METABOLIC CORE LABORATORY
I. Description: The main services of the Metabolic Core are gene expression analysis using real-time quantitative PCR and tissue procurement/preparation and processing for analysis of phosphorylation and expression of key post-receptor insulin signaling proteins that regulate insulin sensitivity and lipid metabolism at the cellular level. The Core also provides selected assays for specific hormones and metabolites. This Core provides an array of assays over a broad range of species (rat, mouse, human, sheep) and tissue types (fat, muscle, liver), for multiple investigators studying the clinical consequences of nutrition-related disorders. The Metabolic Core lab also provides members of the Center with reagents, access to equipment, and exposure to new techniques (insulin clamping, insulin signaling, real-time PCR) that have helped to expand the quantity and quality of nutrition research in the CNRU. The services of the Metabolic Core Laboratory have been used by many investigators to extend their research beyond the specific hypotheses proposed in their RO1 grants. For example, many investigators who did not previously assess insulin action in their own studies have taken advantage of this core laboratory to obtain these measures using the insulin signaling assays. Thus, the Metabolic Core of the Nutrition Center achieves an overall cost-savings and provides more important investigator time in their own laboratories for their own experiments. II. Organization and Description of Services: The Core services the needs of the CNRU on a reduced fee-for service basis including:
New investigators, particularly CNRU pilot awardees, are encouraged to consult with Dr. Friedman in advance to help plan experiments involving the services listed above. The Metabolic Core lab is also a resource for providing expertise and technology for analysis of genetically defined mouse and rat models relevant to nutrition research. We provide experimental support and technical advice regarding measurement of insulin sensitivity in vivo (insulin clamps) and end organ-based metabolism (e.g. glucose and fatty acid uptake, oxidation, and synthesis) and metabolic biochemistry (cytokines, receptor proteins) and gene expression (RNA analysis) These services promote interaction between investigators and foster multidisciplinary research training in clinical nutrition and obesity across the UCD campus. Facilities: The Metabolic Core Laboratory is located in the Center for Human Nutrition within basic science Division of Adult Endocrinology in Dr. Friedman’s laboratory (Research Complex 1, 7 th floor at Anschutz Medical Campus). The laboratory is 1150 sq. ft. and includes all of the major equipment needed for research in molecular nutrition (listed below). The lab is adjacent to a satellite animal facility set up for performing chronic animal surgeries, energy balance, and hormone infusion studies in mice. This section of the lab includes a general work area, surgery set-up, lamps, Harvard infusion pumps and metabolic chamber. Within the PI’s laboratory is a designated area with water bath for performing glucose transport studies. Across the hallway is a 150 sq. ft. tissue culture facility used for growing human and non-human mammalian cells, including tissue culture supplies, light microscope, CO 2 incubators, liquid nitrogen storage tank, sink, and two laminar flow hoods. The following major items of equipment are within the PI’s lab: real-time PCR detection system, automated RNA and protein analysis system, two PCR thermal cyclers, high-speed refrigerated centrifuge, microfuges, horizontal and vertical gel electrophoresis systems and power supplies, gel documentation camera equipment and computer software for image quantification, UV spectrophotometer, walk-in cold room, three -20C freezers, and two ultra-cold -80C freezers for storing biological samples. The following major pieces of shared equipment are located on the floor of the PI’s laboratory: Several refrigerated ultracentrifuges and high-speed rotors, Beckman liquid scintillation counter, autoclaves, film developer, fluorometer, luminometer, and distilled water purification system. The PI shares the maintenance contract on these items with other members of the department and has full access to this equipment.
III. Management of Core Operation – Cost Effectiveness/Quality Control: The extent of resource support requested by the investigator is discussed with the PI during the planning stages of the project and reviewed at a meeting with the PI prior to initiation of the project. The extent of support provided by core staff will depend largely on the type of extramural support available for the project, with priority given to those projects for which funds for personnel expenses are limited (pilot/exploratory studies, career development awards, etc). Detailed plans for prioritizing samples are based on the source of funding and the type of award, as follows:
The mechanisms for monitoring budgetary overlap of current funded projects and the Metabolic Core lab are handled by Dr. Friedman in consultation with other core directors. This Core continues to provide services at no cost to new investigators without grant funds and at subsidized rates for pilot awardees in order to help them compete at the national level for independent funding. The Core lab thus provides members of the Center with reagents, access to equipment, and exposure to new techniques that have expanded the quantity and quality of nutrition research in the CNRU. Because CNRU investigators continue to submit new grant applications that propose the use of the core facility, we suggest that core activity be planned in advance whenever possible. Consultation /Research training: Quality control is part and parcel of the services provided by this core. Core personnel are actively involved with investigators in providing assistance with the selection of assessment methods and the design of experimental protocols to insure the samples we receive are of high quality. Investigators receive assistance in determining the most appropriate assay for a specific research study. In helping each investigator to decide on which assay is best to use, the following issues are addressed: 1) appropriateness of assay to the study, 2) appropriateness of a given assay to the scientific question, and 3) cost. To insure the samples we receive are treated appropriately, when assaying insulin sensitivity in human muscle biopsies for example, we routinely send an assistant with the investigator to ensure the first biopsies are properly dissected, frozen immediately, or placed in RNAlater for future use. For animal studies, we make sure the time of fasting is controlled for, and that to the extent possible, hormone treatments are carried out in conjunction with assay of proteins to examine phosphorylation patterns. In terms of assay quality control, for hormones we assay known standards to ensure our inter-assay and intra-assay variability is within 5%. For Western blotting, we use internal controls (either manufacturer’s standard or excess sample from the study) on every blot to account for inter-blot variability, and use GAPDH as a loading control to control pipeting error and/or transfer of protein. For gene expression analyses, we run all quantitative real-time PCR reactions in duplicate and normalize the data to reference genes.
Service information: Click here for our current Metabolic Core price list. List of Reagents: For a working list of antibodies available in the Friedman laboratory, click here. We have experience with performance of most if not all of these antibodies. After viewing the list you may want to refer to the link to the Manufacturer's web site that is provided. For a working list of real-time probes and primer sets available in the Core, click here . If you are interested in any other proteins or real-time probes not on these lists please contact Dr. Friedman at the number provided. Contact: Jacob E. (Jed) Friedman, Ph.D. For body composition/calorimetry and assays of LPL and HSL, contact: Robert Eckel, MD
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