Single-cell profiling reveals the origin of phenotypic variability in adipogenesis

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Phenotypic heterogeneity within clonal cell populations is common but poorly understood.
• Non-mutative factors contributing to cell variability during differentiation require investigation.

Purpose of the Study:

• To investigate non-mutative causes of phenotypic heterogeneity during 3T3-L1 cell differentiation into adipocytes.
• To elucidate the role of insulin signaling in lipid droplet formation variability.

Main Methods:

• Employed single-cell analysis using coherent anti-Stokes Raman scattering microscopy and flow cytometry.
• Simultaneously visualized adipogenic gene expression, insulin signaling, and lipid droplet accumulation.

Main Results:

• Adipogenic gene expression (PPARgamma, C/EBPalpha, aP2, LP2) occurred in all differentiating cells.
• Lipid droplet formation varied significantly, indicating gene expression alone is insufficient.
• Insulin signaling pathway kinetics (sensitivity, kinase activity, glucose import, insulin degradation) dictated lipid droplet accumulation.
• Prolonged insulin stimulation enhanced lipid droplet formation in all cells.

Conclusions:

• Cell-to-cell variability in adipogenesis stems from differences in insulin signaling cascade responses.
• The kinetics of insulin signaling, not just gene expression, drive phenotypic heterogeneity in adipogenesis.
• Single-cell profiling offers insights into the origins of variability in drug-inducible adipogenesis.