The role of the insulin receptor in mature human adipocytes

<p dir="ltr">Obesity is a global health crisis, affecting millions of individuals worldwide and imposing substantial burdens on healthcare systems. The prevalence of obesity has reached epidemic proportions, with roughly 12.5% of adults worldwide classified as obese. Obesity is closely linked to the development of insulin resistance, type 2 diabetes (T2D) and other metabolic disorders. While adipose tissue plays a crucial role in metabolic homeostasis, the precise molecular mechanisms underlying insulin resistance in adipocytes remain incompletely understood. This thesis presents three interconnected studies that provide new insights into adipocyte biology, with a focus on developing advanced methodologies to investigate insulin receptor (INSR) dynamics in the context of obesity and metabolic dysfunction.</p><p dir="ltr">(<b>I</b>) The first study introduces a novel approach for detecting cellular senescence using reflected light microscopy. Senescence is an important cellular program involved in development, tissue repair, cancer and aging. More recently, a premature, metabolically induced senescence has been described in multiple tissues and is associated with obesity and T2D. However, characterizing and quantifying senescent cells at the single-cell level has been challenging, particularly in large primary cells like human adipocytes. This study introduces a technique that utilizes reflected light for accurate senescence-associated beta- galactosidase (SABG) staining measurements in adipocytes. By leveraging confocal microscopy to detect X-gal crystals using reflected light, this approach achieves superior sensitivity over traditional brightfield techniques. It allows for the capture of all X-gal precipitates in SABG-stained samples and can detect diverse staining patterns. The method can be integrated with immunofluorescence and is compatible with primary mature adipocytes from both human and mouse, as well as differentiated 3T3-L1 cells. Importantly, this technique outperforms western blot analysis for detecting and quantifying senescence in mature human adipocytes. This methodological advancement provides a powerful tool for investigating the role of cellular senescence, or other metabolic phenotypes, in adipose tissue dysfunction at a single-cell level.</p><p dir="ltr">(<b>II</b>) The second study addresses methodological challenges in protein quantification using western blotting, a crucial technique for analyzing protein expression in adipocyte research. This study investigates the robustness of housekeeping proteins and total protein (TP) as normalization references for western blotting in primary mature human adipocytes. TP exhibited the lowest variance among technical replicates and was superior as a normalization reference for glucose transporter 4 (GLUT4), a key protein involved in insulin- stimulated glucose uptake. TP also demonstrated the closest alignment with expected values when loaded as a protein gradient and showed lower intra- and inter-patient variability compared to housekeeping proteins across metabolically similar patients. The study concludes that TP normalization is the preferred method for reliable protein expression analysis in primary mature human adipocytes. This methodological improvement enhances the accuracy of protein quantification in adipocyte research, which is crucial for understanding the nuanced molecular changes associated with obesity and insulin resistance.</p><p dir="ltr">(<b>III</b>) These two methodological advancements (I, II) serve as foundational tools for the third and principal study, which investigates INSR and insulin-like growth factor 1 receptor (IGF1R) expression and dynamics in primary mature human adipocytes from individuals with various metabolic profiles. By leveraging the high-precision confocal imaging technique (I), plasma membrane INSR negative (INSR-) adipocytes were observed primarily in obese hyperinsulinemic (OB/HI) males at a single-cell resolution. Further analysis of protein lysates using the optimized western blot technique (II) revealed that although total INSR levels remained unchanged, phosphorylated INSR and the activation of downstream signaling pathways were elevated in OB/HI males compared to non-obese (NOB) male controls. Notably, these patterns were sex-specific, with significant correlations between the fraction of INSR+ adipocytes and markers of insulinemia observed exclusively in obese males. In vitro differentiated adipocytes partially recapitulated the INSR- phenotype, and subsequent mass spectrometry indicated alterations in the endosomal trafficking proteins, providing a potential mechanistic explanation for the observed INSR- adipocytes.</p><p dir="ltr">Together, these three studies advance our understanding of adipocyte biology and explore the molecular mechanisms underlying obesity-associated metabolic dysfunction. The novel senescence method and improved western blotting normalization strategy serve as powerful tools for investigating adipocyte biology with enhanced precision and accuracy, enabling the exploration of INSR dynamics and revealing a potential new mechanism of insulin resistance in mature human adipocytes. These insights open new avenues for targeted therapies, and future research based on these methodological and mechanistic advances may improve strategies for preventing and treating obesity-related metabolic dysfunction in adipose tissue. Moreover, the techniques developed here may find broader application in studying other aspects of adipocyte biology and disease mechanisms beyond insulin resistance, T2D, and obesity.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Dedic, B.</b>, Westerberg, L., Mosqueda Solís, A., Dumont, K. D., Ruas, J. L., Thorell, A., Näslund, E., & Spalding, K. L. (2024). Senescence detection using reflected light. Aging cell,23(11), e14295. <a href="https://doi.org/10.1111/acel.14295">https://doi.org/10.1111/acel.14295</a></p><p dir="ltr">II. Westerberg, L. J. S., <b>Dedic, B.</b>, Näslund, E., Thorell, A., & Spalding, K. L. (2025). Superior normalization using total protein for western blot analysis of human adipocytes. PloS one, 20(7), e0328136. <a href="https://doi.org/10.1371/journal.pone.0328136">https://doi.org/10.1371/journal.pone.0328136</a></p><p dir="ltr">III. <b>Dedic, B.</b>, Westerberg, L. J. S., Cutler, H. B., Thorell, A., James, D. E., & Spalding, K. L. Plasma Membrane Insulin Receptor-Negative Adipocytes in Hyperinsulinemic Males Exhibit Persistent Metabolic Activation. [Manuscript]</p>