Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Center for Biotechnology
Non Technical Summary
Conjugated linoleic acid causes dramatic fat loss in mice and adipocytes. Our goal is to understand the signaling pathways that cause this fat loss. We recently discovered that AMP-activated protein kinase (AMPK) is a critical step in the fat loss process and that a drug, metformin, that also activates AMPK, works to increase fat loss with conjugated linoleic acid. The proposed research seeks to further identify components of the fat loss response and how they work together with AMPK to achieve this. Results from this work could provide new methods of controling fat in humans and animals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
High calorie foods and inactive life styles are increasing the frequency of obesity, and increasing the associated health risks including diabetes, atherosclerosis and cardiovascular diseases [1]. Current estimates indicate the body weights of 66% of Americans are above their optimal weight and 32% are obese. Dieting, exercise, and developing the will power to resist consuming extra calories are valid methods to control weight but are difficult to effectively implement. Therapeutic drugs that help reduce adiposity are also needed to control weight gain, but only drugs of limited efficacy are currently available in the US. Conjugated isomers of dietary linoleic acid are found in dairy products [2]. Considerable research has established that the trans-10, cis-12 isomer of conjugated linoleic acid (t10c12 CLA) causes dramatic reduction in white adipose tissue (WAT) in mice. In mice on non-restrictive diets containing 0.5% t10c12 CLA, reductions of WAT of up to 75% occur within two weeks. However, t10c12 CLA has had limited effects in human trials [3]. Our research seeks to contribute to the discoveries of the mechanisms by which t10c12 CLA causes fat loss in mice and to use this information for applications for reducing adiposity in humans. Secondary effects of CLA-induced lipodystrophy have been seen but a slower rate of delipidation of adipose tissue was found to produce a healthier result [4]. In this latter study, a lower dose of the mixture of the c9t11 and t10c12 CLA isomers was used during a 35-day period in mice. As expected, this produced a smaller but still respectable 35% reduction in WAT without noticeable lipodystrophy. None of the other tissues examined, including the brown adipose tissue, pancreas, gastrocnemius muscle, liver, or stomach showed any difference in the weights of these tissues when compared to those from control animals. More importantly, the levels of glucose, insulin, insulin sensitivity, plasma fatty acids, and macrophage levels were all the same as control animals while adiponectin, leptin, and resistin levels showed modest reductions [4]. This study indicates a more gradual rate of WAT reduction is more consistent with normal physiological functioning. This research seeks to better define the AMPK-dependent mechanisms by which t10c12 CLA reduces WAT with the goal of using this information to predict how to better reduce adiposity in humans. The impact would be an important improvement in human health due to weight reduction through improved nutritional products. This research could also lead to improved methods for manipulating fat in meat/animal products. Outcomes: Measurements of output: Publications, symposia/meeting reports, conference presentations, seminars, and patents. Projected impacts: Improved methods for weight control
Project Methods
3T3-L1 cell model and AMPKα1β1γ1 cDNAs An early passage of 3T3-L1 cells were obtained from the Green laboratory [10], and cryopreserved after an initial propagation. These cryopreserved cells will be used as the source of cells, after brief propagation, for differentiation into adipocytes. We use a rapid five day differentiation procedure that produces adipocytes that are highly responsive to t10c12 CLA [10]. Our recent microarray analysis of the response of 3T3-L1 adipocytes and mouse WAT indicated these 3T3-L1 adipocytes are a good model for mouse WAT for the response to t10c12 CLA [10]. AMPKα1β1γ1 cDNA clones will be made from 3T3-L1 adipocyte mRNA, cloned, and sequenced. AMPKα1 clone will be mutated to construct the variants described below. Virus vectors for 3T3-L1 cells 3T3-L1 adipocytes are not transfected efficiently enough with the standard DNA transfection methods for the high efficiency transient assays this project will require. Most of the publications using D/N-AMPK or CA-AMPKα overexpression methods have used adenovirus vectors for gene delivery [15, 16]. For constructs we make, we plan to use the Invitrogen Gateway-based Virapower Adenovirus system. We will use an Ad/CMV/GFP vector as a control to verify this replication-defective adenovirus does not significantly affect the relevant adipocyte biology (one of the criteria will be the Ad/CMV/GFP infected adipocytes treated with metformin should produce the same amount of triglycerides as the metformin treated adipocytes). If adenovirus infection does significantly affect the adipocyte responses we will use the Invitrogen Virapower Lentiviral system for transient assays. In the unexpected event that neither virus system works efficiently, stably transformed cells lines will be made, screened and analyzed for high expression of the transgenes. The genetic treatment that produces the highest level of AMPK activity will be used in part IV.B. IV.A. Use a D/N-AMPK to test whether AMPK is necessary for the delipidation response An adenovirus vector expressing a mouse D/N-AMPK will be requested or constructed. If we are unable to obtain this vector, we will construct the D/N mutation [16] by site-directed mutagenesis using overlap PCR of the AMPKα1 cDNA, clone the product into a Gateway ENTR vector, sequence the resulting mutant, and recombine this into the Gateway-based Virapower Adenovirus system. High titer adenovirus stocks will be prepared on 293A host cells and titered on these cells. 3T3-L1 adipocytes will be infected with a 50 MOI of control GFP or D/N-AMPK adenovirus stocks on the fourth day of differentiation and then treated with t10c12 CLA on the following day. Triglyceride (TG) levels are normally reduced by 50% after 24 hrs of t10c12 CLA treatment in our experiments, and inhibition of AMPK activity by D/N-AMPK expression should produce higher levels of TG if AMPK is necessary for the response. The effectiveness of AMPK inhibition will be measured by the amount of phosphorylated ACC, which should decrease if there is less AMPK activity (e.g., see Figure 3).