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

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.