DOI: 10.5061/dryad.np5hqbzxx
title: Data of multiple different high-fat diets

description: Background and Objectives: The spectrum of non-alcoholic fatty liver
 disease (NAFLD), known as non-alcoholic steatohepatitis (NASH), can lead
 to advanced liver disease. It is known that a variety of diets play a
 significant role in the development of NAFLD/NASH. The goal of this study
 is to determine the most appropriate composition of diet to induce NASH in
 an animal model. Methods: This research used Rattus norvegicus strain
 Wistar (n=27), which were divided into four groups and given each diet for
 12 weeks: normal diet (ND, n=7), high-fat diet (HFD, n=6), Western diet
 (WD, n=7), high-fat-high-fructose diet (HFHFD, n=7). Subjects were
 documented for body weight. Blood samples were taken for biochemical
 analysis: low-density lipoprotein (LDL), triglyceride, alanine
 aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
 phosphatase (ALP), hepatic lipase, tumor necrosis factor-alpha (TNF-α),
 interleukin-6 (IL-6), and lipopolysaccharide (LPS). Feces were taken for
 short-chain fatty acid (SCFA) analysis. Liver histology was analyzed using
 NAS (NAFLD activity score). The comparison test was carried out using the
 one-way ANOVA or Kruskal–Wallis test. Results: The highest mean of body
 weight was in the WD group (346.14 g). Liver enzymes, LDL, triglyceride,
 propionic acid, and acetic acid in each group were not significantly
 different. TNF-α, IL-6, and hepatic lipase were significant (p = 0.000; p
 = 0.000; p = 0.004) and the highest was in the HFD group. Butyrate level
 was significant (p = 0.021) and the least was in the HFHFD group (4.77
 mMol/g). Only WD and HFHFD had an NAS ≥ 5 (14% and 14%). The highest
 percentage of borderline NAS was found in WD (57%). Conclusions: The HFD
 group induced significant liver inflammation but did not produce NASH
 histologically, whereas the WD and HFHFD groups had the potential to
 develop NASH because the diets affected metabolic and inflammatory
 parameters as well as liver histology.
description: The Ethical Committee of the
 Faculty of Medicine, Universitas Brawijaya reviewed and approved all
 procedures (No.
 66/EC/KEPK/02/2021). Twenty seven male Wistar rats were obtained from the
 Universitas Gadjah Mada.
 Rat inclusion criteria
 included: male rat with shiny white fur, healthy, active, and have normal
 behavior; about 8–12 weeks old; the average body weight was 150–180 grams.
 Exclusion criteria included: the appearance of dull fur, loss and
 baldness; less or inactive activity; rats that during the study did not
 want to eat; weight loss &gt;10% after adaptation period; Disabled,
 sick and/or dead rat. In minimizing pain and discomfort to experimental
 animals, this research uses the refinement principle for ensuring the
 welfare of experimental animals until the end of the study. We provided
 food and drink regularly every day with a certain type of diet according
 to the type of treatment. Cage maintenance, cage cleaning, and husk
 replacement were carried out every day with attention to light,
 temperature, and humidity. We monitored and evaluated rats condition every
 day and placed them individually in each cage. Before being treated, the
 rats were acclimatized for 2 weeks, given a standard diet, and placed
 inside cages at the Pharmacology Laboratory, Faculty of Medicine,
 University Brawijaya. The Wistar rats were randomly assigned using table
 of random numbers, then categorized into four groups: normal diet (ND)
 (67% carbohydrate, 21% protein, 7% fat, 5% fiber); HFD (67.1%
 carbohydrate, 16.5% fat, 16.4% protein), WD (52% carbohydrate, 16.1%
 protein, 31.7% fat), and HFHFD (41.5% carbohydrate, 10.3% fat, 10.2%
 protein, 38% fructose). All diets were given for 12 weeks. All four groups
 were euthanized with ketamine–xylazine intravenously to relieve pain on
 the same day before surgery was performed. The entire liver was taken out and weighed.
 For further analysis, the livers were either collected and stored at -20°C
 or fixed in 10% paraformaldehyde. Rat serum was used to analyze biochemical
 parameters in the Clinical Pathology Laboratory, Universitas
 Brawijaya,
 Indonesia. Serum
 alanine aminotransferase
 (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)
 were analyzed chemically using colorimetric analysis (<em>ADVIA 2400
 Clinical Chemistry System (Siemens, Germany</em>).
 Serum hepatic lipase, tumor necrosis factor-alpha (TNF-α),
 interleukin-6 (IL-6), and lipopolysaccharide (LPS) were analyzed with the
 sandwich enzyme-linked immunosorbent assay (ELISA)
 method.  A total of 0.5 gram fecal samples from the colon
 were used, and a dilution ratio of 1:4 between samples and sterilized
 water was established. We subsequently vortexed the samples for 1 min and
 centrifuged the homogenate at 10000 x g for 10 min at 4°C. Filtration of
 SCFA-containing supernatant was completed by using cellulose acetate
 membrane (GyroDisc, Orange Scientific, CA, US) and it was stored at -80°C
 until further analysis was performed. At the time
 of analysis, 20 mg of the faecal sample supernatant poured into a 1.5 ml
 microtube and then added 200uL of distilled water homogenized using an
 iron spatula. 200 µL of sample, 200 µL of a mixture of N-butanol,
 tetrahydrofuran and acetonitrile at a ratio of 50:30:20 40 µl 0.1 M HCl,
 20 mg of citric acid and 40 mg of sodium chloride were added to 1.5 mL
 microtubules. The microtubules were then shaken using a vortex for 1
 minute. Sample centrifuged at 14870 x g at room temperature for 10
 minutes. Supernatant transferred using a micropipette to the
 chromatography vial for later Gas Chromatography analysis was carried out.
 The results of the analysis are entered into the software EZchrome and GC
 solution. SCFA analysis
 was performed at Food Technology and Agricultural Products Laboratory,
 Universitas Gadjah Mada, Indonesia.  
 The liver was sliced, fixed
 with 10% buffered formalin, embedded in paraffin, and stained with
 hematoxylin–eosin (HE) stain at a thickness of 5 μm (11). The sample preparation was performed at the
 Anatomical Pathology Laboratory of Universitas Brawijaya, Indonesia. Liver histopathology was
 used to find the NAFLD Activity Score (NAS). Three parameters (steatosis
 score 0–3; lobules inflammation score 0–3; ballooning score 0–2) were used
 to know NAFLD staging. Scores of 0–2 are defined non NASH, scores of 3–4
 are defined as borderline, while scores ≥ 5 are considered diagnostic of
 NASH.  Data were presented as the mean
 ± standard deviation and were analyzed with SPSS
 25.0 (RRID:SCR_002865) for Windows. A one way ANOVA was carried out when the data were
 normally distributed and then continued with the Tukey Honest Significant
 Difference (HSD) <em>post hoc</em> test if the data were
 significant. The Kruskal–Wallis test was used when the data distribution
 was not normal. If the results were significant then the Mann Whitney test was performed. When p
 &lt;0.05, data were
 considered significant.
description: Software that is required to open the data files in format .sav
 is SPSS.