Adipose tissue inflammation, hepatic fat accumulation and insulin resistance

Insulin resistance is the central feature of a variety of metabolic disorders, such as type 2 diabetes and the metabolic syndrome. The incidence of these disorders, often together with obesity, is currently increasing throughout the world. Insulin resistance occurs together with obesity, but also when adipose tissue is absent, indicating an important role of normal adipose tissue function for the maintenance of insulin sensitivity. Also, an accumulation of fat in the liver is observed together with both the excess and the absence of adipose tissue and insulin resistance. The interplay between the metabolic disturbances in adipose tissue and accumulation of fat in the liver which may underlie the development of insulin resistance is not well understood. Therefore, the aim of this thesis is to improve our knowledge of the molecular pathways underlying metabolic disturbances in adipose tissue and hepatic fat accumulation as well as the interplay between them in order to better understand the development of insulin resistance and related disorders in man. Twenty type 2 diabetes patients were treated with either rosiglitazone (an insulin sensitizer, an agonist of the transcription factor PPAR , which is predominantly expressed in adipose tissue) or metformin (an insulin sensitizer believed to act mainly in the liver). Rosiglitazone decreased liver fat content in these patients, while metformin did not. Biopsies from subcutaneous adipose tissue were taken before and after 16 weeks of treatment. Expression of 50 genes previously shown to be altered by TZDs in experimental models was measured. We found that rosiglitazone, but not metformin treatment, increased expression of genes involved in fatty acid synthesis and storage and in glucose uptake and decreased some macrophage and inflammation markers. This suggests that together with an improvement in insulin sensitivity and a decrease in liver fat content, rosiglitazone increases lipid storage and decreases inflammation within subcutaneous adipose tissue. In order to investigate changes in the liver when it becomes fatty, we analyzed gene expression in liver biopsies from 24 subjects with histologically determined liver fat content ranging from normal (n=8) to non-alcoholic hepatic steatosis (n=16). We found that expression of genes involved in fatty acid storage and inflammation was grater in livers of subjects with high liver fat content, indicating an alteration of hepatic gene expression towards an adipogenic and inflammatory profile upon excess fat accumulation. Next, we investigated whether adipose tissue is inflamed in subjects with increased liver fat content independently of the degree of obesity. Twenty obese, but otherwise healthy women with a wide range of liver fat contents were recruited and divided in normal liver fat (n=10) and high liver fat (n=10) groups. Biopsies from subcutaneous adipose tissue were analyzed by quantitative PCR, immunohistochemistry and lipidomic analysis. Expression of inflammatory markers was higher in the adipose tissue of women with high liver fat. Immunohistochemical analysis revealed increased macrophage infiltration in adipose tissue of women with high liver fat. These macrophages were mostly found surrounding dead adipocytes. Lipidomic analysis showed higher concentrations of long-chain TGs and some sphingomyelins and ceramides in the adipose tissue of women with high liver fat. In order to investigate the increase in ceramides in the inflamed adipose tissue of women with high liver fat content, we characterized ceramide metabolism within adipose tissue. Gene expression analysis suggested that the increase in ceramide might be explained by an increase in sphingomyelin hydrolysis by sphingomyelinases, especially by SMPD3, rather than by de novo synthesis of ceramide. Sphingomyelinases were expressed by both adipocytes and macrophages, but strongest expression was found in and around blood vessels within adipose tissue. In summary, the work contained in this thesis demonstrates the importance of inflammation in adipose tissue and liver and the degree of hepatic fat accumulation, but not the degree of obesity per se, in the development of insulin resistance. Sphingomyelinmediated ceramide production, which shares some features with the process of atherosclerosis, offers a link between the accumulation of fat in the liver and the development of inflammation in adipose tissue. LIST OF PUBLICATIONS I. Maria Kolak, Hannele Yki-Jarvinen, Katja Kannisto, Mirja Tiikkainen, Anders Hamsten, Per Eriksson, Rachel M. Fisher. Effects of chronic rosiglitazone therapy on gene expression in human adipose tissue in vivo in patients with type 2 diabetes. The Journal of Clinical Endocrinology & Metabolism,2007, 92:720-724 II. Jukka Westerbacka, Maria Kolak, Tuula Kiviluoto, Perttu Arkkila, Jukka Siren, Anders Hamsten, Rachel M. Fisher and Hannele Yki-Jarvinen. Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects. Diabetes, 2007, 56:2759-2765 III. Maria Kolak, Jukka Westerbacka, Vidya R. Velagapudi, Dick Wagsater, Laxman Yetukuri, Janne Makkonen, Aila Rissanen, Anna-Maija Hakkinen, Monica Lindell, Robert Bergholm, Anders Hamsten, Per Eriksson, Rachel M. Fisher, Matej Oresi , and Hannele Yki-Jarvinen. Adipose tissue inflammation and increased ceramide content characterize subjects with high liver fat content independent of obesity. Diabetes, 2007, 56:1960-1968 IV. Maria Kolak, Jukka Westerbacka, Anders Hamsten, Scott A. Summers, Anders Franco-Cereceda, Jan Liska, Matej Oresi , Hannele Yki-Jarvinen, Per Eriksson, Rachel M Fisher. Characterization of altered ceramide metabolism in inflamed adipose tissue. Manuscript.