The importance of the Niemann Pick C-1 Like 1 (NPC1L1) protein in cholesterol absorption by the intestine is well recognized. However, whether NPC1L1 is a membrane bound protein serving as a transporter for cholesterol uptake by enterocytes or an intracellular protein responsible for cholesterol trafficking from the apical plasma membrane to intracellular compartments for lipoprotein assembly remains controversial. The NPC1L1 is also expressed at high levels in human liver, but its expression in rodent liver is minimal. Hence, the importance of hepatic NPC1L1 is still unclear. The goals of this study are (i) to identify the physiological mechanism by which NPC1L1 participates in cholesterol absorption in the intestine, and (ii) to delineate the role of liver NPC1L1 in lipoprotein metabolism and homeostasis. Preliminary Results showed that whereas NPC1LT''mice were defective in cholesterol absorption, no difference in cholesterol transport kinetics was observed between intestinal brush border membrane vesicles obtained from NPC1L1+/+ and NPC1LT''mice. Moreover, fluorescent cholesterol was efficiently taken up from intestinal lumen into absorptive epithelial cells of the intestine in both NPC1L1+/+ and NPC1LT''mice, but not in mice treated with the cholesterol absorption inhibitor ezetimibe. These results provided strong support for the hypothesis that NPC1L1 is not the high affinity membrane transporter but an intracellular cholesterol transport protein. Our data also showed that suppression of NPC1L1 activity in the liver lowered VLDL production and inhibited selective uptake of HDL-cholesterol, suggesting that hepatic NPC1L1 may modulate plasma lipoprotein homeostasis.
Specific Aim 1 will identify the functional role of NPC1L1 in intestinal lipid transport by mating NPC1L1*/+ and NPC1LT''mice to ABCG5G8'''mice and then determining the distribution and site of cholesterol accumulation in the intestine to further test the hypothesis that NPC1L1 is an intracellular lipid trafficking protein targeting cholesterol after apical uptake to intracellular compartments for lipoprotein assembly.
Specific Aim 2 will use in vitro cell culture model to test the hypothesis that NPC1L1 expression is necessary but not sufficient without expression of cell surface cholesterol transporters such as SR-BI and/or CD36 in promoting cholesterol absorption and intracellular transport.
Specific Aim 3 will produce transgenic mice expressing NPC1L1 in the liver, to similar extent as its expression level in human liver, to identify the functional significance of hepatic NPC1L1 in plasma lipid homeostasis, VLDL production, selective uptake of HDL-cholesterol, and reverse cholesterol transport. Taken together, these studies will provide a better understanding on the physiological importance and mechanism of action of NPC1L1, potentially offering alternative targets for therapeutic treatment of hypercholesterolemia and lowering the risk of lipid-induced metabolic disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK076907-04
Application #
7902092
Study Section
Special Emphasis Panel (ZRG1-EMNR-G (03))
Program Officer
Grey, Michael J
Project Start
2007-08-01
Project End
2012-07-31
Budget Start
2010-08-01
Budget End
2012-07-31
Support Year
4
Fiscal Year
2010
Total Cost
$310,270
Indirect Cost
Name
University of Cincinnati
Department
Pathology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Adams, Michelle R; Konaniah, Eddy; Cash, James G et al. (2011) Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine. Am J Physiol Gastrointest Liver Physiol 300:G164-9
Perez-Tilve, Diego; Hofmann, Susanna M; Basford, Joshua et al. (2010) Melanocortin signaling in the CNS directly regulates circulating cholesterol. Nat Neurosci 13:877-82
Yang, Li; Li, Xiaoming; Ji, Yong et al. (2010) Effect of ezetimibe on incretin secretion in response to the intestinal absorption of a mixed meal. Am J Physiol Gastrointest Liver Physiol 299:G1003-11
Labonte, Eric D; Camarota, Lisa M; Rojas, Juan C et al. (2008) Reduced absorption of saturated fatty acids and resistance to diet-induced obesity and diabetes by ezetimibe-treated and Npc1l1-/- mice. Am J Physiol Gastrointest Liver Physiol 295:G776-83
Hui, David Y; Labonte, Eric D; Howles, Philip N (2008) Development and physiological regulation of intestinal lipid absorption. III. Intestinal transporters and cholesterol absorption. Am J Physiol Gastrointest Liver Physiol 294:G839-43
Labonte, Eric D; Howles, Philip N; Granholm, Norman A et al. (2007) Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles. Biochim Biophys Acta 1771:1132-9