The more things change, the more they stay the same: the Amylin liver NASH (AMLN) Diet


A wide variety of high fat diet-induced non-alcoholic steatohepatitis (NASH) models have been used to recapitulate certain etiological aspects of the disease, with refinements in dietary formulations made over the past decade.

Amylin liver NASH model

The Amylin liver NASH (AMLN) model was described by Trevaskis et al. in 2012 as a diet-induced model which displayed both aspects of NASH (nonalcoholic steatohepatitis) and metabolic syndrome. The authors compared two diets high in fat, fructose and cholesterol: one version with trans fat (Research Diets D09100301) and the other with lard, and concluded that "the addition of trans-fat better mirrored pathophysiological features of NASH (e.g., hepatomegaly, hepatic lipid, and fibrosis)." In both Lepob/Lepob and C57BL/6J mice, the high trans fat diet induced elevated plasma ALT and hepatic steatosis after 8 weeks (Lepob/Lepob) or 12 weeks (C57BL/6J) exposure, with both experiencing increased liver weight relative to body weight. After just 4 weeks exposure to the high trans fat diet, the Lepob/Lepob mice displayed fibrosis; in contrast, minimal fibrosis was seen in C57BL/6J mice exposed to the same diet for 16 weeks1. Further studies extended the time on diet to 12 weeks (Lepob/Lepob) or 26 weeks (C57BL/6J) and used liver biopsy to confirm development of steatosis and fibrosis prior to intervention, with both strains displaying steatosis and fibrosis at the indicated time point2. NASH: Nonalcoholic Steatohepatitis The AMLN diet (D09100301) used with either the Lepob/Lepob or C57BL/6J strain was thus adopted for drug studies directed at NASH therapeutic development by many researchers, in part due to its clinically translatable etiology. However, the unintended consequences of a decision by the US Food and Drug Administration (FDA) resulted in confusion and disarray among preclinical NASH scientists.

FDA ban on trans fats impacts preclinical research

Trans fats are unsaturated fatty acids with trans rather than cis configurations. Although trans fats are found naturally in meat and dairy, a significant source of trans fat in human diets has been in the form of partially hydrogenated oils, which are made industrially via hydrogenation of vegetable oils to generate a product which is solid at room temperature. Numerous studies have shown negative health impacts associated with consumption of trans fats, with increases in mortality due to all causes, as well as coronary heart disease, sudden cardiac death and fatal cancers3.

With mounting evidence that the consumption of trans fats has a negative overall impact on human health, regulatory bodies made changes designed to limit consumption of artificially produced trans fats in the human diet. The FDA reclassified partially hydrogenated oils as not Generally Recognized as Safe (GRAS) in 2015 and banned use of them for most food applications as of June 18, 2018, with a complete phase out of partially hydrogenated oils in other food applications by January 1, 20214. This ban impacted the availability of Primex, a shortening containing trans fats which was a key component of the AMLN diet (D09100301).

Primex ceased being used in laboratory animal diet formulations in 2017. While many researchers stockpiled diets containing Primex prior to the discontinuation of those diets, it provided only a short-term solution. Alternative diets were required for future research endeavors. Various fat sources considered for alternative diets included: partially hydrogenated corn oil, Primex Z (a solid shortening with no trans fats) and palm oil.

"That was a challenging time for scientists studying diet-induced NASH models. It wasn't clear what the optimal replacement for the Primex diet would be," according to Dr. Matthew Ricci, Vice President and Science Director at Research Diets. "We worked with our customers to evaluate various options, including diets with different fat sources which matched the Primex diet for palmitic acid levels, trans fat levels or both. In the end, the feedback we received from most labs was that D09100310, which contains palm oil, gave similar results compared to the old Primex diet. We also think this diet may be more relevant to current human nutrition. Because of the regulatory environment, most humans are not currently eating high levels of trans fat. The original Primex shortening was replaced with a non-trans fat shortening containing a mixture of palm oil and hydrogenated soybean oil."

Modified AMLN diet

Scientists from both Gubra and Medimmune recently presented a poster at the International Liver Congress 2019 which characterized Research Diets D09100310 diet (containing palm oil in place of Primex) as compared to the original ALMN diet D09100301 (see a comparison of the two diets) for its ability to induce a NASH phenotype in both C57BL/6J and Lepob/Lepob mice. Although some differences were seen in weight gain in Lepob/Lepob mice, the researchers concluded that the NASH phenotype induced in both strains was similar between the two diets. In C57BL/6J mice, both diets induced steatosis in all animals sampled, as well as inflammation and fibrosis in nearly all animals exposed to diet for 28 weeks5. Additional data for C57BL/6NTac mice conditioned on D09100310 (see the 'Phenotypic Data' tab) is also now available. These studies support the use of D09100310 as a suitable substitute for the original AMLN diet.

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References:
1. Trevaskis JL et al. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. Am J Physiol Gastrointest Liver Physiol 2012; 302: G762-G772.
2. Kristiansen MN et al. Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy. World J Hepatol. 2016 Jun 8;8(16):673-84.
3. Souza RJD, Mente A, Maroleanu A, et al. Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies. BMJ 2015; 351:h3978.
4. Center for Food Safety and Applied Nutrition Final Determination Regarding Partially Hydrogenated Oils. In: US Food and Drug Administration. Accessed 12 Aug 2019.
5. Veidal S, Feigh M, Boland M, et al. Novel Gubra Amylin NASH diet-induced obese mouse model of biopsy-confirmed non-alcoholic steatohepatitis. Poster presented at the International Liver Congress, April 10-14, 2019, Vienna, Austria.