Abstract
Dysfunction and apoptosis of pancreatic β-cells leads to impaired insulin secretion resulting in the development of type 2 diabetes. This process is partly driven by the environmental stress of high glucose and lipids, termed glucolipotoxicity. High density lipoproteins (HDL) can provide protection from glucolipotoxicity, maintaining β-cell function and survival and resisting the onset of type 2 diabetes.This research aimed to investigate how HDL improves pancreatic β-cell survival during glucolipotoxic challenge, with a focus on the role of microRNA sequences. Three β-cell models were initially considered and evaluated for their suitability: the human hybrid PANC-1 1.1B4 cell line, an insulin-overexpressing 1.1B4 cell line and insulin-expressing cells differentiated from human induced pluripotent stem cells (hiPSCs).
Initial experiments suggested that 1.1B4 cells express many β-cell marker genes and efflux cholesterol to ApoA-I and HDL. However, there was no evidence of glucose-stimulated insulin secretion (GSIS) by 1.1B4 cells, which led to an examination of alternative β-cell models. Transfection of 1.1B4 cells with a pCMV3 vector containing INS gene successfully increased expression of insulin mRNA, however, this did not lead to a functional improvement in GSIS. Human iPSCs that underwent directed differentiation showed successful downregulation of pluripotent factors and upregulation of insulin expression, but the yield proved too low to sustain further investigation. Ultimately, wildtype 1.1B4 cells were the best available model for assessing the change in microRNA expression in β-cells induced by HDL treatment.
Results indicated glucolipotoxic conditions of 30mM glucose and 0.25mM palmitic acid severely reduced 1.1B4 cell viability. The deleterious effects of glucolipotoxicity were reduced by low concentrations of HDL, but not ApoA-I, by protecting cell viability and survival partially by reducing apoptosis. The microRNA profile of 1.1B4 cells following HDL treatment was measured using a microRNA expression microchip array. A total of 64 microRNAs showed altered expression after HDL treatment; selection of biologically relevant microRNAs and removal of ApoA-I sensitive microRNAs identified candidate microRNAs suitable for verification by RT-qPCR. This process discovered that microRNA-21-5p expression is upregulated following HDL treatment, making miR-21-5p a promising candidate for mediating HDL protection of 1.1B4 cells during glucolipotoxicity.
Downstream targets of miR-21-5p (ZNF367, PDCD4, FOXO3a, SMAD7 and STAT3) were identified using bioinformatic analysis and upregulation of miR-21-5p using a mimic led to downregulation of SMAD7 and STAT3. Moreover, HDL treatment reduced the expression of ZNF367, SMAD7 and STAT3. However, experiments using miR-21-5p mimic or inhibitor did not impact cell viability during glucolipotoxicity and inhibition of miR-21-5p did not disrupt HDL protection from glucolipotoxicity.
In conclusion, low concentrations of HDL protect against glucolipotoxicity in human 1.1B4 cells and analysis of miRNA sequences regulated by HDL implicate miR-21- 5p in this process. Exogenous modulation of miR-21-5p levels was insufficient to protect against glucolipotoxicity or inhibit the protection of β-cells by HDL. This demonstrates upregulation of miR-21-5p is not sufficient to replicate HDL protection of 1.1B4 cells during glucolipotoxicity. If miR-21-5p is involved, then it is in concert with a network of miRNAs or other signalling factors.
| Date of Award | 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Annette Graham (Supervisor) |