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Title: Evaluation of bioenergetic changes in acute pancreatitis
Author: Morton, J. C.
ISNI:       0000 0004 7970 3939
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Acute pancreatitis (AP) is a potentially severe inflammatory disease of the exocrine pancreas. It is now well established that the initial site of injury is the pancreatic acinar cell (PAC). AP is primarily caused through biliary disease or alcohol abuse and their resultant mediators, bile salts and non-oxidative metabolites of alcohol, fatty acid ethyl esters (FAEEs), respectively. Mitochondrial dysfunction, and thus ATP production, is a principle pathological feature of AP. Moreover, the mitochondrial permeability transition pore (MPTP) and cyclophilin-D (CypD), a regulator of MPTP, is critical to multiple forms of experimental AP. Recent advances in technology have made it possible to isolate and examine individual populations of circulating leukocytes. Although mitochondrial dysfunction is core to the development of AP, the precise changes that occur are still unclear. Utilising a recent advance in bioenergetic measurements, the Seahorse XF Flux analyser, the overall aim of the study was to investigate mitochondrial bioenergetic alterations in AP, identifying changes in isolated murine PACs and in blood cells from AP patients and healthy subjects. PAC mitochondrial bioenergetics were measured at physiological (low (pM) cholecystokinin (CCK)) and pathophysiological (high (nM) CCK, ethanol, non-oxidative metabolites FAEEs & fatty acids (FAs), and bile acids) concentrations. The distinct modes of action of FAEEs and FAs were evaluated and whether their metabolism was important. Genetic (CypD knockout: PPIF -/-) and pharmacological (cyclosporin A, bongkrekic acid, sangliferin A) inhibition of MPTP were assessed and whether these were tissue specific. Blood cell populations' bioenergetic profiles were analysed and differences between AP patients and healthy volunteers recorded. At physiological concentrations of CCK, the spare capacity of PACs was increased, but not significantly. At pathophysiological CCK significantly decreased ATP-production, but did not result in complete bioenergetic collapse. Our data suggests ethanol sensitises PACs to the effects of CCK via an increase in the glycolytic pathway. Bile acid did not alter mitochondrial bioenergetics. FAEES and FAs were both extremely detrimental to the bioenergetics of PACs. Both toxins caused a significant reduction in ATP-linked production as well as a significant increase in proton leak. FAs also significantly reduced maximal respiration and spare capacity. Preventing the breakdown of FAEEs to FAs and inhibiting uncoupling protein 2 (UCP2), which allows for proton leak independently of ATP synthase, did not alleviate the detrimental effects of FAEEs and FAs on PAC mitochondrial bioenergetics. FAs were shown to increase proton leak across complex III of the mitochondria. Genetic and chemical inhibition of MPTP formation did not prevent the detrimental effects of FAEES and FAs. However, the genetic inhibition of CypD significantly reduced basal, maximal ATP-linked production, spare capacity and proton leak in PACs but not hepatocytes. The oxidative burst was shown to be increased in monocytes and lymphocytes, but decreased in the neutrophils from patients compared to healthy volunteers, respectively. Moreover, the spare capacity and maximal respiration were significantly reduced in the lymphocytes of patients compared to healthy volunteers. The study has demonstrated clear alterations of mitochondrial bioenergetics in physiological and pathophysiological conditions relevant to AP. In addition, it has been discovered that CypD has a potential role in normal cellular production of ATP in PACs. Finally, bioenergetic alterations are detectable in blood cell subpopulations of AP patients, opening up the potential for further work aimed at understanding the inflammatory response in the pathophysiology of AP and detection of clinical progression.
Supervisor: Criddle, David ; Tepikin, A. V. ; Sutton, R. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral