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