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

With a global prevalence of 10–12%, chronic kidney disease (CKD) is a major health burden associated with increased morbidity, mortality, and reduced quality of life^1,2. When patients with CKD have progressed to end-stage kidney disease they exhibit high cardiovascular (CV) mortality^3,4. CKD manifests with a progeric phenotype⁵ and the survival of incident dialysis patients is lower than that of patients with most solid-organ cancers^6,7. Some well-established mechanisms linking CKD with CV morbidity and mortality are inflammation⁸, oxidative stress⁹, cellular senescence¹⁰ and metabolic dysfunction¹¹.

Gaseous TMA is absorbed into the circulation and subsequently oxidized to TMAO in the liver by flavin monooxygenases¹⁴. While high choline and TMAO independently predicted the risk for CVD in patients undergoing elective cardiac evaluations, betaine insufficiency is associated with an adverse vascular risk profile in the metabolic syndrome^15,16. Moreover, low betaine was associated with components of the metabolic syndrome in healthy men and women¹⁷ and betaine supplementation reduced body fat¹⁸. Importantly, TMAO levels are increased in CKD, are associated with mortality risk, and induces renal fibrosis^19,20. While positive associations with CKD and CVD have been validated especially for TMAO^21–23, betaine’s and choline’s associations with CVD are mediated by TMAO²⁴, a finding recently validated in a meta-analysis²⁵.

Based on ingenious solutions developed in some animal species during evolution, we have suggested²⁶ that expanding biomedical investigation to new approaches based on a broad awareness of the diversity of animal life and comparative physiology can accelerate innovations in human health care. Natural comparative animal models, such as different nutritional, seasonal and long-time fasting (such as hibernation) patterns and other physiologic adaptions to unfavourable environmental conditions can be used for studying metabolic regulation of the microbiota^27,28. Here, we investigated circulating levels of the three metabolites betaine, choline and TMAO (1) in patients with CKD stage 3 and 5 and healthy controls; (2) in different animal species with various dietary habits; (3) during hibernation and active periods in free-ranging brown bears and captive garden dormice (Eliomys quercinus); (4) in free-ranging brown bears (Ursus arctos) and captive brown bears; and (5) cross-sectionally between CKD-prone felids compared to hibernating brown bears and human CKD patients.

In the present study, we applied a comparative biomimetic approach to study circulating levels of betaine, choline, and TMAO in healthy humans, CKD patients and different mammalian species, such as captive felids (susceptible to CKD) and free-ranging hibernating bears (protected against CKD). We report substantial differences in betaine, choline, and TMAO levels between species, dietary habits and hibernation status that provide useful insights when designing studies of susceptibility and protection of CKD, and other burden of lifestyle diseases, in relation to human lifestyle and diet (Fig. 2).

The main finding of the study was the exceptional, more than fourfold, increase in circulating betaine observed in free-ranging brown bears during hibernation compared to betaine levels both in the active summer period of free-ranging brown bears, healthy humans and other mammals (Figs. 2 and 3). Generation of betaine during hibernation may have multiple protective effects. During hibernation, bears are typically anuric and it has been reported that their renal blood flow decreases by 36%, while glomerular filtration rate (GFR) declines by 68% compared with the active state²⁹. Bears have developed an amazing capacity to protect their kidneys during hibernation, and in the spring renal function returns to normal within a couple of weeks^30,31. Better understanding of organ protective mechanisms during the vulnerable hibernation period may also provide insights into potential therapies for critically ill patients and organ injury during cold storage and reimplantation during transplantation³².

Betaine is an anti-inflammatory and anti-oxidant essential osmoprotective nutrient involved in liver function, cellular reproduction, and cell volume regulation³³. Since a high-quality genome assembly shows that anti-inflammatory pathways are activated in hibernating bears³⁴, the anti-inflammatory potential of betaine in CKD deserves further study. In free-eating healthy human adults there is a negative association between intake of betaine (and choline) and the inflammatory process³⁵. As methyl groups are important for numerous cellular functions, such as DNA methylation, phosphatidylcholine synthesis and protein synthesis, the methyl-donor properties of betaine have clinical implications. Moreover, since betaine enhances lipid metabolism and improves insulin resistance in laboratory mice (Mus musculus f. domesticus) fed a high-fat diet³⁶, high betaine levels may play a role in the healthy obese phenotype³⁷ observed in bears preparing for hibernation³⁸. As betaine has protective effects on isoprenaline-induced renal failure in rats (Rattus norvegica f. domesticus) via a decrease in tumour necrosis factor and nitric oxide synthase³⁹, high betaine levels may also protect kidneys during hibernation. Our observation of lower betaine levels in CKD (Fig. 2) and the finding that betaine inhibits cell proliferation and extracellular matrix deposition⁴⁰ supports a renal protective effect for betaine. During hibernation bears should be at high risk (physiologic kidney insufficiency + no intake of B-vitamin) of hyperhomocysteinemia; an established risk factor for CV, cerebrovascular and neurodegenerative disease⁴¹. However, as we observed no significant increase in homocysteine levels during hibernation (Table 1), high betaine may protect against hyperhomocysteinemia by re-methylation of homocysteine⁴². Accordingly, homocysteine does not elevate in bat (Myotis pilosus) brains during torpor due to increased expression of betaine-homocysteine S-methyltransferase⁴³. As betaine is an osmolyte that protects proteins, cells and enzymes from environmental stress, such as extreme temperature and lack of water in animals⁴⁴ and plants⁴⁵, betaine may have protective effects when organisms are under environmental stress.

It has been reported that betaine improves animal performance and increases lean body mass, meat quality, and reduces fat deposits in meat-producing cattle, pigs and poultry^18,46,47. Thus, high betaine during hibernation may protect the bears’ lean body mass and enhance lipolysis, important for energy supply. In humans, high dietary choline and betaine intake have been significantly associated with favourable body composition⁴⁸. The impact of the endogenous microbiota may be significant in this respect in man. While humans have a synthetic capacity for betaine, this is supplemented via nutritional acquisition of substrates and subsequent conversion to betaine by gut microbes. Seasonal changes in the gut microbiota in bears, with the ability to transfer seasonal metabolic characteristics to germ-free mice via fecal transplants have previously been reported⁴⁹. As choline and other methyl donors regulate methylation of genes related to memory and cognitive functions⁵⁰, high betaine and choline levels may also protect their brain during hibernation. In addition to serving as an osmolyte and metabolic intermediate, betaine influences pathways of inhibitory neurotransmitter production-recycling⁵¹.

The reason(s) why free-ranging bears can give birth and lactate (conditions associated with high metabolic demand) during hibernation⁵², are likely physiological and behavioural adaptations through which offspring survive harsh seasonal conditions, such as inclement weather or low food availability^53–55. The successful development of the fetus depends on optimal dietary intake of folate, choline and betaine involved in one-carbon transfer methylation and related epigenetic effects on gene expression⁵⁶. Moreover, maternal and postnatal dietary methyl nutrients have effects on the epigenetic machinery that in adulthood may lead to non-communicable diseases⁵⁷. As the supply of methyl groups is vital during pregnancy, we speculate that high levels of betaine protect the fetuses in pregnant bears during hibernation. Indeed, betaine plays an important role in physiologic pregnancy⁵⁸, fetal development^59,60 and DNA methylation⁶¹. Furthermore, in dairy cattle, betaine supplementation increase lactation performance⁶².

The mechanism(s) by which betaine and choline levels increase during the long period of fasting are unclear. However, in part, the increase may reflect an excessive intake of betaine-rich food in the autumn in preparation for hibernation. As betaine is found in microorganisms, plants, and animal muscle, it is a significant component of many foods. The daily intake of betaine in the human diet ranges from an average of 1 g/d to a high of 2.5 g/d. Nutrients rich in betaine include wheat bran (1339 mg/100 g), wheat germ (1241 mg/100 g), spinach (645 mg/100 g), shrimp (218 mg/100 g), and wheat bread (201 mg/100 g)⁶³. Although some bears in our study area do eat wheat during spring and early summer⁶⁴, betaine-rich foods are not part of the typical diet of free-ranging Scandinavian brown bears, which on an annual basis usually consists of 50% protein (ants, ungulate carcasses) that is mainly consumed in spring, and 50% carbohydrates (berries) that is mainly consumed in summer and fall⁶⁵. The betaine content in bilberries is rather low (0.2 mg/100 g)⁶³. However, since free-ranging bears during hyperphagia can eat a massive amount of different berries per day a significant amount of betaine will be ingested in the peak season. As increased intake of betaine- and choline-rich food in the autumn is unlikely to increase circulating betaine as much as fourfold, a metabolic switch that increase endogenous production of choline is likely to occur before and under hibernation. Since ≈30% of phosphatidylcholine can be produced by the sequential methylation of phosphatidyl-ethanolamine (via PE-methyltransferase), the body has the capacity to make choline ´de novo’⁶⁶. Similarly, Olthof et al.⁶⁷ have shown that supplementation of choline, as phosphatidylcholine, increases the pool of choline for the production of betaine. Thus, since phospholipids and phosphatidyl-ethanolamine could be used as an endogenous source for choline, further studies need to investigate these metabolic events in hibernating mammals⁶⁸.

Beside bears, only few mammals and birds are known to give birth and lactate during hibernation⁶⁹. To investigate if high levels of betaine during hibernation are specific for bears or a feature of temperature changes, we studied garden dormice—another hibernating species. In contrast to bears’ constant hibernation body temperature, dormice have to periodically rewarm from cold torpid states to warm interbout intervals⁷⁰. We find that whereas betaine and choline are not generated in dormice at 5 °C they are rapidly generated at 37 °C despite fasting (Fig. 4). This supports the hypothesis of a metabolic switch with endogenous betaine production as cell-protective substance during warming-up periods. The torpor-arousal switch is characterized by the accumulation of metabolites of nitrogen (glutamine) and phospholipid (betaine) catabolism in late torpor with the capacity to act as protective osmolytes⁷¹.

In contrast to free-ranging hibernating bears, which are protected against CKD and the CKD complications observed in the human uremic phenotype, such as osteoporosis, sarcopenia and arteriosclerosis³⁰, wild and domestic felids are susceptible to CKD⁷² and arteriosclerosis⁷³. The prevalence of CKD in domestic cats (Felis catus) is high (estimated to affect 1/3 of older cats) and has increased in recent decades⁷². The tubulointerstitial changes, including fibrosis, that are present in the early stages of feline CKD become more severe in advanced disease⁷². As transforming growth factor (TGF)-β1 is involved in epithelial cell de-differentiation, growth arrest and apoptosis in feline CKD, it has been suggested that feline CKD is a useful, naturally occurring model of human CKD⁷⁴. The prevalence of CKD in wild felids is not well studied. However, a recent thesis concluded that CKD is a prevalent disease affecting non-domestic felids throughout zoos in Australia⁷⁵. In accordance, a study of captive wild felids at German zoos, showed that renal lesions were detected in 33 out of 38 animals⁷⁶. A variety of factors—including ageing, ischemia, comorbidities and high phosphorus overload due to a carnivorous diet have been implicated as factors that promote felid CKD⁷².

Gut microbial metabolism of dietary choline, a nutrient abundant in the Western diet, produces TMA and the atherothrombosis- and fibrosis-promoting metabolite TMAO. High TMAO levels are mechanistically linked to atherosclerosis⁷⁷ and increase the risk for premature CVD⁷⁸. The high TMAO levels reported in CKD are due to both impaired renal function²⁰ and gut dysbiosis with higher percentages of opportunistic pathogens⁷⁹. As TMAO promotes interstitial nephritis⁸⁰, we measured TMAO in felids (Fig. 6). High intake of red meat (especially processed and red meat) has been associated with increased risk of CKD⁸¹ and premature ageing in the general population⁸². Chronic dietary exposures of red meat increase TMAO levels via modulation of the gut microbiota⁸³. We compared CKD 3 patients with felids and hibernating brown bears with comparable levels of S-creatinine (Table 2) and report major differences in betaine and TMAO levels (Fig. 6). Both fasting⁸⁴ and hibernation⁸⁵ alter the gut microbiota. As TMAO was not detectable in free-ranging active bears (except one) this suggests that free-ranging brown bears do not synthesize TMA due a “healthy” gut microbiota. It is noticeable that of the eight species of bacteria from two main phyla (Firmicutes and Proteobacteria) that use choline for production of TMA⁸⁶ none was reported in a study of bear microbiota composition in relation to seasonal changes in energy metabolism⁴⁹. TMAO promotes vascular calcification⁸⁷ and the absence of TMAO in free-ranging bears may contribute to their low risk of arteriosclerosis despite massive hypercholesterolemia⁶⁸.

In contrast to free-ranging bears, TMAO was measurable in all captive bears, likely related to different food habits associated with altered gut microbiota. As captivity and domestication have dramatically reshaped the gastrointestinal microbiota in numerous species, e.g. in wild and domestic equids⁸⁸, free-ranging and captive Andean bears (Tremarctos ornatus)⁸⁹, and primates⁹⁰, loss of diversity (and maybe infection of human microbiota from animal keepers) most likely occurs in captive brown bears, which contributes to the generation of TMAO. Higher Firmicutes to Bacteroidetes enrichment leads to a greater response to dietary TMAO precursor intake⁹¹. Thus, differences in microbial composition between humans and the other species likely contribute to the observed difference. As fermented food lowers TMAO levels in healthy adults⁹², a difference in the amount of ingested fermented food could also contribute to observed differences. To the best of our knowledge, circulating TMAO levels have not previously been measured in a comparative human–animal study. However, high levels of TMAO in muscle tissue have been reported in marine mammals⁹³. As muscle TMAO contents increase linearly with depth in six fish families⁹⁴, it has been suggested that TMAO counteracts protein-destabilizing forces in deep-sea water. We report that circulating TMAO levels are higher in omnivorous healthy humans compared to other species (Fig. 2C). As humans are the only species in this study that eats fish on a regular basis, this could contribute to higher TMAO levels. Plasma TMAO concentrations were directly associated with dairy consumption and low-grade inflammation⁹⁵ and in vegans/vegetarians the transformation into TMA via low-abundance microbiota occurs at a markedly lower extent compared to omnivores⁹⁶.

The results of this descriptive study should be interpreted taking a number of caveats into account. Firstly, the number of healthy controls, CKD patients and animal species are limited. However, even with this rather limited number we have observed major differences between the species. Our sample numbers were, however, too small to enable sex-specific statistics. Secondly, the free-ranging and captive bears were not matched for weight and age. Thirdly, as we do not have information on dietary habits in healthy controls, CKD patients and free ranging bears we cannot relate our findings to dietary exposures. Our study would also have benefited by faecal or circulatory microbiota cultures in the different species. Creatinine, a by-product of muscle catabolism excreted by the kidneys, is considered a reliable index for kidney function. However, as S-creatinine is also affected by muscle mass and meat intake it is possible that higher muscle mass and the carnivorous diet contributes to high S-creatinine levels in tigers and lions.

In conclusion, this comparative biomimetic study suggests that a metabolic switch leading to endogenously produced betaine protects bears and garden dormice during the vulnerable hibernation period. Our data also show that carnivorous eating habits in the animal kingdom are linked to higher TMAO levels. Furthermore, we show a strong association between body temperature and the endogenous production of betaine and the depletion of TMAO. The characterization and manipulation of a metabolic switch that divert choline to generation of betaine instead of TMAO would likely have a major impact in prevention of not only CKD but also other burden of lifestyle diseases.