An investigation into the mechanism of long-chain fatty acid uptake by pancreatic β-cells

Clavelo-Farrow, Christina (2023). An investigation into the mechanism of long-chain fatty acid uptake by pancreatic β-cells. University of Birmingham. M.Sc.

Preview

ClaveloFarrow2023MScbyResearch.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (12MB) | Preview

Abstract

Background and aims: Pancreatic β-cell dysfunction and death is the central aspect during the development of type 2 diabetes (T2D). Although T2D is a major public health concern worldwide, the underlying cause of the disease is not yet fully established. Obesity is a primary risk factor for T2D, however, the relationship between the two conditions remains unclear. One feature observed in both obesity and T2D is an elevated concentration of free fatty acids (FFA) in the blood. It is widely believed that FFA can exert different effects in pancreatic β-cells depending on their degree of saturation, such that long-chain saturated fatty acids (LC-SFA) are toxic, whereas monounsaturated fatty acids (LC-MUFA) are not and may even offer protection against the toxic effects of LC-SFAs. However, the mechanism of long-chain free fatty acid (LC-FFA) uptake into pancreatic β-cells, where they seemingly exert these effects, is poorly understood. Identifying the mechanism of uptake is important to produce therapeutics to regulate their entry, thereby preventing the lipotoxic effects of LC-SFAs and possibly slowing the development of obesity induced T2D.

Methods: Evidence for candidate LC-FFA transport proteins in mammalian cells was investigated using a scoping review. The gene expression of candidate membrane-associated LC-FFA transport proteins in β-cells was studied using bioinformatic analysis of pre-published bulk RNA-sequencing data. Experimental studies used the human-derived EndoC-βH1 and rat-derived INS-1 832/13 pancreatic β-cell lines. LC-FFAs were conjugated to bovine serum albumin (BSA) and acute uptake was examined using a fluorescent intracellular pH indicator, pHrodo Green AM, and confocal microscopy. Inhibition of dynamin was achieved using Dyngo-4a. Membrane fluidity was determined using the fluorescent probe 2-Dimethylamino-6-lauroylnaphthalene (Laurdan) and confocal microscopy.

Results: The results of the scoping review revealed that cluster of differentiation 36 (CD36) and fatty acid transport proteins (FATP) have the most evidence to support a role in facilitating LC-FFA uptake in a range of human cell types. A bioinformatic analysis showed that long-chain acyl-CoA synthetase (ACSL) 1, ACSL3, ACSL4, FATP4, TBC1 domain family member (TBC1D) 1 and TBC1D4 are the most highly expressed candidate LC-FFA transporters in both ex-vivo human pancreatic islets and human-derived EndoC-βH1 cells. In both human-derived EndoC-βH1 cells and rat-derived INS-1 832/13 cells, the uptake of the LC-SFA, C16:0, was saturable, and inhibited in EndoC-βH1 cells in the presence of a dynamin inhibitor, indicating a protein-mediated process. BSA, the vehicle control, was also taken up into INS-1 832/13 and EndoC-βH1 cells in a manner similar to C16:0. Exposing EndoC-βH1 cells to a combination of 375μM C16:0 and 125μM C18:1 or 125μM C16:0 and 375μM C18:1 increased the lipid order of the plasma membrane. Exposure of EndoC-βH1 cells to 250μM C16:0 and 250μM C18:1, however, did not influence membrane order. The acute uptake of C16:0 into EndoC-βH1 cells was different for cells exposed to BSA alone vs. cells cultured in 250μM C16:0 and 250μM C18:1. Moreover, uptake of BSA into EndoC-βH1 cells was different to the uptake of C16:0 when cells were exposed to varying ratios of LC-SFA:LC-MUFA. BSA uptake was inhibited in EndoC-βH1 cells, but C16:0 uptake was promoted when cells were cultured in 1% BSA.

Conclusion: The mechanism of LC-FFA uptake into human β-cells appears to involve protein-mediated transport, although the exact mechanism of uptake is still unclear. Future work should aim to investigate the function of candidate LC-FFA transporters in pancreatic β-cells.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):