All animal husbandry and animal experiments complied with regulations of the University of Tsukuba for animal experiments and were approved by the Animal Experiment Committee of the University of Tsukuba. Seven-week-old C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan). Mice were housed in colony cages under a 12 h light/12 h dark cycle, with unlimited access to food and water. At 10 weeks of age, mice were randomly divided into the following four groups: chow group (fed on standard chow with vehicle treatment), HFD group (fed with vehicle treatment), the HFD + octacosanol group (60 mg/kg/day; Sigma-Aldrich, Tokyo, Japan), and HFD + policosanol group (60 mg/kg/day; NOF, Tokyo, Japan). octacosanol and policosanol were dissolved in 10 mg/ml Acacia-gum water. octacosanol, policosanol, or the same volume of vehicle was administered via oral gavage once a day for four weeks. At the end of four weeks, all mice were sacrificed in the early light phase in a non-fasting state. HFD was obtained from Oriental Yeast (Tokyo, Japan) and the composition of HFD is shown in Supplementary Table S1 . The policosanol extracted from rice bran oil was obtained from NOF (Tokyo, Japan). The composition of aliphatic alcohols in policosanol is C22; 1%, C24; 9%, C26; 9%, C28; 18%, C30; 21%, C32; 9%, C34; 16%, C36; 14%, and others; 3%.
High fat diet (hfd)
Manufactured by Oriental Yeast
Sourced in Japan
The HFD is a piece of laboratory equipment designed for high-frequency drying. It is a specialized device used to rapidly dry various materials or samples in controlled conditions. The core function of the HFD is to efficiently remove moisture from substances through the application of high-frequency electromagnetic radiation.
Automatically generated - may contain errors
Lab products found in correlation
Zoledronic acid za, by Merck Group (1 mentions)
Octacosanol, by Merck Group (1 mentions)
High fat diet (hfd), by Merck Group (1 mentions)
Hamilton syringe, by Merck Group (1 mentions)
M80 stereomicroscope, by Leica (1 mentions)
One touch ultra, by LifeSpan BioSciences (1 mentions)
Clodronate containing liposomes, by Formumax Scientific (1 mentions)
Stereotaxic frame, by Narishige (1 mentions)
Control diet, by Oriental Yeast (1 mentions)
C57bl 6 mice, by Japan SLC (1 mentions)
14 protocols using high fat diet (hfd)
1
Effect of Octacosanol and Policosanol on High-Fat Diet-Induced Obesity in Mice
2
High-Fat Diet and Metabolic Syndrome Mitigation
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C57BL/6 J mice were housed in a vivarium in accordance with the guidelines of the animal facility center of Kumamoto University. The light/dark cycle was set at 12 h, and the temperature was kept at a room temperature of 20 °C. To prepare diabetes model with HFD loading, an HFD purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan) was used as previously described23 (link). HFD was composed of 14% lard, 14% beef tallow, 25% casein, 20% sucrose, 15% cornstarch, 5% cellulose.
Five-week-old mice were randomly divided into 4 groups and given the following diet for 10 weeks: control diet for 10 weeks (n = 6); HFD for 10 weeks (n = 6); HFD for first 6 weeks and HFD supplemented with MSE powder 500 mg/kg/day for last 4 weeks (n = 6); and HFD for the first 6 weeks and HFD supplemented with MSE powder 1,000 mg/kg/day for the last 4 weeks (n = 6). HFD was orally administered to mice. Adipose tissue, muscle and liver samples were collected from mice that completed the diet. Since MSE powder has been reported to be well tolerated at an oral administration of 5,000 mg/day in humans5 (link), the dose of 1,000 mg/kg/day for mice was calculated using a human equivalent dose (5,000 mg/60 kg/day in human)24 .
All experiments on mice were approved by the Animal Welfare Committee of Kumamoto University and were conducted in adherence to institutional regulations and guidelines.
Five-week-old mice were randomly divided into 4 groups and given the following diet for 10 weeks: control diet for 10 weeks (n = 6); HFD for 10 weeks (n = 6); HFD for first 6 weeks and HFD supplemented with MSE powder 500 mg/kg/day for last 4 weeks (n = 6); and HFD for the first 6 weeks and HFD supplemented with MSE powder 1,000 mg/kg/day for the last 4 weeks (n = 6). HFD was orally administered to mice. Adipose tissue, muscle and liver samples were collected from mice that completed the diet. Since MSE powder has been reported to be well tolerated at an oral administration of 5,000 mg/day in humans5 (link), the dose of 1,000 mg/kg/day for mice was calculated using a human equivalent dose (5,000 mg/60 kg/day in human)24 .
All experiments on mice were approved by the Animal Welfare Committee of Kumamoto University and were conducted in adherence to institutional regulations and guidelines.
3
High-fat diet and Sunchlon extract effects
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Mice were fed a HFD (Oriental Yeast Co., Tokyo, Japan; 60% calories, 5.06 kcal/g; protein [casein, L-cysteine] 23.0%, carbohydrate [corn, maltodextrin, sucrose] 25.3%, fat [lard, soybean oil] 35.0%, other) or normal diet (CE-2; Clea Japan, Inc., Tokyo, Japan; 3.59 kcal/g; protein [soybean waste, whitefish meal, yeast] 24.9%, carbohydrate [wheat flour, corn, Milo] 51.0%, fat [cereal germ, soybean oil] 4.6%, other) for a total of 12 weeks. After 8 weeks, the mice were divided into control groups (CE-2 and HFD), which received saline, and treatment groups that were given SE (Sunchlon Co. Ltd., Nagano, Japan) (CE-2 + SE and HFD + SE). The 0.2 mL dose of SE is equivalent to 0.564 g of S. veitchii leaves, as previously described.24 ) Treatment with saline or SE, 0.2 mL twice per day by oral gavage, lasted 4 weeks.
Body weight and food intake were measured every week throughout the study. Food intake per day was calculated as total food intake / number of mice. Following the experiment, mice from each group were fasted for 16 h, euthanized using pentobarbital, and bled for plasma samples, which were stored at –80 °C pending assays. Liver and epididymal adipose weights were determined. Separate samples from each liver were stored at –80 °C or fixed in 15% neutral buffered formalin (pH 7.2).
Body weight and food intake were measured every week throughout the study. Food intake per day was calculated as total food intake / number of mice. Following the experiment, mice from each group were fasted for 16 h, euthanized using pentobarbital, and bled for plasma samples, which were stored at –80 °C pending assays. Liver and epididymal adipose weights were determined. Separate samples from each liver were stored at –80 °C or fixed in 15% neutral buffered formalin (pH 7.2).
4
NASH Model in Rats Treated with AMSC-EVs
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The NASH model was generated by feeding rats (n = 22) on a HFD (Oriental Yeast, Tokyo, Japan) for four weeks, whereas rats in the control group (n = 6) were given a normal diet (Oriental Yeast). AMSC-EVs (15 μg/kg) suspended in 200 μL PBS were intravenously injected into the HFD rats (HFD + AMSC-EV group, n = 11) through the penile vein at weeks 3 and 4. Conversely, 200 μL PBS was injected via the same route in control rats and those treated with HFD (HFD group, n = 11). All rats were sacrificed at five weeks after HFD initiation (Figure 1(a) ).
5
Comparative Analysis of Genetically Modified Mice
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WT C57BL/6J mice were purchased from Clea Japan. Dscr-1−/− and ApoE−/− mice with C57BL/6J backgrounds have been previously described (17 (link), 33 (link)). Hypoxanthine phosphoribosyltransferase (Hprt) locus targeted Tie2 promoter-driven human DSCR-1 short isoform expressed mice and DSCR-1 promoter-driven lacZ-expressing mice have also been previously described (13 (link), 18 (link)). All animal care and experimental procedures followed instructions from the committee of both the University of Tokyo and Kumamoto University. All animals were allowed free access to water and a normal chow diet (CE-2; CLEA Japan) or a high-fat diet (HFD, ORIENTAL YEAST) containing 35% fat, 30% maltose, 22% casein, 7% dextrin, and 3.5% minerals. Littermate mice were fed HFD for 12 weeks from 8 weeks of age.
6
Dietary Modulation of Osteoclast Apoptosis
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Wild-type mice (C57BL/6N) were obtained from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). Eight-week-old female mice were maintained on a normal diet (12.0% fat, 28.9% protein, and 59.1% carbohydrate; Oriental Yeast Co., Tokyo, Japan) (ND) or high-fat diet (62% fat, 18.2% protein, and 19.6% carbohydrate; Oriental Yeast Co.) (HFD) for one week before mating, as well as throughout pregnancy and lactation. All the pups were weaned at 5 weeks of age and fed an ND until the age of 8 weeks. Zoledronic acid (ZA, 100 μg/kg) (Sigma Aldrich, St Louis, MO, USA) was intravenously injected just after CaCl2 application to induce osteoclast apoptosis and inhibit osteoclast function in the pups [17 (link)].
The animals were housed in a room maintained at 22 °C under a 12-h light/dark cycle and provided with drinking water ad libitum. At 1, 4, and 8 weeks after CaCl2 application, the mice were euthanized by transcardial perfusion under anesthesia induced by isoflurane (2%; 0.2 mL/min). The total number of mice used was 366, not including the preliminary experiments.
The animals were housed in a room maintained at 22 °C under a 12-h light/dark cycle and provided with drinking water ad libitum. At 1, 4, and 8 weeks after CaCl2 application, the mice were euthanized by transcardial perfusion under anesthesia induced by isoflurane (2%; 0.2 mL/min). The total number of mice used was 366, not including the preliminary experiments.
7
Dual p62 and Nrf2 Knockout Mouse Model
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p62 and Nrf2 DKO mice were produced by crossing p62-KO and Nrf2-KO mice and regenotyping [33 (link)]. All mice were maintained under specific pathogen-free conditions in an environmentally controlled clean room at the Laboratory Animal Resource Center, University of Tsukuba. Mice were housed at an ambient temperature of 23 ± 2.5 °C with a daily light/dark cycle from 05:00 to 19:00. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committees of the University of Tsukuba. Male mice were used in all experiments.
The mice were fed a normal chow (NC: 5.1% fat, 23.1% protein, 360 kcal/100 g from Oriental Yeast, Tokyo, Japan) or a 60% high-fat diet (HFD: 60% fat, 24.5% protein, 640 kcal/100 g from Oriental Yeast, Tokyo, Japan) for 26 weeks from 6 to 32 weeks of age. During the same period with HFD feeding, the mice had free access to drinking water with SMAPoTN (10 mg/day) or without SMAPoTN.
The mice were fed a normal chow (NC: 5.1% fat, 23.1% protein, 360 kcal/100 g from Oriental Yeast, Tokyo, Japan) or a 60% high-fat diet (HFD: 60% fat, 24.5% protein, 640 kcal/100 g from Oriental Yeast, Tokyo, Japan) for 26 weeks from 6 to 32 weeks of age. During the same period with HFD feeding, the mice had free access to drinking water with SMAPoTN (10 mg/day) or without SMAPoTN.
8
Generating p62-Deficient and Muscle-Specific Rescue Mice
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Mice on C57BL/6 background were used. We generated p62-knock-in mice (p62KIKI), in which a transcription termination signal flanked by loxp sequences and a polyA-FRT-neomycin resistance gene cassette (neo)-FRT sequence were inserted into the intron between exons 1 and 2 of the p62 gene (Figure 1 ). These mice have whole-body p62 deficiency. Muscle-specific p62 gene rescue mice (p62KIKI:ACTA1-Cre/+: p62-mRes) were obtained by crossing p62KIKI and human alpha-skeletal actin (ACTA1)-Cre mice using the cre/loxp system (Sauer, 1987 (link)). The mice were housed at 20–23°C under a 12-h light/dark cycle. Male 5-week-old p62KIKI and p62-mRes mice were fed normal chow (NC) or a 60% high-fat/high-sucrose diet (HFD, Oriental Yeast, Tokyo, Japan) for 20 weeks. When the mice were 25 weeks of age, liver, skeletal muscle, epididymal adipose tissue (WAT), and blood samples were collected under isoflurane anesthesia. Blood samples were centrifuged at 1,500 × g for 10 min to collect serum. All the samples collected were stored at −80°C. The study was approved by the Animal Care and Use Committee of the University of Tsukuba, and was conducted in accordance with the Regulations on Animal Care and Use in Research; the Welfare and Management of Animals Act, and the Standards for Husbandry, Housing, and Pain Relief of Experimental Animals.
9
Corneal Macrophage Depletion in Diabetic Mice
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C57BL/6 mice (Mus musculus) were purchased from Japan CLEA (Tokyo, Japan) and fed a control diet (CE-2; Japan CLEA). Six-week-old BKS.Cg-+Lepr db /+Lepr db /J (db/db) mice (Mus musculus) was purchased from Jackson Laboratory Japan (Kanagawa, Japan). To induce T2D, 6-week-old male db/db mice were fed a high-fat diet (HFD; Oriental Yeast, Tokyo, Japan) for 6 weeks. After HFD feeding, the development of T2D was monitored by investigating fasting blood glucose and HbA1c levels using a OneTouch Ultra (LifeSpan, Tokyo, Japan) and Quo-Lab HbA1c Analyzer (EKF Diagnostic PLC, Cardiff, UK), respectively, according to the manufacturers' instructions.
To deplete the corneal macrophages, 0.05 mg/10 μL clodronate-containing liposomes (Formumax Scientific, Sunnyvale, CA, USA) was subconjunctivally injected Briefly, mice were anesthetized and placed in a stereotaxic frame (Narishige, Tokyo, Japan). All injection procedures were performed using a 5-μL Hamilton syringe with a 24-G needle (Merck, Darmstadt, Germany) under a Leica M80 stereo microscope (Leica, Wetzlar, Germany). On Day 5 from the primary injection of clodronate, IL-1β, and IL-34, mice were euthanized to perform enucleation of the eyeball (Figure 1) [11] .
To deplete the corneal macrophages, 0.05 mg/10 μL clodronate-containing liposomes (Formumax Scientific, Sunnyvale, CA, USA) was subconjunctivally injected Briefly, mice were anesthetized and placed in a stereotaxic frame (Narishige, Tokyo, Japan). All injection procedures were performed using a 5-μL Hamilton syringe with a 24-G needle (Merck, Darmstadt, Germany) under a Leica M80 stereo microscope (Leica, Wetzlar, Germany). On Day 5 from the primary injection of clodronate, IL-1β, and IL-34, mice were euthanized to perform enucleation of the eyeball (Figure 1) [11] .
10
Exploring Dietary Interventions for Obesity in Mice
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The composition of different diets fed to mice are listed in Table 1
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