Pex11β knockdown decreases peroxisome abundance and reverses the inhibitory effect of palmitate on pancreatic beta-cell function

Abstract

12 13 14 *Corresponding Author: 15 Mark.walker@ncl.ac.uk 16 2 17 Aims18 Reactive oxygen species generated by the peroxisomes and mitochondria contribute to 19 lipotoxicity in pancreatic beta-cells. Through targeted Pex11β knockdown and peroxisome 20 depletion, our aim was to investigate the specific contribution of peroxisomes to palmitate 21 mediated pancreatic beta-cell dysfunction.22 Methods 23 MIN6 cells were transfected with probes targeted against Pex11β, a regulator of peroxisome 24 abundance, or with scrambled control probes. Peroxisome abundance was measured by 25 PMP-70 protein expression. 48hrs post transfection, cells were incubated with or without 26 250μM palmitate for a further 48hrs before measurement of reactive oxygen species, 27 mitochondrial respiratory function, and glucose stimulated insulin secretion. Results29 Pex11β knockdown decreased target gene expression by more than 80% compared with the 30 scrambled control (P<0.001), leading to decreased PMP-70 expression (p<0.01). Pex11β 31 knockdown decreased palmitate mediated generation of reactive oxygen species (P<0.001), 32 but with no effect on mitochondrial respiratory function. At 25mM glucose, palmitate 33 treatment decreased insulin secretion in the control cells (2.54±0.25 vs 7.07±0.83 34 [mean±SEM] ng/hr/µg protein; P<0.001), with a similar pattern in the Pex11β knockdown 35 cells. However, in the presence of palmitate, insulin secretion was significantly higher in the 36 Pex11β knockdown versus control cells (4.04±0.46 vs 2.54±0.25 ng/hr/µg protein; p<0.05). Conclusion38 Pex11β knockdown decreased peroxisome abundance, decreased palmitate mediated ROS 39 generation, and reversed the inhibitory effect of palmitate on insulin secretion. These 40 findings highlight a specific and independent role for peroxisomes in pancreatic beta-cell 41 lipotoxicity. 42 43 44 3 45 46 Introduction47 Increased adiposity is a risk factor for type 2 diabetes. Ectopic fat deposition arises when 48 excess lipid is stored in tissues other than adipose tissue, such as the liver, muscle and 49 pancreas. Lipid excess in the pancreas is associated with pancreatic beta-cell dysfunction 50 and impaired insulin secretion (1-4). In vitro, chronic treatment with the long chain 51 saturated fatty acid palmitate has been shown to reduce insulin secretion in human, rat and 52 mouse islets (5-7) and pancreatic beta-cell lines (7-10). 53 Fatty acid β-oxidation in animals and man is conducted by both peroxisomes and 54 mitochondria (11, 12). Peroxisomes have a role in β-oxidation of long and medium chain 55 fatty acids, with the intermediate products transported to the mitochondria for complete 56 oxidation (12, 13). The first step of peroxisomal β-oxidation generates reactive oxygen 57 species (ROS), principally hydrogen peroxide (H 2 O 2 ). In the majority of cells, this is 58 catabolised by oxidoreductase catalase. However, pancreatic β-cells are essentially deficient 59 in this enzyme (14-16) leaving them at risk of lipotoxicity from excess H 2 O 2 production. 60 Elsne...