**Background and rationale** Calorie restriction (eating fewer calories than the daily metabolic requirement) has been known for almost 100 years to result in extension of life and a reduction in the rate of ageing/age related disease prevalence (reviewed in Speakman and Mitchell 2011: see uploaded pdf). It is effective in a wide range of organisms from yeast and *C.elegans*, to spiders, *Drosophila*, rodents, dogs and cows. Although the effects on lifespan in non-human primates is contested there is a consistent effect in such studies on healthspan. A randomised controlled trial in humans (the CALERIE project) concluded that after 2 years of CR at a level of 15% humans were showing many of the same changes in key biomarkers that are shown in animal studies. CR therefore represents one of the most promising interventions that will slow aging and reduce the age related burden of disease. Despite decades of study the mechanism(s) that underpin the life enhancing effects of CR remain obscure. The rationale of the current project is that in rodents there is a direct positive linear relationship between the extent of restriction and the extension of lifespan (reviewed in Speakman and Hambly, 2007; see uploaded pdf and updated review in Speakman et al 2016 see uploaded pdf). Hence the most probable factors that underpin the life enhancing impacts of CR are likely to be also linearly related to the extent of restriction. **Project goals** The goal of the project therefore is to expose mice to graded levels of restriction from 0% to 40% in 10% increments. Mice aged 5 months will be exposed to restriction for two periods – a short term restriction of 3 months, and a long term restriction of 19 months. The mice will be extensively phenotyped to find responses to restriction that mimic the linear nature of the lifespan response. This is an observational hypothesis generating experiment, that will also allow us to test whether some of the current ideas for the mechanism underpinning the CR response are consistent with the graded response paradigm. **Detailed Methods** *Overall design* We characterised the response to CR (and protein restriction :PR) in C57BL/6 male mice, a strain known to have a positive lifespan response under CR. The time-point at which CR is started has an impact on the lifespan effect. Initiation of CR at 4 weeks of age shortened lifespan (Harrison and Archer 1987) while CR introduced at 6 weeks increased lifespan (Weindruch and Walford 1982). Nevertheless both these early start points impact development, and are probably unrealistic models for implementation of CR in humans. In our project mice were introduced to CR or PR at 20 weeks of age, approximately equivalent to early human adulthood, and close to the time when mice reach skeletal maturity (Somerville, et al. 2004). This start time avoids impacts of CR on developmental processes. Previous studies suggested CR begun at six months was as effective at increasing lifespan as starting at 6 weeks (Yu, et al. 1985). We exposed mice to 5 different levels of CR: 0, 10, 20, 30 and 40 % lower calories than their own individual intakes measured over a baseline period (BL) of 14 days prior to introducing the restricted diets. Mice on restriction were individually housed and fed daily at lights out (1830h). There is a potential issue with an appropriate control group in CR studies (Speakman and Mitchell 2011; Sohal and Forster 2014). Animals that are fed completely ad libitum (AL) may become obese and hence the comparison of CR to AL animals may simply reflect an anti-obesity effect of CR. This is less of an issue when graded levels of CR are used instead of a single comparison of one CR level to AL animals. A further problem however occurs in relationship to terminal measurements. When animals are under CR they generally consume their food during the first few hours after it has been provided (see eg Acosta-Rodriguiz et al 2017). They then have a protracted period without food before the next daily allocation of food arrives. AL animals in contrast can by definition eat at any time throughout the 24h period. Although normally they consume most food in the dark phase most AL animals also have occasional meals during the light phase as well. Consequently, when it comes to culling animals to perform molecular biology work the CR animals may have been starving for 10-16 h, while the AL animals may have eaten in the hour immediately prior to culling. The CR v AL comparison may then be confounded by an immediate ‘time since last meal’ effect. To avoid these issues we used 2 ‘control groups’ exposed to 0% CR. For the first group (24AL) we allowed them 24 h access to food without restriction. For the second group (12AL) we allowed them unrestricted access to food for the 12h of darkness but then removed the food at lights on (0630h), replacing it 12h later at lights off when the CR animals were also fed. Hence these animals, like the CR animals, had been starving for at least 7.5 h when we came to cull them between 1400 and 1800h. The 12AL group protocol is equivalent to a 'time restricted feeding' paradigm. All animals were fed a high carbohydrate open source diet (D12450B: Research diets, NJ, USA) which contains 20 % protein, 70 % carbohydrate and 10 % fat (by energy). For the animals on PR we started with the same diet containing 20 % protein as the control group. We then modified this diet by reducing its protein level and replacing the missing protein with carbohydrate to achieve protein levels of 16, 14 and 12 % protein. Animals on these protein diets were prevented from overeating to compensate for the reduced protein and were fed a fixed weight of food equivalent to their own individual BL intake on the 20 % protein diet. Hence their energy intakes were the same as during the BL period but their protein intakes were restricted by 20, 30 and 40 %, to match the protein levels consumed by the 20, 30 and 40 % CR groups. To match the CR protocol these animals were also only fed in darkness. Two separate experiments were performed involving calorie restriction for 3 months and 19 months. Protein restriction was only conducted for 3 months. The overall aim of the study was to collect extensive phenotype data across the 7-9 animals in each group. These data included transcriptomic, proteomic and metabolomic profiles in multiple tissues, physiological, endocrinological and behavioural responses, as well as morphological changes, changes in daily food intake (FI), body mass (BM), digestive efficiency, Dual-energy x-ray absorptiometry (DXA) measures throughout the restriction period, and detailed aspects of the body composition changes. We have included an integrated overview of the 3 month CR study below. ![Integrated overview of the graded CR project][1] *Animals* C57BL/6 mice were purchased from Charles River (Ormiston, UK). Free access to water was provided. BM and FI were recorded daily, immediately prior to feeding. Over a 2 week BL period a number of measures were taken among which are reported here: DXA, and digestive efficiency measures. Mice were allocated into experimental groups matched for BM. Prior to culling all parameters measured at BL were repeated and referred to as the final measures (F). Mice were killed approximately 4 hours prior to lights out from 1400 to 1800 h by a terminal CO2 overdose. After death a blood sample was collected by heart puncture. Brains were removed, weighed and frozen in isopentane over dry ice. All remaining tissues were rapidly removed (~10mins), weighed, divided appropriately for future analysis and snap frozen in liquid nitrogen. The liver was divided into 7 pieces and individually frozen in cryovials to avoid freeze/thaw artefacts. Any apparent disease states were recorded. *Analysis of mechanical properties of bones* The tibia and femur of the right leg were preserved by wrapping in phosphate-buffered saline (PBS) soaked tissue, sealed in plastic bags and stored at -20°C for analysis of mechanical properties. The tibia and femur of the left leg were fixed in 3.7% formaldehyde, and scanned by micro-computed tomography (micro-CT). For full details on methods refer to (Aspden 2003; Goodyear and Aspden 2012). Precise measurements of length and diameter of both tibia and femur were recorded using a digital micrometer (± 0.01 mm) (RS 572-044, Mitutoyo, Andover, UK) and the mechanical properties were evaluated by three-point bending using an Instron 5564 testing machine (Instron, High Wycombe, UK). MathCAD software was used for analysis of data (Mathsoft Engineering and Education Inc., Cambridge, MA, USA). Ultrasound was used to measure the speed of sound in a bone slice using a pulser receiver (Model 5052 PR, Panametrics Inc, Waltham, MA, USA) and an oscilloscope (Hitachi V-665A, Tokyo, Japan). The density of the cortical bone was determined using Archimedes’ principle. Finally, the water, organic and mineral contents of the bones were calculated from wet, dry (24h at 105 oC) and ashed (24h at 600 oC) weights. The left tibia and femur of 12AL control (n=5) and 30CR mice (n=4) were analysed by three-dimensional micro-CT using Skyscan 1072 X-ray Microtomograph Scanner (Skyscan, Aartselaar, Belgium). Skyscan Nrecon software was used to reconstruct the images using a modified Feldkamp algorithm to obtain a three-dimension image which was then analysed using the software CTAN. The fractional bone volume, (i.e. the percentage of bone volume relative to the total volume (BV/TV), trabecular thickness (Tb Th), trabecular separation (Tb Sp), trabecular number (Tb N), trabecular pattern factor (Tb Pf), the structural model index (SMI) and the degree of anisotropy (DoA) were recorded).   *Dual Energy X-ray Absorptiometry (DXA)* Fat mass (FM), fat free mass (FFM), bone mineral density (BMD), content (BMC) and bone area (BA) were quantified using DXA (GE PIXImus2 Series Densitometers installed with software version 1.46.007) (GE Medical Systems Ultrasound and BMD, UK) (Johnston, et al. 2005). Measurements were taken at BL, 4 and 8 weeks after restriction started and 3-4 days prior to the final kill. *Bomb Calorimetry* Faeces collected over 6 days during BL and following 11-12 weeks of restriction, were carefully separated from sawdust, weighed and dried along with a sample of each diet. Gross energy content for each diet or faecal sample was measured by bomb calorimetry (Parr 6100 calorimeter using a semi-micro 1109 oxygen bomb 1109A, Scientific and Medical Products Ltd, Cheadle, UK) with a minimum of three replicates, within ±0.25 kJ. Metabolisable energy intake (MEI) (kJ/day) was calculated from the gross energy intake (GEI) and energy output assuming a 3 % energy loss via urine (Drozdz 1975; Krol and Speakman 2003). The apparent energy absorption efficiency (AEAE) was calculated as the percentage of the ingested food taken up by the body. *Plasma assays* To avoid the level of hunger affecting the concentration of adipokines and hormones measured, all mice were fasted prior to kill. Food was removed from both the 12 and 24AL groups at 0630 on the day prior to the kill at ~1400-1700h. Circulating insulin, leptin, tumor necrosis factor (TNF)-α, resistin and interleukin (IL)-6 were measured in plasma using the MilliplexTM mouse adipokine panel (MADPK-71K, Millipore, Watford, UK). Fasted plasma IGF-1 was detected using a mouse specific Enzyme Linked Immunoassay (ELISA) (R&D Systems Europe Ltd, Abingdon, UK). *Intraperitoneal glucose tolerance tests (IPGTT) and the homeostatic model assessment (HOMA2)* IPGTT were performed twice, firstly during baseline then after ~ 11 weeks of restriction. Mice were fasted for at least 8 hours prior to IPGTT. Fasted glucose was measured from tail blood immediately prior to a 2 mg/g intraperitoneal glucose injection. Following injection glucose was measured at 15, 30, 60 and 120 minutes using Johnson and Johnson’s OneTouch® Ultra Blood Glucose Monitoring System. Insulin sensitivity and insulin resistance were evaluated according to the HOMA2 Calculator© (The University of Oxford) (fasting blood glucose (mmol/L) x fasting insulin (µU/ml)/22.5) using fasting glucose measurements taken immediately prior to kill. *Biomarkers of oxidative damage* As a measure of oxidative stress DNA, lipid and protein damage was evaluated in the liver along with the activity of antioxidants, catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the blood. Protocols are briefly described below. *8-Hydroxy-2′-deoxyguanosine (8-OHdG)* levels, widely used as biomarker of oxidative DNA damage, were measured using the Highly Sensitive Competitive ELISA kit (Japan Institute for the Control of Aging (JAICA), Shizuoka, Japan). The intra-assay and inter-assay variations, as reported by the manufacturer, were 1.4-2.1% and 1.9-7.1%. To decrease the risk of artificial DNA oxidation, the extraction process was optimized to include sodium iodide and enzymatic DNA digestion. *Protein Carbonyls* were measured by ELISA (BioCell, Papatoetoe, New Zealand). The measurement of Liver *F2-isoprostanes* was carried out at the Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center at San Antonio under the kind supervision of Wenbo Qi in the lab of Holly Van Remmen. The process involves lipid extraction, thin layer chromatography (TLC) purification, and quantification by gas chromatography–mass spectrometric analysis. Antioxidant enzyme activities were measured using direct spectrophotometric methods. First homogenates from ~50 mg of liver were prepared in ice-cold 50 mM phosphate buffer (PB) (pH7.4), centrifuged and supernatants collected. All reactions were carried out in triplicate and absorbance read at 25°C using the SpectraMax Plus384 spectrophotometer with SoftMax software. *Catalase activity* was measured on the day of homogenization. Incubation with 1% Triton X ensured the release of catalase activity from the peroxisomes. Samples were run alongside blank (buffer) and standard (water) reactions. 6 mM hydrogen peroxide (H2O2) was added to sample and blank followed by 2 mM potassium permanganate to all. Reactions were stopped after 3 min by the addition of 3 M sulphuric acid. The residual potassium permanganate color from the catalase driven peroxidation was measured at 480 nm at 25 ºC. *Glutathione peroxidase (GPx) activity* was determined by a modification of the method of Paglia and Valentine. Reaction mixtures consisted of 1 mM Ethylenediaminetetraacetic acid (EDTA), 4 mM sodium azide, 0.2 mM Nicotinamide adenine dinucleotide 2-phosphate reduced tetrasodium salt hydrate (NADPH), 1.083 U/ml glutathione reductase and 4 mM reduced glutathione in ice cold 50 mM PB. Reactions were initiated by the addition of H2O2 and monitored for 1min at 340 nm. Absorbances were recorded in a quartz cuvette at 340 nm and activity calculated from the oxidation rate of NADPH to NADP. One GPx unit is directly proportional to the amount of NADPH consumed in nmol per minute at 23–25°C. *Total superoxide dismutase (SOD) activity* was measured using rapid inhibition of the auto-oxidation of pyrogallol (1,2,3-benzenetriol) by SOD. Reactions were performed in a 50 mM Trizma buffer containing 1 M of the iron chelator diethylenetriaminepentaacetic acid (DPTA). The reaction rate, in the presence and absence of sample, was measured as the change in absorbance at 420 nm over a 2 minute interval. This method cannot determine between the different forms of SOD, ie CuZn, Mn and FE SODs. Specific antioxidant activities were calculated on the basis of concentration of protein per assay. Protein content was detected using the Bradford assay. Standard curves were created from known concentrations of bovine serum albumin (BSA). Briefly Bradford Reagent Working solution (BioRad, Hemel Hempstead, UK) was added to 10 µl of sample, in a 96 well plate, and absorbance read at 595 nM. Plasma antioxidant potential and reactive oxygen metabolites (ROM) were evaluated in plasma samples using the OXY-Adsorbent (OxyD) and d-ROM kits respectively (Diacron international, Grossetto, Italy). Antioxidant power is expressed as µmol of HCIO/ml and ROMs, primarily hydroperoxides, results are expressed as Carratelli Units (CARR U). *Major urinary proteins (MUPs)* Urine was collected immediately prior to kill by scruffing the mice over a Petri dish and gently massaging the bladder. Urine samples were transferred to eppendorfs and stored at -20 °C. Briefly proteins were measured using the Coomassie plus®protein assay reagent kit from Perbio Science UK Ltd (Cramlington, Northumberland, UK). To correct for urinary dilution, urinary creatinine was measured using the alkaline picrate assay (Sigma Chemicals, UK). *RNA-seq analysis* Liver, adipose tissue and hypothalamus were removed as part of the overall dissection, weighed, and were immediately snap frozen in liquid nitrogen and stored in -80°C. RNA was isolated by homogenizing in Tri-Reagent (Sigma Aldrich, UK) according to manufacturer’s instructions. Prior to RNA quantification by Agilent RNA 6000 Nano Kit samples were denatured at 65 °C. Isolated RNA was sent to Beijing Genomic Institute (BGI, Hong Kong) for RNA sequencing. Library preparation was conducted by enriching total RNA by using oligo(dT) magnetic beads. The fragmentation buffer was added to obtain short fragments from the RNA. The mRNA was used as template for the random hexamer primers which synthesizes the first strand of cDNA. The second strand was synthesized by adding buffer dNTPs, RNase and DNA polymerase. QiaQuick PCR extraction kit was used to purify the double stranded cDNA and washed with an elution buffer for end repair and single nucleotide A addition. The fragments were ligated with sequencing adaptors and purified by agarose gel-electrophoresis to obtain the correct fragments. These were enriched by PCR amplification. During the quality control step, Agilent 2100 Bioanalyser and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products were sequenced using an Illumina Hi-seq 2000 resulting in 50 bp single ended reads (standard protocol BGI, Hong Kong). Standard primers and barcodes developed by BGI were used. Prior to alignment to the reference genome, FASTQ files were quality controlled to identify the presence of adaptors or low quality sequences using fastQC. To ensure high sequencing quality, the reads were trimmed with a cut-off phred score of 28 using Trimmomatic. Reads were aligned to the reference genome obtained from the National Center for Biotechnology Information (NCBI) database (Mus musculus, version MGSCv37, 2010/09/23). The reference genome was indexed using Bowtie2 and reads aligned with Tophat2 using default settings. Multi mapped reads were removed using the Sequence Alignment/Map (SAM) tool before proceeding to quantification of the reads. Aligned sequencing reads were counted with HTSeq-count by identification of how many reads mapped onto a single feature (genes containing exons). To remove any genes that exhibited no or a very low number of mapped reads, only genes that had more than 1 count per million in at least 4 samples across all treatments were retained for further analysis. Read counts were normalized using the trimmed mean of M values (TMM normalization) to account for highly expressed genes consuming a substantial proportion of the total library size. This composition effect would cause remaining genes to be under sampled. Differential gene expression was modelled using the edgeR package in R (version 3.1.2) and pairwise comparisons were conducted between 12AL and 24AL and between 12AL and each level of CR. **Grant funding** The project has been generously supported by the following grant agencies. Between 2009 and 2013 the work was supported by a standard responsive mode UK BBSRC grant **(grant: BB/G009953/1)** and from 2012 to 2016 by a BBSRC China partnering award **(BB/JO20028/1)**. Zhanhui Tang was a visiting scientist from Jinan University who worked on the project between 2010-2011 supported by a visiting scholar grant from the national Science Foundation of China (NSFC). Davina Derous was supported to work on the project between 2013 and 2017 by PhD studentship awarded by the Centre for Genome Enabled Biology at the University of Aberdeen, and Cara Green was supported from 2013 to 2107 by a BBSRC-EastBio PhD studentship. An additional grant from the BBSRC under the BBSRC-SFI scheme was awarded in January 2017 to follow up aspects of the body composition effects of CR (in collaboration with Kanishka Nilaweera from Teagasc, Ireland) (**BB/P009875/1**): total 1.1M GBP. From January 2018 the project will be supported by a grant from the Chinese Academy of Sciences under the Lujiazi International Collaborative Group Program scheme (350k GBP). The following students have worked on the project as exchange students from Agrosup Dijon and/or on the EU Erasmus program Celine Kebois Camille Deville Penelope Konstantopedos Aurelie Bruel Ophelie Prevot **Additional collaborators on the project include** Prof Dean Jones and Dr Quinlyn Soltow (Emory University, Georgia). Serum metabolomics. Prof Jane Hurst (University of Liverpool, UK) Analysis of MUPs in urine (data in pdf 436) Dr Will Cawthorn (University of Edinburgh) Estimation of marrow fat store sizes from bone measurements Prof Richard Aspden and Dr Simon Goodyer (University of Aberdeen) Bone health (data in pdf 428) Dr Marco Demaria (Medical University of Groningen, Netherlands) and Prof Luigi Fontana (Washington School of Medicine, Missouri) Senescent cells in the lungs and colon. **Outcomes** To date (June 2021) we have published 17 papers on the mice that were included in the short-term exposure experiment. These are as follows. See uploaded pdfs. Garcia-Flores, L.A., Green, C.L., Mitchell, S.E., Promislow, D.E.L., Lusseau, L., Douglas, A. and SPEAKMAN, J.R. (2021: in press) The effects of graded levels of calorie restriction XVII: multi-tissue metabolomics reveals synthesis of carnitine and NAD, and tRNA charging as key pathways modulated by calorie restriction Proceedings of the National Academy of Sciences Green, C.L., Mitchell, S.E., Derous, D., García-Flores, L.A., Wang, Y.C., Chen, L., Han, J.D.J., Promislow, D.E.L., Lusseau, D., Douglas, A. and SPEAKMAN, J.R. (2020) The effects of graded levels of calorie restriction: XVI. Metabolomic changes in the cerebellum indicate activation of hypothalamo-cerebellar connections driven by hunger responses. Journals of Gerontology A: Biological Sciences 75: 601-610 See also commentary article on this paper Garcia-Flores, L.A. and Green, C.L. (2021) Of mice and men: Impacts of calorie restriction on metabolomics of the cerebellum. Journals of gerontology A Biological Sciences 76: 547-551 Sun, D., Liu, F., Mitchell, S.E., Ma, H.F., Derous, D., Wang, Y., Han, J.D., Promislow, D.E.L., Lusseau, D., Douglas, A., SPEAKMAN, J.R. and Chen, L. (2020) The effects of graded levels of calric restriction XV: Phase space attractors reveal distinct behavioural phenotypes. Journals of Gerontology A 75: 858-866. Green, C.L., Mitchell, S.E., Derous, D., Wang, Y.C., Chen, L., Han, J.D.J., Promislow, D.E.L., Lusseau, D., Douglas, A., and SPEAKMAN, J.R. (2019) The effects of graded levels of calorie restriction: XIV. Global metabolomics screen reveals brown adipose tissue changes in amino acids, catecholamines and antioxidants after short-term restriction in C57BL/6 mice. Journals of Gerontology series A: Biological Sciences and Medical Sciences Green CL, Soltow QA, Mitchell SE, Derous D, Wang Y, Chen L, Han J-DJ, Promislow DEL, Lusseau D, Douglas A, Jones DP, SPEAKMAN JR. (2018, in print) The Effects of Graded Levels of Calorie Restriction: XIII. Global Metabolomics Screen Reveals Graded Changes in Circulating Amino Acids, Vitamins, and Bile Acids in the Plasma of C57BL/6 Mice. *Journals Gerontol Ser A*; Fontana L, Mitchell SE, Wang B, Tosti V, van Vliet T, Veronese N, Bertozzi B, Early DS, Maissan P, SPEAKMAN JR, Demaria M. (2018) The effects of graded caloric restriction: XII. Comparison of mouse to human impact on cellular senescence in the colon. *Aging Cell* **17**: e12746. Derous, D., Mitchell, S.E., Green, C.L., Wang, Y.C., Han, J.D.J, Chen, L.N., Promislow, D.E.L., Lusseau, D., Douglas, A. and SPEAKMAN, J.R. (2017) The Effects of Graded Levels of Calorie Restriction: XI. Evaluation of the main hypotheses underpinning the life extension effects of CR using the hepatic transcriptome *Aging-US* **9**:1770-1824 Derous, D., Mitchell, S.E., Green, C.L., Wang, Y.C., Han, J.D.J, Chen, L.N., Promislow, D.E.L., Lusseau, D., Douglas, A. and SPEAKMAN, J.R. (2017) The Effects of Graded Levels of Calorie Restriction: X. Transcriptomic Responses of Epididymal Adipose Tissue *Journals Gerontol Ser A* **73**:279-288 Green, C.L., Mitchell, S.E., Derous, D., Wang, Y.C., Han, J.D.J., Chen, L., Promislow, D.E.L., Lusseau, D., Douglas, A. and SPEAKMAN, J.R. (2017) The effects of graded levels of calorie restriction: IX. Global metabolomics screen reveals modulation of carnitines, sphingolipids and bile acids in the liver of C57BL/6 mice. *Aging Cell* **16**: 529-540 Mitchell, S.E., Tang, Z.H., Kerbois, C., Delville, C., Derous, D., Green, C.L., Wang, Y.C., Han, J.D.J., Chen, L., Douglas, A., Lusseau, D., Promislow, D.E.L. and SPEAKMAN, J.R. (2017) The effects of graded levels of calorie restriction: VIII. impact of short term calorie and protein restriction on basal metabolic rate in the C57BL/6 mouse. *Oncotarget* Derous, D., Mitchell, S.E., Green, C.L., Wang, Y.C., Han, J.D.J., Chen, L., Promislow, D.E.L., Lusseau, D., SPEAKMAN, J.R. and Douglas, A. (2016) The Effects of Graded Levels of Calorie Restriction: VII. Topological Rearrangement of Hypothalamic Aging Networks *Aging-US* **8**: 917-32 Mitchell, S.E., Derous, D., Green, C.L., Chen, L., Han, J.D.J., Wang, Y.C., Promislow, D.E.L., Lusseau, D., Douglas, A. and SPEAKMAN, J.R. (2016) The Effects of Graded Levels of Calorie Restriction: V. Impact of Short-term Graded Calorie Restriction on physical activity in the C57BL/6 mouse. *Oncotarget* **7:** 19147-19170 Derous, D., Mitchell, S.E., Green, C.L., Chen, L., Han, J.D.J., Wang, Y.C., Promislow, D.E.L., Lusseau, D., SPEAKMAN, J.R. and Douglas, A. (2016) The Effects of Graded Levels of Calorie Restriction: VI. Impact of Short-term Graded Calorie Restriction on Transcriptomic Responses of the Hypothalamic Hunger and Circadian Signaling Pathways *Aging-US* **8**: 642-663 Lusseau, D., Mitchell, S.E., Barros, C., Derous, D., Green, C., Chen, L., Han, J.D.J., Wang, Y.C., Promislow, D.E.L., Douglas, A. and SPEAKMAN, J.R. (2015) The effects of graded levels of caloric restriction, IV: Non-linear change in behavioural phenotype of male C57BL/6 mice in response to short-term graded caloric restriction *Scientific reports* **5**: 13198 Mitchell, S.E., Delville, C., Konstantopedos, P., Hurst, J., Derous, D., Green, C., Chen, L.N., Han, J.D.J., Wang, Y.C., Promislow, D.E.L., Lusseau, D., Douglas, A., and SPEAKMAN, J.R. (2015). The effects of graded levels of calorie restriction: III. Impact of short term calorie and protein restriction on mean daily body temperature and torpor use in the C57BL/6 mouse. *Oncotarget* **6**: 18314-18337 Mitchell, S.E., Delville, C., Konstantopedos, P., Hurst, J., Derous, D., Green, C., Chen, L., Han, J.D.J., Wang, Y.C., Promislow, D.E.L., Lusseau, D., Douglas, A., and SPEAKMAN, J.R. (2015). The effects of graded levels of calorie restriction: II. Impact of short term calorie and protein restriction on circulating hormone levels, glucose homeostasis and oxidative stress in male C57BL/6 mice. *Oncotarget* **6**：23213-23227 Mitchell, S.E., Tang, Z.H., Kerbois, C., Delville, C., Konstantopedos, P., Bruel, A., Derous, D., Green, C., Aspden, R.M., Goodyear, S.R., Chen, L.N., Han, J.J.D., Wang, Y.C., Promislow, D.E.L., Lusseau, D., Douglas, A. and SPEAKMAN, J.R. (2015) The effects of graded levels of calorie restriction: I. Impact of short term calorie and protein restriction on body composition in the C57BL/6 mouse. *Oncotarget* **6**: 15902-15930 [1]: https://mfr.osf.io/export?url=https://osf.io/d6qcg/?action=download&mode=render&view_only=5a663065b5454330989de78cb315eaed&direct&initialWidth=848&childId=mfrIframe&format=1200x1200.jpeg