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2000
Volume 33, Issue 2
  • ISSN: 0929-8673
  • E-ISSN: 1875-533X

Abstract

Introduction

Fructose, like other sugars and sugar metabolites, is capable of glycating protein. Insulin's fructosylation leads to the generation of Advanced Glycation End Products (AGEs). Reducing sugars reaction with proteins to form Schiff’s bases, which are characterized by the presence of an imine (C=N) bond. The Schiff bases then undergo irreversible rearrangements, followed by the production of much more stable compounds called Amadori products. These Amadori products can further undergo oxidation, dehydration, cyclization, and condensation to form highly toxic advanced glycation end-products (AGEs). These processes are accompanied by oxidative stress, secondary structural perturbations, and altered morphology, progressing toward amyloidogenesis. Metformin, a biguanide, is the most common drug used to treat type 2 Diabetes Mellitus (T2DM).

Aim

The aim of this study was to evaluate the protective effect of metformin against fructosylation-induced cross-β structures and amyloid aggregations of human insulin.

Methods

UV-absorbance and fluorescence spectroscopy, determination of carbonyl content, free lysine and arginine residues, determination of fructosamine content, SDS-PAGE, circular dichroism (CD) spectroscopy, dynamic light scattering, and scanning and transmission electron microscopy.

Results

Physicochemical studies in the presence or absence of metformin revealed a concentration-dependent structural restoration of fructosylated insulin. Results from the thioflavin-T fluorescence assay suggested that metformin limited the transition of insulin from native to fibrillar state, which was validated by scanning and transmission electron microscopy. Metformin lowered the ThT fluorescence intensity in a concentration-dependent manner. The ThT-specific fluorescence intensity was reduced to 114 and 112.5%. The fluorescence intensity at 2.5 mM metformin was close to native insulin. Electron microscopy revealed that insulin fructosylated by 25 mM fructose in the presence of 2.5 mM metformin suppressed the formation of fibrillar structures. Dynamic light scattering data revealed the potential of metformin to conserve and reinstate the increased hydrodynamic radii (R) of fructosylated insulin close to the native conformer. The R values of native, fructosylated insulin and insulin incubated with fructose and metformin were found to be 2.65 ± 0.28, 307.6 ± 24.19 nm, and 110.1 ± 4.08 nm, respectively. This study also identified metformin as an antioxidant by protecting critical amino acid residues of the insulin domain.

Discussion

The study reports the protective effects of metformin on insulin structure, conformation, and function. The findings suggest a potential role for metformin in improving the risk profile associated with insulin resistance due to altered structure or the accumulation of protein aggregates. Interaction studies between insulin and metformin presented here are due to the chemical effect; hence, further in-depth studies are required to identify the molecular mechanism of insulin sensitivity and changes in cellular processes and pathways.

Conclusion

The results suggest that metformin safeguards against fructosylation-induced structural, conformational, morphological, and amyloidogenic aggregating tendencies of insulin. Protein aggregation has been linked to several neurological and metabolic diseases. Hence, metformin may be crucial in preserving the biological activity of insulin by maintaining and protecting its structural integrity and minimizing the associated comorbidities. The study may further be extended to identify the role of metformin in controlling the gradual insulin resistance in T2DM at the molecular level.

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References

  1. ChoN.H. ShawJ.E. KarurangaS. HuangY. da Rocha FernandesJ.D. OhlroggeA.W. MalandaB. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045.Diabetes Res. Clin. Pract.201813827128110.1016/j.diabres.2018.02.02329496507
     [Google Scholar]
  2. NowotnyK. JungT. HöhnA. WeberD. GruneT. Advanced glycation end products and oxidative stress in type 2 diabetes mellitus.Biomolecules20155119422210.3390/biom501019425786107
     [Google Scholar]
  3. DarenskayaM.A. KolesnikovaL.I. KolesnikovS.I. Oxidative stress: Pathogenetic role in diabetes mellitus and its complications and therapeutic approaches to correction.Bull. Exp. Biol. Med.2021171217918910.1007/s10517‑021‑05191‑734173093
     [Google Scholar]
  4. JakasA. KatićA. BiondaN. HorvatŠ. Glycation of a lysine-containing tetrapeptide by d-glucose and d-fructose—influence of different reaction conditions on the formation of Amadori/Heyns products.Carbohydr. Res.2008343142475248010.1016/j.carres.2008.07.00318656854
     [Google Scholar]
  5. TakeuchiM. IwakiM. TakinoJ. ShiraiH. KawakamiM. BucalaR. YamagishiS. Immunological detection of fructose-derived advanced glycation end-products.Lab. Invest.20109071117112710.1038/labinvest.2010.6220212455
     [Google Scholar]
  6. GugliucciA. Formation of fructose-mediated advanced glycation end products and their roles in metabolic and inflammatory diseases.Adv. Nutr.201781546210.3945/an.116.01391228096127
     [Google Scholar]
  7. BrayG.A. NielsenS.J. PopkinB.M. Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity.Am. J. Clin. Nutr.200479453754310.1093/ajcn/79.4.53715051594
     [Google Scholar]
  8. ZamanA. ArifZ. Moinuddin AlamK. Fructose-human serum albumin interaction undergoes numerous biophysical and biochemical changes before forming AGEs and aggregates.Int. J. Biol. Macromol.201810989690610.1016/j.ijbiomac.2017.11.06929133088
     [Google Scholar]
  9. GugliucciA. Formation of fructose-mediated advanced glycation end products and their roles in metabolic and inflammatory diseases.Adv. Nutr.201781546210.3945/an.116.01391228096127
     [Google Scholar]
  10. ZhaoH.R. SmithJ.B. JiangX.Y. AbrahamE.C. Sites of glycation of β B2-crystallin by glucose and fructose.Biochem. Biophys. Res. Commun.1996229112813310.1006/bbrc.1996.17688954094
     [Google Scholar]
  11. GabbayK.H. KinoshitaJ.H. Mechanism of development and possible prevention of sugar cataracts.Isr. J. Med. Sci.197288155715614647820
     [Google Scholar]
  12. HintonD.J.S. AmesJ.M. Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose.Amino Acids200630442543410.1007/s00726‑006‑0269‑216583308
     [Google Scholar]
  13. ScivittaroV. GanzM.B. WeissM.F. AGEs induce oxidative stress and activate protein kinase C-β II in neonatal mesangial cells.Am. J. Physiol. Renal Physiol.20002784F676F68310.1152/ajprenal.2000.278.4.F67610751230
     [Google Scholar]
  14. GraciaK.C. Llanas-CornejoD. HusiH. CVD and oxidative stress.J. Clin. Med.2017622210.3390/jcm602002228230726
     [Google Scholar]
  15. ChellanP. NagarajR.H. Protein crosslinking by the Maillard reaction: dicarbonyl-derived imidazolium crosslinks in aging and diabetes.Arch. Biochem. Biophys.199936819810410.1006/abbi.1999.129110415116
     [Google Scholar]
  16. VerzijlN. DeGrootJ. ZakenC.B. Braun-BenjaminO. MaroudasA. BankR.A. MizrahiJ. SchalkwijkC.G. ThorpeS.R. BaynesJ.W. BijlsmaJ.W.J. LafeberF.P.J.G. TeKoppeleJ.M. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: A possible mechanism through which age is a risk factor for osteoarthritis.Arthritis Rheum.200246111412310.1002/1529‑0131(200201)46:1<114::AID‑ART10025>3.0.CO;2‑P11822407
     [Google Scholar]
  17. IghodaroO.M. Molecular pathways associated with oxidative stress in diabetes mellitus.Biomed. Pharmacother.201810865666210.1016/j.biopha.2018.09.05830245465
     [Google Scholar]
  18. CaugheyB. LansburyP.T.Jr. Protofibrils, pores, fibrils, and neurodegeneration: Separating the responsible protein aggregates from the innocent bystanders.Annu. Rev. Neurosci.200326126729810.1146/annurev.neuro.26.010302.08114212704221
     [Google Scholar]
  19. LaurénJ. GimbelD.A. NygaardH.B. GilbertJ.W. StrittmatterS.M. Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.Nature200945772331128113210.1038/nature0776119242475
     [Google Scholar]
  20. LiS. LeblancR.M. Aggregation of insulin at the interface.J. Phys. Chem. B201411851181118810.1021/jp410120224328184
     [Google Scholar]
  21. SchofieldC.J. SutherlandC. Disordered insulin secretion in the development of insulin resistance and Type 2 diabetes.Diabet. Med.201229897297910.1111/j.1464‑5491.2012.03655.x22443306
     [Google Scholar]
  22. RazaA. MahmoodR. HabibS. TalhaM. KhanS. HashmiM.A. MohammadT. AliA. Fructosylation of human insulin causes AGEs formation, structural perturbations and morphological changes: An in silico and multispectroscopic study.J. Biomol. Struct. Dyn.202341125850586210.1080/07391102.2022.209882035869652
     [Google Scholar]
  23. HunterR.W. HugheyC.C. LantierL. SundelinE.I. PeggieM. ZeqirajE. SicheriF. JessenN. WassermanD.H. SakamotoK. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase.Nat. Med.20182491395140610.1038/s41591‑018‑0159‑730150719
     [Google Scholar]
  24. BaileyC.J. Metformin: Effects on micro and macrovascular complications in type 2 diabetes.Cardiovasc. Drugs Ther.200822321522410.1007/s10557‑008‑6092‑018288595
     [Google Scholar]
  25. ReganT.J. JyothirmayiG.N. LahamC. JainA. Left ventricular diastolic dysfunction in diabetic or hypertensive subjects: Role of collagen alterations.Adv. Exp. Med. Biol.200149812713210.1007/978‑1‑4615‑1321‑6_1711900360
     [Google Scholar]
  26. TanakaY. UchinoH. ShimizuT. YoshiiH. NiwaM. OhmuraC. MitsuhashiN. OnumaT. KawamoriR. Effect of metformin on advanced glycation endproduct formation and peripheral nerve function in streptozotocin-induced diabetic rats.Eur. J. Pharmacol.19993761-2172210.1016/S0014‑2999(99)00342‑810440084
     [Google Scholar]
  27. Twarda-ClapaA. OlczakA. BiałkowskaA.M. KoziołkiewiczM. Advanced glycation end-products (AGEs): Formation, chemistry, classification, receptors, and diseases related to AGEs.Cells2022118131210.3390/cells1108131235455991
     [Google Scholar]
  28. AlenaziF. SaleemM. KhajaA.S.S. ZafarM. AlharbiM.S. HagbaniT.A. AshrafJ.M. QamarM. RafiZ. AhmadS. Metformin encapsulated gold nanoparticles (MTF-GNPs): A promising antiglycation agent.Cell Biochem. Funct.202240772974110.1002/cbf.373836098489
     [Google Scholar]
  29. TalhaM. MirA.R. HabibS. AbidiM. WarsiM.S. IslamS. Moinuddin. Hydroxyl radical induced structural perturbations make insulin highly immunogenic and generate an auto-immune response in type 2 diabetes mellitus.Spectrochim. Acta A Mol. Biomol. Spectrosc.202125511964010.1016/j.saa.2021.11964033744841
     [Google Scholar]
  30. LevineR.L. GarlandD. OliverC.N. AmiciA. ClimentI. LenzA.G. AhnB.W. ShaltielS. StadtmanE.R. Determination of carbonyl content in oxidatively modified proteins.Methods Enzymol.199018646447810.1016/0076‑6879(90)86141‑H1978225
     [Google Scholar]
  31. KhanM.Y. AlouffiS. AhmadS. Immunochemical studies on native and glycated LDL – An approach to uncover the structural perturbations.Int. J. Biol. Macromol.201811528729910.1016/j.ijbiomac.2018.04.01629634967
     [Google Scholar]
  32. SmithR.E. MacQuarrieR. A sensitive fluorometric method for the determination of arginine using 9,10-phenanthrenequinone.Anal. Biochem.197890124625510.1016/0003‑2697(78)90029‑5727468
     [Google Scholar]
  33. ChayaratanasinP. BarbieriM.A. SuanpairintrN. AdisakwattanaS. Inhibitory effect of Clitoria ternatea flower petal extract on fructose-induced protein glycation and oxidation-dependent damages to albumin in vitro.BMC Complement. Altern. Med.20151512710.1186/s12906‑015‑0546‑2
     [Google Scholar]
  34. TufailN. AbidiM. WarsiM.S. KausarT. NayeemS.M. Computational and physicochemical insight into 4-hydroxy-2-nonenal induced structural and functional perturbations in human low-density lipoprotein.J. Biomol. Struct. Dyn.20244252698271310.1080/07391102.2023.220823437154523
     [Google Scholar]
  35. AhmadR. WarsiM.S. AbidiM. HabibS. SiddiquiS. KhanH. NabiF. Moinuddin. Structural perturbations induced by cumulative action of methylglyoxal and peroxynitrite on human fibrinogen: An in vitro and in silico approach.Spectrochim. Acta A Mol. Biomol. Spectrosc.202430712350010.1016/j.saa.2023.12350037989033
     [Google Scholar]
  36. WarsiM.S. HabibS. TalhaM. KhanS. SinghP. MirA.R. AbidiM. AliA. 4-chloro-1,2-phenylenediamine induced structural perturbation and genotoxic aggregation in human serum albumin.Front. Chem.202210101635410.3389/fchem.2022.101635436199663
     [Google Scholar]
  37. AhmadS. ShahabU. BaigM.H. KhanM.S. KhanM.S. SrivastavaA.K. SaeedM. Moinuddin. Inhibitory effect of metformin and pyridoxamine in the formation of early, intermediate and advanced glycation end-products.PLoS One201389e7212810.1371/journal.pone.007212824023728
     [Google Scholar]
  38. BeisswengerP. Ruggiero-LopezD. Metformin inhibition of glycation processes.Diabetes Metab2003294 Pt 26S9510310.1016/s1262‑3636(03)72793‑114502106
     [Google Scholar]
  39. IannuzziC. IraceG. SirangeloI. Differential effects of glycation on protein aggregation and amyloid formation.Front. Mol. Biosci.20141910.3389/fmolb.2014.0000925988150
     [Google Scholar]
  40. dos SantosM.M. PrestesA.S. de MacedoG.T. EckerA. BarcelosR.P. BoligonA.A. SouzaD. de BemA.F. da RochaJ.B.T. BarbosaN.V. Syzygium cumini leaf extract inhibits LDL oxidation, but does not protect the liproprotein from glycation.J. Ethnopharmacol.2018210697910.1016/j.jep.2017.08.03328844679
     [Google Scholar]
  41. SzwergoldB. A hypothesis: Fructosamine-3-kinase-related-protein (fn3krp) catalyzes deglycation of maillard intermediates directly downstream from fructosamines.Rejuvenation Res.202124431031810.1089/rej.2021.000934314247
     [Google Scholar]
  42. AliS.M. NabiF. FurkanM. HisamuddinM. MalikS. ZakariyaS.M. RizviI. UverskyV.N. KhanR.H. Tuning the aggregation behavior of human insulin in the presence of luteolin: An in vitro and in silico approach.Int. J. Biol. Macromol.202323712421910.1016/j.ijbiomac.2023.12421936990415
     [Google Scholar]
  43. YamagishiS. MatsuiT. Role of hyperglycemia-induced advanced glycation end product (AGE) accumulation in atherosclerosis.Ann. Vasc. Dis.201811325325810.3400/avd.ra.18‑0007030402172
     [Google Scholar]
  44. AdesharaK.A. BangarN.S. DoshiP.R. DiwanA. TupeR.S. Action of metformin therapy against advanced glycation, oxidative stress and inflammation in type 2 diabetes patients: 3 months follow-up study.Diabetes Metab. Syndr.20201451449145810.1016/j.dsx.2020.07.03632769032
     [Google Scholar]
  45. SzkudlarekA. PentakD. PlochA. PożyckaJ. Maciążek-JurczykM. In vitro investigation of the interaction of tolbutamide and losartan with human serum albumin in hyperglycemia states.Molecules20172212224910.3390/molecules2212224929258218
     [Google Scholar]
  46. LopesJ.L.S. MilesA.J. WhitmoreL. WallaceB.A. Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses.Protein Sci.201423121765177210.1002/pro.255825262612
     [Google Scholar]
  47. Pereira MoraisM.P. MarshallD. FlowerS.E. CauntC.J. JamesT.D. WilliamsR.J. WaterfieldN.R. van den ElsenJ.M.H. Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis.Sci. Rep.201331143710.1038/srep0143723531746
     [Google Scholar]
  48. KangY.J. JeongH.C. KimT.E. ShinK.H. Bioanalytical method using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPL-CHRMS) for the detection of metformin in human plasma.Molecules20202520462510.3390/molecules2520462533050662
     [Google Scholar]
  49. RoyH. BrahmaC. NandiS. ParidaK. Formulation and design of sustained release matrix tablets of metformin hydrochloride: Influence of hypromellose and polyacrylate polymers.Int. J. Appl. Basic Med. Res.201331556310.4103/2229‑516X.11224223776841
     [Google Scholar]
  50. MandalB.M. Conducting polyaniline: Dispersions, nanoparticles and nanocomposites.J. Indian Chem. Soc.199775121129
     [Google Scholar]
  51. RoyT.T. SarbadhikaryS.B. GuhaA.K. MandalB.M. BhattacharyyaS.N. On the estimation of biological oxygen demand of collected water samples with special reference to that of the Ganges water.J. Indian Chem. Soc.199673472475
     [Google Scholar]
  52. BhattacharyyaC. BhattacharyyaS.N. MandalB.M. Poly (vinyl propionate) and Poly (ethyl acrylate)-A miscible polymer pairt.J. Indian Chem. Soc.198663157160
     [Google Scholar]
  53. FloryP.J. Thermodynamics of high polymer solutions.J. Chem. Phys.1942101516110.1063/1.1723621
     [Google Scholar]
  54. FloryP.J. Principles of Polymer Chemistry.Cornell University PressIthaca, NY1953
     [Google Scholar]
  55. KlueppelbergJ. HandgeU.A. ThommesM. WinckJ. Composition dependency of the flory–huggins interaction parameter in drug–Polymer phase behavior.Pharmaceutics20231512265010.3390/pharmaceutics1512265038139992
     [Google Scholar]
  56. RanaD. BagK. BhattacharyyaS.N. MandalB.M. Miscibility of poly(styrene-co-butyl acrylate) with poly(ethyl methacrylate): Existence of both UCST and LCST.J. Polym. Sci., B, Polym. Phys.200038336937510.1002/(SICI)1099‑0488(20000201)38:3<369::AID‑POLB3>3.0.CO;2‑W
     [Google Scholar]
  57. RanaD. MandalB.M. BhattacharyyaS.N. Analogue calorimetric studies of blends of poly (vinyl ester)s and polyacrylates.Macromolecules19962951579158310.1021/ma950954n
     [Google Scholar]
  58. RanaD. MandalB.M. BhattacharyyaS.N. Analogue calorimetry of polymer blends: poly(styrene-co-acrylonitrile) and poly(phenyl acrylate) or poly(vinyl benzoate).Polymer199637122439244310.1016/0032‑3861(96)85356‑0
     [Google Scholar]
  59. RanaD. MandalB.M. BhattacharyyaS.N. Miscibility and phase diagrams of poly(phenyl acrylate) and poly(styrene-co-acrylonitrile) blends.Polymer19933471454145910.1016/0032‑3861(93)90861‑4
     [Google Scholar]
  60. HuangS.J. GokulkumarK. GovindasamyM. AlbaqamiM.D. WabaidurS.M. Nanoarchitectonics of europium vanadate nanoparticles decorated carbon nanofibers for electrochemical detection of fungicide in fruits.J. Taiwan Inst. Chem. Eng.2024161105563
     [Google Scholar]
  61. RemilaA. ShallyV. ParvathirajaC. DarwinT. DharshiniM.P. JayamT.G. WabaidurS.M. SiddiquiM.R. Superior performance of nickel doped vanadium pentoxide nanoparticles and their photocatalytic, antibacterial and antioxidant activities.Res. Chem. Intermed.20245073009303110.1007/s11164‑024‑05316‑3
     [Google Scholar]
  62. LiL. DaiX. LuM. GuoC. WabaidurS.M. WuX.L. LouZ. ZhongY. HuY. Electron-enriched single-Pd-sites on g-C3N4 nanosheets achieved by in-situ anchoring twinned Pd nanoparticles for efficient CO2 photoreduction.Advanced Powder Materials20243210017010.1016/j.apmate.2024.100170
     [Google Scholar]
  63. El-BazY.G. MoustafaA. AliM.A. El-DesokyG.E. WabaidurS.M. IqbalA. Green synthesized silver nanoparticles for the treatment of diabetes and the related complications of hyperlipidemia and oxidative stress in diabetic rats.Exp. Biol. Med. (Maywood)2023248232237224810.1177/1535370223121425838205769
     [Google Scholar]
  64. MishraP. FaruquiT. AkhtarS. NadeemI. KhanI. WabaidurS.M. KaziM. RahimM. RafiZ. KhanS. Antiproliferative activity of gold and silver nanoparticles fabricated using bark extract of Murraya koenigii.J. Drug Deliv. Sci. Technol.20238910501410.1016/j.jddst.2023.105014
     [Google Scholar]
  65. El-BazY.G. MoustafaA. AliM.A. El-DesokyG.E. WabaidurS.M. FaisalM.M. An analysis of the toxicity, antioxidant, and anti-cancer activity of cinnamon silver nanoparticles in comparison with extracts and fractions of Cinnamomum cassia at normal and cancer cell levels.Nanomaterials202313594510.3390/nano1305094536903823
     [Google Scholar]
  66. PrabulaS.S. HentryC. RoseB.L. ParvathirajaC. ManiA. WabaidurS.M. EldesokyG.E. IslamM.A. Synthesis of silver nanoparticles by using Cassia auriculata flower extract and their photocatalytic behavior.Chem. Eng. Technol.202245111919192510.1002/ceat.202200082
     [Google Scholar]
  67. KučukN. PrimožičM. KnezŽ. LeitgebM. Sustainable biodegradable biopolymer-based nanoparticles for healthcare applications.Int. J. Mol. Sci.2023244318810.3390/ijms2404318836834596
     [Google Scholar]
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Metformin Protects Human Insulin from Fructosylation: An In Vitro Biochemical Study
Curr. Med. Chem. 33, 461 (2026); https://doi.org/10.2174/0109298673397564250529061139
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  • Article Type:     Research Article
Keyword(s): Advanced glycation end products; amyloid aggregation; diabetes; electron microscopy; fructosamine; insulin resistance

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