I have been culturing human podocytes in normal media (RPMI+10%FBS+ITS+L-glut) and high glucose media (same, but final glucose concentration of 30 mM). Every time I have seen that high glucose increases phospho-AMPK levels (Thr172) in Western blot. I have tried this several times with several incubation times. On the other hand, AICAR increases p-AMPK levels. Do you have any idea what's wrong with these cells?
It is possible that 30 mM glucose is a stressful condition for your cell. Have you measured any marker for autophagy as a consequence of exposition to high glucose?
I haven't checked the total AMPK levels each time. However, when I have checked them they have been pretty constant. I'll anyway check that possibility.
Autophagy is not greatly changed.
Do 30 mM Mannitol induce the same elevation of pAMPK(Thr172) levels in your cells? 30 mOsm do not sound much but we saw e.g. p38MAPK elevation when we didn`t correct for the change in osmolarity between the media. If Mannitol also induces AMPK phosphorylation you may see an effect of hyperosmolarity.
Normal blood glucose level in humans is about 5.5 mM (5.5 mmol/L or 100 mg/dL, i.e. milligrams/deciliter), so 30 mM can be perceived as very high. Indeed, I have observed similar effect on liver and muscle cells in vitro, and it is glucose concentration dependent one. I tend to agree with Erik and Maria - you are inducing osmotic stress, and cell responds with cry for help by inducing a stress response via AMPK. You might want to go down quite a bit in your glucose levels. Hope it helps. Good luck!
Another, eminently testable hypothesis: with so much fuel floating around, your mitochondria went into overdrive with oxphos and, hense, oxygen consumption, leading to intracellular oxygen level collapse followed by sequential oxphos slowdown and switch to glycolysis as an alternative. Less ATP produced as a result of the switch means higher AMP, change in AMP/ATP ratio and activation of the AMPK. Glycolysis leads to drop in pH and increase in lactate production. Both can be easily quantified. Does your medium gets yellowish in high glucose? If it does, then your pH is low - another stressor of the cell. As for the oxygen levels - look into HIF1alpha expression by western. That what I would do first - HIF1alpha levels. There are good antibodies out there. In mild hypoxia (I suspect your case) HIF1alpha levels will go up.
Hi, thanks for your suggestions! I could easily check the effect of mannitol. I haven't notice any change in the colour of the media with high glucose but I'll keep that in mind.
The RPMI contains 11 mM glucose, so it is more than the normal blood concentration, unfortunately. But perhaps I have to consider lower glucose concentration in the high glucose media.
Okay, 30 mM glucose clearly increases p-AMPK/AMPK (as well as p-AMPK/AMPK/actin, ~70-100%) ratio whereas 30 mM mannitol has only a minor increasing effect (~20%). Probably I'll still try 20 mM glucose instead of 30 mM...
Hi Sonja,
sorry that my "osmotic stress hypothesis" was wrong!
I looked up normal glucose levels in mice and they are 4-6 mM, from 9 mM on mouse researchers speak of hyperglycaemic, but you can find 40 mM and more in the literature. As far as I understood you its the basal levels that are too high, so I would suggest to prepare RPMI 1640 (or a comparable medium) with 5 mM glucose (as control) and 30 mM as test condition and supply the 5 mM glucose with mannitol to keep your conditions iso-osmolar.
Hi Sonja,
In your high glucose condition, OGT must be activated, and AMPK could be activated by N-GluNacylation. Your P-AMPK level could be due to this post-translational modification... You can try to block this glucNacylation pathway (using Azaserine for example) and check again p-AMPK level.
^^ Anyway, that option had to be ruled out! I think Sigma is selling RPMI only with 11 mM glucose but some other company was selling it without glucose. I could consider that. The thing is that these cells have been cultured in 11 mM glucose media forever because it is recommended by the person who made them...
^ That's totally new to me but I'll read about it.
Hi Sonja! Again... Immortalized cells of all shape and form tolerate changes in the conditions such as glucose, HEPES, etc. quite readily. I wouldn't hesitate to adjust your glucose level down a couple folds. Alternatively, you can starve them for a couple hours prior the assay, whatever your assay is. Curious what happens to MTT assay with these cells in the presence of high and low glucose. A poor person's test of ATP (and hence respiration or ETC) production - it's not just a viability assay! Sorry for continuously chiming in, but I find AMPK pathway fascinating. Good luck with solving your mysteries - AMPK and beyond!
Hi,
Can you please suggest me whether the AICAR activation of AMPK is not much effective in DMEM(25mM) rather than RPMI1640(11mM) and MEM(5mM)?
This is most likely due to something called the Crabtree effect where excess glucose inhibits mitochondrial OXphos. check your cells' Oxygen consumption if its possible. Crabtree effect is very well established and that is why doing metablosim studies at 25-30 mM glucose is not physiologically relevant
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