Progress 08/01/02 to 07/31/05
Outputs
The most productive aspect of this grant centered on AIM 3. Contrary to our initial hypothesis, plasma clearance of lipids in aberrant chylomicrons from CEL-/- mice do not differ from lipids in chylomicrons from control mice. However, lipids carried by chylomicrons from normal animals are cleared from circulation more rapidly in CEL-/- mice as compared to control mice. These results indicated that lack of CEL causes systemic changes in lipoprotein metabolism. This phenomenon was pursued in detail and became the primary focus of the project. We found that more chylomicron triglycerides were present in heart and fat pads of CEL-/- mice as compared to control mice. Consistent with this, lipoprotein lipase mRNA was more abundant in heart (40%) and fat pads (2.5 fold) of CEL-/- mice. Because CEL is known to be expressed and secreted by the liver, hepatic lipoprotein metabolism was studied in detail. We found that CEL directly affects selective uptake of cholesteryl esters
(CE) from HDL by hepatocytes. CEL interacts either directly with SR-BI (scavenger receptor class B type I) or with the process mediated by it. We also found that hydrolysis of HDL-CE is impaired and that utilization of this cholesterol is changed in CEL-/- mice. Overall, less HDL-CE is used for bile acid synthesis and more is secreted into bile without hydrolysis. In addition, bile acid composition changes because use of an alternative pathway increases, resulting in more muricholate and chenodeoxycholate. A consequence of these changes is reduced excretion (in feces) of cholesterol. The physiological impact of this result is two-fold. First, reverse cholesterol transport - removal of excess cholesterol from the body - is decreased by the absence of CEL. Second, plasma cholesterol is higher in CEL-/- mice. Interestingly, changes in bile acid composition, cholesterol excretion, and plasma cholesterol are specific to females. Analyses of liver mRNA and protein indicated that lack of CEL
causes complex changes in expression of several genes relevant to cholesterol metabolism. Most of these changes, however, occur only in male mice. AIM 2: When fed chow diet, male CEL-/- mice were observed to eat less and gain less weight than male controls. Their energy expenditure was also lower. No differences were seen between control and CEL-/- females fed chow. When fed "Western diet, however, CEL-/- females ate more and gained weight more rapidly than control females. No differences were seen between CEL-/- and control males fed Western diet. AIM 1: Focus was on the ceramidase activity and lysophospholipase activities of CEL. Experiments showed more ceramide in postprandial intestinal microsomes of CEL-/- mice as compared to controls. Serum lysophospholipid is also elevated postprandially. Infusion of mutant CEL with only ceramidase activity or a bacterial lysophospholipase during lipid administration, followed by chylomicron analysis was to be performed. Because of the
extensive time and resources required, these studies were delayed due to focus on AIM 3. Transgenic mice carrying mutant and normal CEL are now available from a collaborator and will be used instead.
Impacts
This study has revealed that CEL impacts several aspects of lipoprotein metabolism with direct effects on bile secretion, reverse cholesterol transport, and plasma cholesterol levels. These effects are most pronounced in female mice. The CEL-/- mice studied in this project as well as CEL transgenic mice with elevated expression in the liver or pancreas, which are under development, will be useful tools for further investigating these aspects of lipid metabolism and especially for understanding the known differences between men and women with respect to cholesterol and lipid metabolism and the associated diseases - atherosclerosis, obesity, and diabetes. New avenues for therapeutic intervention and/or new markers for disease susceptibility may be revealed by these studies.
Publications
- Burkart, C., Camarota, L., Zheng, S., Tso, P., Hui, D. and Howles P. 2003. Altered metabolism of postprandial lipoproteins by carboxyl ester lipase knockout mice: possible relation to diet-induced obesity. Gastroenterology 124(4)suppl.1:A70
- Camarota, L., Chapman, J., Hui, D. and Howles, P. 2004. Carboxyl ester lipase cofractonates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of choelsteryl esters from HDL3. J. Biol. Chem. 279(26):27599-27606.
- Hui, D. and Howles, P. 2005. Molecular mechanisms of cholesterol absorption and transport in the intestine. Sem. in Cell & Dev. Biol. 16:183-192
- Camarota, L., Tso, P. and Howles, P. 2005. Gender-specific effects of carboxyl ester lipase on bile acid composition and biliary secretion of HDL-cholesteryl esters in mice. Gastroenterology 128(4)suppl.2:A691
- Camarota, L. and Howles, P. 2005. HDL cholesteryl ester metabolism and fecal sterol excretion are altered in carboxyl ester lipase deficient mice. Biochim. Biophys. Acta (submitted)
|
Progress 08/01/03 to 07/31/04
Outputs
Significant and exciting progress was made during year 2 of this project. AIM 1- Determine which CEL lipase activity is needed for normal chylomicron synthesis: Mammalian cell lines secreting the needed mutant forms of the enzyme are now available and purification systems established. In addition, transgenic mice with pancreas-specific expression of these mutants are also available. These reagents were made in collaborator D.Y. Hui's laboratory. The enzyme infusion experiments described in the proposal can now be performed and will be a major focus in year 3. AIM 2- Food intake/weight gain/satiety: Male CEL knockout mice were observed to eat less and gain less weight on a chow diet. However, the initial observation of greater weight gain by CEL knockout mice eating Western diet was not seen in a second study. The study will be repeated on another cohort of animals during year 3. Effects of Western, atherogenic and chow diets will be compared. AIM 3- Hepatic and tissue
clearance of chylomicron lipids; systemic vs. intestinal effects of CEL; changes in gene expression: This has been a very productive and exciting part of the project. The year 1 discovery that CEL has systemic effects on lipid metabolism has been pursued in detail. We have demonstrated that CEL interacts with the SR-BI pathway to significantly enhance selective uptake and hydrolysis of HDL cholesteryl esters. This study was published in J.Biol.Chem. during year 2. We have now discovered that lack of CEL significantly increases secretion of cholesteryl ester into hepatic bile while decreasing the concentration of bile acids. Bile acid composition is also changed indicating decreased use of the cyp8B1 dependent pathway. A manuscript describing the bile analysis results is being prepared for the Journal of Lipid Research. Preliminary data suggest changes in expression of SREBP's-1 & -2, cyp7A1 and cyp8B1 genes. Interestingly, several of the differences between CEL knockout and control
mice are specific to females. This project has been further developed in an R01 application to the NIH. Initial reviews were favorable and the proposal has been resubmitted for Feb.'05 review. Year 3 will be focused on finishing the chylomicron synthesis and preparing manuscripts.
Impacts
CEL has marked effects on triglyceride and cholesterol metabolism and is known to be polymorphic in humans. A thorough understanding of how CEL affects dietary triglyceride metabolism will facilitate the development of more appropriate strategies for treating and preventing obesity. Determining how CEL affects liver HDL, cholesterol, and bile acid metabolism will impact strategies for increasing reverse cholesterol transport for the treatment of cardiovascular disease.
Publications
- Camarota, L.C., Chapman, J.C., Hui, D.Y., and Howles, P.N. 2004. Carboxyl ester lipase cofractionates with scavenger receptor B-I in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3. J. Biol. Chem. 279, 27599-27606.
|
Progress 08/01/02 to 07/31/03
Outputs
Aim 1: Efforts during year 1 of the grant were focused primarily on Aims 2 and 3. Analysis of intestinal tissues from control and knockout mice indicates greater ceramide present in knockout mice, suggesting that ceramidase is the key activity supplied by CEL. Cloning and expression of mutant forms of CEL for injection back into mice will be developed in year 2. Aim 2: Signinificant progress was made toward completion of Aim 2. A cohort of age-matched control and knockout mice of both sexes was generated for the food intake and energy metabolism studies. Their weight gain and food intake was monitored for 2 weeks while they were being fed a chow diet. At the end of that period, their metabolism was measured using indirect calorimetry, a service provided by the Mouse Metabolic Phenotype Center at the University of Cincinnati. Thereafter, the same mice were fed a diet high in fat and cholesterol for several weeks. Their weight gain, food intake and energy metabolism
were again measured. It was found that male CEL-/- mice ate significantly less food and gained weight less rapidly than control mice. Interestingly, their metabolism was also lower as compared to control mice. Similar trends were found in female mice but differences were not significant. Preliminary data had shown that CEL-/- mice gain weight more rapidly when fed an atherogenic diet, which contains cholic acid to suppress bile acid synthesis. The current data suggest that this diet affects liver cholesterol metabolism differently in control and CEL-/- mice. This phenomenon is now being investigated as part of Aim 3. Aim 3: Major progress has been made toward completion of aim 3. CEL-/- mice were found to clear triglycerides from postprandial intestinal lipoproteins 70% more rapidly than control mice (P < 0.05). Triglycerides in conrol and CEL-/- chylomicrons were cleared similarly when injected into control mice. Thus, differences in postprandial lipid metabolism appear to result
from chronic effects of lacking CEL systemically rather than differences in the particles that carry the lipid. More dietary lipid was found in adipose tissue rather than liver in the knockout mice. Northern blot analyses of RNA from liver and various fat pads indicate that mRNA for fatty acid synthase and lipoprotein lipase is more abundant in adipose tissue in CEL-/- mice than in control mice. Additional experiments must be performed to confirm these differences in tissue distribution and gene expression. Because, metabolic differences between CEL-/- and control mice appear to result from chronic lack of CEL rather than acute differences in chylomicron properties, changes in liver cholesterol metabolism have been investigated as part of this aim. Data demonstrate that CEL plays a key role in selective uptake and hydrolysis of HDL cholesteryl esters and secretion of biliary cholesterol. These findings have led to a new area of investigation for which other sources of funding are
being sought.
Impacts
CEL has marked effects on triglyceride and cholesterol metabolism and is known to be polymorphic in humans. A thorough understanding of how CEL affects dietary triglyceride metabolism will facilitate the development of more appropriate strategies for treating and preventing obesity. Determining how CEL affects liver HDL, cholesterol, and bile acid metabolism will impact strategies for increasing reverse cholesterol transport for the treatment of cardiovascular disease.
Publications
- Camarota L.C., Chapman J.M., Hui D.Y., Howles P.N. (2004) Carboxyl Ester Lipase Cofractionates with Scavenger Receptor-BI in Hepatocyte Lipid Rafts and Enhances Selective Uptake and Hydrolysis of Cholesteryl Esters from HDL3. Journal of Biological Chemistry (second revision submitted)
|
|