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Reframing Nutritional Microbiota Studies To Reflect an Inherent Metabolic Flexibility of the Human Gut: a Narrative Review Focusing on High-Fat Diets

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There is a broad consensus in nutritional-microbiota research that high-fat (HF) diets are harmful to human health, at least in part through their modulation of the gut microbiota. However, various studies also support the inherent flexibility of the human gut and our microbiota's ability to adapt to a variety of food sources, suggesting a more nuanced picture. In this article, we first discuss some problems facing basic translational research and provide a different framework for thinking about diet and gut health in terms of metabolic flexibility. We then offer evidence that well-formulated HF diets, such as ketogenic diets, may provide healthful alternative fuel sources for the human gut. We place this in the context of cancer research, where this concern over HF diets is also expressed, and consider various potential objections concerning the effects of lipopolysaccharides, trimethylamine-N-oxide, and secondary bile acids on human gut health. We end by providing some general suggestions for how to improve research and clinical practice with respect to the gut microbiota when considering the framework of metabolic flexibility.
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Reframing Nutritional Microbiota Studies To Reect an Inherent
Metabolic Flexibility of the Human Gut: a Narrative Review
Focusing on High-Fat Diets
Jonathan Sholl,
a
Lucy J. Mailing,
b
Thomas R. Wood
c,d,e
a
Université Bordeaux, CNRS, ImmunoConcEpT, UMR 5164, Bordeaux, France
b
Independent Researcher, Milwaukee, Wisconsin, USA
c
Institute for Human and Machine Cognition, Pensacola, Florida, USA
d
Center on Human Development and Disability, University of Washington, Seattle, Washington, USA
e
Department of Pediatrics, University of Washington, Seattle, Washington, USA
Jonathan Sholl and Lucy J. Mailing contributed equally. Author order was based on relative contributions to drafting and editing the manuscript.
ABSTRACT There is a broad consensus in nutritional-microbiota research that high-
fat (HF) diets are harmful to human health, at least in part through their modulation
of the gut microbiota. However, various studies also support the inherent exibility
of the human gut and our microbiotas ability to adapt to a variety of food sources,
suggesting a more nuanced picture. In this article, we rst discuss some problems fac-
ing basic translational research and provide a different framework for thinking about
diet and gut health in terms of metabolic exibility. We then offer evidence that well-
formulated HF diets, such as ketogenic diets, may provide healthful alternative
fuel sources for the human gut. We place this in the context of cancer research,
where this concern over HF diets is also expressed, and consider various potential
objections concerning the effects of lipopolysaccharides, trimethylamine-N-oxide,
and secondary bile acids on human gut health. We end by providing some gen-
eral suggestions for how to improve research and clinical practice with respect to
the gut microbiota when considering the framework of metabolic exibility.
KEYWORDS cancer, gut health, high-fat diets, metabolic exibility, microbiota
It is generally accepted that diet is a major factor shaping both the composition and
the function of the human gut microbiota. However, much debate focuses on the
health effects of dietary components, with ber generally being seen as not only bene-
cial but necessary and animal fat (and sometimes protein) from high-fat (HF) diets
being singled out as detrimental to the gut microbiota (19). As a result, concerns over
HF diets feature heavily in the framing of studies on the microbiota and health. For
instance, HF or even high-protein, low-carbohydratediets are often suggested to play a
causal role in various forms of cancer, cardiovascular disease, immunological dysregulation,
and diabetes, through a variety of mechanisms (1014). This concern is expressed by inter-
national authorities on gut health, e.g., the European Society of Neurogastroenterology
and Motility (15), and in consensus statements by groups like the International Cancer
Microbiome Consortium (16).
It seems safe to say that the consensus is that HF diets are harmful to human health,
at least in part through their modulation of our gut microbiota. Put differently, the pri-
mary substance that feeds benecialgut microbes is microbiota-accessible carbohy-
drates(17), and in the absence of these, protein and fat will deteriorate our gut health.
One of the most cited studies used to support this consensus is that of David et al.
(18). While this study demonstrates how quickly the human gut microbiota adapts to
Citation Sholl J, Mailing LJ, Wood TR. 2021.
Reframing nutritional microbiota studies to
reect an inherent metabolic exibility of the
human gut: a narrative review focusing on
high-fat diets. mBio 12:e00579-21. https://doi
.org/10.1128/mBio.00579-21.
Editor Danielle A. Garsin, University of Texas
Health Science Center at Houston
Copyright © 2021 Sholl et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Jonathan Sholl,
jonathan.sholl@u-bordeaux.fr, or Thomas R.
Wood, tommyrw@uw.edu.
Published 13 April 2021
March/April 2021 Volume 12 Issue 2 e00579-21 ®mbio.asm.org 1
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dietary changes, what is less clear is how this should be interpreted (9). As we will dis-
cuss, this study highlights the need to consider the metabolic exibility of the gut
(19, 20). We are still far from being able to precisely dene a healthygut microbiota
(2125), and it is quite likely that the human gut and its microbial symbionts evolved
to adapt to a variety of macronutrient patterns. Acknowledging this exibility will
help to expand research and guide clinical interventions.
Here, we suggest one way in which translational research on nutrition and the
microbiota can be misleading (1st section) and provide a way to reframe this research
in terms of metabolic exibility (2nd section). We then offer evidence supporting the
potential healthfulness of alternative fuel sources derived from HF ketogenic diets
(KDs) (3rd section) and question the harmful role of these diets in diseases such as can-
cer (4th section). After addressing some likely objections (5th section), we end by rais-
ing the concern that the consensus on dietary fat may reect a research bias more
than physiological reality and provide suggestions for future research.
LOST IN TRANSLATION: OF MICE AND JUNK FOOD
We are not the rst to point out the limitations of preclinical nutritional microbiota
research or the ubiquitous problem of HF diets in animal models. These diets are typi-
cally composed of soybean oil, lard, rened sugar, and little to no ber (26, 27), which
Craig Warden called the mouse equivalent of pork rinds, ribs, and coke(28). The clas-
sic animal HF diet is therefore much more reective of the standard American diet
than nutritionally replete high-fat diets, such as therapeutic KDs (29, 30). Evidence for
the role of simple sugars in harmfully disrupting the gut microbiota is growing (31),
and this alone should provide ample reason not to draw conclusions based solely on
fat content without considering other macronutrients or dietary quality.
While human metabolism can adapt to diets higher in either fats or carbohydrates,
the natural diet of a mouse is low in fat and high in carbohydrates. It is therefore unsur-
prising that mice develop issues when eating a species-inappropriate diet. The strain
of mice commonly used for such studies, C57BL/6, has also been genetically selected
for its ability to gain weight in response to a HF diet. While humans are capable of
weight loss or gain on a variety of dietary patterns (3234), C57BL/6 mice have greater
weight gain and metabolic disruptions on low-carbohydrate diets (35). Consequently,
...rodent models of obesity may be most valuable in the understanding of how meta-
bolic mechanisms can work in ways different from the effect in humans(35). Broadly
translating ndings from inbred mice fed a highly rened HF diet to humans is there-
fore fraught with potential for misunderstanding.
Similar problems exist in the clinical literature examining effects of the diet on the
gut microbiota and associated disease risk. For instance, in the Malmö Offspring Study,
Ericson et al. identied health-consciousand sugar and high-fat dairydietary pat-
terns associated with decreased and increased risk of having prediabetes, respectively
(36). The latter pattern was characterized by high intakes of pastry/desserts, high-fat
milk/cream, low-ber bread, potatoes, and processed/red meat, with the overarching
assumption that these components all equally and signicantly contribute to the
potential negative effects of this dietary pattern. Though these foods may cluster to-
gether frequently on the population level, we cannot assume that they contribute equally
toanyassociatedhealthoutcomessinceeachofthemwouldbeexpectedtohaveverydif-
ferent effects on both the gut microbiota and on general health. As highlighted in the com-
mentary accompanying reference 36, the association between the health-conscious dietary
pattern and prediabetes was lost after adjusting for body mass index (BMI) (2), suggesting
that a primary driver of differences between dietary patterns may be caloric intake. Any
attempt to assess the effects of dietary components on health must consider food process-
ing and energy density, both of which appear to contribute to increased caloric intake
beyond the effect of individual macronutrients (37, 38). One must also consider whether it
is the presence or absence of certain foods that drives downstream effects (2). Without a
nuanced approach examining dietary quality and individual dietary components, we are
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left making assumptions about fat in human diets similar to those made when we attrib-
ute the effects of HF diets in rodents purely to fat content.
We largely agree that the Westerndiet full of processed food causes problems for
both the mouse and the human gut microbiota. However, there are a variety of ways
to construct an HF diet, with data from humans suggesting that well-formulated thera-
peutic KDs, which in some clinical trials contain between 3 and 5 servings of non-
starchy vegetables per day (39, 40), may be more benecial for our gut and overall health
than some animal studies suggest (30, 41, 42). What is needed, then, is a way to reframe
the debate to better reect the overall evidence.
REFRAMING A HEALTHY GUT IN TERMS OF EVOLVED FLEXIBILITY
New technologies and greater interest in gut health in recent years have dramati-
cally increased our understanding of gut microbes. Nevertheless, we are still unable to
dene a healthygut microbiota (22, 25). On average, any two individuals share only
about a third of their gut microbiota, with the other two-thirds varying depending on
genetics, geographical location, history of antibiotic and medication use, mode of
delivery at birth, diet, and other undetermined factors (43, 44). It is even possible that
two otherwise-healthy individuals can show no overlap in microbiota composition
(44). Thus, outside clear instances of dysbiosis, we have insufcient information to say
that one individualstwo-thirdsis any better than anothers.
While it is generally believed that diversity and community stability are key compo-
nents of a healthy gut ecosystem, even these can sometimes be associated with dis-
eased states (45, 46). Some of the keystone microbes commonly considered crucial for
gut health, such as Bidobacterium, are completely absent from the guts of traditional
cultures, like the Hadza, who are otherwise virtually free of chronic disease (47). Gut
health and dysbiosis thus remain vague and sometimes contested concepts (25, 48,
49); if there is a healthy coremicrobiota, it may be at the level of microbial functions,
not species (23, 43). Part of this relative lack of insight may result from technologies
such as 16S rRNA sequencing, which do not provide accurate information beyond the
genus level and provide little insight into microbial functions (50). While there is hope
that higher-resolution technologies (e.g., metagenomics, metabolomics), larger data
sets, and advanced computing techniques will bring us closer to dening a healthy micro-
biota, many researchers call for moving away from cataloguing species and toward an
approach that considers the intricate nature of microbiota-host interactions (22, 51).
While technological advances are eagerly awaited, some initial clarity might come
from placing the human microbiota in its evolutionary context. Our relationship with
our gut microbes is the product of thousands of generations of close coevolution (52,
53). The environments in which we evolved also required regular adaptation to chang-
ing conditions. Our ancestors may not always have had steady access to food and
would likely have undergone occasional bouts of signicant deprivation when food
was scarce (54, 55). Similarly, diets changed seasonally and geographically, as is
reected by the seasonal changes in the guts of traditional populations, like the Hadza
(56), or in the specic adaptations in cultures known to eat relatively few plant foods,
such as the Inuit (57, 58). This variability can be explained in terms of metabolic exibil-
ity (19), which is the evolved ability to shift our metabolism to changes in dietary intake:
to burn and use carbohydrates when they are plentiful and to turn dietary fat or stored
body fat into ketones for energy when food or carbohydrates are scarce. Consequently,
it seems likely that our guts also exhibit the exibility to adapt to changing food sources
rather than suffer signicant gut dysfunction whenever ber is absent.
In line with this evolutionary perspective on the compositional and functional
adaptability of our gut and its microbiota, David et al. write (18),
Our ndings that the human gut microbiome can rapidly switch between
herbivorous and carnivorous functional proles may reect past selective pressures
during human evolution. Consumption of animal foods by our ancestors was likely
volatile, depending on season and stochastic foraging success, with readily available
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plant foods offering a fallback source of calories and nutrients. Microbial communities
that could quickly, and appropriately, shift their functional repertoire in response to
diet change would have subsequently enhanced human dietary exibility.
In other words, a healthygut microbiota adapts to a wide range of food sources
and does not necessarily become more or less pathogenic depending on the amount
of carbohydrate or fat in the diet. Moreover, while short-term dietary changes tend to
produce signicant changes in the gut microbiota (18), long-term studies suggest a rel-
ative resilience of the microbiota to shifts in diet (59). Due to the aforementioned fac-
tors shaping gut microbiota (43, 44), we should consider whether a dietary change
must produce signicant physiological changes in the host before a new microbial sta-
bility is achieved, with diet-induced uctuations merely an expression of the guts abil-
ity to adapt to ensure optimal function. If human guts are inherently metabolically ex-
ible, short-term diet-induced changes in microbiota composition could be considered
a potential hallmark of gut health (60). We should thus determine whether the short-
and long-term taxonomic changes resulting from this metabolic exibility are predic-
tive of overall health outcomes and how/whether the microbiota drives those out-
comes (6163).
EVIDENCE FOR HIGH-FAT KETOGENIC DIETSCONSIDERING ALTERNATIVE
PATHWAYS
Gut bacteria metabolize complex carbohydrates to produce short-chain fatty acids
(SCFAs), like acetate, propionate, and butyrate, with the last being the preferred fuel
source for gut epithelial cells. Published estimates suggest that butyrate provides
about 70% of colonic epithelial cell energy requirements (64), with a regular supply of
butyrate required to maintain gut barrier function. What remains to be seen is how dif-
ferent diets modulate SCFA production and whether this results in different down-
stream health effects.
Animal-based diets. The work of David et al. (18) has been instrumental in high-
lighting how quickly and reliably the human gut microbiota adapts to dietary changes.
What is unclear is whether this study should be used to support the avoidance of diets
high in fat or protein. Ten healthy human volunteers were placed on a short-term
plant-based diet (PBD) consisting of 300 g of carbohydrate per day from cereal, vegeta-
bles, rice, lentils, and fruit or on an animal-based diet (ABD) consisting of less than 3 g
of carbohydrates per day, with 30% of calories from protein and 70% of calories from
fat from eggs, meat, and cheese. Subjects on the ABD group were conrmed to be in
ketosis by day 2 of the diet, with distinct gut microbial communities emerging in both
diet groups within 3 days.
The most interesting and perhaps contentious nding was that there was no signi-
cant change in alpha-diversity in either group (18). Those on the ABD saw an increase
in the relative abundance of bile-tolerant microorganisms, like Bilophila,Alistipes,and
Bacteroides spp., and a decrease in the relative abundance of microbes known to metab-
olize complex dietary plant bers, such as Roseburia,Eubacterium,andRuminococcus
spp. While often cited as evidence that an ABD is harmful, this is far from conclusive. The
PBD, despite being supposedly uniquely capable of producing butyrate from micro-
biota-accessible carbohydrate metabolism, produced only slightly more butyrate than
did the ABD, with the ABD also resulting in signicantly greater production of isovalerate
and isobutyrate (Fig. 1) (18). Isobutyrate has been shown to activate many of the same
receptors as butyrate (see Considering alternative pathwaysbelow), weakening the
notion that PBDs are signicantly betterfor the gut due to butyrate/SCFA production.
Mucus barrier. Another recent study by Ang et al. conrmed that a ketogenic diet
(KD) can alter the structure and function of the gut microbiota (65). In humans, among
the most signicant changes of the fecal microbiota following a KD was a dramatic
reduction in the abundance of several Bidobacterium species. In controlled-feeding
studies of mice, the researchers found that KDs had a unique impact on the gut micro-
biota relative to conventional HF diets, with the abundance of Bidobacterium organ-
isms decreasing with increasing carbohydrate restriction. Further experiments found
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that both a KD or ketone ester supplementation increased beta-hydroxybutyrate
(
b
HB) in the lumen of the gut and in colon tissue (65), with ketone bodies directly in-
hibiting the growth of Bidobacterium. Ketosis was also associated with a reduction in
small intestinal Th17 cells, which help maintain the gut mucosal barrier and contribute
to pathogen clearance at mucosal surfaces. However, Th17 cells have also been impli-
cated in autoimmune and inammatory disorders (66).
Next, Ang et al. (65) sought to determine whether the change in Th17 cells was de-
pendent on the ketone-induced changes in the microbiota. Mice that received a fecal
transplant of the ketone-fed microbiota from human donors had signicantly fewer in-
testinal Th17 cells. Contrary to previous ndings that mice fed ber-free diets had a sig-
nicant breakdown of the colonic mucus layer (67, 68), Ang et al. write, A ketogenic
diet maintains a robust mucus layer despite the lack of fermentable carbohydrates
(65). The KD maintained not only the thickness of the mucus layer but also the expres-
sion of Muc2, the primary constituent of gut mucus. Nutritional ketosis might actually
support the gut mucus layer.
Multiple sclerosis and epilepsy. There are various levels of support for therapeutic
KDs on the gut and overall health in longer-term studies, for instance, the long-term
effects of a KD on the fecal microbiota in 25 patients with multiple sclerosis (MS) (69).
Like many autoimmune diseases, MS is associated with gut pathologies, with gut dys-
biosis and intestinal permeability potentially preceding the development of autoim-
munity (70). Swidsinski et al. (69) found that patients with MS tended to have reduced
numbers of Roseburia,Bacteroides, and Faecalibacterium prausnitzii organisms at base-
line than healthy individuals. The effects of a 6-month therapeutic KD were biphasic:
In the short term, bacterial concentrations and diversity were further reduced. They
FIG 1 Short-chain fatty acid (SCFA) production in humans eating low-fat, plant-based and high-fat,
animal-based diets. (a) Plant-based diets result in roughly twice the production of acetate and butyrate.
(b) Animal-based diets result in roughly twice the production of isovalerate and isobutyrate, which
have metabolic functions overlapping those of the traditional SCFAs acetate and butyrate. (Republished
from reference 18 with the permission of the publisher.)
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started to recover at week 12 and exceeded signicantly the baseline values after 23 to
24 weeks on the ketogenic diet(69). Such studies are inconclusive since they are rela-
tively uncontrolled, but they nevertheless further support the need to consider the
time course of dietary adaptation before determining whether a diet is benecial or
detrimental for the gut microbiota.
In another context, researchers investigated whether the benecial effects of a ther-
apeutic KD on epilepsy are mediated through the gut microbiota (71). The KD reduced
microbial diversity but increased the abundance of Akkermansia muciniphila and
Parabacteroides spp. By treating mice fed a normal high-carbohydrate diet with these
specic microbes, the researchers demonstrated that these taxa were at least partly re-
sponsible for the antiepileptic effects of the KD. Similar microbial changes, as well as
increases in butyrate and propionate, have been observed when using a modied
Mediterranean ketogenic diet in Alzheimers patients (72).
Together with the David et al. study (18), these ndings suggest that, while short-
term dietary changes can rapidly shift the composition of the gut microbiota, these
changes may not be detrimental and may provide benet. They also underline the
need to look at long-term dietary changes and collect samples at multiple time points
to determine the true effect of an intervention like a therapeutic KD, including whether
the benets or any risks are mediated through changes in the microbiota.
Considering alternative pathways. Multiple strands of evidence question an
assumption about normalmetabolic pathways in the gut. Alongside the SCFAs men-
tioned above, there are several other molecules that can serve as sources of fuel for
gut epithelial cells. The very idea of a preferredfuel source may be skewed from
studying people (and rodents) who eat a large amount of microbiota-accessible carbo-
hydrates. In other words, while butyrate production may be reduced on a KD, other
molecules can potentially take butyrates place to help maintain gut barrier function.
This shift to an alternative pathway is what we might expect from the perspective
of metabolic exibility, where we see a potential analogy between the butyrate-gut
connection and glucose-brain connection. While glucose is a necessary fuel for the
brain, we have known for some time that in the (relative) absence of carbohydrates,
the body will shift its metabolism from glucose to fatty acids, producing ketone bodies,
FIG 2 The many substrates and pathways that contribute to energy production in the intestinal epithelium. TCA, tricarboxylic
acid.
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such as
b
HB, to support brain metabolism (73). This kind of fatty acid-based metabo-
lism appears to have numerous neurological benets and may be a preferredfuel for
the brain during both development (74) and aging (75). Similarly, while researchers
repeatedly stress that gut epithelial cells are uniquely fueled by the butyrate produced
by our resident microbiota after consuming microbiota-accessible carbohydrates
(17), here too the body is exible, with
b
HB also being capable of supporting energy
requirements in the gut.
In fact, there are at least four molecules that can replace butyrate: isobutyrate, ace-
toacetate,
b
HB, and bile-derived acylcarnitines (Fig. 2). Isobutyrate is a metabolite of
protein fermentation that is typically produced at lower levels than butyrate. When bu-
tyrate is less abundant, isobutyrate can be absorbed from the gut lumen by gut epithe-
lial cells and metabolized for energy (76). Fecal isobutyrate was found to be elevated
in humans consuming a KD (18). Moreover, isobutyrate can stimulate the same recep-
tors as butyrate in the gut (GPR41, GPR43, and GPR109a) to inuence mucus secretion,
antimicrobial peptide release, and immune regulation (77), and while isobutyrate may
be produced at lower levels on a moderately high-protein diet than butyrate would be
produced on a high-carbohydrate diet (18), isobutyrate appears to be a more potent
stimulator of butyrate receptor GPR41 (FFAR3) than butyrate itself (78). In other words,
what isobutyrate lacks in concentration relative to butyrate, it may make up for in po-
tency. Relatedly, this may provide a reason not to confuse high-protein and rened HF
diets, since the most abundant end products of protein fermentation or catabolism are
SCFAs, such as isobutyrate (79). As suggested by David et al., it might be the overall
context in which protein fermentation occurs that is important to downstream health
outcomes rather than the protein fermentation itself.
Like butyrate,
b
HB can also stimulate GPR109a, reducing intestinal inammation (80,
81). Most notably, however, both
b
HB and its related ketone body acetoacetate are inter-
mediates in the pathway for butyrate metabolism; when butyrate is taken up by gut epithe-
lial cells, it is converted into
b
HB rst and then acetoacetate before being broken down fur-
ther for energy (Fig. 2) (82). Gut epithelial cells express the monocarboxylate transporter
MCT1, a primary ketone body transporter on the basolateral surface (83), and several papers
suggest that gut epithelial cells are capable of utilizing ketone bodies from the vascular bed
(84, 85). As gastrointestinal inammation and mucosal damage can impair butyrate uptake
from the intestinal lumen (86, 87), circulating ketones may provide a potential therapeutic
option in certain patients with gastrointestinal disease.
To our knowledge, no studies have assessed the effects of ketones or a KD on gut
barrier function. However, a recent study found that ketone body signaling regulates
the normal function of intestinal stem cells (ISC) and their ability to respond to injury
(88). In this study,
b
-hydroxy
b
-methylbutyryl-coenzyme A (HMG-CoA) synthase 2
(HMGCS2), a rate-limiting step in ketone production, was enriched in small intestinal
stem cells. Ablating the Hmgcs2 gene in mice diminished
b
HB levels in the crypts,
compromising ISC function and regeneration of the gut epithelium after injury.
Exogenous
b
HB rescued ISC function and partially restored intestinal regeneration. A
KD also increased HMGCS2 expression and ISC number, function, and postinjury
regeneration. In contrast, a glucose-supplemented diet suppressed ISC ketogenesis
and skewed the differentiation of ISC toward goblet and Paneth cells. Notably, once
stem cells had differentiated into mature epithelial cells and migrated out of the
crypt, they expressed very little HMGCS2. This suggests that mature epithelial cells
do not possess the ability to generate large amounts of ketones through the classical
ketogenic pathway (via condensation of two molecules of acetyl-CoA), though we
know that they have the ability to utilize ketones.
Thus, it follows that if (i) a KD produces high levels of ketones in mature intestinal
epithelial cells (as Ang et al. [65] found) and (ii) these are not being generated in
mature epithelial cells (as is suggested by the lack of HMGCS2 in the work of Chen et
al. [88]), then the ketones are almost certainly coming from circulation. Along these
lines, the authors write, Because exogenous ketones rectify Hmgcs2 loss in vitro and in
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vivo, liver or other nonintestinal sources of ketones may substitute or supplement ISC-
generated ketones in KTD-mediated regeneration, where circulating ketone levels are
highly elevated(88).
Lastly, it has recently been shown that not only can colonic epithelial cells oxidize
both short- and long-chain fatty acids but also, through mitochondrial metabolism,
these cells can oxidize medium- and long-chain acyl-carnitines that are delivered from
biliary secretions (89). This has clinical relevance in that a reduction in these metabo-
lites may contribute to colonic inammation. Importantly, this provides yet another
host-derived energy source for the epithelium during low-carbohydrate diets to com-
plement the three discussed above. We will return to this below since bile acid secre-
tion is a common concern with HF diets.
To conclude, we suggest that a more nuanced picture of how HF diets impact gut
and overall health is required, with particular attention being paid to therapeutic KDs.
By considering the alternative pathways by which ketones and KDs can inuence gut
function, we can move toward a more evolutionarily consistent picture of human gut
variability. Nevertheless, research needs to clarify whether the benets of lower carbo-
hydrate or KDs come directly from increasing
b
HB, reducing inammation, modifying
insulin and glucose metabolism, reducing caloric intake, altering the gut microbiota, or
other undetermined factors.
HIGH-FAT DIETS, THE GUT, AND CRCSETTLED SCIENCE?
We now briey discuss colorectal cancer (CRC), where the HF diet-microbiota link is
commonly highlighted. While the 2019 consensus statement by the International
Cancer Microbiome Consortium acknowledges the need for better human studies
into how the microbiota inuences carcinogenesis (16), it nevertheless implicates a
high-fat, low-ber Western-style diet in changes in mucosal biomarkers of cancer risk
(90). This follows the WHOs 2015 classication of redandprocessedmeatas a
(class 2A) probable carcinogen, which relies heavily on preclinical and mechanistic
data due to our current inability to isolate the effects of individual foods in clinical
epidemiological studies (91, 92). A recent review in Nature echoes the WHOs position
on processed and red meat and cancer, aiming to establish an oncogenicCRC-asso-
ciated microbiota (93).
In their Nature review, Janney et al. rely on the nding that CRC etiology is largely
environmental, potentially accounting for 70 to 90% of the disease risk (93). Here,
dietis strongly implicated, and the authors stress those diets that are low in ber and
high in fat and red meat.However, in the two references used to support this claim,
only one mentions red meat(94) and only in the context of epidemiological associa-
tions, which are often plagued by healthy-user bias and signicant reporting error (95,
96). Moreover, Janney et al. (93) vacillate between the terms high-fatand Westernized
high-fat,which introduces confounders. Some mechanistic studies in mice do appear to
support this HF diet-cancer link (97100), but this might be tumor type specic (101,
102). As the HF dietsstudied are admixtures of rened sugars and/or hydrogenated
oils, as mentioned above, this conates a junk food-mimicking diet with any diet high in
fat. Such results thus remain inconclusive (103), as we discuss below. Some of the sug-
gested mechanisms connecting diet and CRC are protein fermentation, secondary bile
acids, and increased levels of reactive oxygen species and reactive nitrogen species due to
increased bile acid, heme iron, decreases in SCFAs, and specicmicrobialchanges,e.g.,an
increased Bilophila abundance.Asdiscussedaboveandexploredinmoredetailbelow,the
rst three mechanisms are not clearly pathogenic, the putative antitumorigenic properties
of butyrate (104) can also be obtained through alternative mechanisms, the relevance of
heme iron remains to be seen (105, 106), and the signicance of microbial changes
depends on the broader physiological context and the relative abundances of microbes.
Since various conditions, such as irritable bowel syndrome (IBS), ulcerative colitis, and
Crohns disease, appear to increase ones risk for developing CRC (107109), further
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research might consider the role of KDs in these contexts (110). Unfortunately, few such
studies have been performed, and we are often left with case studies (111, 112).
We can, however, piece together various strands of evidence suggesting a more
nuanced picture on animal fats as CRC risk factors. First, some mouse models and pre-
clinical studies show KDs or ketones to be cancer suppressive (113, 114), perhaps pri-
marily through glucose restriction (103) and by increasing intratumoral oxidative stress,
leading to tumor cell apoptosis (115). Variations of therapeutic KDs might provide ben-
ets for breast cancer by decreasing tumor necrosis factor alpha (TNF-
a
) and insulin
while increasing interleukin 10 (IL-10) (116) and may be a promising adjuvant therapy
for various cancers (117121). However, as many of these studies are in animal models,
caution is warranted (122, 123). At the least, these studies and recent reviews suggest
a variety of mechanisms by which animal-food-based KDs may have benecial effects
on colorectal and other cancers.
This evidence converges with studies suggesting that reducing red meat and total
fat consumption, while increasing fruit and grain consumption, does not reduce the
risk for polyp reoccurrence even after 8 to 16 years (124126) and has unclear risk ben-
ets for CRC or any kind of cancer (127129). Animal models suggesting that beef
consumption does not promote cancer, that bacon may be protective, and that un-
saturated fat may have carcinogenic effects (130, 131) all increase the likelihood that
strong statements on animal foods and cancer are premature. Similarly, a growing
number of reviews and meta-analyses weaken the links between meat consumption
and cancer (132136) and possibly overall health (137, 138), with some showing
inverse correlations between meat intake and overall mortality in speciccohorts
(139) and lower rates of CRC in meat eaters than in vegetarians (140). Studies that
correlate meat intake with CRC also suggest a complex etiology due to contributing
factors, such as obesity and hyperinsulinemia (141). As most of these studies have
limitations, more research will be needed (142, 143), with the overall balance of evi-
dence not currently appearing to support an independent effect of animal-based
foods on the incidence of CRC. Given meats long-term presence in the hominid diet
(144147), it is more likely that modern dietary components and cooking techniques
are driving cancer risk factors through their effects on our guts and general
physiology.
POSSIBLE OBJECTIONS AND CONCERNS
We acknowledge that there are likely to be various objections concerning the
effects of fat and protein on our gut microbiota. We will address three of these, lipo-
polysaccharides, trimethylamine-N-oxide (TMAO), and secondary bile acids, and end
with a cautionary note concerning KDs and hydrogen sulde (H
2
S).
LPS. High-fat diets are commonly said to increase intestinal absorption of lipopoly-
saccharides (LPS), which are a group of endotoxins found in the cell walls of Gram-neg-
ative bacteria. If LPS gets into circulation, it can cause low-grade systemic inammation
(148), with the type and extent of the response dependent on the microbial source
and LPS subtype (149). When we consume more long-chain fatty acids, our body
makes more chylomicrons, which can carry LPS (150). Indeed, fat-enriched meals have
been shown to moderately increase serum levels of LPS in both mice and humans
(151, 152). While worth considering, we believe that, for several reasons, this is unlikely
to contribute signicantly to systemic inammation in those consuming KDs.
First, several studies suggest that the transport of LPS by chylomicrons may confer
an advantage because it favors the clearance of LPS by the liver, reducing LPS toxicity
(153, 154). Moreover, chylomicrons have an innate ability to inactivate LPS (155), and
the increased absorption of LPS appears to reduce inammation in the gut mucosa
(156). Similar benecial adaptations can be seen with exercise, which increases LPS
translocation but also LPS clearance, for instance via upregulation of anti-LPS immuno-
globulins (157). This is important since the primary mode of systemic exposure to LPS
is not through fat absorption but through reduced gut barrier function (158). When
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the gut is permeable, large amounts of LPS can leak into the submucosa and blood-
stream, causing localized gut immune responses and systemic inammation (159). This
is likely to be a consequence, rather than a cause, of the metabolic endotoxemia asso-
ciated with metabolic syndrome and cardiovascular disease, with poor systemic health
subsequently impairing gut barrier function (160). Certain LPS subtypes have also been
suggested to have benecial immunomodulating roles (161). In other words, com-
pared to the intestinal permeability associated with inammatory gastrointestinal con-
ditions, chylomicron-induced LPS absorption is likely minimal. One hypothesis emerg-
ing from these various studies that can be tested in humans is whether, for patients
dealing with severe intestinal permeability, chylomicron-induced detoxication of LPS
reduces inammation enough to facilitate healing of the gut epithelium.
Importantly, many of the above-described studies are preclinical, but they neverthe-
less point to promising mechanisms that are being pursued in human studies to better
contextualize the various roles of LPS. If fat-induced LPS absorption were an issue, we
would expect to see increased systemic inammation in those fed a KD. In contrast,
humans consuming therapeutic KDs generally experience a reduction in systemic inam-
mation (162), with possible anti-inammatory mechanisms, including NLRP3 inamma-
some inactivation (163), modulation of TNF-
a
, IL-6, IL-8, MCP1, E-selectin, I-CAM, and
PAI-1 (all studied in a registered clinical trial [164]), and an improved cytosolic NADH/
NAD
1
ratio (165). Taken together, these studies should assuage some concerns of LPS
absorption following fat intake.
TMAO. Conventional nutrition science has long considered diets rich in animal-
based foods a risk factor for cardiovascular disease. A recent mechanism of interest is
TMAO (61). Increased concentrations of TMAO in circulation have been shown to con-
tribute to atherosclerosis in animal models and correlate with cardiovascular disease
risk in human studies (166). Certain gut bacteria convert choline and carnitine, both
prominent in animal foods, to trimethylamine (TMA), which is then absorbed and oxi-
dized in the liver to TMAO. However, some in vitro and animal evidence points to an
altered small intestinal microbiota characterized by an overabundance of choline-con-
suming, TMA-producing Escherichia coli as the culprit for high TMAO, rather than
excess consumption of animal products (167).
More importantly, a recent study suggests that gut microbiota composition can
inuence the amount of TMAO produced with an animal-based diet. Bacteria in the
genus Bilophila,whichtendtoincreaseinsubjectsonananimal-baseddiet,maybe
able to help circumvent TMAO production by degrading TMA to dimethylamine
(DMA) (168). Further analysis revealed that in a human cohort, Bilophila was signi-
cantly more abundant in the microbiotas of healthy individuals than in those with
cardiovascular disease. As such, Bilophilas pathogenicity may be context dependent,
and it may even be benecial for mitigating cardiovascular disease. Additionally,
recent Mendelian randomization studies have suggested that increased TMAO in
those at risk of cardiovascular disease may be a consequence of metabolic dysfunc-
tion, rather than an independent risk factor for disease risk (169). For multiple rea-
sons, we thus believe that TMAO may not be a signicant independent contribution
to cardiovascular disease, with gut and overall health more likely to be the critical
drivers of any associations.
Bile acids. It is commonly argued that an HF diet might be detrimental to the gut
microbiota and gut barrier because it stimulates increased secretion of secondary bile
acids (170). While some studies have shown that sustained exposure of the gut barrier
to high concentrations of bile acids (above 400
m
M) results in intestinal permeability
(171), physiologic doses of bile acids (which may be nontoxic up to 50 to 100
m
M
[172]) have several potential benets. For instance, bile acids have been shown to sup-
port barrier function by inducing the secretion of mucus from goblet cells, promoting
epithelial cell migration, and boosting gut innate immune defenses (173). They can
have antimicrobial properties, helping to regulate the gut microbiota, and may protect
against small intestinal dysbiosis (174, 175). Several studies even suggest that bile acids
activate enteroendocrine cells to release serotonin, which helps promote gut motility
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(176). Evidence for the physiological role of biliary secretions in producing an alterna-
tive fuel (acyl-carnitines) for gut epithelial cells was discussed above (89).
Exploring the complexities of every type of conjugated and deconjugated bile acid
is beyond the scope of this article (177), but this should be sufcient to question the
assumption that bile acid secretion resulting from the consumption of animal foods is
inherently pathogenic.
H
2
S. There is one important caveat concerning KDs and individuals with H
2
S-associ-
ated bacterial overgrowth. H
2
S is normally produced in the body and acts as an impor-
tant signaling molecule. Certain gut bacteria can also produce H
2
S, which at low con-
centrations has been shown to protect the gut against injury, stimulate gut motility,
and support ulcer healing (178). However, an overabundance of these bacteria can
lead to excess H
2
S, which has been linked to diarrhea, gut hypersensitivity, IBS, irritable
bowel disease (IBD), and colorectal cancer (179), thereby suggesting pleotropic and
dose-dependent effects (178). Some of the common H
2
S producers in the human gut,
Desulfovibrio spp., Bilophila wadsworthia, and Fusobacterium nucleatum, tend to thrive
on a diet that is high in animal protein and fat (180, 181). Thus, in patients with an
overabundance of these microbes, it is probably best to avoid a ketogenic or high-fat
diet until they can address this issue. Adding ber to the diet (e.g., Brassica vegetables)
may reduce the abundance of sulfate-reducing bacteria (182), further suggesting that
the dietary context accompanying protein and fat consumption be considered.
Overall, we do not believe that there is sufcient evidence to suggest that the pro-
duction of TMAO or LPS following animal protein/fat consumption or the physiologic
increase of bile acids seen on a KD is harmful to the gut microbiota or gut barrier func-
tion. These metabolites might exacerbate ongoing pathological conditions of dysbio-
sis, but there are reasons to believe that they are not harmful under physiological
conditions.
LOOKING AHEAD: REDIRECTING RESEARCH FOR NUANCE
We conclude by suggesting how nutritional microbiota research might proceed in
terms of what questions need to be asked or answered and how studies could be car-
ried out.
1. Researchers should be more explicit about the kind of HF diets used. A diet
mimicking a Western diet is not nutritionally equivalent to all HF diets, which vary
in terms of fat sources and overall diet quality. Even changing the language in
articles from high-fatto high-fat, high-sugarwould better reect the diet
studied and could alter perceptions. From there, it will be helpful to study levels
of ber in HF or animal-based diets, which represent variations on the theme of
nutritionally replete low-carbohydrate or therapeutic KDs.
2. While studies are starting to focus on different fat sources (4, 183, 184), still more
are needed with as few confounders as possible. Some of the most problematic
for mechanistic studies include the use of rened/hydrogenated fats and seed
oils, which likely have rather different metabolic effects than fats from whole
plant and animal foods, especially when these oils are mixed with rened sugars.
Nutritional epidemiology should not only account for fat sources and dietary
patterns/context but should explicitly address (un)healthy-user biases. The
problem is not that such biases exist, as some may be unavoidable. The problem
is that they remain under-discussed.
3. We should clearly acknowledge the limitations of animal research to inform
human health/nutrition and our limited knowledge of what constitutes a
healthygut microbiota. Since it is likely that gut health encompasses more
variability than is often acknowledged, we need to further test the evidence that
humans evolved to tolerate, adapt to, and perhaps thrive on a variety of dietary
patterns, with varying proportions of ber, protein, and fat. An interdisciplinary
approach may better elucidate the health effects of diet-gut interactions.
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4. Researchers should explicitly state whether their interpretation considers the
physiological context or makes claims based solely on isolated mechanisms and
nutritional epidemiology. Properly labeling evidence and placing observations in the
broader context of research can help prevent potentially biased interpretations.
5. Similarly, researchers might carefully consider the differences in host and
microbial metabolism. For instance, studies relying on fecal samples might skew
towards microbes found in the large intestine or colon, where carbohydrates are
digested and metabolized. This may obscure the role of microbial and host
responses in the small intestine (9, 185), thereby underrepresenting the microbes
that are more involved in fatty acid catabolism or the production of lipase
coenzymes in the jejunum. Similarly, by focusing largely on microbial metabolism
of carbohydrates and ber, we might be overlooking host-specic metabolism,
which appears to be highly adaptable to relative levels of dietary fat and protein.
6. Clinicians can also remain open-minded to alternative dietary approaches. One
implication of considering alternative energy sources is that in the presence of a
healthymicrobiota and gut mucosa, butyrate is probably sufcient to fuel the
gut. However, if patients (i) have ulcerative colitis or other mucosal damage, with
impaired butyrate uptake, (ii) have gut dysbiosis characterized by a lack of butyrate
producers, or (iii) are on a restrictive diet, such as a low-FODMAP (fermentable
oligosaccharides, disaccharides, monosaccharides, and polyols) diet or the specic
carbohydrate diet (SCD), resulting in reduced butyrate production, it may be wise
for clinicians to consider nontraditionaltherapeutic options, such as KDs, to
support gut epithelial metabolism, at least until treating the underlying gut
pathologies and healing the gut mucosa.
7. Finally, it will be important to objectively weigh the evidence concerning plants,
animal fats, and proteins. Conventional wisdom holds the belief that plants are
denitively healthful and animal products are at least potentially harmful. The
effects of this belief can lead to conicts of interest in nutrition studies more
generally (186) and may inuence the decision-making leading to dietary
guideline statements that provide strong recommendations despite abundant
evidence supporting the idea that humans can thrive on a diverse range of
diets (187).
In the end, we hope that more time and research will help to uncover these biases
and lead to a more accurate depiction of the responsiveness of the human gut and its
microbes to nutritional variations.
ACKNOWLEDGMENTS
Jonathan Sholl received funding from the European Research Council (ERC) under
the European Unions Horizon 2020 research and innovation program (grant agreement
637647), IDEM. Once this grant was completed, he received funding from the Université
de Bordeaux, Région Nouvelle-Aquitaine, and SIRIC BRIO.
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... Secondary bile acids have complex and unelucidated biological functions. Studies have illustrated that secondary bile acids regulated by high fat diet has adverse metabolic effects (146). DCA tends to increase in diet-related or genetic childhood obesity (147). ...
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An amendment to this paper has been published and can be accessed via the original article.
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