In a new study published in Frontiers in Cardiovascular Medicine, our research team at the University of California, Davis reveals how a single 36-hour water-only fast can lead to major changes in fat molecules in the blood. In an earlier study, we showed that going without food for 36 hours causes big changes to the thousands of small molecules circulating in blood plasma. We found that certain chemicals produced by the body during a 36-hour fast helped strengthen immune cells called macrophages. These fasting chemicals also increased the typical lifespan of a worm species by as much as 96%. In the new paper, we analyzed the blood samples of the same 20 healthy, young people after an overnight fast, after eating, after a 36-hour water-only fast, and after eating again after the 36-hour fast. As expected, we found that 36 hours of fasting markedly increased free fatty acids, molecules that can be used for energy, while decreasing triglycerides, a type of fat found in blood that when elevated can increase the risk of cardiovascular disease. Fasting for 36 hours also decreased two types of fat molecules called lysophospholipids, which are linked to inflammation, cardiovascular disease, Alzheimer’s disease, and other health conditions when elevated. According to our findings, even a short fast of a day and a half can “vastly remodel” fats in the bloodstream, which may have benefits for cardiometabolic health and healthy aging. Bouts of 36 hours of fasting may be useful to “reset” plasma lipids toward a more beneficial profile in individuals whose blood lipids put them at risk for cardiovascular disease, neurodegenerative disease and other metabolic conditions. However, researchers do not know yet how many bouts of prolonged fasting would be beneficial in different individuals, how frequently these fasts should be performed for maximal benefit, and how best to manage the potential downsides of prolonged fasting, for example, making sure to minimize the loss of muscle mass and micronutrient deficiency.
The beneficial effects of fasting have been recognized and appreciated for a long time across cultures. Some of the health benefits of prolonged fasting (defined as lasting >24 hours and practiced over a variety of intervals, from alternate day, to once weekly, to quarterly) have been described in the literature. Prolonged fasting has been shown to beneficially modulate the immune system, and counteract the process of “inflammaging,” the process by which immune system function is diminished with age, accompanied by increased inflammation. Perhaps most striking is the finding that fasting extends the life of every model organism that has ever been studied, from fruit flies to mice. There is something fundamentally beneficial and innate about the program that gets turned on in all living organisms during an extended fasting period beyond just the typical overnight fast. In the Zivkovic Lab, we wanted to know: what are the acute effects of a single 36h water-only fast on the plasma metabolome, providing a full picture of the metabolic changes that occur in response to fasting, and on the function of innate immune cells, specifically macrophages.
In this study our team recruited 20 healthy, young individuals (10 males, 10 females), who participated in a 3-day intervention including 4 time points: Day 1, after a standard overnight fast (8AM, Baseline), Day 1, 2 hours after their evening meal (8PM, Fed), Day 3, after a 36h water-only fast (8AM, Fasted), and Day 3, again 2 hours after the evening meal (8PM, Refed). With this study design we were able to compare the effects of the 36h fast to a standard overnight fast, as well as to the postprandial state, and we were also able to assess whether the 36h fasting period had effects that carried over into the next feeding period.
We were not surprised to find that the effects of fasting were universal and profound: the absence of nutrition for this length of time triggered an expected metabolic shift toward the utilization of stored fat and away from carbohydrate metabolism, which was clearly illustrated by the increase in free fatty acids in blood. Also expected was the increase in ketone bodies, which also confirmed that our study participants all adhered to the 36h fast and did not “cheat” by eating foods during the entire 36h period. What was somewhat surprising was the very strong anti-inflammatory effect that 36h fasted plasma had on macrophages, which are part of our innate immune protection arsenal. Compared to not just the postprandial state, which is known to be pro-inflammatory, but also to the overnight fasted state, 36h of fasting profoundly decreased the synthesis of reactive oxygen species, the production of pro-inflammatory signaling molecules, and shifted the phenotype of macrophages from a pro-inflammatory state to what is thought of as a pro-resolving state. We next wanted to find out which specific molecules in plasma contributed to these immunomodulatory effects in macrophages. We performed metabolomic analysis of the plasma and found that >300 metabolites were significantly changed by 36h of fasting, including as mentioned previously, changes in expected molecules such as fatty acids, but also molecules that had not been previously been reported to be altered in this state. Specifically, we were interested in the effects of 4 metabolites that were significantly increased by fasting: spermidine, 1-methylnicotinamide (NMA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA).
We were surprised to find that not only did each of these 4 metabolites individually have a stronger anti-inflammatory effect in our cell model than the well-known ketone body beta-hydroxy butyrate, but the combination of all 4 metabolites had a synergistic effect that reduced the production of pro-inflammatory cytokines even more, such that the effect of the combination was indistinguishable from the negative control in the assay.
We thought this might be too good to be true so we reached out to Dr. JoAnne Engebrecht, a professor of Molecular and Cellular Biology whose research program using C. elegans as a model organism focuses on fundamental aspects of cellular biology and genetics. When we first reached out to her with the idea that we wanted to study the effects of our metabolites on longevity she was unimpressed and frankly, skeptical that it could work. But she was kind enough to give it a shot. A few weeks later we received the results and saw that each of the individual metabolites prolonged the life of C. elegans, but the really striking finding was the synergistic effect of the combination of the 4 metabolites, which made the worms live nearly twice as long as the unsupplemented worms.
The findings from our study highlight that this study design may be a very powerful discovery tool for finding molecules naturally induced during fasting that may be beneficial to human health. Our study also paves the way for a more thorough mechanistic understanding of all the metabolic programs that are tuned on and off in the prolonged fasted state. Further work is now under way to better understand the effects of fasting on a number of additional endpoints, including the structure and function of HDL, and the plasma lipidome. Findings from this study were also translated to a supplement, which is now available through a biotech startup from this lab, headed by lead author and former graduate student Chris Rhodes. Check out Chris’s company and supplement at Mimio.
Alzheimer’s disease is the most common form of dementia and is growing in prevalence. Because Alzheimer’s disease starts many decades before symptoms appear, and because many drugs attempting to treat Alzheimer’s disease once symptoms have already started show either no or very small effects, the search is on for new ways to treat and prevent this devastating disease.
Glycosylation is a biological process where different types of sugars are added to proteins. This process is important for the proteins to fold correctly and function properly. Scientists have noticed changes in the glycosylation patterns in the brains of people with Alzheimer’s disease. In our recently published paper our group explored how glycosylation is affected in the brain of individuals with Alzheimer’s disease, in the hope that our findings might lead to future discoveries for potential treatments.
In this study, we performed four different types of analyses to understand the main pathways of glycosylation that are altered in the brains of Alzheimer’s disease patients. First, we mined publicly available transcriptomic data to determine how gene expression of the genes specifically involved in glycosylation was different in Alzheimer’s patients compared with controls. We then performed quantitative PCR on a subset of those genes in a separate set of brain samples to determine whether the gene expression changes observed in the transcriptomic data could also be seen by qPCR. We used an additional set of brain samples to measure the actual glycans (the sugars themselves) by mass spectrometry to see if the changes predicted by gene expression could be measured directly in the resulting glycans. Finally, we used computational approaches and databases to explore the possible regulatory factors that control the specific glycosylation pathways that were found to be altered by transcriptomics and glycomics.
We found two genes, MGAT1 and B4GALT1, involved in complex N-linked glycan formation and galactosylation, upregulated in the brains of Alzheimer’s patients. Concentrations of glycans that are synthesized by these genes were also increased in Alzheimer’s patients. We also observed that the isoforms of particular enzymes changed differently in AD. For example, ST6GALNAC2, 3, and 5 are isoforms of alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GALNAC), but the expression of ST6GALNAC2 and 3 increased in AD, while ST6GALNAC5 decreased. A similar pattern was also found in isoforms of polypeptide N-acetylgalactosaminyltransferase (GALNT). These isoforms perform the same function (i.e. add a sialic acid to the end of the glycan structure in the case of the ST6GALNAC isoforms) but they act on different target proteins. These results suggested that the glycosylation changes observed in Alzheimer’s disease patient brains are highly specific. Additionally, we noticed that certain genes related to glycolipids (UGT8 and PIGM) were more active in AD brains. Considering the regulation of these enzymes, we predicted that certain transcription factors, such as STAT1 and HSF5, and microRNAs, such as hsa-miR-1-3p and hsa-miR-16-5p, may play a crucial role in regulating the expression of glycosyltransferases.
These findings help us gain insights into the underlying mechanisms of Alzheimer’s disease and may contribute to the development of potential treatments in the future. This study was a collaboration with Carlito Lebrilla and his team, who specialize in the measurement of glycans by mass spectrometry, and the laboratories of Lee-Way Jin and Izumi Maezawa, who are experts in neurodegenerative disease.
Two of our graduate students, Cynthia Tang and Jack Zheng, received the Rucker Family Fellowship! This is a great honor and a wonderful recognition of Cynthia and Jack’s accomplishments as PhD candidates in Nutritional Biology. Dr. Rucker was and still is one of my most valued mentors. When I was a graduate student in the program Dr. Rucker was still actively involved in research and teaching, and was doing wonderful work in the discovery and characterization of the impacts of PQQ on human health. Dr. Rucker was ahead of his time and was a strong supporter and advocate of my work in the area of what was then called “personalized nutrition” and is now referred to as Precision Nutrition. This was at a time when this concept of personalizing nutrition was not generally appreciated or accepted in the broader nutrition community. Dr. Rucker’s support was invaluable in helping me to achieve my goals and dreams. It is therefore even more of an honor to have some of my own students now receiving recognition in the form of the Rucker Family Fellowship for their work.
Cynthia is a computational guru who is exploring the contributions of glycosylation pathways in the etiology of Alzheimer’s disease, and how nutritional components may be involved in regulating these pathways. She is using cutting edge computational biology tools to explore the complex regulatory networks of glycosylation machinery in the human brain integrating RNA sequencing and -omic data. She is also interested in the interconnections between glycosylation and lipid metabolism and how this impacts brain health.
Jack is reviving the use of a tried but true imaging technology to study the most complex and difficult class of nanoparticles, high-density lipoproteins (HDL). Although electron microscopy (EM) is not new, it is still one of the only ways to “see” these tiniest of nanoparticles, which are only 5-12 nm in diameter, and therefore too small to image using most other techniques. EM is traditionally considered too cumbersome and difficult to image more than a few samples at a time. Jack is developing methods to use EM to image hundreds of clinical samples, including tens of thousands of HDL particles for every sample, to understand the biological variability in particle size and morphology.
Congratulations Cynthia and Jack!!
In our newly published narrative review in the special issue “Alzheimer’s disease- 115 Years After Its Discovery” in the journal Biomedicines we discuss the ins and outs of cholesterol handling in microglia, the immune cells of the brain, and discuss implications for Alzheimer’s disease.
In this review article we explore what is known about the effects of high and low cholesterol concentrations on microglia phenotype and function, and areas of research that still need to be explored to better understand this aspect of biology. Given the importance of microglia in driving the neuroinflammation that is associated with neurodegenerative diseases like Alzheimer’s disease, improving microglia function and decreasing microglia-associated inflammation are priority targets for finding new, effective treatments for Alzheimer’s disease. Understanding the role of cholesterol in this process may be a key for finding therapeutic solutions.
High density lipoproteins (HDL) are difficult to study: with a diameter range of 5-12nm they are too small to be studied by many tools that are routinely used to count, characterize and image other nanoparticles such as extracellular vesicles and cells, yet they are multi-molecular complexes that perform a wide array of functions which are dependent on their structure and composition. Despite more than 60 years of research, predominantly in the cardiovascular field due to the importance of HDL in clearing excess cholesterol arterial plaques, we understand very little about the complex biology of HDL particles and their myriad critical functions. We all know that not enough HDL is bad (low HDL-cholesterol is a part of the diagnostic criteria for metabolic syndrome), but recently it was found that too much HDL may also be bad (HDL-cholesterol concentrations >100 mg/dL are linked with higher mortality). What’s more, the amount of HDL in circulation (measured as HDL-cholesterol) only explains about 40% of the variability in the ability of HDL to perform their flagship function of cholesterol efflux, or removal of cholesterol from lipid-loaded macrophages. This means that measuring HDL as the amount of cholesterol carried in the particles is really only telling us a very small piece of the story. And it turns out that HDL composition – the proteins, lipids, lipid soluble components, and even RNA that they transport – HDL structure, and HDL particle size distribution, are all critical factors in how these particles do their jobs of protecting us from infection, blocked arteries, hyper inflammatory responses, and a number of additional functions. Yet we do not know how to improve HDL and truly even how to measure them. In the recently funded grant from the National Institutes of General Medical Sciences, the Zivkovic Lab will develop and optimize new technologies to study HDL particles and their complex biology so that we can harness this vast army of nanoparticles (over 6 quadrillion particles in every millimeter of plasma!) to improve and optimize health.
Dr. Zivkovic has published a new paper in Scientific Reports about how HDL glycoprotein composition affects HDL’s immunomodulatory function.