1a. Objectives (from AD-416)
1. Define the role and mechanisms of adipocyte death in obesity-associated inflammation and metabolic disorders using genetic and nutritional models of adipocyte growth and death. 2. Determine the role of the macrophage in modulating adipocyte death and associated adipose tissue inflammation using genetically altered animal models. 3. Determine the mechanisms by which alterations in Lipid Droplet (LD) proteins modulate lipolysis and risk of developing metabolic disorders.
1b. Approach (from AD-416)
The role of adipocyte death in obesity will be investigated using a combination of transgenic and knockout mouse models and bone-marrow transplantation in mice fed different diets to understand the influence of obesity. In vivo and in vitro studies will investigate glucose and insulin homeostasis complemented by histological, immunohistological, electron microscopic, gene expression, FACS analysis, adipocyte lipolysis and Akt signaling studies. For studies investigating lipid droplet proteins, we will use both adenovirus expression vectors and possibly transgenic animals to determine how alterations in expression and intracellular signaling regulate protein expression, metabolic pathways, and lipolysis in cultured cells and animals. Depending upon which tissue is studied, we will examine lipolysis and protein expression, alterations in cytokine, lipid accumulation, signal transduction pathways, and oxidative gene expression.
3. Progress Report
Performed studies investigating our line of FAT-ATTAC mice in which we can induce in mice the expression of a protein, which induces cell death (apoptosis).Importantly, we confirmed that the expression of the protein that induces fat cell (adipocyte) death was substantially adipocyte specific which allowed us to move forward without performing bone marrow transplantation procedures in our studies. We also established conditions where the rates of fat cell death in FAT-ATTAC mice were comparable to that which occurs with diet-induced obesity (DIO) in mice.We then isolated the immune cells called macrophages, which accompanied fat cell death in the fat tissue of FAT-ATTAC and DIO mice. We are presently analyzing the data from these studies. Another mouse model, which we proposed to study, was a line of aP2-alpha2-AR transbeta3 AR-/- mice, which was reported to become obese due to increase numbers of small fat cells. In two separate studies we have not been able to replicate the published study that this line of mice develops obesity with increased numbers of small fat cells. For objective 2, we have established a colony of backcrossed Ikbkbdelta myeloid mice in which macrophage inflammation is reduced which we can now use for our proposed studies in year 4. In objective 3 of our plan we proposed to study how proteins found at the surface of stored fat (called lipid droplets) in fat cells regulate fat storage and breakdown. We have investigated whether an important lipid droplet associated protein called perilipin protein expression was regulated postranslationally by the activation of an intracellular signaling pathway involving the protein, Akt. We had proposed that Akt modifies perilipin at amino acid 385 (a serine) to regulate its expression. We have not been able to demonstrate that serine 385 is important for perilipin protein expression. We continue to study the role of serine 385 in perilipin’s action to regulate fat cell metabolism. We did generate a mouse that had increased levels of perilipin in fat cells (adipocytes). Remarkably we have found that increased expression of perilipin reduced fat cell size and fat accumulation, provides protection against becoming obese on a high caloric diet, and increases the expression of genes and protein that burn fat within fat cells. Additionally we have been investigating the lipid droplet associated protein FSP27, developing tools to increase its expression or reduce its expression, and a protein that breakdowns fat in hepatocytes called adipose tissue triglyceride lipase (ATGL). Previously we noted that a reduction in ADRP protein expression in liver cells resulted in reduced fat accumulation and increased expression of fat burning genes. ADRP protein expression as well as FSP27 may block the actions of the lipase ATGL, which breakdowns and increases fat burning. Using all of the above tools and models, we have been able to demonstrate that a reduction in ATGL in liver cells increases accumulation of fat and that increased expression of ATGL resulted in increased burning of fat and reduced fat accumulation.
1. Factors Identified in Cells that Protect Against Diet-induced Obesity. Since high caloric diets promote obesity in animals and humans ARS-funded researchers at JMUSDA-HNRCA at Tufts University, Boston, MA, identified factors or proteins in cells that will protect against diet-induced obesity. We challenged two different lines of mice with a high caloric diet, one with a lower and the other with a higher expression of the protein, perilipin, in fat cells. They found that mice with increased expression of the protein perilipin in their fat cells (adipocytes) were protected against the development of obesity as compared to mice with reduced expression of perilipin. They demonstrated that increased levels of perilipin in fat cells resulted in increased rates of fat burning and increased levels of proteins that promote burning of fat. These studies indicate that researchers should continue efforts at identifying nutrients and foods that increase perilipin protein expression in fat cells to help ameliorate the epidemic of obesity.