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 inhibition³. Adipose tissue is one of insulin's most critical target organs. Obesity causes fat cells to become insensitive to insulin, reducing glucose uptake, inhibiting triglyceride synthesis, while simultaneously increasing lipolysis and releasing more free fatty acids, thereby exacerbating systemic insulin resistance^4,5.

In physiological conditions, insulin activates the PI3K-AKT pathway to translocate glucose transporter 4 (GLUT4) to the cell membrane, promoting glucose uptake and triglyceride synthesis in adipocytes. Simultaneously, insulin inhibits the activity of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) on the surface of lipid droplets, thereby blocking lipolysis⁶. When IR occurs, impaired insulin signaling weakens lipolysis inhibition, leading to massive release of free fatty acids (FFAs) into the bloodstream. This results in massive release of FFAs into the bloodstream, causing ectopic triglyceride deposition in organs such as the liver, skeletal muscle, and pancreas. This induces lip toxicity and chronic inflammation, ultimately contributing to the onset and progression of T2DM⁷. Therefore, establishing a mature adipocyte model that reproduces in vivo IR characteristics is crucial for elucidating the molecular mechanisms of IR and developing novel intervention strategies.

Currently, the 3T3-L1 mouse embryonic fibroblast cell line has become a classic tool for in vitro studies of adipocyte IR due to its short induction cycle and good reproducibility^8,9. However, the embryonic origin of 3T3-L1 cells, their uniform genetic background, and susceptibility to phenotypic drift after long-term passage result in significant differences in metabolic behavior compared to adult in situ adipocytes, limiting their translational medical value^10,11. In contrast, the stromal vascular fraction (SVF) isolated from rodent subcutaneous adipose tissue is rich in multipotent adipose stem cells (ASCs). Mature adipocytes differentiated from these ASCs exhibit morphological, functional, and transcriptional characteristics that more closely resemble those of native white adipocytes than the widely used 3T3-L1 cell line. This makes SVF-derived adipocytes a complementary model for studying insulin signaling and resistance in a context that better approximates primary tissue complexity¹².

This protocol is most suitable for researchers aiming to study insulin action in a primary adipocyte model that retains greater tissue heterogeneity than immortalized cell lines. However, users should note that SVF is a mixed population containing endothelial and immune cells; therefore, readouts reflect the behavior of a culture where adipocytes are a major but not exclusive component. This model may be less suitable for studies requiring a pure, synchronized adipocyte population.

This study proposes a protocol for constructing an insulin-resistant cell model using mature adipocytes derived from SVF. The protocol details the entire process of SVF isolation, culture, differentiation, and induction of insulin resistance, successfully mimicking the insulin-resistant state of adipocytes in vivo. Model evaluation will be conducted at the levels of glucose consumption, cellular morphology, and molecular mechanisms. This provides a technical foundation for investigating the molecular mechanisms underlying insulin resistance in adipocytes.

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 biosafety cabinet. Wear sterile gloves and masks.
3. Adipose tissue harvesting: Euthanize mice by carbon dioxide inhalation followed by cervical dislocation. Immediately immerse in 75% ethanol for 10 min for surface disinfection. Using sterile ophthalmic scissors, make a circular incision along the midline of the chest and abdomen. Perform blunt dissection to expose the bilateral inguinal white adipose tissue, located on the inner thigh and extending toward the testicular region in a V shape. Carefully remove any visible lymph nodes. Collect the adipose tissue into pre-chilled PBS and perform three rapid rinses.
4.  Collagenase digestion: Immediately transfer the washed adipose tissue to a sterile culture dish and mince into 1-2 mm³ fragments. Add 0.2% Type II collagenase (dissolved in Dulbecco's PBS, pH 7.4, pre-warmed to 37 °C) at a volume 1.5x-2x that of the tissue. Incubate at 37 °C on a constant-temperature shaker at 100 rpm for 10 min. Gently invert every 3 min to avoid over-digestion.
5. Termination and filtration: Upon completion of digestion, add an equal volume of pre-warmed complete medium (DMEM/F-12 + 10% FBS + 1% penicillin-streptomycin) at 37 °C to terminate the reaction. Gently pipette 5x-8x using a 1 mL pipette tip. Filter through a 70 µm cell strainer to remove undigested tissue fragments and mature adipocytes.
6. Centrifugation and washing: Centrifuge the filtrate at 300 x g for 5 min at 25 °C. Carefully discard the supernatant containing mature adipocytes. Resuspend the pellet in complete medium. Centrifuge again at 300 x g for 5 min at 25 °C. Repeat this washing step 2x to remove residual collagenase and any remaining mature adipocytes.
7. Seeding and culture: Resuspend the final pellet in 1 mL of complete medium. Seed at a density of 4 mice inguinal fat samples per 10 cm cell culture dish. Incubate at 37 °C with 5% CO₂. Perform the first medium change after 24 h to remove non-adherent cells. Subsequently, change the medium every 48 h. Passage the cells when they reach 80%-90% confluence.
   NOTE: Do not passage cells when confluence exceeds 90%, as the cells have reached contact inhibition and their proliferative capacity is diminished.

2. Preparation of type II Collagenase working solution

1. Reagents and consumables: Obtain type II collagenase (activity ≥ 120 U/mg); BSA (Fatty-acid-free ≥ 98%); HEPES; Dulbecco's PBS (containing Ca²⁺ 1.8 mmol/L, Mg²⁺ 0.9 mmol/L), pH 7.2-7.4; 0.22 µm sterile low-protein-binding filter.
2. Preparation of 0.2% type II Collagenase solution: Weigh 60 mg type II Collagenase powder, 800 mg BSA, and 48 mg HEPES into a 50 mL centrifuge tube. Dilute to 40 mL with Dulbecco's PBS. Adjust pH to 7.4 ± 0.1 using 1 M NaOH or HCl. Sterilize by filtration through a 0.22 µm filter. Aliquot 5 mL per tube and store at -20 °C. Avoid repeated freeze-thaw cycles.
3. Induce differentiation when cells reach 100% confluence post-passage. The optimal differentiation state is indicated by cells exhibiting a vortex-like morphology. Use induction solution I for the first 48 h, then switch to induction solution II (Table 1).
4. Confirm successful differentiation by the following criteria: by day 4 of induction, oil red O staining reveals lipid droplet accumulation in most cells (approximately 50%), accompanied by a statistically significant increase (p < 0.05) in the expression of key adipogenesis markers.

3. Establishment of the Insulin resistance model

1. Select differentiation day 4 as the starting point for inducing insulin resistance. Confirm under the microscope that all cells exhibit a morphological transition from spindle-shaped to round, with the cytoplasm of most cells filled with high-refractive-index lipid droplets.
2. Replace the medium with induction medium II containing 1 µmol/L Dex and continue culturing for 48 h (Table 1). Following this procedure, consider the insulin-resistant adipocyte model established. Note that these cells exhibit no further morphological alterations. Use these cells directly for glucose uptake/consumption assays and for the detection of insulin signaling pathway proteins.
3. Criteria for successful insulin resistance model establishment: Following 48 h of dexamethasone treatment, confirm that glucose uptake capacity in most cells stimulated with 100 nM insulin decreases by approximately 40%. Also, confirm a statistically significant reduction (p < 0.05) in the phosphorylation levels of key insulin signaling pathway proteins.
   NOTE: This process requires continuous addition of induction maintenance medium II .

4. Detection of residual glucose in culture supernatant

1. Collect cell culture supernatants at the designated time point. Remove cells by centrifugation at 4 °C and 300 x g for 5 min.
2. Measure glucose concentrations in the supernatants using the glucose oxidase-peroxidase (GOD-POD) assay kit. Calculate concentrations using the formula:
   Glucose concentration (mmol/L) = (A_(sample) - A_(blank)) / (A_(standard) - A_(blank)) * C_(standard)
   Glucose consumption (mmol/L) = Glucose concentration in cell-free medium (mmol/L) - Glucose concentration in cell supernatant (mmol/L)
   Normalized Glucose Consumption (mmol/mg protein) = Raw Glucose Consumption (mmol/L) / Total Cellular Protein Concentration (mg/L)

5. Western blot

1. Stimulate cells at the designated time point with 100 nM insulin in complete medium for 10 min at 37 °C to activate the PI3K-AKT pathway. Subsequently, add 200 µL of cell lysis buffer to each 3.5 cm cell culture dish. Incubate on ice for 30 min. Centrifuge at 13,040 x g for 10 min at 4 °C. Transfer the supernatant to a new 1.5 mL microcentrifuge tube. Quantify the protein concentration using the BCA working solution.
2. Load samples onto an 8% SDS-PAGE gel. Run the gel at 80 V until the samples reach the stacking/separating gel interface. Increase the voltage to 120 V and run until the marker bands are fully resolved. Perform transfer at a constant current of 200 mA for 2 h.
3. Incubate the membrane at room temperature with 5% skim milk or BSA for 2 h for blocking. Wash the membrane 3x with TBST. Subsequently, incubate with primary antibody overnight at 4 °C. Primary antibodies (Dilution): AKT: 1:1000, p-AKT (Ser473): 1:1000, β-Actin: 1:1000, PI3K p110α: 1:1000, Phospho-PI3K p110: 1:1000, C/EBPα: 1:2000, FABP4: 1:2000.
4. Wash the membrane 3x with TBST. Subsequently, incubate with HRP-conjugated secondary antibody at room temperature for 1 h. Secondary Antibodies (Dilution): Goat anti-mouse IgG-HRP: 1:5000, Goat anti-rabbit IgG-HRP: 1:5000.
5. Detect the signal using ECL chemiluminescence. Visualize and record the experimental results using a Western blot imager.
6. Analyze the grayscale values of the resulting bands using ImageJ software.

6. Glucose uptake staining assay

1. Starve the cells at the two specified time points separately for 12 h using RPMI 1640 containing 1% FBS to eliminate interference from basal glucose.
2. Following 10 min of stimulation with 100 nM insulin, add 1 mL of 1x 2-NBDG solution to each well. Incubate the plate at 37 °C for 1 h, protected from light.
3. Wash the cells 2x-3x. Add 1 mL of ready-to-use Hoechst staining solution to each well. Incubate the plate at 37 °C in the dark for 10 min. Add the staining solution slowly along the side wall of the culture vessel, avoiding direct contact with the cell layer to prevent detachment of cells.
4. Wash the cells 2x-3x, then add PBS. Capture fluorescent images of the cells using an inverted fluorescence microscope.
5. Perform quantitative analysis of the images using ImageJ software. Use ImageJ to separate each channel, isolating the glucose uptake signal (green channel) from the nuclear fluorescence signal (blue channel).
6. Convert all images into 8-bit grayscale images. Apply the Default automatic thresholding algorithm to define regions of positive signal.
7. Measure the average fluorescence intensity within these designated regions. Normalize the glucose uptake fluorescence signal by dividing the average fluorescence intensity of the green channel by the corresponding average fluorescence intensity of the blue channel.

7. Oil red O staining

1. Dilute the Oil Red O stock solution with RNase-free water at a 3:2 ratio. Filter the solution sequentially through filter paper and a 0.22 µm filter to prepare the working solution.
2. Aspirate the cell culture medium and gently wash the cells 2x with PBS. Add 4% paraformaldehyde to each well and fix the cells at room temperature for 30 min.
3. Add the fixative and wash the cells with double-distilled water. Add 1 mL of Oil Red O working solution to each well. Incubate the plate at room temperature in the dark for 25 min.
4. After staining, aspirate the staining solution. Thoroughly wash the cells with distilled water to remove background and stain artifacts. Add 1 mL of distilled water to cover the cells. Immediately capture brightfield images using an inverted microscope.

8. BODIPY

1. Dilute the BODIPY stock solution with sterile water at a 1:200 volume ratio to prepare the working solution.
2. Wash the cell coverslips with sterile PBS. Add 30 µL of 4% paraformaldehyde solution to each coverslip and fix at room temperature for 30 min.
3. Aspirate the fixative and wash 3x with PBS. Add 30 µL of BODIPY working solution to each cell coverslip. Incubate at room temperature in the dark for 30 min.
4. Wash the coverslips 3x with PBS. Add 30 µL of DAPI stain to each coverslip. Incubate at room temperature in the dark for 10 min to label cell nuclei.
5. Wash the coverslips three more times with PBS. Proceed with mounting: Add 10 µL of anti-fade mounting medium onto a clean microscope slide. Using forceps, gently invert the cell coverslip (cell side down) onto the mounting medium. Allow the mountant to dry in the dark.
6. Immediately capture images using a laser scanning confocal microscope.

9. Statistical analysis

1. Each experiment included three biological replicates. Analyze data using GraphPad Prism 10.0 software. Present quantitative data, meeting the criteria for normal distribution as mean ± standard deviation (mean ± SD). For two groups satisfying normality and homogeneity of variance, apply the independent samples t-test. For multiple groups, perform one-way ANOVA, p < 0.05 is considered statistically significant.

10. Safety and waste disposal

1. Handling of animal tissues
   1. Perform all procedures involving primary cells in a Class II biosafety cabinet under sterile conditions.
   2. Classify all animal tissue waste, unused organs, and contaminated consumables as biohazardous waste. Place such waste in designated biohazard bags, processed through autoclaving, and finally disposed of.
2. Handling of Dexamethasone
   1. Wear gloves, medical masks, and lab coats when weighing Dex powder. Conduct solution preparation within a chemical fume hood.
   2. Collect waste contaminated with Dex separately and dispose of it as chemical waste.
3. Handling of biohazardous materials
   1. Thoroughly disinfect work surfaces and the interior of biosafety cabinets with 70% ethanol before experimental procedures can commence.
   2. Ensure all liquid waste generated from cell culture is autoclaved to render it non-infectious before disposal.

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