Modeling Adipocyte Insulin Resistance Using Mouse Subcutaneous Adipose Tissue-derived Stromal Vascular Fraction

Summary

This study presents an experimental protocol utilizing stromal vascular fraction derived from mouse subcutaneous adipose tissue and treated with dexamethasone to simulate insulin resistance. By retaining adipose tissue complexity, it offers a physiologically authentic platform for mechanistic research.

Abstract

Insulin resistance in adipose tissue is a central feature of metabolic disorders such as type 2 diabetes, yet many in vitro models rely on immortalized cell lines that incompletely reflect the cellular complexity of native adipose tissue. The goal of this protocol is to establish a reproducible and experimentally accessible method for modeling adipocyte insulin resistance using primary adipocytes derived from the stromal vascular fraction (SVF) of mouse subcutaneous adipose tissue. The protocol describes the isolation of SVF cells by enzymatic digestion, their adipogenic differentiation into lipid-laden mature adipocytes, and the subsequent induction of insulin resistance using dexamethasone. Insulin resistance is operationally defined and validated through functional and molecular readouts, including reduced insulin-stimulated glucose uptake and consumption, as well as decreased phosphorylation of key insulin signaling proteins in the PI3K-AKT pathway. By retaining SVF-derived cellular heterogeneity, this approach provides a primary-cell-based system that supports investigation of adipocyte insulin signaling under conditions that more closely approximate adipose tissue physiology than conventional cell lines. This protocol is intended for researchers seeking a standardized platform to study mechanisms of adipocyte insulin resistance or to evaluate metabolic interventions, while acknowledging that readouts reflect responses from a mixed SVF-derived culture rather than a purified adipocyte population.

Introduction

Type 2 diabetes mellitus (T2DM) has become one of the fastest-growing noncommunicable metabolic diseases globally, with the total number of patients now exceeding 537 million and projected to surpass 783 million by 2045¹. Its core pathophysiological features manifest as peripheral tissue insulin resistance (IR), accompanied by progressive β-cell dysfunction². The essence of IR lies in the significantly diminished biological effects triggered by physiologically concentrated insulin in target organs, leading to reduced glucose uptake, increased hepatic glucose output, and impaired lipolysis inhibition

Protocol

All animal experiments were approved by the Animal Experimental Ethics Committee of Xinjiang Medical University (Ethical Approval No.: IAGUC-20240228-41).

1. SVF isolation

1. Experimental animals: Use 4-6-week-old SPF-grade C57BL/6J mice weighing 9-11 g. House them at 22 ± 2 °C under a 12 h light-dark cycle with free access to food and water.
2. Sterile preparation: Autoclave all instruments and consumables at 121 °C for 30 min. Prior to experimentation, spray the laminar flow hood with 75% ethanol and irradiate with UV light for ≥15 min. Conduct all procedures within a Class II biosafet....

Results

SVF cells isolated from the iWAT of C57BL/6J mice exhibited a punctate suspension upon initial seeding in culture dishes. Approximately 40% of cells completed primary attachment after 18 h of seeding; by day 5 post-seeding, cells were fully spread, with confluence reaching 95% (Figure 1A,B). When cells reached confluence and exhibited a characteristic vortex-like arrangement, the differentiation medium was replaced. By day 4 of induction, numerous refractile lipid droplets w.......

Discussion

Extensive research indicates that SVF cell-based models offer unique value in simulating pathological features of adipose tissue. However, the lengthy construction cycle of these models, which typically requires approximately 14 days for tissue isolation, primary culture, induced differentiation, and Dex treatment, poses challenges for experimental continuity and cellular state stability. To address these challenges, this study adopted and optimized an experimental workflow centered on the following key measures: (1) Sel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm filter	Biosharp	BSF-XS-0232-01
2-NBDG	Beyotime	S0561M
3-Isobutyl-1-methylxanthine	Sigma	15879
4% Polyformaldehyde	Biosharp	BL1278A
70 μm cell strainer	Biosharp	BS-70-CS
Anti-AKT	Proteintech	10175
Anti-C/EBPα	Abclone	ab40764
Anti-FABP4	Cell Signaling Technology	2120S
Anti-pAKT (Ser473)	Proteintech	28731
Anti-PI3K	Bioss	10657R
Anti-pPI3K (p110)	Bioss	6417R
Anti-β-ACTIN	Proteintech	60008
BCA Protein Assay Kit	Thermo Fisher	23225
BODIPY 493/503	Invitrogen	D3922
BSA	Sigma	A7030
Cell climbing slices Biosharp	Biosharp	BS-14-RC
Cell Culture Dish, 100mm x 20mm	Corning	430167
Cell Culture Dish, 35mm x 10mm	Corning	430165
DAPI Solution	Solarbio	28718-90-3
Dexamethasone	Sigma	D2915
DMEM/F-12	Gibco	C11330500BT
Dulbecco’s PBS	Cytiva	SH30264.02
Eppendorf tube	Corning	MCT-175-C
FBS	Life-iLab	AC03L0555
Fluorescence microscope	Leica	DMi8
Glucose oxidase-peroxidase	Nanjing Jiancheng	F006-1-1
Goat anti-mouse IgG-HRP	Beijing Zhongshan Jinqiao	ZB-2305
Goat anti-rabbit IgG-HRP	Beijing Zhongshan Jinqiao	ZB-2301
GraphPad Prism	Version 10.0.0	/
Hepes	BioFroxx	1112GR025
Hochest	biosharp	BL803A
ImageJ	Version 1.54p	/
Insulin	Solarbio	18040
Laser scanning confocal microscope	Nikon	Ti-E
Mounting Medium antifading	Solarbio	S2100
Oil red O staining solution	Sigma	O0625-25G
Penicillin-Streptomycin	hyclone	SV30010
RIPA Buffer	Thermo Fisher	89901
Rosiglitazone	Sigma	R2408
RPMI 1640	VivaCell	C3010-0500
Skim Milk Powder	BioFroxx	1132GR500
TBS Buffer	Solarbio	T1060
Type II collagenase	Sigma	C6885