Insights in the regulation of trimetylamine N-oxide production using a comparative biomimetic approach suggest a metabolic switch in hibernating bears

Chronic kidney disease (CKD), affecting an estimated 10–12% of the global population, is associated with high morbidity, mortality, and cardiovascular (CV) risk. Mechanisms linking CKD with adverse outcomes include inflammation, oxidative stress, cellular senescence, and metabolic dysfunction. The gut microbiota has emerged as a contributor to CKD and CVD via metabolism of dietary nutrients into bioactive metabolites. A central pathway involves microbial conversion of choline and L-carnitine into trimethylamine (TMA), followed by hepatic oxidation to trimethylamine N-oxide (TMAO), which has been linked to atherosclerosis, thrombosis, vascular calcification, and renal fibrosis. Conversely, choline can be metabolized to betaine, an osmoprotective methyl donor with putative beneficial effects. While high choline and TMAO predict CVD risk, low betaine associates with adverse metabolic profiles. Given the remarkable physiological adaptations of hibernating mammals, the study aimed to leverage a comparative biomimetic approach to evaluate circulating betaine, choline, and TMAO across humans with CKD, multiple mammalian species with differing diets, and hibernation states, to identify regulatory patterns (metabolic switches) that might confer protection from CKD and cardiometabolic disease.

Prior work has shown: (1) the gut microbiota and its dysbiosis in CKD promote increased TMA/TMAO generation; (2) elevated TMAO associates with incident CVD, mortality, and induces renal fibrosis; (3) choline and betaine intakes correlate with inflammatory markers and body composition, with betaine insufficiency linked to adverse metabolic profiles; (4) hibernators demonstrate profound physiological adaptations including reduced renal function during torpor without permanent damage; and (5) captivity and diet can reshape gut microbiota composition across species, influencing TMAO precursor metabolism. These findings underpin the hypothesis that species- and state-specific metabolic routing of choline toward betaine rather than TMAO could be protective.

Design: Comparative observational study assessing circulating betaine, choline, and TMAO among human cohorts and multiple mammalian species, including longitudinal sampling across hibernation and active periods. Human cohorts: Fasting plasma from CKD stage 3 (eGFR 30–60 ml/min; n=30) and CKD stage 5 (eGFR<15 ml/min; n=121) patients from two prospective cohorts; 80 age- and sex-matched population controls from Stockholm, Sweden. Ethical approvals obtained; informed consent collected. Animal cohorts: (a) Free-ranging brown bears (Ursus arctos): n=15 subadults sampled during hibernation (Feb–Mar) and again in summer (June), Sweden. (b) Captive brown bears: n=34 from European zoos, standard mixed diet; single fasting sample per animal. (c) Garden dormice (Eliomys quercinus): active n=23; hibernation torpor early n=14, late n=9; interbout interval early n=11, intermediate n=8; animals kept under controlled temperature/photoperiod. (d) Captive felids: lions n=11, tigers n=11; standard red meat-based carnivore diet; fasting samples during health checks. (e) Wild boars (Sus scrofa): n=10 adult females maintained under naturalistic conditions; fasting samples. Sampling/processing: Blood collected (jugular vein in large mammals; cardiac post-mortem in dormice), processed and stored at −70/−80 °C as appropriate. Assays: Quantification of betaine, choline, and TMAO via LC–MS/MS using deuterated internal standards (TMAO-D9, betaine-D9, choline-D9). Chromatography on Acquity UPLC Amide column; detection on Agilent 6490 Triple Quadrupole. Additional biochemical parameters measured using standard clinical analyzers; insulin by ELISA; sulfur amino acids by HPLC. Statistics: Non-parametric analyses. Group differences by Kruskal–Wallis with Bonferroni post hoc tests; paired hibernation vs summer in free-ranging bears by Wilcoxon signed-rank; free-ranging vs captive bears by Mann–Whitney U. Significance threshold p<0.05.

- Humans: Median betaine 8.3 [7.8] ng/µl; decreased with worsening renal function (p<0.001). CKD5 had the lowest betaine vs healthy (p<0.001) and vs CKD3 (p=0.003). Median choline 7.2 [2.7] ng/µl; CKD5 showed highest choline vs healthy (p=0.07) and vs CKD3 (p=0.002). Median TMAO 2.3 [5.2] ng/µl; CKD5 had ~13-fold higher TMAO vs healthy (p<0.001) and ~7-fold vs CKD3 (p<0.001). - Across species: Significant interspecies differences for all three metabolites (overall p<0.001). Active dormice had highest betaine; active free-ranging bears had lowest choline, while active dormice had highest choline. TMAO was detectable in only 1/15 free-ranging bears but measurable in all captive bears. - Bears, hibernation vs summer (free-ranging): During hibernation, betaine increased by 422% (47.7 vs 11.3 ng/µl; p<0.001); choline increased by 18% (4.0 vs 3.4 ng/µl; p=0.009); TMAO was below detection in all hibernating bears. Physiologic changes included lower body weight, liver enzymes, urea, and higher albumin, total protein, triglycerides, creatinine, and insulin in winter (all p<0.05). - Garden dormice: During hibernation (torpor/interbout), betaine (4.7 vs 88.9 ng/µl; p<0.05) and choline (3.6 vs 21.4 ng/µl; p<0.001) were significantly lower than in active animals, paralleling body temperature changes. TMAO was low overall; active, late torpor, and early interbout had higher TMAO than early torpor and intermediate interbout (all p<0.05). - Free-ranging vs captive brown bears (active state): Free-ranging bears had higher betaine (11.3 vs 6.4 ng/µl; p<0.001) and lower choline (3.4 vs 10.2 ng/µl; p<0.001). TMAO detected in 1/15 free-ranging vs all captive bears (median ~0.04 ng/µl; p<0.001). - CKD-like comparison (CKD3 humans vs felids vs hibernating bears): Hibernating bears had ~4-fold higher betaine than CKD3 patients and felids (p<0.001), significantly lower choline (p<0.001), and undetectable TMAO, whereas felids and CKD3 patients had significantly higher TMAO (p<0.001).

Findings support a state- and species-specific metabolic routing of choline metabolism. In free-ranging bears, hibernation is associated with a robust rise in circulating betaine and depletion of TMAO to undetectable levels, consistent with a metabolic switch favoring endogenous betaine generation and reduced microbial TMA production. This switch may confer renal and cardiometabolic protection during prolonged anuria and reduced GFR, aligning with betaine’s roles as an osmoprotectant, methyl donor, anti-inflammatory and metabolic modulator. By contrast, carnivorous felids and humans with CKD exhibit elevated TMAO, likely reflecting gut microbiota composition and diet favoring TMA production, which may contribute to CKD susceptibility and vascular pathology. Captivity appears to shift brown bear microbiota and metabolite profiles toward higher choline and detectable TMAO, implicating environment and diet in modulating these pathways. In garden dormice, metabolite patterns track body temperature state, suggesting thermally linked regulation of endogenous betaine production and TMAO depletion during hibernation cycles. Collectively, the data highlight the potential of manipulating choline metabolic routing and gut microbial TMA production as therapeutic targets for CKD and cardiometabolic disease.

A comparative biomimetic analysis reveals a hibernation-associated metabolic switch in free-ranging brown bears that diverts choline metabolism toward betaine synthesis and away from TMA/TMAO production. This is associated with markedly elevated betaine, undetectable TMAO, and may underlie renal and cardiometabolic protection during hibernation. Carnivorous diets correlate with higher TMAO levels across species, and captivity is associated with detectable TMAO in bears, suggesting environmental and dietary impacts on gut microbiota and metabolite pathways. Understanding and leveraging this metabolic switch—enhancing betaine generation and suppressing TMAO—could inform novel strategies to prevent or mitigate CKD and related lifestyle diseases. Future research should characterize mechanistic drivers (microbiota composition, hepatic and phospholipid metabolism, thermoregulation), evaluate causality, and test interventions that mimic the hibernation-associated metabolic routing in humans.

- Sample sizes of human subgroups and animal species were modest, limiting power for some comparisons and precluding sex-specific analyses. - Free-ranging and captive bears were not matched for age and weight. - Detailed dietary information for healthy controls, CKD patients, and free-ranging bears was unavailable, limiting dietary adjustment. - No direct microbiome profiling (stool or circulating) was performed to link microbial taxa to metabolite levels. - Serum creatinine, influenced by muscle mass and meat intake, may confound cross-species comparisons of kidney function (notably in felids).