Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

Many physiological processes are regulated with a 24-h periodicity to anticipate the environmental changes of daytime to nighttime and vice versa. These 24-h regulations, commonly termed circadian rhythms, among others control the sleep–wake cycle, locomotor activity and preparation for food availability during the active phase (daytime for humans and nighttime for nocturnal animals). Disturbing circadian rhythms at the organ or whole-body level by social jetlag or shift work, increases the risk to develop chronic metabolic diseases such as type 2 diabetes mellitus. The molecular basis of this risk is a topic of increasing interest. Mitochondria are essential organelles that produce the majority of energy in eukaryotes by converting lipids and carbohydrates into ATP through oxidative phosphorylation. To adapt to the ever-changing environment, mitochondria are highly dynamic in form and function and a loss of this flexibility is linked to metabolic diseases. Interestingly, recent studies have indicated that changes in mitochondrial morphology (i.e., fusion and fission) as well as generation of new mitochondria are dependent on a viable circadian clock. In addition, fission and fusion processes display diurnal changes that are aligned to the light/darkness cycle. Besides morphological changes, mitochondrial respiration also displays diurnal changes. Disturbing the molecular clock in animal models leads to abrogated mitochondrial rhythmicity and altered respiration. Moreover, mitochondrial-dependent production of reactive oxygen species, which plays a role in cellular signaling, has also been linked to the circadian clock. In this review, we will summarize recent advances in the study of circadian rhythms of mitochondria and how this is linked to the molecular circadian clock.

Obesity‐induced insulin resistance and type 2 diabetes mellitus can ultimately result in various complications, including diabetic cardiomyopathy. In this case, cardiac dysfunction is characterized by metabolic disturbances such as impaired glucose oxidation and an increased reliance on fatty acid (FA) oxidation. Mitochondrial dysfunction has often been associated with the altered metabolic function in the diabetic heart, and may result from FA‐induced lipotoxicity and uncoupling of oxidative phosphorylation. In this review, we address the metabolic changes in the diabetic heart, focusing on the loss of metabolic flexibility and cardiac mitochondrial function. We consider the alterations observed in mitochondrial substrate utilization, bioenergetics and dynamics, and highlight new areas of research which may improve our understanding of the cause and effect of cardiac mitochondrial dysfunction in diabetes. Finally, we explore how lifestyle (nutrition and exercise) and pharmacological interventions can prevent and treat metabolic and mitochondrial dysfunction in diabetes.

Impairment of the protective properties of the blood-brain barrier (BBB) is a key event during numerous neurological diseases, including multiple sclerosis (MS). Under these pathological conditions, the specialized brain endothelial cells (BECs) lose their protective function leading to neuroinflammation and neurodegeneration. To date, underlying mechanisms for this loss of function remain unclear. Endothelial to mesenchymal transition (EndoMT) is a dynamic process by which endothelial cells (ECs) dedifferentiate into mesenchymal cells and as a result lose their specific phenotype and function. As yet, little is known about the involvement of this process in the impaired function of the BECs under pathological conditions such as MS. Interestingly, several signaling pathways that can induce EndoMT are also involved in different central nervous system (CNS) pathologies associated with BBB dysfunction. In this review, we first discuss the structure and function of the BBB highlighting the changes that occur during MS. Next, we will summarize recent findings on the pathways underlying EndoMT, and finally, we will discuss the potential role of EndoMT during BBB dysfunction in neurological disorders. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Desynchronization between the master clock in the brain, which is entrained by (day) light, and peripheral organ clocks, which are mainly entrained by food intake, may have negative effects on energy metabolism. Bile acid metabolism follows a clear day/night rhythm. We investigated whether in rats on a normal chow diet the daily rhythm of plasma bile acids and hepatic expression of bile acid metabolic genes is controlled by the light/dark cycle or the feeding/fasting rhythm. In addition, we investigated the effects of high caloric diets and time-restricted feeding on daily rhythms of plasma bile acids and hepatic genes involved in bile acid synthesis. In experiment 1 male Wistar rats were fed according to three different feeding paradigms: food was available ad libitum for 24 h (ad lib) or time-restricted for 10 h during the dark period (dark fed) or 10 h during the light period (light fed). To allow further metabolic phenotyping, we manipulated dietary macronutrient intake by providing rats with a chow diet, a free choice high-fat-high-sugar diet or a free choice high-fat (HF) diet. In experiment 2 rats were fed a normal chow diet, but food was either available in a 6-meals-a-day (6M) scheme or ad lib. During both experiments, we measured plasma bile acid levels and hepatic mRNA expression of genes involved in bile acid metabolism at eight different time points during 24 h. Time-restricted feeding enhanced the daily rhythm in plasma bile acid concentrations. Plasma bile acid concentrations are highest during fasting and dropped during the period of food intake with all diets. An HF-containing diet changed bile acid pool composition, but not the daily rhythmicity of plasma bile acid levels. Daily rhythms of hepatic Cyp7a1 and Cyp8b1 mRNA expression followed the hepatic molecular clock, whereas for Shp expression food intake was leading. Combining an HF diet with feeding in the light/inactive period annulled CYp7a1 and Cyp8b1 gene expression rhythms, whilst keeping that of Shp intact. In conclusion, plasma bile acids and key genes in bile acid biosynthesis are entrained by food intake as well as the hepatic molecular clock. Eating during the inactivity period induced changes in the plasma bile acid pool composition similar to those induced by HF feeding. ARTICLE HISTORY

Background: Disturbance of immunometabolic signaling is a key process involved in the progression of obesity. Microglia—the resident immune cells in the brain, initiate local immune responses. It is known that hypercaloric diets lead to microglial activation. Previously, we observed that hypothalamic microglial cells from mice fed high-fat diet (HFD) lose their day/night rhythm and are constantly activated. However, little is known about daily rhythmicity in microglial circadian, immune and metabolic functions, either in lean or obese conditions. Therefore, we hypothesized that HFD disturbs microglial immunometabolism in a day/night-dependent manner. Methods: Obesity was induced in Wistar rats by feeding them HFD ad libitum for the duration of 8 weeks. Microglia were isolated from HFD- and chow-fed control animals at six time points during 24 h [every 4 h starting 2 h after lights on, i.e., Zeitgeber Time 2 (ZT2)]. Gene expression was evaluated using quantitative RT-PCR. JTK_Cycle software was used to estimate daily rhythmicity. Statistical analysis was performed with two-way ANOVA test. Results: Consumption of the obesogenic diet resulted in a 40 g significantly higher body weight gain in week 8, compared to chow diet ( p < 0.0001), associated with increased adiposity. We observed significant rhythmicity of circadian clock genes in microglia under chow conditions, which was partially lost in diet-induced obesity (DIO). Microglial immune gene expression also showed time-of-day differences, which were disrupted in HFD-fed animals. Microglia responded to the obesogenic conditions by a shift of substrate utilization with decreased glutamate and glucose metabolism in the active period of the animals, and an overall increase of lipid metabolism, as indicated by gene expression evaluation. Additionally, data on mitochondria bioenergetics and dynamics suggested an increased energy production in microglia during the inactive period on HFD. Finally, evaluation of monocyte functional gene expression showed small or absent effect of HFD on peripheral myeloid cells, suggesting a cell-specific microglial inflammatory response in DIO. Conclusions: An obesogenic diet affects microglial immunometabolism in a time-of-day dependent manner. Given the central role of the brain in energy metabolism, a better knowledge of daily rhythms in microglial immunometabolism could lead to a better understanding of the pathogenesis of obesity.

The effects of feeding behavior and diet composition, as well as their possible interactions, on daily (clock) gene expression rhythms have mainly been studied in the liver, and to a lesser degree in white adipose tissue (WAT), but hardly in other metabolic tissues such as skeletal muscle (SM) and brown adipose tissues (BAT). We therefore subjected male Wistar rats to a regular chow or free choice high-fat-high sugar (fcHFHS) diet in combination with time restricted feeding (TRF) to either the light or dark phase. In SM, all tested clock genes lost their rhythmic expression in the chow light fed group. In the fcHFHS light fed group rhythmic expression for some, but not all, clock genes was maintained, but shifted by several hours. In BAT the daily rhythmicity of clock genes was maintained for the light fed groups, but expression patterns were shifted as compared with ad libitum and dark fed groups, whilst the fcHFHS diet made the rhythmicity of clock genes become more pronounced. Most of the metabolic genes in BAT tissue tested did not show any rhythmic expression in either the chow or fcHFHS groups. In SM Pdk4 and Ucp3 were phase-shifted, but remained rhythmically expressed in the chow light fed groups. Rhythmic expression was lost for Ucp3 whilst on the fcHFHS diet during the light phase. In summary, both feeding at the wrong time of day and diet composition disturb the peripheral clocks in SM and BAT, but to different degrees and thereby result in a further desynchronization between metabolically active tissues such as SM, BAT, WAT and liver.

Aims/hypothesisThe central pacemaker of the mammalian biological timing system is located within the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. Together with the peripheral clocks, this central brain clock ensures a timely, up-to-date and proper behaviour for an individual throughout the day–night cycle. A mismatch between the central and peripheral clocks results in a disturbance of daily rhythms in physiology and behaviour. It is known that the number of rhythmically expressed genes is reduced in peripheral tissue of individuals with type 2 diabetes mellitus. However, it is not known whether the central SCN clock is also affected in the pathogenesis of type 2 diabetes. In the current study, we compared the profiles of the SCN neurons and glial cells between type 2 diabetic and control individuals.MethodsWe collected post-mortem hypothalamic tissues from 28 type 2 diabetic individuals and 12 non-diabetic control individuals. We performed immunohistochemical analysis for three SCN neuropeptides, arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP) and neurotensin (NT), and for two proteins expressed in glial cells, ionised calcium-binding adapter molecule 1 (IBA1, a marker of microglia) and glial fibrillary acidic protein (GFAP, a marker of astroglial cells).ResultsThe numbers of AVP immunoreactive (AVP-ir) and VIP-ir neurons and GFAP-ir astroglial cells in the SCN of type 2 diabetic individuals were significantly decreased compared with the numbers in the SCN of the control individuals. In addition, the relative intensity of AVP immunoreactivity was reduced in the individuals with type 2 diabetes. The number of NT-ir neurons and IBA1-ir microglial cells in the SCN was similar in the two groups.Conclusions/interpretationOur data show that type 2 diabetes differentially affects the numbers of AVP- and VIP-expressing neurons and GFAP-ir astroglial cells in the SCN, each of which could affect the daily rhythmicity of the SCN biological clock machinery. Therefore, for effectively treating type 2 diabetes, lifestyle changes and/or medication to normalise central biological clock functioning might be helpful.Electronic supplementary materialThe online version of this article (10.1007/s00125-019-4953-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.