Multi-omics profiling reveals rhythmic liver function shaped by meal timing.

Post-translational modifications (PTMs) couple feed-fast cycles to diurnal rhythms. However, it remains largely uncharacterized whether and how meal timing organizes diurnal rhythms beyond the transcriptome. Here, we systematically profile the daily rhythms of the proteome, four PTMs (phosphorylation, ubiquitylation, succinylation and N-glycosylation) and the lipidome in the liver from young female mice subjected to either day/sleep time-restricted feeding (DRF) or night/wake time-restricted feeding (NRF). We detect robust daily rhythms among different layers of omics with phosphorylation the most nutrient-responsive and succinylation the least. Integrative analyses reveal that clock regulation of fatty acid metabolism represents a key diurnal feature that is reset by meal timing, as indicated by the rhythmic phosphorylation of the circadian repressor PERIOD2 at Ser971 (PER2-pSer971). We confirm that PER2-pSer971 is activated by nutrient availability in vivo. Together, this dataset represents a comprehensive resource detailing the proteomic and lipidomic responses by the liver to alterations in meal timing.

Daytime-restricted feeding enhances running endurance without prior exercise in mice.

Physical endurance and energy conservation are essential for survival in the wild. However, it remains unknown whether and how meal timing regulates physical endurance and muscle diurnal rhythms. Here, we show that day/sleep time-restricted feeding (DRF) enhances running endurance by 100% throughout the circadian cycle in both male and female mice, compared to either ad libitum feeding or night/wake time-restricted feeding. Ablation of the circadian clock in the whole body or the muscle abolished the exercise regulatory effect of DRF. Multi-omics analysis revealed that DRF robustly entrains diurnal rhythms of a mitochondrial oxidative metabolism-centric network, compared to night/wake time-restricted feeding. Remarkably, muscle-specific knockdown of the myocyte lipid droplet protein perilipin-5 completely mimics DRF in enhancing endurance, enhancing oxidative bioenergetics and outputting rhythmicity to circulating energy substrates, including acylcarnitine. Together, our work identifies a potent dietary regimen to enhance running endurance without prior exercise, as well as providing a multi-omics atlas of muscle circadian biology regulated by meal timing.

Adipocyte reconstitution of Npy4r gene in Npy4r silenced mice promotes diet-induced obesity.

Neural regulation of adipose tissue is crucial in the homeostasis of energy metabolism. Adipose tissue neuropeptide Y (NPY) and its receptors contribute to the development of diet-induced obesity. NPY1R and NPY2R are major receptors for NPY in peripheral tissues including the adipose tissue. NPY receptor 4 (Npy4r) gene is expressed in adipose tissue. However, it is unknown whether Npy4r is involved in the development of diet-induced obesity. Here, we established an immunofluorescence microscopy technique and generated an adipocyte-reconstituted Npy4r gene knockout mouse. Among six adipose depots, we found that NPY is highly expressed around the vasculature in a dot-like fashion in interscapular brown fat and subcutaneous fat, and NPY receptors are expressed in a depot-specific manner. NPY1R is highly expressed in epidydimal fat, interscapular and peri-aortic brown fat, NPY2R in both interscapular and peri-aortic brown fat, and NPY4R in both brown fat and epidydimal fat. Next, we showed that adipocyte-reconstituted expression of Npy4r promoted diet-induced obesity in mice (P < 0.0001). Overall, this study defines the abundance and distribution of NPY and its receptors 1, 2, and 4 in mouse adipose depots, and demonstrates in an adipocyte-reconstituted gene knockout model that adipocyte Npy4r is sufficient to promote diet-induced obesity.

Circadian signatures of anterior hypothalamus in time-restricted feeding.

Background: Meal timing resets circadian clocks in peripheral tissues, such as the liver, in seven days without affecting the phase of the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Anterior hypothalamus plays an essential role in energy metabolism, circadian rhythm, and stress response. However, it remains to be elucidated whether and how anterior hypothalamus adapts its circadian rhythms to meal timing. Methods: Here, we applied transcriptomics to profile rhythmic transcripts in the anterior hypothalamus of nocturnal female mice subjected to day- (DRF) or night (NRF)-time restricted feeding for seven days. Results: This global profiling identified 128 and 3,518 rhythmic transcripts in DRF and NRF, respectively. NRF entrained diurnal rhythms among 990 biological processes, including 'Electron transport chain' and 'Hippo signaling' that reached peak time in the late sleep and late active phase, respectively. By contrast, DRF entrained only 20 rhythmic pathways, including 'Cellular amino acid catabolic process', all of which were restricted to the late active phase. The rhythmic transcripts found in both DRF and NRF tissues were largely resistant to phase entrainment by meal timing, which were matched to the action of the circadian clock. Remarkably, DRF for 36 days partially reversed the circadian clock compared to NRF. Conclusions: Collectively, our work generates a useful dataset to explore anterior hypothalamic circadian biology and sheds light on potential rhythmic processes influenced by meal timing in the brain (www.circametdb.org.cn).

[Feeding rhythm entrains circadian metabolism genes, but not the circadian clock, in brown adipose tissue].

The central circadian clock and feeding rhythm coordinately reset peripheral circadian clocks. Emerging evidence suggests that feeding rhythm resets peripheral circadian clocks in a tissue-specific manner. This study aimed to determine whether and how feeding rhythm regulates circadian rhythms of the circadian clock and metabolic genes in brown adipose tissue (BAT). We applied different regimens of time-restricted feeding (TRF) in wildtype and Per1/2 deficient C57BL/6 mice, and quantified the effects of sex, treatment duration, constant light, and circadian clock on circadian rhythms of the BAT circadian clock and metabolic genes by RT-qPCR; Representative circadian clock genes are Bmal1, Nr1d1, Dbp, and Per2, and representative metabolic genes are uncoupling protein 1 (Ucp1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) that controls the flux through glycolysis, pyruvate dehydrogenase kinase isozyme 4 (Pdk4) gating the tricarboxylic acid cycle, and carnitine palmitoyltransferase 1A (Cpt1a) that controls mitochondrial fatty acid oxidation. The results showed that, daytime-restricted feeding (DRF) moderately shifted the phase of the BAT circadian clock in female mice within 7 or 36 d, and resulted in the loss of circadian rhythm in Dbp and Per2 transcripts in males. DRF induced de novo oscillation of the Ucp1 transcript, and shifted the phase of representative metabolic genes, such as Pfkfb3, Pdk4, and Cpt1a, more than 7 h. Constant light is known to disrupt the synchrony of the central circadian clock. The results showed that constant light promoted phase entrainment of the circadian clock by DRF in BAT, but abolished the oscillation of the metabolic genes (except for Pdk4). Despite combined treatment with Per1/2 deficiency and constant darkness, DRF was sufficient to drive circadian rhythms of Bmal1 and Dbp, but not those of Nr1d1, Ucp1, Pfkfb3, and Cpt1a. Overall, the circadian clock of BAT has weak adaptation to altered feeding rhythms and sex differences. The central circadian clock antagonizes DRF in the entrainment of the BAT circadian clock, whereas DRF resets circadian rhythms of metabolic genes, such as Ucp1, Pfkfb3, and Cpt1a, in a circadian clock-dependent manner.

Targeting leptin-positive adipocytes by expressing the Cre recombinase transgene under the endogenous leptin gene.

Adipose tissue plays an important role in metabolic physiology through energy storage and endocrine functions. Spatial transcriptomics is revealing the complexity of cell types and their interaction in the adipose tissue with regards to development, homeostasis and disease. Emerging evidence suggests the existence of different subtypes of mature adipocytes that may have distinct functions, the markers of which include leptin (LEP), adiponectin (ADIPOQ), perilipin-1/4 (PLIN), and serum amyloid A (SAA), marking different adipocyte subtypes. Currently, Adipoq-Cre is widely used to study adipocyte biology, however, there is no Cre line that specifically targets LEP+ adipocytes. Here, we report the construction and validation of a Lep-Cre mouse line, which has the endogenous Lep gene edited by the CRISPR-Cas9 technology to generate the Lep-peptide 2A (P2A)-Cre fusion gene. P2A induces an auto-hydrolysis of the fusion protein, leading to expression of the Cre recombinase by the Lep gene activity. The activity of Lep-Cre in different depots of adipose tissues and non-adipose tissues was visualized by the immunofluorescence microscopy in the Lep-Cre Rosa26-loxP-Stop-loxP-tdTomato mice. We showed that Lep-Cre marked white/beige adipose depots extensively, followed by brown adipose depots. Leaky activity was observed in varying degrees among peripheral organs but not in the paraventricular nucleus of the hypothalamus. In summary, we have constructed a new adipocyte-targeting Cre mouse line that would be useful to study the development and physiology of LEP+ adipocytes.

Knockdown of hepatocyte Perilipin-3 mitigates hepatic steatosis and steatohepatitis caused by hepatocyte CGI-58 deletion in mice.

Comparative gene identification-58 (CGI-58), also known as α/ß hydrolase domain containing 5, is the co-activator of adipose triglyceride lipase that hydrolyzes triglycerides stored in the cytosolic lipid droplets. Mutations in CGI-58 gene cause Chanarin-Dorfman syndrome (CDS), an autosomal recessive neutral lipid storage disease with ichthyosis. The liver pathology of CDS manifests as steatosis and steatohepatitis, which currently has no effective treatments. Perilipin-3 (Plin3) is a member of the Perilipin-ADRP-TIP47 protein family that is essential for lipid droplet biogenesis. The objective of this study was to test a hypothesis that deletion of a major lipid droplet protein alleviates fatty liver pathogenesis caused by CGI-58 deficiency in hepatocytes. Adult CGI-58-floxed mice were injected with adeno-associated vectors simultaneously expressing the Cre recombinase and microRNA against Plin3 under the control of a hepatocyte-specific promoter, followed by high-fat diet feeding for 6 weeks. Liver and blood samples were then collected from these animals for histological and biochemical analysis. Plin3 knockdown in hepatocytes prevented steatosis, steatohepatitis, and necroptosis caused by hepatocyte CGI-58 deficiency. Our work is the first to show that inhibiting Plin3 in hepatocytes is sufficient to mitigate hepatocyte CGI-58 deficiency-induced hepatic steatosis and steatohepatitis in mice.

Circadian signatures of adipose tissue in diet-induced obesity.

High-fat diet (HFD) feeding rewires circadian rhythms of peripheral organs including the liver and adipose tissue. While the liver has been extensively studied, it remains largely unknown whether and how HFD organizes circadian biology in adipose tissue. Here, we took a systems approach to profile the diurnal transcriptome of adipose tissue in diet-induced obese mice either fed a low-fat diet (LFD) that reduces weight or still fed HFD. We detected about 200 and 2,500 diurnal genes in HFD and LFD, respectively. Pathway analysis revealed that rhythmic pathways in HFD are represented by circadian rhythm, ribosome biogenesis, and nucleosome organization, whereas those in LFD are represented by myeloid cell function. Remarkably, the majority of the circadian clock genes, except Clock, exhibited robust diurnal rhythm in the adipose tissue of HFD-fed mice. Analysis of mRNAs and proteins in another cohort of HFD-fed mice confirmed that Clock lost rhythmicity at the transcript, but not protein level. Diet reversal to LFD specifically restored diurnal difference of the Clock transcripts in adipose tissue. We matched transcriptomics data with global profiling of neutral lipids and found that lipid metabolism catalyzed by triglycerol hydrolase Ces1d is a key circadian feature that is activated by diet reversal. Together, our work defines the circadian signatures in the adipose tissue of diet-induced obese mice, and their flexibility upon dietary intervention, thereby shedding light on potential clock-modulated tissue-specific pathways during obesity.

Regulation of the urea cycle by CPS1 O-GlcNAcylation in response to dietary restriction and aging.

O-linked N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of intracellular proteins is a dynamic process broadly implicated in age-related disease, yet it remains uncharacterized whether and how O-GlcNAcylation contributes to the natural aging process. O-GlcNAc transferase (OGT) and the opposing enzyme O-GlcNAcase (OGA) control this nutrient-sensing protein modification in cells. Here, we show that global O-GlcNAc levels are increased in multiple tissues of aged mice. In aged liver, carbamoyl phosphate synthetase 1 (CPS1) is among the most heavily O-GlcNAcylated proteins. CPS1 O-GlcNAcylation is reversed by calorie restriction and is sensitive to genetic and pharmacological manipulations of the O-GlcNAc pathway. High glucose stimulates CPS1 O-GlcNAcylation and inhibits CPS1 activity. Liver-specific deletion of OGT potentiates CPS1 activity and renders CPS1 irresponsive to further stimulation by a prolonged fasting. Our results identify CPS1 O-GlcNAcylation as a key nutrient-sensing regulatory step in the urea cycle during aging and dietary restriction, implying a role for mitochondrial O-GlcNAcylation in nutritional regulation of longevity.

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.

Circadian Clock-Controlled Checkpoints in the Pathogenesis of Complex Disease.

The circadian clock coordinates physiology, metabolism, and behavior with the 24-h cycles of environmental light. Fundamental mechanisms of how the circadian clock regulates organ physiology and metabolism have been elucidated at a rapid speed in the past two decades. Here we review circadian networks in more than six organ systems associated with complex disease, which cluster around metabolic disorders, and seek to propose critical regulatory molecules controlled by the circadian clock (named clock-controlled checkpoints) in the pathogenesis of complex disease. These include clock-controlled checkpoints such as circadian nuclear receptors in liver and muscle tissues, chemokines and adhesion molecules in the vasculature. Although the progress is encouraging, many gaps in the mechanisms remain unaddressed. Future studies should focus on devising time-dependent strategies for drug delivery and engagement in well-characterized organs such as the liver, and elucidating fundamental circadian biology in so far less characterized organ systems, including the heart, blood, peripheral neurons, and reproductive systems.

Time to eat reveals the hierarchy of peripheral clocks.

Meal timing resets trillions of cellular circadian clocks in the body. Recent advances in multiomics demonstrate that clocks in peripheral tissues are differentially reset by feeding rhythm, and modulated by the central clock and the liver clock. This highlights the essential roles of tissue-specific regulation and intercellular signaling in clock synchronization.

Protocol for setup and circadian analysis of inverted feeding in mice.

Inverted feeding is a paradigm to study synchronization of circadian clocks by feeding rhythm in tissues more directly. Here, we provide a protocol for performing inverted feeding in mice and analyzing circadian rhythmicity in mouse tissues. We describe setting up inverted feeding and performing tissue dissection, followed by RNA extraction and gene expression analysis, and lastly R software-based analysis of circadian rhythmicity. This protocol can be combined with the use of CircaMetDB database for mechanistic studies of inverted feeding. For complete details on the use and execution of this protocol, please refer to Xin et al. (2021).

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

Time 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).

O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity.

Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.

O-GlcNAcase targets pyruvate kinase M2 to regulate tumor growth.

Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked ß-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.

O-GlcNAc transferase suppresses necroptosis and liver fibrosis.

Worldwide, over a billion people suffer from chronic liver diseases, which often lead to fibrosis and then cirrhosis. Treatments for fibrosis remain experimental, in part because no unifying mechanism has been identified that initiates liver fibrosis. Necroptosis has been implicated in multiple liver diseases. Here, we report that O-linked ß-N-acetylglucosamine (O-GlcNAc) modification protects against hepatocyte necroptosis and initiation of liver fibrosis. Decreased O-GlcNAc levels were seen in patients with alcoholic liver cirrhosis and in mice with ethanol-induced liver injury. Liver-specific O-GlcNAc transferase-KO (OGT-LKO) mice exhibited hepatomegaly and ballooning degeneration at an early age and progressed to liver fibrosis and portal inflammation by 10 weeks of age. OGT-deficient hepatocytes underwent excessive necroptosis and exhibited elevated protein expression levels of receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), which are key mediators of necroptosis. Furthermore, glycosylation of RIPK3 by OGT is associated with reduced RIPK3 protein stability. Taken together, these findings identify OGT as a key suppressor of hepatocyte necroptosis, and OGT-LKO mice may serve as an effective spontaneous genetic model of liver fibrosis.

Setting fire to fat.

Adropin is a liver-secreted peptide that is crucial for metabolic health. However, the molecular functions and clinical significance of adropin have not been adequately explored. Butler et al. now investigate adropin expression profiles and links to cardiometabolic disease risk in two nonhuman primate models, increasing our translational and mechanistic understanding of this fascinating hormone.

Adipocyte OGT governs diet-induced hyperphagia and obesity.

Palatable foods (fat and sweet) induce hyperphagia, and facilitate the development of obesity. Whether and how overnutrition increases appetite through the adipose-to-brain axis is unclear. O-linked beta-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) couples nutrient cues to O-GlcNAcylation of intracellular proteins at serine/threonine residues. Chronic dysregulation of O-GlcNAc signaling contributes to metabolic diseases. Here we show that adipocyte OGT is essential for high fat diet-induced hyperphagia, but is dispensable for baseline food intake. Adipocyte OGT stimulates hyperphagia by transcriptional activation of de novo lipid desaturation and accumulation of N-arachidonyl ethanolamine (AEA), an endogenous appetite-inducing cannabinoid (CB). Pharmacological manipulation of peripheral CB1 signaling regulates hyperphagia in an adipocyte OGT-dependent manner. These findings define adipocyte OGT as a fat sensor that regulates peripheral lipid signals, and uncover an unexpected adipose-to-brain axis to induce hyperphagia and obesity.