Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
NUTRITIONAL SCIENCES
Non Technical Summary
The prevalence of obesity has reached epidemic proportions in the U.S. The rising rate in obesity is alarming because obesity poses a major risk for many serious diet-related chronic diseases, including diabetes, cardiovascular disease, stroke, and certain types of cancer. In addition to financial and emotional tolls on individuals, obesity and its associated conditions have a significant economic impact on the U.S. health care system. Wisconsin is no exception in the obesity trend and is not free from its associated costs. The medical expenditures attributable to obesity in Wisconsin alone were estimated to be as high as 1.5 billion dollars, even though this estimate did not include indirect costs such as absenteeism and decreased productivity attributed to obesity and related diseases. Obesity is characterized by excess body fat resulting from energy imbalance, in which more calories are consumed than are expended over a period of time. The extra energy not used is
then metabolized and accumulated as body fat. A variety of genetic, behavioral, and environmental factors contribute to energy imbalance, and the causes of the obesity epidemic are complex. One major factor is the overconsumption of food high in fat, which has a high energy density and, after absorption, can be readily converted to body fat for storage. Our laboratory aims to advance the understanding of fat metabolism and energy balance, focusing on an enzyme involved in the absorption of dietary fat. The absorption of fat involves several steps that are not well characterized at the molecular level. Molecules involved in fat absorption may be targets for preventive or therapeutic interventions. We have recently identified one such molecule, MGAT2. After generating mice lacking the enzyme, we showed that MGAT2-deficiency delays the absorption of dietary fat, increases body temperature, and promotes energy expenditure. These findings suggest that MGAT2 may prove to be a useful target
for nutrition or drugs to combat human obesity. However, many important issues need to be addressed, such as what functions MGAT2 has in tissues besides intestine and whether blocking MGAT2 causes undesirable outcomes. MGAT2 is also highly expressed in human liver and its functions there are not known. We hypothesize that MGAT2 in the liver is involved in the making of lipoproteins, which carry fats, including triglyceride and cholesterol, to appropriate tissues for metabolism. Disorders in the making of these lipoproteins lead to common human diseases, such as hyperlipidemia and fatty liver disease. We will test our hypothesis using both cultured cells and genetically modified mice. The results from this project will contribute to our understanding of fundamental processes involved in lipoprotein assembly and secretion and help to determine whether MGAT2 is a feasible pharmaceutical target for modulating fat absorption and treating obesity and related diseases.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to determine the physiological functions of the triacylglycerol (triglyceride) synthesis enzyme monoacylglycerol acyltransferase 2 (MGAT2) in the liver. We have recently cloned and identified this enzyme believed to be important for the absorption of dietary fat, a major source of metabolic energy, fatty acids, and other essential nutrients. After generating knockout mice deficient in this enzyme, we found that MGAT2 deficiency delays fat absorption and reduces metabolic efficiency in response to dietary fat. Mice lacking MGAT2 did not gain weight like normal mice, while consuming a similar amount of a high-fat diet, and were protected from diet-induced obesity. Inhibiting intestinal MGAT, thus, may prove to be a useful intervention to combat human obesity. However, unlike mouse MGAT2, which is primarily expressed in the small intestine, human MGAT2 is more highly expressed in the liver. The functions of MGAT2 in the liver are not
known. Both the liver and the small intestine secrete the triacylglycerol-rich lipoproteins [very low density lipoprotein (VLDL) and chylomicron, respectively]. These lipoproteins are crucial in the transport and metabolism of dietary and endogenously generated lipids, including triacylglycerol and cholesterol. Disorders in the metabolism of these lipoproteins lead to common human diseases, such as hyperlipidemia and fatty liver disease. We hypothesize that hepatic MGAT2 mediates the resynthesis of triacylglycerol channelled to VLDL assembly and secretion. We will test this hypothesis with two aims: 1) using liver cell lines, we will determine if expression of MGAT2 correlates with triacylglycerol synthesis and VLDL secretion; 2) using transgenic mice, we will determine if expressing MGAT2 specifically in liver promotes triacylglycerol synthesis for VLDL secretion. The results from this project will contribute to our understanding of fundamental processes involved in lipoprotein
assembly and secretion and help to determine whether MGAT2 is a feasible pharmaceutical target for modulating fat absorption and treating obesity and related diseases.
Project Methods
We have recently cloned and identified MGAT2, providing the information to generate molecular tools needed to define the physiological functions of MGAT2 in the liver. Specifically, we will test the hypothesis that MGAT2 mediates the resynthesis of triacylglycerol in the liver and that the resynthesized triacylglycerol is channelled to lipoprotein (VLDL) assembly and secretion. In Aim 1, we will determine if MGAT2 expression correlates with triacylglycerol synthesis and secretion using hepatic cell lines. We will measure the levels of MGAT2 expression and MGAT activity in various cell lines, including human HepG2 cells and rat FTO2B cells and determine if MGAT2 expression is induced when these cells are exposed to fatty acids, a condition that promotes triacylglycerol synthesis and VLDL secretion. We will also establish stable cell lines that overexpress human MGAT2 by transfecting CMV-based plasmid vectors (pIRESneo2) and selecting with neomycin (the plasmid has an
internal ribosomal entry site and expresses neomycin resistance in clones that are successfully transfected). We will select low-expressing and high-expressing clonal cell lines to compare with control LacZ-transfected cells. The amount of triacylglycerol synthesis and secretion in stable cell lines will be assessed by metabolic labeling in cultured cells. In Aim 2, we will determine if expressing MGAT2 specifically in the liver promotes hepatic triacylglycerol resynthesis and VLDL secretion using genetically modified mice. We will generate transgenic mice that overexpress human MGAT2 in the liver and their wildtype littermate controls and compare them for VLDL secretion and hepatic lipid accumulation, and fatty acid oxidation under two conditions associated with hepatic steatosis. Since starting our laboratory at UW-Madison, we have made the construct for driving human MGAT2 expression specifically in the liver. This construct contains the promoter and the liver-specific enhancer
from the human apoE gene locus, which was developed by Dr. J. Taylor at the Gladstone Institutes and has been used for liver-specific expression of many genes. The construct was microinjected into the pronucleus of fertilized single-cell embryos and implanted into pseudo-pregnant female mice. Offspring will be screened by Southern analysis and PCR. These results will be primarily disseminated to other scientists through peer-reviewed publications and presentations at national and international conferences.