Studies on novel interactors in human adipose tissue

Abstract: Obesity is associated with a mild inflammation and altered protein secretion in adipose tissue. These changes have been suggested to contribute to the development of obesity-associated disorders including, insulin resistance, dyslipidemia, type 2 diabetes and cardiovascular disease. The aim of this thesis was to identify novel factors expressed in the adipose tissue that could be important in the development of obesity and obesity-associated disorders. In paper I we studied the transcriptional control of the human CIDEA gene and the effects of TNFα. CIDEA has previously been shown to be differentially expressed in lean and obese subjects and repressed by tumor necrosis factor α (TNFα), however the human CIDEA promoter has not been investigated. Reporter assays demonstrated that the minimal transcriptional activity of the human CIDEA promoter was confined to a region 97 bp upstream of the transcriptional start site (TSS). TNFα attenuated the transcriptional activity of CIDEA and this regulation was confined to a region between 244 and 123 bp upstream of the TSS. Electrophoretic mobility shift assays and mutational analysis suggested that the regulation by TNFα is mediated by a nuclear factor (NF)-κB binding site. In paper II and III we investigated the function of the twist1 gene in human white adipocytes and its relationship with obesity. Twist1 is involved in various processes, such as bone development, cancer progression, inflammation and brown adipose tissue function, but the role in white adipose tissue is not known. In paper II we assessed the expression of twist1 in human white adipose tissue, liver, skeletal muscle and pancreas as well as in different cell types in adipose tissue. Twist1 mRNA expression was significantly higher in adipose tissue compared to the other organs and within adipose tissue; the expression was highest in the adipocyte fraction. In vitro silencing of twist1 by RNA interference was used to investigate the role of twist1 in lipid turnover and inflammation in human adipocytes. In contrast to mice where twist1 reduces fatty acid oxidation, reduction of twist1 expression in human in vitro differentiated adipocytes lead to a reduction in fatty acid oxidation. Furthermore, expression of the rate-limiting enzyme for fatty acid oxidation, carnitine palmitoyl transferase 1, was also reduced when twist1 was silenced, suggesting that twist1 is required for fatty acid oxidation in human white adipocytes. We were also able to demonstrate that twist1 regulates the expression of inflammatory proteins, including interleukin (IL) -6 and monocyte chemoattractant protein 1. Chromatin immunoprecipitation demonstrated direct binding to sequences in the promoter regions of these genes. In paper III, we further explored the expression of twist1 in non-obese and obese subjects and correlated the expression with different clinical parameters of insulin resistance. Interestingly and in contrast to studies in mice, low twist1 expression associated with a higher BMI and a more adverse clinical profile, characterized by higher HOMA-IR values, large adipocytes and an increased secretion of pro-inflammatory factors. We also assessed the effect of twist1 silencing on TNFα-induced cytokine and chemokine expression and secretion in human adipocytes. Twist1 knockdown accentuated the pro-inflammatory effects of TNFα- suggesting that twist1 may have a protective role in adipose inflammation. In conclusion, we have defined the minimal promoter needed for transcription of human CIDEA and a region in the promoter regulated by TNFα. We have also demonstrated that twist1 is expressed in white adipocytes and that low expression of twist1 associates with high BMI, markers of insulin resistance and an increased secretion of pro-inflammatory factors. Additionally, silencing of twist1 in human in vitro differentiated adipocytes demonstrates a role for this transcription factor in fatty acid oxidation and inflammation. We hypothesize that low twist1 expression in obesity renders the adipose tissue more vulnerable to the pro-inflammatory effects of TNFα, which could be of importance in the development of obesity-associated insulin resistance and type 2 diabetes.