Diets were purchased from Specialty Feeds (Perth, Western Australia) and formulated to have the same total energy content (isocaloric) but different ratios in protein to carbohydrate with fixed fat ( Table 1 ). Each diet was based on the rodent diet AIN-93G (Specialty Feeds) and formulated to contain all essential vitamins, minerals, and amino acids for growth in mice. The primary dietary protein component was casein, the main carbohydrate component was starch, and the main fat component was soy oil.

Animals were purchased from the Animal Resource Centre (Perth, WA) and housed four per cage on a 12-hour light/dark cycle at 22-24°C at the Charles Perkins Centre at The University of Sydney. All animals were given free access to water and randomly assigned to experimental groups. Ad-libitum animals were given free-access to food while CR animals were given an allotment with 20% fewer calories than the average intake of their ad-libitum 19% protein counterparts daily at 3.00pm. Mice were weaned at 3 weeks of age and diets were started at approximately three months of age. Energy intake from each macronutrient was determined and averaged daily from 12 – 15 months of age. Body weights were taken every two weeks and animals were routinely monitored every week for general health. The ethics in this study were approved by the University of Sydney, animal ethics number 2014/752.

METHOD DETAILS

Animal sacrifice and tissue collection Valencak et al., 2017 Valencak T.G.

Osterrieder A.

Schulz T.J. Sex matters: The effects of biological sex on adipose tissue biology and energy metabolism. At 15 months of age a subset (n = 12 males, n = 12 females per group) of mice were culled. All animals were sacrificed between the hours of 10am and 12 noon. Animals were sacrificed in randomized order to minimize experimental bias. After deep anesthetizing with xylazine-ketamine (10mg/g bw), approximately 1 mL of blood was taken via cardiac puncture. The liver was removed, weighed, and flash-frozen in liquid nitrogen. The mice were then decapitated and the brain was carefully removed. One-half of the brain was immediately washed with ice-cold double-deionized water and placed into Golgi-Cox solution. The whole hippocampus was carefully dissected from the other half, immediately snap-frozen in liquid-nitrogen, and moved to −80°C until further processing. The blood was placed into a ice-cold tube and placed directly into wet ice before centrifugation at 14,000 rpm for 10 minutes for plasma collection. The plasma was used for subsequent metabolic and systemic measurements. Males and females were analyzed separately on metabolic measurements due to innate metabolic differences between sexes ().

Fat mass and lean mass Fat mass and lean mass were measured by magnetic resonance imaging (EchoMRI 900 – EchoMRI LLC, Houston, Texas, USA). The mice were awake during the process and snuggly put into a plastic tube before being placed inside the machine for approximately 1 minute. The machine calculated the fat mass (g) and lean mass (g) per each mouse.

Metabolic measurements The respiratory quotient was measured by Metabolic Cage (Promethion, Sable Systems International, Las Vegas, NV, USA). Briefly, individual mice were placed in each cage and acclimatized for 8 hours then left for 48 hours (2 dark and 2 light cycles). Calorimetric data were calculated with the software and the average respiratory quotient was obtained during the dark cycle. Data are presented as CO 2 eliminated/O 2 consumed (Respiratory quotient; RQ).

Glucose tolerance Glucose tolerance was measured at 15 months of age. Mice were fasted for 6 hours before the test which took place in the afternoon at 2.00 pm. Mice were orally gavaged with glucose (2 g/kg lean mass) and blood glucose levels were read by tail snip at time 0, 15 min, 30 min, 45 minutes, 60 minutes, and 90 minutes (Accu-Check Performa, Roche, Australia). The total area under the curve (AUC) was calculated and data are presented as mm/l.min.

Insulin determination Total fasting insulin levels were measured with an ultra-sensitive mouse insulin ELISA kit per manufacturer’s instructions (Chrystal Chem, Elk Grove Village, IL, USA). Whole blood was added to each well in the 96-well plate containing 90 μL of the provided diluent “G.” 10 μL of blood was collected by tail snip and added to each well. The “mouse insulin standard” was reconstituted and the standards were mixed (from 0.1 ng/mL to 6.4 ng/mL). 95 μL of the provided diluent and 5 μl of each standard was added into each well, the plate was covered, and incubated overnight at 4°C. The plate was removed from 4°C and washed 5 times with 250 μl wash buffer and 100 μl of the anti-inulin conjugate was added to each well. The plate was covered and incubated for 30 minutes at room temperature and subsequently washed 7 times with 250 μL wash buffer in each well. 100 μl of the enzyme substrate was added to each well, covered in the dark for 40 minutes, and the reaction ended with the addition of 100 μl stop solution “F.” The plate was read at an absorbance of A450-A630 and insulin concentration was determined by linear fit. Data are presented as ng/mL.

FGF-21 assay The Fibroblast Growth Factor-21 assay was analyzed by ELISA (Biovendor, Karasek, Czech Republic). The mouse master standards were reconstituted with the provided dilution buffer (40 pg/mL – 2568 pg/mL). 100 μl of standards and diluted samples (serum diluted in dilution buffer) were added to each appropriate well. The plate was incubated at room temperature for one hour with shaking at 300 rpm and washed three times with the provided wash buffer. 100 ul of biotin-labeled antibody was subsequently added to each well and incubated for one hour at room temperature with shaking. The wells were washed 3x with the wash buffer and 100 μL of Sterptavidin-HRP conjugate was added to each well and incubated with shaking for 30 minutes. The plate was washed 3x with wash buffer and 100 μl of substrate solution was added to each well, covered in foil, and incubated at room temperature for 10 minutes, and the reaction was stopped by the addition of 100 μl of stop solution. The absorbance of each well was determined by reading on a microplate reader at 550 – 650 nm and concentrations were determined with the standard curve. Data are presented serum FGF-21 concentration in pg/mL.

Adiponectin determination Adiponectin concentrations were determined using a mouse adiponectin ELISA kit (Crystal Chem, Elk Grove Village, Il, USA). The mouse standards were reconstructed from the provided stock (0.025 – 1 ng/mL). Serum samples were diluted to the appropriate concentration (1:10,000) in the dilution buffer. 100 μL of sample and each standard were added to each well and incubated for 1 hour at RT with shaking for 350 RPM. The plate was washed 3x and 100ul of the provided antibody conjugate was added to each well and incubated for 1 hour with shaking at 350 rpm. The plate was washed 3x with wash buffer, 100 μL of substrate solution was added and incubated for 30 minutes. 100 μL of stop solution was added and the OD was measured at 460/630 nm. Data are presented as the Adiponectin concentration in ng/mL.

Leptin determination Leptin concentrations were determined using a mouse leptin ELISA kit (Crystal Chem, Elk Grove Village, Il, USA). The mouse standards were reconstructed from the provided stock (0.0 – 12.8 ng/mL) and serum samples were diluted to the appropriate concentrations. 100 μL of each standard or sample was added to each well and incubated overnight at 4°C. The plate was then washed and 100 μL of the conjugate solution was added followed by 4 hours of incubation at 4°C. The plate was washed and 100 μL of the substrate solution was added, incubated for 30 minutes, and 100 μL of stop solution was added to each well. The plate was read on a spectrometer at an OD of 450/630 nm. Data are presented as serum leptin levels in ng/mL.

Cholesterol, Albumin, ALT, Urea, and Triglycerides Serum cholesterol, albumin, ALT, urea, and triglyceride levels were analyzed by a Cobas 8000, c702 photometric module (Hitachi, Japan) and all reagents were provided by Roche (Roche diagnostics, Germany). All analyses were completed at Concord Hospital (NSW, Australia).

RNA isolation and processing RNA was isolated using the Trizol method. 1mL of ice-cold TRIzol reagent (Sigma Aldrich, St. Louis, MO, USA) was added to fresh-frozen whole hippocampus. Tissue was homogenized using the bead method (QIAGEN, Hilden, Germany) for 30 s at 50 Hz. After samples were let to sit on ice for 10 minutes, 200 μl of ice-cold chloroform was added and samples were let to sit at room temperature for an additional 3 minutes. Samples were centrifuged at 14,000 rpm for 20 minutes and approximately 500 μL of the supernatant was collected and added to an equal volume of ice-cold 2-propanol. Samples were mixed, placed directly on ice, and centrifuged at 14,000 RPM for 20 minutes. The supernatant was removed and the remaining pellet was washed with 500 μL of ice-cold ethanol 3 times before air-drying. The pellet was resuspended in 15 μL nuclease-free water and cleaned with DNase (Invitrogen, Carlsbad, CA, USA). RNA purity was assessed using a nanodrop spectrometer (Thermofisher Scientific Australia) before freezing at −80°C until further analysis.

RNA sequencing and analysis st strand cDNA synthesis was randomly primed followed by second strand cDNA synthesis. cDNA library preparation was prepared first by DNA fragment end repair followed by 3′ adenylation of DNA fragments and subsequent sequencing adaptor ligation. Finally, the library was amplified by PCR. Samples were processed by The Australian Genome Research Facility (AGRF - Victoria, Australia; http://www.agrf.org.au/ ) with Illumina TruSeq stranded mRNA sample preparation and technology. RNA integrity was initially assessed by a bioanalyzer and all samples passed quality control with RIN values ≥ 8.0 and an A260/280 ratio of 1.8 – 2.0. Briefly, mRNA was purified via oligo(dT) beads followed by fragmentation of mRNA with divalent cations and heat. 1strand cDNA synthesis was randomly primed followed by second strand cDNA synthesis. cDNA library preparation was prepared first by DNA fragment end repair followed by 3′ adenylation of DNA fragments and subsequent sequencing adaptor ligation. Finally, the library was amplified by PCR. Primary sequence data were generated using the Illumina bcl2fastq 2.19.0.12 pipeline and sequence reads from all samples were analyzed per AGRF quality control measures. Briefly, cleaned sequence reads were aligned against the Mus musculus genome (Build version mm10). The Tophat aligner (v2.0.14) was used to map reads to the genomic sequences. The raw gene reads were generated by featureCounts (v1.4.6) and the differential gene expression was analyzed by DESeq2 (v1.16.1) in R (package v3.4.0). The Gene Set Enrichment analysis (GSEA) was completed by a SetRank method (PMID: 28259142 ). The heatmaps and Venn Diagrams were generated in R (package v3.4.0). The heatmaps were generated from FPKM values of the genes that were considered significant compared to the 19% protein group. For each heatmap, the red, white, and blue colors indicate higher than mean, close to mean, and lower than mean expression of a particular gene, respectively, as measured by the row of standardized Z-scores. The rows are organized by hierarchical clustering using agglomerative clustering with complete linkage and Euclidian distance metric. The volcano plot and Venn diagrams were constructed by a Pearson calculation with the top 2.5% of genes in either direction considered significant. All genetic data are available at the online database GEO: GSE111778

Protein isolation Whole frozen hippocampus was homogenized using the bead method for 50hz for 30 s (QIAGEN TissueLyser LT) in 500 μL ice-cold RIPA buffer containing Tris-HCl, NaCl, Triton X-100, Na-deoxycholate, SDS, and fresh protease and phosphatase inhibitor tablets (Roche cOmplete, EDTA-free Protease Inhibitor Cocktail). The tissue was let to sit on ice for 10 minutes followed by 20 minutes of centrifugation at 14,000 rpm. The supernatant was transferred to a fresh ice-cold tube and protein concentrations were assessed using a bicinchoninic acid (BCA) assay (Thermo Scientific, Pierce BCA Protein Assay Kit). Samples were then diluted to approximately the same concentrations before being stored at minus 80°C until further analysis. Protein lysates were used for subsequent enzymatic activity assays, Milliplex Map Panels, and western blots.

Western blots Eaton et al., 2013 Eaton S.L.

Roche S.L.

Llavero Hurtado M.

Oldknow K.J.

Farquharson C.

Gillingwater T.H.

Wishart T.M. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. Lysates were prepared for bis/tris polyacrylamide gel electrophoresis under reduced conditions. Proteins were transferred to nitrocellulose membranes (Invitrogen) and immediately stained with Ponceau S dye (Sigma Aldrich, St. Louis, MO, USA) for rapid and reversible visualization of total protein per lane. Protein expression was detected using specific primary antibodies. Antibodies raised against p-MTOR (#2971; 1:000), MTOR (#2983; 1:1000), SIRT1 (#9475; 1:750), and β-ACTIN (#4970; 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies raised against DREBRIN (ab60933; 1:000), GAPDH (ab9485; 1:1000), and PGC1-α (ab54481; 1:1000) were purchased from abcam (Cambridge, UK). All antibodies were detected using secondary rabbit IgG-horse radish peroxidase (#7074; 1:5000) purchased from Cell-Signaling and visualized by enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA). Levels of specific proteins were normalized to total protein as visualized to ponceau S staining () and band densitometric values analyzed with ImageJ (National Institutes of Health, Bethesda, MD). Loading controls GAPDH and ACTIN bands are shown along with their representative blots solely for comparison purposes.

Milliplex map panel enzymatic activity assays Levels of IL-6, IL-10, and TNFα were assessed using the mouse cytokine/chemokine magnetic bead panel 96-Well Plate Assay (Cat # MCYTOMAG – 70K, EMD Millipore Corporation, Billerica, MA, USA) per manufacturer’s instructions. After a plate wash, 25ul of each standard or control was added to the appropriate wells followed by 25 μL of assay buffer to the sample wells. 25 μL of whole hippocampal tissue lysates were added to the appropriate wells followed by 25 μL of premixed cytokine panel beads. The plate was sealed, covered with aluminum foil, and incubated overnight at 4°C. The following day the plate was removed from 4°C and washed 2 times with the provided wash buffer. 25 μL of detection antibodies were added to each well and the plate was covered with foil and incubated with shaking for 1 hour at RT. 25 μL of Streptavidin-Phycoerythrin was then added to each well, covered with foil, and incubated with shaking for 30 minutes. The plate was then washed 2 times and 150 μL of the provided drive fluid was added to each well followed by 5 minutes of shaking. The plate was run and read on MAGPIX® machine with xPonenent® Software (EMD Millipore Corporation, Billerica, MA, USA). The data were analyzed using Xponent® software. Briefly, the software analyzed median fluorescent intensity data using 5-parameter logistic or spline curve-fitting method for calculating each cytokine concentration in samples. Data are presented as ng/mg protein.

BDNF enzymatic activity assay The BDNF ImmunoAssay Elisa was performed per manufacturer’s instructions (Promega Corporation, Madison, WI, USA). Flat-bottom 96-well plates were coated with Anti-BDNF Monoclonal Antibody overnight at 4°C to bind soluble antibody. After the addition of 25 μL of each standard or lysate, the plate was incubated for 2 hours and secondary antibody was added. The plate was washed 3 times with TBS-T and the amount of bound pAb was detected using an anti-IgY horseradish peroxidase antibody. Unbound conjugate was removed by washing 5 times with TBST, a chomogenic substrate was added, and color was measured on a spectrometer at a wavelength of 450nm. Data are presented as mg BDNF/μg protein.

Golgi Staining Zaqout and Kaindl, 2016 Zaqout S.

Kaindl A.M. Golgi-Cox staining step by step. A rapid Golgi Stain was performed per manufacturer’s instructions (FD Neurotechnologies, Inc, Columbia, MD, USA). Brains were removed and rinsed with ice-cold double deionized water. The complete right section was submerged in an impregnation solution consisting of equal parts of solution ‘A’ and solution ‘B’. Samples were stored at room temperature in the dark for 2 weeks and then moved to solution ‘C’ for 3 days. They were then cut on the midsagittal plane with a vibratome at 100 μm to visualize and quantify dendritic spines (). Samples were left to dry before the staining protocol. Sections were rinsed in distilled water before placing into a mixture consisting of 1 part solution ‘D’ and 1 part solution ‘E’. After an additional rinse, sections were dehydrated in increasing 50%, 75%, and 95% ethanol solutions before clearing in xylene and coverslipping in DPX mounting media for histology (Sigma Aldrich, St. Louis, MO, USA).

Dendritic spine quantification Orlowski and Bjarkam, 2012 Orlowski D.

Bjarkam C.R. A simple reproducible and time saving method of semi-automatic dendrite spine density estimation compared to manual spine counting. Orlowski and Bjarkam, 2012 Orlowski D.

Bjarkam C.R. A simple reproducible and time saving method of semi-automatic dendrite spine density estimation compared to manual spine counting. Jacobs et al., 2014 Jacobs B.

Johnson N.L.

Wahl D.

Schall M.

Maseko B.C.

Lewandowski A.

Raghanti M.A.

Wicinski B.

Butti C.

Hopkins W.D.

et al. Comparative neuronal morphology of the cerebellar cortex in afrotherians, carnivores, cetartiodactyls, and primates. Stranahan et al., 2009 Stranahan A.M.

Lee K.

Martin B.

Maudsley S.

Golden E.

Cutler R.G.

Mattson M.P. Voluntary exercise and caloric restriction enhance hippocampal dendritic spine density and BDNF levels in diabetic mice. Slides were imaged on an Olympus VS-120 Slide Scanner at 40X magnification which gave good resolution for spine counting purposes (). 25 z-slices of 1 μm each were imaged from each hippocampus. Dendritic spines were quantified using NeuronStudio software (Mount Sanai School of Medicine, available at http://research.mssm.edu/cnic/tools-ns.html ) and blindly counted (DW). It has been demonstrated that the manual counting method by an investigator does not produce significantly different results when compared to other software or semi-automatic counting methods (). Spines were quantified in the dentate gyrus of each hippocampus and secondary and tertiary branches of each neuron were analyzed. A total of 8 - 12 segments were quantified from each hippocampus depending on the quality of the stain. The quantified dendritic branch segments were required to have the characteristics as previously described (): (1) completely and darkly stained near the center of the 100 μm section, (2) contain no broken sections with complete spines, and (3) isolated without interference or overlap from other structures. Data are presented at the number of spines per 10 microns ().

Immunofluorescence Brains were carefully removed and cut on a midsagittal plane. The left side was placed in 4% formalin for 24 hours followed by 30% sucrose cryoprotection for 24 hours at 4°C. Brains were embedded in OCT (Siltera-Finetele, Inc, USA, Torrance, CA) before slowly freezing on dry ice and storing at −80°C until further use. They were sliced at 30 μm on a midsagittal plane on a cryostat. 5 sections were taken from each brain and mounted on slides. For Iba1 staining, sections were post-fixed with 4% PFA before quenching with 50 mM NH 4 CL. For antigen retrieval, a commercially available Proteinase K solution was used for three minutes (Sigma Aldrich, St. Louis, MO, USA) and tissue was blocked for 30 minutes with 10% goat serum in PBS. Slides were incubated with primary antibodies Iba1 (1:250; GeneTex Irvine, CA, USA) overnight at 4°C. Sections were thoroughly washed and incubated with the fluorescent secondary antibody (1:1000, Cyanine 3, ThermoFisher Scientific) for 1 hour before washing and coverslipping with vectashield mounting media containing DAPI (Vector Laboratories, Burlingame, CA, USA). For GFAP, slides were washed with PBST before blocking for 30 minutes in 3% goat serum in PBS. Slides were placed into primary antibody (1:2000, abcam, United States) diluted in 3% goat serum in PBS. Slides were incubated overnight at 4 degrees followed by washing with PBST and a 1 hour incubation in secondary antibody (1:1000; Cyanine3, ThermoFisher Scientific) for one hour. Slides were imaged on a on an Olympus VS-120 Slide Scanner at 20X magnification (Iba1) or a Leica confocal microscope with white light laser (WLL), coupled with a 20X HC PL APO CS2 NA 0.75 lens (GFAP). Images were quantified using ImageJ. Data are presented as the number of Iba1+ cells per 1mm2 in the hippocampus or the mean corrected total hippocampus fluorescence (GFAP).

Behavioral testing Tian et al., 2017 Tian Q.

An Y.

Resnick S.M.

Studenski S. The relative temporal sequence of decline in mobility and cognition among initially unimpaired older adults: results from the Baltimore Longitudinal Study of Aging. Simpson and Kelly, 2012 Simpson J.

Kelly J.P. An investigation of whether there are sex differences in certain behavioural and neurochemical parameters in the rat. Animals were handled extensively before and during the testing phase to acclimatize them to human interaction and minimize potential anxiety caused by interference. Mice received a minimum 2-hours of room habituation before commencing each test. Animals were tested in random order during each testing period and equipment was thoroughly cleaned with 80% ethanol between trials to minimize scents. To test memory during aging animal performance was assessed on both the Novel Object Recognition and Barnes Maze memory tasks. The same animals were tested at young and old age in a longitudinal manner, similar to human dementia and memory studies (). The two-time points were 13 months of age (young) and 23 months of age (old). Animals received a minimum 3 days rest in between the two tests. Male and female results were analyzed separately because of innate behavioral and response differences between sexes ().

Novel Object Recognition (NOR) Bevins and Besheer, 2006 Bevins R.A.

Besheer J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ‘recognition memory’. The NOR task was performed in a custom made white opaque plexiglass box (40cmx40cmx40cm; City West Plastic, Sydney, NSW). The task was performed as previously described () with an inter-trial interval of 24 hours. On day 1, mice were placed into the box and allowed to explore (habituation period) for 5 minutes followed by one hour of rest before the first trial. Two identical non-toxic and odourless objects (cell culture flasks filled with sand) were placed into the box (5cm from each wall) and the mice could explore for 5 minutes. After an inter-trial interval of 24 hours the mice were placed back into the box but one of the old objects was replaced with a new one (tower of Legos blocks) of similar shape, size, and height. The trial ended once a total exploration time of 20 s was reached, or 5 minutes elapsed. A recognition index (RI) was calculated as the time the mouse spent exploring the new object over total object exploration time. Mice were excluded from the analysis if they did not reach the 5-minute exploration criteria. All trials were recorded with an AnyMaze USB camera at 30 frames per second and quantified with a stopwatch by two independent and skilled reviewers.