Circadian Metabolic Rhythms Research Articles

Pterostilbene targets the molecular oscillator RORγ to restore circadian rhythm oscillation and protect against sleep restriction induced metabolic disorders

BackgroundConsiderable researches have directed toward metabolic disorders caused by sleep restriction (SR). SR-induced disruption of circadian metabolic rhythmicity is identified as an important pathophysiological mechanism. The flavonoid pterostilbene (PTE) is abundant in the traditional Chinese medicine dragon's blood with protective efficacy against obesity-related metabolic dysfunctions. Our previous study found that PTE ameliorates exercise intolerance and clock gene oscillation in the skeletal muscles subjected to SR. PurposeThis study aimed to explore whether PTE improves SR-induced metabolic disorders and delineate the relationship between PTE and the circadian clock. Study design and methodsTwo hundred male C57/B6J mice were kept awake for 20 h/d over five consecutive days and concurrently gavaged with 50, 100, or 200 mg/kg·bw/d PTE. Food consumption and body weight were monitored, and the metabolic status of the mice was evaluated by performing OGTT and ITT, measuring the serum lipid profiles and liver histopathology in response to SR. Daily behavior was analyzed by Clocklab™. The circadian rhythms of the liver clock genes and metabolic output genes were evaluated by cosine analysis. Binding between PTE and RORα/γ or NR1D1/2 was investigated by molecular docking. A luciferase reporter assay was used to determine the impact of PTE on Bmal1 transcription in SR-exposed mice co-transfected with Ad-BMAL1-LUC plus Ad-RORγ-mCherry or Ad-NR1D1-EGFP. ResultsPTE significantly ameliorated abnormal glucose and lipid metabolism (p < 0.05) in SR-exposed mice. PTE improved circadian behavior (p < 0.05) and rescued the circadian rhythm oscillation of the liver clock (p < 0.05) and metabolic output genes (p < 0.05) under SR condition. Molecular docking disclosed that PTE might interact with RORs, and PTE was found to increase Bmal1 promoter luciferase activity with RORE elements in the presence of Ad-RORγ-mCherry (p < 0.05). ConclusionsPTE may protect against SR-induced metabolic disorders by directly modulating RORγ to maintain circadian metabolic rhythm. The findings provide valuable insights into the potential use of PTE in the treatment of metabolic disorders associated with disruptions in the circadian rhythm.

Circadian rhythm of carbohydrate metabolism in health and disease

The article presents a review of the main circadian mechanisms regulating carbohydrate metabolism and their role in maintenance of energy homeostasis; the molecular genetic structure of the circadian system is also discussed. The role of adipose tissue and other organs and systems in the maintenance of circadian rhythm of carbohydrate metabolism, both in health and in obesity and diabetes, is highlighted. Particular attention is paid to diurnal rhythms of endocrine factors responsible for metabolic patterns of hormones such as cortisol, growth hormone and melatonin. Gender differences in the circadian regulation of energy and carbohydrate metabolism are also discussed, as well as their changes in different age periods. Article provides detailed review of the mechanisms of glucose utilization, reactivity of the pancreatic islets and peripheral insulin sensitivity shifts at different time periods of the day in people with normal body weight, android and gynoid types of obesity, both in women and men. Protective factors of energy metabolism circadian regulation structure preventing the development of diabetes mellitus and cardiovascular disease in individuals with so-called “metabolically healthy” obesity type are discussed. Article provides a review of various pathways of circadian rhythm disturbances, mechanisms of their development, as well as exogenous and endogenous factors leading to carbohydrate metabolic circadian rhythm misalignment, such as shift work, untiming of natural and artificial lighting, jet lags, sleep disorders. Represented data contribute to a new look at the pathogenesis of obesity and carbohydrate metabolism disorders in various types of obesity in men and women, that provides basis for searching for new effective methods of prevention and treatment of these conditions, elaboration of evidence-based diets and physical activity recommendations, as well as approaches to their medical treatment.

Co-expression of diurnal and ultradian rhythms in the plasma metabolome of common voles (Microtus arvalis).

Metabolic rhythms include rapid, ultradian (hourly) dynamics, but it remains unclear what their relationship to circadian metabolic rhythms is, and what role meal timing plays in coordinating these ultradian rhythms in metabolism. Here, we characterized widespread ultradian rhythms under ad libitum feeding conditions in the plasma metabolome of the vole, the gold standard animal model for behavioral ultradian rhythms, naturally expressing ~2-h foraging rhythms throughout the day and night. These ultradian metabolite rhythms co-expressed with diurnal 24-h rhythms in the same metabolites and did not align with food intake patterns. Specifically, under light-dark entrained conditions we showed twice daily entrainment of phase and period of ultradian behavioral rhythms associated with phase adjustment of the ultradian cycle around the light-dark and dark-light transitions. These ultradian activity patterns also drove an ultradian feeding pattern. We used a unique approach to map this behavioral activity/feeding status to high temporal resolution (every 90min) measures of plasma metabolite profiles across the 24-h light-dark cycle. A total of 148 known metabolites were detected in vole plasma. Supervised, discriminant analysis did not group metabolite concentration by feeding status, instead, unsupervised clustering of metabolite time courses revealed clusters of metabolites that exhibited significant ultradian rhythms with periods different from the feeding cycle. Two clusters with dissimilar ultradian dynamics, one lipid-enriched (period=3.4h) and one amino acid-enriched (period=4.1h), both showed co-expression with diurnal cycles. A third cluster solely comprised of glycerophospholipids (specifically ether-linked phosphatidylcholines) expressed an 11.9h ultradian rhythm without co-expressed diurnal rhythmicity. Our findings show coordinated co-expression of diurnal metabolic rhythms with rapid dynamics in feeding and metabolism. These findings reveal that ultradian rhythms are integral to biological timing of metabolic regulation, and will be important in interpreting the impact of circadian desynchrony and meal timing on metabolic rhythms.

Rest phase snacking increases energy resorption and weight gain in male mice

ObjectiveSnacking, i.e., the intake of small amounts of palatable food items, is a common behavior in modern societies, promoting overeating and obesity. Shifting food intake into the daily rest phase disrupts circadian rhythms and is also known to stimulate weight gain. We therefore hypothesized that chronic snacking in the inactive phase may promote body weight gain and that this effect is based on disruption of circadian clocks. MethodsMale mice were fed a daily chocolate snack either during their rest or their active phase and body weight development and metabolic parameters were investigated. Snacking experiments were repeated in constant darkness and in clock-deficient mutant mice to examine the role of external and internal time cues in mediating the metabolic effects of snacking. ResultsChronic snacking in the rest phase increased body weight gain and disrupted metabolic circadian rhythms in energy expenditure, body temperature, and locomotor activity. Additionally, these rest phase snacking mice assimilated more energy during the inactive phase. Body weight remained increased in rest phase snacking wildtype mice in constant darkness as well as in clock-deficient mutant mice under a regular light–dark cycle compared to mice snacking in the active phase. Weight gain effects were abolished in clock-deficient mice in constant darkness. ConclusionsOur data suggest that mistimed snacking increases energy resorption and promotes body weight gain. This effect requires a functional circadian clock at least under constant darkness conditions.

Dietary Protein Quality Influences Liver Circadian Rhythms in a GCN2‐dependent Manner

Circadian disruption correlates with a wide range of metabolic and neurological disorders. General control non‐derepressible 2 (GCN2) activation, the primary sensory mechanism of amino acid insufficiency, also plays an integral role in maintaining the circadian clock in peripheral tissues such as the liver. The objective of this work is to examine the regulation of the circadian clock via GCN2‐mediated sensing of dietary protein quality. We hypothesized that low protein quality feeding will alter the circadian clock in a GCN2 dependent manner. To test this, wild type (WT) and whole body GCN2 knockout (GCN2KO) mice were housed in total darkness within an environmentally‐controlled indirect calorimetry cabinet and provided a diet either devoid of leucine (LeuD) or amino acid replete (Control) for 8‐days and then killed at 2 circadian time (CT) points 12‐hours apart (CT3, CT15). Following the treatment period, LeuD‐fed GCN2KO mice displayed a 40% reduction in the circadian oscillation of their energy expenditure (p&lt;.001) relative to Controls whereas no such difference was present in their WT counterparts. Measurements of the 12‐hour expression pattern of various circadian oscillatory genes within the liver revealed that GCN2 is necessary for circadian oscillation of Bmal2 mRNA expression independent of diet (p&lt;0.01). Furthermore, LeuD feeding alters the expression pattern of the clock‐controlled genes (CCGs) Ciart, Klf10and Noctby over 5‐, 10‐, and 2‐fold respectively (p&lt;0.05) in a GCN2 dependent manner. Together, these findings demonstrate that dietary protein quality plays a role in the maintenance of circadian rhythms within the liver. Furthermore, GCN2 is required to maintain whole body metabolic rhythms when challenged with a diet of poor protein quality. In observing the alterations in the circadian expression pattern of these CCGs with feeding a low protein quality diet, our findings expand our understanding of the role of dietary amino acid intake and the maintenance of circadian metabolic rhythms within the liver.

Influence of photoperiod and protocol length on metabolic rate traits in ballan wrasse Labrus bergylta.

In this study, ballan wrasse Labrus bergylta were subjected to either a conventional 1-day or an extended 5-day respirometry protocol. Additionally, in the 5-day protocol the fish were subjected to a 12 h light-dark cycle to assess the effects of photoperiods on metabolic rates (ṀO2 ). Diurnal patterns in routine and resting ṀO2 were not observed, suggesting that circadian rhythms in metabolism largely are driven by activity patterns rather than being of endogenous origin. Moreover, lack of a detectable circadian ṀO2 may be an adaptation to lower costs of living in ballan wrasse. Protocol length influenced standard metabolic rates (SMR) where estimates decreased by 13% and 17% when using 48 h and 5 days, respectively, compared to 24 h. The maximum metabolic rate (MMR) and the derived absolute aerobic scope (MMR-SMR) were unaffected by protocol length. However, factorial scopes (MMR/SMR) were reduced from 8.5 to 6.4 in the 5-day protocol, showing that factorial scopes are more sensitive to how SMR are obtained. The critical oxygen tension (Pcrit ) was reduced from 15% PO2 in the 1-day group to 11% PO2 in the 5-day group. However, ṀO2 in response to decreasing PO2 was similar, which together with a similar oxygen extraction coefficient, α (ṀO2 /PO2 ), suggested that the higher Pcrit in the 1-day group was an artefact of overestimating SMR. Finally, α was 12% lower at MMR compared to at Pcrit , which either means that MMR was underestimated in proportion to this difference or that α is not constant in the entire PO2 range. In summary, this study found that a conventional 1-day respirometry protocol may overestimate SMR and thereby alter the derived Pcrit and aerobic scope, while α is unaffected by protocol length. Moreover, alternating light conditions in the absence of other stressors did not influence ṀO2 in ballan wrasse.

Clock-modulated checkpoints in time-restricted eating.

Time-restricted eating (TRE), which limits the daily meal timing to a window of 6-12 h, has been shown to reduce the risks of cardiometabolic diseases through consolidating circadian rhythms of metabolism and physiology. Recent advances indicate that canonical circadian clocks are dispensable for the actions of TRE in the liver, and that meal timing entrains circadian rhythms in peripheral tissues in a tissue-specific manner (e.g., the liver and fat are readily entrainable, whereas the heart and kidneys are resistant). Here, we propose that TRE engages clock-modulated checkpoints (CCPs) to reset circadian rhythms of tissue functions. Elucidation of CCPs would reveal the mechanistic basis of tissue responsiveness to TRE, and facilitate the use of TRE in precision medicine for cardiometabolic diseases.

A multi-tissue multi-omics analysis reveals distinct kineztics in entrainment of diurnal transcriptomes by inverted feeding

SummaryTime of eating synchronizes circadian rhythms of metabolism and physiology. Inverted feeding can uncouple peripheral circadian clocks from the central clock located in the suprachiasmatic nucleus. However, system-wide changes of circadian metabolism and physiology entrained to inverted feeding in peripheral tissues remain largely unexplored. Here, we performed a 24-h global profiling of transcripts and metabolites in mouse peripheral tissues to study the transition kinetics during inverted feeding, and revealed distinct kinetics in phase entrainment of diurnal transcriptomes by inverted feeding, which graded from fat tissue (near-completely entrained), liver, kidney, to heart. Phase kinetics of tissue clocks tracked with those of transcriptomes and were gated by light-related cues. Integrated analysis of transcripts and metabolites demonstrated that fatty acid oxidation entrained completely to inverted feeding in heart despite the slow kinetics/resistance of the heart clock to entrainment by feeding. This multi-omics resource defines circadian signatures of inverted feeding in peripheral tissues (www.CircaMetDB.org.cn).

A Multi-Tissue Multi-Omics Analysis Reveals Distinct Kinetics in Entrainment of Diurnal Transcriptomes by Inverted Feeding

Time of eating synchronizes circadian rhythms of metabolism and physiology. Inverted feeding reverses peripheral circadian clocks, but privileges the light-responsive central clock located in the suprachiasmatic nucleus. However, system-wide changes of circadian metabolism and physiology entrained to inverted feeding in peripheral tissues remain largely unexplored. Here, we performed a 24-h global profiling of transcripts and metabolites in mouse peripheral tissues to study the transition kinetics during inverted feeding, and revealed distinct kinetics in phase entrainment of diurnal transcriptomes by inverted feeding, which graded from fat tissue (near-completely entrained), liver, kidney, to heart. Phase kinetics of tissue clocks tracked with those of transcriptomes, and was gated by light cue. Integrated analysis of transcripts and metabolites demonstrated that fatty acid oxidation entrained completely to inverted feeding in heart despite the slow kinetics of the heart clock. This multi-omics resource defines circadian signatures of inverted feeding in peripheral tissues (www.CircaMetDB.org.cn).

Can your diet change your clock?

High-fat diets disturb circadian metabolic rhythms in the brain.

A molecular circadian clock operates in the parathyroid gland and is disturbed in chronic kidney disease associated bone and mineral disorder

Circadian rhythms in metabolism, hormone secretion, cell cycle and locomotor activity are regulated by a molecular circadian clock with the master clock in the suprachiasmatic nucleus of the central nervous system. However, an internal clock is also expressed in several peripheral tissues. Although about 10% of all genes are regulated by clock machinery an internal molecular circadian clock in the parathyroid glands has not previously been investigated. Parathyroid hormone secretion exhibits a diurnal variation and parathyroid hormone gene promoter contains an E-box like element, a known target of circadian clock proteins. Therefore, we examined whether an internal molecular circadian clock is operating in parathyroid glands, whether it is entrained by feeding and how it responds to chronic kidney disease. As uremia is associated with extreme parathyroid growth and since disturbed circadian rhythm is related to abnormal growth, we examined the expression of parathyroid clock and clock-regulated cell cycle genes in parathyroid glands of normal and uremic rats. Circadian clock genes were found to be rhythmically expressed in normal parathyroid glands and this clock was minimally entrained by feeding. Diurnal regulation of parathyroid glands was next examined. Significant rhythmicity of fibroblast-growth-factor-receptor-1, MafB and Gata3 was found. In uremic rats, deregulation of circadian clock genes and the cell cycle regulators, Cyclin D1, c-Myc, Wee1 and p27, which are influenced by the circadian clock, was found in parathyroid glands as well as the aorta. Thus, a circadian clock operates in parathyroid glands and this clock and downstream cell cycle regulators are disturbed in uremia and may contribute to dysregulated parathyroid proliferation in secondary hyperparathyroidism.

Feeding Rhythms and the Circadian Regulation of Metabolism.

The molecular circadian clock regulates metabolic processes within the cell, and the alignment of these clocks between tissues is essential for the maintenance of metabolic homeostasis. The possibility of misalignment arises from the differential responsiveness of tissues to the environmental cues that synchronize the clock (zeitgebers). Although light is the dominant environmental cue for the master clock of the suprachiasmatic nucleus, many other tissues are sensitive to feeding and fasting. When rhythms of feeding behavior are altered, for example by shift work or the constant availability of highly palatable foods, strong feedback is sent to the peripheral molecular clocks. Varying degrees of phase shift can cause the systemic misalignment of metabolic processes. Moreover, when there is a misalignment between the endogenous rhythms in physiology and environmental inputs, such as feeding during the inactive phase, the body's ability to maintain homeostasis is impaired. The loss of phase coordination between the organism and environment, as well as internal misalignment between tissues, can produce cardiometabolic disease as a consequence. The aim of this review is to synthesize the work on the mechanisms and metabolic effects of circadian misalignment. The timing of food intake is highlighted as a powerful environmental cue with the potential to destroy or restore the synchrony of circadian rhythms in metabolism.

The Disruption of Liver Metabolic Circadian Rhythms by a Cafeteria Diet Is Sex-Dependent in Fischer 344 Rats.

Circadian rhythms are ~24 h fluctuations of different biological processes that are regulated by the circadian clock system. They exert a major influence on most of the metabolism, such as the hepatic metabolism. This rhythmicity can be disrupted by obesogenic diets, fact that is considered to be a risk factor for the development of metabolic diseases. Nevertheless, obesogenic diets do not affect both genders in the same manner. We hypothesized that the circadian rhythms disruption of the hepatic metabolism, caused by obesogenic diets, is gender-dependent. Male and female Fischer 344 rats were fed either a standard diet or a cafeteria diet and sacrificed at two different moments, at zeitgeber 3 and 15. Only female rats maintained the circadian variations of the hepatic metabolism under a cafeteria diet. Most of those metabolites were related with the very low density lipoprotein (VLDL) synthesis, such as choline, betaine or phosphatidylcholine. Most of these metabolites were found to be increased at the beginning of the dark period. On the other hand, male animals did not show these time differences. These findings suggest that females might be more protected against the circadian disruption of the hepatic metabolism caused by a cafeteria diet through the increase of the VLDL synthesis at the beginning of the feeding time.

Early Time-Restricted Feeding Reduces Appetite and Increases Fat Oxidation But Does Not Affect Energy Expenditure in Humans.

ObjectiveEating earlier in the daytime to align with circadian rhythms in metabolism enhances weight loss. However, it is unknown whether these benefits are mediated through increased energy expenditure or decreased food intake. We therefore performed the first randomized trial to determine how meal timing affects 24-hour energy metabolism when food intake and meal frequency are matched.MethodsEleven overweight adults practiced both early time-restricted feeding (eTRF; eating from 8 am-2 pm) and a control schedule (eating from 8 am-8 pm) each for four days. On the fourth day, 24-hour energy expenditure and substrate oxidation were measured by whole-room calorimetry, in conjunction with appetite and metabolic hormones.ResultseTRF did not affect 24-hour energy expenditure (Δ=10±16 kcal/day; p=0.55). Despite the longer daily fast (intermittent fasting), eTRF decreased mean ghrelin levels by 32±10 pg/ml (p=0.006), made hunger more even-keeled (p=0.006), and tended to increase fullness (p=0.06-0.10) and decrease the desire to eat (p=0.08). eTRF also increased metabolic flexibility (p=0.0006) and decreased the 24-hour non-protein respiratory quotient (Δ=−0.021±0.010; p=0.05).ConclusionsMeal timing interventions facilitate weight loss primarily by decreasing appetite rather than by increasing energy expenditure. eTRF may also increase fat loss by increasing fat oxidation.

Diet-Induced Circadian Enhancer Remodeling Synchronizes Opposing Hepatic Lipid Metabolic Processes

Overnutrition disrupts circadian metabolic rhythms by mechanisms that are not well understood. Here, we show that diet-induced obesity (DIO) causes massive remodeling of circadian enhancer activity in mouse liver, triggering synchronous high-amplitude circadian rhythms of both fatty acid (FA) synthesis and oxidation. SREBP expression was rhythmically induced by DIO, leading to circadian FA synthesis and, surprisingly, FA oxidation (FAO). DIO similarly caused a high-amplitude circadian rhythm of PPARα, which was also required for FAO. Provision of a pharmacological activator of PPARα abrogated the requirement of SREBP for FAO (but not FA synthesis), suggesting that SREBP indirectly controls FAO via production of endogenous PPARα ligands. The high-amplitude rhythm of PPARα imparted time-of-day-dependent responsiveness to lipid-lowering drugs. Thus, acquisition of rhythmicity for non-core clock components PPARα and SREBP1 remodels metabolic gene transcription in response to overnutrition and enables a chronopharmacological approach to metabolic disorders.

Effect of Daytime Blue-enriched LED Light on the Nighttime Circadian Melatonin Inhibition of Hepatoma 7288CTC Warburg Effect and Progression.

Liver cancer is the second leading cause of cancer death worldwide. Metabolic pathways within the liver and liver cancers are highly regulated by the central circadian clock in the suprachiasmatic nuclei (SCN). Daily light and dark cycles regulate the SCN-driven pineal production of the circadian anticancer hormone melatonin and temporally coordinate circadian rhythms of metabolism and physiology in mammals. In previous studies, we demonstrated that melatonin suppresses linoleic acid metabolism and the Warburg effect (aerobic glycolysis)in human breast cancer xenografts and that blue-enriched light (465-485 nm) from light-emitting diode lighting at daytime (bLAD) amplifies nighttime circadian melatonin levels in rats by 7-fold over cool white fluorescent (CWF) lighting. Here we tested the hypothesis that daytime exposure of tissue-isolated Morris hepatoma 7288CTC-bearing male rats to bLAD amplifies the nighttime melatonin signal to enhance the inhibition of tumor growth. Compared with rats housed under a 12:12-h light:dark cycle in CWF light, rats in bLAD light evinced a 7-fold higher peak plasma melatonin level at the mid-dark phase; in addition, high melatonin levels were prolonged until 4 h into the light phase. After implantation of tissue-isolated hepatoma 7288CTC xenografts, tumor growth rates were markedly delayed, and tumor cAMP levels, LA metabolism, the Warburg effect, and growth signaling activities were decreased in rats in bLAD compared with CWF daytime lighting. These data show that the increased nighttime circadian melatonin levels due to bLAD exposure decreases hepatoma metabolic, signaling, and proliferative activities beyond what occurs after normal melatonin signaling under CWF light.

Early Time-Restricted Feeding Improves Insulin Sensitivity, Blood Pressure, and Oxidative Stress Even without Weight Loss in Men with Prediabetes

Intermittent fasting (IF) improves cardiometabolic health; however, it is unknown whether these effects are due solely to weight loss. We conducted the first supervised controlled feeding trial to test whether IF has benefits independent of weight loss by feeding participants enough food to maintain their weight. Our proof-of-concept study also constitutes the first trial of early time-restricted feeding (eTRF), a form of IF that involves eating early in the day to be in alignment with circadian rhythms in metabolism. Men with prediabetes were randomized to eTRF (6-hr feeding period, with dinner before 3 p.m.) or a control schedule (12-hr feeding period) for 5weeks and later crossed over to the other schedule. eTRF improved insulin sensitivity, β cell responsiveness, blood pressure, oxidative stress, and appetite. We demonstrate for the first time in humans that eTRF improves some aspects of cardiometabolic health and that IF's effects are not solely due to weight loss.

Diurnal Variation in PDK4 Expression Is Associated With Plasma Free Fatty Acid Availability in People.

Many biological pathways involved in regulating substrate metabolism display rhythmic oscillation patterns. In rodents, clock genes regulate circadian rhythms of metabolic genes and substrate metabolism. However, the interrelationships among substrate metabolism, metabolic genes, and clock genes have not been fully explored in people. We tested the hypothesis that the diurnal expression pattern of pyruvate dehydrogenase kinase 4 (PDK4), a key metabolic enzyme involved in fuel switching between glucose and free fatty acids (FFAs), is associated with plasma FFA concentration and clock genes. We analyzed peripheral blood mononuclear cells (PBMCs), subcutaneous adipose tissue, and plasma samples obtained serially during 24 hours from metabolically healthy women (n = 10) and evaluated the interrelationships among PDK4, plasma FFA, and clock genes. We also determined the potential mechanisms responsible for PDK4 transcriptional regulation by using primary human PBMCs and adipocytes. We found that PDK4 diurnal expression patterns were similar in PBMCs and adipose tissue (ρ = 0.84, P < 0.001). The diurnal variation in PBMC PDK4 expression correlated more strongly with plasma FFA and insulin (ρ = 0.86 and 0.63, respectively, both P < 0.001) concentrations than clock genes. Data obtained from primary culture experiments demonstrated that FFAs directly induced PDK4 gene expression, at least in part through activation of peroxisome proliferator-activated receptor α. Our results suggest that plasma FFA availability is an important regulator of diurnal expression patterns of PDK4, and we identify a novel interaction between plasma FFA and cellular diurnal rhythms in regulating substrate metabolism.

PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms.

Magnesium (Mg2+) plays pleiotropic roles in cellular biology, and it is essentially required for all living organisms. Although previous studies demonstrated intracellular Mg2+ levels were regulated by the complex of phosphatase of regenerating liver 2 (PRL2) and Mg2+ transporter of cyclin M (CNNMs), physiological functions of PRL2 in whole animals remain unclear. Interestingly, Mg2+ was recently identified as a regulator of circadian rhythm-dependent metabolism; however, no mechanism was found to explain the clock-dependent Mg2+ oscillation. Herein, we report PRL2 as a missing link between sex and metabolism, as well as clock genes and daily cycles of Mg2+ fluxes. Our results unveil that PRL2-null animals displayed sex-dependent alterations in body composition, and expression of PRLs and CNNMs were sex- and circadian time-dependently regulated in brown adipose tissues. Consistently, PRL2-KO mice showed sex-dependent alterations in thermogenesis and in circadian energy metabolism. These physiological changes were associated with an increased rate of uncoupled respiration with lower intracellular Mg2+ in PRL2-KO cells. Moreover, PRL2 deficiency causes inhibition of the ATP citrate lyase axis, which is involved in fatty acid synthesis. Overall, our findings support that sex- and circadian-dependent PRL2 expression alter intracellular Mg2+ levels, which accordingly controls energy metabolism status.

Principles for circadian orchestration of metabolic pathways

Circadian rhythms govern multiple aspects of animal metabolism. Transcriptome-, proteome- and metabolome-wide measurements have revealed widespread circadian rhythms in metabolism governed by a cellular genetic oscillator, the circadian core clock. However, it remains unclear if and under which conditions transcriptional rhythms cause rhythms in particular metabolites and metabolic fluxes. Here, we analyzed the circadian orchestration of metabolic pathways by direct measurement of enzyme activities, analysis of transcriptome data, and developing a theoretical method called circadian response analysis. Contrary to a common assumption, we found that pronounced rhythms in metabolic pathways are often favored by separation rather than alignment in the times of peak activity of key enzymes. This property holds true for a set of metabolic pathway motifs (e.g., linear chains and branching points) and also under the conditions of fast kinetics typical for metabolic reactions. By circadian response analysis of pathway motifs, we determined exact timing separation constraints on rhythmic enzyme activities that allow for substantial rhythms in pathway flux and metabolite concentrations. Direct measurements of circadian enzyme activities in mouse skeletal muscle confirmed that such timing separation occurs in vivo.