Hypoxia and Adiponectin in Adipose Tissue Inflammation

Prevalence rates of obesity and associated metabolic disturbances like insulin resistance and type 2 diabetes are increasing and pose a major health problem to Western societies. Importantly, obesity, insulin resistance, and diabetes are pro-inflammatory states: inflammatory markers are increased in these disorders and macrophages as well as other cells of the immune system were found in hypertrophic adipose tissue of obese animals and humans. Furthermore, adipocytes themselves can secrete and produce mediators which may contribute to the pro- inflammatory state. Adiponectin, which belongs to the family of adipocytokines, is most abundantly produced by adipose tissue. Recently, this adipocyte-derived hormone has also been reported to be involved in the clearance of early apoptotic cells by macrophages. Adiponectin levels are decreased in obese patients. Also, reductions of adiponectin levels have been reported in hypoxic white adipose tissue of obese mice. However, the reason behind decreased adiponectin in hypoxic white adipose tissue is unknown. We aim at investigating [1] the influence of hypoxia on adiponectin secretion, apoptosis and necrosis in the cross- talk of human adipocytes with macrophages. In addition, we aim at determining [2] the influence of hypoxia and adiponectin on phenotypic changes and phagocytotic activity of macrophages held in co-culture with adipocytes. Understanding the mechanisms of adipose tissue inflammation is an important step in developing new strategies for the treatment of obesity and obesity-associated diseases.

White fat tissue is an important endocrine organ, secreting a variety of protein factors (adipokines). In obese people some areas are poorly oxygenated due to fat tissue oversize. In this research project, the VIVIT team focused on the impact of oxygen deficiency (hypoxia) on human fat cells (adipocytes) and on the obesity-related dysregulation of adipokines, which are involved in initiation of inflammation and diseases including type II diabetes mellitus and the metabolic syndrome. Amongst others, we could demonstrate that hypoxic cultivation of human adipocytes significantly induces expression and secretion of the adipokine apelin and that this is dependent on HIF-1a, the key factor for hypoxic response. Moreover, we went on investigating the effect of hypoxia on the gene expression in human adipocytes. For this we performed a genome-wide gene expression microarray analysis (GeneChip U133 Plus 2.0, Affymetrix) and this was done in cooperation with the "Gene Discovery Core Facility Expression Profiling Unit" at the medical university Innsbruck using SGBS cells (adipocytes derived from a Simpson Golabi-Behmel-Syndrom patient). Comparing the gene expression profiles of these cells under normoxic cultivation with those cultivated under hypoxia (1% O2, 5% CO2 and 94% N2) for 3, 6 and 16 h, eight genes (ADM, ANKRD37, DDIT4, KDM3A; PFKFB4, PPP1R3C, VEGFA, ZNF395) displayed a significantly increased expression rate at all three mentioned time points. This hypoxia-induced overexpression has been shown for the very first time in adipocytes. In further analysis, we revealed binding sites for transcription factors HIF-1, HIF-2, as well as for the FOXO-family, within promoters of these genes. As some of them are overlapping, a gene expression control under hypoxia seems most likely, allowing for a time-dependent regulation which may preclude an overwhelming reaction. Additionally, we demonstrated a significantly increased transcription rate of transcription factors ATF3, FOSL2, JUN, and KLF7 and of genes involved in glycolysis and the insulin pathway. Most clearly, this was true for enolase 2 and phospho-fructo-kinase. In both cases, the about 100 fold upregulation turned out to be HIF-1-dependent, as the inhibition of HIF-1 resulted in a 20 fold reduction of gene expression. Obviously, hypoxia heavily impacts metabolic and glycolytic gene expression. We hypothesize that this directs glycolysis to enforced glycogen and glutathione/NADPH generation, representing a kind of hypoxia protection molecules or mechanisms and shielding cells from reactive oxygen species (ROS). Most interesting, further analyzing this hypoxia-induced gene regulation pattern of adipocytes revealed a striking match between the set of upregulated genes and disease biomarkers for diabetes mellitus but also cancer. With growing intensity, scientists are investigating the impact of anti-inflammatory plant compounds on T2DM and metabolic syndrome with respect to prophylaxis and therapy. We ascertained for some of these compounds a mitigating or even compensating action directed against hypoxia- induced effects. Of note, these compounds also interact with the mitochondrial electron transfer chain, known as the hypoxia-sensing machinery in the cell. These findings are summarized in our recent review article and are the basis for our new research task: Elucidating the molecular interplay between hypoxia and plant compounds and its impact on the inflammatory cascade in obese tissue. If facilitated by a FWF grant, we now are willing to further expand this interesting issue to elucidate the molecular interplay of hypoxia and the so called phytochemicals. Hence we hope to get new insights relevant for obesity associated diseases and novel prophylaxis and therapy concepts.