Hypoglycemic, anti-aging, anti-cancer bottlenecks, what to do?
Why don't you ask about the magical metformin?
Metformin, as a hypoglycemic drug, has been "serving" type 2 diabetics for decades and is currently the most widely used hypoglycemic drug in the world [1].
In addition to reducing sugar, in recent years, metformin has also been found a variety of other potential effects, such as cholesterol reduction, anti-aging life extension, anti-cancer and so on. Widely used, cheap prices, and proper civilian gods of war have made many people shout "I can!" (Prescription drug warnings are mandatory here).
However, although this civilian god of war is said to have achieved a lot of results, there are still many opinions about its role target.
Current studies have shown that metformin acts directly on the liver, kidneys, and intestines, and after transport to cells, exerts important biological functions such as hypoglycemic performance, primarily by activating the AMPK signaling pathway [2,3]. This AMPK is the main switch of the body's metabolism and plays an important role in the homeostasis regulation of cellular energy. But to ask how metformin turns on this switch, the key targets and related mechanisms in the middle are really unclear.
The team of Academician Lin Shengcai of Xiamen University and the team of Deng Xianming worked together to clear the cloud of doubt about the target of metformin and further reveal the mechanism of action of metformin [4].
They found that a protein called PEN2 was a key target. After interacting with PEN2, metformin inhibits the activity of the lysozyme mass sub-pump v-ATPase, thereby activating the AMPK signaling pathway and reducing glucose levels, reducing liver fat content, and prolonging life.
The article was published yesterday in the top issue of Nature.
Screenshot of the first page of the paper
While studying the mechanism by which metformin activates AMPK, scientists discovered a strange phenomenon.
As the highly conserved and most important energy receptor in eukaryotic cells, AMPK can sense changes in glucose levels within cells, which are activated when amplification and ADP/ATP ratios increase. In other words, in order for metformin to turn on the AMPK switch, it must first let the ratio of intracellular AMP/ATP and ADP/ATP fluctuate [5,6].
However, this change can only be observed in high-dose oral experiments in mice [7]. Studies have shown that when metformin is used clinically, the concentration of metformin in the blood is not enough to cause an increase in the amp/ATP and ADP/ATP ratios in cells, but metformin is also good [3].
As a result, the typical pathway to activating AMPK doesn't fully apply to metformin, which may go off the beaten path and activate AMPK with a new, non-AMP-dependent pathway.
In this regard, in 2017, the team of academician Lin Shengcai proposed a lysosomal pathway and named it "Lin Pathway". They argue that metformin activates AMPK through this pathway that does not depend on AMP, which points to a new direction for the study of the mechanisms associated with metformin and even AMPK, but fails to fully reveal the full picture of the lysosomal pathway [8].
After the unremitting exploration of the research team and the removal of layers of fog, the lysosomal pathway of metformin has finally been made up by their own hands.
Let's take a look at how the academician Lin Shengcai team and Deng Xianming's team found this molecular target.
First of all, it is also the highlight of this study - fishing.
This fishing refers to molecular probe technology that can be used to study specific interactions with specific target molecules. This method is perfect for catching mysterious targets.
After a series of explorations, the researchers successfully synthesized metformin's molecular probe (Met-P) and incubated it with protein extracts from lysosomals. In the pond of lysosomal, metformin is used as bait, the unknown target molecule is hooked, and the rod of biotin molecule is completed to complete the stitching, and the metformin-protein complex is recovered together. By mass spectrometry and other methods, a total of 113 proteins specifically bound to metformin were screened in lysosomal protein extracts.
Fishing, anything
But anglers know one thing, and that is to catch and release and only take what they need.
This time, the researchers are looking for the target that plays a key role in the activation of the AMPK pathway in metformin. The rest of the protein and metformin combined to do something, which is an additional price to be handed over to follow-up research.
In mouse embryonic fibroblasts and human primary liver cells, they knocked down the expression of these 113 proteins, cultured them in vitro, and observed which protein expression can regulate the activation of metformin on the ampkk molecule in the cell.
The results showed that the PEN2 protein was the fish that the researchers were really looking for, and it was the key target for metformin to activate the lysosomal pathway and activate AMPK.
When PEN2 expression is knocked down or the Pen2 gene is knocked out, the activation of the intracellular AMPK signaling pathway by metformin is inhibited. Key active sites F35 and E40 associated with metformin on PEN2 were identified.
When PEN2 expresses knockout, metformin cannot activate AMPK at low doses
The researchers further confirmed the identity of PEN2 in animals.
After knocking out the PEN2 of mice and Caenorhabditis elegans, they found that metformin could no longer activate AMPK, and the metformin-induced effects of lowering glucose levels, lowering liver fat content, and prolonging life were also gone. These are enough to show that PEN2 is an indispensable signaling molecule in the mechanism of action of metformin.
a, c, g: metformin induced to reduce glucose levels, reduce liver fat content, and prolong life
Finally, from a mechanistic point of view, metformin induces PEN2 to bind to a subunit of v-ATPase, ATP6AP1, after being transferred to cells and binding to PEN2, thereby inhibiting the activity of v-ATPase. Once the activity of v-ATPase is inhibited, the AMPK signaling pathway is activated.
In summary, Academician Lin Shengcai's team and Deng Xianming's team jointly discovered the key molecular target of metformin - PEN2. Metformin combines with PEN2 to activate the signaling pathway and activate the lysosomal "glucose sensor" AMPK through ATP6AP1, thereby reducing glucose levels, reducing liver fat content, and prolonging life.
In this regard, the researchers also deliberately emphasized that this pathway does not rely on AMP and does not disturb intracellular AMP/ADP levels, which may explain why metformin does not have significant side effects while exerting its therapeutic benefits.
There are still many unsolved mysteries about the mechanism of metformin, and I really don't know what "divine power" this civilian god of war has?
bibliography:
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[2] Foretz, M., Guigas, B., Bertrand, L., Pollak, M. & Viollet, B. Metformin: from mechanisms of
action to therapies. Cell Metab. 20, 953–966 (2014).
[3] Graham, G. G. et al. Clinical pharmacokinetics of metformin. Clin. Pharmacokinet. 50,
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[4]https://www.nature.com/articles/s41586-022-04431-8
[5] El-Mir, M. Y. et al. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted
on the respiratory chain complex I. J. Biol. Chem. 275, 223–228 (2000).
[6] Owen, M. R., Doran, E. & Halestrap, A. P. Evidence that metformin exerts its anti-diabetic
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[7] Hunter, R. W. et al. Metformin reduces liver glucose production by inhibition of fructose-1-
6-bisphosphatase. Night. 24, 1395–1406 (2018).
[8] Zhang CS, et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature. 2017 Aug 3;548(7665):112-116. doi: 10.1038/nature23275. Epub 2017 Jul 19.
This article is written by | Eddie Zhang