title: The differential regulation of the adiponectin system in response to lipopolysaccharide and sepsis
Creators: name: Hall, Alison M
Publisher: name: University of Liverpool Repository
Year: 2013

description: Background Sepsis is a condition characterised by a massive acute inflammatory response and insulin resistance. Several inflammatory mediators involved in the immune response during sepsis have been identified. Recently it has become clear that adipose tissue contributes to the production of pro- and anti-inflammatory mediators, which have been termed adipokines. Adiponectin is an adipokine that has anti-diabetic, anti-atherogenic and anti-inflammatory properties. Its role in chronic inflammatory diseases, such as type II diabetes mellitus (DM) and obesity has been extensively studied. Generally, adiponectin is down-regulated in these conditions which are characterised by insulin resistance. Adiponectin, previously thought to be exclusively expressed in and secreted from adipocytes, has now been shown to be released from other tissues such as skeletal muscle, cardiac muscle and bone. Adiponectin from adipose tissue is down-regulated in mouse models of sepsis, however, no information is available about the role of adiponectin receptors. In chronic insulin resistance, adiponectin receptor gene expression is decreased, suggesting a down-regulation of the ‘adiponectin system’. Adiponectin gene expression appears to be partially regulated by NFκB, a transcription factor co-ordinating the release of inflammatory mediators in response to an appropriate stimulus, such as lipopolysaccharide. Other signalling mechanisms may also be involved, in particular the HIF-1α pathway. HIF-1α is another transcription factor with a large number of target genes, many of which are involved in the inflammatory process. Although HIF-1α was initially discovered as a cellular regulator of hypoxia, the pathway has now been shown to be activated by other non-hypoxic mechanisms of up-regulation, including bacterial infection. HIF-1α is expressed in immune cells, however, its role in adipose tissue during sepsis remains unclear. Methods Three different lines of experiments used in this thesis. The animal model used high dose LPS injected intra-peritoneally (under general anaesthesia) into 8-10 week old male C57BL/6J mice. Mice were killed at 4 or 24 hours after injection and tissues (Peri-renal, subcutaneous and epididymal fat, liver, muscle, small bowel and spleen) were removed for analysis. Adiponectin and adiponectin receptor gene expression was determined by quantitative real-time PCR (qPCR). The cell culture model used cell lines, 3T3-L1 adipocytes and C2C12 myocytes, grown in culture and then treated with varying concentrations of LPS. Cells were harvested at 4 and 24 hours and qPCR was performed to ascertain adiponectin and adiponectin receptor gene expression. The same animal and cellular models were utilised for the HIF-1α investigations with protein determination carried out using ELISA. Finally, twenty-one septic patients were recruited from the Intensive care unit at the Royal Liverpool University Hospital, following ethical approval and written consent. Blood samples were taken on days 1 and 2 and day of discharge and serum levels of total and HMW adiponectin were determined by ELISA. Results Alterations in adiponectin and its receptors expression in murine endotoxaemia Adiponectin receptors were down-regulated following LPS injection. The greatest changes acutely were in muscle, liver and peri-renal fat (adipoR1) and liver, muscle, peri-renal and sub-cutaneous fat (adipoR2). There were no significant changes in the other tissue depots. After 24 hours, there were fewer changes in gene expression with adipoR1 being down-regulated in liver and skeletal muscle and AdipoR2 in skeletal muscle only. Down-regulation of adiponectin gene expression following LPS was confirmed in the adipose tissue depots. We demonstrated that the adiponectin gene was expressed in skeletal muscle and sequencing of the PCR product confirmed a 100% match for adiponectin mRNA. C2C12 myocytes were then used to verify the presence of adiponectin mRNA in skeletal muscle cells. In tissue depots, adiponectin gene expression was significantly reduced in skeletal muscle in both the 4 and 24 hour cohorts respectively. Alterations in adiponectin and its receptors expression in cell lines In the cell lines, the inflammatory response to LPS was confirmed using IL-6 as a reference gene. This also confirmed methodological success. Adiponectin gene expression from 3T3-L1 adipocytes was acutely reduced following treatment with high dose LPS but there were no changes in expression in cells treated with lower concentrations of LPS. There were no changes at 24 hours. Adiponectin receptors were down-regulated but not consistently with dose and these changes were only observed in the cells harvested after 4 hours. In C2C12 myocytes, there was a significant reduction in adiponectin gene expression following high doses of LPS but there were minimal changes in adiponectin receptor expression in the C2C12 myocytes. Human Study There was a significant increase in both total and HMW adiponectin between day 1 and day of discharge and the ratio of HMW adiponectin to total adiponectin also increased between admission and discharge. There were no changes in total or HMW adiponectin or their ratio between day 1 and day 2 of admission. HIF-1α HIF-1α gene expression was up-regulated in liver and spleen 4 hours post LPS injection. The changes persisted 24 hours after LPS treatment with increased expression in liver, small bowel and spleen. Protein levels were elevated in skeletal muscle after 4 hours and liver after 24 hours and spleen. Discussion These results increase the evidence that adipose tissue is not an inert storage medium for fatty acids but a sophisticated endocrine organ. The ‘adiponectin system’, including adiponectin and its two receptors, is down-regulated in-vivo and in-vitro models of sepsis. This may play a role in the metabolic derangements such as hyperglycaemia and insulin resistance. In addition, hypoadiponectinaemia may have a significant role in the disordered inflammatory process known to occur in sepsis, possibly impacting on mortality as shown in some animal studies. Adiponectin is not exclusively adipose tissue derived and interestingly we have demonstrated the presence of adiponectin mRNA in other tissue such as skeletal muscle. The effect of reduced gene expression from extra-adipose tissue depots is yet to be established but may have a paracrine or autocrine effect rather than an endocrine role. Low total and HMW adiponectin levels during human sepsis have also been identified. Whilst hypoadiponectinaemia in sepsis has been observed in previous studies, increases in HMW adiponectin associated with clinical improvement have not been previously demonstrated. A further signalling pathway investigated in these models was HIF-1α. These results demonstrate a global up-regulation of HIF-1α gene expression across tissue depots and cellular models. This may reflect tissue hypoxia but also may reflect non-hypoxic up-regulation by LPS and inflammatory mediators. HIF-1α is known to play a part in the inflammatory process and, like adiponectin, has links to the NFκB signalling pathways.