Novel aspects of metabolic regulation and inflammation in human adipocytes

The significance of adipose tissue and obesity has been recognized in numerous pathologies. However, the mechanisms behind this connection are not yet completely understood. The aim of this thesis was to investigate the roles of Liver X Receptor (LXR), V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) and Salt-inducible kinases (SIKs) in primary human adipocytes with focus on metabolic regulation and inflammation. Our overall hypothesis was that these factors may influence metabolism and inflammation in human white adipose tissue (WAT). In paper I, we studied the effects of LXR activation on human basal lipolysis. We show that stimulation of LXR using the LXR agonist GW3965 enhances spontaneous lipolysis, measured as glycerol released into culture medium. We demonstrate that hormone-sensitive lipase (HSL) and perilipin 1 (PLIN1), two proteins important for lipolysis, are downregulated on both mRNA and protein level by GW3965 treatment, which changes the morphology of the lipid droplet coating. Using chromatin immunoprecipitation we show that LXR binds to the proximal promoters of both LIPE (encoding HSL) and PLIN1. Furthermore, we demonstrate that LXRα is the isoform mediating increased lipolysis. PLIN1 knockdown in combination with GW3965 treatment indicate that PLIN1 downregulation was the main mechanism by which LXR activation increase glycerol release in human adipocytes. In paper II we examined the role of LXR in human adipocyte glucose uptake. We identified three proteins important for insulin signalling – AKT2, c-CBL associated protein (CBL) and caveolin 1 (CAV1) – to be downregulated by LXR activation. LXR activation reduced AKT2 phosphorylation and GLUT4 translocation to the plasma membrane, which resulted in reduced insulin-stimulated glucose uptake. In addition, AKT2 and SORBS1 (encoding CAP) expression in human WAT was inversely correlating with BMI and severity of insulin resistance. We concluded that LXR is an inhibitor of insulin-stimulated glucose uptake in human adipocytes, likely via transcriptional suppression of AKT2, SORBS1 and CAV1. In paper III, we investigated the role of MAFB in human WAT. MAFB mRNA expression was upregulated in obesity and downregulated after weight loss in adipose tissue from four different human cohorts. We found that the expression of MAFB is increased during adipocyte differentiation and that it mediates effects of TNFα-stimulation on lipid accumulation and basal lipolysis. In turn, knockdown of MAFB in adipocytes resulted in reduced expression of pro-inflammatory factors. MAFB was differentially regulated in response to TNFα treatment in adipocytes (downregulated) vs. monocytes (upregulated). MAFB was mainly expressed in adipose tissue macrophages and its expression correlated positively with macrophageand pro-inflammatory markers in WAT, and negatively with insulin-stimulated lipogenesis and stimulated lipolysis in human mature adipocytes, suggesting that it might be involved in the development of unhealthy glucoseand lipid metabolism. In paper IV, we aimed to identify the role of SIKs in human obesity and adipose tissue. We found that expression of SIK2 and SIK3 mRNA in human WAT was downregulated in obesity, upregulated after weight loss and correlated negatively with insulin resistance. SIK2 was the most highly expressed SIK isoform in human adipocytes. Both basal and insulin-stimulated glucose uptake were inhibited by SIK inhibitor. SIK inhibition also lead to reduced phosphorylation of histone deacetylase 4 (HDAC4) and CREB regulated transcription coactivator 1 (CRTC2), which may potentially mediate the attenuation of glucose uptake. Taken together, SIK2 may have a protective role in obesityinduced loss of insulin sensitivity. In summary, we have identified the roles of three factors in human adipocytes, all of which were previously uncharacterised in human WAT. We show that LXR enhances spontaneous lipolysis while inhibiting insulin-stimulated glucose uptake in human adipocytes, two features associated with an unfavourable metabolic profile. We also found that MAFB and SIK2 expression is regulated by obesity and inflammation. Finally, we demonstrate that MAFB is itself a regulator of inflammation in human adipocytes and that SIK2 positively regulates glucose uptake in human adipocytes. Our findings contribute to the understanding of the metabolic syndrome and its development in obesity. LIST OF SCIENTIFIC PAPERS I. Britta M. Stenson, Mikael Rydén, Nicolas Venteclef, Ingrid Dahlman, Annie M. L. Pettersson, Aline Mairal, Gaby Åström, Lennart Blomqvist, Victoria Wang, Johan W. E. Jocken, Karine Clément, Dominique Langin, Peter Arner, and Jurga Laurencikiene. Liver X Receptor (LXR) Regulates Human Adipocyte Lipolysis Journal of Biological Chemistry, 2011; vol 286; page 370-379 II. Annie M. L. Pettersson, Britta M. Stenson, Silvia Lorente-Cebrián, Daniel P. Andersson, Niklas Mejhert, Johan Krätzel, Gaby Åström, Ingrid. Dahlman, Alexander V. Chibalin, Peter Arner and Jurga Laurencikiene. LXR is a negative regulator of glucose uptake Diabetologia, 2013; vol 56; page 2044-2055 III. Annie M.L. Pettersson*, Juan R. Acosta*, Johan Krätzel, Britta Stenson, Lennart Blomqvist, Nathalie Viguerie, Dominique Langin, Peter Arner and Jurga Laurencikiene. MAFB as a novel regulator of human adipose tissue inflammation Manuscript * Shared first authorship IV. Johanna Säll, Annie M.L. Pettersson, Emma Henriksson, Fredrik Linder, Yuedan Zhou, Ola Hansson, Daniel P. Andersson, Mikael Ekelund, Eva Degerman, Jurga Laurencikiene and Olga Göransson. Salt-inducible kinase 2 (SIK2) is downregulated in obesity and is required for glucose uptake in human adipocytes Manuscript