28 research outputs found
The role of the insulin receptor in mature human adipocytes
No full textObesity 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.(I) 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.(II) 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.(III) 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.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.List of scientific papersI. Dedic, 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. https://doi.org/10.1111/acel.14295II. Westerberg, L. J. S., Dedic, 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. https://doi.org/10.1371/journal.pone.0328136III. Dedic, 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
Victimization in Childhood as a Suicide Risk Factor in Adults
No full textObjectiveLittle is known about the predictive association between victimization among children and adolescents with depression and suicidal ideation in adults. The aim of our study was to examine whether victimization in childhood and early adolescence increases the likelihood of self-harming in adults.MethodThe sample consisted of 82 patients, 65 females and 17 males, aged 38.02 ± 11.05 years on average, hospitalized in Daily hospital. The juvenile victimization questionnaire (JVQ), defense style questionnaire (DSQ) and Beck depression inventory were applied in 48 patients following suicide attempt and in 34 patients who were on psychotherapeutic treatment due to various life crises not resulting in suicide attempt. According to indication, we excluded patients with psychosis, substances abuse and dementia. The examinees of both groups were matched by age, education, professional and marital status. Comparison of the patient groups was done by t-test.ResultsThe suicide attempters were depressed (Beck depression inventory 19.13 ± 10.20), using immature defense mechanisms (P < 0.005). The JVQ established statistically differences in two general areas: Peer and Sibling Victimization (P < 0.001) and sexual victimization (P < 0.05).ConclusionEven after controlling for lifetime factors known to increase the risk of suicidal behavior, adults who reported peer and sibling and sexual victimization in childhood were still more likely than other adults to attempt suicide later in life. Psychotherapists must understand that history of childhood victimization is important to put suicide attempt in a psychodynamic context which can help them in their work with patients’ psychotherapeutic crisis intervention following suicide attempt.Disclosure of interestThe author has not supplied his/her declaration of competing interest.</jats:sec
Optimization of project planning in the construction industry
No full textI varje projekt finns det brister i kommunikation som kan leda till att kostnad, tid och kvalitét påverkas. Varje projekt är en tillfällig organisation med olika företag och olika personer med varierande erfarenheter inom projektering. Detta medför att varje projekt är i sig unikt och hur alla ska kommunicera med varandra kan vara stora frågetecken under projekteringens förlopp. Denna rapport tittar närmare på vilka brister och svårigheter som kan uppkomma i projekteringskedet
Optimization of project planning in the construction industry
No full textI varje projekt finns det brister i kommunikation som kan leda till att kostnad, tid och kvalitét påverkas. Varje projekt är en tillfällig organisation med olika företag och olika personer med varierande erfarenheter inom projektering. Detta medför att varje projekt är i sig unikt och hur alla ska kommunicera med varandra kan vara stora frågetecken under projekteringens förlopp. Denna rapport tittar närmare på vilka brister och svårigheter som kan uppkomma i projekteringskedet
Author response
No full textCholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera.</p
Characterization of moose intestinal glycosphingolipids
No full textAs a part of a systematic investigation of the species-specific expression of glycosphingolipids, acid and non-acid glycosphingolipids were isolated from three small intestines and one large intestine of the moose (Alces alces). The glycosphingolipids were characterized by binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays, and by mass spectrometry. The non-acid fractions were complex mixtures, and all had glycosphingolipids belonging to the lacto- and neolactoseries (lactotriaosylceramide, lactotetraosylceramide, neolactotetraosylceramide, Galα3-Lex hexaosylceramide, and lacto-neolactohexaosylceramide), globo-series (globotriaosylceramide and globotetraosylceramide), and isogloboseries (isoglobotriaosylceramide). Penta- and heptaglycosylceramides with terminal Galili determinants were also characterized. Furthermore, glycosphingolipids with terminal blood group O determinants (H triaosylceramide, H type 2 pentaosylceramide, H type 1 penta- and heptaosylceramide) were characterized in two of the moose small intestines, and in the one large intestine, while the third small intestine had glycosphingolipids with terminal blood group A determinants (A tetraosylceramide, A type 1 hexa- and octaosylceramide, A dodecaosylceramide). The acid glycosphingolipid fractions of moose small and large intestine contained sulfatide, and the gangliosides GM3, GD3, GD1a, GD1b, and also NeuGc and NeuAc variants of the Sda ganglioside and the sialyl-globopenta/SSEA-4 ganglioside. In humans, the NeuAc-globopenta/SSEA-4 ganglioside is a marker of embryonic and adult stem cells, and is also expressed in several human cancers. This is the first time sialyl-globopentaosylceramide/SSEA-4 has been characterized in a fully differentiated normal tissue, and also the first time NeuGc-globopentaosylceramide has been characterized. © 2015 The Author(s)
High-power RF conditioning and 700 keV beam commissioning of the revised RFQ for the Frankfurt Neutron Source
No full text
