WUSTL Metabolic Tissue Function Core

Overview

Diabetes mellitus comprises a group of disorders characterized by an absolute or relative deficiency of insulin. Diabetes can be associated with impaired function in many tissues regulating metabolism, including pancreatic β-cells, adipose tissue, skeletal muscle, and the hypothalamus. Many issues fundamental to understanding the etiology and consequences of diabetes are best understood at the level of tissue pathology, cellular structure and function. The purpose of the Metabolic Tissue Function Core is to improve the efficiency of diabetes-related research by providing services related to the structural and functional analyses of tissues important in the pathogenesis and complications of diabetes. A major goal of the core is to assist DRC investigators in obtaining metabolic tissues, including β-cell lines, rodent and human primary islets, and iPSCs for differentiation into diabetes relevant tissues as well as β-cells. The Core will also assist investigators in conducting functional and metabolic analysis of cells and tissues relevant to the pathogenesis of diabetes. These goals are accomplished through consultations, provision of expert service, and access to state-of-the-art equipment.

Services

Metabolic Tissue Acquisition

• Advice for design of studies using islets or β-cells: Drs. Urano and Remedi provide consultations to researchers who are interested in using islets or β-cells but lack experience working with or generating these metabolic tissues.
• Rodent islet isolation: The Core assists DRC investigators in the isolation of primary islets from mouse models of diabetes and metabolic disorders. The procedure can be performed by Core personnel or they can provide training to personnel from DRC member labs, so that they can carry out their own isolations.
• Procurement of human primary islets: The Core assists DRC investigators in the isolation of primary islets from mouse models of diabetes and metabolic disorders. The procedure can be performed by Core personnel or they can provide training to personnel from DRC member labs, so that they can carry out their own isolations.
• Procurement of human primary islets: The Core assists investigators in preparing effective applications to the NIDDK-sponsored Integrated Islet Distribution Program (IIDP). Upon arrival, islets are tested for quantity and purity by Dithizone staining and by fluorescein diacetate/propidium iodide staining for viability, according to IIDP standard operating procedures. Results are compared to data obtained prior to shipping and feedback is provided to IIDP.
• Supply of β-cell lines: The Core maintains stocks of established β-cell lines, including MIN6, RINm5F, INS-1, βTC and βTC6. Upon request, investigators receive a frozen vial and standard operating procedures for culture.
• Generation of iPSC and genetically-modified cell lines: The Core leverages the well-established, high functioning Washington University Genome Engineering and iPSC Center (GEiC) to obtain iPSCs and genetically-modified cell lines for study of diabetes and its complications. DRC members are eligible for a one-time $1000 scholarship toward a diabetes-related project.

Metabolic Tissue Analyses

• Static Islet Secretory Responses: For rapid and cost-effective screening of insulin, glucagon, and somatostatin secretion in response to secretagogues, the Core offers static incubations in a 24 well plate format. Hormones are typically quantified by ELISA.
• Dynamic perifusion testing for quantification of physiological function of islets: Dynamic perifusion testing provides the most accurate assessment of regulated hormonal (i.e. insulin and glucagon) release ex-vivo to monitor islet function and physiology and enables pharmacological manipulation to dissect out signaling mechanisms and kinetics (e.g., first and second phase insulin secretion, pulsatile glucagon secretion) underlying release of different hormones.
• Islet Morphometry: The Core provides rigorous quantification of β-cell mass using histological staining and systematic imaging and quantification of serial pancreas sections. Insulin and glucagon primary antibodies and appropriate secondary fluorescent antibodies are provided. If other staining is desired, the requestor should provide their own antibodies in sufficient amounts to complete their project.
• Quantification of Metabolic Rates in Diabetes-Related Tissues and Cells: The Core supports analysis of cellular respiration and glycolysis and the real-time kinetics of bioenergetic pathways using Seahorse XF Extracellular Flux Analyzers. In addition to providing access to instrumentation (24 and 96 well formats), the core also provides training in application of this platform for the study of islets and β-cells.
• Frozen Section Insulin Quantitation: The Core provides insulin extraction from frozen pancreatic sections and insulin ELISA assay.
• Quantification of Metabolic Rates in Diabetes-Related Tissues and Cells: The Core supports analysis of cellular respiration and glycolysis and the real-time kinetics of bioenergetic pathways using Seahorse XF Extracellular Flux Analyzers. In addition to providing access to instrumentation (24 and 96 well formats), the core also provides training in application of this platform for the study of islets and β-cells.
• Analysis of Endoplasmic Reticulum Stress in Tissues: The Core has a battery of antibodies that can be used for ER stress analysis by Western Blot. These may be used individually or in groups of 4.