A Combinatorial Mouse Model of Circadian Disruption and Dietary Stress for Investigating Lifestyle-Accelerated Aging Pathophysiology

Summary

Here, we present a protocol to establish a mouse model of disordered circadian rhythm plus high-fat, high-sugar (HFHS) diet, which mimics unhealthy lifestyles and induces metabolic disorders and aging phenotypes.

Abstract

The goal of this protocol is to establish a standardized mouse model that recapitulates the multifactorial damage induced by modern, unhealthy lifestyles. To this end, we developed a novel C57BL/6J model combining randomized circadian rhythm disruption and a high-fat, high-sugar (HFHS) diet, with single-factor HFHS and circadian disruption groups as controls. Compared with controls, the composite model showed more severe metabolic disorders, including increased body weight, elevated fasting glucose and lipid levels, and marked hepatic steatosis, as well as obvious cognitive and motor impairments. The composite model also exhibited more significant accelerated aging, as confirmed by the frailty index and hematoxylin and eosin (HE) staining. This integrated model reliably mimics lifestyle-related metabolic-cognitive decline and premature aging. This step-by-step reproducible protocol supports mechanistic studies on lifestyle-mediated aging and the evaluation of targeted therapeutic strategies.

Introduction

Accelerated modern lifestyles increasingly expose populations to rhythm-disrupting behaviors and obesogenic diets, with compelling evidence linking these factors to systemic functional decline and chronic disease pathogenesis¹. Epidemiological data reveal striking comorbidities: shift workers show a high prevalence of metabolic syndrome markers, while long-term intake of a high-fat and high-sugar diet is strongly associated with an elevated risk of dementia^2,3. These insults converge to drive multiorgan pathology—metabolic dysfunction frequently coincides with accelerated cognitiv....

Protocol

All experimental procedures were approved by the Animal Research Ethics Committee of Hebei Yiling Pharmaceutical Research Institute (Approval No: N2024043). Thirty-two 8-month-old male C57BL/6J mice (see Table of Materials) were housed in a specific pathogen-free (SPF) animal facility affiliated with the New Drug Evaluation Center, Hebei Yiling Pharmaceutical Research Institute. The rearing environment was maintained under standardized conditions: ambient temperature ranged from 20–26 °C, relative humidity was controlled at 40%–70%, and a 12-h light/12-h dark cycle was implemented. The mice had free access to a standard laboratory rod....

Results

Synergistic exacerbation of metabolic pathology in dual-exposure cohorts
Longitudinal body weight monitoring revealed distinct growth trajectories among cohorts. The model group and the HFHS group exhibited accelerated weight gain compared to both the Control and CR groups. Notably, the CR group demonstrated modest acceleration relative to Control (Figure 3A). Further evidence of metabolic dysfunction was provided by the observation that both fasting blood glucose and bl.......

Discussion

Contemporary research has unequivocally confirmed that circadian disruption and a high-fat, high-sugar diet are two independent drivers of systemic functional decline in the organism. Our data demonstrates that the pathological damage induced by the combined action of these two factors is far more severe than the simple additive effect of either alone. Compared with the single-factor intervention groups, the composite model group showed no significant difference in adiposity relative to the HFHS group, but exhibited a ma.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ANY-maze	Science	SA225
Accu-Chek Active Blood Glucose Monitor	Switzerland, Roche Diagnostics	accu check active
C75BL/6J mice	BEIJING HFK BIOSCIENCE CO.,LTD	No.110324241101250228
Grip strength meter	USA, Columbus Instruments	1027CSM-E54
High-fat and high-sucrose diet	China, Xietong Biotechnology Co., Ltd.	XT303
HistoCore MULTICUT - Semi-Automated Rotary Microtome	Germany, Leica	RM2245
Oil Red O solution	China, Wuhan Servicebio Technology Co., Ltd.	G1260
Optimal cutting temperature compound	Japan, Sakura	4583
Programmable Timer Socket	China, GONEO	GND-1
Tissue Tech Prisma Plus	Japan, Sakura	20B2X00014000034
Tissue-Tek TEC 6 Embedding Module	Japan, Sakura	M01-021E-02
Total Cholesterol (TC) Assay Kit	China, Suzhou Grace Biotechnology Co., Ltd.	G0909W