Nava Chapnik

Timed high-fat diet resets circadian metabolism and prevents obesity

Disruption of circadian rhythms leads to obesity and metabolic disorders. Timed restricted feeding (RF) provides a time cue and resets the circadian clock, leading to better health. In contrast, a high‐fat (HF) diet leads to disrupted circadian expression of metabolic factors and obesity. We tested whether long‐term (18 wk) clock resetting by RF can attenuate the disruptive effects of diet‐induced obesity. Analyses included liver clock gene expression, locomotor activity, blood glucose, metabolic markers, lipids, and hormones around the circadian cycle for a more accurate assessment. Compared with mice fed the HF diet ad libitum, the timed HF diet restored the expression phase of the clock genes Clock and Cry1 and phase‐advanced Per1, Per2, Cry2, Bmal1, Rorα, and Rev‐erbα. Although timed HF‐diet‐fed mice consumed the same amount of calories as ad libitum low‐fat diet‐fed mice, they showed 12% reduced body weight, 21% reduced cholesterol levels, and 1.4‐fold increased insulin sensitivity. Compared with the HF diet ad libitum, the timed HF diet led to 18% lower body weight, 30% decreased cholesterol levels, 10% reduced TNF‐α levels, and 3.7‐fold improved insulin sensitivity. Timed HF‐diet‐fed mice exhibited a better satiated and less stressed phenotype of 25% lower ghrelin and 53% lower corticosterone levels compared with mice fed the timed low‐fat diet. Taken together, our findings suggest that timing can prevent obesity and rectify the harmful effects of a HF diet.—Sherman, H., Genzer, Y., Cohen, R., Chapnik, N., Madar, Z., Froy, O. Timed high‐fat diet resets circadian metabolism and prevents obesity. FASEB J. 26, 3493–3502 (2012). www.fasebj.org

High α-defensin levels in patients with systemic lupus erythematosus

Innate immunity plays a role in systemic lupus erythematosus (SLE). Our objective was to determine the levels of defensins, which are antimicrobial and immunomodulatory polypeptides, in SLE. Sera from SLE patients and healthy controls were tested for pro‐inflammatory human β‐defensin 2 (hBD‐2) and for α‐defensin human neutrophil peptide 1 (HNP‐1). hBD‐2 could not be detected by enzyme‐linked immunosorbent assay (ELISA) and its mRNA levels were low in SLE patients and similar to those found in controls. In contrast, the mean α‐defensin level in the sera of all SLE patients (11·07 ± 13·92 ng/μl) was significantly higher than that of controls (0·12 ± 0·07 ng/μl). Moreover, 60% of patients demonstrated very high serum levels (18·5 ± 13·36 ng/μl) and 50% showed elevated gene expression in polymorphonuclear cells. High α‐defensin levels correlated with disease activity, but not with neutrophil count. Thus, activation and degranulation of neutrophils led to α‐defensin secretion in SLE patients. Given the immunomodulatory role of α‐defensins, it is possible that their secretion may activate the adaptive immune system leading to a systemic response.

Metformin affects the circadian clock and metabolic rhythms in a tissue-specific manner.

Metformin is a commonly-used treatment for type 2 diabetes, whose mechanism of action has been linked, in part, to activation of AMP-activated protein kinase (AMPK). However, little is known regarding its effect on circadian rhythms. Our aim was to evaluate the effect of metformin administration on metabolism, locomotor activity and circadian rhythms. We tested the effect of metformin treatment in the liver and muscle of young lean, healthy mice, as obesity and diabetes disrupt circadian rhythms. Metformin led to increased leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was similar leading to AMPK activation either by liver kinase B1 (LKB1) and/or other kinases in the muscle. AMPK activation resulted in the inhibition of acetyl CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis. Metformin also led to the activation of liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock. This effect was mainly of phase advances in the liver and phase delays in the muscle in clock and metabolic genes and/or protein expression. In conclusion, our results demonstrate the differential effects of metformin in the liver and muscle and the critical role the circadian clock has in orchestrating metabolic processes.

The circadian clock is functional in eosinophils and mast cells

Allergic diseases are frequently exacerbated between midnight and early morning, suggesting a role for the biological clock. Mast cells (MC) and eosinophils are the main effector cells of allergic diseases and some MC‐specific or eosinophil‐specific markers, such as tryptase or eosinophil cationic protein, exhibit circadian variation. Here, we analysed whether the circadian clock is functional in mouse and human eosinophils and MC. Mouse jejunal MC and polymorphonuclear cells from peripheral blood (PMNC) were isolated around the circadian cycle. Human eosinophils were purified from peripheral blood of non‐allergic and allergic subjects. Human MC were purified from intestinal tissue. We found a rhythmic expression of the clock genes mPer1, mPer2, mClock and mBmal1 and eosinophil‐specific genes mEcp, mEpo and mMbp in murine PMNC. We also found circadian variations for hPer1, hPer2, hBmal1, hClock, hEdn and hEcp mRNA and eosinophil cationic protein (ECP) in human eosinophils of both healthy and allergic people. Clock genes mPer1, mPer2, mClock and mBmal1 and MC‐specific genes mMcpt‐5, mMcpt‐7, mc‐kit and mFcεRI α‐chain and protein levels of mMCPT5 and mc‐Kit showed robust oscillation in mouse jejunum. Human intestinal MC expressed hPer1, hPer2 and hBmal1 as well as hTryptase and hFcεRI α‐chain, in a circadian manner. We found that pre‐stored histamine and de novo synthesized cysteinyl leukotrienes, were released in a circadian manner by MC following IgE‐mediated activation. In summary, the biological clock controls MC and eosinophils leading to circadian expression and release of their mediators and, hence it might be involved in the pathophysiology of allergy.

Long-lived mice exhibit 24 h locomotor circadian rhythms at young and old age.

αMUPA transgenic mice exhibit spontaneously reduced eating and increased life span compared with their wild type (WT) control FVB/N mice. αMUPA mice also show high-amplitude circadian rhythms in food intake, body temperature, and hepatic clock gene expression. Here we examined young and aged WT and αMUPA mice for the period of locomotor activity (tau) under total darkness (DD). We show that tau changed in WT mice from a period <24 h at 8 months to a period >24 h at 18 months. However, the period of αMUPA mice was ~24 h at both 8 and 18 months. As deviation of tau from 24 h has been found to be inversely related to life span in a large number of rodents, our results suggest that the sustainable endogenous period of ~24 h in αMUPA mice may contribute to their prolonged life span.

Effect of intermittent fasting on circadian rhythms in mice depends on feeding time

Calorie restriction (CR) resets circadian rhythms and extends life span. Intermittent fasting (IF) also extends life span, but its affect on circadian rhythms has not been studied. To study the effect of IF alongside CR, we imposed IF in FVB/N mice or IF combined with CR using the transgenic FVB/N alphaMUPA mice that, when fed ad libitum, exhibit spontaneously reduced eating and extended life span. Our results show that when food was introduced during the light period, body temperature peak was not disrupted. In contrast, IF caused almost arrhythmicity in clock gene expression in the liver and advanced mPer2 and mClock expression. However, IF restored the amplitudes of clock gene expression under disruptive light condition regardless whether the animals were calorically restricted or not. Unlike daytime feeding, nighttime feeding yielded rhythms similar to those generated during ad libitum feeding. Taken together, our results show that IF can affect circadian rhythms differently depending on the timing of food availability, and suggest that this regimen induces a metabolic state that affects the suprachiasmatic nuclei (SCN) clock.

The suprachiasmatic nuclei are involved in determining circadian rhythms during restricted feeding.

The circadian clock in the suprachiasmatic nuclei (SCN) responds to light and regulates peripheral circadian rhythms. Feeding regimens also reset the clock, so that time-restricted feeding (RF) dictates rhythms in peripheral tissues, whereas calorie restriction (CR) affects the SCN clock. To better understand the influence of RF vs. CR on circadian rhythms, we took advantage of the transgenic alphaMUPA mice that exhibit spontaneously reduced eating, and can serve as a model for CR under ad libitum feeding, and a model for temporal CR under RF compared with wild type (WT) mice. Our results show that RF advanced and generally increased the amplitude of clock gene expression in the liver under LD in both mouse types. However, under disruptive light conditions, RF resulted in a different clock gene phase in WT mice compared with alphaMUPA mice, suggesting a role for the reduced calories in resetting the SCN that led to the change of phase in alphaMUPA mice. Comparison of the RF regimen in the two lighting conditions in WT mice revealed that mPer1, mClock, and mBmal1 increased, whereas mPer2 decreased in amplitude under ultradian light in WT mice, suggesting a role for the SCN in determining clock gene expression in the periphery during RF. In summary, herein we reinforce a role for calorie restriction in resetting the SCN clock, and unravel a role for the SCN in determining peripheral rhythms under RF.

Relationship between calorie restriction and the biological clock: lessons from long-lived transgenic mice.

The master clock located in the brain regulates circadian rhythms in mammals. Similar clocks are found in peripheral tissues. Life span has been independently increased by reset circadian rhythms and caloric restriction (CR). The mechanisms by which CR extends life span are not well understood. We found that alphaMUPA transgenic mice that exhibit reduced eating and live longer show high amplitude, appropriately reset circadian rhythms in clock gene expression, and clock-controlled output systems, such as feeding time and body temperature. As CR resets circadian rhythms, and the circadian clock controls many physiological and biochemical systems, we suggest that the biological clock could be an important mediator of longevity in calorically restricted animals.

A superactive leptin antagonist alters metabolism and locomotion in high-leptin mice

Transgenic alpha murine urokinase-type plasminogen activator (αMUPA) mice are resistant to obesity and their locomotor activity is altered. As these mice have high leptin levels, our objective was to test whether leptin is responsible for these characteristics. αMUPA, their genetic background control (FVB/N), and C57BL mice were injected s.c. every other day with 20  mg/kg pegylated superactive mouse leptin antagonist (PEG-SMLA) for 6 weeks. We tested the effect of PEG-SMLA on body weight, locomotion, and bone health. The antagonist led to a rapid increase in body weight and subsequent insulin resistance in all treated mice. Food intake of PEG-SMLA-injected animals increased during the initial period of the experiment but then declined to a similar level to that of the control animals. Interestingly, αMUPA mice were found to have reduced bone volume (BV) than FVB/N mice, although PEG-SMLA increased bone mass in both strains. In addition, PEG-SMLA led to disrupted locomotor activity and increased corticosterone levels in C57BL but decreased levels in αMUPA or FVB/N mice. These results suggest that leptin is responsible for the lean phenotype and reduced BV in αMUPA mice; leptin affects corticosterone levels in mice in a strain-specific manner; and leptin alters locomotor activity, a behavior determined by the central circadian clock.

Caffeine alters circadian rhythms and expression of disease and metabolic markers

The circadian clock regulates many aspects of physiology, energy metabolism, and sleep. Restricted feeding (RF), a regimen that restricts the duration of food availability entrains the circadian clock. Caffeine has been shown to affect both metabolism and sleep. However, its effect on clock gene and clock-controlled gene expression has not been studied. Here, we tested the effect of caffeine on circadian rhythms and the expression of disease and metabolic markers in the serum, liver, and jejunum of mice supplemented with caffeine under ad libitum (AL) feeding or RF for 16 weeks. Caffeine significantly affected circadian oscillation and the daily levels of disease and metabolic markers. Under AL, caffeine reduced the average daily mRNA levels of certain disease and inflammatory markers, such as liver alpha fetoprotein (Afp), C-reactive protein (Crp), jejunum alanine aminotransferase (Alt), growth arrest and DNA damage 45β (Gadd45β), Interleukin 1α (Il-1α), Il-1β mRNA and serum plasminogen activator inhibitor 1 (PAI-1). Under RF, caffeine reduced the average daily levels of Alt, Gadd45β, Il-1α and Il-1β mRNA in the jejunum, but not in the liver. In addition, caffeine supplementation led to decreased expression of catabolic factors under RF. In conclusion, caffeine affects circadian gene expression and metabolism possibly leading to beneficial effects mainly under AL feeding.