C57BL/6J mice (The Jackson Laboratory, United States) were genetically modified via the Cre-loxP recombination system (Ozgene, WA, Australia) and ubiquitous genetic knock-in of the human gene encoding OST48 (DDOST) at the ROSA26 locus with removal of the delivery neomycin cassette, as described in Supporting Experimental Procedure 1 and Supporting Fig. 1 . DDOSTflox/flox mice are referred to as the wild-type (WT) background and DDOSTflox•Cre/flox are labelled as the genetic OST48 heterozygous knock-in mouse model (DDOST+/−). Eight week old male DDOST+/− mice and littermate controls (WT) were randomised to be fed either AIN-93G (low AGE diet; Specialty Feeds, Perth, Australia)31 (link) or baked AIN-93G (1 hour at 100 °C; high AGE diet), which contained a 5-fold higher content of the AGEs, N(ε)-(carboxymethyl)lysine (CML), N(ε)-(carboxyethyl)lysine and methylglyoxal32 (link) for 24 weeks. Heat labile vitamin contents (vitamin A and thiamine) were not decreased by the heating protocol32 (link). Mice were allowed access to food and water ad libitum and were maintained on a 12 hour light:dark cycle at 22 °C. All mouse experiments were performed following approval from the AMREP Animal Ethics Committee and as per guidelines from the National Health and Medical Research Council of Australia.
Ain93g
Manufactured by Specialty Feeds
Sourced in Australia
AIN93G is a laboratory equipment product designed for use in animal nutrition research. It serves as a standard rodent diet formulation, providing a balanced nutrient profile for laboratory animals. The core function of AIN93G is to support the nutritional needs of research animals in a controlled and consistent manner.
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Lab products found in correlation
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Pyrogene recombinant factor c kit, by Lonza (1 mentions)
Sf09 028, by Specialty Feeds (1 mentions)
Sf03 020, by Specialty Feeds (1 mentions)
L citrulline, by Specialty Feeds (1 mentions)
L arginine, by Specialty Feeds (1 mentions)
L alanine, by Merck Group (1 mentions)
L arginine, by Merck Group (1 mentions)
L citrulline, by Merck Group (1 mentions)
Sf00 219, by Specialty Feeds (1 mentions)
12 protocols using ain93g
1
Genetic OST48 Knock-In Mouse Model
2
Maternal High-Fat Diet and Offspring Metabolic Health
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Animal husbandry and experimental diet details have been previously published [15 (link)]. Briefly, female Sprague-Dawley breeder rats were fed either a standard chow diet (C, 7% total fat– 0.5% saturated fat, 10% sucrose wt/wt, 16.1 MJ/Kg; AIN93G, Specialty Feeds, Glen Forest, WA, Australia) or a diet rich in saturated fat (sourced mainly from animal lard) and sucrose (HF, 23.5% total fat– 9.83% saturated fat, 20% sucrose wt/wt, 19.6MJ/Kg; SF08-023, Specialty Feeds) for the 3 weeks prior to mating and throughout pregnancy and lactation. Male offspring of control (n = 6) and HF (n = 5) dams were weaned at postnatal day 21 and maintained on a standard chow diet (ad libitum) until they reached 12 months. Blood was collected via cardiac puncture and glucose determined by handheld glucometer (Accu-Chek Roche, Castle Hill, NSW, Australia), plasma assayed for insulin by ELISA (Kit 90010, Crystal Chem, Downers Grove, IL, USA).
3
Macronutrient Modulation of Metabolism
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Animals were fed ad libitum on either a high protein, moderately low carbohydrate, moderately low-fat diet (P60, C20, F20; henceforth, HP); a low protein, high carbohydrate, moderately low-fat diet (P5, C75, F20; henceforth, HC), or a low protein, moderately low carbohydrate, high-fat diet (P5, C20, F75; henceforth, HF). These three diets represented the extremes (apices) from a larger set of 10 diet compositions (Supplementary Table 1 ) that were used in a subset of the experiments, encompassing a macronutrient range of protein (5–60%), carbohydrate (20–75%), and fat (20–75%) chosen using nutritional geometry to comprehensively sample dietary macronutrient mixture space39 (link). All diets were isocaloric at 14.5 MJ/kg and based on a modification to the semi-purified AIN93G formulation and purchased from Specialty Feeds, Glenn Forest, Australia (SF17-188-SF17-197) and the HP/HC/HF used for most experiments had very low endotoxin levels as tested with the PyroGene Recombinant Factor C kit (Lonza).
4
Titanium Dioxide Exposure in Mice
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Five to six week-old male C57BL/6JAusb mice from Australian Bio Resources were maintained under specific-pathogen-free conditions. All experimental procedures involving animals were approved by the University of Sydney Animal Ethics Committee under protocol number 2014/696. Mice were cohoused with water and food (AIN93G; Specialty Feeds) access ad libitum. Titanium dioxide (E171) was added to water and sonicated daily. TiO2 was administered in drinking water at doses of 0, 2, 10, and 50 mg TiO2/kg BW/day, which was calculated based on the water intake measured per cage. At week 4, mice were euthanized using CO2 asphyxiation.
5
Dietary n-3 PUFA Modulation of Oxidative Stress
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Animals were randomly assigned to n-3-LC-PUFA diet condition {Specialty Feeds, Cat No. SF09-109 5% Fat High N3 Modified Rodent Diet, Glen Forest, WA, Australia [EPA 20:5 n-3 5% of total free fatty acid (FFA), DHA 22:6 n-3 23.8% of total fatty acids]}, selected as it has previously been demonstrated to reduce oxidative stress in the rat model (39 (link)) or basal diet condition (Specialty Feeds, Cat No. AIN93G; Glen Forest, WA, Australia).
6
Gpr43-/- Mice Acetate Supplementation
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C57BL/6J and Gpr43−/−age and sex-matched male mice of 6–8 weeks old (backcrossed to C57BL/6J > 13 generations) were obtained from Monash Animal Services and housed under specified pathogen-free (SPF) conditions in Monash University Animal Research Laboratories. Germ-free (GF) male C57BL/6J mice at 7 weeks old were obtained from Walter and Eliza Hall Institute of Medical Research Animal Facility. Following transportation, mice were acclimatized for a minimum period of 7 days before use. Mice were housed in a 12-hour light-dark cycle in a temperature controlled environment with free access to food and water. Where indicated, mice were supplemented with 200mM acetate in the drinking water, or removed from their normal chow and fed either control diet (Ctrl; AIN93G, consisting of 4.7% crude fibre; Specialty Feeds) or no fibre diet (NF; SF09-028, consisting 0% fibre, modified from AIN93G) for 2 weeks prior to experimentation. All animal experiments were approved by the Monash University Animal Ethics Committee.
7
Rat Offspring Metabolic Programming
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The University of Melbourne Animal Ethics Committee, (AEC number 1212639) approved all the animal experimental procedures. Female F0 Wistar-Kyoto rats (initial generation, F0) were purchased from the Biological Research Facility (The University of Melbourne) at 8 weeks of age, and they were cohoused (6 rats/box) in a temperature-controlled room (22 °C), with a 12 h light-dark cycle and access to standard rat chow and tap water ad libitum. At 16 weeks of age, the F0 females were mated with breeder males. On gestation day 18 (E18), the dams were allocated to either uteroplacental insufficiency (bilateral uterine vessel ligation; offspring termed: restricted) or sham (offspring termed: control) surgery, as previously described in Reference [19 (link)]. The dams delivered pups (F1, first generation) at term on E22. At 5 weeks of age, the F1 control and restricted sibling females (maximum 2 siblings/litter/diet) were randomly allocated to a chow diet (AIN93G containing 7% fat) or HFD (SF01-028 containing 23% fat, and SF03-020 containing 23% fat and 0.19% cholesterol) obtained from Specialty Feeds, Australia. The females consumed these allocated diets for the remainder of the experiment.
8
Calorie Restriction and Macronutrient Ratios in Mice
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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.
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.
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 (
9
Calorie Restriction and Macronutrient Ratios in Mice
10
Dietary Amino Acid Supplements in Mice
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Four-week-old C57BL/10 and mdx mice were administered L-alanine (n = 12), L-citrulline (n = 12), or L-arginine (n = 12; Sigma-Aldrich, St. Louis, MO, USA) via their food, a method that results in elevated concentrations of L-citrulline and L-arginine in plasma and skeletal muscle.43 (link) Following a 1-week acclimation period during which mice received standard laboratory chow and water ad libitum, boxes of 4–5 mice were randomly allocated to receive a purified diet (AIN93G; Specialty Feeds, Glen Forrest, WA, Australia), supplemented with either 1% L-alanine, L-citrulline or L-arginine for 8 weeks. Control mice received the purified diet without amino acid supplementation (n = 12). Based on the typical food consumption of 5 g of dry food per day for an about 25 g C57BL/10 mouse, this equated to a dose of about 1 g/kg/day. It is important to note that dietary protein intake in mice is about 27.2 g/kg/day, and the dose provided represents only about 3% higher amino acid intake.
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