Ammonium Hydroxide Enhancement of Dietary Protein in High-Fat Diets Modulates Liver Metabolism Signaling in a Sex and Age-Dependent Manner in C3H/HeJ Mice

Sep 30, 2026

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Dietary lifestyle changes to prevent MASLD remain difficult, motivating strategies that improve risk without major compliance barriers. This study tests ammonium hydroxide enhancement (AHE) of dietary proteins as molecular evidence that MASLD development may be attenuated. C3H/HeJ female and male mice received long-term high-fat diets containing beef (HFB) or casein (HFC) with or without AHE. At multiple timepoints, hepatic targeted metabolomics and immunoblotting were applied, with RNA sequencing at 18 months. AHE reduced β-catenin and CYP3A4 in males and improved metabolic and inflammatory signaling patterns depending on sex. Further evaluation of AHE as a dietary intervention is warranted.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages19

Reading Time48 min

AccessFree to download

Introduction

MASLD and disease progression

Current treatments and barriers

Food as medicine and dietary intervention rationale

Article Ammonium Hydroxide Enhancement of Dietary Protein in High-Fat Diets Modulates Liver Metabolism Signaling in a Sexand Age-Dependent Manner in C3H/HeJ Mice Benjamin R. Barr 1, Indhu Subramaniyan 2, Li Li 2, Danielle E. Levitt 3 and Lauren S. Gollahon 1, * Academic Editors: Luís Fernando Barbisan and Miguel Walter Fornés Received: 25 November 2025 Revised: 23 December 2025 Accepted: 25 December 2025 Published: 30 December 2025 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. 1 Department of Biological Sciences, Texas Tech University, 2500 Broadway, Lubbock, TX 79409, USA; [benjamin.barr@ttu.edu](benjamin.barr@ttu.edu) 2 Departments of Pharmacy Practice and Pharmaceutical Sciences, North Texas Clinical Pharmacology Cancer Core, Texas Tech University Health Sciences Center, 5920 Forest Park Ln, Dallas, TX 75235, USA; [indhumathy.subramaniyan@ttuhsc.edu](indhumathy.subramaniyan@ttuhsc.edu) (I.S.); [li.li@ttuhsc.edu](li.li@ttuhsc.edu) (L.L.) 3 Department of Kinesiology and Sport Management, Texas Tech University, 2500 Broadway, Lubbock, TX 79409, USA; [danielle.levitt@ttu.edu](danielle.levitt@ttu.edu) * Correspondence: [lauren.gollahon@ttu.edu](lauren.gollahon@ttu.edu) Abstract (1) Lifestyle changes to modify unhealthy dietary patterns with the goal of preventing MASLD have proven challenging. Here, dietary proteins and their modification with ammonium hydroxide enhancement (AHE) provide molecular evidence that this novel approach may attenuate the development of MASLD without undue dietary adjustments, potentially bypassing non-compliance. (2) High-fat diets containing dietary beef (HFB) orcasein (HFC) + AHE (HFBN and HFCN, respectively) were fed to 256 C3H/HeJ female and male mice long term. At 6, 12, or 18 months, hepatic samples were analyzed with targeted metabolomics (glucose, lactate, alanine, glutamine, carnitine) and Western analysis (β-catenin, glutamine synthetase, CYP3A4) . RNA sequencing was performed on samples collected at 18 months (n = 3; male HFC n = 2) . (3) Metabolomics results showed that at 18 months, hepatic glutamine was greater in HFBN versus HFCN in females, whereas in males, hepatic glutamine, glucose and lactate were lower in HFBN versus HFCN. Additionally, diets with AHE decreased β-catenin and CYP3A4 protein expression in males. Ingenuity pathway analysis (IPA) of RNA-seq data predicted that HFBN activates PPARα signaling in the liver in both sexes compared to HFCN. Inflammatory activity showed predicted activation for females in the HFBN:HFCN comparison. In males, the inflammatory pathway molecular mechanisms of cancer was predicted as deactivated in HFBN:HFCN. (4) Dietary protein source impacts outcomes, and these outcomes improved with AHE. The HFBN diet improves signaling associated with lipid utilization for females and males, and improved inflammatory signaling for males compared with HFCN. Further exploration of AHE as a dietary intervention in high-fat diets is warranted. Keywords: dietary protein; ammonium hydroxide enhancement; MASLD; MASH; HCC; diet-induced obesity; metabolites; dietary intervention 1. Introduction Metabolic dysfunction-associated steatotic liver disease (MASLD) has been designated as the most common cause of chronic liver disease [1] . MASLD is characterized by the accumulation of lipid within the liver in association with systemic metabolic dysfunction [2] . As lipids accumulate, the disease progresses into metabolic dysfunction-associated steatohepatitis (MASH), further into cirrhosis, and/or potentially hepatocellular carcinoma (HCC) [2] . Key in the transition from MASLD to MASH is an increase in hepatic inflammation connected with lipid accumulation [2] . Additionally, during the progression to MASH, inflammation promotes fibrosis resulting in further damage to the liver, inc

FAQs

What problem does the study target regarding MASLD prevention?

Preventing MASLD through lifestyle change is challenging, so the study explores a dietary modification that may reduce MASLD development without requiring major dietary adjustments.

How was ammonium hydroxide enhancement (AHE) evaluated in the animal model?

Female and male C3H/HeJ mice were fed long-term high-fat diets containing beef or casein with or without AHE, followed by hepatic targeted metabolomics, Western analysis, and RNA sequencing at 18 months.

What molecular effects did AHE show in males and how did outcomes vary by sex?

AHE decreased β-catenin and CYP3A4 protein expression in males, while metabolomics and pathway predictions differed by sex, indicating sex- and time-dependent signaling changes.

Sep 30, 2026

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