Abstract
Intracellular cholesterol accumulation can have a toxic effect on pancreatic (B-cells, by impairing insulin secretion. Stimulation of cholesterol efflux can improve pancreatic function by preventing cholesterol accumulation in pancreatic B-cells.The aim of this study was to investigate the pancreatic expression of mitochondrial cholesterol trafficking and metabolising proteins in two rodent models of obesity and examine how these relate to the diabetic status of these animals. In addition, the cholesterol trafficking protein CYP27A1, in the presence or absence of its redox partners ADX and ADXR, was reconstituted in a rodent insulinoma cell line and the effect of this manipulation on cholesterol efflux and insulin secretion was examined.
Plasma glucose, cholesterol, triglycerides and insulin were measured in six month old control and high fat diet (HFD)-fed Wistar rats and in four month old lean (Fa/?)and obese ifa/fa) male and female Zucker rats. Protein expression of CYP27A1, ADX, ADXR, TSPO, STARD1, LXRa and ABCA1 was measured using immunoblotting. The expression of Ins2 in the CYP27A1 overexpressing cells was measured with qPCR. BRIN-BD11 rodent insulinoma cells overexpressing CYP27A1 or CYP27A1, ADX and ADXR were generated. LDL-cholesterol was utilised to “load” the cells. Dibutyryl-cAMP and FGIN-1-27 were utilised to target STARD1 and TSPO, respectively. [ H]cholesterol was utilised to measure the lipidation of ApoA-I, HDL and human serum by these cells and [l4C]acetate was used to measure the biosynthesis of cholesterol and cholesteryl esters. The secretion of insulin into the media was measured using an ELISA kit.
The study demonstrated that in obese (fa/fa) male but not female Zucker rats, hyperinsulinaemia is associated with elevated pancreatic protein levels of CYP27A1, ADXR, TSPO and LXRa. Furthermore, the obese (fa/fa) Zucker rats of both sexes displayed elevated plasma cholesterol and triglycerides. In the FIFD-fed Wistar rats,obesity was associated with hyperglycaemia and reduced pancreatic ABCA1 protein levels. Overexpression of CYP27A1 in BRIN-BD11 cells led to increased lns2 gene expression and enhanced cholesterol efflux to ApoA-I, HDL and human serum in the presence of a cholesterol “load”. Overexpression of CYP27A1, ADX and ADXR inthe same cells led to enhanced cholesterol efflux to ApoA-I and human serum in both the basal and cholesterol “loaded” states.
Genetic obesity alters the pancreatic expression of cholesterol trafficking complex and metabolising protein members, suggesting that their modulation could impact on insulin secretion. Reconstitution of CYP27A1 alone or alongside ADX and ADXR enhanced cholesterol efflux by these cells, suggesting that enhancing cholesterol flux through CYP27A1 could have a beneficial role in the prevention or treatment of diabetes.
| Date of Award | 2017 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Annette Graham (Supervisor) |