Development and application of advanced 3T3-L1 adipocyte culture models to enhance drug discovery

Vita, Luca (2026) Development and application of advanced 3T3-L1 adipocyte culture models to enhance drug discovery. PhD thesis, University of Glasgow.

Abstract

Obesity and type 2 diabetes are among the most pressing health challenges worldwide, driven by insulin resistance and the failure of adipose tissue to maintain metabolic function. Adipocytes not only store and release energy but also function as endocrine regulators of systemic homeostasis. However, the in vitro models most commonly used to study adipocytes remain limited, with conventional two-dimensional (2D) monolayers often lacking the architecture and complexity of adipose tissue. At the same time, drug discovery efforts require models that are both physiologically relevant and compatible with scalable, quantitative readouts. This thesis aimed to develop advanced adipocyte platforms that integrate improved culture formats with genetically encoded biosensors, enabling direct interrogation of receptor signalling and insulin-regulated glucose transport.

Initial work established and validated the differentiation of 3T3-L1 preadipocytes in 2D culture, confirming lipid accumulation, expression of canonical adipogenic markers, and functional outputs including lipolysis and insulin-stimulated glucose uptake. Building on this, a scaffold-free spheroid system was implemented to capture elements of adipose architecture. These 3D cultures underwent adipogenic differentiation, upregulated adipocyte genes, and preserved functional responses, demonstrating that spheroids can reproduce features of adipose biology while remaining experimentally tractable.

A conformational intramolecular BRET sensor for FFA4 was stably expressed in 3T3-L1 adipocytes. The sensor reports receptor active-state transitions in real time, enabling quantitative comparison of endogenous fatty acids and TUG-891, and it revealed ligand-dependent kinetics with a modest basal tone consistent with endogenous lipid agonists. The biosensor line retained adipogenic competence and the signal was maintained after differentiation in both 2D monolayers and 3D spheroids, allowing direct assessment of culture-context effects on FFA4 pharmacology.

A luminescent GLUT4-HiBiT translocation biosensor was designed to measure insulin signalling in living cells. Introduced into 3T3-L1 cells, the line was characterised and retained adipogenic competence; the sensor reported insulin-dependent, concentration-graded GLUT4 surface delivery in both undifferentiated and differentiated states. The assay was then miniaturised and automated for screening, and a pilot high-throughput screen was completed under a submaximal insulin challenge. Although this library produced no clear insulin-sensitising hits, the workflow ran reliably at scale and establishes a screen-ready platform for larger or targeted libraries and follow-up studies.

Together, these studies show how combining established 2D monolayers, emerging 3D spheroid formats, and genetically encoded biosensors can generate adipocyte models that are both physiologically relevant and discovery-ready. By providing live, quantitative readouts of receptor activation and insulin-regulated glucose transport, this work advances the toolkit available for metabolic disease research. The platforms developed here bridge the gap between reductionist culture systems and complex in vivo physiology, offering new opportunities for mechanistic insight and for accelerating the identification of therapeutic strategies to combat insulin resistance and type 2 diabetes.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QR Microbiology > QR180 Immunology; R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Hudson, Dr Brian, Dalby, Professor Matthew and Berry, Dr Catherine
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-85773
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 20 Feb 2026 11:55
• Last Modified: 21 Feb 2026 12:56
• Thesis DOI: 10.5525/gla.thesis.85773
• URI: https://theses.gla.ac.uk/id/eprint/85773

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