Elevated trimethylamine-N-oxide is an established cardiovascular and metabolic risk
factor
To date, at least five prospective cohort studies have concluded that increased plasma
levels of trimethylamine N-oxide (TMAO) predict increased risk for major adverse cardiovascular
(CV) events in patients with pre-existing coronary heart disease.1–5 Moreover, though
some epidemiology does not support a connection between plasma TMAO and CV risk,6
7 a recent meta-analysis of 11 prospective cohort studies concludes that higher plasma
TMAO correlates with a 23% increase in risk for CV events (HR 1.23, 95% CI 1.07 to
1.42), as well as a 55% increase in all-cause mortality.8 The possibility that TMAO
may be a mediating factor in this regard is raised by rodent studies in which plasma
levels have been raised either by direct oral administration of TMAO, or by administration
of very high doses (proportionately very much higher than would be employed in human
supplementation) of its precursors phosphatidylcholine and carnitine; in these studies,
in which the achieved plasma level of TMAO was at least an order of magnitude higher
than commonly observed in humans, a proatherogenic effect was documented.9–14 In vitro
studies, likewise employing supraphysiological concentrations of TMAO, have demonstrated
effects suggesting proatherogenic potential.12 13 15–17
In case–control epidemiology, elevated TMAO has also been linked to substantially
increased risk for type 2 diabetes and metabolic syndrome.18–20 Indeed, the correlations
between TMAO and diabetes risk appear to be stronger than those for CV risk.
Nutritional intakes of TMAO and its precursors do not correlate with CV risk
Yet the notion that TMAO acts as a human vascular toxin at the plasma concentrations
seen in people with reasonably normal renal function is difficult to square with other
recent findings. Preformed TMAO is notably high in fish, in which it serves to maintain
osmotic balance; levels tend to be higher in deep-sea fish, which must survive at
higher pressures.21–24 This TMAO can be directly absorbed after fish consumption.25
However, at least in those who do not ingest a very large amount of fish, a high proportion
of their plasma TMAO arises from bacterial metabolism of dietary choline (usually
ingested as phosphatidylcholine) and carnitine; trimethyllysine also makes a minor
contribution in this regard.9 10 26 Certain gut bacteria can metabolise these compounds
to trimethylamine (TMA) via TMA lyase activity; inhibition of this lyase activity
prevents induction of atherosclerosis in mice fed high-dose choline.11 27 28 This
TMA can then be absorbed; its subsequent oxidation by hepatic flavin-containing monooxygenases
(FMOs) converts it to TMAO.29 30 Unless choline has cardioprotective properties, we
are currently unaware of, a diet relatively rich in choline would be expected to increase
CV risk if physiological levels of plasma TMAO can indeed provoke CV disease or CV
events. Yet a recent meta-analysis of prospective epidemiological studies concluded
that dietary choline intake has no significant impact on risk for incident CV disease
or CV mortality; with respect to CV mortality, only two pertinent studies were available,
so the conclusion in this respect might not be definitive.31 Likewise, a recent meta-analysis
failed to associate consumption of eggs–a rich source of phosphatidylcholine–with
increased CV risk.32
With respect to carnitine and CV risk, a meta-analysis of prospective clinical trials
in patients who had recently experienced a myocardial infarction (MI) concluded that
carnitine supplementation is markedly protective with respect to total mortality,
ventricular arrhythmias and new-onset angina; trends for lower incidence of reinfarction
or heart failure did not achieve statistical significance, possibly owing to the modest
size of the studies included.33 Clinical trials have also reported favourable effects
of supplemental carnitine or carnitine esters on angina, intermittent claudication
and heart failure.34–39 Moreover, rodent atherogenesis studies, in which carnitine
has been administered in doses reasonably proportional to the supplementation doses
used clinically, have found that carnitine is antiatherogenic, despite its propensity
to raise TMAO.22 40–42 With respect to fish, the primary dietary source of preformed
TMAO, a meta-analysis found that fish consumption correlates dose dependently with
CV protection, likely because of the long-chain omega-3 content of fish.43 While it
might be argued that the benefits of omega-3 ingestion are masking a genuine adverse
impact of TMAO on CV risk, the impact of moderate supplemental intakes of fish omega-3
on this risk seems to be rather modest in the context of current drug therapy, primarily
influencing risk for sudden death arrhythmias.44–46 Hence, these benefits would seem
unlikely to overwhelm the adverse effects of TMAO if these were of important magnitude.
In aggregate, these findings are difficult to square with the notion that TMAO is
a mediating CV risk factor, at least in commonly occurring levels, since increased
ingestion of choline, carnitine or fish would be expected to increase TMAO levels,
but is not associated with increased CV risk.
It is, therefore, reasonable to suspect that moderately elevated TMAO, rather than
being a mediator of the associated CV risk, is a marker for factors which both promote
CV events and increase plasma TMAO.47 48 Indeed, after a plethora of multicentre supplementation
trials, we have learnt something precisely comparable about moderately elevated homocysteine
and coronary risk.49 50 Whereas the highly elevated homocysteine levels seen in genetic
hyperhomocysteinaemia are evidently directly pathogenic to the vascular system, and
homocysteine at comparably high levels exerts proinflammatory effects on vascular
cells in vitro, we were never presented with evidence that the moderately elevated
levels of homocysteine associated with increased CV risk–roughly an order of magnitude
lower–exerted important effects in vitro. Currently, TMAO appears to be in an analogous
position.22
Diminished renal function can markedly elevate TMAO
Evidently, an increase in plasma TMAO can reflect an increase in TMAO synthesis or
a reduction in its renal clearance. A promising lead is offered by the observation
that plasma TMAO levels are highly dependent on renal function. A study examining
plasma TMAO levels in patients with varying degrees of renal compromise and in healthy
controls found that TMAO averaged 5.8 µM/L in the controls (with average measured
glomerular filtration rate [mGFR]–83 mL/min), 14.6 µM/L in patients with stages 3–4
kidney disease (mGFR 28 mL/min) and 75.5 µM/L (mGFR 7 mL/min) in stage 5 patients.51
Hence, as mGFR falls, plasma TMAO tends to rise almost proportionally.
Although it is well known that severe kidney disease is associated with a considerable
increase in CV risk, a meta-analysis of general population cohort studies has found
that even a mild reduction in estimated GFR (eGFR) is a risk factor for CV mortality.52
Thus, whereas risk for CV mortality was found to be relatively flat for eGFRs in the
range of 75–120 mL/min, a significantly higher risk was seen at eGFR 60 mL/min, and
this mortality rose progressively as eGFR fell. Hence, even relatively modest reductions
of eGFR sometimes considered to be within the ‘normal’ range of kidney function (eGFR
of 60 mL/min or greater) are associated with increased CV risk. This increased risk
could presumably reflect an impact of suboptimal kidney function per se (leading to
increased levels of phosphate or other uraemic toxins), as well as of vasculotoxic
factors inducing reduction of kidney function. These may not have been adequately
corrected for in epidemiological analyses focusing on TMAO.
Nonetheless, there is good reason to believe that, whereas uncorrected correlations
of TMAO with CV risk are explained in part by the CV risk associated with diminished
renal function, this is not the sole explanation for the utility of TMAO as a risk
factor. That is because the five cohort studies cited above-included analyses which
adjusted for eGFR as a covariate; while this correction markedly decreased the calculated
CV risk associated increased TMAO, it by no means eliminated it.1–5 We can, therefore,
conclude that factors, which boost TMAO synthesis, are mediators of some of the risk
associated with elevated TMAO.
Are bad bacteria the culprit?
Two steps are involved in the synthesis of TMAO: generation of TMA from certain dietary
precursors–most notably, choline and carnitine–by the TMA lyase activity of gastrointestinal
(GI) bacteria; and oxidation of circulating TMA to TMAO by hepatic FMOs, by far the
most active of which in this regard is FMO3.29 With respect to GI bacteria, rodent
studies have led to increasing awareness of the fact that microbiota can notably modulate
metabolic health.53–56 Is it possible that certain commonly occurring GI bacteria
are quite proficient at generating TMA, while simultaneously increasing CV risk by
certain mechanisms–for example, by suppressing incretin synthesis or maximising bile
acid reabsorption (which might elevate LDL cholesterol)? Or could some dietary factor–soluble
fibre, perhaps–suppress the capacity of GI bacteria to generate TMA, while simultaneously
protecting CV health?
While this is an intriguing hypothesis that merits further follow-up, research studies
to date provide little support for it. Controlled clinical studies of supplementation
with probiotic micro-organisms linked to improved intestinal health–Lactobacillus
casei Shirota and another preparation providing a Lactobacillus, Bifidobacterium and
Streptococcus thermophilus–have so far failed to demonstrate reductions in plasma
TMAO.57 58 Faecal microbiota transplantation from vegan donors to recipients with
metabolic syndrome, while it did succeed in altering the latter’s GI flora, did not
lower their plasma TMAO levels.59 Administration of the cholesterol-lowering prebiotic
glucaro-1,4-lactone to rats fed a high-fat diet, which markedly boosted intestinal
levels of Lactobacillus, Bifidobacteria and Enterococcus, while suppressing Escherichia
coli, was associated with an increase of TMAO in urine.60 Supplementation of mouse
diets with either galacto-oligosaccharides/inulin or polydextrose and insoluble bran
fibre increased serum TMAO levels, whereas supplementation with both simultaneously
failed to influence TMAO.61
In one mouse study, supplementation with soluble fibre from wheat bran did lower colonic
TMA lyase activity as well as serum cholesterol.62 However, it seems unlikely that
an increased intake of protective soluble fibre explains the association of TMAO with
vascular risk, since very ample intakes of soluble fibre are required to achieve a
modest reduction in Low-density lipoprotein (LDL) cholesterol–intakes which very few
people ingest; and in any case the associated risk persists after adjustment for lipid
risk factors such as LDL cholesterol.63
While it is feasible to produce mice whose intestines have been colonised with bacteria
with limited capacity to generate TMA, there so far is no evidence that this confers
any special vascular protection on these mice when they eat normal diets.27
Elevated hepatic FMO3 activity can reflect hepatic insulin resistance
Which brings us to the alternative thesis: that modulation of hepatic FMO3 activity
by certain factors that can influence CV health, can rationalise the epidemiology
of TMAO. The regulation of hepatic FMO3 requires much further research, but several
intriguing findings have emerged. Insulin suppresses FMO3 expression at both the messenger
RNA (mRNA) and protein level; conversely, glucagon elevates FMO3 expression.64 Also,
the FXR receptor, for which many bile acids serve as activating ligands, stimulates
transcription of the FMO3 gene.29 65 With respect to the impact of insulin, genetically
modified mice in which hepatic expression of the insulin receptor has been selectively
ablated (Liver-specific insulin receptor knockout mice) have greatly enhanced hepatic
expression of FMO3.64 These mice develop marked hypercholesterolaemia and are exceptionally
prone to atherosclerosis when fed a proatherogenic diet, and also understandably have
an elevated hepatic glucose output.66 The pertinence of these findings to humans has
been clarified by a study in which liver biopsies were obtained both from obese subjects
and lean controls; mRNA expression of FMO3 was about twice as high in the obese subjects,
likely reflecting hepatic insulin resistance in the context of hyperinsulinaemia.64
Recent studies suggest that the hepatic insulin resistance associated with obesity
and metabolic syndrome is mediated by increased hepatic influx of free fatty acids
(FFAs), giving rise to increased levels of diacylglycerol; the latter promotes activation
of protein kinase C-epsilon, which in term hampers the tyrosine kinase activity of
the insulin receptor by phosphorylating threonine-1160 of the beta-chain.67–69 Other
kinase or phosphatase activities stimulated by lipid overload may also impair insulin
signalling at points downstream from the insulin receptor.70 71 Excess FFA influx
also drives increased triglyceride synthesis, giving rise to the hepatic steatosis
often associated with hepatic insulin resistance. However, increased hepatic triglyceride
levels per se may not promote hepatic insulin resistance; such resistance correlates
with hepatocyte levels of diacylglycerol, rather than of triglycerides.69 72
Hepatic insulin resistance and its common concomitant hepatic steatosis are associated
with increased CV risk, as well as elevated risk for type 2 diabetes—risks likewise
associated with elevated TMAO.66 73–77 It is, therefore, straightforward to postulate
that TMAO can serve as a marker for hepatic insulin resistance, and that this explains
at least a portion of the risk for CV events and diabetes linked to TMAO. Although
studies establishing TMAO as an independent CV risk factor have often corrected for
certain correlates of obesity, such as body mass index or diabetes, it is unlikely
that such corrections fully capture the impact of hepatic insulin resistance.
Correcting hepatic insulin resistance
This analysis suggests that healthful measures which tend to correct hepatic insulin
resistance may favourably impact the vascular and metabolic health of subjects with
high TMAO. Evidently, sustained remediation of the visceral obesity which often underlies
hepatic insulin resistance should be helpful in this regard; nonetheless, it is easier
to recommend this than to achieve it! By improving the insulin sensitivity of hypertrophied
adipocytes, thiazolidinediones such as pioglitazone tend to improve hepatic insulin
resistance in people with diabetes by quelling excessive fatty acid efflux from adipocytes,
even though they tend to increase body fat mass somewhat.78–81
Hormones and medications which boost hepatic AMPK activity tend to improve impaired
hepatic insulin sensitivity. AMPK achieves this, at least in part, by downregulating
mTORC1 activity, which acts indirectly to promote phosphorylations of insulin receptor
substrate-1 that impede transmission of the insulin signal.82 Also, by promoting oxidative
disposal of FFAs while suppressing lipogenesis, AMPK could be expected to lessen hepatic
diacylglycerol synthesis, thereby getting to the root of hepatic insulin resistance.83
84 The favourable impact of metformin on hepatic insulin resistance in diabetes is
thought to be mediated by activation of AMPK.85–88 The phytochemical nutraceutical
berberine, widely used in China for the management of type 2 diabetes, is likewise
thought to improve glycaemic control via activation of AMPK, and has been shown to
counter hepatic insulin resistance in diabetic hamsters.89–93
Both adiponectin and glucagon-like peptide-1 (GLP-1) act on the liver to stimulate
AMPK activity; moreover, they have been shown to combat hepatic insulin resistance,
and work in various ways to promote vascular and metabolic health.94–106 Hence, elevated
TMAO may often be a marker for suboptimal adiponectin and/or GLP-1 activity. The antidiabetic
drug pioglitazone tends to boost the diminished adiponectin secretion of hypertrophied
adipocytes.107 108 It seems likely that plant-based diets of rather low-protein content
can increase adiponectin production, as these boost the liver’s production of fibroblast
growth factor-21, one of whose major functions is to promote adiponectin secretion
by adipocytes.109 110 Such diets are also useful for preventing or correcting the
obesity that often underlies hepatic insulin resistance.111–113
With respect to GLP-1, acarbose, dietary lente carbohydrate, bile acid sequestrants
and certain prebiotics can boost GLP-1 production, drugs inhibiting plasma dipeptidyl
peptidase-4 can prolong its half-life, and injectable GLP-1 receptor agonists can
mimic its bioactivity.114–117
PPARalpha agonists, such as fenofibrate, also promote hepatic fatty acid oxidation,
owing to induction of a range of mitochondrial enzymes (including carnitine palmitoyl
transferases-1a and -2, fatty acyl-CoA dehydrogenase, UCP-2) which catalyse such oxidation.118
119 Moreover, PPARalpha agonism also acts indirectly to stimulate AMPK in the liver
and other tissues by boosting adiponectin production in adipose tissue; PPARalpha
enhances hepatic synthesis and release of fibroblast growth factor-21, which in turn
stimulates adiponectin synthesis in adipocytes.120–123 Not surprisingly, fenofibrate
has been shown to decrease hepatic levels of diacylglycerol and alleviate hepatic
insulin resistance in rodents fed diets high in fat and/or fructose.124–128 Moreover,
fenofibrate therapy has been shown to reduce risk for CV events in patients with metabolic
syndrome.118
There is recent evidence that the carotenoid antioxidant astaxanthin can also serve
as a PPARalpha agonist, and, both in rodents and humans, alleviate the dyslipidaemia
associated with metabolic syndrome.129–135 In obese mice, astaxanthin has been reported
to improve hepatic insulin resistance.136 Krill oil provides esterified forms of astaxanthin
which have superior bioavailability, as well as health-protective omega-3 fatty acids,
oxidised forms of which likewise serve as PPARalpha agonists.137–140 Moreover, krill
oil supplementation has been found to beneficially modulate serum lipid profile–including,
intriguingly, a reduction in LDL cholesterol–in controlled clinical trials.141 Krill
oil, even when compared with fish oil, suppresses hepatic steatosis in rodents.142–144
This may be due to its astaxanthin content, which is not found in fish oil. Moreover,
krill oil, but not fish oil, reduces diacylglycerol and ceramide content in the liver.145
The phospholipid fraction of krill oil has also been noted to reduce hepatic glucose
production, unlike fish oil.146 Thus, krill oil, being a source of highly bioavailable
form of astaxanthin, appears to have additional advantages for reducing hepatic steatosis
and hepatic insulin resistance compared with fish oil.
In brief, if this analysis is accurate, various measures which alleviate hepatic insulin
resistance–correction of visceral obesity, activation of 5' adenosine monophosphate-activated
protein kinase (AMPK) with metformin or berberine, activation of PPARalpha with fenofibrate
or astaxanthin, amplification of adiponectin production with pioglitazone or plant-based
diets, and clinical strategies which boost the production or bioactivity of GLP-1,
could be expected to decrease elevated TMAO while also decreasing the risk for vascular
events and diabetes associated with this risk factor. Figure 1 summarises these relationships.
Figure 1
Measures which increase adiponectin, increase GLP-1 activity, control metabolic syndrome
and activate hepatic AMPK or PPARalpha may decrease elevated TMAO and associated vascular/metabolic
risk. GLP-1, glucagon-like peptide-1; TMAO, trimethylamine-N-oxid.
FMO3 might also mediate risk associated with elevated TMAO
One intriguing observation to emerge from TMAO research is that elevated hepatic expression
of FMO3 boosts hepatic lipogenesis and gluconeogenesis, independent of its impact
on TMAO levels; this might reflect FMO3’s ability to somehow support expression of
FoxO1.30 64 This raises the interesting prospect that drugs selectively targeting
FMO3 might have some utility in diabetes and hyperlipidaemia, particularly when elevated
TMAO levels suggest that hepatic FMO3 expression is high. However, since FMO3 plays
a systemic role in catecholamine metabolism, suppressing its function might not prove
to be innocuous; genetic absence of FMO3 activity has been associated with hypertension.147
In any case, when hepatic insulin resistance is present, correcting this should lessen
hepatic FMO3 expression.
Overview
Accumulating evidence points to elevated plasma TMAO as a risk factor for both atherosclerosis,
CV events and type 2 diabetes, and rodent studies have found that extremely high dietary
intakes of TMAO per se or its dietary precursors choline and carnitine are proatherogenic.
Moreover, supraphysiological concentrations of TMAO exert proinflammatory effects
in cell culture studies. These findings have led some observers to recommend that
dietary or supplementary consumption of choline and carnitine should be minimised–although
these analysts have rarely recommended abstinence from fish, the richest dietary source
of preformed TMAO. In fact, a meta-analysis of pertinent nutritional epidemiology
has failed to observe an impact of dietary choline on CV risk. Supplemental use of
carnitine has been found to reduce mortality and diminish risk for arrhythmias and
new-onset angina in patients who have suffered a previous MI, has shown clinical utility
in angina, intermittent claudication and heart failure, and exerts antiatherogenic
effects in rodents when fed at moderate levels comparable to human supplemental intake.
And, fish consumption correlates dose dependently with favourable vascular outcomes.
These findings point ineluctably to the conclusion that TMAO is not a mediating risk
factor, at least in the concentrations seen in people whose renal function is not
severely defective.
Hence, moderately elevated TMAO must be viewed as a marker for other factors that
both raise TMAO and confer increased risk for vascular disease and diabetes. Plasma
levels of TMAO are highly reflective of renal function, and hence a portion of the
risk associated with elevated TMAO is mediated either by impaired renal function,
or renotoxic factors that are also vasculotoxic or promote diabetes. Nonetheless,
TMAO remains predictive of vascular risk after statistical correction for eGFR; factors
influencing TMAO synthesis evidently mediate some of this risk. While it is theoretically
possible that certain strains of GI bacteria possessing high TMA lyase activity exert
adverse effects on vascular and metabolic health, this remains to be demonstrated,
and efforts to lower plasma TMAO with probiotics thought to be health protective have
so far failed.
Factors which upregulate hepatic expression and activity of FMO3, chiefly responsible
for conversion of TMA to TMAO, therefore, fall under suspicion. In this regard, it
is notable that subnormal hepatic insulin activity reflecting hepatic insulin resistance
has been found to boost hepatic FMO3 expression. Hepatic insulin resistance is typically
induced by the excessive FFA influx associated with metabolic syndrome and visceral
obesity, well-known risk factors for vascular disease and diabetes. This excessive
FFA influx also gives rise to hepatic steatosis; although excessive accumulation of
triglycerides in the liver does not appear to mediate hepatic insulin resistance,
it serves as a marker for the increased FFA influx that does. Subnormal activities
of either adiponectin or GLP-1–both of which exert favourable vascular and metabolic
effects–can also promote hepatic insulin resistance. It is, therefore, reasonable
to speculate that lifestyle measures which reverse visceral obesity, or nutraceutical/drug/dietary
measures which boost the production or bioactivity of adiponectin and/or GLP-1, will
alleviate the risk associated with elevated TMAO by ameliorating hepatic insulin resistance.
Activation of AMPK with metformin or berberine, or of PPARalpha with fenofibrate or
astaxanthin, could also be expected to have a favourable impact in this regard, in
part by accelerating the oxidative disposal of excessive hepatic FFAs. Finally, elevated
FMO3 activity per se may mediate some of the risk associated with high TMAO via upregulation
of hepatic lipogenesis and gluconeogenesis.
Importantly, this analysis does not exclude the possibility that TMAO might be directly
pathogenic at the very elevated levels typically seen in severe kidney dysfunction.
Indeed, cell culture studies suggest that TMAO can be proinflammatory in the plasma
concentrations achieved during kidney failure. It generally is wise to minimise the
consumption of nitrogenous compounds in this context.
In conclusion, there is a reason to suspect that the elevated risk for vascular events
and type 2 diabetes associated with elevated TMAO, after correction for recognised
risk factors, is mediated largely by hepatic insulin resistance and the metabolic
factors which induce it. This implies that a range of measures which typically improve
hepatic insulin sensitivity, as catalogued above, could be expected to decrease elevated
TMAO–a proposition that is readily clinically testable–while ameliorating the vascular
and metabolic risk associated with high TMAO.