Induction of microRNA hsa-let-7d-5p, and repression of HMGA2, contribute protection against lipid accumulation in macrophage 'foam' cells.

Accumulation of excess cholesterol and cholesteryl ester in macrophage 'foam' cells within the arterial intima characterises early 'fatty streak' atherosclerotic lesions, and is accompanied by epigenetic changes, including altered expression of microRNA sequences which determine of gene and protein expression. This study established that exposure to lipoproteins, including acetylated LDL, induced macrophage expression of microRNA hsa-let-7d-5p, a sequence previously linked with tumour suppression, and repressed expression of one of its target genes, high mobility group AT hook 2 (HMGA2). A let-7d-5p mimic repressed expression of HMGA2 (18%; p < 0.05) while a marked increase (2.9-fold; p < 0.05) in expression of HMGA2 was noted in the presence of let-7d-5p inhibitor. Under these conditions, let-7d-5p mimic significantly (p < 0.05) decreased total (10%), free (8%) and cholesteryl ester (21%) mass, while the inhibitor significantly (p < 0.05) increased total (29%) and free cholesterol (29%) mass, compared with the relevant controls. Let-7d-5p inhibition significantly (p < 0.05) increased endogenous biosynthesis of cholesterol (38%) and cholesteryl ester (39%) pools in macrophage 'foam' cells, without altering the cholesterol efflux pathway, or esterification of exogenous radiolabelled oleate. Let-7d-5p inhibition in sterol-loaded cells increased the level of HMGA2 protein (32%; p < 0.05), while SiRNA knockdown of this protein (29%; p < 0.05) resulted in a (21%, p < 0.05) reduction in free cholesterol mass. Thus, induction of let-7d-5p, and repression of its target HMGA2, in macrophages is a protective response to the challenge of increased cholesterol influx into these cells; dysregulation of this response may contribute to atherosclerosis and other disorders such as cancer.

Protection against Glucolipotoxicity by High Density Lipoprotein in Human PANC-1 Hybrid 1.1B4 Pancreatic Beta Cells: The Role of microRNA.

High-density lipoproteins provide protection against the damaging effects of glucolipotoxicity in beta cells, a factor which sustains insulin secretion and staves off onset of type 2 diabetes mellitus. This study examines epigenetic changes in small non-coding microRNA sequences induced by high density lipoproteins in a human hybrid beta cell model, and tests the impact of delivery of a single sequence in protecting against glucolipotoxicity. Human PANC-1.1B4 cells were used to establish Bmax and Kd for [3H]cholesterol efflux to high density lipoprotein, and minimum concentrations required to protect cell viability and reduce apoptosis to 30mM glucose and 0.25 mM palmitic acid. Microchip array identified the microRNA signature associated with high density lipoprotein treatment, and one sequence, hsa-miR-21-5p, modulated via delivery of a mimic and inhibitor. The results confirm that low concentrations of high-density lipoprotein can protect against glucolipotoxicity, and report the global microRNA profile associated with this lipoprotein; delivery of miR-21-5p mimic altered gene targets, similar to high density lipoprotein, but could not provide sufficient protection against glucolipotoxicity. We conclude that the complex profile of microRNA changes due to HDL treatment may be difficult to replicate using a single microRNA, findings which may inform current drug strategies focused on this approach.

Genetic obesity increases pancreatic expression of mitochondrial proteins which regulate cholesterol efflux in BRIN-BD11 insulinoma cells.

Pancreatic ß-cells are sensitive to fluctuations in cholesterol content, which can damage the insulin secretion pathway, contributing to the aetiology of type 2 diabetes mellitus. Cholesterol efflux to (apo)lipoproteins, via ATP-binding cassette (ABC) transporter A1 (ABCA1), can prevent intracellular cholesterol accumulation; in some peripheral cells, ABCA1-dependent efflux is enhanced by promotion of cholesterol trafficking to, and generation of Liver X receptor (LXR) ligands by, mitochondrial sterol 27-hydroxylase (Cyp27A1 (cytochrome P450 27 A1/sterol 27-hydroxylase)) and its redox partners, adrenodoxin (ADX) and ADX reductase (ADXR). Despite this, the roles of mitochondrial cholesterol trafficking (steroidogenic acute regulatory protein [StAR] and 18-kDa translocator protein [TSPO]) and metabolising proteins in insulin-secreting cells remain wholly uncharacterised. Here, we demonstrate an increase in pancreatic expression of Cyp27A1, ADXR, TSPO and LXRα, but not ADX or StAR, in obese (fa/fa) rodents compared with lean (Fa/?) controls. Overexpression of Cyp27A1 alone in BRIN-BD11 cells increased INS2 expression, without affecting lipid metabolism; however, after exposure to low-density lipoprotein (LDL), cholesterol efflux to (apo)lipoprotein acceptors was enhanced in Cyp27A1-overexpressing cells. Co-transfection of Cyp27A1, ADX and ADXR, at a ratio approximating that in pancreatic tissue, stimulated cholesterol efflux to apolipoprotein A-I (apoA-I) in both basal and cholesterol-loaded cells; insulin release was stimulated equally by all acceptors in cholesterol-loaded cells. Thus, genetic obesity increases pancreatic expression of Cyp27A1, ADXR, TSPO and LXRα, while modulation of Cyp27A1 and its redox partners promotes cholesterol efflux from insulin-secreting cells to acceptor (apo)lipoproteins; this response may help guard against loss of insulin secretion caused by accumulation of excess intracellular cholesterol.