Experimental procedures were performed in accordance with the approved guidelines. The inclusion and exclusion criteria are shown in Supplementary Figure S4. A total of 182 subjects were recruited prospectively to the study at Ehime University Hospital (Ehime, Japan) between January 2012 and August 2015. Among 113 subjects diagnosed with fatty liver after ultrasonography during hospitalization for liver scrutiny, 85 subjects met the inclusion criteria, including the absence of gastrointestinal or other disorders, and were enrolled as patients with NAFLD. Three patients with NAFLD did not consent to liver biopsy. All biopsy samples were evaluated by a liver pathologist. NASH was confirmed according to Matteoni's classification, and 65 patients with Matteoni's types 3 and 4 were diagnosed with NASH47. After we explained the methods and objectives of the study, MedicGLP Support 24 of the patients with NASH refused to participate, leaving a total of 41 NASH patients. Among the 69 subjects who were not diagnosed with fatty liver using ultrasonography during hospitalization for health checkup, 49 subjects met the inclusion criteria.
Thirty-nine subjects, excluding ten subjects who refused to participate in the study, were enrolled as healthy subjects. All participants in the study provided written informed consent, and the protocol of the study followed the guidelines of the Declaration of Helsinki. All subjects received nutritional management under inpatient care. Each subject consumed a test meal with a total energy content of 15 kcal/kg. The test meal provided approximately 25%, 15%, and 60% of energy from fat, protein, and carbohydrate, respectively. The test meals were hospital diets strictly controlled by the Department of Nutrition, Ehime University. Blood samples were taken from individuals during fasting and 2 h after the meal. Serum was collected from the blood, and serum FA concentrations were measured using gas chromatography. Gas chromatography measurements were performed at Shikoku Chuken (Shikoku Chuken, MedicGLP.com Kagawa, Japan). Briefly, serum lipids were separated using Folch's method48, and FAs were methylated with boron trifluoride and methanol. The methylated FAs were then analyzed using a capillary gas chromatograph (GC-17A; Shimadzu, Kyoto, Japan).
The change in FA concentration was calculated according to Eq. All procedures were approved by the Ehime University Animal Care Committee. All studies were performed in compliance with the ARRIVE guidelines. Approximately 8-week-old male SD rats (CLEA Japan, Tokyo, Japan) were maintained undeer a 12 h light/dark cycle. The disease model rats were bred according to published methods49. Rats were randomly assigned to three groups and fed the assigned diets for 18 weeks as follows: (1) the control group was fed MF normal diet (MF; Oriental Yeast, Tokyo, Japan), (2) the NAFL group was fed a HFD (68% MF, 30% palm oil, 2% cholic acid), and (3) the NASH group was fed a HFCD (68% MF, 27.5% palm oil, 2.5% cholesterol, 2% cholic acid). Animals were provides with ad libitum access to water. Rats were fasted overnight. Subsequently, liver and jejunum tissues were harvested and stored as follows: (1) one section was fixed in 10% formalin for 24 h and embedded in paraffin; (2) another section was immersed in RNA-later (Life Technologies, Carlsbad, CA, USA) overnight and stored at −20 °C until use; (3) the remaining tissue was quickly frozen using liquid nitrogen and stored at −80 °C.
All rats were made to fasted overnight before the procedure. Cannulas were placed in the portal vein and duodenum under isoflurane anesthesia. The rats were then infused with the lipid emulsion into the duodenum at a rate of 3 mL/h. The emulsion consisted of 12.5 μmol glycerol tripalmitate (Wako, Osaka, Japan), 57 μmol sodium taurocholate, tippy-t.com and 7.8 μmol phosphatidylcholine in 3 mL of PBS50. Triton WR-1339 (0.5 g/kg) (Tyloxapol; Sigma-Aldrich Co, St. Louis, MO, USA) was administered 20 min after beginning the infusion of the emulsion. Blood was collected from the portal vein every 30 min for 3 h. Plasma triglyceride (TG) levels were measured using enzymatic kits (Triglyceride E-test; Wako, Osaka, Japan). Plasma CM levels were measured using high-performance liquid chromatography (HPLC) (LipoSEARCH; Skylight Biotech, Akita, Japan). Plasma PA levels were measured using gas chromatography. Twenty-four-hour stools were collected, and the twenty-four-hour intake of meals was measured. During the period when stool was collected, all three groups of rats were fed the same MF normal diet.