Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Biochemistry
Non Technical Summary
The overall objective of our research program is to dissect the signaling pathways that are induced by treatment of AGEs in preadipoctes. The predicted results will contribute to understanding basic mechanisms of obesity. Based on such information, we might be able to come up better approaches to prevent or treat obesity in the future.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
It has been well-accepted that increased consumption of sugary drinks can contribute to obesity, a serious public health problem that increases morbidity, mortality and has substantial long term economic and social costs. In order to come up with better approaches to prevent unhealthy diet-associated obesity, we need to understand the underlying molecular mechanisms. The overall objective of my proposed research program is to dissect basic signaling pathways that are responsible for regulation of lipid metabolism. The expected results of the proposed research will contribute to understanding development of obesity.During thermal food processing, the cooking style widely used for Western food, carbonyl groups of reducing sugars and amino residues on proteins can generate advanced glycation end products (AGEs) via a non-enzymatic glycation browning reaction. A prolonged non-enzymatic glycation reaction between glucose and amino residues under a hyperglycemic condition can also form glycated proteins in mammalian cells. It has been documented that AGEs are involved in the development of aging-related diseases, such as diabetes and vascular diseases. In other words, it is the specific components, not the absolute amount of calories, that affect the fatty acid accumulation.At the molecular level, AGEs interact with and activate its receptors (RAGE), subsequently resulting in oxidative stress of cells. Thus, the so called "junk food" (like sugary drinks) components selectively elevate AGEs, which activate RAGE, causing increased oxidative stress and increased activation of lipid biosynthesis pathways compared to normal cells that are exposed to lower levels of AGEs in people eating healthier diets. The downstream pathway activated by RAGE and oxidative stress involves PI3 kinase and AKT, but the details concerning how these stimuli activate this pathway in adipocytes are not well understood. Our research program focuses on the role of Polo-like kinase 1 (Plk1), a critical regulator of many cell cycle-related events, in the RAGE signaling and its implication in obesity. We will use state-of-the-art molecular, cellular and biochemical approaches to study how Plk1 regulates adipogenesis, a cellular event that has to be accurately controlled to prevent obesity. Our preliminary data show that 1) activation of the RAGE pathway activates Plk1; 2) loss of Plk1 reduces lipid accumulation in adipocytes; 3) oxidative stress activates the PI3K/AKT/mTOR survival pathway, which has a documented role in lipid biosynthesis via the SREBP (the sterol regulatory element binding proteins) pathway; and 4) oxidative stress-induced activation of the PI3K/AKT/mTOR pathway is Plk1 dependent and that Plk1 is essential for lipid biosynthesis. Therefore, we hypothesize that elevation of Plk1 plays a critical role in AGEs-associated signaling pathways, thus eventual development of obesity. Our working model is as follows: accumulation of AGEs due to uptake of excess unhealthy food results in oxidative stress, which leads to elevation of Plk1. Increased Plk1 activates the PI3K/AKT/mTOR pathway, subsequently contributing to increased lipid biosynthesis via the SREBP pathway and development of obesity.To test our central hypothesis, we propose three aims.Aim 1: Validate Plk1 as a target for the RAGE pathway and dissect how Plk1 phosphorylation of PTEN activates the PI3K pathway. We will establish that Plk1 is a target for the RAGE pathway and then provide a detailed mechanism to understand how elevation of Plk1 contributes to activation of the PI3K pathway.Aim 2: Examine how Plk1-associated kinase activity regulates lipid biosynthesis via the SREBP pathway. The expected results will provide evidence to support the hypothesis that elevation of Plk1 plays a critical role in lipid accumulation.Aim 3: Test whether Plk1 elevation contributes to lipid accumulation in vivo. We will provide experimental data to show that overexpression of Plk1 results in lipid accumulation in mice.
Project Methods
Aim 1: Validate Plk1 as a target for the RAGE pathway and dissect how Plk1 phosphorylation of PTEN activates the PI3K pathway1.1. We asked whether Plk1 is involved in the RAGE pathway. For that purpose, we exposed 3T3-L1 murine preadipocytes to glycated BSA during adipogenesis, and found that Plk1 was elevated upon GA-BSA treatment, suggesting that Plk1 might be a target in the RAGE pathway (Fig 1A).More significantly, depletion of Plk1 inhibits adipogenesis as indicated by a reduced level of total lipid in 3T3-L1 cells (Fig 1B). We will further validate these important observations by the following experiments. First, 3T3-L1 preadipocytes will be treated with 300 ?g/ml of GA-BSA during an 8-day period of adipogenesis, and harvested for anti-RAGE Western blotting to make sure the RAGE pathway is activated. Second, 3T3-L1 cells will be depleted of RAGE with lentivirus-based RNAi, treated with GA-BSA during an 8-day period of adipogenesis and harvested for anti-Plk1 Western blotting to test whether depletion of RAGE abolishes GA-induced elevation of Plk1. Third, because activation of the RAGE pathway causes increased oxidative stress, we want to directly test whether oxidative stress also drives activation of Plk1. 3T3-L1 cells will be treated with H2O2 or diamide and harvested for anti-Plk1 Western blotting. Fourth, because NF-kB has a well-established role in oxidative stress-induced cellular response, we will ask whether NF-kB affects AGE-associated activation of Plk1. 3T3-L1 cells will be treated with GA-BSA, in the presence or absence of NF-kB inhibitor PDTC, during an 8-day period of adipogenesis and harvested for anti-Plk1 IB.1.2 Plk1 phosphorylates PTEN at S385. To understand how Plk1 regulates the PI3K pathway and lipid biosynthesis (Fig 1B), we identified PTEN as a Plk1 substrate (Fig 2A) and mapped S385 as the phosphorylation site (Fig 2B). Plk1 phosphorylation of PTEN at S385 in vitro was confirmed by anti-phospho-PTEN-S385 immunoblotting (Fig 2C). Moreover, the level of pS385 (phospho-PTEN-S385) was inhibited by BI2536, indicating that phosphorylation of PTEN-S385 is dependent on Plk1 activity in vivo (Fig 2D). Considering that CK2 is a PTEN kinase in vitro, we then depleted either Plk1 or CK2 with RNAi in cells expressing GFP-PTEN. Depletion of Plk1 significantly reduced the signal of pS385, confirming that Plk1 is indeed a kinase responsible for phosphorylation of PTEN-S385. In contrast, CK2 depletion did not affect the level of pS385, but slightly reduced the signal of pS370 (phospho-PTEN-S370), suggesting that CK2 might phosphorylate PTEN-S370, but not S385 (Fig 2E). Further, anti-pS385 identified the phosphorylated PTEN in mitotic cell lysates, but not from Plk1-inhibited cells, suggesting that endogenous PTEN is phosphorylated at S385 by Plk1 (Fig 2F).Nedd4-1 is the major E3 ubiquitin ligase that controls PTEN stability, as Nedd4-1-associated ubiquitination results in degradation of PTEN (Trotman et al., 2007). Furthermore, Ndfip1 enhances PTEN ubiquitination by Nedd4-1 via direct binding to PTEN (Figure 3A). Thus, we will test several possibilities to dissect how Plk1-associated phosphorylation might regulate PTEN.First, we will ask whether Nedd4-1/Ndfip1 binding of PTEN is affected by various mutations at S385 in a cellular context (i.e., in the presence of other proteins). Accordingly, 3T3-L1 cells will be infected with retrovirus expressing different forms of PTEN (WT, S385A, S385D), and harvested for anti-GFP immunoprecipitation (IP), followed by immunoblotting (IB) with antibodies against Nedd4-1 and Ndfip1. Second, we will ask whether Plk1 phosphorylation of PTEN regulates its ubiquitination. Cells expressing various GFP-PTEN constructs will be transfected with His-ubiquitin, treated with a proteasome inhibitor MG132, and harvested for anti-GFP IP, followed by anti-ubiquitin IB. To further confirm that Plk1 regulates PTEN stability, cells expressing GFP-PTEN constructs will be treated with cycloheximide, an inhibitor of protein translation, for different times, and harvested for anti-GFP IB to measure half-lives of different GFP-PTEN proteins. Further, we will ask whether Plk1 phosphorylation of PTEN at S385 controls the PI3K pathway. Accordingly, cells will be expressed with different GFP-PTEN constructs and harvested for IB against p-AKT and p-S6.Aim 2: Analyze how Plk1 affects lipid biosynthesis via the SREBP pathway.First, if PTEN is the major Plk1 target in regulation of lipid metabolism, overexpression of PTEN is expected to rescue Plk1 overexpression-induced increased lipid accumulation. Accordingly, 3T3-L1 cells will be infected with adenovirus expressing Plk1, re-infected with retrovirus expressing different forms of GFP-PTEN (WT, S385A, S385D), and harvested for Oil Red O staining to follow the levels of total lipids or measurement of cholesterol levels with Raman spectroscopy. Second, we will ask whether PTEN depletion rescues inhibition of Plk1-induced metabolic phenotypes. 3T3-L1 cells will be depleted of PTEN with lentivirus-based RNAi, treated ± BI2536 for 60h, and harvested for measurement of total lipids or cholesterol as described above. Third, to probe the mechanism, cells will be infected with adenovirus expressing Plk1, re-infected with retrovirus expressing different forms of PTEN (WT, S385A, S385D), and harvested for IB against proteins that play key roles in regulating lipid synthesis (FAS, fatty acid synthase) and cholestderol metabolism, namely those involved in (i) cholesterol homeostasis: SREBP-1a, -1c, and -2; (ii) cholesterol uptake: LDL receptor (LDL-R); (iii) cholesterol de novo synthesis, HMG-CoA reductase (HMGCoA-R); (iv) cholesterol efflux, ABCA1; (v) cholesterol esterification, Acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1); and (vi) CE hydrolysis, nCEH.Aim 3: Test whether Plk1 affects lipid biosynthesis in vivo.We reason that if PTEN is the major mediator in Plk1 expression-induced metabolic phenotypes, Plk1 transgenic mice will have similar metabolic defects to PTEN knockout mice. To test this possibility, we have recently generated Rosa26-CreER/PTENflox/flox mice by crossing Rosa26-CreER mice with PTENflox/flox mice. Upon tamoxifen injection of adult mice, we will obtain PTEN-/- mice. In addition, we also generated LSL-Plk1 transgenic mice (WT and T210D) (Fig 4), which will be crossed with Rosa26-CreER mice to induce Plk1 expression. To ensure we have enough control and treatment groups to reach a solid conclusion, six groups will be included as we will have three genotypes (Plk1tg, Plk1-TDtg, PTEN-/-). Based on previous experience with power analysis, 12 mice per group are required to reach statistical significance. We also need to compare mice at different ages (5 months vs 20 months). Thus, we will need 12 x 6 x 2 = 144 mice for this part. We will perform the following metabolic analyses. First, we will examine body composition by determining percentages of fat and lean mass by EchoMRI. Second, we will ask whether both Plk1 overexpression and PTEN knockout affects energy expenditure in a similar fashion. In brief, activity, food consumption, oxygen consumption rate (VO2), carbon dioxide release (VCO2), respiratory exchange ratio (RER; VCO2/VO2), and energy expenditure per kg of body weight will be measured using the Oxymax/CLAMS system. Studies will be started after 2 to 4 days of acclimation to the metabolic chamber using an air flow of 1 l/min. VO2 will be measured in individual mice at 15-min intervals during a 48-hr period and normalized with respect to body weight. Activity will be measured on x, y, and z axes by using infrared beams to count the beam breaks during a specified measurement period.