-maximal exposure. These features may not be strictly correct.

If we wanted to construct an imaginary dose response curve it would look like this, here we are converting the above graph in to one showing the amount of phosphorylation of AKT produced by a given concentration of insulin. Again, the curve will not be accurate but the principle will be. As a rule of thumb 1000pM of insulin, ie 1.0nM, is peak insulin exposure after an high carbohydrate meal in an healthy person, which lets me put some very approximate absolute levels of insulin exposure:

It is quite possible to move the horizontal red line of response to supra maximal insulin exposure up or down. If you are insulin "resistant" you will have less pAKT at supramaximal insulin exposure. If you are insulin sensitive you will have more.

Now it's very simple. If you wanted a single measure of "insulin resistance" just look at the maximum level of pAKT under extreme hyperinsulinaemic conditions. If pAKT is low this signifies inadequate maximal insulin signalling and so insulin resistance.

If pAKT is high this signifies insulin sensitivity. This is the concept encapsulated by the hyperinsulinaemic euglycaemic clamp from back in the days when measuring AKT and pAKT involved more than buying a kit from Sigma-Aldrich.

All of which is missing the point. Completely.

What is actually important is the level of ROS generation from the mitochondria.

Metformin: What does it do? At pharmacological plasma levels it inhibits the action of the glycerophosphate shuttle. It reduces the conversion of NADH to FADH2 by this shuttle. Less FADH2 means less reverse electron transport (RET) as judged by the FADH2:NADH ratio. If this results in a pharmacologiocally reduced level of ROS under metformin this will allow more glucose signalling (ie pAKT) before cellular "satiety" kicks in due to generation of high physiological ROS to finally shut down IRS1 functionality. Like this

So given that single measurement of pAKT (or the rate of glucose infusion needed for euglycaemia under the last 40 minutes of an hyperinsulinaemic clamp) then metformin is, absolutely, an insulin sensitising agent. But that's because you are looking at pAKT, not ROS.

The level of ROS at for both plateaux in the above graph will be identical. That is what evolution has determined to be the best peak "target" level of ROS. Metformin blunts ROS production so allows more pAKT to be formed before ROS generation becomes high enough to shut down insulin signalling.

I could suggest that metformin allows more insulin mediated ROS at peak physiological (or above) insulin exposures. That seems quite simple.

But is the above metformin graph actually correct? Partial reduction of ROS by inhibiting mtG3Pdh to allow a greater peak insulin effect is one thing. But what about reducing ROS from physiological levels of insulin exposure, where ROS are activating to insulin signalling? We are now looking at modifying this red arrow process:

So if we lower ROS under these circumstances using metformin we will decrease insulin signalling. So our graph should actually look like this:

The blue section of the metformin curve has reduced ROS so signals less insulin pathway activation compared to control cells. ROS never peak anyway, and so are simply proportionally reduced under metformin.

So "normal" people, who run their metabolism on the blue dashed part of the metformin curve will show as "paradoxically" worsened insulin signalling. As we saw here:

Now let's consider DMT2. At its simplest level diabetes is the over distension of adipocytes secondary to the insulin sensitising effects of linoleic acid in combination with an insulinogenic diet. Once adipocytes are large enough basal lipolysis allows FFA release which cannot be suppressed by insulin.

If you have elevated fatty acid oxidation which cannot be reduced by insulin acting on adipocytes then ROS will be being generated at all times. There will be elevated baseline ROS, being generated from this fatty acid oxidation. If we add glucose and insulin, as in an OGTT or an hyperinsulinaemic euglycaemic clamp, the additional ROS from this calorie source will not have to generate very much extra ROS to shut down insulin signalling at IRS1 and so limit pAKT.

I repeat, it's the ROS that count. Oxidising fatty acid generates ROS without phosphorylating AKT. There is then only limited "scope" in the ROS budget before insulin signalling (hence pAKT and/or glucose infusion under clamp) has to be shut down. Not because the cells are "insulin resistant", it's because they have largely already met their ROS quota from fat. Which should not be there, fat supply should shut down immediately with even a tiny increase in adipocyte insulin exposure. But excess FFA will always be supplied (and oxidised) if there is unstoppable basal lipolysis.

Under these circumstances pAKT will be low because the ROS quota is nearly full to begin with. Adding metformin will reduce the generation of ROS from the glycerophosphate shuttle and so allow more "room" in the ROS budget which will allow more AKT phosphorylation and more glucose uptake before the ROS quota is used up. Things appear to improve for DMT2 under metformin's action.

TLDR: Is metformin insulin sensitising? Wrong question. Ask instead what metformin does to the generation of ROS. You can ask the same question about BAM15, DNP and even semaglutide.

Balancing the ROS budget explains everything.

I'll stop now.

If anyone has a better explanation I'm all ears.

Peter