Complex physiology and clinical implications of time-restricted eating.

Time-restricted eating (TRE) is a dietary intervention that limits food consumption to a specific time window each day. The effect of TRE on body weight and physiological functions has been extensively studied in rodent models, which have shown considerable therapeutic effects of TRE and important interactions among time of eating, circadian biology, and metabolic homeostasis. In contrast, it is difficult to make firm conclusions regarding the effect of TRE in people because of the heterogeneity in results, TRE regimens, and study populations. In this review, we 1) provide a background of the history of meal consumption in people and the normal physiology of eating and fasting; 2) discuss the interaction between circadian molecular metabolism and TRE; 3) integrate the results of preclinical and clinical studies that evaluated the effects of TRE on body weight and physiological functions; 4) summarize other time-related dietary interventions that have been studied in people; and 4) identify current gaps in knowledge and provide a framework for future research directions.

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.

HAF prevents hepatocyte apoptosis and progression to MASH and hepatocellular carcinoma through transcriptional regulation of the NF-κB pathway.

BACKGROUND: HCC incidence is increasing worldwide due to the obesity epidemic, which drives metabolic dysfunction-associated steatohepatitis (MASH) that can lead to HCC. However, the molecular pathways driving MASH-HCC are poorly understood. We have previously reported that male mice with haploinsufficiency of hypoxia-associated factor, HAF (SART1+/-) spontaneously develop MASH-HCC. However, the cell type(s) responsible for HCC associated with HAF loss are unclear. RESULTS: We generated SART1-floxed mice, which were crossed with mice expressing Cre-recombinase within hepatocytes (Alb-Cre; hepS-/-) or myeloid cells (LysM-Cre, macS-/-). HepS-/- mice (both male and female) developed HCC associated with profound inflammatory and lipid dysregulation suggesting that HAF protects against HCC primarily within hepatocytes. HAF-deficient hepatocytes showed decreased P-p65 and P-p50 and in many components of the NF-κB pathway, which was recapitulated using HAF siRNA in vitro. HAF depletion also triggered apoptosis, suggesting that HAF protects against HCC by suppressing hepatocyte apoptosis. We show that HAF regulates NF-κB activity by regulating transcription of TRADD and RIPK1. Mice fed a high-fat diet (HFD) showed marked suppression of HAF, P-p65 and TRADD within their livers after 26 weeks, but showed profound upregulation of these proteins after 40 weeks, implicating deregulation of the HAF-NF-κB axis in the progression to MASH. In humans, HAF was significantly decreased in livers with simple steatosis but significantly increased in HCC compared with normal liver. CONCLUSIONS: HAF is novel transcriptional regulator of the NF-κB pathway and is a key determinant of cell fate during progression to MASH and MASH-HCC.

Time-Restricted Feeding Reduces Atherosclerosis in LDLR KO Mice but Not in ApoE Knockout Mice.

BACKGROUND: Dyslipidemia increases cardiovascular disease risk, the leading cause of death worldwide. Under time-restricted feeding (TRF), wherein food intake is restricted to a consistent window of <12 hours, weight gain, glucose intolerance, inflammation, dyslipidemia, and hypercholesterolemia are all reduced in mice fed an obesogenic diet. LDLR (low-density lipoprotein receptor) mutations are a major cause of familial hypercholesterolemia and early-onset cardiovascular disease. METHODS: We subjected benchmark preclinical models, mice lacking LDLR-knockout or ApoE knockout to ad libitum feeding of an isocaloric atherogenic diet either ad libitum or 9 hours TRF for up to 13 weeks and assessed disease development, mechanism, and global changes in hepatic gene expression and plasma lipids. In a regression model, a subset of LDLR-knockout mice were ad libitum fed and then subject to TRF. RESULTS: TRF could significantly attenuate weight gain, hypercholesterolemia, and atherosclerosis in mice lacking the LDLR-knockout mice under experimental conditions of both prevention and regression. In LDLR-knockout mice, increased hepatic expression of genes mediating ß-oxidation during fasting is associated with reduced VLDL (very-low-density lipoprotein) secretion and lipid accumulation. Additionally, increased sterol catabolism coupled with fecal loss of cholesterol and bile acids contributes to the atheroprotective effect of TRF. Finally, TRF alone or combined with a cholesterol-free diet can reduce atherosclerosis in LDLR-knockout mice. However, mice lacking ApoE, which is an important protein for hepatic lipoprotein reuptake do not respond to TRF. CONCLUSIONS: In a preclinical animal model, TRF is effective in both the prevention and regression of atherosclerosis in LDLR knockout mice. The results suggest TRF alone or in combination with a low-cholesterol diet can be a lifestyle intervention for reducing cardiovascular disease risk in humans.

Time-restricted feeding combined with resistance exercise prevents obesity and improves lipid metabolism in the liver of mice fed a high-fat diet.

Nonalcoholic fatty liver disease (NAFLD), a condition characterized by the accumulation of fat in the liver, is estimated to be the most common liver disease worldwide. Obesity is a major risk factor and contributor, and, accordingly, weight loss can improve NAFLD. Previous studies in preclinical models of diet-induced obesity and fatty liver disease have shown the independent benefits of resistance exercise training (RT) and time-restricted feeding (TRF) in preventing weight gain and hepatic build-up of fat. Here, we tested the combined effect of TRF and RT on obesity and NAFLD in mice fed a high-fat diet. Our results showed that both TRF-8-h food access in the active phase-and RT-consisting of three weekly sessions of ladder climbing-attenuated body weight gain, improved glycemic homeostasis, and decreased the accumulation of lipids in the liver. TRF combined with RT improved the respiratory exchange rate, energy expenditure, and mitochondrial respiration in the liver. Furthermore, gene expression analysis in the liver revealed lower mRNA expression of lipogenesis and inflammation genes along with increased mRNA of fatty acid oxidation genes in the TRF + RT group. Importantly, combined TRF + RT was shown to be more efficient in preventing obesity and metabolic disorders. In conclusion, TRF and RT exert complementary actions compared with isolated interventions, with significant effects on metabolic disorders and NAFLD in mice.NEW & NOTEWORTHY Whether time-restricted feeding (TRF) combined with resistance exercise training (RT) may be more efficient compared with these interventions alone is still unclear. We show that when combined with RT, TRF provided additional benefits, being more effective in increasing energy expenditure, preventing weight gain, and regulating glycemic homeostasis than each intervention alone. Thus, our results demonstrate that TRF and RT have complementary actions on some synergistic pathways that prevented obesity and hepatic liver accumulation.

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.

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.

Conserved Noncoding Cis-Elements Associated with Hibernation Modulate Metabolic and Behavioral Adaptations in Mice.

Our study elucidates functional roles for conserved cis-elements associated with the evolution of mammalian hibernation. Genomic analyses found topologically associated domains (TADs) that disproportionately accumulated convergent genomic changes in hibernators, including the TAD for the Fat Mass & Obesity (Fto) locus. Some hibernation-linked cis-elements in this TAD form regulatory contacts with multiple neighboring genes. Knockout mice for these cis-elements exhibit Fto, Irx3, and Irx5 gene expression changes, impacting hundreds of genes downstream. Profiles of pre-torpor, torpor, and post-torpor phenotypes found distinct roles for each cis-element in metabolic control, while a high caloric diet uncovered different obesogenic effects. One cis-element promoting a lean phenotype influences foraging behaviors throughout life, affecting specific behavioral sequences. Thus, convergent evolution in hibernators pinpoints functional genetic mechanisms of mammalian metabolic control.

Long-term ketogenic diet causes hyperlipidemia, liver dysfunction, and glucose intolerance from impaired insulin trafficking and secretion in mice.

A ketogenic diet (KD) is a very low-carbohydrate, very high-fat diet proposed to treat obesity and type 2 diabetes. While KD grows in popularity, its effects on metabolic health are understudied. Here we show that, in male and female mice, while KD protects against weight gain and induces weight loss, over long-term, mice develop hyperlipidemia, hepatic steatosis, and severe glucose intolerance. Unlike high fat diet-fed mice, KD mice are not insulin resistant and have low levels of insulin. Hyperglycemic clamp and ex vivo GSIS revealed cell-autonomous and whole-body impairments in insulin secretion. Major ER/Golgi stress and disrupted ER-Golgi protein trafficking was indicated by transcriptomic profiling of KD islets and confirmed by electron micrographs showing a dilated Golgi network likely responsible for impaired insulin granule trafficking and secretion. Overall, our results suggest long-term KD leads to multiple aberrations of metabolic parameters that caution its systematic use as a health promoting dietary intervention.

Finding balance: understanding the energetics of time-restricted feeding in mice.

Over the course of mammalian evolution, the ability to store energy likely conferred a survival advantage when food became scarce. A long-term increase in energy storage results from an imbalance between energy intake and energy expenditure, two tightly regulated parameters that generally balance out to maintain a fairly stable body weight. Understanding the molecular determinants of this feat likely holds the key to new therapeutic development to manage obesity and associated metabolic dysfunctions. Time-restricted feeding (TRF), a dietary intervention that limits feeding to the active phase, can prevent and treat obesity and metabolic dysfunction in rodents fed a high-fat diet, likely by exerting effects on energetic balance. Even when body weight is lower in mice on active-phase TRF, food intake is generally isocaloric as compared with ad libitum fed controls. This discrepancy between body weight and energy intake led to the hypothesis that energy expenditure is increased during TRF. However, at present, there is no consensus in the literature as to how TRF affects energy expenditure and energy balance as a whole, and the mechanisms behind metabolic adaptation under TRF are unknown. This review examines our current understanding of energy balance on TRF in rodents and provides a framework for future studies to evaluate the energetics of TRF and its molecular determinants.

Diurnal transcriptome landscape of a multi-tissue response to time-restricted feeding in mammals.

Time-restricted feeding (TRF) is an emerging behavioral nutrition intervention that involves a daily cycle of feeding and fasting. In both animals and humans, TRF has pleiotropic health benefits that arise from multiple organ systems, yet the molecular basis of TRF-mediated benefits is not well understood. Here, we subjected mice to isocaloric ad libitum feeding (ALF) or TRF of a western diet and examined gene expression changes in samples taken from 22 organs and brain regions collected every 2 h over a 24-h period. We discovered that TRF profoundly impacts gene expression. Nearly 80% of all genes show differential expression or rhythmicity under TRF in at least one tissue. Functional annotation of these changes revealed tissue- and pathway-specific impacts of TRF. These findings and resources provide a critical foundation for future mechanistic studies and will help to guide human time-restricted eating (TRE) interventions to treat various disease conditions with or without pharmacotherapies.

Reciprocal regulation between the molecular clock and kidney injury.

Tubulointerstitial fibrosis is the common pathological substrate for many etiologies leading to chronic kidney disease. Although perturbations in the circadian rhythm have been associated with renal disease, the role of the molecular clock in the pathogenesis of fibrosis remains incompletely understood. We investigated the relationship between the molecular clock and renal damage in experimental models of injury and fibrosis (unilateral ureteral obstruction, folic acid, and adenine nephrotoxicity), using genetically modified mice with selective deficiencies of the clock components Bmal1, Clock, and Cry We found that the molecular clock pathway was enriched in damaged tubular epithelial cells with marked metabolic alterations. In human tubular epithelial cells, TGFß significantly altered the expression of clock components. Although Clock played a role in the macrophage-mediated inflammatory response, the combined absence of Cry1 and Cry2 was critical for the recruitment of neutrophils, correlating with a worsening of fibrosis and with a major shift in the expression of metabolism-related genes. These results support that renal damage disrupts the kidney peripheral molecular clock, which in turn promotes metabolic derangement linked to inflammatory and fibrotic responses.

Cellular senescence and disrupted proteostasis induced by myotube atrophy are prevented with low-dose metformin and leucine cocktail.

Aging coincides with the accumulation of senescent cells within skeletal muscle that produce inflammatory products, known as the senescence-associated secretory phenotype, but the relationship of senescent cells to muscle atrophy is unclear. Previously, we found that a metformin + leucine (MET+LEU) treatment had synergistic effects in aged mice to improve skeletal muscle structure and function during disuse atrophy. Therefore, the study's purpose was to determine the mechanisms by which MET+LEU exhibits muscle atrophy protection in vitro and if this occurs through cellular senescence. C2C12 myoblasts differentiated into myotubes were used to determine MET+LEU mechanisms during atrophy. Additionally, aged mouse single myofibers and older human donor primary myoblasts were individually isolated to determine the translational potential of MET+LEU on muscle cells. MET+LEU (25 + 125 µM) treatment increased myotube differentiation and prevented myotube atrophy. Low concentration (0.1 + 0.5 µM) MET+LEU had unique effects to prevent muscle atrophy and increase transcripts related to protein synthesis and decrease transcripts related to protein breakdown. Myotube atrophy resulted in dysregulated proteostasis that was reversed with MET+LEU and individually with proteasome inhibition (MG-132). Inflammatory and cellular senescence transcriptional pathways and respective transcripts were increased following myotube atrophy yet reversed with MET+LEU treatment. Dasatinib + quercetin (D+Q) senolytic prevented myotube atrophy similar to MET+LEU. Finally, MET+LEU prevented loss in myotube size in alternate in vitro models of muscle atrophy as well as in aged myofibers while, in human primary myotubes, MET+LEU prevented reductions in myonuclei fusion. These data support that MET+LEU has skeletal muscle cell-autonomous properties to prevent atrophy by reversing senescence and improving proteostasis.

Reliability estimates for assessing meal timing derived from longitudinal repeated 24-hour dietary recalls.

BACKGROUND: Regulating meal timing may have efficacy for improving metabolic health for preventing or managing chronic disease. However, the reliability of measuring meal timing with commonly used dietary assessment tools needs characterization prior to investigating meal timing and health outcomes in epidemiologic studies. OBJECTIVES: To evaluate the reliability of estimating meal timing parameters, including overnight fasting duration, the midpoint of overnight fasting time, the number of daily eating episodes, the period with the largest percentage of daily caloric intake, and late last eating episode (> 09:00 pm) from repeated 24-h dietary recalls (24HRs). METHODS: Intraclass correlation coefficients (ICC), Light's Kappa estimates, and 95% CIs were calculated from repeated 24HR administered in 3 epidemiologic studies: The United States-based Interactive Diet and Activity Tracking in AARP (IDATA) study (n = 996, 6 24HR collected over 12-mo), German EPIC-Potsdam Validation Study (European Prospective Investigation into Cancer and Nutrition Potsdam Germany cohort) (n = 134, 12 24HR collected over 12-mo) and EPIC-Potsdam BMBF-II Study (Federal Ministry of Education and Research, "Bundesministerium für Bildung und Forschung") (n = 725, 4 24HR collected over 36 mo). RESULTS: Measurement reliability of overnight fasting duration based on a single 24HR was "poor" in all studies [ICC range: 0.27; 95% CI: 0.23, 0.32 - 0.46; 95% CI: 0.43, 0.50]. Reliability was "moderate" with 3 24HR (ICC range: 0.53; 95% CI: 0.47, 0.58 in IDATA, 0.62; 95% CI: 0.52, 0.69 in the EPIC-Potsdam Validation Study, and 0.72; 95% CI: 0.70-0.75 in the EPIC-Potsdam BMBF-II Study). Results were similar for the midpoint of overnight fasting time and the number of eating episodes. Reliability of measuring late eating was "fair" in IDATA (Light's Kappa: 0.30; 95% CI: 0.21, 0.39) and "slight" in the EPIC-Potsdam Validation study and the EPIC-Potsdam BMBF-II study (Light's Kappa: 0.19; 95% CI: 0.15, 0.25 and 0.09; 95% CI: 0.06, 0.12, respectively). Reliability estimates differed by sex, BMI, weekday, and season of 24HR administration in some studies. CONCLUSIONS: Our results show that ≥ 3 24HR over a 1-3-y period are required for reliable estimates of meal timing variables.

Weight loss increases skeletal muscle mitochondrial energy efficiency in obese mice.

Weight loss from an overweight state is associated with a disproportionate decrease in whole-body energy expenditure that may contribute to the heightened risk for weight regain. Evidence suggests that this energetic mismatch originates from lean tissue. Although this phenomenon is well documented, the mechanisms have remained elusive. We hypothesized that increased mitochondrial energy efficiency in skeletal muscle is associated with reduced expenditure under weight loss. Wildtype (WT) male C57BL6/N mice were fed with high fat diet for 10 weeks, followed by a subset of mice that were maintained on the obesogenic diet (OB) or switched to standard chow to promote weight loss (WL) for additional 6 weeks. Mitochondrial energy efficiency was evaluated using high-resolution respirometry and fluorometry. Mass spectrometric analyses were employed to describe the mitochondrial proteome and lipidome. Weight loss promoted ~50% increase in the efficiency of oxidative phosphorylation (ATP produced per O2 consumed, or P/O) in skeletal muscle. However, weight loss did not appear to induce significant changes in mitochondrial proteome, nor any changes in respiratory supercomplex formation. Instead, it accelerated the remodeling of mitochondrial cardiolipin (CL) acyl-chains to increase tetralinoleoyl CL (TLCL) content, a species of lipids thought to be functionally critical for the respiratory enzymes. We further show that lowering TLCL by deleting the CL transacylase tafazzin was sufficient to reduce skeletal muscle P/O and protect mice from diet-induced weight gain. These findings implicate skeletal muscle mitochondrial efficiency as a novel mechanism by which weight loss reduces energy expenditure in obesity.

When a calorie is not just a calorie: Diet quality and timing as mediators of metabolism and healthy aging.

An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.

Mechanisms of STAT protein activation by oncogenic KIT mutants in neoplastic mast cells.

Mutations in the c-kit gene occur in the vast majority of mastocytosis. In adult patients as well as in the cell line derived from mast cell neoplasms, the mutations occur almost exclusively at amino acid 816 within the kinase domain of KIT. Among the downstream effectors of KIT signaling, STAT3 and STAT5 have been shown to be critical for cell proliferation elicited by the KIT-Asp(816) mutant protein. However, little is known about the mechanisms of activation of STAT proteins. In this study, we identify and clarify the contribution of various STAT kinases in two widely used neoplastic mast cell lines, P815 and HMC-1. We show that STAT1, -3, and -5 proteins are activated downstream of the KIT-Asp(816) mutant. All three STAT proteins are located in the nucleus and are phosphorylated on serine residues. KIT-Asp(816) mutant can directly phosphorylate STATs on the activation-specific tyrosine residues in vitro. However, within cells, SRC family kinases and JAKs diversely contribute to tyrosine phosphorylation of STAT proteins downstream of the KIT mutant. Using a panel of inhibitors, we provide evidence for the implication or exclusion of serine/threonine kinases as responsible for serine phosphorylation of STAT1, -3, and -5 in the two cell lines. Finally, we show that only STAT5 is transcriptionally active in these cells. This suggests that the contribution of STAT1 and STAT3 downstream of KIT mutant is independent of their transcription factor function.

Pediatric mastocytosis-associated KIT extracellular domain mutations exhibit different functional and signaling properties compared with KIT-phosphotransferase domain mutations.

Compared with adults, pediatric mastocytosis has a relatively favorable prognosis. Interestingly, a difference was also observed in the status of c-kit mutations according to the age of onset. Although most adult patients have a D(816)V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus KIT-PTD mutants were introduced into rodent Ba/F3, EML, Rat2, and human TF1 cells to investigate their biologic effect. Both ECD and PTD mutations induced constitutive receptor autophosphorylation and ligand-independent proliferation of the 3 hematopoietic cells. Unlike ECD mutants, PTD mutants enhanced cluster formation and up-regulated several mast cell-related antigens in Ba/F3 cells. PTD mutants failed to support colony formation and erythropoietin-mediated erythroid differentiation. ECD and PTD mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT, whereas AKT was only activated by ECD mutants. Consistently, AKT inhibitor suppressed ECD mutant-dependent proliferation, clonogenicity, and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EML-PTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes.

Time-Restricted Feeding and Caloric Restriction: Two Feeding Regimens at the Crossroad of Metabolic and Circadian Regulation.

In addition to diet quality and quantity, the "timing" of food intake recently emerged as a third key parameter in nutritional and metabolic health. The link between nutrition timing and metabolic homeostasis is in part due to the regulation of daily feeding:fasting cycles and metabolic pathways by the circadian clock. Preclinical feeding regimen studies in rodents are invaluable to further define the modalities of this relationship and get a better understanding of its mechanistic underpinnings. Time-restricted feeding (TRF) and caloric restriction (CR) are examples of feeding regimen at the crossroads of metabolic and circadian regulation. Here we propose methods to implement TRF and CR highlighting the parameters that are relevant to the study of circadian and metabolic health. We also provide methods to determine their impact on the output of the circadian clock by analyzing diurnal expression profiles using 24 h time-series collection as well as their impact on metabolic homeostasis using a glucose tolerance test (GTT).