Synergistic effects of metformin with liraglutide against endothelial dysfunction through GLP-1 receptor and PKA signalling pathway
Authors: Wenfang Hou
Tianpei Hong
Qing Tian
Ye Liu
Ran Lu
Jing Ke
Rui Wei
Guang Wang
Jin Yang
Journal: Scientific Reports (2017)
Discipline: Multidisciplinary
Keywords: Metformin; GLP-1 analogue; liraglutide; cardiovascular benefits; vasculature
Affiliations: Department of Endocrinology and Metabolism, Peking University Third Hospital, Beijing, China., Department of Endocrinology and Metabolism, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.

Abstract:  Metformin or glucagon-like peptide-1 (GLP-1) analogue liraglutide has cardiovascular benefits. However, it is not clear whether their combined treatment have additive or synergistic effects on the vasculature. In this study, human umbilical vein endothelial cells (HUVECs), exposed to palmitic acid (PA) to induce endothelial dysfunction, were incubated with metformin, liraglutide or their combination. High fat diet (HFD)-fed ApoE-/- mice were randomized into control, metformin, liraglutide, and combination treatment groups. Results showed that in PA-treated HUVECs and HFD-fed ApoE-/- mice, combination of metformin and liraglutide at lower dose significantly improved endothelial dysfunction compared with the single treatment. Metformin upregulated GLP-1 receptor (GLP-1R) level and protein kinase A (PKA) phosphorylation. However, PKA inhibition but not GLP-1R blockade eliminated the protective effects of metformin on endothelial function. Furthermore, AMPK inhibitor compound C abolished the metformin-mediated upregulation of GLP-1R level and PKA phosphorylation. In conclusion, combination of metformin and liraglutide has synergistic protective effects on endothelial function. Moreover, metformin stimulates GLP-1R and PKA signalling via AMPK-dependent pathway, which may account for its synergistic protective effects with liraglutide. Our findings provide new insights on the interaction between metformin and GLP-1, and provide important information for designing new GLP-1-based therapy strategies in treating type 2 diabetes. 

Retracted: No

PubPeer comments:
 Bands in figure 3a and 3b appeared to be reused, although flipped horizontally. Could the authors please post uncropped original scans for all of the Western blots in Figure 3? You may post those here or use a link to an external site (such as Imgur.com or Google Drive) for larger files. *** ![file] , 
 Dear Actinopolyspora Biskrensis, Thank you very much for your comment. We have carefully checked our experimental records and original data, and found the uncropped original scans for the Western blots in Figure 3. Bands in Figure 3a and 3b look highly similar to each other at the first glance, but there are distinct differences. The images in Figure 3a had been rotated in order to keep the brands in the same order. The marked original scan is shown as below. ![file] , 
 #2 Thank you for your reply. Another reader prepared this illustration suggesting that the u201coriginal scanu201d confirms the original concerns. ![file] , 
 Dear Actinopolyspora Biskrensis, Thank you and the other reader for the kind reminding. We have carefully checked the figure again and found that we did reuse the same image. As you can see, there are distinct differences in the bands of original western blot scan between the two experiments. However, the person who edited the figure copied and pasted the same section from original western blot images repeatedly, leading to the reuse of the same bands. We should have noticed such a low-level mistake during proofreading the manuscript, but we didnu2019t. We apologize for this mistake. Also, we will submit the marked original scan to the journal and make the corrigendum. Thanks again for your kind comment. , 
 #4 I was unable to locate a correction on this paper. Could the authors please provide an update or a link to the correction? In reviewing the supplemental files today, I believe I ve identified an additional issue with gel slices in Supplemental Figure 5a. *** ![file] , 
 Dear Actinopolyspora Biskrensis, Thanks for your comments. We have carefully referred to the experimental records and would like to show the original gels here. Experiment #1 ![file] Experiment #2 ![file] Experiment #3 ![file] Experiment #4 ![file] The results in Experiment #3 were shown in the published paper. After we carefully reviewed the original gels, we found that the gels of GAPDH in Experiments #2, #3 and #4 were wrong actually. In the gels shown above, the GAPDH in Experiments #2, #3 and #4 appeared to be the PKA, since the gels of GAPDH in Experiments #2, #3 and #4 are highly similar to those of PKA, and even with the same molecular weight. In our experiments, the western blotting process was as follows: The membrane was first incubated with the p-PKA antibody and its second antibody for the detection of p-PKA. Next, the membrane was stripped with the stripping buffer, and then incubated with the PKA antibody and its second antibody. After stripping for the second time, the membrane was subsequently incubated with the GAPDH antibody and its second antibody. During this process, we did not realize that the PKA antibody was still there and the GAPDH antibody was somehow not working. The molecular weights of PKA (42 kD) and GAPDH (36 kD) were so similar that we did not realize our mistakes. Nevertheless, we normalized the protein level of p-PKA with PKA (but not GAPDH) in our paper. Therefore, the wrong GAPDH band did not change the results and conclusions substantially. By the way, we will present this mistake together with the older one to the journal, and ask for a corrigendum again. Thank you very much for your carefulness. ,