Effect of Dietary Lipids on the Gut Microbiome: What Do We Know?

Welcome to today's webcast. My name is Angela Zivkovic, and I'm an assistant professor in the department of Nutrition at the University of California at Davis. Today, I will be discussing our current state of knowledge on the effects of dietary lipids on the gut microbiome. I have no conflicts of interest to disclose. Our gastrointestinal tract is home to a complex community of microorganisms, the gut microbiota or the gut microbiome. These intestinal microbes are lower in abundance in the upper intestinal tract, but increase in abundance to about 10 to 12 microbes per gram of pieces in the distal colon. We're still very much at the beginning stages of understanding the complexity of the gut microbiome, how the different organisms interact with each other, and the ecological networks they form. But because sequencing methods are getting better and better and the costs of sequencing are becoming lower every day, we are quickly learning about the various inputs that affect our gut microbiome and the effects these microbes in term have on our health. We know that interactions between our diets, our genotype and our gut microbes affect our health or phenotype. In particular, in this webcast I will be focusing on what we're uncovering about the effects of dietary lipids or fats on the gut microbiome and how, in turn the microbiome affects our intestinal barrier function, immune function and metabolic function, both directly and through the microbial metabolites that are produced and enter the circulation. So first, I'd like to address what we think we know about dietary fat and the gut microbiome. The textbooks will tell you that dietary triglycerides, or fat, represent 30 to 35% of total daily calories, and that we are very efficient at digesting and absorbing those dietary fats. About 4 to 5 grams of fat is thought to reach the colon. And we think that about 3 to 5 grams of fat are excreted in the stool. But are these assumptions correct and reasonable? So addressing first, the assumption that dietary fat represents 30 to 35% of total daily calories, that is the recommended range recommended by the US dietary guidelines. But we know that over 90% of all Americans eat more added fat than is recommended. And also the recent popularity of low carb diets, Atkins diet and even ketogenic diets really is changing that. There's also been a backlash against the low fat diet phase. And we have new cholesterol recommendations that basically state that we do not need to limit dietary cholesterol. So more and more people are eating higher levels of fat. The second assumption is that we digest and absorb dietary fat very efficiently. But we do know that there are populations that have fat malabsorption. For example, in early infancy, the gastrointestinal tract is not developed. In liver and gallbladder disease there can be a lack of bile or there can be obstructions that prevent bile from getting down to the intestinal tract. In pancreatic disease, there's a lack of lipases that can digest those fats. And in cases of mucosal damage. So, for example, in celiac disease, inflammatory bowel disease, enteric infections, in all of these cases, there will be reduced fat absorption. There are also individuals with disorders of lipid metabolism. So all of these populations will have fat malabsorption and will have higher levels of fat getting to the colon. We also know that there are differences in fat absorption depending on a number of factors. So from day to day we can have different efficiency of absorption. Just variability. Time of day also affects how well we digest and absorb fat, for example, there's a study that showed that at dinner there was a slower rate of fat absorption than there is earlier in the day. Meal size can affect how well fat is absorbed, the type of fat, the food matrix, or what other kinds of foods or or ingredients are being consumed with the meal. And genetics and other components, for example, developmental components can affect how well we digest and absorb fat. We also think that about 4 to 5 grams of fat reaches the colon, but that really depends on a number of factors. So if we go through a couple of cases or examples here, we see that if an individual is following the dietary recommendations and is consuming a 30% fat diet and eating about 2000 calories a day, that means that they're consuming about 67 grams of fat. And if they really do absorb at about 95% efficiency, then they really are seeing about 3 to 4 grams of fat reaching the colon. But even just reducing the absorption efficiency to 90% will increase or double the amount of fat that's reaching the colon. And another individual who's eating a higher fat diet, 60% fat on the same number of total calories or 2000 calories per per day, they will be consuming about 133 grams of fat. And if they're absorbing efficiently, they're seeing about 6 to 7 grams of fat reaching the colon and double as much as 13 grams if they're absorption efficiency is reduced to 90%. And then if you have an individual who is consuming a 60% fat diet and eating a higher number of total calories, so about 3500 calories per day, they are eating as much as 233 grams of fat per day. And then we can see as much as 12 and even 24 grams of fat reaching the colon. So we start to realize that there could be significant amounts of fat reaching the colon and they could, in fact, have some significant effects on the microbiome. We know that about 3 to 5 grams of fat are excreted in the stool. And we, in fact, have a way to diagnose fat malabsorption, the fecal fat test. Basically we have individuals consume about 100 grams per day of fat for three days, and if they have malabsorption, we expect to see more than 40 grams of fat excreted. But fecal fat measurement is not a very popular clinical diagnostic. It's not something that's done very routinely. So we actually don't know very much about the variability in the population and how much fat excretion is happening in individuals depending on diet. So the real question is, is there evidence that fat in the diet influences the gut microbiome? One seminal study showed that when we look at the microbe functions by examining the metagenome of stool samples and we compare US populations versus populations that have more traditional diets that are higher in traditional foods that are plant based. So in this case, the researchers looked at Malawian and Amerindian populations. They found that the differences in the microbiomes paralleled the difference in carnivorous versus herbivorous mammals. In other words, the US microbiota looked more like the microbiomes of carnivores like tigers and the microbiomes of Malawian and Amerindian individuals looked more like herbivorous mammals or zebras. So what is this telling us? That the US gut metagenome reflects the Western diet. So degradation of amino acids breaking down of simple sugars, metabolism of xenobiotics, for example, various medications that we take, and also bile salt metabolism functions were enriched, which basically reflects a diet richer in fats. So we can see that the amount of fat in the diet is influencing our gut microbiome. So what do high fat, low carbohydrate diets mean for the microbiota? First of all, with high fat, low carbohydrate diets, there is a reduction in fermentable substrate derived from the carbohydrates. And this means that there's less energy sources for the microbes that specialize in breaking down the fiber. So because humans lack the enzymes needed to break down dietary fibers, we have saccharolytic bacteria that do this for us. So in the small intestine, we absorb those digestible carbohydrates that we can break down. And then the non digestible carbohydrates become fermentable substrates in the large intestine. In the proximal colon, active bacterial growth happens. We have carbohydrate fermentation and high short chain fatty acid production. And then as we move down to the distal colon, there is slower bacterial growth and lower short chain fatty acid production. And you can see that the pH changes across the length of the colon. So with high fat, low carbohydrate diets, as fermentation decreases, so do the byproducts of fermentation. In this case, short chain fatty acids. And when short chain fatty acids decrease, they change the acidity of the intestinal environment. So when short chain fatty acids decrease, we have an increase in the pH of the colonic environment. So in other words, the colon becomes less acidic. So what is the problem with that? Well, just like in certain environments, like in in very cold environments, polar bears prefer to live. But in warmer climates, we have different kinds of mammals that prefer to live. The same thing happens with microbes in a less acidic environment, acid sensitive groups of bacteria will thrive. And unfortunately, these are typically the bad guys, the proteobacteria. Like different strains of E.coli, salmonella, Vibrio Helicobacter. At the same time beneficial microbes that do well in acidic environments drop in abundance. So the butyrate producers and acetate producers, for example, the bifidobacteria will decrease and this will actually become a sort of feedback loop. Those beneficial microbes don't do as well in a more alkaline or less acidic environment, and they will therefore produce less short chain fatty acids. And the production of the lower production of short chain fatty acids will lead to an increase in pH, which again will accommodate the more pathogenic bacteria. We know that high fat diet shifts the gut microbiota. So, for example, a study in mice fed a high fat diet showed an increase in firmicutes, which includes Clostridia Proteobacteria and a decrease in Bacteroidetes. These microbiota changes were seen independent of obesity. So at first we thought that high fat diets cause obesity, and that's why we see the shifts in the gut microbiome. But in fact, this was true even in knockout mice, which do not get obese in a high fat diet. We saw the same changes in the microbiome independent of weight gain. So it's the high fat diet itself that is actually shifting the gut microbiota. The decreases in short chain fatty acids in response to a high fat diet also decrease the beneficial effects of these compounds on the host. So what do short chain fatty acids do? Among other things, they improve insulin sensitivity. They have effects on distal organs, including the brain. They increase satiety in both muscle and adipose tissue. They actually increase insulin sensitivity as well as in the liver. And so these short chain fatty acids are have been shown to be very important to maintaining good metabolic health. High fat diets also induce endotoxemia and diabetes. So in some seminal experiments from about ten years ago, it was shown that high fat diet, high fat diets actually are increasing intestinal permeability. And what we mean by this is that the tight junctions between epithelial cells that line the intestinal layer, become less good at keeping things out from getting between the cells. Usually the correct way for things to get into our body is through transcellular uptake. In other words, those things we intend to absorb, we absorb through cells, through receptor mediated processes. Those things that are sneaking between the cells through paracellular uptake typically are not supposed to be absorbed. And so when we have increases in intestinal intestinal permeability, we have things that are not supposed to be getting in, sneaking between those cells and causing problems. So the high fat diet is increasing the intestinal permeability. And this means that bacterial components such as LPS or Lipopolysaccharide are getting in into the circulation. Lipopolysaccharide then in turn leads to inflammation, both in the adipose tissue and this causes macrophage infiltration and a lot of other effects that are not beneficial to the host. And this in turn has effects on insulin resistance. These effects can be ameliorated by prebiotic supplementation. So in a high fat diet, when we add a prebiotic, in this case it was oligo fructose. We see that we can promote the growth of beneficial bacteria or in this case bifidobacteria. And these bifidobacteria were negatively correlated with the amount of LPS that was getting into the system. So in other words, the beneficial microbes are having direct effects on our gut barrier integrity. They're promoting better tight junction efficiency. But how relevant is all this for humans? So, so far we're seeing effects in mouse models. But is this something that we can actually observe in humans eating real diets? There was one study that was done that showed that in healthy adults, when inhibitor of fat absorption is given, it's called orlistat, w can have a lot of different a lot of variability in the effects of that inhibitor. In this case, when individuals were given the Orlistat, you can see that the variability in the amount of fecal fat that was excreted went all the way from 4 grams to 35 grams. So individuals had a very variable response. The increased fecal fat with the Orlistat treatment was ameliorated with addition of a prebiotic. So what they also covered in the study is that there were no consistent changes in that with higher fat in the colon. So not only was there variability in terms of how much fat made it down to the colon and how much was excreted, there was also a lot of variability in terms of what the effects were and in fact, phylum or family level microbiota changes were not significant because there was so much variability in the effects. Short chain fatty acid production, antioxidant activity, inflammation markers, intestinal barrier function. All of these things were not significantly different. But is this because people were free to choose the source of fat that they were eating? So in this study, people were just told to eat a higher level of fat, but they were not given any specific instructions as to what type of fat they should eat. And does this make a difference? Well, it turns out that it probably does. So in a very nice study that was performed a couple of years ago, it was discovered that there is a big difference whether you eat lard or fish oil. In this study, they used a mouse model and they fed 70% fat. They found that lard was pro-inflammatory and increased gut permeability, whereas fish oil did not. What they found specifically was that lard enriched for bad bacteria such as clostridia and bilophila wadsworthia, whereas fish oil enriched for good bacteria such as Bifidobacteria and Akkermansia muciniphila. So it's not just how much fat you eat, it's what type of fat. What was interesting about this study was that they actually showed the mechanism and how exactly this worked. They found that the lard enriched for certain microbes which increased the intestinal permeability in the mice, and this increase in intestinal permeability meant that there were more microbial factors that were getting into the serum. And once these microbial factors were circulating, they actually triggered inflammation. They showed with a very nice mechanistic detail that in fact the specific activation of TNF-α and the CCL2 gene were involved and they showed that these change were induced through changes in the gut microbiota. But it's even more complicated than that. It's not just the type of fat. It's also the source of fat that matters. So there's also been studies that have shown that what type of saturated fat we eat makes a difference in this case, a a milk fat was fed and the milk fat specifically promoted the growth of a bacterium called bilophila wadsworthia, which is a type of delta proteobacteria. This bilophila wadsworthia consumes the bile acids that are produced to absorb milk, fat and yes, they are found in humans who consume high fat diets. So it's not just something that's happening in mice. What they did find in this study is that mice that were susceptible, so these were IL-10 knockout mice, this bloom of bilophila wadsworthia that happened on the high milk fat diet led to the development of colitis. So this doesn't mean that we should all stop eating milk fat because it's going to give us colitis. It means that certain kinds of fat can cause the growth of specific types of microbes that can then have particular deleterious effects in individuals who are susceptible. But the fatty acid composition of foods is not that simple. So is all milk fat the same? Is all saturated fat the same? It's not. If we look, for example, at the average composition of pork, fat or lard, we see that although it's considered a saturated fat, it actually has about as much, if not a little more monounsaturated fat, depending on exactly what the pigs are fed. Butter, also, it has high levels of saturated fat, but it is not pure saturated fat. It does have monounsaturated and polyunsaturated fat in it as well. And then coconut oil has even higher levels of saturated fat. But interestingly, even just calling the fatty acid saturated versus unsaturated fat is also not quite as details as we need to be because in fact we have different saturated saturation levels, but we also have different lengths of chains. So long chain saturated fats like 16:0 and 18:0 are different from medium chain saturated fats, like 12:0 and 14:0. And so these vary in composition depending on the kind of food that we're looking at. Coconut oil is particularly enriched and medium chain saturated fats, which are different in their effects than the longer chain saturated fatty acids. We have evidence from epidemiological studies that the source of saturated fat matters. In fact, in 2012, we saw that although the intake of saturated fat derived from meat was positively associated with cardiovascular disease risk, dairy derived saturated fat were inversely associated with cardiovascular disease risk. In fact, each 5% unit increase in energy from dairy caused a 40% reduction in risk of cardiovascular disease. So not all saturated fat is the same. And as if it were not complicated enough, dietary lipids are not just triglycerides. We also get fat in the form of phospholipids, sterols, and sphingolipids. And we know that the specific kind of fat phosphatidylcholine is very important for a microbial for the microbial effects. Choline is a precursor for microbial produced trimethylamine. So choline is a compound that our gut microbiota convert to trimethylamine. That trimethylamine is readily absorbed, and once it is in the circulation, our liver mono oxygenases convert that TMA to TMAO or trimethylamine oxide. And it's been shown that trimethylamine oxide or TMAO reduces reverse cholesterol transport and bile acid synthesis, and it's directly related to atherosclerosis. So TMAO independently predicts cardiovascular outcomes even after adjusting for traditional risk factors. As you can see on this graph, the highest quartile of TMAO levels in the bloodstream were associated with significantly higher risk of myocardial infarct, infarction, stroke and, uh so where are these cholines found? Choline and carnitine are both precursors for TMA, and they're found in meats, organ meats, eggs, soy lecithin is also enriched in phosphatidylcholine. Certain dairy products, although not all of them are enriched in phosphatidylcholine and even certain vegetables and mushrooms can be enriched in Phosphatidylcholine. Trimethylamine is also found in fish and seafood and can also contribute to the levels of TMAO in the bloodstream. But there is still a lot we don't know about this. The background diet determines the ability to produce trimethylamine. So we've seen that vegans and vegetarians actually cannot produce TMA. So in this graph, what I'm showing is that labeled trimethylamine oxide was measured in individuals after consuming a meal that had eggs that were labeled with this D3 choline. And what they found was that vegans, vegetarians did not produce any choline in the postprandial state or for the 24 hours following consumption of of this labeled choline. Omnivores, on the other hand did produce TMAO. But as you can see, there was high inter individual variability in the production among the omnivores. So whereas some individuals had very low levels of production that were very similar to vegans or vegetarians, other individuals had 30 times higher levels of production of this metabolite. And we still have not yet fully answered the question whether TMAO production is inherent to an individual's microbiome or whether it changes in response to diet. So the effects of long term diets on changing levels of the amount of TMAO produced have not been fully examined. We do know that in studies that have been published recently, they've basically described that although plasma choline response to three eggs per day increased, the plasma levels of TMAO were not significantly changed. But if you look closely at the graphs, what you'll notice is that there is a high degree of individual variability in the levels of TMAO that were produced. And so it's possible that the reason there were no significant changes seen is that it increased in some individuals but not others, and we still don't know what it is that exactly is controlling. Whether you have a response to dietary modulation of choline levels. Foods may also affect the gut microbiota function, even if they do not affect composition. Studies have shown that when we look at the effects of yogurt, for example, it may not actually change the levels of lactobacillus or other yogurt derived microbes as expected, but it might change the function. So in this particular study, the researchers fed female monozygotic twins fermented milk product or yogurt, and they measured the meta-transcriptomes of the microbes before and after this treatment. What they found was that although the microbial ecology did not change, so the relative abundance of the various species did not change before versus after treatment. The transcription profiles of the microbiota present did change. So what the microbes do. And the same changes occurred in mice consuming the same fermented milk product. So by feeding ourselves, we are feeding our gut microbiota and in turn their metabolism affects us. There are primary plant fiber degrades or saccharolytic bacteria that break down those fibers that we cannot digest and they generate metabolites that secondary glycan integrators can then consume. This is how they form these ecological networks we were talking about. They produce metabolites that then affect the growth of other bacteria and those metabolites can have direct effects on our metabolic tissues. In summary, dietary fat can increase intestinal permeability and increase inflammation. Dietary fat can change the microbiota composition and function, but the effects of dietary fat depend on the source and the composition of that dietary fat, as well as individual, underlying genetic and other factors. All of this is very hard to study in humans. Individuals vary significantly in how much and what type of fat they eat, absorb and digest, and how much this fat intake affects their gut microbiota composition and function. Some people are more sensitive, others are less sensitive to the effects of dietary fat on the microbiota depends on many factors so whether fiber is present, the type of fat, the specific fatty acid composition, the source. And we need more stories of how interactions of diet, genotype and phenotype affect our gut microbiota and in turn how these changes in the gut microbiome affect our specific health outcomes.