Insulin-regulated serine and lipid metabolism drive peripheral neuropathy.

Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves1,2. Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia3-7, aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes8-10. Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome10-14, but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy15,16. Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.

Revival of light signalling in the postmortem mouse and human retina.

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

Pharmacological activation of REV-ERBs is lethal in cancer and oncogene-induced senescence.

The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer and chronic circadian rhythm disruption predisposes individuals to tumour development. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. REV-ERBs, the nuclear hormone receptors REV-ERBα (also known as NR1D1) and REV-ERBß (also known as NR1D2), are essential components of the circadian clock. Here we show that two agonists of REV-ERBs-SR9009 and SR9011-are specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and have no effect on the viability of normal cells or tissues. The anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as HRAS, BRAF, PIK3CA and others) and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 has a critical role in evoking an apoptotic response in malignant cells. Notably, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective antitumour strategy, identifying a class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are inhibitors of autophagy and de novo lipogenesis, with selective activity towards malignant and benign neoplasms.

A genome-wide microRNA screen identifies the microRNA-183/96/182 cluster as a modulator of circadian rhythms.

The regulatory mechanisms of circadian rhythms have been studied primarily at the level of the transcription-translation feedback loops of protein-coding genes. Regulatory modules involving noncoding RNAs are less thoroughly understood. In particular, emerging evidence has revealed the important role of microRNAs (miRNAs) in maintaining the robustness of the circadian system. To identify miRNAs that have the potential to modulate circadian rhythms, we conducted a genome-wide miRNA screen using U2OS luciferase reporter cells. Among 989 miRNAs in the library, 120 changed the period length in a dose-dependent manner. We further validated the circadian regulatory function of an miRNA cluster, miR-183/96/182, both in vitro and in vivo. We found that all three members of this miRNA cluster can modulate circadian rhythms. Particularly, miR-96 directly targeted a core circadian clock gene, PER2. The knockout of the miR-183/96/182 cluster in mice showed tissue-specific effects on circadian parameters and altered circadian rhythms at the behavioral level. This study identified a large number of miRNAs, including the miR-183/96/182 cluster, as circadian modulators. We provide a resource for further understanding the role of miRNAs in the circadian network and highlight the importance of miRNAs as a genome-wide layer of circadian clock regulation.

Predicting energy intake with an accelerometer-based intake-balance method.

Nutritional interventions often rely on subjective assessments of energy intake (EI), but these are susceptible to measurement error. To introduce an accelerometer-based intake-balance method for assessing EI using data from a time-restricted eating (TRE) trial. Nineteen participants with overweight/obesity (25-63 years old; 16 females) completed a 12-week intervention (NCT03129581) in a control group (unrestricted feeding; n 8) or TRE group (n 11). At the start and end of the intervention, body composition was assessed by dual-energy X-ray absorptiometry (DXA) and daily energy expenditure (EE) was assessed for 2 weeks via wrist-worn accelerometer. EI was back-calculated as the sum of net energy storage (from DXA) and EE (from accelerometer). Accelerometer-derived EI estimates were compared against estimates from the body weight planner of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Mean EI for the control group declined by 138 and 435 kJ/day for the accelerometer and NIDDK methods, respectively (both P ≥ 0·38), v. 1255 and 1469 kJ/day, respectively, for the TRE group (both P < 0·01). At follow-up, the accelerometer and NIDDK methods showed excellent group-level agreement (mean bias of -297 kJ/day across arms; standard error of estimate 1054 kJ/day) but high variability at the individual level (limits of agreement from -2414 to +1824 kJ/day). The accelerometer-based intake-balance method showed plausible sensitivity to change, and EI estimates were biologically and behaviourally plausible. The method may be a viable alternative to self-report EI measures. Future studies should assess criterion validity using doubly labelled water.

Daytime light exposure is a strong predictor of seasonal variation in sleep and circadian timing of university students.

In the absence of electric light, sleep for humans typically starts soon after dusk and at higher latitudes daily sleep timing changes seasonally as photoperiod changes. However, access to electric light shields humans from natural photoperiod changes, and whether seasonal changes in sleep occur despite this isolation from the natural light-dark cycle remains a matter of controversy. We measured sleep timing in over 500 university students living in the city of Seattle, WA (47.6°N) throughout the four seasons; we show that even when students are following a school schedule, sleep timing is delayed during the fall and winter. For instance, during the winter school days, students fell asleep 35 min later and woke up 27 min later (under daylight-savings time) than students during the summer school days, a change that is an hour larger relative to solar midnight. Furthermore, chronotype defined by mid-sleep on free days corrected for oversleep (MSFc), an indirect estimate of circadian phase, was more than 30 min later in the winter compared with the summer. Analysis of the effect of light exposure showed that the number of hours of light exposure to at least 50 lux during the daytime was a stronger predictor of MSFc than the exposure time to this illuminance after dusk. Specifically, MSFc was advanced by 30 min for each additional hour of light exposure during daytime and delayed by only 15 min for each additional hour of postdusk exposure to light. Additionally, the time of the day of exposure to high light intensities was more predictive of MSFc when daytime exposure was considered than when exposure for the full 24-h day was considered. Our results show that although sleep time is highly synchronized to social time, a delayed timing of sleep is evident during the winter months. They also suggest that daily exposure to daylight is key to prevent this delayed phase of the circadian clock and thus circadian disruption that is typically exacerbated in high-latitude winters.

Assessing temporal eating pattern in free living humans through the myCircadianClock app.

The quality and quantity of nutrition impact health. However, chrononutrition, the timing, and variation of food intake in relation to the daily sleep-wake cycle are also important contributors to health. This has necessitated an urgent need to measure, analyze, and optimize eating patterns to improve health and manage disease. While written food journals, questionnaires, and 24-hour dietary recalls are acceptable methods to assess the quantity and quality of energy consumption, they are insufficient to capture the timing and day-to-day variation of energy intake. Smartphone applications are novel methods for information-dense real-time food and beverage tracking. Despite the availability of thousands of commercial nutrient apps, they almost always ignore eating patterns, and the raw real-time data is not available to researchers for monitoring and intervening in eating patterns. Our lab developed a smartphone app called myCircadianClock (mCC) and associated software to enable long-term real-time logging that captures temporal components of eating patterns. The mCC app runs on iOS and android operating systems and can be used to track multiple cohorts in parallel studies. The logging burden is decreased by using a timestamped photo and annotation of the food/beverage being logged. Capturing temporal data of consumption in free-living individuals over weeks/months has provided new insights into diverse eating patterns in the real world. This review discusses (1) chrononutrition and the importance of understanding eating patterns, (2) the myCircadianClock app, (3) validation of the mCC app, (4) clinical trials to assess the timing of energy intake, and (5) strengths and limitations of the mCC app.

Eating architecture in adults at increased risk of type 2 diabetes: associations with body fat and glycaemic control.

Eating architecture is a term that describes meal frequency, meal timing and meal size and the daily variation in each of these. The aim of this study was to determine the relationship between components of eating architecture on body fat and markers of glycaemic control in healthy adults at increased risk of type 2 diabetes (T2DM). Participants (n 73, 39 males, age 58·8 (8·1) years, BMI 33·4 (4·4) kg/m2) recorded food intake and wore accelerometers and continuous glucose monitors (CGM) for 7-14 d under free-living conditions. Body fat and glycated Hb (HbA1c) were also measured. The mean and day-to-day variation (calculated as the standard deviation during the monitoring period) of each component of eating architecture were calculated. Multivariable linear regression models were constructed for three separate outcome variables (body fat mass, mean CGM glucose and HbA1c) for each component of eating architecture before and after adjustment for confounders. Higher variability in the time of first meal consumption was associated with increased body fat mass after adjusting for confounders (ß = 0·227, 95 % CI: 0·019, 0·434, P = 0·033). Increased variability in the time lag from waking to first meal consumption was also positively associated with increased HbA1c after adjustment (ß = 0·285, 95 % CI: 0·040, 0·530, P = 0·023). Low day-to-day variability in first meal consumption was associated with lower body fat and improved glucose control in adults at increased risk of T2DM. Routine consumption of meals may optimise temporal regulation to anticipate and respond appropriately to a glucose challenge.

Time-Restricted Eating to Prevent and Manage Chronic Metabolic Diseases.

Molecular clocks are present in almost every cell to anticipate daily recurring and predictable changes, such as rhythmic nutrient availability, and to adapt cellular functions accordingly. At the same time, nutrient-sensing pathways can respond to acute nutrient imbalance and modulate and orient metabolism so cells can adapt optimally to a declining or increasing availability of nutrients. Organismal circadian rhythms are coordinated by behavioral rhythms such as activity-rest and feeding-fasting cycles to temporally orchestrate a sequence of physiological processes to optimize metabolism. Basic research in circadian rhythms has largely focused on the functioning of the self-sustaining molecular circadian oscillator, while research in nutrition science has yielded insights into physiological responses to caloric deprivation or to specific macronutrients. Integration of these two fields into actionable new concepts in the timing of food intake has led to the emerging practice of time-restricted eating. In this paradigm, daily caloric intake is restricted to a consistent window of 8-12 h. This paradigm has pervasive benefits on multiple organ systems.

Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-ß.

The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-α and REV-ERB-ß have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-ß genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-ß cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-ß function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-ß with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

Time-restricted feeding for prevention and treatment of cardiometabolic disorders.

The soaring prevalence of obesity and diabetes is associated with an increase in comorbidities, including elevated risk for cardiovascular diseases (CVDs). CVDs continue to be among the leading causes of death and disability in the United States. While increased nutritional intake from an energy-dense diet is known to disrupt metabolic homeostasis and contributes to the disease risk, circadian rhythm disruption is emerging as a new risk factor for CVD. Circadian rhythms coordinate cardiovascular health via temporal control of organismal metabolism and physiology. Thus, interventions that improve circadian rhythms are prospective entry points to mitigate cardiometabolic disease risk. Although light is a strong modulator of the neural circadian clock, time of food intake is emerging as a dominant agent that affects circadian clocks in metabolic organs. We discovered that imposing a time-restricted feeding (TRF) regimen in which all caloric intakes occur consistently within ≤ 12 h every day exerts many cardiometabolic benefits. TRF prevents excessive body weight gain, improves sleep, and attenuates age- and diet-induced deterioration in cardiac performance. Using an integrative approach that combines Drosophila melanogaster (fruit fly) genetics with transcriptome analyses it was found that the beneficial effects of TRF are mediated by circadian clock, ATP-dependent TCP/TRiC/CCT chaperonin and mitochondrial electron transport chain components. Parallel studies in rodents have shown TRF reduces metabolic disease risks by maintaining metabolic homeostasis. As modern humans continue to live under extended periods of wakefulness and ingestion events, daily eating pattern offers a new potential target for lifestyle intervention to reduce CVD risk.

Meal frequency and timing in health and disease.

Although major research efforts have focused on how specific components of foodstuffs affect health, relatively little is known about a more fundamental aspect of diet, the frequency and circadian timing of meals, and potential benefits of intermittent periods with no or very low energy intakes. The most common eating pattern in modern societies, three meals plus snacks every day, is abnormal from an evolutionary perspective. Emerging findings from studies of animal models and human subjects suggest that intermittent energy restriction periods of as little as 16 h can improve health indicators and counteract disease processes. The mechanisms involve a metabolic shift to fat metabolism and ketone production, and stimulation of adaptive cellular stress responses that prevent and repair molecular damage. As data on the optimal frequency and timing of meals crystalizes, it will be critical to develop strategies to incorporate those eating patterns into health care policy and practice, and the lifestyles of the population.

Circadian timing in central and peripheral tissues in a migratory songbird: dependence on annual life-history states.

Predictable seasonal change in photoperiod triggers a sequential change in the daily activity-rest pattern, adaptive for migration in several bird species. The night-migratory black-headed bunting (Emberiza melanocephala) is day active under short photoperiods (8 h light:16 h dark, short day sensitive). Under long photoperiods (16 h light:8 h dark), the buntings are initially day active (long day premigratory) but subsequently become intensely night active (long day migratory) and after few weeks again return to a day active pattern (long day refractory). However, it is unclear how the daily expression of circadian genes changes during photoperiod-induced seasonal life-history states (LHSs). We measured period 2 (Per2), cryptochrome 1 (Cry1), brain and muscle arnt-like protein 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) mRNA expressions in various neural and peripheral tissues of buntings in different LHSs and discovered differences of ∼2 to 6 h in the phase and 2- to 4-fold in amplitude of circadian oscillations of Per2, Cry1, and Bmal1 between photoperiod-induced LHSs. Phase relationship in mRNA oscillations was altered between oscillator components in the circadian pacemaker system (retina, pineal, hypothalamus) as well as in the peripheral (liver, muscle) tissues. These results show for the first time altered waveforms of clock gene expressions in all tissues in parallel with behavioral shifts and suggest the involvement of circadian system in photoperiod induction of seasonal LHSs in a migratory species.

Co-expression of VAL- and TMT-opsins uncovers ancient photosensory interneurons and motorneurons in the vertebrate brain.

The functional principle of the vertebrate brain is often paralleled to a computer: information collected by dedicated devices is processed and integrated by interneuron circuits and leads to output. However, inter- and motorneurons present in today's vertebrate brains are thought to derive from neurons that combined sensory, integration, and motor function. Consistently, sensory inter-motorneurons have been found in the simple nerve nets of cnidarians, animals at the base of the evolutionary lineage. We show that light-sensory motorneurons and light-sensory interneurons are also present in the brains of vertebrates, challenging the paradigm that information processing and output circuitry in the central brain is shielded from direct environmental influences. We investigated two groups of nonvisual photopigments, VAL- and TMT-Opsins, in zebrafish and medaka fish; two teleost species from distinct habitats separated by over 300 million years of evolution. TMT-Opsin subclasses are specifically expressed not only in hypothalamic and thalamic deep brain photoreceptors, but also in interneurons and motorneurons with no known photoreceptive function, such as the typeXIV interneurons of the fish optic tectum. We further show that TMT-Opsins and Encephalopsin render neuronal cells light-sensitive. TMT-Opsins preferentially respond to blue light relative to rhodopsin, with subclass-specific response kinetics. We discovered that tmt-opsins co-express with val-opsins, known green light receptors, in distinct inter- and motorneurons. Finally, we show by electrophysiological recordings on isolated adult tectal slices that interneurons in the position of typeXIV neurons respond to light. Our work supports "sensory-inter-motorneurons" as ancient units for brain evolution. It also reveals that vertebrate inter- and motorneurons are endowed with an evolutionarily ancient, complex light-sensory ability that could be used to detect changes in ambient light spectra, possibly providing the endogenous equivalent to an optogenetic machinery.

RNA Dynamics in the Control of Circadian Rhythm.

The circadian oscillator is based on transcription-translation feedback loops that generate 24 h oscillations in gene expression. Although circadian regulation of mRNA expression at the transcriptional level is one of the most important steps for the generation of circadian rhythms within the cell, multiple lines of evidence point to a disconnect between transcript oscillation and protein oscillation. This can be explained by regulatory RNA-binding proteins acting on the nascent transcripts to modulate their processing, export, translation and degradation rates. In this chapter we will review what is known about the different steps involved in circadian gene expression from transcription initiation to mRNA stability and translation efficiency. The role of ribonucleoprotein particles in the generation of rhythmic gene expression is only starting to be elucidated, but it is likely that they cooperate with the basal transcriptional machinery to help to maintain the precision of the clock under diverse cellular and environmental conditions.

Local circadian clock gates cell cycle progression of transient amplifying cells during regenerative hair cycling.

Regenerative cycling of hair follicles offers an unique opportunity to explore the role of circadian clock in physiological tissue regeneration. We focused on the role of circadian clock in actively proliferating transient amplifying cells, as opposed to quiescent stem cells. We identified two key sites of peripheral circadian clock activity specific to regenerating anagen hair follicles, namely epithelial matrix and mesenchymal dermal papilla. We showed that peripheral circadian clock in epithelial matrix cells generates prominent daily mitotic rhythm. As a consequence of this mitotic rhythmicity, hairs grow faster in the morning than in the evening. Because cells are the most susceptible to DNA damage during mitosis, this cycle leads to a remarkable time-of-day-dependent sensitivity of growing hair follicles to genotoxic stress. Same doses of γ-radiation caused dramatic hair loss in wild-type mice when administered in the morning, during mitotic peak, compared with the evening, when hair loss is minimal. This diurnal radioprotective effect becomes lost in circadian mutants, consistent with asynchronous mitoses in their hair follicles. Clock coordinates cell cycle progression with genotoxic stress responses by synchronizing Cdc2/Cyclin B-mediated G2/M checkpoint. Our results uncover diurnal mitotic gating as the essential protective mechanism in highly proliferative hair follicles and offer strategies for minimizing or maximizing cytotoxicity of radiation therapies.

T-box transcription regulator Tbr2 is essential for the formation and maintenance of Opn4/melanopsin-expressing intrinsically photosensitive retinal ganglion cells.

Opsin 4 (Opn4)/melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a major role in non-image-forming visual system. Although advances have been made in understanding their morphological features and functions, the molecular mechanisms that regulate their formation and survival remain unknown. Previously, we found that mouse T-box brain 2 (Tbr2) (also known as Eomes), a T-box-containing transcription factor, was expressed in a subset of newborn RGCs, suggesting that it is involved in the formation of specific RGC subtypes. In this in vivo study, we used complex mouse genetics, single-cell dye tracing, and behavioral analyses to determine whether Tbr2 regulates ipRGC formation and survival. Our results show the following: (1) Opn4 is expressed exclusively in Tbr2-positive RGCs; (2) no ipRGCs are detected when Tbr2 is genetically ablated before RGC specification; and (3) most ipRGCs are eliminated when Tbr2 is deleted in established ipRGCs. The few remaining ipRGCs display abnormal dendritic morphological features and functions. In addition, some Tbr2-expressing RGCs can activate Opn4 expression on the loss of native ipRGCs, suggesting that Tbr2-expressing RGCs may serve as a reservoir of ipRGCs to regulate the number of ipRGCs and the expression levels of Opn4.

Temporal orchestration of circadian autophagy rhythm by C/EBPß.

Temporal organization of tissue metabolism is important for maintaining nutrient and energy homeostasis in mammals. Autophagy is a conserved cellular pathway that is activated in response to nutrient limitation, resulting in the degradation of cytoplasmic components and the release of amino acids and other nutrients. Here, we show that autophagy exhibits robust circadian rhythm in mouse liver, which is accompanied by cyclic induction of genes involved in various steps of autophagy. Functional analyses of transcription factors and cofactors identified C/EBPß as a potent activator of autophagy. C/EBPß is rhythmically expressed in the liver and is regulated by both circadian and nutritional signals. In cultured primary hepatocytes, C/EBPß stimulates the program of autophagy gene expression and is sufficient to activate autophagic protein degradation. Adenoviral-mediated RNAi knockdown of C/EBPß in vivo abolishes diurnal autophagy rhythm in the liver. Further, circadian regulation of C/EBPß and autophagy is disrupted in mice lacking a functional liver clock. We have thus identified C/EBPß as a key factor that links autophagy to biological clock and maintains nutrient homeostasis throughout light/dark cycles.

Small-molecule antagonists of melanopsin-mediated phototransduction.

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image-forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several central nervous system responses, including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. New sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod- and cone-mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses, including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.