Insulin Signaling in Human Adipocytes a Systems Biology Approach

Obesity and a sedentary life style are associated with type 2 diabetes, a disease starting with insulin resistance in the adipose tissue, which spreads to the whole body. Despite large research efforts to understand the insulin signaling system, there is little knowledge of the mechanisms behind insulin resistance and type 2 diabetes developments. We have herein focused on the insulin signaling in adipocytes, elucidating mechanisms for early signaling. We have also modeled isolated adipocytes and data from the in vivo, whole bodysituation, concurrently. We also mapped and quantitatively described differences in the insulin signaling of adipocytes from type 2 diabetics and non-diabetics. In paper I we show that neither insulin degradation, receptor internalization, nor feedback signals can as separate explanations cause the overshoot in tyrosine phosphorylation of IRS1, while an endocytosis-dependent feedback mechanism explains all available data. In paper II we show that it is not possible to scale up the experimentally determined glucose uptake by isolated human adipocytes to match the glucose uptake profile of the whole adipose tissue in vivo. Other insulin effects need to be accounted for. In paper III we show that attenuation of the positive feedback to serine 307 phosphorylation of IRS1 can explain the insulin resistance in the insulin signaling in adipocytes seen in type 2 diabetes. However, to fully explain both the signaling and the glucose uptake, a reduction in the amount of Glut4 is also needed

LIST OF ORIGINAL PAPERS.......................................................................... 8 PAPERS NOT INCLUDED IN THESIS............................................................ 10

ISBN: 978-91-7519-789-0 ISSN: 0345-0082 Published articles were reprinted with permission from the copyright holder. During the course of the research underlying this thesis, Cecilia Brännmark was enrolled in Forum Scientium, a multidisciplinary doctoral program at Linköping University, Sweden. Den vinner som är trägen, den förlorar som ger upp. Lars WinnerbäckABSTRACT..................................................................................................

Proteomic profiling identifies novel inflammation-related plasma proteins associated with ischemic stroke outcome

Abstract Background The inflammatory response to cerebral ischemia is complex; however, most clinical studies of stroke outcome focus on a few selected proteins. We, therefore, aimed to profile a broad range of inflammation-related proteins to: identify proteins associated with ischemic stroke outcome that are independent of established clinical predictors; identify proteins subsets for outcome prediction; and perform sex and etiological subtype stratified analyses. Methods Acute-phase plasma levels of 65 inflammation-related proteins were measured in 534 ischemic stroke cases. Logistic regression was used to estimate associations to unfavorable 3-month functional outcome (modified Rankin Scale score > 2) and LASSO regressions to identify proteins with independent effects. Results Twenty proteins were associated with outcome in univariable models after correction for multiple testing (FDR < 0.05), and for 5 the association was independent of clinical variables, including stroke severity (TNFSF14 [LIGHT], OSM, SIRT2, STAMBP, and 4E-BP1). LASSO identified 9 proteins that could best separate favorable and unfavorable outcome with a predicted diagnostic accuracy (AUC) of 0.81; three associated with favorable (CCL25, TRAIL [TNFSF10], and Flt3L) and 6 with unfavorable outcome (CSF-1, EN-RAGE [S100A12], HGF, IL-6, OSM, and TNFSF14). Finally, we identified sex- and etiologic subtype-specific associations with the best discriminative ability achieved for cardioembolic, followed by cryptogenic stroke. Conclusions We identified candidate blood-based protein biomarkers for post-stroke functional outcome involved in, e.g., NLRP3 inflammasome regulation and signaling pathways, such as TNF, JAK/STAT, MAPK, and NF-κB. These proteins warrant further study for stroke outcome prediction as well as investigations into the putative causal role for stroke outcome

A Hierarchical Whole-body Modeling Approach Elucidates the Link between in Vitro Insulin Signaling and in Vivo Glucose Homeostasis

Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.This research was originally published in: Elin Nyman, Cecilia Brännmark, Robert Palmér, Jan Brugård, Fredrik Nyström, Peter Strålfors and Gunnar Cedersund, A Hierarchical Whole-body Modeling Approach Elucidates the Link between in Vitro Insulin Signaling and in Vivo Glucose Homeostasis, 2011, Journal of Biological Chemistry, (286), 29, 26028-26041. http://dx.doi.org/10.1074/jbc.M110.188987 © the American Society for Biochemistry and Molecular Biology http://www.asbmb.org/</p

Intracellular K(+) Determination With a Potentiometric Microelectrode Based on ZnO Nanowires

The fabrication and application of an intracellular K(+)-selective microelectrode is demonstrated. ZnO nanowires with a diameter of 100-180 nm and a length of approximately 1.5. m are grown on a borosilicate glass microcapillary. The ZnO nanowires were coated by a K(+)-ionophore-containing membrane. The K(+)-selective microelectrode exhibited a K(+)-dependent potentiometric response versus an Ag/AgCl reference microelectrode that was linear over a large concentration range (25 . M-125 mM) with a minimum detection limit of 1 . M. The measured K(+) concentrations in human adipocytes and in frog oocytes were consistent with values of K(+) concentrations reported in the literature. The sensor has several advantages including ease of fabrication, ease of insertion into the cells, low cost, and high selectivity features that make this type of sensor suitable to characterize physiologically relevant ions within single living cells.©2011 IEEE. Personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works must be obtained from the IEEE. Syed Usman Ali, Muhammad Asif, Alimujiang Fulati, Omer Nur, Magnus Willander, Cecilia Brännmark, Peter Strålfors, Ulrika Englund, Fredrik Elinder and Bengt Danielsson, Intracellular K(+) Determination With a Potentiometric Microelectrode Based on ZnO Nanowires, 2011, IEEE transactions on nanotechnology, (10), 4, 913-919. http://dx.doi.org/10.1109/TNANO.2010.2089696</p

Gene expression.

<p>Gene expression in preadipocytes (denoted Undiff) and differentiated human preadipocytes in 2D aligned and random fibers Analyzed data is ΔCt values from technical duplicates. A) PCA analysis of the variation between samples. B) A heatmap was created from mean expression levels in three donors, and the expression profiles were analyzed with hierarchical clustering (UPGMA method with Euclidian method for distance measure).</p