Microbiome Nonsense: response to “Chowing Down On Meat”

Response to "Chowing Down On Meat"

As the claim that animal protein and saturated fat is unhealthy becomes less and less tenable, those who have the intuition that animal-based nutrition must be bad for you are looking elsewhere.
There was great excitement at the end of 2014 about a study posted in Nature demonstrating the rapid changes in human gut microbes in response to animal-based vs. plant-based diets [1].
The paper is very interesting, and it has a lot of original data of a kind we’ve often wished for.
The authors then go on to interpret their findings without apparent restraint.
A report on the study on NPR called Chowing Down On Meat, Dairy Alters Gut Bacteria A Lot, And Quickly gets right to the point:

"Looks like Harvard University scientists have given us another reason to walk past the cheese platter at holiday parties and reach for the carrot sticks instead: Your gut bacteria will thank you."

and finally:

""I mean, I love meat," says microbiologist Lawrence David, who contributed to the study and is now at Duke University. "But I will say that I definitely feel a lot more guilty ordering a hamburger … since doing this work," he says."

That’s right.
The excitement in the blog-o-sphere was not so much about the clear results — that the changes in the gut flora in response to diet are fast and large — but about the authors’ opinions that the observed changes support a link between meat consumption and inflammatory bowel disease (IBD).
We take exception to these claims, as they are not well-founded by the data in the study, or in any other study.
The data to support them do not warrant the conclusion.
We consider it irresponsible at best to suggest that a dietary practice is harmful to health when the evidence is weak, especially when one is in a position of authority and subject to high publicity.
Here are the points we address:

The Claims about Inflammatory Bowel Disease

Here are some quotes from the paper stressing the possible dangers of a carnivorous diet based on a supposed link to IBD — inflammatory bowel disease.
Notice that they use language that implies the claims are proven, when as we will show, they are not.

"increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease [6]" — Abstract
"Bile acids have been shown to cause inflammatory bowel disease in mice by stimulating the growth of the bacterium Bilophila[6], which is known to reduce sulphite to hydrogen sulphide via the sulphite reductase enzyme DsrA (Extended Data Fig. 10)." — from figure 5, page 4.
"Mouse models have also provided evidence that inflammatory bowel disease can be caused by B. wadsworthia, a sulphite-reducing bacterium whose production of H2S is thought to inflame intestinal tissue [6]. Growth of B. wadsworthia is stimulated in mice by select bile acids secreted while consuming saturated fats from milk. Our study provides several lines of evidence confirming that B. wadsworthia growth in humans can also be promoted by a high-fat diet. First, we observed B. wadsworthia to be a major component of the bacterial cluster that increased most while on the animal-based diet (cluster 28; Fig. 2 and Supplementary Table 8). This Bilophila-containing cluster also showed significant positive correlations with both long-term dairy (P , 0.05; Spearman correlation) and baseline saturated fat intake (Supplementary Table 20), supporting the proposed link to milk-associated saturated fats[6]. Second, the animal-based diet led to significantly increased faecal bile acid concentrations (Fig. 5c and Extended Data Fig. 9). Third, we observed significant increases in the abundance of microbial DNA and RNA encoding sulphite reductases on the animal-based diet (Fig. 5d, e). Together, these findings are consistent with the hypothesis that diet-induced changes to the gut microbiota may contribute to the development of inflammatory bowel disease." — last paragraph, emphasis ours.

This concern is prominent in the paper;
they start with it and end with it.
It is based on a single citation to a study in mice.

Reasons those claims are not warranted

Let’s look at that study (Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice [2]):

Here’s the abstract (emphasis ours):

"The composite human microbiome of Western populations has probably changed over the past century, brought on by new environmental triggers that often have a negative impact on human health1. Here we show that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance, sulphite-reducing pathobiont, Bilophila wadsworthia2. This was associated with a pro-inflammatory T helper type 1 (TH1) immune response and increased incidence of colitis in genetically susceptible Il10−/−, but not wild-type mice. These effects are mediated by milk-derived-fat-promoted taurine conjugation of hepatic bile acids, which increases the availability of organic sulphur used by sulphite-reducing microorganisms like B. wadsworthia. When mice were fed a low-fat diet supplemented with taurocholic acid, but not with glycocholic acid, for example, a bloom of B. wadsworthia and development of colitis were observed in Il10−/− mice. Together these data show that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis that can perturb immune homeostasis. The data provide a plausible mechanistic basis by which Western-type diets high in certain saturated fats might increase the prevalence of complex immune-mediated diseases like inflammatory bowel disease in genetically susceptible hosts."

They took some mice who were particularly susceptible to colitis, and also some regular mice, and fed them one of three different diets: a low fat diet (if we’re reading it correctly they used the AIN-93M Purified Diet from harlan, which is about 10% fat), or a diet with 37% fat which was either polyunsaturated, or saturated milk fat. They didn’t specify the amount of carbohydrate or protein, but we assume the diets were about 10-15% protein, leaving about 50% carbohydrate.
The mice who had the high milk-fat diet had a significant increase in the gut bacteria called Bilophila wadsworthia.
The susceptible mice on the high milk-fat diet got colitis at a high rate (more than 60% in 6 months).
The other susceptible mice, those on low-fat or polyunsaturated fat also got colitis, but at a lower rate (25-30%).
The regular mice didn’t get colitis, even on the high milk-fat diet.
What’s the problem with knockout mice?
The mice that got colitis were susceptible because they were genetically manipulated to not function normally.
Specifically, they couldn’t produce something called interleuken-10 (IL-10).
IL-10 has many complex actions including fighting against inflammation in multiple ways.
The argument made by the scientists is that Bilophila wadsworthia must induce inflammation, and that colitis probably comes about in people who are less effective at fighting that inflammation, just like the knockout mice.
This seems intuitive, but it is certainly not proven by the experiment.

Look at it this way:
Suppose we didn’t know the cause of phenylketonuria, a genetic disorder that makes the victim unable to make enzymes necessary to process the amino acid phenylalanine. We could knockout that gene in an animal, feed it phenylalanine, watch it suffer retardation and seizures, and conclude that phenylalanine must promote brain problems. This would be a mistake, of course. Phenylalanine is an essential amino acid occurring in breast milk. As far as we know, there is nothing unhealthy about it, as long as you don’t have a genetic mutation interfering with its metabolism.

It is, of course, possible that Bilophila wadsworthia inflames the colon.
As a hypothesis, based on this study, it is not by itself objectionable.
What we object to is the leap to citing Bilophila wadsworthia as causing colitis, as in the second excerpt above, which we repeat here:

"Bile acids have been shown to cause inflammatory bowel disease in mice by stimulating the growth of the bacterium Bilophila[6], which is known to reduce sulphite to hydrogen sulphide via the sulphite reductase enzyme DsrA (Extended Data Fig. 10)." — from figure 5, page 4.

In fact, Bilophila did not appear to affect the normal mice at all!
There is no claim that the genetic mutation in the mice has any relation to genetic susceptibility to IBS in humans,
yet it is implied that natural human susceptibility might work the same way.
Hydrogen Sulfide
In the knockout mice study, a second experiment was done to determine whether the Bilophila wadsworthia seen in the milk-fat condition came from a particular bile acid, taurocholic acid.
They fed the knockout mice a low fat diet supplemented with either taurocholic acid (TC), or glycocholic acid (GC).
They confirmed that Bilophila wadsworthia was increased by taurocholic acid and not by glychocholic acid.
What else do we know about taurocholic acid?
According to the authors of this study, it is "a rich source of organic sulphur, […] resulting in the formation of H2S [hydrogen sulfide]".
In one figure they even demonstrated the presence of Bilophila wadsworthia by the presence of H2S.
But H2S can be beneficial:

  • There is emerging evidence that H2S has diverse anti-inflammatory effects, as well as pro-inflammatory effects, possibly only at very high levels [3].
  • The levels needed for harm are probably higher than occurs naturally [4]
  • H2S levels in the blood are associated with high HDL, low LDL, and high adiponectin in humans [5], all considered good things.

Moreover, there is now evidence that colon cells in particular can actually use H2S as fuel, and lots of it.
Other researchers have used a a similar argument in the opposite way.
They claim that eating fiber is healthy,
because of the butyrate generated from it in the colon, which colons cells then use as fuel.
While we have problems with that argument,
it shows a pervasive bias:
Using it when it supports plants, but ignoring it when it doesn’t.
Taking all this into account, it is not at all clear that the higher levels of sulfite reducing bacteria seen in the meat and cheese eaters was unhealthy.

What would happen if a human sufferer of IBS went on an animal foods only diet?

It’s clear that these researchers are not studying IBS at all.
They were studying gut bacteria, found an association, and cherry-picked one study suggesting that what they found in the animal diet results might be unhealthy.
If they were studying IBS, they might have noticed reasons to hypothesise that a diet low in fiber [6], [7], carbohydrates [8], or fermentable carbohydrates [9] would help IBS sufferers.
If humans who are susceptible to IBS are susceptible in the same way as the knockout mice in the cited study, then these results might be surprising.
Instead, these results in combination with the animal diet paper, should further decrease our belief that the mice results have any relevance at all.
Moreover, unless the authors are advocating a diet of low-fiber, low-carb plants (can’t think of any plants like that off the top of my head…), they are encouraging IBS sufferers to eat foods that may worsen their condition.
We don’t know what would happen in an all meat trial for IBS, but we’d love to find out.

In Sum

The supposed link between the animal diet and inflammatory bowel disease is composed of a chain of weak links:
A kind of bacteria they found in those eating meat and cheese was also found in a mouse study that suggested a link between the bacteria and IBS.

  • It used animals that were genetically engineered to not function normally.
  • It did not and cannot establish causality between the observed gut bacteria changes and the increased level of disease.
  • It was merely an observation of the two coinciding along with a plausible mechanism, i.e. a clever story about how this might be a causal relationship.

This plausible mechanism is not as clean a story as it appears. Presenting it as such is downright misleading.


[1] Diet rapidly and reproducibly alters the human gut microbiome

Lawrence A. David, Corinne F. Maurice, Rachel N. Carmody, David B. Gootenberg, Julie E. Button, Benjamin E. Wolfe, Alisha V. Ling, A. Sloan Devlin, Yug Varma, Michael A. Fischbach, Sudha B. Biddinger, Rachel J. Dutton & Peter J. Turnbaugh
Nature (2013) doi:10.1038/nature12820
[2] Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice

Suzanne Devkota, Yunwei Wang, Mark W. Musch, Vanessa Leone, Hannah Fehlner-Peach, Anuradha Nadimpalli, Dionysios A. Antonopoulos, Bana Jabri & Eugene B. Chang
Nature (2012) doi:10.1038/nature11225
[3] Evidence type: review and non-human animal experiment

Wallace JL.
Trends Pharmacol Sci. 2007 Oct;28(10):501-5. Epub 2007 Sep 19.

The notion of H2S being beneficial at physiological concentrations but detrimental at supraphysiological concentrations bears similarity to the situation with nitric oxide (NO), another gaseous mediator, which shares many biological effects with H2S. Also in common with NO, there is emerging evidence that physiological concentrations of H2S produce anti-inflammatory effects, whereas higher concentrations, which can be produced endogenously in certain circumstances, can exert pro-inflammatory effects [5]. Here, I focus on the anti-inflammatory effects of H2S, and on the concept that these effects can be exploited in the development of more effective and safer anti-inflammatory drugs. "

[4] Evidence type: review and non-human animal experiment

Wallace JL.
Trends Pharmacol Sci. 2007 Oct;28(10):501-5. Epub 2007 Sep 19.

(Emphasis ours)
"How much H2S is physiological?
"H2S is present in the blood of mammals at concentrations in the 30–100 m M range, and in the brain at concentrations in the 50–160 m M range [1–3]. Even after systemic administration of H2S donors at doses that produce pharmacological effects, plasma H2S concentrations seldom rise above the normal range, or do so for only a very brief period of time [24,27]. This is, in part, due to the efficient systems for scavenging, sequestering and metabolizing H2S. Metabolism of H2S occurs through methylation in the cytosol and through oxidation in mitochondria, and it is mainly excreted in the urine [1]. It can be scavenged by oxidized glutathione or methemoglobin, and can bind avidly to hemoglobin. Exposure of certain external surfaces andtissues to H2S can trigger inflammation [28], perhaps because of a relative paucity of the above-mentioned scavenging, metabolizing and sequestering systems. The highest concentrations of H2S in the body occur in the lumen of the colon, although there is some disagreement [29] as to whether theconcentrations of ‘free’ H2S really reach the millimolar concentrations that have been reported in some studies [30,31]. Although often alluded to [32,33], there is no direct evidence that H2S causes damage to colonic epithelial cells. Indeed, colonocytes seem to be particularly well adapted to use H2S as a metabolic fuel [4].
"There have been several suggestions that H2S might trigger mutagenesis, particularly in the colon. For example, one recent report [33] suggested that the concentrations of H2S in ‘the healthy human and rodent colon’ are genotoxic. Despite the major conclusion of that study, the authors observed that exposure of cultured colon cancer epithelial cells (i.e. transformed cells) to concentrations of Na2S as high as 2 mM for 72 hours did not cause any changes consistent with a genotoxic effect (nor cell death). It was only when the experiments were performed in the presence of two inhibitors of DNA repair, and only with a concentration of 2 mM, that they were able to detect a significant genotoxic signal. It is also important to bear in mind that the concentrations of H2S used in studies such as that described above are often referred to as those found in the ‘healthy’ colon. Clearly, if concentrations of H2S in the healthy colon do reach the levels reported, and if H2S has the capacity to produce genotoxic changes and/or to reduce epithelial viability, there must be systems in place to prevent the putative untoward effects of this gaseous mediator – otherwise, the colon would probably not be ‘healthy’"

[5] Evidence type: observational

Jain SK, Micinski D, Lieblong BJ, Stapleton T.
Atherosclerosis. 2012 Nov;225(1):242-5. doi: 10.1016/j.atherosclerosis.2012.08.036. Epub 2012 Sep 10.

"Hydrogen sulfide (H2S) is an important signaling molecule whose blood levels have been shown to be lower in certain disease states. Increasing evidence indicates that H2S plays a potentially significant role in many biological processes and that malfunctioning of H2S homeostasis may contribute to the pathogenesis of vascular inflammation and atherosclerosis. This study examined the fasting blood levels of H2S, HDL-cholesterol, LDL-cholesterol, triglycerides, adiponectin, resistin, and potassium in 36 healthy adult volunteers. There was a significant positive correlation between blood levels of H2S and HDL-cholesterol (r=0.49, p=0.003), adiponectin (r=0.36, p=0.04), and potassium (r=0.34, p=0.047), as well as a significant negative correlation with LDL/HDL levels (r= -0.39, p=0.02). "

[6] Evidence type: preliminary experiment

J. T. Woolner and G. A. Kirby
Journal of Human Nutrition and Dietetics Volume 13, Issue 4, pages 249–253, August 2000

Introduction High-fibre diets are frequently advocated for the treatment of irritable bowel syndrome (IBS) although there is little scientific evidence to support this. Experience of patients on low-fibre diets suggests that this may be an effective treatment for IBS, warranting investigation.
Methods Symptoms were recorded for 204 IBS patients presenting in the gastroenterology clinic. They were then advised on a low-fibre diet with bulking agents as appropriate. Symptoms were reassessed by postal questionnaire 4 weeks later. Patients who had improved on the diet were advised on the gradual reintroduction of different types of fibre to determine the quantity and type of fibre tolerated by the individual.
Results Seventy-four per cent of questionnaires were returned. A significant improvement (60–100% improvement in overall well-being) was recorded by 49% of patients.
Conclusion This preliminary study suggests that low-fibre diets may be an effective treatment for some IBS patients and justifies further investigation as a full clinical trial."

[7] Evidence type: Review

Eswaran S1, Muir J, Chey WD.
Am J Gastroenterol. 2013 May;108(5):718-27. doi: 10.1038/ajg.2013.63. Epub 2013 Apr 2.

Despite years of advising patients to alter their dietary and supplementary fiber intake, the evidence surrounding the use of fiber for functional bowel disease is limited. This paper outlines the organization of fiber types and highlights the importance of assessing the fermentation characteristics of each fiber type when choosing a suitable strategy for patients. Fiber undergoes partial or total fermentation in the distal small bowel and colon leading to the production of short-chain fatty acids and gas, thereby affecting gastrointestinal function and sensation. When fiber is recommended for functional bowel disease, use of a soluble supplement such as ispaghula/psyllium is best supported by the available evidence. Even when used judiciously, fiber can exacerbate abdominal distension, flatulence, constipation, and diarrhea."

[8] Evidence Type: uncontrolled experiment

Austin GL, Dalton CB, Hu Y, Morris CB, Hankins J, Weinland SR, Westman EC, Yancy WS Jr, Drossman DA.
Clin Gastroenterol Hepatol. 2009 Jun;7(6):706-708.e1. doi: 10.1016/j.cgh.2009.02.023. Epub 2009 Mar 10.

Background & Aims
Patients with diarrhea-predominant IBS (IBS-D) anecdotally report symptom improvement after initiating a very low-carbohydrate diet (VLCD). This is the first study to prospectively evaluate a VLCD in IBS-D.
Participants with moderate to severe IBS-D were provided a 2-week standard diet, then 4 weeks of a VLCD (20 grams of carbohydrates/day). A responder was defined as having adequate relief (AR) of gastrointestinal symptoms for 2 or more weeks during the VLCD. Changes in abdominal pain, stool habits, and quality of life (QOL) were also measured.
Of the 17 participants enrolled, 13 completed the study and all met the responder definition, with 10 (77%) reporting AR for all 4 VLCD weeks. Stool frequency decreased (2.6 ± 0.8/day to 1.4 ± 0.6/day; p<0.001). Stool consistency improved from diarrheal to normal form (Bristol Stool Score: 5.3 ± 0.7 to 3.8 ± 1.2; p<0.001). Pain scores and QOL measures significantly improved. Outcomes were independent of weight loss.
A VLCD provides adequate relief, and improves abdominal pain, stool habits, and quality of life in IBS-D."

[9] Evidence type: review

Suma Magge, MD and Anthony Lembo, MDcorresponding author
Gastroenterol Hepatol (N Y). 2012 Nov; 8(11): 739–745.

A low-FODMAP diet appears to be effective for treatment of at least a subset of patients with IBS. FODMAPs likely induce symptoms in IBS patients due to luminal distention and visceral hypersensitivity. Whenever possible, implementation of a low-FODMAP diet should be done with the help of an experienced dietician. More research is needed to determine which patients can benefit from a low-FODMAP diet and to quantify the FODMAP content of various foods, which will help patients follow this diet effectively."

similarities between germ-free mice and ketogenic humans

similarities between germ-free mice and ketogenic humans

tracing a chain of ideas

Sometimes the assumptions that scientists start with about what is “good”, “healthy”, or “normal”
can cause them to interpret results in a completely different way than someone starting with different assumptions would have.
Then, the resulting conclusions become the assumptions in the next round of interpretation,
leading to a chain of logic in which one questionable assumption leads to another.
We recently read a paper in which the authors made a series of logical steps,
and it became almost comical to us how at each step we would have interpreted
their results in an opposite way than they did.
When the results of their experiment are looked at from our perspective, it
suggests an intriguing hypothesis:
Maybe some of the health benefits a ketogenic diet are due, not just to the
diet being low in digestible carbohydrate and thus leading to ketosis, but
also to being low in indigestible fiber and thus starving certain gut

Or, to phrase the same hypothesis differently, maybe one mechanism by which a
glycolytic or high-fiber diet causes health problems is that it feeds harmful gut bacteria,
and the presence of those bacteria causes the health problems.
If that hypothesis were true, it would imply that if you are eating a
low-carb diet, then including a lot of low-carb vegetables would feed these
hypothesized harmful gut bacteria and reduce some of the potential health benefits of
a low-carb diet.

in brief

The purpose of this article is two-fold:

  • First, to compare the authors’ interpretations of the observations to ours, given what we know about the metabolic effects of ketogenic
    We draw attention to the fact that the metabolism of germ-free mice is strikingly similar to that of ketogenic dieters.
    This similarity holds at the whole-body level in terms of behaviour and physical characteristics, as well as the level of mitochondrial
    We show that these characteristics appear to be beneficial.
  • Second, to raise the following questions:
    Are some of the benefits of a ketogenic diet mediated by starving gut bacteria, and if so,
    does eating fiber (i.e. low-carb vegetables) reduce some of the health benefits of a keto diet?
    Would eating a carbohydrate- and fiber- free diet confer some keto-like benefits even in the absence of ketosis?

the end of the chain

It all started when we saw the following statement on the Wikipedia page about butyrate :

"Butyrates are important as food for cells lining the mammalian colon
(colonocytes). Without butyrates for energy, colon cells undergo
autophagy (self digestion) and die.[1] Short-chain fatty acids, which
include butyrate, are produced by beneficial colonic bacteria
(probiotics) that feed on, or ferment prebiotics, which are plant
products that contain adequate amounts of dietary fiber."

The topic of butyrate is exciting to some scientists, because they have the idea that eating indigestible fiber is good for human health.
Epidemiological studies have found correlations between high fiber intake and relatively less disease.
However, randomised controlled trials have repeatedly failed to confirm the hypotheses that the fiber intake was actually protective. [1].
Therefore when a new idea comes up that might explain how eating fiber would be good for human health, scientists still hoping for such evidence latch onto it.
Butyrate is an example of such a candidate mechanism for how eating fiber would be good for human health.
To us, since we think that eating fiber is useless (at best) for health,
the statement above poses a challenge and a mystery.
Almost the only source of butyrate in the human body is, as the wikipedia page explains in the excerpt above,
from gut bacteria digesting fiber that you ate.
(There’s also some butyrate in butter, presumably made the same way in cows.)
If butyrate is necessary for the health and even the survival of colon cells,
wouldn’t that mean that a low-fiber diet — such as an all-meat diet — would
be very unhealthy?
Amber hasn’t eaten any significant amount of indigestible fiber in more than four years;
does this mean that her colon cells have died off?
So we set out to investigate what led to that statement on wikipedia.
Our investigation ultimately led to an intriguing hypothesis about a candidate mechanism to explain some of the health benefits of a keto diet.
The paper referenced as "[1]" on the wikipedia page is Donohoe-2011-“The Microbiome and Butyrate Regulate Energy Metabolism and Autophagy in the Mammalian Colon”. We read it with interest.
The authors of this paper performed a good experiment, made precise measurements, got interesting results, and clearly reported their results.
But when it came to interpretation, they started with some assumptions we don’t think are warranted,
and therefore produced a chain of reasoning that eventually led them and their readers, such as the authors of the wikipedia page, to conclusions that are opposite from ours.

similarities between germ-free mice and ketogenic dieters

If you have followed the debates about low-carb diets conferring a metabolic advantage (demonstrated by their superior performance as weight loss diets [2], [3])
then the above description of germ-free mice should sound familiar.
Compared to low-fat dieters, ketogenic dieters tend to be leaner (i.e. have a higher ratio of muscle to body fat), and have lower insulin and blood glucose levels.
This can happen despite similar caloric intake.
Their liver glycogen levels are also lower; ketogenesis may depend on low glycogen levels [4].
However, the germ-free mice are not ketogenic, presumably because they are consuming regular, glucose-plentiful diets.
In fact they are less ketogenic than the conventional mice, as measured by beta-hydroxybutyrate in the blood.
So what is the cause of the similarity in metabolism between germ-free mice and ketogenic humans?

There is one other important way in which the germ-free mice were different from the conventional mice.
They had lower NADH/NAD+ ratios and ATP levels (per mitochondrion) in their colon cells, but not in the liver, heart, or kidneys.
Note that the heart, liver and kidneys favour fat metabolism, even in glycolytic (non-ketogenic) dieters [5].
The authors took this to be further evidence that the mice were in a state of energy deprivation,
even though by all accounts they appeared to be using substantially more energy.
They even previously mentioned this in connection with the reduced fatness:

"[Germ-free] mice exhibit increased locomotor activity. Therefore, the increased food consumption and decreased body fat of germ-free mice may simply be due to increased energy expenditure." — from Donohoe-2011

However, it is a mistake to assume that lower NADH/NAD+ ratios and ATP levels per mitochondrion corresponds to less cellular energy.
In fact, it is likely to be the opposite.

mitochondrial energetics is the commonality

There are three other conditions we know of that reduce the NADH/NAD+ ratio: calorie restriction [6], ketogenic diets [7], and the diabetes drug metformin [8].
Ketogenic diets share mechanisms with caloric restriction.
Indeed, it seems likely that benefits of calorie restriction come from the activation of ketone bodies [9].
When you use fat and ketones for fuel instead of glucose, you produce fewer free radicals through reducing the NADH/NAD+ ratio [6].
This is probably the main mechanism by which it achieves the neuroprotection we mentioned in a recent post.
Similarly, the reduction of the NADH/NAD+ ratio is probably one of the mechanisms by which calorie restriction can increase lifespan [5].
Calorie restriction also preserves ATP production, but it does this by increasing the number of mitochondria to match or exceed the lower ATP yield per mitochondrion [10], [11].
There is preliminary evidence that a ketogenic diet also increases mitochondrial number [12], [13].
So the idea of Donohoe et al. — that total ATP production is compromised because the NADH/NAD+ ratio in the individual mitochondrion has lowered ATP output per mitochondrion — seems unwarranted.
Instead, there is likely to be a compensatory increase in mitochondrial number.
That would be consistent with the fact that the mice appear to have more energy, not less.
Ketogenic dieters have also been measured to have more energy expenditure than low-fat dieters [14].
So, a reduction of NADH/NAD+ ratio is associated with health benefits, and proposed longevity mechanisms.
As you might now suspect, previous studies have shown that germ-free animals have increased lifespans [15].
(They also show decreased anxiety [16] and increased bone mass [17]. Once again, to us this sounds like a better kind of mouse to be!)

fiber-free for better health?

Given this observation — that some of the benefits of ketogenic diets are present in mice that don’t have gut bacteria which process dietary fiber, even though the mice are not in ketosis — it raises the following questions:

  1. Could the starvation of gut bacteria be a part of the mechanism of the benefits of ketogenic diets?
  2. Since butyrate restores the mitochondrial working of the cells to be like the conventional controls (which, from our perspective, is a worse physiological state), could fiber be actually counter-productive to a ketogenic diet?
  3. In analogy to the way the putative benefits of fiber may simply be that they displace refined carbohydrates in the diet, could the reason probiotics can lead to improved health be not because they are beneficial, but because they push out more harmful strains [18]?
  4. Could a diet free of carb and fiber (i.e. one extremely low in plants) have benefits independent of its tendency to be ketogenic?

We don’t have enough evidence to settle these questions, but they are interesting hypotheses that come directly from the results of this study.

in sum

  • Contrary to the conclusions of the authors and Wikipedia editors that butyrate is necessary for cell energy, we interpret the results as showing improved cellular energy in the absence of butyrate.
  • We now have another source for making hypotheses about potentially important cellular metabolism. Before we learned about germ-free mice, we could already use mechanisms discovered from caloric restriction and compare them to mechanisms of ketogenic diets. Now we can compare and contrast mechanisms from caloric restriction, ketogenic diets, and germ-free animals.
  • This raises some interesting (perhaps even provocative) questions about the health effects of dietary fiber and gut flora on human health.


[1] Evidence type: review

Carla S Coffin, MD FRCPC and Eldon A Shaffer, MD FRCPC
Can J Gastroenterol. 2006 April; 20(4): 255–256.

(emphasis ours)
"A recent pooled analysis of 13 prospective cohort studies (6) found that dietary fibre was not associated with a reduced risk of colorectal cancer after adjusting for other dietary risk factors. The Cochrane collaboration (7) systematically reviewed five studies of over 4000 subjects for the effect of dietary fibre on the incidence or recurrence of colorectal adenomas and incidence of colorectal cancer over a two-to four-year period. The population included all subjects that had adenomatous polyps but no history of colorectal cancer or a documented ‘clean colon’ at baseline with follow-up colonoscopy. Study interventions included soluble and insoluble dietary fibre or a comprehensive dietary intervention with high fibre whole food sources. The combined data showed no outcome difference between the intervention and control groups in the number of subjects with at least one adenoma or a new diagnosis of colorectal cancer. The Cochrane reviewers (7) concluded that there was no evidence from randomized controlled trials to suggest that increased dietary fibre intake would reduce the incidence or recurrence of adenomatous polyps.
"Widespread popular media advertisements have purported the benefits of soluble fibre in lowering the risk of atherosclerotic coronary artery disease, mainly by modifying the main coronary artery disease risk factors (ie, dyslipidemia, diabetes and obesity). As for diabetes, high fibre diets slow the postprandial rise in blood glucose and thus, improve glycemic control (8). In dyslipidemic patients, pundits have proposed that psyllium lowers serum cholesterol by binding bile acids in the intestinal lumen resulting in decreased absorption and increased fecal excretion. The ensuing bile acid depletion increases hepatic demand for the de novo synthesis of bile acids from cholesterol. Investigating this mechanism, Van Rosendaal et al (9) found that fibre administration had no effect and certainly did not lower serum cholesterol. Similarly, an earlier study (10) comparing the effect of wheat bran on serum cholesterol of hyperlipidemic and normolipidemic controls showed no change in total cholesterol or ratio of low density lipoprotein to high density lipoprotein cholesterol. Another trial (11) of intensive dietary advice regarding fat, cereal fibre and fish intake on diet and mortality of men with a recent history of myocardial infarction did not find any substantial long-term benefit. The authors admitted to limitations of dietary data in the study (ie, only short-term period of advice and limited number of questions), but there was no evidence to guide decisions about value of dietary advice to increase fish or cereal fibre by people with coronary disease. We await the results of three Cochrane protocols undertaken to review the evidence of dietary fibre in fruits and vegetables, wholegrain cereals or high-fat, low fibre dietary intervention in the prevention of coronary heart disease (12–14). Any conclusions regarding the effectiveness of fibre for the prevention of heart disease appear premature."
"In one of the first randomized, placebo-controlled trials of the role of bran in patients with [diverticular disease] (17), the authors concluded that dietary fibre supplements do nothing more than relieve constipation, and the impression that fibre helps [diverticular disease] is “simply a manifestation of western civilization’s obsession with the need for frequent defecation”. Recent systematic reviews (18,19) of the role of dietary fibre and [diverticular disease] (both asymptomatic diverticulosis and symptomatic diverticulitis) conclude that most of the positive evidence of the effects of fibre supplementation in treating or preventing disease is from retrospective analyses with inherent limitations and high risk of bias."
"Systematic reviews have shown that the treatment of IBS patients with fibre is controversial. One recent meta-analysis of 17 randomized controlled trials (20) quantified the effectiveness of different types of fibre. The reviewers found that fibre was only marginally effective in terms of global symptom improvement or constipation and there was no effect in IBS related abdominal pain. Fibre has a role in treating constipation but its value for IBS, pain and diarrhea is controversial. Any effectivenss of fibre in the long-term management of IBS remains questionable. Clinically, bran is no better than placebo in the relief of the overall symptoms of IBS, and is possibly worse than a normal diet for some symptoms."

[2] Evidence type: review of randomised controlled trials in humans.

Hession M, Rolland C, Kulkarni U, Wise A, Broom J.
Obes Rev. 2009 Jan;10(1):36-50. doi: 10.1111/j.1467-789X.2008.00518.x. Epub 2008 Aug 11.

There are few studies comparing the effects of low-carbohydrate/high-protein diets with low-fat/high-carbohydrate diets for obesity and cardiovascular disease risk. This systematic review focuses on randomized controlled trials of low-carbohydrate diets compared with low-fat/low-calorie diets. Studies conducted in adult populations with mean or median body mass index of > or =28 kg m(-2) were included. Thirteen electronic databases were searched and randomized controlled trials from January 2000 to March 2007 were evaluated. Trials were included if they lasted at least 6 months and assessed the weight-loss effects of low-carbohydrate diets against low-fat/low-calorie diets. For each study, data were abstracted and checked by two researchers prior to electronic data entry. The computer program Review Manager 4.2.2 was used for the data analysis. Thirteen articles met the inclusion criteria. There were significant differences between the groups for weight, high-density lipoprotein cholesterol, triacylglycerols and systolic blood pressure, favouring the low-carbohydrate diet. There was a higher attrition rate in the low-fat compared with the low-carbohydrate groups suggesting a patient preference for a low-carbohydrate/high-protein approach as opposed to the Public Health preference of a low-fat/high-carbohydrate diet. Evidence from this systematic review demonstrates that low-carbohydrate/high-protein diets are more effective at 6 months and are as effective, if not more, as low-fat diets in reducing weight and cardiovascular disease risk up to 1 year. More evidence and longer-term studies are needed to assess the long-term cardiovascular benefits from the weight loss achieved using these diets."

[3] Evidence type: review of randomised controlled trials in humans.

Although this analysis is not peer-reveiwed, it is thorough, appears to be accurate, and does not omit any counter-evidence as far as we are aware.
Kris Gunnars
October 15, 2013
Authority Nutrition

(emphasis ours)
"In this article, I have analyzed the data from 23 of these studies comparing low-carb and low-fat diets.
"All of the studies are randomized controlled trials, the gold standard of science. All are published in respected, peer-reviewed journals.

"The majority of studies achieved statistically significant differences in weight loss (always in favor of low-carb). There are several other factors that are worth noting:

  • The low-carb groups often lost 2-3 times as much weight as the low-fat groups. In a few instances there was no significant difference.
  • In most cases, calories were restricted in the low-fat groups, while the low-carb groups could eat as much as they wanted.
  • When both groups restricted calories, the low-carb dieters still lost more weight (7, 13, 19), although it was not always significant (8, 18, 20).
  • There was only one study where the low-fat group lost more weight (23) although the difference was small (0.5 kg – 1.1 lb) and not statistically significant.
  • In several of the studies, weight loss was greatest in the beginning. Then people start regaining the weight over time as they abandon the diet.
  • When the researchers looked at abdominal fat (the unhealthy visceral fat) directly, low-carb diets had a clear advantage (5, 7, 19).
[4] Evidence type: review of experiments

McGarry JD, Foster DW.
Arch Intern Med. 1977 Apr;137(4):495-501.

A two-site, bihormonal concept for the control of ketone body production is proposed. Thus, ketosis is viewed as the result of increased mobilization of free fatty acids from adipose tissue (site 1) to the liver (site 2), coupled with simultaneous enhancement of the liver’s capacity to convert these substrates into acetoacetic and beta-hydroxybutyric acids. The former event is believed to be triggered by a fall in plasma insulin levels while the latter is considered to be effected primarily by the concomitant glucagon excess characteristic of the ketotic state. Although the precise mechanism whereby elevation of the circulating [glucagon]:[insulin] ratio stimulates hepatic ketogenic potential is not known, activation of the carnitine acyltransferase reaction, the first step in the oxidation of fatty acids, is an essential feature. Two prerequisites for this metabolic adaptation in liver appear to be an elevation in its carnitine content and depletion of its glycogen stores. Despite present limitations the model (evolved mainly from rat studies) provides a framework for the description of various types of clinical ketosis in biochemical terms and may be useful for future studies."

[5] Evidence type: authority

El Bacha, T., Luz, M. & Da Poian, A. (2010)
Nature Education 3(9):8

"[M]any different cells do oxidize fatty acids for ATP production. Between meals, cardiac muscle cells meet 90% of their ATP demands by oxidizing fatty acids. Although these proportions may fall to about 60% depending on the nutritional status and the intensity of contractions, fatty acids may be considered the major fuel consumed by cardiac muscle.

Other organs that use primarily fatty acid oxidation are the kidney and the liver."

[6] Evidence type: controlled non-human animal experiments

Su-Ju Lin, Ethan Ford, Marcia Haigis, Greg Liszt, and Leonard Guarente.
Genes Dev. 2004 January 1; 18(1): 12–16.

"Our studies show that a switch to oxidative metabolism during CR increases the NAD/NADH ratio by decreasing NADH levels. NADH is a competitive inhibitor of Sir2, implying that a reduction in this dinucleotide activates Sir2 to extend the life span in CR. Indeed, overexpression of the NADH dehydrogenase specifically lowers NADH levels and extends the life span, providing strong support for this hypothesis. Regulation of the life span by NADH is also consistent with the earlier finding that electron transport is required for longevity during CR (Lin et al. 2002). The NAD/NADH ratio reflects the intracellular redox state and is a readout of metabolic activity. Our findings suggest that this ratio can serve a critical regulatory function, namely, the determination of the life span of yeast mother cells. It remains to be seen whether this ratio will serve related regulatory functions in higher organisms."

[7] Evidence type: controlled non-human animal experiments

Marwan Maalouf, Patrick G. Sullivan, Laurie Davis, Do Young Kim, and Jong M. Rho Neuroscience.
2007 March 2; 145(1): 256–264.

"[W]e demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress."

[8] Evidence type: controlled non-human animal experiment

Paul W Caton, Nanda K Nayuni, Julius Kieswich, Noorafza Q Khan, Muhammed M Yaqoob and Roger Corder
J Endocrinol April 1, 2010 205 97-106

(emphasis ours)
Metformin increases SIRT1 in db/db mice
Systemic activation of SIRT1 with the activator SRT1720 is reported to lower blood glucose and improve insulin sensitivity in Zucker rats and diet-induced obese mice in part through inhibition of hepatic gluconeogenesis (Milne et al. 2007). Therefore, we investigated whether metformin inhibited gluconeogenesis through changes in hepatic SIRT1. Eight-week-old db/db or control (db/m) mice were administered metformin (250 mg/kg per day; 7 days). Levels of SIRT1 protein, activity and NAD+/NADH ratio were significantly increased in metformin-treated db/db mice compared with the controls and untreated db/db mice (Fig. 1A, C and D). Despite increased protein levels, Sirt1 mRNA levels were unchanged following metformin treatment (Fig. 1B). Levels of SIRT1 protein and activity as well as NAD+/NADH levels were unchanged between the control and untreated mice (Fig. 1A–C). Metformin had no effect on SIRT1 in control mice (data not shown). Furthermore, incubation of HepG2 cells with metformin (2 mM) also resulted in increased levels of SIRT1 protein and activity and NAD+/NADH ratio (Fig. 1E–G). This indicates that increasing SIRT1 protein and activity could be a key mechanism by which metformin inhibits gluconeogenic gene expression."

[9] Evidence type: review of experiments

Marwan A. Maalouf, Jong M. Rho, and Mark P. Mattson
Brain Res Rev. 2009 March; 59(2): 293–315.

"Calorie restriction and the ketogenic diet share two characteristics: reduced carbohydrate intake and a compensatory rise in ketone bodies. The neuroprotective effects of reduced carbohydrate per se are being investigated by several research groups (Mattson et al. 2003; Ingram et al. 2006). We have evaluated the possibility that ketone bodies might mediate the neuroprotective effects of calorie restriction and of the ketogenic diet. An expanding body of evidence indicates that ketone bodies are indeed neuroprotective and that the underlying mechanisms are similar to those associated with calorie restriction – specifically at the mitochondrial level."

[10] Evidence type: review of clinical reports

G. López-Lluch, N. Hunt, B. Jones, M. Zhu, H. Jamieson, S. Hilmer, M. V. Cascajo, J. Allard, D. K. Ingram, P. Navas, and R. de Cabo
Proc Natl Acad Sci U S A. 2006 February 7; 103(6): 1768–1773.

"[M]itochondria under CR conditions show less oxygen consumption, reduce membrane potential, and generate less reactive oxygen species than controls, but remarkably they are able to maintain their critical ATP production. In effect, CR can induce a peroxisome proliferation-activated receptor coactivator 1α-dependent increase in mitochondria capable of efficient and balanced bioenergetics to reduce oxidative stress and attenuate age-dependent endogenous oxidative damage."

[11] Evidence type: review of controlled experiments

Marwan A. Maalouf, Jong M. Rho, and Mark P. Mattson
Brain Res Rev. 2009 March; 59(2): 293–315.

(emphasis ours)
"Slowing of brain aging in calorie-restricted animals was originally believed to result from reduced metabolic activity and, hence, decreased production of reactive oxygen species, a natural byproduct of oxidative metabolism (Wolf 2006). Several studies revealed that calorie restriction was associated with energy conservation (Gonzales-Pacheco et al. 1993; Santos-Pintos et al. 2001) and that mitochondria isolated from calorie-restricted animals produced less ATP than those from controls fed ad libitum, a finding compatible with increased UCP activity (Sreekumar et al. 2002; Drew et al. 2003). However, separate investigations in rodents have suggested that, when adjusted for body weight, metabolic rate does not decrease with calorie restriction (Masoro et al. 1982; McCarter et al. 1985; Masoro 1993). More importantly, calorie restriction prevents the age-related decline in oxidative metabolism in muscle (Hepple et al. 2005; Baker et al. 2006). These data are supported by recent studies indicating that, in contrast to isolated mitochondria, ATP synthesis in intact myocytes and in vivo does not decrease following calorie restriction (Lopez-Lluch et al. 2006; Zangarelli et al. 2006). Additional support is provided by the finding that, in yeast, oxidative metabolism increases with calorie restriction (Lin et al. 2002). […] Although the effects of calorie restriction on ATP generation might appear to contradict those on uncoupling proteins, this discrepancy can be explained by the fact that calorie restriction also promotes mitochondrial biogenesis, thereby enhancing total metabolic output per cell while decreasing mitochondrial production of reactive oxygen species (Diano et al. 2003; Nisoli et al. 2005; Civitarese et al. 2007)."

[12] Evidence type: in vitro non-human animal experiment

Bough KJ, Wetherington J, Hassel B, Pare JF, Gawryluk JW, Greene JG, Shaw R, Smith Y, Geiger JD, Dingledine RJ.
Ann Neurol. 2006 Aug;60(2):223-35.

The full anticonvulsant effect of the ketogenic diet (KD) can require weeks to develop in rats, suggesting that altered gene expression is involved. The KD typically is used in pediatric epilepsies, but is effective also in adolescents and adults. Our goal was to use microarray and complementary technologies in adolescent rats to understand its anticonvulsant effect.
Microarrays were used to define patterns of gene expression in the hippocampus of rats fed a KD or control diet for 3 weeks. Hippocampi from control- and KD-fed rats were also compared for the number of mitochondrial profiles in electron micrographs, the levels of selected energy metabolites and enzyme activities, and the effect of low glucose on synaptic transmission.
Most striking was a coordinated upregulation of all (n = 34) differentially regulated transcripts encoding energy metabolism enzymes and 39 of 42 transcripts encoding mitochondrial proteins, which was accompanied by an increased number of mitochondrial profiles, a higher phosphocreatine/creatine ratio, elevated glutamate levels, and decreased glycogen levels. Consistent with increased energy reserves, synaptic transmission in hippocampal slices from KD-fed animals was resistant to low glucose.
These data show that a calorie-restricted KD enhances brain metabolism. We propose an anticonvulsant mechanism of the KD involving mitochondrial biogenesis leading to enhanced alternative energy stores."

[13] Evidence type: controlled non-human animal experiments

Srivastava S, Kashiwaya Y, King MT, Baxa U, Tam J, Niu G, Chen X, Clarke K, Veech RL.
FASEB J. 2012 June; 26(6): 2351–2362.

(Emphasis ours)
"We measured the effects of a diet in which d-β-hydroxybutyrate-(R)-1,3 butanediol monoester [ketone ester (KE)] replaced equicaloric amounts of carbohydrate on 8-wk-old male C57BL/6J mice. Diets contained equal amounts of fat, protein, and micronutrients. The KE group was fed ad libitum, whereas the control (Ctrl) mice were pair-fed to the KE group. Blood d-β-hydroxybutyrate levels in the KE group were 3-5 times those reported with high-fat ketogenic diets. Voluntary food intake was reduced dose dependently with the KE diet. Feeding the KE diet for up to 1 mo increased the number of mitochondria and doubled the electron transport chain proteins, uncoupling protein 1, and mitochondrial biogenesis-regulating proteins in the interscapular brown adipose tissue (IBAT). [18F]-Fluorodeoxyglucose uptake in IBAT of the KE group was twice that in IBAT of the Ctrl group. Plasma leptin levels of the KE group were more than 2-fold those of the Ctrl group and were associated with increased sympathetic nervous system activity to IBAT. The KE group exhibited 14% greater resting energy expenditure, but the total energy expenditure measured over a 24-h period or body weights was not different. The quantitative insulin-sensitivity check index was 73% higher in the KE group. These results identify KE as a potential antiobesity supplement."

[14] Evidence type: randomised controlled clinical trial

Cara B. Ebbeling, PhD; Janis F. Swain, MS, RD; Henry A. Feldman, PhD; William W. Wong, PhD; David L. Hachey, PhD; Erica Garcia-Lago, BA; David S. Ludwig, MD, PhD
JAMA. 2012;307(24):2627-2634. doi:10.1001/jama.2012.6607.

"The results of our study challenge the notion that a calorie is a calorie from a metabolic perspective. During isocaloric feeding following weight loss, REE was 67 kcal/d higher with the very low-carbohydrate diet compared with the low-fat diet. TEE differed by approximately 300 kcal/d between these 2 diets, an effect corresponding with the amount of energy typically expended in 1 hour of moderate-intensity physical activity."

[15] Evidence type: review of non-human animal experiments

H A Gordon and L Pesti.
Bacteriol Rev. 1971 December; 35(4): 390–429.

"Two attempts have been made to construct life tables and to determine lesions at natural death in germ-free and conventional animals. One study (105) was conducted in genetically closely linked Swiss Webster mice and included over 300 germ-free and the same number of conventional controls which were introduced into the colony at the age of 12 months (to eliminate the effect of early losses). At natural death, the ages of the mice were (means and standard errors in days 19; females, are given): germ-free males, 723 681 i 12; conventional males, 480 i 10; females, 516 i 10. This pattern of survival rates seemed to continue throughout the course of the experi- ment. In the second study (335), approximately 50 germ-free and the same number of conven- tional ICR mice were introduced into the colony after weaning. At natural death, the age of the mice was (using the same mode of expression): germ-free males, 556 i 43; females, 535 + 46; 41; females, 547 ± conventional males, 536 45. In the first trimester of life, the survival rate was essentially the same in the germ-free and conventional control groups. In the middle third, the germ-free mice displayed an increased survival rate (e.g., 40% cumulative mortality was reached for the combined group of germ-free males and females only at the age of approximately 580 days, whereas for the conventional controls this value was approximately 410 days). Increased mortality of the germ-free group at more advanced age resulted in the similarity of mean ages between the opposing animal groups when all animals participating in the study were considered."

[16] Evidence type: controlled non-human animal experiment

Rochellys Diaz Heijtz, Shugui Wang, Farhana Anuar, Yu Qian, Britta Björkholm, Annika Samuelsson, Martin L. Hibberd, Hans Forssberg, and Sven Pettersson
Proc Natl Acad Sci U S A. 2011 February 15; 108(7): 3047–3052.

"Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior."

[17] Evidence type: controlled non-human animal experiment

Sjögren K, Engdahl C, Henning P, Lerner UH, Tremaroli V, Lagerquist MK, Bäckhed F, Ohlsson C.
J Bone Miner Res. 2012 Jun;27(6):1357-67. doi: 10.1002/jbmr.1588.

The gut microbiota modulates host metabolism and development of immune status. Here we show that the gut microbiota is also a major regulator of bone mass in mice. Germ-free (GF) mice exhibit increased bone mass associated with reduced number of osteoclasts per bone surface compared with conventionally raised (CONV-R) mice. Colonization of GF mice with a normal gut microbiota normalizes bone mass. Furthermore, GF mice have decreased frequency of CD4(+) T cells and CD11b(+) /GR 1 osteoclast precursor cells in bone marrow, which could be normalized by colonization. GF mice exhibited reduced expression of inflammatory cytokines in bone and bone marrow compared with CONV-R mice. In summary, the gut microbiota regulates bone mass in mice, and we provide evidence for a mechanism involving altered immune status in bone and thereby affected osteoclast-mediated bone resorption. Further studies are required to evaluate the gut microbiota as a novel therapeutic target for osteoporosis."

[18] Evidence type: review of experiments and hypotheses

Parvez S, Malik KA, Ah Kang S, Kim HY.
J Appl Microbiol. 2006 Jun;100(6):1171-85.

(emphasis ours)
Probiotics are usually defined as microbial food supplements with beneficial effects on the consumers. Most probiotics fall into the group of organisms’ known as lactic acid-producing bacteria and are normally consumed in the form of yogurt, fermented milks or other fermented foods. Some of the beneficial effect of lactic acid bacteria consumption include: (i) improving intestinal tract health; (ii) enhancing the immune system, synthesizing and enhancing the bioavailability of nutrients; (iii) reducing symptoms of lactose intolerance, decreasing the prevalence of allergy in susceptible individuals; and (iv) reducing risk of certain cancers. The mechanisms by which probiotics exert their effects are largely unknown, but may involve modifying gut pH, antagonizing pathogens through production of antimicrobial compounds, competing for pathogen binding and receptor sites as well as for available nutrients and growth factors, stimulating immunomodulatory cells, and producing lactase. Selection criteria, efficacy, food and supplement sources and safety issues around probiotics are reviewed. Recent scientific investigation has supported the important role of probiotics as a part of a healthy diet for human as well as for animals and may be an avenue to provide a safe, cost effective, and ‘natural’ approach that adds a barrier against microbial infection. This paper presents a review of probiotics in health maintenance and disease prevention."