PROJECT SUMMARY A common pathological state strongly associated with both obesity and aging is insulin resistance (IR) in which cells become resistant to the effects of insulin. IR is a hallmark of prediabetes, affecting a third of Americans. It also represents a major risk factor for type 2 diabetes mellitus, physical dysfunction, heart disease, and dementia. Other than exercise and diet, limited mechanism-based strategies exist to improve IR. Another shared feature of obesity and aging is accumulation of p21Cip1–highly-expressing (p21high) cells in various tissues. However, the roles of p21high cells in IR and physical dysfunction remain largely unknown. To examine the relationship between p21high cells and IR, we have generated and validated a novel “p21-Cre” transgenic mouse model containing a p21 promoter driving a Cre fused to a tamoxifen-inducible estrogen receptor (ER) element. This novel model enables us to monitor, kill or modulate p21high cells in vivo without affecting other cells. In our preliminary studies, we find that intermittent clearance of p21high cells in obese mice significantly alleviates IR, indicating that strategies targeting these cells could result in novel approaches for managing IR and metabolic dysfunction. Based on these findings, we will test our central hypothesis that targeting p21high cells will alleviate metabolic and physical dysfunction associated with obesity. We will use p21-Cre mouse models to examine the role (Aim 1) and underlying mechanism (Aim 2) of p21high cells in IR and physical dysfunction. We will also leverage powerful single cell transcriptomics (SCT) technology to reveal the heterogeneity and conserved transcriptomic features of these p21high cells in tissues with obesity. This project will have a broad impact on both aging and obesity research by determining how p21high cells contribute to IR. Using multiple in vivo models, coupled with the powerful approach of single cell transcriptomics, we expect to gain a comprehensive understanding of p21high cells (at both functional and expression levels) in vivo. Results from this work will also enable future testing of pharmacological interventions that eliminate these cells to treat not only metabolic dysfunction, but also a wide range of age-related diseases.