To stay up to date on the cutting-edge of health and performance, H.V.M.N. Research Lead Dr. Brianna Stubbs tends to read a lot of scientific literature...a lot. Every month, she will dive into the latest and most exciting research papers by walking us through the experiment process, dissecting the results and implications, and candidly sharing her own thoughts on the study and subject as a whole.

Today, we’ll be going down one of Brianna's personal favorite rabbit holes: How ketones (from both through the ketogenic diet and exogenous ketones) affect inflammation in the body.

Is the ketogenic diet as a whole responsible for the reduction of inflammation? Or is it the specific ketone body BHB (beta hydroxybutyrate) that has the most impact? This is a major question that Brianna brings up and explores in this episode.

Referenced Studies

1. Comparison of Low Fat and Low Carbohydrate Diets on Circulating Fatty Acid Composition and Markers of Inflammation
2. The Ketone Metabolite BHB Blocks NLRP3 Inflammasome-Mediated Inflammatory Disease
3. The Activation of Retinal HCA2 Receptors by Systemic BHB Inhibits Diabetic Retina Damage Through Reduction of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome

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Transcription

Overview

Today, we’ll be going down one of my personal favorite rabbit holes: The way that ketones affect inflammation. Specifically, the ketone body BHB - beta hydroxybutyrate. It’s one of the three ketone bodies, and it’s the present at the highest concentration in the blood. We’ll go through three studies on this topic, where I’ll walk you through the research process, discuss and analyze the results, and muse over the potential implications.

Let’s start from the top with a bit of background on inflammation. I’m sure you’ve heard a lot about it over the years, with it often being portrayed in a negative light. It seems like we are always trying to reduce inflammation. We’ll take a step back and paint a balanced picture, as every process in the body has its purpose.

Inflammation is the body's natural response to injury. A sequence of complicated, interrelated events work to defend the body, ultimately bringing plasma proteins and phagocytes (white blood cells that engulf and consume foreign material and debris) to the injured area for the purpose of initiating tissue repair. Inflammation has also long been a well-known symptom of many infectious diseases, but molecular and epidemiological research increasingly suggests that it is also intimately linked with a broad range of non-infectious diseases...perhaps even all of them.

The association of inflammation with modern human diseases, (like obesity, cardiovascular disease, type 2 diabetes, mellitus, cancer) remains an unsolved mystery of current biology and medicine. The inflammatory response evolved as a protective response to noxious stimuli (which is the fancy way of saying, stuff that might harm us), but inflammation unavoidably occurs at a cost to normal tissue function. This fundamental tradeoff between the cost and benefit of the inflammatory response has been optimized over evolutionary time for specific environmental conditions.

The rapid change of the human environment that has occurred in the last 100 years or so outpaces genetic adaptation through natural selection, leading increasingly to a mismatch between the modern environment and selected traits. Consequently, the multiple tradeoffs that were made over evolutionary time and affect human physiology are not optimized to the modern environment, leading to increased disease susceptibility.

An inflammatory response can be triggered by a variety of noxious stimuli, including infection and injury. Accordingly, inflammatory responses are highly variable in terms of the cell types and molecular mediators involved. Inflammation can be classified in several ways: acute versus chronic and local versus systemic. Despite this complexity, all inflammatory responses can be broken down into four common components that align in a universal configuration of the inflammatory pathway: inducers, sensors, mediators, and target tissues.

Inflammatory inducers can be exogenous signals (e.g. bacteria, viruses or toxins) or endogenous signals (e.g. ATP or urate crystals- which are fine for us if they are in the right place in our body, but trigger inflammation when damage means they leak into places they shouldn’t be). These signals report on tissue stress, injury, or malfunction. Sensors can be cells, such as tissue-resident macrophages and mast cells, or special protein complexes such as the ‘inflammasome’. Sensors detect inducers with specific receptors and respond by producing inflammatory mediators. Depending on the nature of the inducers, sensors produce different combinations and amounts of mediators, creating a unique mediator signature for the inducer. These inflammatory mediators, in turn, act on target tissues and alter their functional states, promoting elimination of the inducers, adaptation to the noxious state, and restoration of normal tissue function.

So...do we want and need inflammation to survive? The answer is YES, absolutely. But, it is one of those things that I like to call a ‘Goldilocks’ problem- we don’t want too much, we don’t want too little- it needs to be just right. Now that we have the basics fresh in our minds, let’s take a look at the three papers demonstrating positive effects of ketones on inflammation.

Comparison of Low Fat and Low Carbohydrate Diets on Circulating Fatty Acid Composition and Markers of Inflammation

The first paper we will look at today addresses how long-standing, low-level inflammation that bubbles away in people with metabolic disease can be improved through a low carb, ketogenic diet. It was written in 2008 by some of the leading researchers in the keto space- Dr Jeff Volek and Dr Stephen Phinney, who are pioneering the widespread adoption of the ketogenic diet through telemedicine, as well as conducting cutting edge research studies into the effects of the diets.

Published in the journal ‘Lipids’, this study looked at the effects of a 12 week long ketogenic diet on blood biomarkers in 40 overweight men and women who had metabolic syndrome.

Another quick bit of important background here. Metabolic syndrome is generally defined by high fasting glucose, high triglycerides, high blood pressure and waist circumference, and low HDL cholesterol. New markers that appear to be associated with metabolic syndrome include disturbed circulating fatty acid composition, perturbed lipid metabolism and increased oxidative stress and inflammation. Fatty acids themselves contribute to overall inflammatory balance by several mechanisms. In a subset of sensor cells called macrophages, fatty acids activate receptor signaling leading to activation of a transcription factor that regulates over 100 genes. Many of these downstream genes have a role in inflammatory responses and atherosclerosis, and may therefore represent a crucial link between fatty acids, metabolic syndrome and atherogenesis.

Another way fatty acids contribute to inflammation is through conversion into pro-inflammatory metabolites, either by enzymes or by reactions with oxygen based free radicals. One example is arachidonic acid in membranes, which is commonly thought to have a deleterious effect on overall inflammatory balance.

Now, back to the study. The participants were asked to follow a diet that was either low fat, with less than 10% of calories from fat, or a diet that was around 60% of energy from fat...so nearly keto. They had weekly follow up counselling, and kept week long diet diarys for week 1, 6 and 12 of the study. They had blood samples taken in the morning after an overnight fast at the start and end of the study.

This paper describes the changes in the amounts and types of fatty acids in the blood pre and post diet, along with the level of inflammatory mediators that could be related to these fatty acids. In terms of overall changes in their metabolic syndrome, the researchers found there was a clear advantage of the low carbohydrate diet over the low fat diet. The keto diet group lost more weight, more fat mass, had better glycemic control, improved insulin sensitivity, and better blood work specifically with regard to triglycerides and HDL (which is known informally as the ‘healthy’ kind of cholesterol). Cholesterol is a hefy and nuanced topic, make sure to check out our podcast episode with Dave Feldman. I’ve listened twice already, and each time I learn something new.

The researchers also looked at biomarkers that might indicate that someone was at risk for cardiovascular disease, with a detailed analysis of lipoprotein types and ratios. These markers also went in favor of the ketogenic diet group. Now, what about the main area of interest here: Inflammation?

Despite the two diet groups consuming roughly the same caloric intake and all losing at least some weight, there were larger reductions in the keto group in many biomarkers of inflammation. The levels of the mediator molecules TNF-a, IL-8, MCP-1, PAI-1, E-selectin and I-CAM all went down. These markers showed little change on the low fat group, suggesting that it is the macronutrient composition not weight loss or caloric reduction that is key. The researchers noted that most of the inflammatory markers did not correlate with weight loss. A correlation would not have proved that weight loss caused change in inflammatory markers, but the lack of correlation makes it extremely unlikely.

I’m going to throw out a curve ball here. One result that doesn’t fit the expected picture is that the low fat dieters had LOWER levels of the pro-inflammatory fatty acid that we mentioned earlier: arachadonic acid. Levels were actually increased in the keto group. The researchers suggest that the increase in plasma arachidonic acid with the keto group is best explained by decreased degradation, which is presumably due to less interaction with reactive oxygen species. If more arachadonic acid is consumed in a higher fat diet and less arachadonic acid is broken down into those pro-inflammatory downstream end products, the net effect would be less inflammation and more arachadonic acid in the membranes. The paper suggests that, rather than being a negative factor within lipid membranes, increased arachidonic acid appears to be a beneficial outcome of weight- reducing diets.

This paper is now often cited as one of the first studies that really started to unpick the beneficial non-weight loss effects of the ketogenic diet, and there certainly were some striking observations.

The Ketone Metabolite BHB Blocks NLRP3 Inflammasome-Mediated Inflammatory Disease

On to the second paper!

So, we have seen that the ketogenic diet can alter overall inflammation status, but the previous paper focused in on the role of the fatty acids from the diet on blood lipid profiles, presumably because fat consumption increases so much on the ketogenic diet.

But in 2015, a new paper was published in the journal ‘Nature Medicine; that revealed that it might not just be the type and amount of fat and carbs in the diet that could regulate inflammation. In the study described in the paper, the key endpoint biomarker of the ketogenic diet, beta-hydroxybutyrate (or BHB) itself, was found to directly affect inflammation.

Through a series of elegant studies, the researchers described how the compound BHB directly inhibits NLRP3, which is part of a complex set of proteins called the inflammasome that are a part of our innate immune system. The inflammasome drives the inflammatory response in several disorders including autoimmune diseases, type 2 diabetes, Alzheimer’s disease, atherosclerosis, and autoinflammatory disorders.

This was exciting as BHB is a key biomarker that links the anti-inflammatory effects we just heard about with respect to the ketogenic diet, with the well known inflammation busting effects of fasting and calorie restriction. Up until this study pointed the finger at BHB, it was unclear how immune cells adapt to reduced availability of glucose in all these states and if the cells can respond to metabolites produced from fat oxidation.

Working with mice and human immune cells, the researchers focused on how macrophages — specialized immune cells that produce inflammation — respond when exposed to ketone bodies and several different types of noxious stimuli and ultimately whether the BHB impacts the inflammasone complex. The cell experiments showed that BHB inhibited the inflammatory cascade in a dose dependant way. What’s more, the doses tested were 1mM to 10mM, in the same range as the levels obtained through fasting, the ketogenic diet or exogenous ketones. They also tested other ketone bodies, acetoacetate and the butyrate molecule, and these compounds did not have a helpful effect. But very interestingly, there was some activity of the non natural form of BHB: L-BHB.

The researchers looked at other types of inflammasome, not just NLRP3- and found that BHB was specific and only interacted with the NLRP3 pathway. They then dug around using different methods to target all the possible ways that BHB might actually have this effect. They looked at oxidative stress and changes in internal metabolites, which were not important, before finding that an effect of BHB on the flux of the ion potassium into the cells was key. Armed with this new understanding from their in vitro experiments, the researchers did animal experiments to see if these effects occurred in a whole organism. Firstly, they took mice and injected them with BHB that was modified so it wasn’t cleared by metabolism as quickly as usual. They then injected a bacterial toxin called LPS, which often strongly activates the inflammasome. In the BHB injected mice, there were fewer white cells that migrated into the site of infection and lower levels of pro-inflammatory mediators. They also looked at a genetically modified mouse that rapidly developed an inflammatory condition of the joints called gout. In these mice, they used an acetoacetate ketone diester to raise ketone levels, and saw that this was protective against the some of the symptoms of gout.

The researchers conclude very neatly: “Our findings suggest that the fasting- or exercise- induced metabolite BHB inhibits the NLRP3 inflammasome in immune cells independently of binding to surface signalling receptors or undergoing mitochondrial oxidation. Thus, in states of extreme energy deficit such as starvation, metabolic signals such as BHB can dampen innate immune responses, sparing energy for the functioning of ketone-dependent organs such as the brain and heart.”

To me, whilst it make sense and is a nice story that we evolutionarily needed to dampen the immune response to spare energy during energy scarcity, the practical utility of the observation in the modern setting is even more impactful. So many pernicious conditions are driven by uncontrolled or unnecessary inflammation. Therefore, the observation that a molecule like BHB, that we can boost in many simple ways could help us to control this problematic process is exciting and could have pretty broad applications. But there is a lot of work still to be done to work out how much BHB could contribute in the many different conditions where inflammation is a problem.

The Activation of Retinal HCA2 Receptors by Systemic BHB Inhibits Diabetic Retina Damage Through Reduction of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome

And lastly, let’s dive into our our third paper.

After the breakthrough study that we just discussed, more and more papers have been published specifically looking at the role of BHB and the NLRP3 inflammasome in several disease models, including gout, neuroinflammation, etc. This final paper was published in January 2019 by the journal PLOS One.

It’s quite a short and sweet set of experiments that used a mouse model of diabetes to look at inflammation in the back of the eye, which is called the retina. The retina is the light sensitive part of our eye that is responsible for seeing detail. Retinal damage is one of the most common complication of diabetes, occuring in about one half of type I and II diabetics, and is a major cause of several visual impairments leading to adult blindness. The potential of going blind as a result of poor blood sugar control and diabetes probably doesn’t get enough air-time, and was something I found personally terrifying when I learned about just how common this is.

Part of what causes diabetes-induced retinal damage is a chronic low-grade inflammatory state. This results in the increased leakiness of the blood-retinal barrier, and ultimately leads to lack of sufficient blood supply and cell damage. In this study, the researchers wanted to find out if BHB had any effect on retinal inflammation in a mouse model of diabetes. They wanted to look to see if there was a role of the NLRP3 pathway, but also if there was an extra effect of BHB binding to a cell surface receptor called HCA2. It was already known that BHB could bind to HCA2, and that the receptor when active had some anti inflammatory and anti-oxidant effects that could also play a protective role.

The researchers made the mice diabetic using an injection of a toxin that killed off cells in the pancreas. They then injected the mice with BHB at increasing doses, reaching blood ketone levels of 0.2 up to 1mM. The first thing that they saw was that the mice with diabetes had higher levels of HCA2 and higher levels of cellular stress markers. In the diabetic mice that had been injected with BHB, there were fewer signs of NLRP3 activation- specifically, the inflammatory mediators IL-1 beta and IL-18 were lower. In essence, this demonstrates that BHB was directly protective against inflammation in the eye that results from diabetes.

To me, this paper is certainly interesting and expands on the potential use cases and indications for methods that elevate BHB. However, the study is overall less complete than the previous paper we discussed. This leaves me with a few unanswered questions.

The researchers spend a while talking about HCA2 and the effect of BHB, but to be sure that there is a link, perhaps they should have either used a pharmacological blocker of HCA2 or else genetically modified their mice to not express HCA2, to see if this changed the degree of benefit offered by BHB, that way they could have been more sure there was a connection. To me, clearly NLRP3 is involved and the results here support that but it’s not too clear about HCA2. Just because it is known that BHB binds, it doesn’t mean that it is the driving factor for the beneficial effects on the retina.

Conclusion

So, there we are. It is pretty tremendous to see how rapidly our understanding of inflammation has grown in the last few decades as analytical techniques have got more and more sophisticated.

To restate some of the points in this episode, we have talked about how changing fatty acid profiles with a ketogenic diet can affect inflammation, but also about how the key endpoint of the ketogenic diet, BHB also directly regulated inflammation. It is interesting to speculate about the relative contribution of diet (for example what types of fat you eat and how much you eat) and BHB on inflammation. As yet there aren’t well run studies that directly contrast the ketogenic diet with the the isolated effect of giving BHB through an exogenous source. For me, this is one of the biggest questions that researchers need to address- in terms of inflammation control, how important is it that you follow the keto diet, or can you use brief periods of fasting or exogenous ketones to get similar health benefits. We have so much work still to do in this field.

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