The relationship of selected biomarkers of gut microbiotadysbiosis with adiposity and metabolic risk factors in nascentmetabolic syndrome patients

Main Article Content

Maysa Suyagh https://orcid.org/0000-0003-2042-8584
Violet Kasabri https://orcid.org/0000-0003-1927-0193
NAILYA Bulatova https://orcid.org/0000-0001-6754-0325
Sumaya AbuLoha https://orcid.org/0000-0003-4742-6212
Jameel Al-Bzour https://orcid.org/0000-0002-8731-7574
Reem AlQuoqa https://orcid.org/0000-0002-2382-2272

Keywords

Microbiome Dysbiosis Signature Determinants: Carnitine; Choline; γButyrobetaine; TMAO; Zonulin; Survivin; LECT2, Antioxidative Stressors: CATALASE; SOD %Inhibition; Trolox Equivalent Total Antioxidant Capacity, Adiposity, Surrogate (Non Insulin Based) Insulin Resistance and Atherogenecity Indices, Cardiometabolic risk factors of nascent metabolic syndrome and prediabetes

Abstract

Background: This study aimed to compare and correlate between non-diabetic MetS, newly diagnosed drug naive pre-diabetic MetS patients vs. lean, apparently healthy and normoglycemic controls the plasma levels of cardiometabolic risk biomarkers’ of pharmacotherapy (appraised using colorimetric and chromatography assays of gut dysbiosis carnitine, choline, γbutyrobetaine, TMAO, Zonulin, survivin, Leukocyte cell-derived chemotaxin 2 (LECT2) and antioxidative stressors (catalase, superoxide dismutase (SOD) and Trolox total antioxidative capacity), adiposity, and atherogenicity with non-insulin based surrogate insulin resistance (sIR) indices. Methods: ANOVA comparisons and Spearman’s rank correlations were conducted in this cross-sectional study of 30 normoglycemic lean subjects (control), 30 nonprediabetic MetS subjects and 30 MetS/pre-diabetic (PreDM) enrolled. Results: MetS-PreDM group presented significantly higher values of FPG (P2<0.001,P3 =0.009) and A1C (P values <0.001) than both normoglycemic MetS and control groups. However, MetS-PreDM and normoglycemic MetS recruits had appreciably higher values of DBP, SBP, TG, and non-HDL-C but significantly lower values of HDL-C (P values <0.001) than the controls. Explicitly no significance in variance was noticeable among any of the study arms (P value < 0.05) for any of the hematological indices. Nevertheless, Both MetS groups (nonprediabetic and PreDM) had substantially higher values for each of adiposity, atherogenecity and surrogate insulin resistance (non insulin based) indices (P2<0.001) vs. controls’ respectively. Both Survivin and LECT2 levels were significantly higher in PreDM MetS group (P value < 0.05 vs. nondiabetic MetS participants). Conversely all 5 gut dysbiosis biomarkers (carnitinine, choline, γBB, TMAo and Zonulin) which proved significantly lower vs. those of either controls (nondiabetic lean or MetS). Surprisingly, a significant variation in all tested 7 biomarkers’ plasma levels were found between nondiabetic MetS and PreDM-MetS groups (P3 < 0.05). Interestingly all 3 antioxidative stressors were on the decline as anticipated; where catalase, SOD % inhibitions and trolox total antioxidative capacities were significantly lower in both MetS recruits vs. controls. Importantly the discrepancy between normoglycemic nonprediabetic MetS vs. the MetS-PreDM (P3 < 0.05) may have not ranked up to significance in indices, clinical parameters or biomarkers. Notably in pooled MetS (both normoglycemic and pre-diabetics participants (N =60)). Most exquisitely survivin with dysbiosis choline and γBB correlated positively and pronouncedly with carnitine in pooled MetS participants. Also in a striking similarity, cardiometabolic LECT2 has a marked direct relation with each of dyasbiosis carnitine and γBB. TMAO, nevertheless, related inversely and significantly with all 3 dysbiosis biomarkers, likewise Zonulin associated disproportionally with both choline and γBB. Exceptionally TMAO- TYG and Zonulin-TYG-WHpR paired in substantial and inverse relations in pooled normoglycemic and preDM MetS participants (n=60). To superbly signify the anticipated deterioration in metabolism via gut microbiota-insulin insensitivity interconnectivity; all dysbiosis biomarkers (carnitine, choline, γBB, TMAO, Zonulin and survivin) correlated highly remarkably and proportionally with all non insulin based surrogate insulin resistance indices in 60 MetS recruits (both normoglycemic and prediabetic; equally). Unequivocally γBB associated directly and pronouncedly with almost all adiposity indices. Surprisingly VAI correlated negatively with Zonulin in the same MetS population. FBG associated exceptionally with carnitine and γButyrobetaine (γBB). Substantially A1c correlated proportionally (P values <0.05) with MetS pooled cases dysbiosis’ carnitine, choline, γBB, and cardiometabolic surviving. Outstandingly both SBP and DBP had direct and marked linkage to LECT2 and so did DBP with choline’s plasma levels. Remarkably TMAO related negatively and pronouncedly with MetS cases levels of FBG, A1c, TG, LDL-C, and so did also zonulin with both A1c and LDL-C. Conclusions: Given the intergroup discrepancies in dysbiosis and cardiometabolic biomarkers along with their elective correlations with MetS-related indices and clinical parameters; our study cannot rule out any potentiality in molecular crosstalk and interplay of those biomarekers with the pathophysiology of MetS and preDM with their related dysregularities. Carnitine, choline, γbutyrobetaine, TMAO, Zonulin, survivin, and LECT2 can be putatively surrogate biomarkers to use as prognostic/predictive tools for the diagnosis/prevention and potential targets for MetS treatment.

Abstract 112 | PDF Downloads 72

References

1. Shoer S, Shilo S, Godneva A et al. Impact of dietary interventions on pre-diabetic oral and gut microbiome, metabolites and cytokines. Nature Communication. 2023; 14:5384. https://doi.org/10.1038/ s41467-023-41042-xBB
2. (a).Wu H, Tremaroli V, Schmidt C, Lundqvist A, Olsson LM, Krämer M, et al. The Gut Microbiota in Prediabetes and Diabetes: A Population-Based Cross-Sectional Study. Cell Metabolism. 2020; 32(3):379-390.e3. https://doi.org/10.1016/j.
cmet.2020.06.011; (b). Zhang Z, Tian T, Chen Z, Liu L, Luo T, Dai J. Characteristics of the gut microbiome in patients with prediabetes and type 2 diabetes. PeerJ. 2021; 9:e10952. https://doi.org/10.7717/peerj.10952; (c).Letchumanan G, Abdullah
N, Marlini M, Baharom N, Lawley B, Omar MR, et al. Gut Microbiota Composition in Prediabetes and Newly Diagnosed Type 2 Diabetes: A Systematic Review of Observational Studies. Frontiers Cellular Infection & Microbiology. 2022; 12: 943427. https:// doi.org/10.3389/fcimb.2022.943427
3. Bourdeau-Julien I, Castonguay-Paradis S, Rochefort G, Perron J, Lamarche B, Flamand N, et al. The diet rapidly and differentially affects the gut microbiota and host lipid mediators in a healthy population. Microbiome. 2023; 11(1):26. https://doi.org/10.1186/s40168-023-01469-2
4. Zhang X, Zhao Y, Xu J, Xue Z, Zhang M, Pang X, et al. Modulation of gut microbiota by berberine and metformin during the treatment of high-fat diet-induced obesity in rats. Scientific Reports. 2015; 5:14405. https://doi.org/10.1038/srep14405
5. (a). Qi Y, Wang X. The Role of Gut Microbiota in High-Fat-Diet-Induced Diabetes: Lessons from Animal Models and Humans. Nutrients. 2023; 15(4):922. https://doi.org/10.3390/nu15040922; (b). Wang Y, Liu H, Zheng M, Yang Y, Ren H, Kong Y, et al.
Berberine Slows the Progression of Prediabetes to Diabetes in Zucker Diabetic Fatty Rats by Enhancing Intestinal Secretion of Glucagon-Like Peptide-2 and Improving the Gut Microbiota. Frontiers in Endocrinology (Lausanne). 2021; 12:609134. https://doi.org/10.3389/fendo.2021.609134
6. (a).Maniar K, Moideen A, Bhattacharyya R, Banerjee D. Metformin exerts anti-obesity effect via gut microbiome modulation in prediabetics: A hypothesis. Medical Hypotheses. 2017; 104:117-120. https://doi.org/10.1016/j.mehy.2017. 06.001; (b). Anavi-Cohen S, Tsybina-Shimshilashvili N, Zandani G, Hovav R, Sela N, Nyska A, et al. Effects of high oleic acid peanuts on mice’s liver and adipose tissue metabolic parameters and gut microbiota composition. Frontiers in Nutrition. 2023; 10:1205377. https://doi.org/10.3389/fnut.2023.1205377
7. Palacios T, Vitetta L, Coulson S, Madigan CD, Lam YY, Manuel R, et al. Targeting the Intestinal Microbiota to Prevent Type 2 Diabetes and Enhance the Effect of Metformin on Glycaemia: A Randomized Controlled Pilot Study. Nutrients. 2020; 12(7):2041.https://doi.org/10.3390/nu12072041
8. Kawano Y, Edwards M, Huang Y, Bilate AM, Araujo LP, Tanoue T, et al. Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome. Cell. 2022; 185(19):3501-3519.e20. https://doi.org/10.1016/j.cell.2022.08.005
9. (a). Jennings T, Janquart M, Washak C, Duddleston K, Kurtz C. What’s gut got to do with it? The role of the microbiota and inflammation in the development of adiposity and obesity. Immunometabolism (Cobham). 2023; 5(3):e00029. https://doi.org/10.1097/IN9.0000000000000029; (b). Suriano F, Vieira-Silva S, Falony G, de Woutersd’Oplinter A, Paone P, Delzenne
NM, et al. Fat and not sugar as the determining factor for gut microbiota changes, obesity, and related metabolic disorders in mice. American Journal of Physiology, Endocrinology & Metabolism. 2023; 324(1):E85-E96. https://doi.org/10.1152/ajpendo.00141.2022
10. Burmeister MA, Smith TE, Fincher TK, Weldon AJ. Evidence for proton-pump inhibitor (PPI)-associated dysbiosis in metabolically unhealthy obesity. Frontiers in Endocrinology (Lausanne). 2023; 14:1205490. https://doi.org/10.3389/fendo.2023.1205490
11. Hu YH, Meyer K, Lulla A, Lewis CE, Carnethon MR, Schreiner PJ, et al. Gut microbiome and stages of diabetes in middle-aged adults: CARDIA microbiome study. Nutrition & Metabolism (Lond). 2023; 20(1):3. https://doi.org/10.1186/s12986-022-00721-0
12. Cui J, Ramesh G, Wu M, Jensen ET, Crago O, Bertoni AG, et al. Butyrate-Producing Bacteria and Insulin Homeostasis: The Microbiome and Insulin Longitudinal Evaluation Study (MILES). Diabetes. 2022; 71(11):2438-2446. https://doi.org/10.2337/db22-0168
13. Gabriel CL, Ferguson JF. Gut Microbiota and Microbial Metabolism in Early Risk of Cardiometabolic Disease. Circulation Research. 2023; 132(12):1674-1691. https://doi.org/10.1161/CIRCRESAHA.123. 322055
14. Roessler J, Leistner DM, Landmesser U, Haghikia A. Modulatory role of gut microbiota in cholesterol and glucose metabolism:Potential implications for atherosclerotic cardiovascular disease. Atherosclerosis. 2022; 359:1-12. https://doi.org/10.1016/j. atherosclerosis.2022.08.018.
15. Jia L, Huang S, Sun B, Shang Y, Zhu C. Pharmacomicrobiomics and type 2 diabetes mellitus: A novel perspective towards possible treatment. Frontiers in Endocrinology (Lausanne). 2023; 14:1149256. https://doi.org/10.3389/fendo. 2023.1149256
16. (a). Liu W, Luo Z, Zhou J, Sun B. Gut Microbiota and Antidiabetic Drugs: Perspectives of Personalized Treatment in Type 2 Diabetes Mellitus. Frontiers Cellular Infection & Microbiology. 2022; 12:853771. https://doi.org/10.3389/fcimb.2022.853771; (b). Su J, Luo Y, Hu S, Tang L, Ouyang S. Advances in Research on Type 2 Diabetes Mellitus Targets and Therapeutic Agents.International Journal of Molecular Sciences. 2023; 24(17):13381. https://doi.org/10.3390/ ijms241713381
17. Luo JQ, Ren H, Chen MY, Zhao Q, Yang N, Liu Q, et al. Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization. iScience. 2023; 26(7):107130. https://doi.org/10.1016/j. isci.2023.107130 
18. Nunes S, Vieira P, Gomes P, Viana SD, Reis F. Blueberry as an Attractive Functional Fruit to Prevent (Pre)Diabetes Progression. Antioxidants (Basel). 2021; 10(8):1162. https://doi.org/10.3390/antiox1008 1162
19. Girdhar K, Soto M, Huang Q, Orliaguet L, Cederquist C, Sundaresh B, Hu J, et al. Gut Microbiota Regulate Pancreatic Growth, Exocrine Function, and Gut Hormones. Diabetes. 2022; 71(5):945-960. https://doi.org/10.2337/ db21-0382
20. Zhang XY, Chen J, Yi K, Peng L, Xie J, Gou X, et al. Phlorizin ameliorates obesity-associated endotoxemia and insulin resistance in high-fat diet-fed mice by targeting the gut microbiota and intestinal barrier integrity. Gut Microbes. 2020; 12(1):1-18. https://doi.org/10.1080/19490976.2020. 1842990
21. Neri-Rosario D, Martínez-López YE, Esquivel-Hernández DA, Sánchez-Castañeda JP, Padron-Manrique C, Vázquez-Jiménez A, et al. Dysbiosis signatures of gut microbiota and the progression of type 2 diabetes: a machine learning approach in a Mexicancohort. Frontiers in Endocrinology. 2023; 14:1170459. https://doi.org/10.3389/fendo.2023.1170459
22. Stonāns I, Kuzmina J, Poļaka I, Grīnberga S, Sevostjanovs E, Liepiņš E, et al. The Association of Circulating L-Carnitine, γ-Butyrobetaine and Trimethylamine N-Oxide Levels with Gastric Cancer. Diagnostics (Basel). 2023; 13(7):1341. https://doi.org/10.3390/diagnostics13071341
23. Wang Sh, Ma W, Yuan Zh, WangSh , YiX, Jia H, et al. Association between obesity indices and type 2 diabetes mellitus among middle-aged and elderly people in Jinan, China: a cross-sectional study. British Medical Journal.2016; 3:6(11): e012742.https://doi.org/10.1136/bmjopen-2016-012742
24. Bene J, Hadzsiev K, Melegh B. Role of carnitine and its derivatives in the development and management of type 2 diabetes. Nutrition Diabetes. 2018; 8:8. https://doi.org/10.1038/s41387-018-0017-1
25. (a).Alipour B, Barzegar A, Panahi F, Safaeian A, Eshaghi M. Effect of L-carnitine supplementation on metabolic status in obese diabetic women with hypocaloric diet. Health Scope. 2014; 3:e14615. https://doi.org/10.17795/jhealthscope-14615; (b).
Fathizadeh H, Milajerdi A, Reiner Z, Kolahdooz F, Asemi Z. The Effects of L-carnitine Supplementation on Glycemic Control: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Excli J. 2019; 18:631-643. https://doi.org/10.17179/excli2019-1447
26. (a).Xu Y, Jiang W, Chen G, Zhu W, Ding W, Ge Z, Tan Y, Ma T, Cui G. L-carnitine treatment of insulin resistance: A systematic review  and meta-analysis. Advanced Clinical & Experimental Medicine. 2017; 26(2):333-338. https://doi.org/10.17219/acem/61609;(b) Kerner J, Hoppel C. Fatty acid import into mitochondria. Biochimica & Biophysica Acta. 2000; 26:1486(1):1-17. https://doi.org/10.1016/ s1388-1981(00)00044-5
27. Malaguarnera M, Vacante M, Avitabile T, Malaguarnera M, Cammalleri L, Motta M. L-Carnitine supplementation reduces oxidized LDL cholesterol in patients with diabetes. American Journal of Clinical Nutrition. 2009; 89(1):71-76. https://doi.org/10.3945/ajcn.2008.26251.E
28. Walford GA, Ma Y, Clish C, Florez JC, Wang TJ, Gerszten RE. Diabetes Prevention Program Research Group. Metabolite profiles of diabetes incidence and intervention response in the diabetes prevention program. Diabetes. 2016; 65(5):1424-1433.https://doi.org/10.2337/db15-1063
29. Li Y, Wang D, Chiuve S, Manson J, Willett W, Hu F, et al. Dietary phosphatidylcholine intake and type 2 diabetes in men and women. Diabetes Care. 2015; 38(2): e13-14. https://doi.org/10.2337/dc14-2093
30. Karalis D, Karalis T, Karalis S, Kleisiari A. L-L-carnitine as a Diet Supplement in Patients with Type II Diabetes. Cureus. 2020;12(5):e7982. https://doi.org/10.7759/cureus.7982
31. (a). Ejaz A, Martinez-Guino L, Goldfine AB, Ribas-Aulinas F, De-Nigris V, Ribo S, et al. Dietary betaine supplementation increases Fgf21 levels to improve glucose homeostasis and reduce hepatic lipid accumulation in mice. Diabetes. 2016; 65(4):902-912. https://doi.org/10.2337/db15-1094; (b). Liepinsh E, Vilskersts R, Loca D, Kirjanova O, Pugovichs O, Kalvinsh I, et al. Inhibitor of Carnitine Biosynthesis, Induces an Increase in Gamma-Butyrobetaine Contents and Cardioprotection in Isolated Rat Heart Infarction. Journal of Cardiovascular Pharmacology. 2006; (6):314-319. https://doi.org/10.1097/01.fjc.0000250077.07702.23; (c). Du J, Shen L, Tan Zh,Zhang P,Zhao X, XuY, et al. Betaine Supplementation Enhances Lipid Metabolism and Improves Insulin Resistance in Mice Fed a High-Fat Diet. Nutrients. 2018; 10(2):131. https://doi.org/10.3390/nu10020131
32. Zhang Z, Zeng H, Lin J, Hu Y, Yang R, Sun J, et al. Circulating LECT2 levels in newly diagnosed type 2 diabetes mellitus and their association with metabolic parameters: an observational study. Medicine. 2018; 97(15):e0354. https://doi.org/10.1097/MD.0000000000010354
33. Wei M, Liu J, Pan H, Zhou Z, Guo K. Plasma Leukocyte Cell-Derived Chemotaxin 2 (LECT2) as a Risk Factor of Coronary Artery Disease: A Cross-Sectional Study. Angiology. 2022; 73(3):265-274. https://doi.org/10.1177/00033197211028023
34. Lan F, Misu H, Chikamoto K, Takayama H, Kikuchi A, Mohri K, et al. LECT2 functions as a hepatokine that links obesity to skeletal muscle insulin resistance. Diabetes. 2014; 63(5):1649-1664. https://doi.org/10.2337/db13-0728
35. (a).Okumura A, Unoki-Kubota H, Matsushita Y, Shiga T, Moriyoshi Y, Yamagoe S, et al. Increased serum leukocyte cell-derived chemotaxin 2 (LECT2) levels in obesity and fatty liver. Bioscience Trends. 2013; 7(6):276-283. PMID: 24390366; (b).Okumura A, Unoki-Kubota H, Yoshida-Hata N, Yamamoto-Honda R, Yamashita S, Iwata M, et al. Reduced serum level of leukocyte cellderived chemotaxin 2 is associated with the presence of diabetic retinopathy. Clinica Chimica Acta.2016; 463:145-149. https://doi.org/10.1016/j.cca.2016.10.031
36. Jung TW, Chung YH, Kim H, Abd El-Aty, A. M., Jeong, J. H. LECT2 promotes inflammation and insulin resistance in adipocytes via P38 pathways. Journal of Molecular Endocrinology. 2018; 61(1):37-45. https://doi.org/10.1530/JME-17-0267
37. Tanisawa K, Taniguchi H, Sun X, Ito T, Kawakami R, Sakamoto S, et al. Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia. PloS One. 2017; 12(3):0173310. https://doi.org/10.1371/journal.pone.0173310
38. Levkau B, Schäfers M, Wohlschlaeger J, et al. Survivin determines cardiac function by controlling total cardiomyocyte number.Circulation. 2008; 117(12):1583-1593. https://doi.org/10.1161/ CIRCULATIONAHA.107.734160
39. (a). Wu X, Zhang Q, Wang X, Zhu J, Xu K, Okada H, et al. Survivin is required for beta-cell mass expansion in the pancreatic duct-ligated mouse model. PLoS One. 2012; 7(8):e41976. https://doi.org/10.1371/journal.pone.0041976; (b). Ejarque M,
Ceperuelo-Mallafré V, Serena C, Pachón G, Núñez-Álvarez Y, Terrón-Puig M, et al. Survivin, a key player in cancer progression,increases in obesity and protects adipose tissue stem cells from apoptosis. Cell Death & Disease. 2017; 8(5):e2802. https://doi.org/10.1038/cddis.2017.209
40. Ju L, Zhang X, Deng Y, Han J, Yang J, Chen S, et al. Enhanced expression of Survivin has distinct roles in adipocyte homeostasis.Cell Death & Disease. 2017; 8(1):e2533. https://doi.org/10.1038/ cddis.2016.439
41. Li Y, Lu W, Yang J, Edwards M, Jiang S. Survivin as a biological biomarker for diagnosis and therapy. Expert Opinion & Biology Therapy. 2021; 21(11):1429-1441. https://doi.org/10.1080/ 14712598.2021.1918672
42. Martini E, Wittkopf N, Günther C, Leppkes M, Okada H, Watson AJ, et al. Loss of Survivin in Intestinal Epithelial Progenitor Cells Leads to Mitotic Catastrophe and Breakdown of Gut Immune Homeostasis. Cell Reports. 2016; 14(5):1062-1073. https://doi.
org/10.1016/j.celrep.2016.01.010
43. (a). Virtanen JK, Tuomainen TP, Voutilainen S. Dietary intake of choline and phosphatidylcholine and risk of type 2 diabetes in men: The Kuopio Ischaemic Heart Disease Risk Factor Study. European Journal of Nutrition, 2020; 59:3857-3861. https://doi.
org/10.1007/s00394-020-02223-2; (b). Malinowska A, Szwengiel A, Chmurzynska A. Dietary, anthropometric, and biochemical factors influencing plasma choline, L-carnitine, trimethylamine, and trimethylamine-N-oxide concentrations. International
Journal of Food Science & Nutrition. 2017; 68(4):488-495. doi:10.1080/09637486.2016.1256379; (c). Barrea L, Annunziata G, Muscogiuri G, Somma C, Laudisio D, Maisto M, et al. Trimethylamine-N-oxide (TMAO) as Novel Potential Biomarker of Early
Predictors of Metabolic Syndrome. Nutrients. 2018; 10:1971. https://doi.org/10.3390/nu10121971; (d). Blaak E, Canfora E. Increased circulating choline, L-carnitine and TMAO levels are related to changes in adiposity during weight loss: role of the
gut microbiota? Annals of Translational Medicine. 2018; 6(Suppl2): S92. https://doi.org/10.21037/ atm.2018.11.11 
44. Trøseid M, Hov J, Nestvold T, Thoresen H, Berge R, Svardal A, et al. Major increase in microbiota dependent proatherogenic metabolite TMAO one year after bariatric surgery. Metabolic Syndrome & Related Disorders. 2016; 14:197-201. https://doi.org/10.1089/met.2015.0120.
45. Zhou D, Zhang J, Xiao C, Mo C, Ding B-S. Trimethylamine-N-oxide (TMAO) mediates the crosstalk between the gut microbiota and hepatic vascular niche to alleviate liver fibrosis in nonalcoholic steatohepatitis. Frontiers in Immunology. 2022; 13:1-14.https://doi.org/10.3389/fimmu.2022.964477
46. Anto L, Blesso CN. Interplay between diet, the gut microbiome, and atherosclerosis: Role of dysbiosis and microbial metabolites on inflammation and disordered lipid metabolism. Journal of Nutrition & Biochemistry. 2022; 105:108991. https://doi.org/10.1016/j.jnutbio.2022.108991
47. (a).Van Mens T, Buller H, Nieuwdrop M. Targeted inhibition of gut microbiota proteins involved in TMAO production to reduce platelet aggregation and arterial thrombosis: a blueprint for drugging the microbiota in the treatment of cardiometabolic disease? Journal of Thrombosis & Haemostasis. 2019; 17(1):3-5. https://doi.org/10.1111/jth.14331; (b). Roncal C, Martínez-
Aguilar E, Orbe J. et al. Trimethylamine-N-Oxide (TMAO) Predicts Cardiovascular Mortality in Peripheral Artery Disease. Scientific Reports. 2019; 9:15580.  https://doi.org/10.1038/s41598-019-52082-z; (c). Shanmugham M, Bellanger S, Leo CH.
Gut-Derived Metabolite, Trimethylamine-N-oxide (TMAO) in Cardio-Metabolic Diseases: Detection, Mechanism, and Potential Therapeutics. Pharmaceuticals. 2023; 16(4):504. https://doi.org/10.3390/ph16040504
48. Chilloux J, Brial F, Everard A, Smyth D, Zhang L, et al. Microbiome Inhibition of IRAK-4 by Trimethylamine Mediates Metabolic and Immune Benefits in High-Fat-Diet-induced Insulin Resistance. bioRxiv. 2018; 277434. https://doi.org/10. 1101/277434
49. (a).Sturgeon G and Fasano A. Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers. 2016; 4(4):e1251384. https://doi.org/10.1080/21688370. 2016.1251384; (b). Ajamian M, Steer D, Rosella G, Gibson PR. Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems.PLoS One. 2019; 14(1): e0210728. https://doi.org/10.1371/journal.pone.0210728; (c). Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals in New York Academy of Science. 2012; 1258(1): 25-33. https://doi.org/10.1111/j.1749-6632.2012.06538.x
50. (a). Hasslacher C, Kulozik F, Platten I, Kraft M, Siege EG. Serum zonulin as parameter of intestinal permeability in longstanding type 2 diabetes: correlations with metabolism parameter and renal function. Journal of Diabetes.Metabolic Disease & Control (JDMDC). 2018; 5(2):58-62. https://doi.org/10.15406/jdmdc.2018.05.00138; (b). Demir E, Ozkan H, Seckin KD, Sahtiyancı
B, Demir B, Tabak O, et al. Plasma Zonulin Levels as a Non-Invasive Biomarker of Intestinal Permeability in Women with Gestational Diabetes Mellitus. Biomolecules. 2019; 11:9(1):24. https://doi.org/10.3390/biom9010024.
51. Güvey H, Çelik S, Çalışkan CS, Yılmaz Z, Yılmaz M, Erten Ö, et al. How Do Serum Zonulin Levels Change in Gestational Diabetes Mellitus, Pregnancy Cholestasis, and the Coexistence of Both Diseases? International Journal of Environmental Research & Public Health. 2021; 18(23):12555. https://doi.org/10.3390/ijerph182312555
52. Khalil MM, Ali HA, Al-Hilo MAH. Role of Zonulin level as new metabolic biomarker in diabetes mellitus patients and associated complications. AIP Conference Proceedings. 2022; 2386(1): 030013. https://doi.org/10.1063/5.0066876
53. Veres-Székely A, Szász C, Pap D, Szebeni B, Bokrossy P, Vannay Á. Zonulin as a Potential Therapeutic Target in Microbiota-Gut-Brain Axis Disorders: Encouraging Results and Emerging Questions. International Journal of Molecular Sciences. 2023; 24(8):7548. https://doi.org/10. 3390/ijms24087548
54. World Medical Association, 2008/DECLARATION OF HELSINKI/Ethical Principles for Medical Research Involving Human Subjects/ https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/doh-oct2008/
55. International Diabetes Federation, 2006. The IDF consensus worldwide definition of the metabolic syndrome. IDF Communications, 1-23.
56. (a). Al-Qudah S, Kasabri V. Uric Acid Relationship with Noninsulin-Based Insulin Resistance Indices in Selected Metabolic Disorders: A Systematic Critical Review. Jordan Journal of Pharmaceutical Sciences. 2021; 14(3): 255-265; (b). Alsoud LO,
Soares NC, Al-Hroub HM, Mousa M, Kasabri V, Bulatova N, et al. Identification of Insulin Resistance Biomarkers in Metabolic Syndrome Detected by UHPLC-ESI-QTOF-MS. Metabolites. 2022; 12(6):508. https://doi.org/10.3390/metabo12060508; (c).
Kasabri V, AlBsoul-Younes A, Suyagh M, Omed K, Yassin G, AlAlawi S, et al. Sirtuin 1 but not Osteocalcin, Correlates with Lipid Accumulation Product, Visceral Adiposity and Atherogenicity Indices in Newly Diagnosed Prediabetes-Metabolic Syndrome
Patients. The Romanian Journal of Diabetes, Nutrition and Metabolic Diseases. 2020; 27(3):220-236. https://doi.org/10.46389/rjd-2020-103; (d). Saber G, Kasabri V, Saleh M, Suyagh M, Halaseh L, Jaber R, et al. Increased irisin versus reduced fibroblast growth factor1 (FGF1) in relation to adiposity, atherogenicity and hematological indices in metabolic syndrome patients with and without prediabetes. Hormone Molecular Biology and Clinical Investigation. 2019; 38(1):20180063. https://doi.org/10.1515/hmbci-2018-0063; (e). Horan A, Albsoul-Younes A, Kasabri V, Suyagh M, Halaseh L, et al. Correlates of resistin and retinol-binding protein 4 in metabolic syndrome patients with and without prediabetes. Hormone Molecular Biology and
Clinical Investigation. 2019; 37(3): 20180051. https://doi.org/10.1515/ hmbci-2018-0051 
57. Ding QY, Tian JX, Li M, Lian FM, Zhao LH, Wei XX, et al. Interactions between Therapeutics for Metabolic Disease, Cardiovascular Risk Factors, and Gut Microbiota. Frontiers in Cell Infection & Microbiology. 2020; 10: 530160. https://doi.org/10.3389/fcimb.2020.530160
58. (a).Zheng P, Li Z, Zhou Z. Gut microbiome in type 1 diabetes: A comprehensive review. Diabetes & Metabolism Research & Reviews. 2018; 34(7):e3043. https://doi.org/10.1002/dmrr.3043; (b). Siljander H, Honkanen J, Knip M. Microbiome and type 1 diabetes. EBioMedicine, 2019; 46:512-521. https://doi.org/10.1016/j.ebiom.2019.06.031; (c). Abela AG, Fava S. Why is the Incidence of Type 1 Diabetes Increasing? Current Diabetes Reviews, 2021; 17(8): e030521193110. https://doi.org/10.2174/157339981766621 050313374759. 
59. Roep BO, Thomaidou S, van Tienhoven R, Zaldumbide A. Type 1 diabetes mellitus as a disease of the β-cell (do not blame the immune system?). Nature Reviews of Endocrinology. 2021; 17(3):150-161. https://doi.org/10.1038/s41574-020-00443-4
60. (a). Jamshidi P, Hasanzadeh S, TahvildariA, et al. Is there any association between gut microbiota and type 1 diabetes? A systematic review. Gut Pathology. 2019; 11: 49. https://doi.org/10.1186/s13099-019-0332-7; (b). Shilo S, Godneva A, Rachmiel
M, Korem T, Bussi Y, Kolobkov D, et al. The Gut Microbiome of Adults with Type 1 Diabetes and Its Association with the Host Glycemic Control. Diabetes Care. 2022; 45(3):555-563. https://doi.org/10.2337/dc21-1656
61. Yuan X, Wang R, Han B, Sun C, Chen R, Wei H, et al. Functional and metabolic alterations of gut microbiota in children with new-onset type 1 diabetes. Nature Communications, 2022; 13(1):6356. https://doi.org/10.1038/s41467-022-33656-4
62. (a).Han H, Li Y, Fang J, Liu G, Yin J, Li T, et al. Gut Microbiota and Type 1 Diabetes. International Journal of Molecular Sciences. 2018; 19(4): 995. https://doi.org/10.3390/ijms19040995; (b). Lau WL, Tran T, Rhee CM, Kalantar-Zadeh K, Vaziri ND. Diabetes
and the Gut Microbiome. Seminars in Nephrology. 2021; 41(2):104-113. https://doi.org/10.1016/j.semnephrol. 2021.03.005 
63. (a).Vatanen T, Franzosa EA, Schwager R, Tripathi S, Arthur TD, Vehik K, et al. The human gut microbiome in early-onset type 1 diabetes from the TEDDY study. Nature. 2018; 562(7728):589-594. https://doi.org/10.1038/s41586-018-0620-2; (b). Calabrese CM, Valentini A, Calabrese G. Gut Microbiota and Type 1 Diabetes Mellitus: The Effect of Mediterranean Diet. Frontiers in Nutrition.2021; 7:612773. https://doi.org/10.3389/fnut.2020.612773
64. Girdhar K, Huang Q, Chow IT, Vatanen T, Brady C, Raisingani A, et al. A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes. Proceedings of National Academy of Sciences in USA. 2022; 119(31):e2120028119. https://doi.org/10.1073/pnas. 2120028119
65. Choi M, Park S, Lee M. L-Carnitine’s Effect on the Biomarkers of Metabolic Syndrome: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Nutrients. 2020; 12(9):2795. https://doi.org/10.3390/nu12092795.
66. Johri AM, Hétu MF, Heyland DK, et al. Progression of atherosclerosis with carnitine supplementation: a randomized controlled trial in the metabolic syndrome. Nutrition & Metabolism. 2022; 19:26. https://doi.org/10.1186/s12986-022-00661-9
67. Gao X, Sun C, Zhang Y, Hu S, Li D. Dietary supplementation of L-carnitine ameliorates metabolic syndrome independent of trimethylamine N-oxide produced by gut microbes in high-fat diet-induced obese mice. Food & Function, 2022; 13(23):12039-12050. https://doi.org/10.1039/d2fo02570a
68. Arias N, Arboleya S, Allison J, Kaliszewska A, Higarza SG, Gueimonde M, et al. The Relationship between Choline Bioavailability from Diet, Intestinal Microbiota Composition, and Its Modulation of Human Diseases. Nutrients. 2020; 12(8):2340. https://doi.org/10.3390/nu12082340
69. Liapi C, Kyriakaki A, Al Humadi H, Al-Saigh R, Al Humadi A, Lazaris A, SAT072. Choline Deprivation Aggravates Hyperglycemia And Fatty Liver In Non-obese Streptozocin-induced Diabetic Rats. Journal of the Endocrine Society. 2023; 7(Supplement_1):bvad114.939. https://doi.org/10.1210/ jendso/bvad114.939
70. Zhan X, Fletcher L, Huyben D, Cai H, Dingle S, Qi N, et al. Choline supplementation regulates gut microbiome diversity, gut epithelial activity, and the cytokine gene expression in gilts. Frontiers in Nutrition. 2023; 10:1101519. https://doi.org/10.3389/fnut.2023.1101519
71. Vilskersts R, Zharkova-Malkova O, Mezhapuke R, Grinberga S, Cirule H, Dambrova M. Elevated vascular γ-butyrobetaine levels attenuate the development of high glucose-induced endothelial dysfunction. Clinical & Experimental Pharmacology & Physiology. 2013; 40(8):518-524. https://doi.org/10.1111/ 1440-1681.12127.
72. Strand E, Rebnord EW, Flygel MR, LysneV, Svingen GFT, et al,Serum Carnitine Metabolites and Incident Type 2 Diabetes Mellitus in Patients With Suspected Stable Angina Pectoris. The Journal of Clinical Endocrinology & Metabolism. 2018; 103(3):1033-1041. https://doi.org/10.1210/jc.2017-02139
73. Lemaitre RN, Jensen PN, Wang Z, Fretts AM, McKnight B, Nemet I, et al. Association of Trimethylamine N-Oxide and Related
Metabolites in Plasma and Incident Type 2 Diabetes: The Cardiovascular Health Study. JAMA Netw Open. 2021; 4(8):e2122844. https://doi.org/10.1001/ jamanetworkopen.2021.22844
74. Skagen K, Trøseid M, Ueland T, Holm S, Abbas A, Gregersen I, et al. The Carnitine-butyrobetaine-trimethylamine-N-oxide pathway and its association with cardiovascular mortality in patients with carotid atherosclerosis. Atherosclerosis. 2016; 247:64-69. https://doi.org/10.1016/j.atherosclerosis.2016.01.033
75. Kim JH. Letter: Increased Serum Angiopoietin-Like 6 Ahead of Metabolic Syndrome in a Prospective Cohort Study Diabetes & Metabolism Journal. 2019; 43(5): 727-728. https://doi.org/10.4093/ dmj.2019.0172
76. Qin L, Wu J, Sun X, Huang X, Huang W, Weng C, et al. The regulatory role of metabolic organ-secreted factors in the nonalcoholic fatty liver disease and cardiovascular disease. Frontiers in Cardiovascular Medicine. 2023; 10:1119005. https://doi.org/10.3389/fcvm.2023.1119005
77. Onishi S, Kitazawa H, Meguro S, Tokimitsu I. Green tea extracts reduce leukocyte cell-Derived chemotaxin 2 and selenoprotein P levels in the livers of C57BL/6J mice fed a high-fat diet. Bioscience Biotechnology Biochemistry. 2018; 82(9):1568-1575. https://doi.org/10.1080/09168451.2018.1480349
78. Yu J, Wang Z, Zhang H, Wang Y, Li DQ. Survivin-positive circulating tumor cells as a marker for metastasis of hepatocellular carcinoma. World Journal of Gastroenterology. 2021; 27(43): 7546-7562. https://doi.org/10.3748/wjg. v27.i43.7546
79. Izquierdo AG, Carreira MC, Rodriguez-Carnero G, Fernandez-Quintela A, Sueiro AM, Martinez-Olmos MA, et al. Weight loss normalizes enhanced expression of the oncogene survivin in visceral adipose tissue and blood leukocytes from individuals with obesity. International Journal of Obesity (Lond). 2021; 45(1):206-216. https://doi.org/10.1038/s41366-020-0630-7
80. Velasquez MT, Ramezani A, Manal A, Raj DS. Trimethylamine N-Oxide: The Good, the Bad and the Unknown. Toxins (Basel).
2016; 8(11):326. https://doi.org/10.3390/toxins8110326 
81. Panyod S, Wu WK, Chen CC, Wu MS, Ho CT, Sheen LY. Modulation of gut microbiota by foods and herbs to prevent cardiovascular diseases. Journal of Traditional & Complementary Medicine. 2021; 13(2):107-118. https://doi.org/10.1016/j.jtcme.2021.09.006.