Insulin and metformin are associated with reduced risk of amyotrophic lateral sclerosis

Insulin and metformin are associated with reduced risk of amyotrophic lateral sclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insulin and metformin are associated with reduced risk of amyotrophic lateral sclerosis Steven Lehrer, Peter Rheinstein This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3860653/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Type 2 diabetes (T2D), but not type 1, protected against ALS. In T2D serum insulin is normal or elevated in the early stages. Type 1 diabetes, characterized by a total lack of insulin, is associated with increased risk of ALS. The antidiabetic metformin also protects against ALS. Connexin 43 (Cx43), an astrocyte protein, operates as an open channel via which toxic substances from astrocytes reach motor neurons to cause ALS. Methods In the current study we analyzed FDA MedWatch data to determine whether insulin or metformin could reduce the risk of ALS. We performed in silico molecular docking studies and molecular dynamics simulation with Cx43 to determine if insulin or metformin dock within the Cx43 channel and can block it effectively, again reducing risk of ALS. Results In MedWatch, Insulin use is associated with a significantly reduced risk of ALS (Proportional Reporting Ratio 0.401). Metformin use is associated with a significantly reduced risk of ALS (PRR 0.567). The Human insulin heterodimer docked within center of the Cx43 channel, effectively blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS. Metformin docks within the Cx43 channel, but the relatively small size of the metformin molecule may not allow it to obstruct the passage of toxic substances from astrocytes to motor neurons. Conclusion MedWatch data indicates that both insulin and metformin reduce risk of ALS. The results of our in silico docking study and molecular dynamics simulation corroborate our previous findings with Cx31. Insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is stable and may be responsible for the protective effect of T2D and insulin on ALS. Metformin probably does not exert its protective effect by blocking the Cx43 channel. Molecular Biology neurodegeneration ALS MedWatch Gromacs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons that affects up to 30,000 people in the United States each year, with 5,000 new cases being diagnosed. Muscles become weaker over time, affecting physical function, and eventually leading to death. The condition has no single cause and no recognized remedy [1]. Type 2 diabetes (T2D), but not type 1, protected against ALS in a Danish population-based study [2]. In T2D serum insulin is normal or elevated in the early stages. A Swedish population study identified a significant inverse association between ALS and T2D, but not type 1 diabetes, with the strongest inverse association 6 years after diabetes onset [3]. An Italian cohort study revealed a significantly reduced ALS risk in T2D (hazard ratio 0.30) with no effect of gender, age, or ALS phenotype [4]. Zhang et al reported that genetically predicted T2D was associated with significantly lower odds of ALS both in European and East Asian populations [5]. Type 1 diabetes, characterized by a total lack of insulin, is associated with increased risk of ALS [2, 3]. Repeat-associated non-AUG (RAN) proteins accumulate in patient brains and contribute to development of neurodegenerative diseases. The antidiabetic biguanide Metformin inhibits RAN translation through PKR pathway and mitigates disease in the C9orf72 ALS/FTD mouse model [6]. Connexin 43 (Cx43), an astrocyte protein, operates as an open channel via which toxic substances from astrocytes reach motor neurons to cause ALS. In a previous study we used in silico docking and molecular dynamics simulation with the three-dimensional structure of Cx31 as a surrogate for Cx43 to show that insulin blocks the open channel and is unlikely to be dislodged, thereby reducing risk of ALS [1]. In the current study we analyzed FDA MedWatch data to determine whether insulin or metformin could reduce the risk of ALS. The three-dimensional structure of Cx43 was deposited in the RCSB Protein Databank in March 2023. We performed in silico molecular docking studies and molecular dynamics simulation with Cx43 to determine if insulin or metformin dock within the Cx43 channel and can block it effectively, again reducing risk of ALS. Methods MedWatch is the Food and Drug Administration (FDA) Safety Information and Adverse Event Reporting Program. MedWatch was organized in 1993 to collect data regarding adverse events in healthcare. An adverse event is any undesirable experience associated with the use of a medical product. The MedWatch system collects reports of adverse reactions and quality problems, primarily due to drugs and medical devices, but also for other FDA-regulated products (e.g., dietary supplements, cosmetics, medical foods, and infant formulas) [7]. Machine-readable data from MedWatch, including adverse drug reaction reports from manufacturers, are part of a public database. We used the publicly available online tool OpenVigil [8] to query the database at https://openvigil.sourceforge.net/. For this study, drug names used were separate: insulin, metformin, and the adverse event was “amyotrophic lateral sclerosis”. Results reported are the rates of the adverse event among users of a particular medication versus the rate of the adverse event among users of all other medications, as well as measures of disproportionality: observed-expected ratios like Relative Reporting Ratio, Proportional Reporting Ratio, and Reporting Odds Ratio. The Relative reporting ratio (RRR) is the ratio of how many adverse drug reactions (ADRs) under exposure were observed over the number of expected events under the assumption that ADR and drug exposure were independent. The proportional reporting ratio (PRR) is the proportion of spontaneous reports for a given drug that are linked to a specific adverse outcome, divided by the corresponding proportion for all or several other drugs. Reporting Odds Ratio (ROR) represents the odds of a certain event occurring with a medicinal product, compared to the odds of the same event occurring with all other medicinal products in the database [9]. A signal is considered when the upper limit of the 95% confidence interval (CI) of the PRR, RRR, or ROR is less than one or the lower limit of the 95% confidence interval is greater than one. Molecular docking was done with AutoDock Vina Extended on the SAMSON platform (OneAngstrom, Grenoble, France). SAMSON is an interface for molecular design that has an open architecture and applicability for drug design [10]. AutoDock Vina Extended achieves approximately 2 orders of magnitude acceleration compared with the molecular docking software AutoDock 4 while also significantly improving the accuracy of the binding mode predictions. Further speed is achieved from parallelism by using multithreading on multicore machines. AutoDock Vina Extended automatically calculates the grid maps and clusters the results in a way transparent to the user [11]. Human insulin in vivo is a heterodimer of an A-chain and a B-chain, which are linked together by disulfide bonds. Heterodimeric human insulin was deposited in the RCSB Protein Data Bank (4EYN) 2012-5-01, released: 2013-05-01 [12]. Cx43 hemi channel in nanodisc (7Z23) was deposited in the RCSB Protein Data Bank (7Z23) 2022-25-2, released: 2023-03-08 [13]. Metformin structure is from PUBCHEM Compound CID: 4091. We used the ClusPro Server for protein-protein docking of human insulin (4EYN) to the Cx43 hexamer. ClusPro (https://cluspro.org) is a widely used tool for protein–protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format [14]. The quality of automated docking by ClusPro is very close to that of the best human predictor groups [15]. We used GROMACS 2021.3 to perform molecular dynamics simulation of human insulin (4EYN) docked to the Cx43 hexamer (7Z23). GROMACS is a molecular dynamics package mainly designed for simulations of proteins, lipids, and nucleic acids. The all-atom OPLS-AA/L force field and SPC/E water model were used for simulations. Energy minimization was performed using the steepest descent method. System Equilibration was done in two phases. The first phase was conducted under an NVT ensemble (constant Number of particles, Volume, and system Temperature). The second phase was conducted under an NPT ensemble, wherein the Number of particles, Pressure, and Temperature are all constant. This ensemble is also called the "isothermal-isobaric" ensemble, and most closely resembles experimental conditions. Results Insulin use is associated with a significantly reduced risk of ALS (PRR 0.401). Table 1 shows MedWatch data to evaluate the relationship of insulin use to ALS in 11,737,133 subjects. 4 men, 1 woman with ALS, Age 59 ± 7.8 (mean ± SD). Rate (DE/D): 0.004%. Chi-Squared with Yates' correction: 3.871. The greater the chi-squared value, the greater the differences. Chi square values greater than 3.84 are considered statistically significant. Measurements of disproportionality (observed-expected ratios RRR, PRR, ROR): Relative Reporting Ratio (RRR) and 95% confidence interval (lower bound; upper bound): 0.404 (0.168; 0.972); Proportional Reporting Ratio (PRR) and 95% confidence interval (lower bound; upper bound): 0.401 (0.167; 0.965); Reporting Odds Ratio (ROR) and 95% confidence interval (lower bound; upper bound): 0.401 (0.167; 0.965). Metformin use is associated with a significantly reduced risk of ALS (PRR 0.567). Table 2 shows MedWatch data to evaluate the relationship of metformin use to ALS in 11,737,133 subjects. 14 men, 1 woman with ALS, Age 68 ± 21. Rate (DE/D): 0.005%. Chi-Squared with Yates' correction: 3.831. Relative Reporting Ratio (RRR) and 95% confidence interval (lower bound; upper bound): 0.572 (0.331; 0.988); Proportional Reporting Ratio (PRR) and 95% confidence interval (lower bound; upper bound): 0.567 (0.328; 0.979); Reporting Odds Ratio (ROR) and 95% confidence interval (lower bound; upper bound): 0.567 (0.328; 0.979). Figure 1A shows metformin docked to Cx43. Figure 1B is a closeup view. Note that metformin docks within the Cx43 channel. Table 3 shows docking parameters calculated by AutoDock Vina Extended for Cx43 to metformin. Lower values of root-mean-square deviations of atomic positions (RMSD) indicate that docking is validated with higher accuracy. RMSD values of 3 or more indicate no docking has occurred. One docking position within the Cx43 channel, mode 1, with RMSD = 0 is highly valid. Figure 2 shows binding affinity (kcal/mol) calculated for 7 docking sites, metformin to Cx43. Only one site within the Cx43 channel with the highest affinity was a valid position. Figure 3A shows the Human insulin heterodimer (4EYN). Figure 3B shows the Human insulin heterodimer (dark blue) docked within center of the Cx43 channel. Figure 4 shows the results from ClusPro with the first six configurations (clusters) of Human Cx43 in its hexameric form with human insulin docked within the hemichannel. Configuration 0, the highest ranked, is shown enlarged and rotated in figure 3B. Note that the human insulin heterodimer (red) is in the identical position in all six configurations, blocking the Cx43 hemichannel. Table 4 shows cluster scores and energies for Human Cx43 in its hexameric form with the hemichannel docked with the human insulin heterodimer. The ligand (insulin) position with the most neighbors within 9 angstroms becomes a cluster center, and its neighbors the members of the cluster. These are then removed from the set and a second cluster center is located, then a third, up to cluster 5. Thus, the cluster rank is determined. Figure 5 shows results of molecular dynamics simulation. 5A. Evolution of the system’s potential energy, Epot, over the Energy Minimization steps. Plot demonstrates the smooth, steady convergence of the potential energy. 5B. Evolution of the system’s temperature over simulation time. Plot demonstrates that the temperature is stabilized around 300 K as set by default in the advanced parameters. 5C & 5D. Once the system’s temperature has stabilized at the desired value, pressure is applied to the system until it reaches the correct density. This second equilibration phase is aimed at stabilizing the system’s density at the desired value by performing equilibration using the NPT ensemble (constant Number of particles, Pressure, and Temperature) also known as “isothermal-isobaric”. The plot demonstrates that the density is stabilized at 1030 kg/m 3 which is close to the experimental value of 1000 kg/m 3 . The expected density of the SPC/E water model is about 1008 kg/m 3 [16]. We can see that the density values are stable over time, indicating that the system is well-equilibrated with respect to pressure and density. 5E. Time series shows the RMSD levels fluctuation of ~0.1 nm (1 Å), indicating that the structure is quite stable. 5G. Radius of gyration (Rg). The reasonably invariant Rg values indicate that the docked protein remains stable over the course of 1 ns. These results suggest that once insulin blocks the Cx43 hemichannel, the block is stable and will remain in place. Discussion Connexin channels are proteins that form gap junctions and hemichannels in astrocytes and play a crucial role in the maintenance of the normal functions of the Central Nervous System (CNS). Alterations of astrocytic connexin expression and function in neurodegenerative diseases have been shown to affect disease progression by changing neuronal function and survival. In ALS, Cx43 gap junctions and hemichannels mediate astrocyte intercellular communication in the CNS under normal conditions and may contribute to astrocyte-mediated neurotoxicity. Targeting connexins can be a plausible therapeutic strategy to manage neurodegenerative diseases, including ALS [17, 18]. Cx43, an astrocyte protein, operates as an open pore via which toxic substances from astrocytes reach motor neurons to cause ALS. We previously performed molecular docking of insulin with monomeric Cx31, monomeric Cx43, and hexameric Cx31 to assess whether insulin might affect the pore. Hexameric Cx31 and hexameric Cx43 are transmembrane hemichannels composed of 6 subunits; they bind together to form gap junction intercellular channels. We used the program AutoDock Vina Extended for the molecular docking study. Cx31 shares amino acid and structural similarity to Cx43, and insulin docks to the same position at the N-terminal domain of monomeric Cx31 and monomeric Cx43. We found that insulin docks within the open hemichannel of hexameric Cx31, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS [1]. MedWatch data presented above in Table 1 confirm the protective effect of insulin. The full Cx43 hexameric structure was deposited in the RCSB Protein Data Bank and released 8 March 2023. The results of our in silico docking study and molecular dynamics simulation confirm our previously reported findings [1]. We found that insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS C-9 ALS is a subtype of ALS caused by a repeat expansion mutation in a gene on chromosome 9, open reading frame 72 (C9orf72). The mutation occurs when six letters of DNA – GGGGCC – are repeated hundreds of times. Besides ALS, mutations in C9orf72 can cause frontotemporal dementia. Some patients with the C9orf72 mutation develop ALS, others develop frontotemporal dementia, and some develop both. MedWatch data (Table 2) corroborate the protective effect of metformin identified in the C9orf72 ALS/FTD mouse ALS model [6]. Metformin inhibits protein kinase R, reduces RAN proteins and improves disease in C9-ALS/FTD mice. Metformin might be therapeutic for this genetic form of ALS and frontotemporal dementia because the C9orf72 mutation makes RAN proteins [6]. But metformin treatment was not effective in mice with a different form of ALS that does not produce RAN proteins [19]. Our finding that metformin docks within the Cx43 channel suggests that in some cases metformin may interfere with the passage of toxins through this channel from glial cells to motor neurons. However, because of its relatively small size compared with insulin, metformin could obstruct the Cx43 channel much less completely than insulin. Our study has weaknesses: A MedWatch report of an adverse event does not establish causation. For any given report, there is no certainty that the drug in question is related to the reaction. The adverse event may have been due to the underlying disease being treated, another drug being taken concurrently, or something else. The MedWatch data are imperfect, with under- and over-reporting, missing denominator (that is, number of doses for a drug), wrong, duplicate and/or missing data in the database [20]. Consequently, the total number of adverse event reports for all drugs and/or the drug in question from OpenVigil can vary slightly from drug to drug and for different adverse events related to the same drug. The imperfect MedWatch data have presented a problem that all analytical software programs, such as OpenVigil, have been forced to confront [21]. Molecular docking studies are a powerful in silico approach for discovering novel therapies for unmet medical needs by predicting drug–target interactions. But molecular docking studies are not a substitute for in vitro studies. In vitro studies are conducted in a controlled environment, such as a test tube or petri dish, and can provide more accurate results than molecular docking studies. In vitro studies can provide information about the drug's efficacy, toxicity, and pharmacokinetics, which are essential for drug development. But molecular docking studies can provide a preliminary assessment of the drug's potential efficacy and binding affinity with the target protein. Molecular docking studies are a valuable tool for drug discovery used in conjunction with in vitro studies to validate the results and ensure the safety and efficacy of the drug [22]. Molecular dynamics simulation is a computational technique that can provide mechanistic understanding of molecular systems and has become a prominent tool in pharmaceutical research. It can provide insight into the behavior of molecules at an atomic level that is difficult to characterize experimentally. However, molecular dynamics simulations are not a replacement for in vitro studies. In vitro studies are conducted in a controlled laboratory environment and can provide more accurate results than simulations. Molecular dynamics simulations can provide valuable insights into molecular systems but should be used in conjunction with in vitro studies to ensure the accuracy of the results [23]. Cx43 (7Z23) structure is presented in the closed position. Therefore, we cannot be certain of the insulin blocking effect when Cx43 is in the open position. Conclusion MedWatch data confirms the protective effect of insulin and metformin against ALS. The results of our in silico docking study and molecular dynamics simulation corroborate our previous findings with Cx31. We now report that insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D and insulin on ALS. Metformin probably does not exert its protective effect by blocking the Cx43 channel. Declarations Data sources described in the article are publicly available. Conflicts of interest: none Competing interests: The authors declare no competing interests. Dr. Lehrer and Dr. Rheinstein contributed equally to the conception, writing, and data analysis of this study. References Lehrer S, Rheinstein PH (2023) Insulin Docking Within the Open Hemichannel of Connexin 43 May Reduce Risk of Amyotrophic Lateral Sclerosis. In Vivo 37 , 539-547. Kioumourtzoglou MA, Rotem RS, Seals RM, Gredal O, Hansen J, Weisskopf MG (2015) Diabetes Mellitus, Obesity, and Diagnosis of Amyotrophic Lateral Sclerosis: A Population-Based Study. JAMA Neurol 72 , 905-911. Mariosa D, Kamel F, Bellocco R, Ye W, Fang F (2015) Association between diabetes and amyotrophic lateral sclerosis in Sweden. Eur J Neurol 22 , 1436-1442. D'Ovidio F, d'Errico A, Carna P, Calvo A, Costa G, Chio A (2018) The role of pre-morbid diabetes on developing amyotrophic lateral sclerosis. Eur J Neurol 25 , 164-170. 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Tables Tables 1 to 4 are available in the Supplementary Files section Additional Declarations The authors declare no competing interests. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3860653","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266879457,"identity":"26e62a7b-84b9-4a8b-be3a-7ed6cfe72861","order_by":0,"name":"Steven Lehrer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYPACCwYDEPWxAUQyNh7Aq5gNTEqAtTDObACyGBgbiNfCzAvWwsCAV4v8/OZjEh93SMiZSx8+9tl2h02dbvthoC01NtG4tBgcY0uTnHlGwtiyLy15du6ZNAmzM4lALcfSchtwaWHjMTbmbZNI3HCGx5g5t+2whNkBoBbGhsM4tci38X82/tsmUb/hDP9nZkuQlvMP8WthOMbD+JixTSLB4AwPMzMjSMsNArYYHEszfNjbJmG44QybMWNvW5rkthtAWxLw+EW++fCDAz/bbOQNzjA/ZgAy+M3Opz988KHGBrfDsIME0pSPglEwCkbBKEADAB7BXPt+oBdNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4850-094X","institution":"Fermata Pharma, Inc.","correspondingAuthor":true,"prefix":"","firstName":"Steven","middleName":"","lastName":"Lehrer","suffix":""},{"id":266879473,"identity":"3addd17f-6866-4243-9e05-f800355be0d5","order_by":1,"name":"Peter Rheinstein","email":"","orcid":"","institution":"Severn Health Solutions","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Rheinstein","suffix":""}],"badges":[],"createdAt":"2024-01-13 16:16:36","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3860653/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3860653/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49660395,"identity":"80d807a2-c9db-4639-a852-773ecf1ef820","added_by":"auto","created_at":"2024-01-16 05:24:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":256631,"visible":true,"origin":"","legend":"\u003cp\u003eA shows metformin docked to Cx43 (arrows). B closeup view. Note that metformin docks within the Cx43 channel.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/43cd8bdc79cb72228fafb7e4.png"},{"id":49660393,"identity":"5afa82f8-7513-43b5-942f-f6e95e7b1148","added_by":"auto","created_at":"2024-01-16 05:24:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30005,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinity (kcal/mol) calculated for 7 docking sites, metformin to Cx43. Only one site with the highest affinity (upper left) was a valid position.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/1e2060ecab67de661295a481.png"},{"id":49660397,"identity":"ab1b8ada-39d1-40c8-b42d-5ce6fabd5ff4","added_by":"auto","created_at":"2024-01-16 05:24:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187380,"visible":true,"origin":"","legend":"\u003cp\u003eA. Human insulin heterodimer. B. Human insulin heterodimer (dark blue) docked within center of Cx43 channel.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/843629ce886df46ac0481162.png"},{"id":49661212,"identity":"946649e8-02f1-4434-8ecc-3b8da2316618","added_by":"auto","created_at":"2024-01-16 05:32:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":363943,"visible":true,"origin":"","legend":"\u003cp\u003eThe results from ClusPro showing the first six configurations (clusters) of Human Cx43 in its hexameric form with human insulin docked within the hemichannel. Configuration 0, the highest ranked, is shown enlarged and rotated in figure 3B. Note that the human insulin heterodimer (red) is in the identical position in all six configurations, blocking the Cx43 hemichannel.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/69fa5cd6d93cb39fc189e928.png"},{"id":49660396,"identity":"6e7e296e-8f25-4676-9a82-f20e9e30f1ad","added_by":"auto","created_at":"2024-01-16 05:24:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90361,"visible":true,"origin":"","legend":"\u003cp\u003eResults of molecular dynamics simulation. 5A. Evolution of the system’s potential energy, Epot, over the Energy Minimization steps. Plot demonstrates the smooth, steady convergence of the potential energy. 5B. Evolution of the system’s temperature over simulation time. Plot demonstrates that the temperature is stabilized around 300 K as set by default in the advanced parameters. 5C \u0026amp; 5D. Once the system’s temperature has stabilized at the desired value, pressure is applied to the system until it reaches the correct density. This second equilibration phase is aimed at stabilizing the system’s density at the desired value by performing equilibration using the NPT ensemble (constant Number of particles, Pressure, and Temperature) also known as “isothermal-isobaric”. The plot demonstrates that the density is stabilized at 1030 kg/m\u003csup\u003e3\u003c/sup\u003e which is close to the experimental value of 1000 kg/m\u003csup\u003e3\u003c/sup\u003e. The expected density of the SPC/E water model is about 1008 kg/m\u003csup\u003e3\u003c/sup\u003e. We can see that the density values are stable over time, indicating that the system is well-equilibrated with respect to pressure and density. 5E. Time series shows the RMSD levels fluctuation of ~0.1 nm (1 Å), indicating that the structure is quite stable. 5F. Radius of gyration (Rg). The reasonably invariant Rg values indicate that the docked protein remains stable over the course of 1 ns. These results suggest that once insulin blocks the Cx43 hemichannel, the block is stable and will remain in place.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/aac76a627b78e00141f517fb.png"},{"id":49661368,"identity":"324c5fa4-43d7-41c9-a83e-c70cc8004c6e","added_by":"auto","created_at":"2024-01-16 05:40:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1065310,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/8cb47948-19cf-4ab0-a4ec-c901c5d1018d.pdf"},{"id":49660392,"identity":"136606ce-3e50-4f67-bd10-a57cf7826052","added_by":"auto","created_at":"2024-01-16 05:24:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16953,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3860653/v1/56ee7817b63546e038f81433.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eInsulin and metformin are associated with reduced risk of amyotrophic lateral sclerosis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons that affects up to 30,000 people in the United States each year, with 5,000 new cases being diagnosed. Muscles become weaker over time, affecting physical function, and eventually leading to death. The condition has no single cause and no recognized remedy\u0026nbsp;[1].\u003c/p\u003e\n\u003cp\u003eType 2 diabetes (T2D), but not type 1, protected against ALS in a Danish population-based study\u0026nbsp;[2]. In T2D serum insulin is normal or elevated in the early stages. A Swedish population study identified a significant inverse association between ALS and T2D, but not type 1 diabetes, with the strongest inverse association 6 years after diabetes onset\u0026nbsp;[3]. An Italian cohort study revealed a significantly reduced ALS risk in T2D (hazard ratio 0.30) with no effect of gender, age, or ALS phenotype\u0026nbsp;[4]. Zhang et al reported that genetically predicted T2D was associated with significantly lower odds of ALS both in European and East Asian populations\u0026nbsp;[5]. Type 1 diabetes, characterized by a total lack of insulin, is associated with increased risk of ALS\u0026nbsp;[2, 3].\u003c/p\u003e\n\u003cp\u003eRepeat-associated non-AUG (RAN) proteins accumulate in patient brains and contribute to development of neurodegenerative diseases. The antidiabetic biguanide Metformin inhibits RAN translation through PKR pathway and mitigates disease in the C9orf72 ALS/FTD mouse model\u0026nbsp;[6].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConnexin 43 (Cx43), an astrocyte protein, operates as an open channel via which toxic substances from astrocytes reach motor neurons to cause ALS. In a previous study we used \u003cem\u003ein silico\u003c/em\u003e docking and molecular dynamics simulation with the three-dimensional structure of Cx31 as a surrogate for Cx43 to show that insulin blocks the open channel and is unlikely to be dislodged, thereby reducing risk of ALS\u0026nbsp;[1].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the current study we analyzed FDA MedWatch data to determine whether insulin or metformin could reduce the risk of ALS. The three-dimensional structure of Cx43 was deposited in the RCSB Protein Databank in March 2023. We performed\u003cem\u003e\u0026nbsp;in silico\u0026nbsp;\u003c/em\u003emolecular docking studies and molecular dynamics simulation with Cx43 to determine if insulin or metformin dock within the Cx43 channel and can block it effectively, again reducing risk of ALS.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMedWatch is the Food and Drug Administration (FDA) Safety Information and Adverse Event Reporting Program. MedWatch was organized in 1993 to collect data regarding adverse events in healthcare. An adverse event is any undesirable experience associated with the use of a medical product. The MedWatch system collects reports of adverse reactions and quality problems, primarily due to drugs and medical devices, but also for other FDA-regulated products (e.g., dietary supplements, cosmetics, medical foods, and infant formulas) [7].\u003c/p\u003e\n\u003cp\u003eMachine-readable data from MedWatch, including adverse drug reaction reports from manufacturers, are part of a public database. We used the publicly available online tool OpenVigil [8] to query the database at https://openvigil.sourceforge.net/. For this study, drug names used were separate: insulin, metformin, and the adverse event was “amyotrophic lateral sclerosis”. Results reported are the rates of the adverse event among users of a particular medication versus the rate of the adverse event among users of all other medications, as well as measures of disproportionality: observed-expected ratios like Relative Reporting Ratio, Proportional Reporting Ratio, and Reporting Odds Ratio. The Relative reporting ratio (RRR) is the ratio of how many adverse drug reactions (ADRs) under exposure were observed over the number of expected events under the assumption that ADR and drug exposure were independent. The proportional reporting ratio (PRR) is the proportion of spontaneous reports for a given drug that are linked to a specific adverse outcome, divided by the corresponding proportion for all or several other drugs. Reporting Odds Ratio (ROR) represents the odds of a certain event occurring with a medicinal product, compared to the odds of the same event occurring with all other medicinal products in the database [9]. A signal is considered when the upper limit of the 95% confidence interval (CI) of the PRR, RRR, or ROR is less than one or the lower limit of the 95% confidence interval is greater than one.\u003c/p\u003e\n\u003cp\u003eMolecular docking was done with AutoDock Vina Extended on the SAMSON platform (OneAngstrom, Grenoble, France). SAMSON is an interface for molecular design that has an open architecture and applicability for drug design [10]. AutoDock Vina Extended achieves approximately 2 orders of magnitude acceleration compared with the molecular docking software AutoDock 4 while also significantly improving the accuracy of the binding mode predictions. Further speed is achieved from parallelism by using multithreading on multicore machines. AutoDock Vina Extended automatically calculates the grid maps and clusters the results in a way transparent to the user [11].\u003c/p\u003e\n\u003cp\u003eHuman insulin \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eis a heterodimer of an A-chain and a B-chain, which are linked together by disulfide bonds.\u0026nbsp;Heterodimeric human insulin\u0026nbsp;was deposited in the RCSB Protein Data Bank (4EYN) 2012-5-01, released: 2013-05-01 [12]. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCx43 hemi channel in nanodisc (7Z23) was deposited in the RCSB Protein Data Bank (7Z23) 2022-25-2, released: 2023-03-08 [13]. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMetformin structure is from PUBCHEM Compound CID: 4091. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe used the ClusPro Server for protein-protein docking of human insulin (4EYN) to the Cx43 hexamer. ClusPro (https://cluspro.org) is a widely used tool for protein–protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format [14]. The quality of automated docking by ClusPro is very close to that of the best human predictor groups [15].\u003c/p\u003e\n\u003cp\u003eWe used GROMACS 2021.3 to perform molecular dynamics simulation of human insulin (4EYN) docked to the Cx43 hexamer (7Z23). GROMACS is a molecular dynamics package mainly designed for simulations of proteins, lipids, and nucleic acids. The all-atom OPLS-AA/L force field and SPC/E water model were used for simulations. Energy minimization was performed using the steepest descent method. System Equilibration was done in two phases. The first phase was conducted under an NVT ensemble (constant Number of particles, Volume, and system Temperature). The second phase was conducted under an NPT ensemble, wherein the Number of particles, Pressure, and Temperature are all constant. This ensemble is also called the \"isothermal-isobaric\" ensemble, and most closely resembles experimental conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eInsulin use is associated with a significantly reduced risk of ALS (PRR 0.401). Table 1 shows MedWatch data to evaluate the relationship of insulin use to ALS in 11,737,133 subjects. 4 men, 1 woman with ALS, Age 59 \u0026plusmn; 7.8 (mean \u0026plusmn; SD). Rate (DE/D): 0.004%. Chi-Squared with Yates\u0026apos; correction: 3.871. The greater the chi-squared value, the greater the differences. Chi square values greater than 3.84 are considered statistically significant. Measurements of disproportionality (observed-expected ratios RRR, PRR, ROR): Relative Reporting Ratio (RRR) and 95% confidence interval (lower bound; upper bound): 0.404 (0.168; 0.972); Proportional Reporting Ratio (PRR) and 95% confidence interval (lower bound; upper bound): 0.401 (0.167; 0.965); Reporting Odds Ratio (ROR) and 95% confidence interval (lower bound; upper bound): 0.401 (0.167; 0.965).\u003c/p\u003e\n\u003cp\u003eMetformin use is associated with a significantly reduced risk of ALS (PRR 0.567). Table 2 shows MedWatch data to evaluate the relationship of metformin use to ALS in 11,737,133 subjects. 14 men, 1 woman with ALS, Age 68 \u0026plusmn; 21. Rate (DE/D): 0.005%. Chi-Squared with Yates\u0026apos; correction: 3.831. Relative Reporting Ratio (RRR) and 95% confidence interval (lower bound; upper bound): 0.572 (0.331; 0.988); Proportional Reporting Ratio (PRR) and 95% confidence interval (lower bound; upper bound): 0.567 (0.328; 0.979); Reporting Odds Ratio (ROR) and 95% confidence interval (lower bound; upper bound): 0.567 (0.328; 0.979).\u003c/p\u003e\n\u003cp\u003eFigure 1A shows metformin docked to Cx43. Figure 1B is a closeup view. Note that metformin docks within the Cx43 channel.\u003c/p\u003e\n\u003cp\u003eTable 3 shows docking parameters calculated by\u0026nbsp;AutoDock Vina Extended for Cx43 to metformin. Lower values of root-mean-square deviations of atomic positions (RMSD) indicate that docking is validated with higher accuracy. RMSD values of 3 or more indicate no docking has occurred. One docking position within the Cx43 channel, mode 1, with RMSD = 0 is highly valid.\u003c/p\u003e\n\u003cp\u003eFigure 2 shows binding affinity (kcal/mol) calculated for 7 docking sites, metformin to Cx43. Only one site\u0026nbsp;within the Cx43 channel\u0026nbsp;with the highest affinity was a valid position.\u003c/p\u003e\n\u003cp\u003eFigure 3A shows the Human insulin heterodimer (4EYN). Figure 3B shows the Human insulin heterodimer (dark blue) docked within center of the Cx43 channel.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 4 shows the results from ClusPro with the first six configurations (clusters) of Human Cx43 in its hexameric form with human insulin docked within the hemichannel. Configuration 0, the highest ranked, is shown enlarged and rotated in figure 3B. Note that the human insulin heterodimer (red) is in the identical position in all six configurations, blocking the Cx43 hemichannel.\u003c/p\u003e\n\u003cp\u003eTable 4 shows cluster scores and energies for Human Cx43 in its hexameric form with the \u0026nbsp;hemichannel docked with the human insulin heterodimer. The ligand (insulin) position with the most neighbors within 9 angstroms becomes a cluster center, and its neighbors the members of the cluster. These are then removed from the set and a second cluster center is located, then a third, up to cluster 5. Thus, the cluster rank is determined.\u003c/p\u003e\n\u003cp\u003eFigure 5 shows results of molecular dynamics simulation. 5A. Evolution of the system\u0026rsquo;s potential energy, Epot, over the Energy Minimization steps. Plot demonstrates the smooth, steady convergence of the potential energy. 5B. Evolution of the system\u0026rsquo;s temperature over simulation time. Plot demonstrates that the temperature is stabilized around 300 K as set by default in the advanced parameters. 5C \u0026amp; 5D. Once the system\u0026rsquo;s temperature has stabilized at the desired value, pressure is applied to the system until it reaches the correct density. This second equilibration phase is aimed at stabilizing the system\u0026rsquo;s density at the desired value by performing equilibration using the NPT ensemble (constant Number of particles, Pressure, and Temperature) also known as \u0026ldquo;isothermal-isobaric\u0026rdquo;. The plot demonstrates that the density is stabilized at 1030 kg/m\u003csup\u003e3\u003c/sup\u003e which is close to the experimental value of 1000 kg/m\u003csup\u003e3\u003c/sup\u003e. The expected density of the SPC/E water model is about 1008 kg/m\u003csup\u003e3\u003c/sup\u003e [16]. We can see that the density values are stable over time, indicating that the system is well-equilibrated with respect to pressure and density. 5E. Time series shows the RMSD levels fluctuation of ~0.1 nm (1 \u0026Aring;), indicating that the structure is quite stable. 5G. Radius of gyration (Rg). The reasonably invariant Rg values indicate that the docked protein remains stable over the course of 1 ns. These results suggest that once insulin blocks the Cx43 \u0026nbsp;hemichannel, the block is stable and will remain in place.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eConnexin channels are proteins that form gap junctions and hemichannels in astrocytes and play a crucial role in the maintenance of the normal functions of the Central Nervous System (CNS). Alterations of astrocytic connexin expression and function in neurodegenerative diseases have been shown to affect disease progression by changing neuronal function and survival. In ALS, Cx43 gap junctions and hemichannels mediate astrocyte intercellular communication in the CNS under normal conditions and may contribute to astrocyte-mediated neurotoxicity. Targeting connexins can be a plausible therapeutic strategy to manage neurodegenerative diseases, including ALS\u0026nbsp;[17, 18].\u003c/p\u003e\n\u003cp\u003eCx43, an astrocyte protein, operates as an open pore via which toxic substances from astrocytes reach motor neurons to cause ALS. We previously performed molecular docking of insulin with monomeric Cx31, monomeric Cx43, and hexameric Cx31 to assess whether insulin might affect the pore. Hexameric Cx31 and hexameric Cx43 are transmembrane hemichannels composed of 6 subunits; they bind together to form gap junction intercellular channels. We used the program AutoDock Vina Extended for the molecular docking study. Cx31 shares amino acid and structural similarity to Cx43, and insulin docks to the same position at the N-terminal domain of monomeric Cx31 and monomeric Cx43. We found that insulin docks within the open hemichannel of hexameric Cx31, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS\u0026nbsp;[1]. MedWatch data presented above in Table 1 confirm the protective effect of insulin.\u003c/p\u003e\n\u003cp\u003eThe full Cx43 hexameric structure was deposited in the RCSB Protein Data Bank and released 8 March 2023. The results of our \u003cem\u003ein silico\u0026nbsp;\u003c/em\u003edocking study and molecular dynamics simulation confirm our previously reported findings\u0026nbsp;[1]. We found that insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC-9 ALS is a subtype of ALS caused by a repeat expansion mutation in a gene on chromosome 9, open reading frame 72 (C9orf72). The mutation occurs when six letters of DNA – GGGGCC – are repeated hundreds of times. Besides ALS, mutations in C9orf72 can cause frontotemporal dementia. Some patients with the C9orf72 mutation develop ALS, others develop frontotemporal dementia, and some develop both. MedWatch data (Table 2) corroborate the protective effect of metformin identified in the C9orf72 ALS/FTD mouse ALS model\u0026nbsp;[6].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Metformin inhibits protein kinase R, reduces RAN proteins and improves disease in C9-ALS/FTD mice. Metformin might be therapeutic for this genetic form of ALS and frontotemporal dementia because the C9orf72 mutation makes RAN proteins [6]. But metformin treatment was not effective in mice with a different form of ALS that does not produce RAN proteins [19]. Our finding that metformin docks within the Cx43 channel suggests that in some cases metformin may interfere with the passage of toxins through this channel from glial cells to motor neurons. However, because of its relatively small size compared with insulin, metformin could obstruct the Cx43 channel much less completely than insulin.\u003c/p\u003e\n\u003cp\u003eOur study has weaknesses:\u003c/p\u003e\n\u003cp\u003eA MedWatch report of an adverse event does not establish causation. For any given report, there is no certainty that the drug in question is related to the reaction. The adverse event may have been due to the underlying disease being treated, another drug being taken concurrently, or something else. The MedWatch data are imperfect, with under- and over-reporting, missing denominator (that is, number of doses for a drug), wrong, duplicate and/or missing data in the database [20]. Consequently, the total number of adverse event reports for all drugs and/or the drug in question from OpenVigil can vary slightly from drug to drug and for different adverse events related to the same drug. The imperfect MedWatch data have presented a problem that all analytical software programs, such as OpenVigil, have been forced to confront [21].\u003c/p\u003e\n\u003cp\u003eMolecular docking studies are a powerful \u003cem\u003ein silico\u003c/em\u003e approach for discovering novel therapies for unmet medical needs by predicting drug–target interactions. But molecular docking studies are not a substitute for \u003cem\u003ein vitro\u003c/em\u003e studies. In vitro studies are conducted in a controlled environment, such as a test tube or petri dish, and can provide more accurate results than molecular docking studies. In vitro studies can provide information about the drug's efficacy, toxicity, and pharmacokinetics, which are essential for drug development. But molecular docking studies can provide a preliminary assessment of the drug's potential efficacy and binding affinity with the target protein. Molecular docking studies are a valuable tool for drug discovery used in conjunction with in vitro studies to validate the results and ensure the safety and efficacy of the drug [22].\u003c/p\u003e\n\u003cp\u003eMolecular dynamics simulation is a computational technique that can provide mechanistic understanding of molecular systems and has become a prominent tool in pharmaceutical research. It can provide insight into the behavior of molecules at an atomic level that is difficult to characterize experimentally. However, molecular dynamics simulations are not a replacement for in vitro studies. In vitro studies are conducted in a controlled laboratory environment and can provide more accurate results than simulations. Molecular dynamics simulations can provide valuable insights into molecular systems but should be used in conjunction with in vitro studies to ensure the accuracy of the results [23].\u003c/p\u003e\n\u003cp\u003eCx43 (7Z23) structure is presented in the closed position. Therefore, we cannot be certain of the insulin blocking effect when Cx43 is in the open position.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMedWatch data confirms the protective effect of insulin and metformin against ALS. The results of our \u003cem\u003ein silico\u0026nbsp;\u003c/em\u003edocking study and molecular dynamics simulation corroborate our previous findings with Cx31. We now report that insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D and insulin on ALS. Metformin probably does not exert its protective effect by blocking the Cx43 channel. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData sources described in the article are publicly available.\u003c/p\u003e\n\u003cp\u003eConflicts of interest: none\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eDr. Lehrer and Dr. Rheinstein contributed equally to the conception, writing, and data analysis of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLehrer S, Rheinstein PH (2023) Insulin Docking Within the Open Hemichannel of Connexin 43 May Reduce Risk of Amyotrophic Lateral Sclerosis. \u003cem\u003eIn Vivo\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 539-547.\u003c/li\u003e\n \u003cli\u003eKioumourtzoglou MA, Rotem RS, Seals RM, Gredal O, Hansen J, Weisskopf MG (2015) Diabetes Mellitus, Obesity, and Diagnosis of Amyotrophic Lateral Sclerosis: A Population-Based Study. \u003cem\u003eJAMA Neurol\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 905-911.\u003c/li\u003e\n \u003cli\u003eMariosa D, Kamel F, Bellocco R, Ye W, Fang F (2015) Association between diabetes and amyotrophic lateral sclerosis in Sweden. \u003cem\u003eEur J Neurol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1436-1442.\u003c/li\u003e\n \u003cli\u003eD\u0026apos;Ovidio F, d\u0026apos;Errico A, Carna P, Calvo A, Costa G, Chio A (2018) The role of pre-morbid diabetes on developing amyotrophic lateral sclerosis. \u003cem\u003eEur J Neurol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 164-170.\u003c/li\u003e\n \u003cli\u003eZhang L, Tang L, Huang T, Fan D (2022) Association between type 2 diabetes and amyotrophic lateral sclerosis. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2544.\u003c/li\u003e\n \u003cli\u003eZu T, Guo S, Bardhi O, Ryskamp DA, Li J, Khoramian Tusi S, Engelbrecht A, Klippel K, Chakrabarty P, Nguyen L, Golde TE, Sonenberg N, Ranum LPW (2020) Metformin inhibits RAN translation through PKR pathway and mitigates disease in C9orf72 ALS/FTD mice. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 18591-18599.\u003c/li\u003e\n \u003cli\u003eGetz KA, Stergiopoulos S, Kaitin KI (2014) Evaluating the completeness and accuracy of MedWatch data. \u003cem\u003eAmerican Journal of Therapeutics\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 442-446.\u003c/li\u003e\n \u003cli\u003eB\u0026ouml;hm R, von Hehn L, Herdegen T, Klein H-J, Bruhn O, Petri H, H\u0026ouml;cker J (2016) OpenVigil FDA - Inspection of U.S. American Adverse Drug Events Pharmacovigilance Data and Novel Clinical Applications. \u003cem\u003ePloS One\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0157753.\u003c/li\u003e\n \u003cli\u003eRothman KJ, Lanes S, Sacks ST (2004) The reporting odds ratio and its advantages over the proportional reporting ratio. \u003cem\u003ePharmacoepidemiology and drug safety\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 519-523.\u003c/li\u003e\n \u003cli\u003eLehrer S, Rheinstein PH (2023) Re: Suppressing c-FOS expression by G-quadruplex ligands inhibits osimertinib-resistant non-small cell lung cancers. \u003cem\u003eJNCI: Journal of the National Cancer Institute\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 1427-1428.\u003c/li\u003e\n \u003cli\u003eTrott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. \u003cem\u003eJ Comput Chem\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 455-461.\u003c/li\u003e\n \u003cli\u003eFavero-Retto MP, Palmieri LC, Souza TA, Almeida FC, Lima LM (2013) Structural meta-analysis of regular human insulin in pharmaceutical formulations. \u003cem\u003eEur J Pharm Biopharm\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 1112-1121.\u003c/li\u003e\n \u003cli\u003eQi C, Acosta Gutierrez S, Lavriha P, Othman A, Lopez-Pigozzi D, Bayraktar E, Schuster D, Picotti P, Zamboni N, Bortolozzi M, Gervasio FL, Korkhov VM (2023) Structure of the connexin-43 gap junction channel in a putative closed state. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eKozakov D, Hall DR, Xia B, Porter KA, Padhorny D, Yueh C, Beglov D, Vajda S (2017) The ClusPro web server for protein\u0026ndash;protein docking. \u003cem\u003eNature protocols\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 255-278.\u003c/li\u003e\n \u003cli\u003eKozakov D, Beglov D, Bohnuud T, Mottarella SE, Xia B, Hall DR, Vajda S (2013) How good is automated protein docking? 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Biotechnol\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 137-138.\u003c/li\u003e\n \u003cli\u003eHauben M, Reich L, DeMicco J, Kim K (2007) \u0026apos;Extreme duplication\u0026apos; in the US FDA Adverse Events Reporting System database. \u003cem\u003eDrug Saf\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 551-554.\u003c/li\u003e\n \u003cli\u003eDnyandev K, Galave V, Kulkarni V, Chandrakant M, Otari K (2021) A Review on Molecular Docking. \u003cem\u003eInternational Research Journal of Pure and Applied Chemistry\u003c/em\u003e, 60-68.\u003c/li\u003e\n \u003cli\u003eShukla R, Tripathi T (2021) Molecular Dynamics Simulation in Drug Discovery: Opportunities and Challenges In \u003cem\u003eInnovations and Implementations of Computer Aided Drug Discovery Strategies in Rational Drug Design\u003c/em\u003e, Singh SK, ed. Springer Singapore, Singapore, pp. 295-316.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"neurodegeneration, ALS, MedWatch, Gromacs","lastPublishedDoi":"10.21203/rs.3.rs-3860653/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3860653/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eType 2 diabetes (T2D), but not type 1, protected against ALS. In T2D serum insulin is normal or elevated in the early stages. Type 1 diabetes, characterized by a total lack of insulin, is associated with increased risk of ALS. The antidiabetic metformin also protects against ALS. Connexin 43 (Cx43), an astrocyte protein, operates as an open channel via which toxic substances from astrocytes reach motor neurons to cause ALS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the current study we analyzed FDA MedWatch data to determine whether insulin or metformin could reduce the risk of ALS. We performed \u003cem\u003ein silico\u003c/em\u003e molecular docking studies and molecular dynamics simulation with Cx43 to determine if insulin or metformin dock within the Cx43 channel and can block it effectively, again reducing risk of ALS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn MedWatch, Insulin use is associated with a significantly reduced risk of ALS (Proportional Reporting Ratio 0.401). Metformin use is associated with a significantly reduced risk of ALS (PRR 0.567). The Human insulin heterodimer docked within center of the Cx43 channel, effectively blocking it. Molecular dynamics simulation showed that the block is highly stable and may be responsible for the protective effect of T2D on ALS. Metformin docks within the Cx43 channel, but the relatively small size of the metformin molecule may not allow it to obstruct the passage of toxic substances from astrocytes to motor neurons.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMedWatch data indicates that both insulin and metformin reduce risk of ALS. The results of our \u003cem\u003ein silico\u003c/em\u003e docking study and molecular dynamics simulation corroborate our previous findings with Cx31. Insulin docks within the open hemichannel of hexameric Cx43, potentially blocking it. Molecular dynamics simulation showed that the block is stable and may be responsible for the protective effect of T2D and insulin on ALS. Metformin probably does not exert its protective effect by blocking the Cx43 channel.\u003c/p\u003e","manuscriptTitle":"Insulin and metformin are associated with reduced risk of amyotrophic lateral sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 05:24:37","doi":"10.21203/rs.3.rs-3860653/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d051c4c-616e-440c-a8d6-32fb81451b2f","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28124059,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2024-01-16T05:24:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 05:24:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3860653","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3860653","identity":"rs-3860653","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}