Probing The Composition Of Mesenteric Lymph During Diabetes And Feeding

Abstract

Diabetes mellitus results from a group of metabolic disorders characterized by hyperglycaemia due to defects in insulin production or action. Intestinal dysfunction is increasingly being implicated in this disease state– for example, abnormalities in incretin secretion, gastric emptying, and chylomicron production have been reported. Mesenteric lymph ML (i.e. lymphatic fluid from the intestine) has recently been shown to be an interesting fluid to study as it contains many factors released from the gut. Given the anatomy of the lymphatic system ML does not get detoxified by the liver and during critical illnesses (e.g. shock, acute pancreatitis) has been shown to be toxic to end organs such as heart, lung and pancreas. However, ML in chronic diseases like diabetes remains unexplored. Furthermore, it is unknown how the compositions of diabetic and normal ML differ during the transition from fasting to the fed state. Therefore, the aim of this study is to provide a deep proteomic, metabolomic, peptidomic and cytokine analysis of diabetic and normal ML in the fed and fasted states. ML was collected during the fasted to fed transition from eight anaesthetised rats randomised to Group 1 (n=8) with streptozotocin (STZ)-induced diabetes and Group 2 (n=8) sham control. Proteomic analysis was performed using immunodepletion of ML followed by ITRAQ (isobaric tags for relative and absolute quantification) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS). Metabolomic analysis was performed using MCF derivatisation followed by GC-MS analysis and cytokine levels analysed using a multiplex cytokine assay. Different peptidome and lipidome extraction methods were tested to establish the initial protocols. The proteomic analysis showed that there was an increase in inflammatory markers, activation of the “complement and coagulation cascades”, altered protease inhibitor activities, increase in immunoglobulins, decrease in carrier proteins and altered lipoprotein metabolism in diabetic ML compared with healthy ML. There was also an increase in cytoskeletal proteins and intracellular proteins in the fed state ML compared to the fasted state ML. Cytokine analysis revealed an increase in proinflammatory cytokines in diabetic ML compared with healthy ML, and an increase in anorexigenic cytokines in fed state ML compared with fasted state ML. Metabolomic analysis revealed altered protein/amino acid, TCA and fatty acid metabolism between diabetic and healthy ML during the fasted-to-fed transition.