Of Planes, Microbes and Clocks

ResearchBlogging.org Human microbiome is definitely one of the trending topics in biological science right now (admittedly, I also have been caught up in this trend and blogged about microbiome before). Each week new research announces it discovered a new role of the microbiome (last week there was a nice paper studying a link between alcohol dependency and gut microbiome) and many researcher now consider the microbiome to be another organ in our body, the functions of which we are only beginning to understand. And here is another great story about the gut microbiome but this time, maybe somewhat unexpectedly, it involves the jet lag. jet_lag Jet lag, as Oxford Dictionary of Phycology defines it, is “A condition of fatigue and disorientation brought about by travelling across several time zones in a short period, especially in an eastward direction, resulting in a mismatch between exogenous temporal cues and endogenous circadian rhythms” Now, for those who have not visited biology class for a long time, a circadian rhythm (also known as biological clock) is “Any 24-hour periodicity in the behaviour or physiology of animals or plants”. Basically, it is what adjust your sleeping and eating patterns to the 24h rhythm. For humans the clock is actually closer to 25h and cues such as light-dark periods help to adjust it to the environment we live in but the circadian rhythms are still maintained even in the absence of environmental cues. Humans and plants are actually not exceptional in this and we now know that both bacteria and archaea also have biological clocks too. Considering the many newly discovered functions of our microbiome the new study questioned if there is a relationship between the biological clock of a host (in this case human or mouse) and its symbiont (the intestinal microbiome.) Initial experiment aimed to determine if the abundance of different types of intestinal microbiota oscillates over time, which would be predicted to happen if it was functioning under a circadian rhythm. To do this, the group collected fecal samples from mice every 6h for two light-dark cycles (i.e. 8 samples collected over 48h) and determined the changes in microbial composition during this period. Indeed, they found that microbiota exhibited oscillation and its patterns were different for different families and species of bacteria (fig.1).

Figure 1. Heat map showing oscillations of different OTUs over time and representative Lactobacillus reuteri oscillations. In the heat map red indicates greater abundance.
Figure 1. Heat map showing oscillations of different OTUs over time and representative Lactobacillus reuteri oscillations. In the heat map red indicates greater abundance.

In addition, when scientists looked at the overall expression patterns for different bacterial metabolic pathways they saw that at least 23% of all pathways also are oscillating over the 24h period. The types of pathways being highly expresses during light and dark periods are also functionally opposing: in light period maintenance genes are more active (genes for detoxification, motility and environment sensing), whereas in dark expression genes for metabolism, DNA-repair and cell growth dominates. Even though microbial oscillations over 24h period were observed, the intestinal microbes themselves were not directly exposed to light and dark periods. Therefore, it is reasonable to ask if the oscillations are a consequence of the host’s circadian rhythm? To know if above is the case, the study used Per1/2-/- mice.  Per1/2-/- mice lack so called ‘Period’ genes, which control expression of other genes involved in establishment of circadian rhythm in an animal, and therefore are deficient in the biological clock. When the fecal material sampled from Per1/2-/- was analysed the oscillations were absent, i.e. the composition of intestinal microbiome and expression of specific genes did not rhythmically fluctuate over sampling period as it did in the wild-type (WT) mice (fig.2).

Figure 2a. Disruption of oscillation in Per1/2-/- mice
Figure 2a. Disruption of oscillation in Per1/2-/- mice
Figure 2b. Disruption of metabolic pathway oscilation in Figure 2 mice
Figure 2b. Disruption of metabolic pathway oscilation in Figure 2 mice

The next step was to determine what links host’s rhythm to the microbial one. One important behaviour that tunes the circadian rhythm is mouse feeding time. Consequently, it is plausible that food also modulates the rhythmicity of microbiome cycling. Normally, mice are more active and tend to eat more during the night, however, the Per1/2-/- mice have abnormal patterns of eating. When WT mice were provided with food only during the dark-period the oscillation of microbiome composition was similar to that observed in previous experiments, however, if the food was available only during the day the oscillations tended to shift by 12h reflecting the role that feeding has on setting the biological clock (fig.3). Moreover, when Per1/2-/- mice were used in the same experimental settings the oscillations of microbial composition were restored in both light and dark-fed mice but not in mice that had food available all the time. In addition, by giving a fecal transplantation of the microbiome of Per1/2-/- mice to germ free mice, researchers showed that they can restore normal microbial oscillations suggesting that the faulty biological clock of the host and not some defects in its microbes lead to the loss of oscillations.

Figure 3a. Shift in oscillations induced by different feeding time
Figure 3a. Shift in oscillations induced by different feeding times
Figure 3b. Increased arrhythmic oscillations in Per1/2-/- mice
Figure 3b. Increased arrhythmic oscillations in Per1/2-/- mice

As anyone who has flown long-distance flights across time zones can attest, jet lag is quite annoying and it can take some time to recover from it. Jet lag, as previously mentioned, is a disruption of a person’s normal biological clock but probably not many have ever considered that it could be linked to the bacteria inside us. Considering such possibility, the study next looked if in mice with induced jet lag the intestinal microbiome is also out of sink. Jet lag was induced in WT mouse by switching time by 8h every 3 days for 4 weeks and the fecal samples were collected afterwards. The samples showed that induction of jet lag led to the loss of oscillations in intestinal microbiota (fig.4). This was probably due to the changes in feeding patterns in mice because although the jet lagged mice ate the same amount of food overall, they tended to consume a similar amount of it during the dark and light phase unlike the WT which ate most of their food during the dark phase.

Figure 4. Loss of bacterial oscillations in jet lagged mice
Figure 4. Loss of bacterial oscillations in jet lagged mice

As jet lag in humans has been linked to cardiovascular diseases, diabetes and obesity the scientists next wanted to see if loss of circadian rhythms could lead to metabolic disruption in the host. WT and jet lagged mice were fed equal amounts of high-fat containing food for 6 weeks to mimic the diet that predisposes humans to the named metabolic dysfunctions. After 6 weeks the jet lagged mice gained greater amount of weight and developed an increased sugar intolerances compared to the WT mice. Moreover, if the mice were given antibiotics during jet lag induction their phenotype was similar that of WT post high fat diet. When germ free-mice were given a fecal transfer of jet-lagged mice microbiota and then were provided with high fat diet they showed increase in body fat and enhanced sugar intolerance just like the jet lagged mice did (fig.5).

Figure 5a. Changes in weight and blood glucose levels in jet lagged mice and mice treated with antibiotics (Abx)
Figure 5a. Changes in weight and blood glucose levels in jet lagged mice and mice treated with antibiotics (Abx)
Figure 5b. Changes in weight and blood glucose in germ-free (GF) mice after fecal transfer from WT and jet lagged mice
Figure 5b. Changes in weight and blood glucose in germ-free (GF) mice after fecal transfer from WT and jet lagged mice

Finally, the study looked at the oscillation patterns in gut microbiome of humans. Samples were collected from two subjects at designated time points and the abundance of different bacteria and activity of different metabolic pathways was tested. Just like in the mouse models, the microbiota showed the predicted oscillation and rhythmic changes in metabolic activities over time (fig.6).

Figure 6. Representative intestinal microbiome oscillations in human samples
Figure 6. Representative intestinal microbiome oscillations in human samples

Next, the two human subjects flew from US to Israel which should have led to induction of 8-10h jet lag and again fecal samples were taken before the jet lag phase, during the jet lag and during the recovery period. It was shown that the abundance of different microbial families has changed in the jet-lagged subjects. Namely there was an increase in the number of bacteria belonging to Firmicutes, which is a phylum that has previously been linked to increased rates of obesity and metabolic dysfunctions in humans. When fecal samples from the two subjects were transferred into the germ free mice, the mice that received the bacteria from the jet lagged samples showed significant increase in weight and greater amount of blood glucose after a glucose challenge compared to mice which received microbiota from non-jet lagged or recovery period samples (fig.7). 

Figure 7. A) Experimental design of human sample collection B) Increased proportion of Firmicutes after jet lag in human samples. C) Experimental design of fecal transfer of human samples to germ-free mice D-E) Changes in weight and blood glucose levels in GF mice after fecal transfer
Figure 7. A) Experimental design of human sample collection B) Increased proportion of Firmicutes after jet lag in human samples. C) Experimental design of fecal transfer of human samples to GF mice D-E) Changes in weight and blood glucose levels in GF mice after fecal transfer

Overall, the study has shown that the host’s biological clock influences the rhythmic fluctuation of the intestinal microbiome. These fluctuations can be regulated by the timing of food intake and if disrupted, as in the case of jet lag, can lead to metabolic dysfunctions in the host. Considering the rather erratic life style that many of us live in, these findings could be of importance. Jet lag is of course mainly a problem for those who have to commute often but the clocks are often disrupted in shift workers and many students who, I can personally attest, sometimes seem to lack any biological clock whatsoever. Our microbiome apparently likes to stick to the schedule so if we want to be healthy and keep our microbes happy we should at least try to stick to it as well. Thaiss, C., Zeevi, D., Levy, M., Zilberman-Schapira, G., Suez, J., Tengeler, A., Abramson, L., Katz, M., Korem, T., Zmora, N., Kuperman, Y., Biton, I., Gilad, S., Harmelin, A., Shapiro, H., Halpern, Z., Segal, E., & Elinav, E. (2014). Transkingdom Control of Microbiota Diurnal Oscillations Promotes Metabolic Homeostasis Cell, 159 (3), 514-529 DOI: 10.1016/j.cell.2014.09.048

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