Integrative Physiology

Circadian physiology and energy homeostasis: the effect of a gap between the period length of endogenous circadian rhythms and the environmental photic cycle, on energy homeostasis

Endogenous rhythms oscillate at a period length (tau) that usually diverts from 24-h. This tau-T-cycle deviation correlates with DIO rates and inversely with lifespan, arguing that a tau-T-cycle deviation results in life-shortening disruptions – i.e., the ‘circadian resonance’ hypothesis20.

However, this was shown in mutated rodents with altered metabolism or under tau-T-cycle deviations preventing entrainment21. Hence, we examined the validity of the hypothesis under more realistic conditions, in which T-cycles slightly divert from tau. This setup better reflects WT mice and humans held under a 'regular' 24-h T-cycle, while their tau is ca. 23.7-h and 24.5-h, respectively. This idea arose from identifying that long-living αMUPA female mice also have a tau of ca. 24-h at young and old age, while the WT shows a tau <24-h at a young age and > 24-h at old age9. As tau's deviation from 24-h is inversely related to life span, our results suggest that the sustainable tau of ~24 h in αMUPA mice contributes to their phenotype. A follow-up study identified this phenotype in αMUPA males with lower sexual dimorphism than their WTs. This suggests that αMUPA’s transgene overrides sex-dependent mechanisms involved in lifespan, energy, and circadian homeostasis22.

We next tested the validity of the ‘circadian resonance’ hypothesis by measuring the propensity to DIO in WT mice held under a T-cycle oscillating at their tau-like period length. As hypothesized, WT mice under this T-cycle do not show the DIO found under the 24-h T-cycle23. Mice fed an HFD under the 24-h T-cycle showed a disrupted pattern of circadian locomotor activity before DIO onset, not found under the tau-like T-cycle23. These results yielded three hypotheses currently being tested – a project funded by a five-year research grant from the Israel Science Foundation. According to the first hypothesis, the reduced DIO under the tau-like T-cycle is due to the lack of need to synchronize the internal clock to the rhythm of the environmental T-cycle. According to the second hypothesis, this lower DIO is due to strengthening the endogenous circadian clock and its period length and preventing the HFD-induced elongating of tau by entraining it to a tau-like T-cycle, i.e., the aftereffect24. According to the third hypothesis, which extends the second one, the lower DIO is due to entrainment to a T-cycle faster than the HFD-elongated tau.

Our preliminary results reject the first hypothesis and imply the validity of the third hypothesis. We will further test these hypotheses, by addressing four objectives:

1. Assessment of HFD-induced metabolic outcomes of holding WT mice under an age-corrected tau-like T-cycle, compared with constant darkness and T-cycles diverting from tau, including the 24-h T-cycle;
2. Assessment of effects of these manipulations on the temporal organization of biochemical regulators related to energy and circadian homeostasis;
3. Identification of mechanisms underlying the metabolic outcomes of these manipulations;
4. Comparison of outcomes of entrainment to a tau-like T-cycle to those obtained under time-restricted-feeding. The latter will be a positive control for the mechanism hypothesized to underlie DIO attenuation under a tau-like T-cycle.

The expected outcomes may provide:

1. Evidence that a tau markedly diverted from 24-h is a risk factor for metabolic disruptions
2. A novel experimental paradigm for developing clock-related therapeutic agents to reduce the tau-24-h deviation and increase the robustness of endogenous cycles.

These enhanced rhythms may attenuate DIO rates among shift workers, who routinely entrain to new T-cycles.

Formulation and examination of nutritional supplements for attenuating metabolic syndrome

My collaborator, Prof. Giora Rytwo (Migal and Tel-Hai College), showed that sepiolite clay efficiently adsorbs hydrophobic molecules30. After that, we showed that this substance highly adsorb edible oil and cholesterol, and mildly attenuates hypercholesterolemia and DIO in mice30,31.

Subsequently, we obtained a three-year grant followed by a four-year grant from The Israeli Ministries of Industry and Science to assess biocomposites' efficacy as dietary supplements for attenuating metabolic syndrome.

We first obtained a biocomposite composed of sepiolite and an indigestible cationic biopolymer with bile-acid-sequestrant properties. We hypothesized that this compound could lower bile-acid reabsorption and fat absorption, reducing DIO and hypercholesterolemia.

Initial results were promising, resulting in a provisional submission. However, further experiments showed that the effect was mild and depended on the biocomposites becoming positively charged under diverse pH conditions (manuscript submitted).

Our future study aims at obtaining a biocomposite that maintains this and other desired properties. Another project, in collaboration with BioRootIn LTD. and Prof. Rachel Amir (Migal and Tel-Hai College) examines whether atherosclerosis development can be attenuated by a dietary supplement obtained from one of the pomegranate tree parts, excluding its juice, which has already been shown to be antiatherogenic. This effect was tested using APO-E mice – known for developing atherosclerosis.

Preliminary results show that the tested dietary supplement is as effective as the pomegranate juice. We are in contact with BioRootIn to examine further collaborations and publication of our results. The knowledge obtained in the project led to another partnership, this time with Prof. Soliman Khatib and Dr. Sanna Musa (Migal and Tel-Hai College) aimed to synthesize natural lipids that attenuate atherosclerosis via improving HDL functions.

The usage of insect-based protein-rich meals for food and feed.

This study aims to create the scientific infrastructure for developing a new industry in Israel of alternative protein production for food and feed by growing black soldier fly larvae (BSFL) on agricultural waste.

Using a three-year grant from the Israeli Ministry of Agriculture, we have found that mice, used as a monogastric animal model, fed with protein meal extracted from BSFL instar 6 had protein digestibility and protein-muscle conversion efficiency resembling casein-based protein. In addition, mechanical removal of the BSFL cuticle, mainly composed of non-digestible fibers, improved the nutritional efficiency of the BSFL meals (manuscript in preparation).

Using a three-year grant from the Israeli Ministry of Economy and Industry, we now aim to:

1. Improve the efficiency of cuticle removal.
2. Better understand the physiological mechanisms underlying the effect of the cuticle on the nutritional properties of the meal.
3. Assess the effect of the BSFL stage (instar five vs. six) on meals’ nutritional properties.

In addition, we will continue to examine the effect of the insect’s diet on its body composition32. This project is a collaboration with entomologists (Dr. Itzik Martinez, Dr. Itai Opatovsky, and Dr. Liora Shaltiel), a food technologist (Dr. Adi Jonas), and a fish breeder (Dr. Avshalom Horowitz) from Migal and Tel-Hai College.

The role of the postnatal leptin surge in susceptibility to high-fat diet-induced obesity

Perturbations in the postnatal surge level or signaling of the hormone leptin are associated with altered energy homeostasis, yet with contradicting results 16,17.

To address the discrepancies, we studied the associations of length and intensity of the leptin surge, with susceptibility to high-fat diet-induced obesity (DIO) using αMUPA mice. Compared to their wild-type (WT), αMUPA mice show lower post-weaning body weight and food intake in conjunction with higher leptin levels than10.

In αMUPA pups, we observed a more extended and higher leptin surge, which is weakly related to fat mass18. Notably, leptin levels in the dams’ serum and breast milk and their pups’ stomach content were higher in αMUPA mice, mitigated by dam removal18.

Subsequently, we found that attenuating postnatal leptin signaling by leptin antagonist reduced αMUPA’s hyperinsulinemia and propensity to DIO under HFD, by mediating energy expenditure19.

Together with our previous findings18, these results suggest that αMUPA’s leptin surge underlies its higher susceptibility to DIO. This highlights the role of leptin-related developmental processes in inducing DIO.

Chronobiology in aid of the dairy industry and the effect of milk’s saturated fatty acid level on atherosclerosis

Exposure to artificial light at night (LAN) disrupts circadian rhythms, yet many dairy cows continue to be exposed to LAN for historical and practical reasons.

In collaboration with Dr. Aviv Asher (Migal), founded by a three-year research grant from the Ministry of Agriculture, we explored the effect of LAN on the production and circadian variation in milk fat composition. Compared to a natural 10-h light 14-h dark light-dark (LD) cycle, LAN reduced the circadian variation in milk fat composition yet did not affect milk production.

Specifically, LAN increased the saturated fatty acids (SFA) level in day milk at the expense of unsaturated fatty acids (UFA). Therefore, LAN has no commercial advantage over the tested natural LD cycle, and conversely, it even shows circadian disruption. Accordingly, this natural LD cycle is preferable over LAN from a cost savings perspective, cows’ well-being, and preserving the natural milk fat profile, as the nutritional value of the day milk is slightly higher. We disseminate our published resutls29 to increase animal welfare and reduce the unneeded usage of electric energy for barn illumination.

The above results merge with another research project related to the dairy industry – a collaboration with Dr. Ofir Benjamin (Tel-Hi College), funded by the Israeli Ministry of Health.

SFA consumption is considered a risk factor for atherosclerosis; hence, its intake is nutritionally beneficial. As milk-fat SFA percentage varies between cows and across milking hours, and it is expected that sorting milk by its SFA percentage during milking will be possible in a few years, we tested the effect of milk-fat-born SFA percentages on atherosclerosis development. APO-E mice fed a low-fat diet with low SFA levels showed increased high-density cholesterol levels compared to their baseline levels, and their atherosclerosis progression was positively related to the dietary SFA percentage.

These results promote milk-sorting, based on its SFA levels, to produce healthier dairy products. We also (in collaboration with Dr. Ari Meerson, Migal) applied a computational approach to analyze hepatic and aorta differential gene expression, highlighting biochemical pathways and interventions for atherosclerosis attenuation.