UCSD-UCLA Metabolic and Molecular Physiology Core

Overview

Novel animal models that closely phenocopy the pathobiology of diabetes and its complications in humans are rapidly increasing in number. These models are critical to understanding the etiology and progression of diabetes and the underlying mechanisms contributing to diabetes complications. Additionally, these genetically engineered model systems provide a platform for the development and testing of new therapeutic strategies to combat metabolic dysfunction for subsequent translation into human use. Among experimental models, mice are unrivaled in offering researchers the power of sophisticated mammalian genetics, particularly the ability to overexpress or ablate genes in a tissue-specific manner. These genetic models allow investigators to interrogate the contribution of gene dosage, the influence of epigenetics, and epistatic interactions among multiple genes to physiological/pathophysiological function. Moreover, recent advances in academic drug discovery have depended critically on both genetic and dietary mouse models of diabetes and obesity. At the same time, recent analyses of diet-induced changes in metabolism coupled to specific genetic models, have allowed for a renewed analysis of interaction between environment or diet and genetics. Development of novel models as well as maximal exploitation of existing models of metabolic dysfunction/type 2 diabetes demands comprehensive, reproducible, as well as innovative phenotyping strategies. Services provided by the Metabolic and Molecular Physiology Core (MMPC) encompass whole animal, tissue and cellular metabolism, insulin action, and inflammatory signaling as well as cardiac, vascular and renal phenotyping associated with diabetes complications research. We also specialize in administering drugs and other substances to mice by different routes in a well-controlled setting.

The primary objectives of the MMPC are to provide DRC investigators with thorough scientific consultation and timely, accurate, and easily accessible mouse, tissue, and cellular phenotyping.

Core goals include the following.

1. Meet scientific objectives in a timely, cost effective, and integrated manner individualized to the specific needs of the investigator
2. Provide sensitive, accurate measurements with superior quality control
3. Maximize organ phenotyping in any specific model, documenting multiple organ damage/tissue cross-talk
4. Expand organ/tissue/primary cell bank for greater use among other DRC users
5. Develop and expand capabilities of existing technology, and
6. evelop, validate, and evolve novel analyses based upon DRC member needs.

Importantly, we have regularly surveyed our users about the quality and timeliness of services, and sought input from investigators about future needs and services. We will continue these surveys on an annual basis and utilize these findings to guide decisions on core innovation, evolution, and budget

Services

Sub-Core A. Insulin Sensitivity and Metabolism (Hevener)

A1. Body Composition.

Body composition can be assessed either by DEXA (Lunar PIXImus Densitometer, GE Medical Systems) or MRI (EchoMRI 3 in 1 Body Composition Analyzer) (25-27). The MRI instrument can also estimate tissue total lipid composition ex vivo for investigators who require immediate and inexpensive analyses in an easily excised organ or tissue bed (25).

A2. In vivo Insulin Sensitivity. Insulin tolerance: IP-ITT or IV-ITT with 2-deoxyglucose.

The insulin tolerance test is a non-terminal test to assess whole body insulin sensitivity. To determine the rate and site of glucose disposal, [3H]-2-deoxy-d-glucose (10µCi/mouse) is infused IV (jugular cannula) into the mouse in combination with insulin. Tissue glucose uptake is determined by the rate of disappearance of 2-[3H]-deoxyglucose from the circulation along with counts of phosphorylated 2-deoxy-D- [2-3H] glucose in individual tissues. Tissues are harvested for post-insulin signal transduction analyses by immunoblotting. Pyruvate tolerance tests and liver perfusion studies to assess gluconeogenic capacity can be performed upon request. Additionally, radiolabeled glucose uptake assays can be conducted under thermal challenge to assess substrate demand in rodent tissues including brown adipose.

Euglycemic-hyperinsulinemic clamp. The euglycemic-hyperinsulinemic clamp technique is the gold standard method for quantification of in vivo insulin sensitivity. Radiolabeled glucose is used to determine tissue insulin sensitivity (muscle, liver, adipose tissue) between genotypes of mice under various dietary conditions. Mice are chronically catheterized using dual lumen cannulas surgically placed into the right jugular vein (25-26, 28-29). Seventy-two hours after surgery glucose turnover is measured at basal and during hyperinsulinemia. The glucose infusion rate (GIR) is determined by the amount of exogenous dextrose necessary to maintain euglycemia. The insulin-stimulated glucose disposal (IS-GDR) and hepatic glucose production (HGP) rates are determined using the Steele equation (30). All individual raw data as well as mean ± SEMs for basal and clamp glucose concentration, clamp circulating plasma and infusate insulin concentrations, basal glucose turnover rate, as well as steady state GIR, IS-GDR, and HGP will be provided. Tissues are harvested post-clamp and insulin signal transduction is assessed by immunoblotting as described (25-26, 28-29).

A3. Ex vivo Tissue Metabolism and Insulin Action.

To quantify insulin action in skeletal muscle, extensor digitorum longus (EDL), tibialis anterior (TA), and soleus (SOL) muscles are harvested from mice (4-6 h fasted condition) and incubated in 2-deoxyglucose in the presence (stimulated) or absence (basal) of physiological insulin and [3H]-2-deoxy-d-glucose (25, 31). Small molecule compounds and hormones (e.g. AICAR, Metformin, SIRT modulators, estradiol, isoproterenol) can be added to the incubation media to challenge metabolism in this controlled setting. A similar approach can be performed on white and brown adipose tissue sections.

A4. Cellular/Molecular Metabolism and Insulin Action.

Skeletal muscle satellite cells are isolated from genetically engineered mice or from human muscle biopsies and differentiated to myotubes. Primary cells can also be obtained from adipose tissue beds (white or brown) and liver. Assays to investigate substrate metabolism and insulin action (insulin-stimulated 2-deoxyglucose uptake and insulin signal transduction) are performed on primary cells under specified conditions (25, 29). In addition, primary cells can be transferred to the Mitochondrial Biology Sub-core for assessment of oxygen consumption by Seahorse Biosciences technology. These techniques to assess insulin action and metabolism are also routinely performed on standard cell lines genetically altered by viral-mediated knockdown approaches. Cellular crosstalk assays to determine whether secreted factors can induce altered metabolism or insulin action of a second cell type can be performed by special request (25, 29). Furthermore, conditioned-media from primary cells can be harvested and transferred to the Inflammatory Signaling Sub-Cores or Core E – TPAC led by Julian Whitelegge and Edward Dennis for factor/lipidomics identification by mass spectral analysis.

Sub-Core B. Oxidative Metabolism in Animals and Tissues (Hevener)

B1. In vivo Oxidative Metabolism and ROS Assessment.

Indirect calorimetry to assess oxygen consumption/caloric expenditure and substrate utilization, as well as movement patterns and food and water consumption during light and dark cycles is performed on mice using Columbus Instruments Oxymax metabolic chambers (normalized to body weight and lean body mass as required). Animals are acclimated for 24 h prior to a 48 h testing period (25-26). ROS can be assessed in tissues by fluroescent probe labeling and posttreatment quantication by fluroescence imaging.

B2. Ex vivo Oxidative Metabolism and ROS Assessment.

Substrate oxidation and ROS assays are performed on small tissue explants and isolated muscle (EDL, soleus or TA) using newly acquired high-resolution respirometry (Oroboros), or in primary myocytes or mitochondrial suspensions prepared according to the methods of (32) with modifications described by Koves et al. (33) and Kim et al. (34). Oxidation rates are determined by radioactive 14CO2 counts assessed by liquid scintillation counting and substrate deposition (esterification and glycogenesis) determined by the tracer incorporation into the storage pool (13, 25).

B3. Exercise Capacity and Performance.

The MMPC has recently acquired 24 mouse running wheels, a rodent treadmill, and instrumentation to test muscle strength and muscular endurance (active hanging and ladder climbing). Dr. Hevener and her team have extensive experience training rodents and are able to deliver comprehensive data sets on metabolic responses to acute and chronic exercise. Tissue/cell fixing and sample delivery to either UCSD or UCLA Histopathology Shared Resource or the UCLA, UCSD EM Core, UCLA BRI or UCLA Mitochondrial Biology Core can also be performed by the MMPC. Reduced fees for microscopy core training and services performed by the UCLA-BRI have been negotiated for all UCSD-UCLA DRC members. Furthermore, Dr. Hevener has obtained numerous tools to assess additional aspects of mitochondrial function and turnover/mitophagy, and these procedures and reagents can be made available upon request.

Sub-Core C. Mitochondrial Biology and Metabolism in Cells (Shirihai)

C1. Cellular Oxygen Consumption.

The Extracellular Flux Analyzer (3xXF-96 and 3xXF-24 Seahorse Biosciences/Agilent Technologies) allows for measurements of oxygen consumption rates (OCR) and extracellular acidificiation rates (ECAR) in a 24 or 96-well plate format in intact cells, permeabilized cells, spheroids, and isolated mitochondria. Intact cell respirometry – the most commonly used respirometry assay is completed in intact cell lines and primary cells. This assay provides first pass information on the changes in mitochondrial function in response to compound treatment, nutrient modifications, or genetic alterations. Isolated mitochondria respirometry – provides more mechanistic insight into the mitochondrial phenotype in response to treatments. Mitochondria are isolated from animal tissues or cell lines immediately before running the respirometry assay. Spheroids or permeabilized cell respirometry in permeabilized cells allows for a more mechanistic approach while maintaining cellular architecture. This assay will be used for more in-depth studies and requires permeabilization reagent exclusively offered through Seahorse Biosciences. Spheroid respiration requires a specialized plate that is available to run assays on 3D cell structures (e.g., pancreatic islets), in order to maintain a more physiological cellular environment.

C2. Cellular Imaging.

The Operetta allows for high content screening, with confocal capabilities, of mitochondrial physiology. The ability to use the Operetta to increase throughput decreases the time and effort involved with running these assays compared to more traditional imaging systems. Additionally, since the Operetta fits on the bench-top, it can be used in the same location as other Core instruments, thus, increasing efficiency. The Operetta also offers specialized Harmony imaging software for improved data analysis.

• Mitochondrial morphology/mass/mitochondrial membrane potential – These parameters are determined with mitochondrial-targeted dyes and proteins for example MitoTracker Green or mitochondrial-targeted GFP. The mitochondrial morphology/mass will be imaged and quantified. Mitochondrial membrane potential will be measured and quantified using mitochondrial-target dyes that are membrane potential –dependent (i.e. TMRE).
• Reactive Oxygen Species/Redox – Depending on the study design and goal, these measurements will be conducted with fluorescent dyes (such as DHE) or more specific genetically-encoded redox probes.
• Turnover – Depending on the study design and goal, this will be conducted with fluorescent dyes (such as lysotracker) or more specific genetically-encoded redox probes (LC3-GFP or mCherry-GFP-Fis).
• Advanced service – includes combined imaging of morphology, mass, mitochondrial membrane potential, and mitochondrial membrane potential heterogeneity. This will employee the combined use of mitochondrial-target dyes that are both membrane potential –dependent (TMRE) and –independent (MTG).

C3. Biochemical Assays.

ATP synthesis measurements can be performed using a Bioluminescence assay run on isolated mitochondria in a plate reader. Total cellular ATP and other high energy substrates can also be measured following cell lysis. The MMPC can also coordinate additional metabolite analyses by providing samples to the UCLA Metabolomics Shared Resource.

Sub-Core D. Inflammatory Signaling and Diabetes Complications (Hevener)

D1. Luminex and MSD MesoScale Multiplexing Assays.

The Inflammatory Signaling sub-core offers a broad range of reliable and sensitive assays to meet the needs of the diverse DRC membership. In order to provide the most value to MMPC users, sample analyses are performed with either MSD MesoScale or Luminex microsphere-based multiplex assays, using standardized quality-controlled biomarkers. The MSD MesoScale QuickPlex instrument offers an expanded broad range, highly sensitive, low sample volume assessment of analytes in biological samples including plasma, tissues, culture media, and cell lysates. This instrument can replace standard ELISA and immunoblotting assays for assessing circulating factors (e.g., hormones, chemokines, and cytokines) and cell or tissue proteins/protein signaling events. Additionally, MSD offers assay development reagents and plates suitable for immunogenicity, PK (pharmacokinetics), serology, and cell binding. MSD provides an open technology platform that allows researchers to develop novel singleplex or multiplex assays quickly using their own in-house or commercially available capture/detection antibody pairs. This is particularly useful for researchers wanting to develop customized multiplex assays or wishing to convert ELISAs or other binding assays to the MSD platform. MSD uses MULTI-ARRAY technology combined with electrochemiluminescence to bring speed and high density of information to biological assays. In combination with MULTI-SPOT plates, this technology enables precise quantitation of multiple analytes in a single sample requiring less time and effort than other assay platforms. Luminex 100 xMAP technology, uses color-coded microsphere sets conjugated with specific antibodies or oligonucleotides to permit the capture and detection of specific analytes from a sample. Microspheres are currently available in 100 different colors, each of which can be used to perform a separate assay. Multiplex assays are performed in 96 well microplates, where samples are incubated with a mixture of analyte-specific, color-coded microspheres, and fluorescently-tagged analyte-specific probes. Multiplex assays offered through the MMPC, and their rationale for inclusion, are listed below and may be customized by request to include any combination of analytes from a given assay panel. Core users may also request custom assay panels that combine components of different assay panels, providing the assay conditions for the merged components are compatible. Multiplex samples are analyzed in duplicate, and most assays require 10–25 µl of serum, plasma or cell culture supernatant per sample well.