Insulin resistance (19) The blue line

First an apology. I thought this post through weeks ago, helped by emails exchanged with Tucker, from which there are various rather exciting studies still to discuss. The problem is that when you retire there are a million things to do. The en-suite bathroom is now almost renovated and a start is made on the main bathroom. I've learned a lot of plumbing. The kids want to go kayak/bodyboard surfing and there as been some swell recently. And the grass is seriously growing and the goat fencing keeps needing moving and I'm still learning Ukrainian and trying to keep the house tidy. My torn hamstring is repairing and, assuming the 24k walk next Saturday goes well, I will start to run again after a two week rest. On a roll in both bouldering and roped climbing. It's been a bit difficult to find time to blog. My bad. Here we go again...

Now it's time to think about how it is possible to reduce hepatic lipid storage by a *reduction* of an ROS signal using α-tocopherol. We're still looking at this study:

α‐Tocopherol suppresses hepatic steatosis by increasing CPT‐1 expression in a mouse model of diet‐induced nonalcoholic fatty liver disease

Here's the blue line:

I work on the basis that the hepatocyte lipid distension noted in the last post is the cause of ALT release and this distention is the result of a failure to develop adequate ROS to resist the fattening signal of insulin. Standard Protons effect of LA.

This makes it easy to explain the difference between the standard diet and the lard based obesogenic diet, even if we do not know the amount of LA in the lard.

What is difficult (for me) is to explain why adding α-tocopherol, an ROS scavenger, blunts this process up to a plasma concentration of 116microM before the standard Protons facilitation process takes over as demonstrated by the red line in the last post.

I had been thinking about an ROS derived product of linoleic acid as a potential explanation around the time that Tucker suggested that this substance might be 4-HNE. This is the obvious candidate.

The simple tenet of Protons is that the oxidation of linoleic acid generates too few ROS to correctly resist insulin when a cell is exposed to a surfeit of substrate.

However, this is not quite as straight forward as it seems. Ultimately a given cell will settle on the correct peak level of ROS generation, which is tightly controlled. In the past few months I have used a fairly arbitrary level of an equivalent to 0.3mM H2O2 as this maximum cut off, though this will certainly not be the actual level in vivo.

The problem with LA is that more caloric substrate will enter a cell before this 0.3mM of H2O2 is generated. Until we get outside of physiology this will still be the limit. Things change in cell culture where ROS generation can be driven above this by manipulating culture conditions and in vivo by supplying excess calories from combined hyperlipidaemia/hyperglycaemia as in diabetes. Then damage  from high ROS occurs.

But, under normal physiology, this does not happen. In simple obesity the failed ability to resist caloric ingress is eventually corrected by rising inner mitochondrial membrane potential and we still have our nice limit of 0.3mM H2O2 as the physiological maximum.

To be clear, the end peak ROS signal is the same under saturated fat, polyunsaturates or even under glucose oxidation. All that differs is the source of those ROS. Predominantly from RET with saturates, a little less so from PUFA and from predominantly from rising delta psi under glucose oxidation.

So, in the absence of diabetes, the peak physiological ROS signal is essentially unchanged.

All that changes under an high fat diet based on modern lard is the amount of linoleate available to form 4-HNE is the presence of that theoretical 0.3mM peak ROS level which is normal physiology (admittedly with pathological caloric ingress in the presence of LA).

It is absolutely and widely accepted that 4-HNE causes insulin resistance. Correct. So how can increasing 4-HNE mediate resisting insulin to cause lipid accumulation?

It can't.

What causes lipid accumulation is insulin signalling. Resisting insulin signalling resists obesity.

Low levels of 4-HNE produced secondarily to physiological ROS exposure generate further ROS which *supplement* insulin signalling. Whether the 4-HNE is a direct insulin mimetic per se in the absence of supplementary low levels of ROS appears to be debatable.

The effect is dose related.

Going to 

The expression and function of Vascular Endothelial Growth Factor in Retinal Pigment Epithelial (RPE) cells is regulated by4-Hydroxynonenal (HNE) and Glutathione s-transferase4-4

we can see that concentrations of 4-HNE in the region of 0.1µM act as agonist for VEGF secretion with progressively less effect with rising concentration until at 5µM there is a peak effect and above this there is progressively less effect until by 40µM there is direct cytotoxicity.

The effect on VEGF secretion is mediated via the phosphorylation of Akt, exactly as insulin phosphorylates Akt to facilitate lipid storage. Work using adipocytes and lipid accumulation usually starts using insulin resisting levels of 4-HNE and works up to cytotoxicity levels, because that's the current paradigm. We've all read

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

where as little as 10µM 4-HNE causes profound failure of adipocytes to accumulate lipid. You know, like this:

To re-emphasis, 4-HNE at 10 microM is highly protective against adipocyte lipid accumulation. It is protective against obesity, whatever collateral damage it causes.

I consider 4-HNE at low levels of around 0.1µM to act as an insulin mimetic and at higher levels to act as an inducer of insulin resistance. We can take the H2O2 graph from Czech et al and substitute with some plausible levels of 4-HNE:

The process of forming agonist levels of 4-HNE from the normal physiological levels of ROS generation by simply increasing the level of LA in the cellular environment is something that a) I find highly plausible and b) likely to be amenable to reversal with α-tocopherol.

This is not using α-tocopherol to lower ROS in bulk. We are looking to reverse the generation of an agonist level of 4-HNE and reduced lipid accumulation from this reduction in 4-HNE. Low doses work for this.

At very high levels of α-tocopherol the ROS scavenging  effect gives a bulk reduction of ROS and, via Protons, imitates the poor ROS generation which facilitates the obesogenic effect of LA.

Low dose vs high dose. The dose makes the poison.

Peter