Studies On The Mechanism Of Metformin Activating AMPK By Spectral Methods

Objective Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). We also found that there is a reduced risk of cancer in patients with type 2 diabetes taking metformin. However, the site of its action, as well as the mechanism to activate AMPK, remains elusive.In this study we determine apparent dissociation constants of the Metformin to AMPK by fluorescence spectral analysis to explore the possible interaction mode and the mechanism at molecular level which will provide the basis for further researching the mechanisms of Metformin and AMPK in tumor and working on gene therapy.Methods Computer molecular mimicry was used to work out the possible sits of Metformin to AMPKγsubunit .We design a pair of primer,which two restriction site with enzyme BglⅡ(invoved in sense primer) and SacⅡ(invoved in antisense primer) , and thirteen pairs of primers, witch with several mutated base pair(bp). The mutanted fragment of AMPKwere cloned by PCR, and the products were extracted, purified, then were inserted to the pMD-18T vactor plasmid. The recombined pMD-AMPKM were identified by analysis of different restriction endonuclease digestion and gene sequence analysis. The purposal fragments were cuted from pMD-AMPKM and then were inserted to the BglⅡand SacⅡsits of pET-3a plasmid, which contained AMPKαandβsubunit . The recombined pET-AMPKM plasmid were transformed into E. coli followed by induction with TPTG. The induced recombinant proteins were identified by SDS-PAGE. Fluorimetric titrations were used to determine apparent dissociation constants of AMPK to pET-AMPK and pET-AMPKM proteins.Results A fragment of 480bp was got by digesting the recombined pMD-AMPKM plasmid with enzyme BglⅡ, which indicated AMPKM had been inserted into pMD-18T successfully. The inserted fragment in the vector was in the right direnction and its sequence was structurally confirmed to be consistent with what we had designed. Two fragments of 480bp and 720bp were got by digesting the recombined pET-AMPKM plasmid with enzyme BglⅡand SacⅡ, which indicated AMPKM had been inserted into pET-3a vector successfully. AMPK and AMPKM protein was purified from the bacterial lysates by Ni2+-affinity chromatography, utilizing a poly-histidine tag fused to the N-terminus of theα-subunit. The three different eluted polypeptides have apparent molecular masses of 63, 39, and 36 kDa, which correspond well to the predicted values of hexahistidinα-taggedα1 (63 kDa) andβ-subunits (39 kDa) andγ-subunit (36kDa). We determined the physical interaction between this compound and Metformin by fluorescence quenching analysis. pET-AMPK displayed maximal fluorescence at 336 nm, whereas Metformin itself had no fluorescence at this wavelength. When the pET-AMPK protein was incubated with increasing amounts of Metformin, the fluorescence intensity gradually decreased , whereas pET-AMPKM 92 95 96和241 242 243 did not have such an effect.Conclusion The recombined pET-AMPKM plasmids were successfully constructed. The physical interaction between AMPK and Metformin is obviously, and they probably binded specifically. The binding domain may be in the AMPKγ-subunit 92,95,96,241,242,243 amino-acid residue . This finding provide the basis for further researching the mechanisms of Metformin and AMPK and AMPK is possibly a valuable molecular marker involved in tumor biotherapy.