Microbiome Rhythm and Metabolic Health

You are when you eat? Perhaps.

Similar to circadian rhythms that help regulate when we naturally fall asleep and wake up, microbial rhythms in our gut are naturally active at certain times of the day to help regulate our digestion.

Researchers from the UC San Diego sought out to track these microbial rhythms to determine whether aligning the times we eat to when our gut microbes are most active can bolster our metabolic health. This alignment is known as time-restricted feeding (TRF).

“Microbial rhythms are daily fluctuations in the composition and function of microbes living in our gut. Much like how our bodies follow an internal clock (circadian rhythm), gut microbes also have their own rhythms, adjusting their activities based on the time of day and when we eat,” said Amir Zarrinpar, MD, PhD, gastroenterologist and associate professor of medicine at UC San Diego School of Medicine, and senior author of the study.

Zarrinpar and his team were particularly interested in observing whether adopting the TRF approach counteracted the harmful metabolic effects often associated with consuming a high-fat diet.

The study is also notable for the team’s use of technology able to observe real-time microbial changes in the gut — something not previously attainable with existing metagenomics.

How the Study Evolved With New Tech

Researchers separated three groups of mice to analyze their microbiome activity: one on a high-fat diet with unrestricted access, another on the same high-fat diet within a TRF window of 8 hours per day, and a control group on a normal chow diet with unrestricted access.

“In mice, [their] microbial rhythms are well-aligned with their nocturnal lifestyle. For example, during their active (nighttime) period, certain beneficial microbial activities increase, helping digest food, absorb nutrients, and regulate metabolism,” said Zarrinpar. As a result, the team made sure the mice’s TRF window was at night or when they would normally be awake.

“We chose an 8-hour feeding window based on earlier research showing this time period allows mice to consume the same total calories as those with unlimited food access,” said Zarrinpar. “By controlling [the] calories in this way, we ensure any metabolic or microbial benefits we observe are specifically due to the timing of eating, rather than differences in total food intake.” 

But before any observations could be made, the team first needed a way to see real-time changes in the animals’ gut microbiomes.

Zarrinpar and his team were able to uncover this, thanks to metatranscriptomics, a technique used to capture real-time microbial activity by profiling RNA transcripts. Compared with the more traditional technique of metagenomics, which could only be used to identify which genes were present, metatranscriptomics provided more in-depth temporal and activity-related context, allowing the team to observe dynamic microbial changes.

“[Metatranscriptomics] helps us understand not just which microbes are present, but specifically what they are doing at any given moment,” said Zarrinpar. “In contrast, metagenomics looks only at microbial DNA, which provides information about what microbes are potentially capable of doing, but doesn’t tell us if those genes are actively expressed. By comparing microbial gene expression (using metatranscriptomics) and microbial gene abundance (using metagenomics) across different diet and feeding conditions in [light and dark] phases, we aimed to identify how feeding timing might influence microbial activity.” 

Because metagenomics focuses on stable genetic material, this technique cannot capture the real-time microbial responses to dietary timing presented in rapidly changing, short-lived RNA. At the same time, the instability of the RNA makes it difficult to test hypotheses experimentally and explains why researchers haven’t more widely relied on metatranscriptomics.

To overcome this difficulty, Zarrinpar and his team had to wait to take advantage of improved bioinformatics tools to simplify their analysis of complex datasets. “It took several years for us to analyze this dataset because robust computational tools for metatranscriptomic analysis were not widely available when we initially collected our samples. Additionally, sequencing costs were very high. To clearly identify microbial activity, we needed deep sequencing coverage to distinguish species-level differences in gene expression, especially for genes that are common across multiple types of microbes,” said Zarrinpar.

What They Found

After monitoring these groups of mice for 8 weeks, the results were revealed.

As predicted, the mice with unrestricted access to a high-fat diet exhibited signs of metabolic dysfunction due to disruptions in their circadian and microbial rhythms. “When mice have free access to a high-fat diet, their normal eating behavior changes significantly. Instead of limiting their activity and feeding to their active nighttime period, these mice begin to stay awake and eat during the day, which is their typical rest phase,” Zarrinpar explained.

“This unusual daytime activity interferes with important physiological processes. Consequently, the animals experience circadian misalignment, a condition similar to what human shift workers experience when their sleep-wake and eating cycles don’t match their internal biological clocks,” he continued. “This misalignment can negatively affect metabolism, immunity, and overall health, potentially leading to metabolic diseases.”

For the mice that consumed a high-fat diet within a TRF window, metabolic phenotyping demonstrated that their specific diet regimen had protected them from harmful high-fat induced effects including adiposity, inflammation, and insulin resistance.

Even more promising, the mice not only were protected from metabolic disruption but also experienced physiological improvements including glucose homeostasis and the partial restoration of the daily microbial rhythms absent in the mice with unrestricted access to a high-fat diet.

While the TRF approach did not fully restore the normal, healthy rhythmicity seen in the control mice, the researchers noted distinct shifts in microbial patterns that indicated time-dependent enrichment in genes attributed to lipid and carbohydrate metabolism.

Better Metabolic Health — and Better Tools for Researching It

Thankfully, the latest advancements in sequencing technology, including long-read sequencing methods, are making metatranscriptomics easier for research. “These newer platforms offer greater resolution at a lower cost, making metatranscriptomics increasingly accessible,” said Zarrinpar. With these emerging technologies, he believes metatranscriptomics will become a more standard, widely used method for researchers to better understand the influence of microbial activity on our health.

These tools, for example, enabled Zarrinpar and the team to delve deeper and focus on the transcription of a particular enzyme they identified as a pivotal influence in observable metabolic improvements: bile salt hydrolase (BSH), known to regulate lipid and glucose metabolism. The TRF approach notably enhanced the expression of the BSH gene during the daytime in the gut microbe Dubosiella newyorkensis, which has a functional human equivalent.

To determine why this happened, the team leveraged genetic engineering to insert several active BSH gene variants into a benign strain of gut bacteria to administer to the mice. The only variant to produce metabolic improvements was the one derived from Dubosiella newyorkensis; the mice who were given this BSH-expressing engineered native bacteria (ENB) had increased lean muscle mass, less body fat, lower insulin levels, enhanced insulin sensitivity, and better blood glucose regulation.

“It is still early to know the full clinical potential of this new BSH-expressing engineered native bacterium,” said Zarrinpar. “However, our long-term goal is to develop a therapeutic that can be administered as a single dose, stably colonize the gut, and provide long-lasting metabolic benefits.” Testing the engineered bacteria in obese and diabetic mice on a high-fat diet would be a next step to determine whether its potential indeed holds up. If proven successful, it could then be used to develop future targeted therapies and interventions to treat common metabolic disorders.

With this engineered bacteria, Zarrinpar and his team are hopeful that it alone can replicate the microbial benefits associated with following a TRF dietary schedule. “In our study, the engineered bacterium continuously expressed the enzyme DnBSH1, independently of dietary or environmental factors. As a result, the bacterium provided metabolic benefits similar to those seen with TRF, even without requiring the mice to strictly adhere to a TRF schedule,” said Zarrinpar.

“This suggests the exciting possibility that this engineered microbe might serve either as a replacement for TRF or as a way to enhance its beneficial effects,” he continued. “Further studies will help determine whether combining this ENB with TRF could provide additional or synergistic improvements in metabolic health.”

Looking Ahead

“As the pioneer of the single anastomosis duodenal switch which separates bile from food until halfway down the GI tract, I agree that bile is very important in controlling metabolism and glucose,” said Mitchell Roslin, MD, FACS, professor of surgery at the Donald and Barbara Zucker School of Medicine, Hempstead, New York, chief director of bariatric and metabolic surgery at Lenox Hill Hospital, who was not involved in the study. “Using enzymes or medications that work in the GI tract without absorption into the body is very interesting and has great potential. It is an early but exciting prospect.”

However, Roslin expressed some reservations. “I think we are still trying to understand whether the difference in microbiomes is the cause or effect/association. Is the microbiome the difference or is a different microbiome representative of a diet that has more fiber and less processed foods? Thus, while I find this academically fascinating, I think that there are very basic questions that need better answers, before we look at the transcription of bacteria.”

Furthermore, translating the metabolic results observed in mice to humans might not be as straightforward. “Small animal research is mandatory, but how the findings convert to humans is highly speculative,” said Roslin. “Mice that are studied are usually bred for medical research, with reduced genetic variation. Many animal models are more sensitive to time-restricted eating and caloric restriction than humans.”

While it requires further research and validation, this UC San Diego study nevertheless contributes to our overall understanding of host-microbe interactions. “We demonstrate that host circadian rhythms significantly influence microbial function, and conversely, these microbial functions can directly impact host metabolism,” said Zarrinpar. “Importantly, we now have a method to test how specific microbial activities affect host physiology by engineering native gut bacteria.”

Roslin similarly emphasized the importance of continued investment in exploring the microbial ecosystem inside us all. “There is wider evidence that bacteria and microbes are not just passengers using us for a ride but perhaps manipulating every action we take.”