Ain 93g

The MCD with 2% carbonyl iron (MCD-Fe) diet was custom-made for this study (Oriental Yeast Co., Ltd, Tokyo, Japan). The components of the diet are shown in Table 1. The normal diet (AIN-93G, Oriental Yeast) included methionine and choline without carbonyl iron. The diet was administered for 12 weeks, beginning when the mice were 4 weeks old.Table 1

Components of the diets.

Table 1

Elements of diet	Normal diet (AIN-93G)	MCD with 2% iron diet
Amino acid mix (without methionine)	-	17.83%
Amino acid mix (with methionine)	18.34%	-
Sucrose	10.00%	10.00%
Lard	10.00%	10.00%
Cellulose	5.00%	5.00%
AIN-93 vitamin mix (without choline)	-	1.00%
AIN-93 vitamin mix (with choline)	1.00%	-
AIN-76 mineral mix	3.50%	3.50%
Tertiary butylhydroquinone	0.002%	0.002%
Corn starch	38.988%	25.948%
Pregelatinized corn starch	13.17%	15.17%
Fe-citrate (Fe17%)	-	11.55%

The HPE used in this study was hydrolysate of human placenta (Laennec; Japan Bio Products Co., LTD, Tokyo, Japan). Mice were intramuscularly administered 0.1 ml of Laennec (3.6 mg/kg) or control saline twice a week during the MCD-Fe diet. Body weights were measured every day at around 10:00 am.

All animal protocols were conducted in accordance with regulations for experiments involving laboratory animals, established by the Animal Use and Care Committee of EN Otsuka Pharmaceutical Co., Ltd., Hanamaki, Japan (approval no. ENDR-15-02, 29 May 2015). We housed 4-week-old male F344/NJcl.Cg-Foxn1rnu rats (CLEA Japan, Inc., Tokyo, Japan) in individual stainless steel metabolic cages under controlled temperature (23 ± 3 °C) and humidity (50 ± 20%) conditions and in a 12 h/12 h light/dark cycle (lights on at 07:00). The rats were fed AIN-93G (Oriental Yeast Co., Ltd., Tokyo, Japan) and provided with drinking water ad libitum. Additionally, the rats were allowed to acclimatize to the laboratory conditions for approximately 1 week before the experiments. The rats were randomly assigned to control or SLC transplantation groups, pair-matched on the basis of body weight.

In the first experiment, the CS +/− (n = 7) and WT (n = 7) mice were fed a low-carbohydrate diet (88% fat, 11% protein, and 1% carbohydrate; 7.2 kcal/g), also called ketogenic diet because it induced ketosis in the subject consuming it, for 8 weeks. In the second experiment, the CS +/− (n = 7) and WT mice (n = 10) were fed a high-fat, high-carbohydrate (42% fat, 18% protein, and 40% carbohydrate; 4.7 kcal/g) for 8 weeks to induce obesity. The ingredients of each diet were purchased from Oriental Yeast Co. (Tokyo, Japan), and the composition of each diet is shown in Supplementary Table 1. Apart from these two main experiments, WT mice (10 weeks old, n = 6) were reared on a normal rodent diet (AIN-93G, Oriental Yeast Co.) for 8 weeks to confirm whether each experimental diet was effective at inducing metabolic changes.
The body weight of the mice was measured every 2 weeks throughout the experimental period. At the end of the experimental period, the mice were fasted for 4 h and anesthetized with isoflurane to collect blood samples into 1.5 mL microcentrifuge tubes. The mice were euthanized by cervical dislocation under anesthesia and immediately dissected to collect tissue samples from the heart and hindlimb muscles. All animal procedures were approved by the Animal Ethics Committee of Ochanomizu University (approval number: 19013 and 20018).

Various doses of powdered fruiting bodies from A. brasiliensis were mixed into the basic synthetic diet AIN-93G (Oriental Yeast Co., Ltd, Tokyo, Japan), and cornstarch was substituted for A. brasiliensis. The doses of KAOD tested were 1, 3, and 10%, whereas KAID and JAID were tested at 3 and 10%, respectively. AIN-93G supplemented with cornstarch was used as the control diet. All feeds were admixed by Oriental Yeast Co., Ltd, and the molding and drying processes were carried out at 60°C or lower. Each test diet was irradiated with 30 kGy γ-rays for sterilization. The ingredients in the solid feed are shown in Table 1. To evaluate the heat stability of KAOD, AIN-93G and KAOD-10% were autoclaved and air-dried at 50°C, and named heat-treated AIN-93G and heat-treated KAOD, respectively.Table 1

Ingredients in solid feed

Ingredient	Content (g/kg diet)
Corn starch	397.486 - R
Casein	200.000
α-Corn starch	132.000
Sucrose	100.000
Soybean oil	70.000
Cellulose powder	50.000
AIN-93G Mineral Mix	35.000
AIN-93G Vitamin Mix	10.000
L-Cystine	3.000
Choline bitartrate	2.500
tert-butylhydroquinone	0.014
KAOD, KAID, or JAID	R

Control diet (AIN-93G): R =0.

KAOD-1%: R =10.

KAOD-3%: R =30.

KAOD-10%: R =100.

KAID-3%: R =30.

JAID-10%: R =100.