My thesis research focuses on understanding retinoid metabolism and physiology in the intestine, primarily in identifying and characterizing an acyl-CoA-dependent enzyme, which was first proposed by the literature over twenty-five years ago to have physiologically important role in catalyzing retinyl ester formation. However, until my work, this enzyme had not been identified or characterized at the molecular level. My studies are also important because they suggest casual linkages between impaired retinyl ester synthesis and storage and the development of two very common chronic diseases, type II diabetes and liver disease.; My studies demonstrate that diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl-CoA-dependent retinyl ester formation in vitro and in vivo. DGAT1 acts as a physiologically and pharmacologically significant intestinal acyl:retinol acyltransferase (ARAT) to facilitate retinyl ester formation and retinoid absorption from the diet. However, other intestinal ARATs also contribute towards intestinal processing of a large pharmacological dose of retinol. Unlike the intestine, DGAT1 does not have a significant role in adipose tissue and liver in acting as an ARAT, under either physiological or pharmacological conditions, to facilitate retinyl ester formation and storage. Moreover, DGAT1 is unable to synthesize retinyl ester that is incorporated into nascent very low density protein (VLDL), whereas lecithin:retinol acyltransferase (LRAT) plays an importance role in this process. My work also demonstrates that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.; Cellular retinol-binding protein, type I (CRBPI) has a role in preventing retinol from degradation in the liver. I obtained this evidence through characterizations of LRAT/CRBPI-deficient mice that I generated for my studies. Moreover, CRBPI may have a previously unsuspected role(s) in adipose tissue since the absence of CRBPI markedly diminishes adipose tissue retinoid storage in LRAT-deficient mice. Intriguingly, two of double knockout mice I generated in my studies, LRAT/DGAT and LRAT/CRBPI-deficient mice displayed hepatic phenotypes that are indicative of the onset of liver disease. The LRAT/DGAT-deficient mice showed excessive accumulation of extracellular matrix in some livers and this was accompanied by an elevation in hepatic CYP26A1 (cytochrome P450 26A1) mRNA expression. These would suggest that these mice are experiencing retinoid toxicity due to the increase in hepatic retinoic acid levels. LRAT/CRBPI-deficient mice exhibit excessive accumulation of triglyceride in their livers. This was evidenced both by histology and a direct increase in hepatic triglyceride levels. The elevated hepatic triglyceride levels were accompanied by an upregulation of hepatic sterol-regulatory element binding protein-1c (SREBP1c), a master regulator of hepatic lipogenesis as well as by an increase in CYP26A1 mRNA expression.; The studies I carried out in the last research chapter of my thesis (Chapter IV) demonstrate that human aldose reductase is a physiologically significant retinaldehyde reductase which catalyzes retinaldehyde reduction to retinol in normal human keratinocytes. The uptake and conversion of retinaldehyde to retinol by normal human keratinocytes is inhibited by a drug, zopolrelstat, which specifically blocks aldose reductase activity. Since aldose reductase activity is highly correlated with the development and progression of diabetic complications, my data suggest a linkage between the complications of diabetes and retinoid physiology, involving aldose reductase. (Abstract shortened by UMI.)
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