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Title: Pancreatic fat accumulation and effects on beta cell function
Author: Pinnick, Katherine Elizabeth
ISNI:       0000 0001 3490 9966
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2009
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Type 2 Diabetes Mellitus (T2DM) is characterised by impaired pancreatic 13-cell function resulting in inadequate insulin secretion. The mechanisms involved in 13-cell dysfunction are largely unknown. Elevated fasting plasma non-esterified fatty acid (NEFA) concentrations have been identified as a risk factor for the development of T2DM. The work in this thesis investigates functional effects of NEFA on the 13-cell. Prolonged exposure to elevated NEFA has previously been associated with impaired insulin secretion, reduced insulin content and altered gene expression and lipid metabolism in the 13-cell. Determining the reversibility of these defects may lead to a greater understanding of the underlying mechanisms. Increased pancreatic fat content is positively associated with body mass index in humans and this may expose the 13-cell to high NEFA concentrations. However, the in vivo concentration and composition of NEFA in the pancreas is not known. An in vitro model of 13-cell 'recovery' from the deleterious effects of fatty acids is presented. The longterm culture (>48h) of mouse islets and INS-1 cells with NEFA (0.5mM) impaired glucose and tolbutamide-stimulated insulin secretion, but this was partially reversed by culture for 24h in the absence of exogenous fatty acids. Culture with oleic acid led to the accumulation of triacylglycerol (TAG) in cytosolic lipid droplets. The protein ADFP was found in close association with these droplets. In contrast, culture with palmitic acid produced large cytoplasmic 'splits'. The removal of exogenous fatty acids from the culture media led to a visible reduction in these morphological features. Extraction of the cellular lipids confirmed an increase in the TAG content following culture with NEFA and demonstrated the incorporation of the experimental fatty acid into the TAG and phospholipid (PL) fractions. Following removal of the fatty acids for 24h, TAG content was reduced and NEFA-induced changes in TAG and PL fatty acid composition were partially reversed. A reduction in TAG content in 'recovering' cells indicated the presence of active Iipases. Culture with NEFA increased lipolysis as shown by the measurement of glycerol in the culture media, but this was reduced in 'recovering' cells. Lipase inhibitors inhibited glycerol release but failed to inhibit a reduction in TAG content, and did not confirm a role for Iipases in the recovery of stimulated insulin secretion. Exposure of INS-1 cells to NEFA increased their oxidative capacity for fatty acids and this remained elevated in 'recovering' cells. Treatment with the CPT-1 inhibitor, etomoxir (10I-lM), impaired the fatty acid oxidative capacity of the 13-cell but did not affect the recovery of insulin secretion. A number of genes were upregulated following prolonged culture with NEFA, these included insulin I and II, CPT-1 and UCP2. These genes all displayed reduced expression in cells cultured further in the absence of exogenous fatty acids. The content and composition of fat in tissues from mice was investigated. The TAG composition reflected the major fatty acids found in the diet, with elevated proportions of palmitic and palmitoleic acid indicating the contribution of de novo lipogenesis and desaturase activity to this fatty acid pool. Pancreatic PL were highly unsaturated compared to liver PL, with arachidonic acid accounting for -25% of the PL fatty acids. In mice fed a high-fat (40%) diet (HFD) which was compositionally matched to a control (5%) diet, a 20-fold increase in pancreatic fat was found by 15 weeks. Adipocytes, which were positively labeled for perilipin were observed in the exocrine tissue of the pancreas in HFD mice and lipid droplets labeled for ADFP were identified in the cytoplasm of exocrine cells. By 15 weeks, the fatty acid composition of the TAG, PL and NEFA fractions showed significant differences between HFD and control mice. Perilipin-positive adipocytes were also identified in human pancreas samples and the percentage adipocyte area in histological sections positively correlated (r=0.64) to total pancreatic TAG content. In conclusion, the in vitro findings show the deleterious effects of fatty acids are not permanent. However, increased fat accumulation in the pancreas, as seen in obesity, could expose the 13-cell to elevated NEFA concentrations which, over many years, may lead to irreversible 13-cell failure.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available