Abstract
Pancreatic β cell dysfunction is an important cause of type 2 diabetes (T2D). Previous studies show trimethylamine N-oxide (TMAO), a gut microbiota-related metabolite, promotes cardiovascular disease and insulin resistance. However, the effect and mechanism of TMAO at pathological concentrations on β cell function remain unknown. The recently published work demonstrates TMAO reduces pancreatic β cell function and glucose homeostasis. Furthermore, TMAO inhibits calcium transients through NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and induced Serca2 loss. Additionally, long-term TMAO exposure promotes β cell endoplasmic reticulum (ER) stress, dedifferentiation and apoptosis. Here, we review the impaired effect of TMAO on β cell function and comment on the implications of this study for understanding the role and mechanism of TMAO in the development of T2D. Further work should focus on screening flavin-containing monooxygenase 3 (FMO3) inhibitors for antidiabetes.
Keywords
Trimethylamine N-oxide, Pancreatic β cell function, Calcium transients, Serca2, Antidiabetes
Commentary
Diabetes is a chronic metabolic disease that seriously harms human health. The main type of diabetes is type 2 diabetes (T2D). T2D is caused by insulin resistance and insufficient insulin secretion resulting from β cell dysfunction and loss. Pancreatic β cell dysfunction and loss play central roles in the initiation and progression of T2D [1]. A decrease in β cell function is characterized by a decrease in glucose-stimulated insulin secretion (GSIS). However, the molecular pathways involved in β cell dysfunction are not fully understood.
Trimethylamine N-oxide (TMAO) is a gut microbiota-dependent metabolite and is related to several metabolic diseases. The dietary precursors choline, carnitine, and phosphatidylcholine are metabolized by the gut microbiota to the intermediate compound trimethylamine (TMA), which is then oxidized to TMAO by hepatic flavin monooxygenase 3 (FMO3) [2]. Importantly, the end product of this nutrient metabolism pathway, TMAO, is linked to cardiovascular disease (CVD) and can be used to predict CVD risk [3]. TMAO promotes atherosclerosis and enhances platelet hyperreactivity and thrombosis risk by enhancing Ca2+ release from platelet intracellular stores [4-6]. High TMAO plasma levels have been linked to diabetes in both mice and humans [7-10]. In mice fed a high-fat diet (HFD), dietary TMAO (~ 17 μM in serum) was shown to exacerbate insulin resistance [11]. The knockdown or inhibition of Fmo3, the TMAO-producing enzyme, prevents insulin resistance in liver insulin receptor knockout insulin-resistant mice and obesity in HFD-fed mice [12,13]. However, the relationship between pathological concentrations of TMAO and β cell dysfunction remains largely unknown.
In this study, the role and mechanism of TMAO in pancreatic β cell function was elucidated [14]. Insulin resistance alone does not lead to hyperglycemia since pancreatic β cells compensate by secreting much more insulin to overcome insulin resistance. It has been reported that β cell function is reduced to 50 to 80% of normal at the time of T2D onset [15]. Therefore, to elucidate the role of TMAO in the pathogenesis of T2D, understanding its effects on β cell function is necessary. This study is the first to show that a pathological dose of TMAO inhibits β cell GSIS and promotes β cell dedifferentiation and apoptosis, revealing a new mechanism for the pathogenesis of diabetes.
In this study, plasma TMAO levels in control and type 2 diabetic mice (db/db mice) or subjects were assessed, revealing that T2D was associated with high circulating levels of TMAO. Hepatic Fmo3, a TMAO-producing enzyme, was more highly expressed in db/db mice. Furthermore, TMAO at a similar concentration to that found in diabetes, inhibited GSIS in a mouse-derived insulin-secreting β cell line MIN6 cells, in a dose-dependent manner, with no change in cell viability or insulin synthesis. Similar to its effect on MIN6 cells, TMAO also inhibited GSIS in mouse and human primary islets. These results demonstrated that in vitro, TMAO reduced the ability of β cells to secrete insulin in response to glucose stimulation. This finding was consistent with several meta-analyses suggesting that elevated TMAO levels are associated with a greater risk of type 2 diabetes [7-9,16-21].
Choline is an important source of TMAO. Therefore, a choline-supplemented chow-based diet (choline diet) is believed to produce more TMAO [3]. For gain-of-function studies, a choline diet was used to increase TMAO levels in C57BL/6J mice. Elevated plasma TMAO levels in mice led to impaired glucose tolerance and decreased insulin and C-peptide levels after intraperitoneal glucose injection. Hyperglycemic clamp confirmed that a choline diet significantly decreased first- and second-phase insulin secretion. The GSIS of isolated primary islets was also inhibited after choline diet feeding. The choline diet impaired islet morphology in mice and reduced the proportion of β cells in the islets and the β cell mass. For loss-of-function studies, genetic knockdown of Fmo3 was performed in choline diet-fed mice to inhibit TMAO production. Fmo3 knockdown increased glucose tolerance and normalized insulin and C-peptide levels after intraperitoneal glucose injection and first-phase insulin secretion in the hyperglycemic clamp test. Staining showed that the proportion of β cells in islets and the β cell mass increased after Fmo3 knockdown. These data showed that TMAO inhibited GSIS in vivo and confirmed the in vitro results.
Previous research has indicated that mitochondrial respiration and cytosolic calcium transients play key roles in GSIS. Endoplasmic reticulum (ER) calcium release is an important contributor to cytosolic calcium transients and depends on the ER calcium recovery channel Serca and the ER release channels Ip3r and Ryr. The results of this study indicated that TMAO reduced ATP content and the ATP/ADP ratio by inhibiting oxidative phosphorylation and increasing glycolysis and interfered with glucose-stimulated calcium transients. RNA-seq revealed that Serca2 is the key protein involved in TMAO-mediated inhibition of GSIS in vitro. qPCR, digital PCR and western blotting confirmed that TMAO reduced Serca2 expression. In the islets of db/db mice, the Serca2 mRNA and protein levels were significantly reduced. TMAO decreased Serca activity and glucose-stimulated ER calcium release, which confirmed that TMAO affects Serca2 function. A Serca2 agonist reversed the TMAO-mediated inhibition of GSIS in vitro and in vivo. Based on the literature, it seems that TMAO has broad and different effects on specific target cells or tissues. Wang et al. suggested that within macrophages, TMAO contributes to the development of atherosclerosis in part by promoting cholesterol accumulation, perhaps by inducing scavenger receptors such as CD36 and SRA1, both of which are involved in the uptake of modified lipoproteins [3]. Zhu et al. showed that the direct exposure of platelets to TMAO enhanced submaximal stimulus-dependent platelet activation induced by multiple agonists through augmented Ca2+ release from intracellular stores [6]. Chen et al. reported that in hepatocytes, the ER stress kinase PERK is the receptor for TMAO and that TMAO selectively activates the PERK branch of the unfolded protein response, induces the transcription factor FoxO1, and leads to insulin resistance [22]. Wu et al. revealed that TMAO-induced allogenic graft-versus-host disease (GVHD) progression results from Th1 and Th17 differentiation, which is mediated by polarized M1 macrophages requiring NLRP3 inflammasome activation [23]. Wang et al. reported that TMAO induces pyroptosis in tumor cells by activating the endoplasmic reticulum stress kinase PERK and thus enhances CD8+ T cell-mediated antitumor immunity in triple-negative breast cancer (TNBC) in vivo [24]. The effect of TMAO on β cells does not occur through PERK since the pathological dose of TMAO did not activate the PERK pathway in β cells. Serca2 is the key mechanism by which TMAO inhibits GSIS. This is the first study to connect TMAO with Serca2.
In β cells, the proinflammatory cytokine IL-1β leads to a loss in Serca2 expression through peroxisome proliferator-activated receptor (PPAR)-γ activation [25]. In macrophages, TMAO activates the NLRP3 inflammasome and increases bioactive IL-1β through mitochondrial ROS and NF-κB [23]. RNA-seq revealed that the ROS pathway was enriched in the TMAO-treated MIN6 cells under high-glucose conditions. The results of this study indicated that TMAO increased mitochondrial ROS and activated the NLRP3 inflammasome to reduce Serca2 expression in β cells.
β-cell loss is another reason for decreased insulin secretion and mainly manifests as β cell dedifferentiation and apoptosis. By evaluating the expression of β cell dedifferentiation markers, β cell apoptosis markers and β cell transcription factors, long-term TMAO treatment was shown to promote β cell dedifferentiation and apoptosis and inhibit β cell transcription factor expression. It has been reported that ER stress drives β cell dedifferentiation and apoptosis [26,27]. Long-term TMAO treatment significantly increases ER stress-related protein levels in β cells and mouse islets. It has been reported that TMAO induces the osteoblast-like transdifferentiation of vascular smooth muscle cells [28] and the innate immune cell transdifferentiation of human aortic endothelial cells [29]. In this study, the choline diet decreased β cell mass and increased α cell mass. This suggests that TMAO may also lead to β cell transdifferentiation to α cells in islets.
In db/db model mice, the inhibition of plasma TMAO production through antisense oligonucleotides to knock down Fmo3 restored Serca2 expression and increased glucose tolerance and GSIS. Fmo3 knockdown in db/db mice also increased the β cell mass, inhibited β cell dedifferentiation and apoptosis, and restored β cell identity.
In insulin-resistant and diabetic db/db mice, TMAO knockdown by Fmo3 antisense oligonucleotides markedly improved β cell function and increased insulin sensitivity. These findings suggest that FMO3 is a promising target for treating diabetes. We are currently developing an FMO3 inhibitor screening method.
Some studies have shown that TMAO (100 mM or 2.78 mM in combination with a 0.25 μl/h infusion through subcutaneous osmotic minipumps) attenuates ER stress and could be beneficial [30,31], which contradicts our results. However, the dosages used in the aforementioned studies are far greater than those used in vivo. An extremely high dose of TMAO functions as a chemical chaperone and corrects protein folding defects indirectly by affecting hydrogen bonds within water molecules [32]. Protein stability is the result of a balance between the intramolecular interactions of protein functional groups and their interactions with the solvent environment. TMAO can enhance the strength of water-water hydrogen bonds, which eventually stabilizes the hydrogen bonds between amide groups in a protein and favors protein folding [33]. This may be one of the reasons for the beneficial effect of TMAO.
This study revealed that the gut microbiota metabolite TMAO plays an important role in the regulation of pancreatic β cell function both in vitro and in vivo. Mechanistically, short-term TMAO treatment inhibits glucose-stimulated calcium transients through NLRP3 inflammasome-related cytokines and Serca2 loss. Long-term TMAO treatment promotes β cell ER stress and dedifferentiation. The inhibition of TMAO/FMO3 might be a potential therapeutic strategy for β cell protection and antidiabetes treatment.
References
2. Howitt MR, Garrett WS. A complex microworld in the gut: gut microbiota and cardiovascular disease connectivity. Nat Med. 2012 Aug;18(8):1188-9.
3. Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011 Apr 7;472(7341):57-63.
4. Wang Z, Roberts AB, Buffa JA, Levison BS, Zhu W, Org E, et al. Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis. Cell. 2015 Dec 17;163(7):1585-95.
5. Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013 May;19(5):576-85.
6. Zhu W, Gregory JC, Org E, Buffa JA, Gupta N, Wang Z, et al. Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk. Cell. 2016 Mar 24;165(1):111-24.
7. Dambrova M, Latkovskis G, Kuka J, Strele I, Konrade I, Grinberga S, et al. Diabetes is Associated with Higher Trimethylamine N-oxide Plasma Levels. Exp Clin Endocrinol Diabetes. 2016 Apr;124(4):251-6.
8. Tang WH, Wang Z, Li XS, Fan Y, Li DS, Wu Y, et al. Increased Trimethylamine N-Oxide Portends High Mortality Risk Independent of Glycemic Control in Patients with Type 2 Diabetes Mellitus. Clin Chem. 2017 Jan;63(1):297-306.
9. Li SY, Chen S, Lu XT, Fang AP, Chen YM, Huang RZ, et al. Serum trimethylamine-N-oxide is associated with incident type 2 diabetes in middle-aged and older adults: a prospective cohort study. J Transl Med. 2022 Aug 18;20(1):374.
10. Huang Y, Wu Y, Zhang Y, Bai H, Peng R, Ruan W, et al. Dynamic Changes in Gut Microbiota-Derived Metabolite Trimethylamine-N-Oxide and Risk of Type 2 Diabetes Mellitus: Potential for Dietary Changes in Diabetes Prevention. Nutrients. 2024 May 30;16(11):1711.
11. Gao X, Liu X, Xu J, Xue C, Xue Y, Wang Y. Dietary trimethylamine N-oxide exacerbates impaired glucose tolerance in mice fed a high fat diet. J Biosci Bioeng. 2014 Oct;118(4):476-81.
12. Miao J, Ling AV, Manthena PV, Gearing ME, Graham MJ, Crooke RM, et al. Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis. Nat Commun. 2015 Apr 7;6:6498.
13. Schugar RC, Shih DM, Warrier M, Helsley RN, Burrows A, Ferguson D, et al.The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue. Cell Rep. 2017 Jun 20;19(12):2451-61.
14. Kong L, Zhao Q, Jiang X, Hu J, Jiang Q, Sheng L, et al. Trimethylamine N-oxide impairs β-cell function and glucose tolerance. Nat Commun. 2024 Mar 21;15(1):2526.
15. DeFronzo RA, Abdul-Ghani MA. Preservation of β-cell function: the key to diabetes prevention. J Clin Endocrinol Metab. 2011 Aug;96(8):2354-66.
16. Lever M, George PM, Slow S, Bellamy D, Young JM, Ho M, McEntyre CJ, Elmslie JL, Atkinson W, Molyneux SL, Troughton RW, Frampton CM, Richards AM, Chambers ST. Betaine and Trimethylamine-N-Oxide as Predictors of Cardiovascular Outcomes Show Different Patterns in Diabetes Mellitus: An Observational Study. PLoS One. 2014 Dec 10;9(12):e114969.
17. Morandi A, Zusi C, Corradi M, Olivieri F, Piona C, Fornari E, et al. Minor diplotypes of FMO3 might protect children and adolescents from obesity and insulin resistance. Int J Obes (Lond). 2018 Jun;42(6):1243-8.
18. Li P, Zhong C, Li S, Sun T, Huang H, Chen X, et al. Plasma concentration of trimethylamine-N-oxide and risk of gestational diabetes mellitus. Am J Clin Nutr. 2018 Sep 1;108(3):603-10.
19. Heianza Y, Sun D, Li X, DiDonato JA, Bray GA, Sacks FM, et al. Gut microbiota metabolites, amino acid metabolites and improvements in insulin sensitivity and glucose metabolism: the POUNDS Lost trial. Gut. 2019 Feb;68(2):263-70.
20. Zhuang R, Ge X, Han L, Yu P, Gong X, Meng Q, et al. Gut microbe-generated metabolite trimethylamine N-oxide and the risk of diabetes: A systematic review and dose-response meta-analysis. Obes Rev. 2019 Jun;20(6):883-94.
21. Kalagi NA, Thota RN, Stojanovski E, Alburikan KA, Garg ML. Association between Plasma Trimethylamine N-Oxide Levels and Type 2 Diabetes: A Case Control Study. Nutrients. 2022 May 17;14(10):2093.
22. Chen S, Henderson A, Petriello MC, Romano KA, Gearing M, Miao J, et al. Trimethylamine N-Oxide Binds and Activates PERK to Promote Metabolic Dysfunction. Cell Metab. 2019 Dec 3;30(6):1141-1151.e5.
23. Wu K, Yuan Y, Yu H, Dai X, Wang S, Sun Z, et al. The gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice. Blood. 2020 Jul 23;136(4):501-15.
24. Wang H, Rong X, Zhao G, Zhou Y, Xiao Y, Ma D, et al. The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer. Cell Metab. 2022 Apr 5;34(4):581-94.e8.
25. Kono T, Ahn G, Moss DR, Gann L, Zarain-Herzberg A, Nishiki Y, et al. PPAR-γ activation restores pancreatic islet SERCA2 levels and prevents β-cell dysfunction under conditions of hyperglycemic and cytokine stress. Mol Endocrinol. 2012 Feb;26(2):257-71.
26. Khin PP, Lee JH, Jun HS. A Brief Review of the Mechanisms of β-Cell Dedifferentiation in Type 2 Diabetes. Nutrients. 2021 May 10;13(5):1593.
27. Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 2011 Mar;13(3):184-90.
28. Filipska I, Winiarska A, Knysak M, Stompór T. Contribution of Gut Microbiota-Derived Uremic Toxins to the Cardiovascular System Mineralization. Toxins (Basel). 2021 Apr 10;13(4):274.
29. Saaoud F, Liu L, Xu K, Cueto R, Shao Y, Lu Y, et al. Aorta- and liver-generated TMAO enhances trained immunity for increased inflammation via ER stress/mitochondrial ROS/glycolysis pathways. JCI Insight. 2023 Jan 10;8(1):e158183.
30. Dumas ME, Rothwell AR, Hoyles L, Aranias T, Chilloux J, Calderari S, et al. Microbial-Host Co-metabolites Are Prodromal Markers Predicting Phenotypic Heterogeneity in Behavior, Obesity, and Impaired Glucose Tolerance. Cell Rep. 2017 Jul 5;20(1):136-48.
31. Achard CS, Laybutt DR. Lipid-induced endoplasmic reticulum stress in liver cells results in two distinct outcomes: adaptation with enhanced insulin signaling or insulin resistance. Endocrinology. 2012 May;153(5):2164-77.
32. Liao YT, Manson AC, DeLyser MR, Noid WG, Cremer PS. Trimethylamine N-oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine. Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):2479-84.
33. Zou Q, Bennion BJ, Daggett V, Murphy KP. The molecular mechanism of stabilization of proteins by TMAO and its ability to counteract the effects of urea. J Am Chem Soc. 2002 Feb 20;124(7):1192-202.