Original Article Neonatal overfeeding during lactation rapidly and permanently misaligns the hepatic circadian rhythm and programmes adult NAFLD Francesc Ribas-Aulinas 1, Silvia Ribo 1, 6, Marcela Parra-Vargas 1, Antonio Fernández-Pérez 2, Judith Cebrià 1, Maria Guardiola-Perello 1, Marta Ramon-Krauel 1, 3, Carles Lerin 1, Ruben Diaz 1, 3, Susana G. Kalko 4, Mario Vallejo 2, Antoni Díez-Noguera 5, Trinitat Cambras 5, Josep C. Jimenez-Chillaron 1, * ABSTRACT Childhood obesity is a strong risk factor for adult obesity, type 2 diabetes, and cardiovascular disease. The mechanisms that link early adiposity with late-onset chronic diseases are poorly characterised. We developed a mouse model of early adiposity through litter size reduction. Mice reared in small litters (SLs) developed obesity, insulin resistance, and hepatic steatosis during adulthood. The liver played a major role in the development of the disease. Objective: To gain insight into the molecular mechanisms that link early development and childhood obesity with adult hepatic steatosis and insulin resistance. Methods: We analysed the hepatic transcriptome (Affymetrix) of control and SL mice to uncover potential pathways involved in the long-term programming of disease in our model. Results: The circadian rhythm was the most significantly deregulated Gene Ontology term in the liver of adult SL mice. Several core clock genes, such as period 1e3 and cryptochrome 1e2, were altered in two-week-old SL mice and remained altered throughout their life course until they reached 4e6 months of age. Defective circadian rhythm was restricted to the periphery since the expression of clock genes in the hypothalamus, the central pacemaker, was normal. The period-cryptochrome genes were primarily entrained by dietary signals. Hence, restricting food availability during the light cycle only uncoupled the central rhythm from the peripheral and completely normalised hepatic triglyceride content in adult SL mice. This effect was accompanied by better re-alignment of the hepatic period genes, suggesting that they might have played a causal role in mediating hepatic steatosis in the adult SL mice. Functional downregulation of Per2 in hepatocytes in vitro confirmed that the period genes regulated lipid-related genes in part through peroxisome proliferator-activated receptor alpha (Ppara). Conclusions: The hepatic circadian rhythm matures during early development, from birth to postnatal day 30. Hence, nutritional challenges during early life may misalign the hepatic circadian rhythm and secondarily lead to metabolic derangements. Specific time-restricted feeding interventions improve metabolic health in the context of childhood obesity by partially re-aligning the peripheral circadian rhythm. Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords Developmental origins of health and disease (DOHaD); Childhood obesity; Circadian rhythm; Litter size reduction; Time-restricted feeding (TRF); Non-alcoholic fatty liver disease (NAFLD) 1. INTRODUCTION disease, kidney failure, and some types of cancer [1e7], which shorten the lifespan of individuals [8]. According to the World Health Organisation, “childhood obesity is one The developmental origins of health and disease (DOHaD) hypothesis of the most serious public health challenges of the twenty-first cen- suggests that early environmental cues, including nutrition, induce tury” [1]. In 2016, more than 41 million children under the age of five long-term physiological effects by permanently altering the expression years were overweight or obese. Obesity is a major risk factor for other of target genes [9]. Childhood overweight/obesity can be considered a chronic diseases, including type 2 diabetes (T2D), cardiovascular part of the DOHaD paradigm because it is strongly associated with 1 Institut de Recerca Sant Joan de Déu (Saint John of God Children’s Hospital Barcelona), Endocrinology, Esplugues, Barcelona, Spain 2Instituto de Investigaciones Bio- médicas Alberto Sols, Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid y Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas CIBERDEM, Madrid, Spain 3Departament de Medicina, Facultat de Medicina, Universitat de Barcelona, Spain 4Moebius Research Ltd, London, UK 5Departament de Bioquímica i Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Spain 6 Current address: VIB-UGent Centre for Inflammation Research, VIB, 9052, Ghent, Belgium. *Corresponding author. Institut de Recerca Sant Joan de Déu (Children’s Hospital Barcelona), Endocrine Division, Santa Rosa 39-57, 4a Planta, Esplugues de Llobregat, Barcelona, 08950, Spain. E-mail: jjimenezc@fsjd.org (J.C. Jimenez-Chillaron). Received October 20, 2020  Revision received December 28, 2020  Accepted January 6, 2021  Available online 7 January 2021 https://doi.org/10.1016/j.molmet.2021.101162 MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 www.molecularmetabolism.com Original Article late-onset metabolic derangements. The molecular mechanisms that Cry1-2 rhythmically accumulate and form a repressor complex that link metabolic adaptations to childhood overweight/obesity with long- interacts with Clock/Bmal1 to inhibit their transcription [21]. Other term metabolic diseases are poorly characterised. Litter size reduction genes, including the nuclear receptor subfamily 1 group D member 1 in mice and rats has been widely used as a model of childhood obesity (Nrd1d1, also known as Rev-erb) and RAR-related orphan receptor and neonatal developmental programming of adult disease (reviewed alpha (Rora), play additional roles in the circadian gene network [22]. in [10,11]). Briefly, rodents raised in small litters (SLs) exhibit rapid The purpose of the circadian circuitry is to produce rhythms in frame weight gain and late-onset metabolic disorders, including obesity, with external cues (light/dark [LD] and feeding/fasting cycles) to insulin resistance, impaired glucose tolerance, hepatic steatosis, and maintain energy homeostasis. In accordance, it has been shown that renal and cardio-metabolic defects. These long-term metabolic de- alterations in circadian systems may contribute to the development of rangements can be due to alterations in either the central nervous metabolic syndrome and obesity-related complications in humans system typically leading to hyperphagia [12e15] or in peripheral tis- [23e26]. Furthermore, genetic ablation of core components of the sues [16e18]. clock system supports a causal role of the circadian rhythm in We developed a mouse model of developmental programming and mediating metabolic disorders, including hyperglycaemia, hyper- early adiposity (i.e. childhood obesity) by litter size reduction [19,20] insulinemia, and impaired lipid metabolism [27e29]. (Figure 1A). Mice reared in SLs showed transient hyperphagia, In this study, we aimed to shed light on the mechanism/pathways that augmented lipid intake, and increased adiposity as early as 2 weeks of may link early overfeeding-associated developmental programming age. Furthermore, the SL mice developed several features of metabolic with late-onset liver dysfunction in our model. Specifically, we con- syndrome later in life, including obesity, hepatic steatosis, insulin ducted an unbiased transcriptomic analysis of the livers of the control resistance, and glucose intolerance [19]. The liver played a major role and SL mice. We found that the circadian rhythm ranked as the most in driving early onset insulin resistance [19,20]. This is an excellent significantly deregulated Gene Ontology term (GO: 0007623). Herein, model of the disease because, similar to human pathophysiology, we provide evidence that (1) early overfeeding rapidly misaligns the developmental metabolic derangements associated to early adiposity hepatic circadian clock, (2) which remains altered throughout the life in mice also lead to late-onset metabolic derangements. course, and (3) this misalignment precedes hepatic steatosis and in- The circadian rhythm is an evolutionarily conserved system that helps sulin resistance in our model in part through a Ppara/Rora-dependent organisms anticipate predictable environmental changes by generating mechanism. near 24-h rhythms in behaviour and physiology. At the molecular level, the core components of the clock system are the transcription factors 2. MATERIALS AND METHODS circadian locomotor output cycles kaput (Clock), neuronal PAS domain protein 2 (Npas2), and brain and muscle aryl hydrocarbon receptor 2.1. Animal care and experimental design nuclear translocator-like protein 1 (Bmal1) (Fig. S1A). Clock hetero- Protocols were approved by the Universitat de Barcelona Animal Care dimerises with Bmal1 or its brain homolog Npas2 and regulates the and Use Committee. ICR mouse strain (ICR-CD1, Envigo Laboratories, transcription of their targets, including the period (Per1, Per2, and Barcelona, Spain) was chosen for this study based on its fast-somatic Per3) and cryptochrome genes (Cry1 and Cry2). In turn, Per1-3 and growth, especially during the neonatal period. Mouse breeding was Figure 1: Early overfeeding altered the hepatic circadian clock in young and adult mice. (A) Mouse model of early adiposity by litter size reduction. Litter size was adjusted at birth to eight pups (control group, C) or four pups per dam (small litter group, SL). (B) David functional clustering enrichment analysis from hepatic gene expression (Affymetrix). The analysis included genes showing statistically significant expression between 4-month-old C and SL mice. Clock genes mRNA levels/expression (qPCR) in the liver of 4-month-old C and SL mice (C) and 15-day-old mice (D). Data represent means  S.E.M. *p < 0.05 vs C mice. Student’s t test. 2 MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com conducted as previously described [20]. Briefly, at birth, the litter size 2.5. Epididymal fat mass assessment was adjusted to eight pups (control group, C) or four pups per dam Fat mass was determined in 4-month-old mice. Epididymal fat depots (small litter group, SL). At weaning, all mice had free access to were dissected and fat mass was calculated as a percentage of wet standard chow (2014 Tekland Global, Harlan Iberica, Barcelona, Spain) tissue per whole-body weight. and water. In this study, metabolic analysis was conducted in males only because SL females were protected against hepatic steatosis- 2.6. Liver TAG analysis hepatic insulin resistance [19]. Food intake was recorded from 2- A total of 100 mg of frozen liver sample was taken and homogenised in month-old individual mice every 24 h and the weight difference was 500 ul of SDS 0.1% using Next Advance Bullet Blender Storm 24. a measure of daily food intake. Average food intake is presented as g/ Then, 350 ul was mixed with 350 ul of methanol. Equal volumes of day/BW at each week over the course of 4 weeks. The mice were chloroform were added and the samples were chilled for 30 min in ice. maintained under constant temperature (21e23  C) and humidity Next, 48 ul of KCl 0.5 M was added and maintained for 30 min on ice. (55  10%). The samples were centrifuged (10 min at 2000 rpm at 4  C), the SN was discarded, and 300 ul of TAG (lower part) was left to evaporate O/ 2.2. Monitoring motor activity/locomotor activity rhythms N under the hood. The pellets were then mixed in 50 ul of pure EtOH. Motor activity was measured in 2-month-old C and SL mice during 30 Liver TAG was measured using specific kits from SigmaeAldrich consecutive days by using activity meters that used two perpendicular (Madrid, Spain): free glycerol reagent (#F6428-40ML), triglyceride crossed infrared beams situated 3 cm above the floor of the cage. Each reagent (#T2449-10ML), and glycerol standard solution (#G7793- beam interruption represented an activity count that was registered 5ML). All samples were normalised by the protein content using Pierce and compiled in 15-min intervals, obtaining a motor activity time se- BCA (Thermo Fisher Scientific, Madrid, Spain). ries. Light dark cycles were recorded simultaneously to the activity in a separate channel connected to a photocell pulse generator. The mice 2.7. Microarray analysis were first maintained on a 12:12-h light/dark (LD) cycle for 10 days Microarray hybridisation and analysis were performed as previously and later on constant darkness (DD) for 20 days. Data from the first 3 described using GeneChip Affymetrix Mouse 430 2.0 whole genome days were not used for the analysis. The mice had ad libitum access to arrays [29]. Briefly, 3 microarrays were hybridised for each group water and a standard chow diet. Motor activity data were first visually (control and SL). Each array contained the pooled RNA from three inspected by double-plotted graphs. Temporal series of motor activity independent mice. Expression values were summarised after back- and light intensity were analysed using El Temps (v293) (Diez- ground correction and normalisation steps using the RMA methodology Noguera, University of Barcelona, Spain, www.el-temps.com), an in- [30]. Differential expression analysis was conducted using the non- tegrated package for chronobiological analysis. Periodogram and parametric Rank Prod approach [31]. Oligonucleotides presenting spectral analyses among others were used to analyse the activity changes between groups with q values lower than 0.1 were consid- rhythms. ered significant. The David tool [32] was used to calculate the func- tional clustering enrichment statistical analysis of the Gene Ontology 2.3. Indirect calorimetry terms and Kegg pathways databases considering the list of significant Indirect calorimetry was carried out using a 16-chamber Phenomaster genes. The data were deposited at the GEO (accession number monitoring system (TSE Systems GmbH, Bad Homburg, Germany). GSE55304). Individually caged mice were acclimated in the measuring room for three days before the onset of the experiments. During the experi- 2.8. Real-time quantitative PCR (qPCR) ments, a 12-h lightedark cycle (lights on at 8:00 am) was kept and the Total RNA was isolated from previously frozen livers (TRIzol, Sigmae room temperature was maintained at 22 þ 2  C. Food and water were Aldrich, Madrid, Spain) of 8-h fasted 4-month-old mice or randomly available ad libitum. Oxygen consumption and CO2 production were fed 15-day-old mice and used for cDNA synthesis (Promega). Tran- directly measured over a period of 48e72 h, and the respiratory ex- script levels were then quantified by qPCR using SYBR Green PCR change ratio (RER) and energy expenditure were calculated from these Master Mix (Promega). Results of the respective genes of interest were values. Locomotor activity was monitored by counting infrared normalised to b-actin and subsequently median normalised to 1. The photocell beam breaks on the X and Y axes. list of primers used is detailed in Supplementary Table 1. 2.4. Time-restricted feeding (TRF) 2.9. Cell culture We conducted a dietary intervention to decouple the central from The murine hepatocarcinoma Hepa1-6 cell line (ATCC CRL-1830) was peripheral clocks. Specifically, the liver is entrained by light:dark cues used to test Per1-3 shRNA and later perform qPCR expression ana- through the hypothalamus and by nutritional cues. Importantly, mice lyses. Cells were maintained in DMEM supplemented with penicillin are crepuscular and consume >80% of their food during the dark (100 units/ml), streptomycin (100 ug/ml), L-glutamine (200 mM), and phase of the lightedark cycle. Hence, we forced them to consume all 10% FBS (SigmaeAldrich, Steinheim, Germany) at 37  C and 5% CO2. their calories during the light phase. Two-month-old C and SL mice The cells were sub-cultured at 90% confluence. were randomly assigned to ad libitum (AL) or time-restricted feeding (TRF) groups. The mice included in the TRF protocol had access to a 2.10. Adenoviral infections (shRNA) chow diet for 8 h per day during the light cycle, from Zeitgeber time 1 Adenoviral vectors were purchased from Vector Biolabs (Malvern, PA, (ZT1, 9 am) to ZT9 (5 pm). ZT0 was lights on (8 am) and ZT12 was USA). The viral vectors contained short hairpin RNAs to inhibit the lights off (8 pm). Food access was controlled via programmable specific expression of Per1 (Ad-GFP-U6-mPER1-shRNA) and Per2 (Ad- automatic feeders (SmartWaiter, Cibertec, Madrid, Spain). The mice GFP-U6-mPER2-shRNA). An adenoviral vector containing a scramble were euthanised 4 weeks after commencing the TRF intervention. The shRNA sequence with the co-expression of green fluorescent protein liver was weighted, rapidly frozen in liquid nitrogen, and stored (GFP) was used as a control (Ad-GFP-U6-shRNA). Next, 80% confluent at 80  C for further analyses (qPCR and TAG content). cells were infected with the corresponding vector at a multiplicity of MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 3 www.molecularmetabolism.com Original Article infection of 10 in DMEM without serum for 2 h. These conditions led to weeks of age [19]. Hence, we measured the expression of clock genes 80e90% viral transduction as assessed by GFP fluorescence. After in the liver of 2-week-old mice (qPCR) and found that Per1, Cry1, Cry2, infection, the cells were washed with PBS and maintained for 48 h in and Rev-erb were already altered at this developmental stage growth media supplemented with FBS (10%). At the end of incubation, (Figure 1D). Again, no changes were observed in the hypothalamus of the cells were washed, RNA was extracted (TRIzol), and the RNA the young SL mice (Fig. S1D). These data were acquired between 9 concentration was analysed by qPCR as previously described. and 11 am (ZT1-ZT3) under static conditions. We then analysed the expression of clock genes during a 24-h cycle (Figure 2 and Table S4). 2.11. Circadian rhythm analysis (cosinor method) At postnatal day 15, only a few clock genes showed rhythmicity in both Liver RNA samples from the C and SL mice were collected at different the control and SL mice. Specifically, in the control mice, significant ZT times along the 24-h cycle and analysed. The number of mice at rhythms (p < 0.05) were detected only for Per3, Cry2, Bmal1, and each time point in the experiments is provided in the figure legends. Rora (Figure 2A and Table S4). In contrast, Cry1, Rev-erb, Bmal1, The presence of rhythmicity in each time series of data was statistically Clock, and Npas2 showed significant rhythmic behaviour in the young evaluated by adjusting the data to a 24-h sinusoidal pattern and sig- SL mice. Thus, by age 15 days, the livers of the SL mice showed an nificance was tested using the cosinor method [34,35] and F test of almost opposite behaviour compared to those of the controls. The sinusoidality [35]. genes that already cycled in the young C mice did not cycle in the SL mice and vice versa. This difference greatly dissipated by 4 months. All 2.12. In silico identification of transcription factors the clock genes showed rhythmicity in both groups (Figure 2B and We first analysed the putative binding sites for transcription factors in Table S4). Although the mean acrophases and amplitudes of Per1, the promoter regions of lipid-related genes. Briefly, we obtained the Per2, Cry2, Reverb, and Npas2 tended to be lower in the SL mice, they nucleotide sequence of 1 kb upstream of the transcription start site of were not statistically significant among the groups. the genes (UCSC Genome Browser; https://genome.ucsc.edu/). We These data suggested that SL-derived developmental programming imported the sequence (FASTA format) into the MEME Suite to eluci- induced rapid and sustained deregulation of clock genes, namely the date the regions containing putative motifs for clock genes (http:// period and cryptochrome genes. Several studies have demonstrated meme-suite.org/). that the circadian rhythm plays a causal role in mediating metabolic dysfunction, including altered hepatic lipid metabolism [37e39]. 2.13. Statistical analysis Therefore, we subsequently determined whether the clock genes The results are expressed as mean  SEM. Statistical analysis was might also play a role in the development of hepatic steatosis and performed using a two-tailed t test or one-way ANOVA as indicated whole-body metabolic dysfunction in our model. (IBM SPSS Statistics 19, Madrid, Spain). A *p value < 0.05 and ***p value < 0.001 was considered significant. The rhythm variables 3.2. Clock genes regulated hepatic lipid metabolism in the SL mice derived from the cosinor analysis, amplitude (difference between the First, we found that the lipid metabolic process was significant in our mesor and peak), and acrophase (clock time of the peak value) were enrichment study (Figure 1B and Table S4). It has been reported that also evaluated. Differences between two rhythms were tested by 15e20% of the liver transcriptome shows some degree of rhythmicity comparisons of cosinors [35]. Indirect calorimetry experiments were [38e42] (Figure 3A, left panel). We estimated how many genes in the analysed by two-way ANOVA followed by Bonferroni’s post hoc test. lipid metabolic ontology process exhibited rhythmic behaviour using the CircadiOmics database (http://circadiomics.ics.uci.edu) [42,43]. 3. RESULTS This database contains more than 230 high-throughput omic datasets corresponding to “over 74 million measurements sampled over 24-h 3.1. Neonatal relative overfeeding induced early and sustained cycles” [43]. Information about the oscillatory trajectory of specific misalignment of hepatic clock genes genes, in particular tissues and species, can be visualised using a We developed a mouse model of early adiposity (childhood obesity) and comprehensive web server. This information includes periodicity sta- adult metabolic dysfunction by litter size reduction (Figure 1A) [19]. In tistics (e.g. period, amplitude, phase, P value, and q value) and this study, we aimed to gain further insight into the molecular oscillatory trajectories. We found that 40% of the genes included in the mechanisms that link developmental adaptations that are related to lipid metabolic term showed rhythmic behaviour in the mouse liver early adiposity with adult insulin resistance/glucose intolerance. We (Figure 3A, right panel). This over-representation (enrichment) sug- previously showed that the liver is the primary mediator of whole-body gested that the circadian clock might have been involved, at least in metabolic dysfunction in adult SL mice. Therefore, we used an unbi- part, in regulating hepatic lipid metabolism in our model. We next ased approach and analysed the hepatic gene expression profile in 4- confirmed (qPCR) that the expression of oscillatory lipid-related genes, month-old control and SL mice (Affymetrix). Overall, 245 annotated including Acox1, Cyp39a1, Cyp7a1, and Pck1, was altered in the liver genes were differentially expressed in the liver of the adult SL mice of the adult SL mice (Figure 3B). Their rhythmic behaviour was mis- (Table S2), with the circadian rhythm being the Gene Ontology term aligned in the young (Figure 3C) and adult SL mice (Figure 3D). with the highest significance (David enrichment analysis) (Figure 1B). How are these lipid-related genes linked to the circadian clock? In The genes included in this ontology (Table S3) encompassed several silico analysis of their promoter region (encompassing 1 kb upstream core Clock genes (Per1, Per3, and Npas2) and Clock Output genes of the transcription start site) showed no consensus-binding motifs for (Rev-erb and Dbp) (Fig. S1A). the core clock genes (Fig. S2A). Instead, clock output nuclear tran- We then confirmed (qPCR) that the expression of some clock genes, scription factors, namely Rev-erb and to a lesser extent Rora, have namely the period and cryptochrome genes, was altered in the livers of putative binding sites for most of them. Although Rev-erb could explain the adult SL mice (Figure 1C). In contrast, the expression of clock the altered expression of the lipid-related genes, it did not appear genes remained largely normal in other tissues, including the hypo- dramatically deregulated in the adult SL mice. The KEGG pathway thalamus and white adipose tissue (Fig. S1B and C). We previously enrichment analysis indicated that PPAR signalling (mmu03320:PPAR reported that young SL mice developed hepatic insulin resistance by 2 signalling pathway) might be a potential upstream regulator of lipid- 4 MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com Figure 2: mRNA expression levels of clock genes during a 24-h cycle in control (blue) and SL (red) (A) 15-day-old mice and (B) 4-month-old mice. The graphs represent double plots in which the 24-h cycles are plotted twice to provide a better graphical view of the rhythmic behaviour. related genes (Fig. S3). Notably, some of them (Pck1, Cyp7a1, free fatty acid esterification, including Mogat1 and Dgat2 (Figure 3A) Cyp39a1, and Acox1) are actually PPAR targets [41]. PPARs are [19]. In the present study, we found that Ad-shPer2, but not Ad- transcription factors that link the circadian rhythm with the regulation shPer1, significantly increased the expression of both of these genes of metabolism [42e45]. Ppara expression was altered in the liver of (Figure 4B). Our data suggested that the period genes likely influenced the 15-day-old SL mice and worsened with age (Figure 3E). In the deregulation of the clock-core system and hepatic lipid-related contrast, Pparg was fairly normal in the adult mice (Figure 3F). These pathways. However, proof of direct regulation (via ChIP-qPCR or data suggested that the misexpression of the lipid-related genes might similar approaches) is currently lacking. This relationship clearly de- have been due to the complex interaction of their upstream nuclear serves investigation. We then addressed whether liver-specific receptors, including Ppara, Rev-erb, and Rora. changes in the clock-component genes influenced whole-body Integrating these data with the previous finding that the period genes rhythmicity. were primarily altered in our model (Figure 1), it is tempting to speculate that the clock genes, namely Per1-3, might influence the 3.3. Endogenous peripheral locomotor activity was misaligned in expression of lipid-related genes, possibly through coordinated action the adult SL mice of Rora/Ppara. To prove a causal relationship between the period We analysed the locomotor activity of the adult control and SL mice genes and lipid metabolism, we selectively inhibited the expression of under a standard LD 12-h:12-h cycle (Figure 5A). Under standard LD Per1 and Per2 in Hepa1-6 cells using adenoviral vectors carrying conditions, the control and SL mice showed no differences in period, specific shRNA constructs. We confirmed that Ad-shPer1 reduced Per1 amplitude, or mean motor activity (Figure 5B). Likewise, during the LD mRNA content by 60% (Figure 4A). Likewise, Per2 and Per3 mRNA cycle, fasting or ad libitum feeding did not significantly influence SL contents were also moderately reduced (20e30%). Per1 down- locomotor activity (Fig. S4A and B). However, under constant dark regulation caused a major impact on the expression of the core clock conditions (DD), the pattern of the activity rhythm was significantly genes: Per2, Per3, Clock, and Bmal1 decreased, whereas Rev-erb was different among the groups (Figure 5C). Specifically, the SL mice significantly elevated (Figure 4A). In contrast, Per1 downregulation did showed a bimodal pattern (12-h rhythm). Of note, the bimodal pattern not influence Ppara and its downstream targets (Figure 4A). However, was quantified using the power of the second harmonic of a spectral Ad-shPer2 effectively reduced Per2 mRNA content by 40% (Figure 4B). analysis. The power of the 12-h rhythm (second harmonic) of the Unlike Per1 inhibition, Per2 downregulation did not impact other clock spectral analysis was higher in the SL group than in the control groups genes, but reduced the expression of Rora-Ppara and some Ppara only in DD stage but not under LD. downstream targets, such as Lpl and Pck1 (Figure 4B). We previously Although we did not find differences in activity during standard LD reported that, in SL mice, hepatic lipid accumulation was largely conditions, some markers of whole-body energy metabolism showed attributable to an increase in the expression of key genes involved in modifications during a specific period of the day. For example, the MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 5 www.molecularmetabolism.com Original Article Figure 3: The lipid metabolism process Gene Ontology term was significantly enriched in genes that showed rhythmic behaviour. (A) Left panel: This graph describes the approximate number of genes that exhibit oscillatory behaviour in the liver. The graph was constructed based on available published information [39e42]. Right panel: The diagram represents the genes from our data set that according to the information included in the CircadiOmics database (http://circadiomics.ics.uci.edu) show hepatic rhythmicity in rodents. (B) Comparison of SL oscillatory lipid-related genes mRNA levels/expression (qPCR) in 4-month-old liver of C and SL mice. (CeD) Oscillatory lipid-related genes mRNA expression levels in 15-day-old (C) and 4-month-old mice (D) Control and SL liver during a 24-h cycle. (E) Ppara and (F) Pparg expression (qPCR) during a 24-h cycle. Data represent means  S.E.M. *p < 0.05 vs C mice;^p < 0.1 vs C mice. Student’s t test. respiratory exchange ratio (RER), which reflects the glucose-to-lipid which indicated the liver as the organ that is likely involved in these oxidation ratio, was moderately elevated in the ad libitum-fed SL effects. mice during the dark phase (Figure 5D). This effect was accompanied by increased food intake during the dark phase of the 24-h cycle 3.4. Time-restricted feeding (TRF) re-aligned clock-controlled (Figure 5E). Furthermore, oxygen consumption and energy expenditure genes and improved lipid metabolism in the adult SL mice (EE) were significantly reduced in the ad libitum-fed SL mice during the In mammals, the hepatic clock is entrained by (1) nutritional cues, such light cycle (Fig. S4C and D). Upon fasting, both the control and SL mice as fasting/feeding or high-fat feeding, and (2) light via the central showed an appropriate decrease in RER, although a minor transient pacemaker (Figure 6A). Hence, we reasoned that restricting food difference was observed during the LD transition among the groups availability during a specific period of time (TRF) might re-programme (Fig. S4E). Likewise, oxygen consumption and EE tended to be lower in the hepatic circadian clock and improve the overall physiology of the the fasted SL mice than in the control mice, (Fig. S4F and G). These adult SL mice. We designed a dietary intervention to uncouple the LD differences were in concordance with the obese phenotype of SL mice. cycle from the nutritional cues. Specifically, the mice included in the Together, the previous data strongly suggested that there was an TRF intervention had access to the diet from 9 am to 5 pm (ZT1-ZT9) intrinsic metabolic alteration affecting different phases of the daily (Figure 6B). During this 8-h window, all mice had unrestricted ad lightedark oscillations in the adult SL mice. This desynchrony was libitum access to food. primarily attributable to the periphery, since the SL mice maintained At the beginning of the intervention, the SL mice were heavier than the normal locomotor activity under standard LD conditions. During the LD, controls and all of the mice were randomly distributed into 4 groups: C- the hypothalamus was able to synchronise whole-body rhythmicity. AL, SL-AL, C-TRF, and SL-TRF (Figure 6C). As described by others However, differences in activity under free running conditions (DD) [48], the average food intake was similar among the 4 groups were likely due to intrinsic alterations in peripheral tissues. This hy- (Figure 6D). However, despite this, the SL-TRF mice stayed leaner than pothesis was supported by the previous expression data (Figure 1), the SL-AL mice and adiposity diminished, although this was not 6 MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com Figure 4: (AeB) Circadian clock and hepatic lipid-related mRNA levels/expression in Hepa-1c cells after infection with adenoviral vectors carrying specific Per1-shRNA (A) and Per2-shRNA (B) constructs. Data represent means  S.E.M. *p < 0.05 vs Scramble-shRNA Hepa1-6 infected cells. Student’s t test. statistically significant (Figure 6E,F). More strikingly, the intervention The liver played a major role in driving these derangements. Indeed, restored the hepatic lipid content to that of the control mice (Figure 6G). young SL mice developed hepatic insulin resistance by 2 weeks of age These data indicated that TRF was an efficient method to improve and fatty liver by a month. These metabolic imbalances persisted until hepatic lipid homeostasis. adulthood (4e6 months). We postulated that TRF-mediated improvements in hepatic lipid ho- In the present study, we sought to gain further insight into the mo- meostasis were partly mediated through the re-programming of pe- lecular mechanisms involved in the development and long-term ripheral clocks. First, the intervention clearly shifted the acrophase maintenance of hepatic dysfunction. To this end, we performed an estimations of Per1, Per2, Per3, Cry2, and Reverb by 6e12 h unbiased transcriptomic analysis using microarrays (Affymetrix) of the compared with the rhythms under ad libitum feeding (Fig. S5 and livers of the adult control and SL mice. While other techniques, such as Table S4). Intriguingly, these were the genes that showed reduced RNA-Seq, are more versatile and offer the possibility of exploring wider amplitudes in the SL-AL mice compared to the controls (Figure 2B). ranges of RNA abundance in biological samples, microarrays are TRF shortened the amplitude differences between the C-TRF and SL- excellent tools to uncover novel unexpected pathways involved in TRF mice, with Reverb significantly surpassing the control-TRF specific physiological processes. Strikingly, we found that the circa- (p < 0.05) (Figure 6H and Table S4). We then explored whether TRF dian rhythm Gene Ontology term was significantly deregulated in our also modified the rhythmicity of Ppara and its downstream targets model. We confirmed that the expression of several hepatic core clock (Figure 7). We found that the rhythmicity of Ppara and some of its genes, namely Per1-3 and Cry1-2, was already altered by 2 weeks of downstream targets, including Cyp7a1, Cyp39a1, Pck1, and Acox1, age and remained as such until adulthood (4e6 months). Our data largely improved in the SL-TRF mice as compared to that in the SL-AL indicated that, in SL mice, the circadian clock might have played a mice (Figure 7A). We also measured the expression of Mogat1 and causal role in mediating hepatic steatosis by influencing in part the Dgat2. They appeared extremely altered in the liver of the SL-AL mice expression of lipid-related genes in a Ppara-dependent manner. (Figure 7B,C). The nutritional intervention partially restored the cycling The circadian system is an endogenous regulatory system that helps of both these genes in the liver of the SL-TRF mice (Figure 7B,C). In organisms anticipate predictable environmental changes by generating summary, these data indicated that TRF might have restored hepatic near 24-h rhythms in behaviour and physiology [21e23]. In humans, lipid content at least in part by re-aligning clock-mediated Ppara alterations of the circadian rhythm have been associated with chronic downstream lipid targets. diseases, including obesity and T2D [25,41,50,51]. Genetic manipu- lation in rodents confirmed a causal role of the circadian rhythm in 4. DISCUSSION mediating metabolic derangements. For example, ablation of Clock/ Bmal1, Per1-3, or Rev-erb leads to whole-body insulin resistance and We previously developed a mouse model of early adiposity (childhood hepatic steatosis [26e28,44,52,53]. The mammalian clock system is obesity) through litter size reduction [19,20]. Mice reared in SLs organised in a hierarchical manner. The master or “central” clock is developed metabolic disorders during adulthood, including obesity, located in the hypothalamus within the suprachiasmatic nucleus (SCN). hepatic steatosis, insulin resistance, and glucose intolerance [19,20]. The central clock acts as an “orchestra director” that coordinates MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 7 www.molecularmetabolism.com Original Article Figure 5: (A) Locomotor activity measurements of 2-month-old C and SL mice under standard light/dark (LD) 12-h:12-h cycles or constant darkness for 21 consecutive days (DD). (B) Pattern of locomotor activity of C and SL mice under LD conditions. (C) Pattern of locomotor activity of C and SL mice under constant dark conditions (DD). Note the bimodal rhythmicity in SL mice (blue arrows). Measurements are means of 21 consecutive days. (D) Respiratory exchange ratio (RER) representation, which reflects the glucose-to-lipid oxidation ratio of C and SL mice during 12-h lightedark cycle upon ad libitum conditions. (E) Feeding behaviour that represents food intake registered every 48 min through a 24-h cycle. Data represent means  S.E.M of animals from the same group. (DeE) A two-way ANOVA analysis was used to test the effect of the animal model (C and SL) on RER and feeding behaviour during day and light periods. Significance at p < 0.05 represented in graphs corresponds to animal model variables, whereas no significant interaction effects were found in the analysed parameters. whole-body temporal physiology by synchronising peripheral clocks the SL mice. Furthermore, the SL mice also showed increased RER that are present in all the body’s organs [54]. Interestingly, in the 2- during the dark period (which is the active phase in rodents), sug- week-old SL mice, the clock genes in the liver were primarily gesting a preference for oxidising carbohydrates over fat. We speculate altered and remained as such until 4 months of age. We did not find that increased RER in the SL mice might have been partly attributed to such alterations in other peripheral tissues or the hypothalamus. These combined elevation of the free fatty acid esterification pathway data suggested that SL-associated signals were extremely specific in (through increased expression of Mogat1 and Dgat2) and impairment programming the periphery (namely the liver) while having no major of fatty acid oxidation (through Ppara misexpression). Further experi- effects on the central pacemaker. ments are needed to fully elucidate the mechanisms leading to Supporting the role of peripheral clocks in our model, we found that physiological defects. In summary, these data strongly supported that whole-body locomotor activity followed a normal pattern when the defective whole-body rhythmicity might have been attributable pri- adult SL mice were maintained under a normal LD cycle. During the marily to peripheral clocks, as the liver is a major player. LD, the hypothalamus, which showed a normal expression of clock Why is the periphery, particularly the liver, affected by early over- genes in the young and adult SL mice, sensed the daily lightedark feeding? This is likely due to the combination of two factors. First, cycle and was able to properly synchronise whole-body rhythmicity. hepatic circadian rhythmicity starts developing at birth and is However, under DD conditions, an endogenous desynchrony man- completed by 30 days of age [55]. Second, it is well known that while ifested in the SL mice. This desynchrony was likely due to intrinsic the central clock is primarily entrained by LD information through the alterations in peripheral tissues because under DD conditions, the retina [57], the peripheral clocks, including that of the liver, are central pacemaker has weaker influence on the periphery and additionally entrained by nutritional cues, such as the feeding/fasting endogenous (abnormal) rhythmicity is partially revealed. Intriguingly, cycle and macronutrient composition [37,58e62]. We previously re- although the SL mice maintained normal activity under LD conditions, ported that SL mice were exposed to high calorie intake and different they still showed mild temporal physiological perturbations. First, the macronutrient composition (high TAG milk concentration) during the SL mice exhibited increased food intake during the dark phase and lactation period [19,20]. Based on these observations, we postulate reduced oxygen consumption and energy expenditure during the light that the hepatic clock was rapidly misaligned in the young SL mice phase. This behaviour contributed to explain the obese phenotype of because they were exposed to complex interactions or early nutritional 8 MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com Figure 6: (A) Schematic representation of the interplay between the central clock in the hypothalamus and peripheral clocks. The central clock is entrained by the light/dark cues through the retina. The hepatic clock is entrained by hypothalamic-derived signals and nutritional cues. (B) TRF intervention. Two-month-old mice had access to a chow diet ad libitum (AL) or for 8 h per day during the light cycle (TRF) from Zeitgeber time (ZT) 1 (9 am) to ZT time 9 (5 pm) for 4 consecutive weeks. (C) Body weight at the beginning of the dietary intervention. (D) Average of food intake measurements (g/day/g body weight) during the 4 weeks of TRF intervention. (E) Final body weight of C and SL mice subjected to ad libitum or TRF at the end of the intervention. (F) Fat mass (epididymal white adipose tissue, eWAT) at the end of the TRF intervention. (G) Means of hepatic lipid content (TAG levels) at ZT0 and ZT12 in C and SL mice. (H) mRNA expression levels of clock genes during a 24-h cycle in control-TRF (light green) and SL-TRF (dark green) at the end of the 4-week intervention. Data represent means  S.E.M of all the animals from the same group. Samples were subjected to a two-way ANOVA analysis in which discordant vowels denote statistical significance. cues, including high calorie intake, differences in macronutrient nutrition imbalances primarily influenced the expression of nutrition- composition, and possibly differences in offspring feeding periodicity. sensitive clock genes. Given the importance of feedingefasting cy- For example, it might have been possible that the SL females fed their cles in mediating temporal regulation of metabolism, we investigated offspring more frequently than the controls, but at the cost of spending whether TRF could improve overall physiology in the adult SL mice less time during the feeding period. While speculative, this hypothesis [65]. TRF refers to the idea of restricting food availability during specific deserves future investigation. windows of the day. During periods when food is available, individuals It was intriguing that the misalignment observed in the young SL mice have ad libitum access to the food [49,66,67]. As previously described, was fairly normalised in the adult SL mice. The difference between the hepatic circadian rhythm is largely entrained by the timing of food both stages was probably due to the fact that, as previously described, consumption [65,68]. Therefore, we conducted a temporal dietary in young mice, hepatic rhythmic behaviour is still under maturation; it intervention in which food was available for 8 h during the light cycle is completed by postnatal day 30 [64]. After ageing, the differences in only, from ZT1 to ZT9. As rodents consume most of their calories rhythmic behaviours are minimal, probably because the central (>70%) during the dark cycle, this TRF forced the animals to exclu- pacemaker is able to re-synchronise peripheral clocks. The endoge- sively consume their food during the light phase. Using this inter- nous defects can be partially revealed through nutritional cues. vention, we could partially uncouple the peripheral (hepatic) oscillators At the molecular level, nutritional cues do not equally influence the from the SCN [58,67]. Our goal was to maintain the light/dark (hy- expression of all of the clock genes. In this regard, the period and pothalamic) signals as they are normally perceived. Therefore, by cryptochrome genes are susceptible to regulation through nutrition changing only the nutritional cues, we could explore the involvement of signals [63], whereas Clock/Bmal1/Npas2 are primarily regulated by the periphery (liver) in our model by comparing its response to in- signals derived from the SCN, which is driven by LD cycles [64]. dividuals maintained ad libitum. Indeed, in our model, Per1-3 and Cry1-2 were altered in both the Strikingly, this TRF was very effective at reducing body fat mass and young and adult SL mice. Therefore, our data indicated that early liver TAG content. These metabolic improvements were associated MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 9 www.molecularmetabolism.com Original Article Figure 7: (A) mRNA expression levels of oscillatory lipid-related genes. (B) Mogat1 expression during a 24-h cycle in the liver of mice fed ad libitum (left panel) or subjected to TRF (right panel). (C) Dgat2 expression during a 24-h cycle in the liver of mice fed ad libitum (left panel) or subjected to TRF (right panel). Data represent means  S.E.M of all the animals from the same group. with better alignment of the hepatic clock genes, suggesting that dramatically misaligned, whereas the clock gene expression was desynchronised peripheral clocks might partially underlie metabolic mildly deregulated. This apparent discrepancy could be partly attrib- deregulation in our model. Together, our interpretation of the TRF data uted to a cascade effect. For example, small changes in clock gene was that the liver of the SL mice had endogenous rhythmic defects expression translate into larger deregulation of clock-controlled tran- (already suggested in Figure 5; DD) that were somewhat masked by scription factor(s) that in turn are further amplified, resulting in striking the hypothalamus (Figure 5; LD). TRF supported that the nutritional misalignment of the final targets (lipid-related genes). We performed cues may have directly influenced the hepatic clock gene expression an in silico analysis of the promoter regions of the chrono-disrupted and therefore the liver rhythmic behaviour and metabolism (Table S4). lipid genes and did not find evidence supporting a direct interaction The TRF protocol we outlined clearly created a desynchrony that did of clock genes in mediating lipid dysfunction. Instead, their effect not pretend to become a therapeutic intervention. It was just a means should have been exerted through an indirect mechanism. In line with to reveal the relative contributions of the peripheral-to-central signals this possibility, one well-known feedback loop connecting clock genes in mediating hepatic behaviour in our models. Likewise, it is very likely and metabolic regulators in the liver is interactions between Per2 and that in the long run, this type of desynchrony would potentially lead to nuclear receptors Ppara and Pparg [71,72]. further abnormal behaviour and additional metabolic defects. Indeed, This observation led us to propose that chrono-disruption of hepatic we found that Clock, Rora, and Npas2 lost significant rhythmicity in the lipid metabolism might occur in part via the Per2-Ppara axis targeting SL mice. However, it is plausible that devising rational therapeutic lipid genes [42,73]. In agreement with this, we found that Ppara, but TRFs can improve overall SL physiology. In agreement with this, other not Pparg, was profoundly chrono-disrupted in the young and adult authors have also reported that different temporal restriction schedules mice. Second, Ppara rhythmicity was partially reset in the SL-TRF may protect mice from hepatic steatosis by re-setting their hepatic mice. We tested this hypothesis through functional assays in vitro. clocks [69,70]. Specifically, we inhibited the expression of Per1 and Per2 in hepato- Our TRF data provide indirect support for the circadian clock playing a cytes in vitro using adenoviral vectors containing specific shRNAs. causal role in hepatic lipid dysregulation in our model. While these data Downregulation of Per1 (60%) induced general derangement of clock are associative, additional lines of evidence support this hypothesis. genes. However, Per2 inhibition (>40%) decreased Ppara expression. First, the lipid metabolic process Gene Ontology term was significantly Importantly, Per2 inhibition also reduced the expression of Ppara target altered in the liver of the adult SL mice. It is known that approximately genes (Lpl and Pck1) and genes involved in free fatty acid esterification 20% of the hepatic transcriptome shows rhythmic behaviour. Strik- (Mogat1 and Dgat2), which were previously associated with the ingly, we confirmed that 40% of the genes included in our gene list development of steatosis in our model [19]. In summary, these data exhibit rhythmic behaviour in the liver [60]. This enrichment strongly indicated that early over-nutrition may lead to hepatic steatosis in part suggested that, to some extent, lipid metabolic deregulation in the through deregulation of clock genes, namely period 1e3, which adult SL mice might have been under the control of the clock genes. secondarily influence the expression of lipid genes partially via Ppara. Interestingly, the rhythmic expression of the lipid-related genes was We recognise that, despite the functional loss-of-function assays, the 10 MOLECULAR METABOLISM 45 (2021) 101162 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com aforementioned interpretation was arrived at through associative ex- the circadian clock in SL mice might deregulate lipid metabolic pro- periments. Further experiments with knock-out models, such as the cesses through Per-Ppara axis-dependent lipid metabolism. This axis period 1e3 KO or the Ppara KO, would help determine the causal contributes to hepatic lipid accumulation in SL mice, which in turn relationship between clock genes (namely period 1e2) and the drives late-onset metabolic syndrome development. Our data support a expression of hepatic lipid-related genes. rationale for designing TRFs for the treatment or prevention of meta- Similar observations in closely related models have been described. bolic derangements associated with early adiposity. It is likely that For example, in utero exposure to an “obesogenic” environment TRFs mediate their beneficial effects though partially re-aligning the altered the hepatic amplitudes of core clock genes and circadian circadian clock. metabolic genes in offspring [76]. These oscillatory derangements were partially attributed to changes in Ppara expression. The authors of ACKNOWLEDGEMENTS this study suggested that offspring of obese dams were unable to adequately “mount a response to metabolic demands that require J.C.J.-C. was supported by grants from FEDER/Ministerio de Ciencia, Innovación y mobilisation of lipids (for example, under fasting of high-fat feeding) via Universidades (SAF2017-84542-R) and Instituto de Salud Carlos III (PI14/00035 and an inability to induce Ppara and its downstream targets” [77]. Another CPII16/00046), co-funded by the European Regional Development Plan, ‘‘A way to model of maternal obesity also led to the development of hepatic make Europe.’’ M.P.-V. was recipient of a postdoctoral fellowship from Mexico’s steatosis in offspring [78]. Again, non-alcoholic fatty liver was partly National Council for Science and Technology (CONACYT), Government of Mexico. attributed to chronic disruption of Clock/Bmal1 and Cry2 transcription, which was characterised by a biphasic rhythmic pattern. These data CONFLICT OF INTEREST indicate that nutritional imbalances during developmental stages (including prenatal and early postnatal life) chrono-disrupt the early None declared. expression of hepatic core clock genes and downstream targets throughout the life course. APPENDIX A. SUPPLEMENTARY DATA The DOHaD hypothesis proposes that early environmental challenges may have long-lasting effects and influence adult physiology [79]. Our Supplementary data to this article can be found online at https://doi.org/10.1016/j. mouse model exposed to high-calorie intake and increased TAG molmet.2021.101162. availability during neonatal development clearly fits within this para- digm. A current key question in the field is the mechanisms that drive REFERENCES these long-term effects. It has been proposed that epigenetic mech- anisms, including DNA methylation, histone modifications, and/or non- [1] Kahn, B.B., Flier, J.S., 2000. Obesity and insulin resistance. 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