Washington Metabolic and Cellular Phenotyping Core

Overview

The Metabolic and Cellular Phenotyping Core provides Diabetes Research Center affiliate investigators with precise measurements of hormone secretion, glucose metabolism, mitochondrial function and intracellular signaling components critically important in diabetes and related disorders. A unique feature of the MCPC is that it enables the study of cellular and (patho)physiological processes in vivo, ex vivo and in vitro by combining methods that span the provision, treatment and analysis of cells/tissue or whole animals.

Services

In Vivo Metabolic Phenotyping

The Core offers in vivo phenotyping of mice to comprehensively assess parameters of metabolism, cellular function, and signaling in tissues critical to and affected by diabetes. Many of these studies can be performed in conscious unrestrained mice using a dual catheter system. Affiliates can also utilize surgical services for continuous glucose monitor implant and pancreatic ductal infusion of AAVs.

The following measurements can be made:

• Hyperinsulinemic-euglycemic clamp
• Intraperitoneal glucose, insulin, and pyruvate tolerance tests
• Intravenous glucose tolerance test (including FSIVGTT)
• Oral glucose tolerance test
• PET/CT imaging
• Islet transplantation

Seahorse XF Analysis

Agilent Seahorse XF analyzers measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells. The Core can perform the Cell Mito Stress Test and Glycolytic Rate Assay using Seahorse FluxPaks purchased from Agilent, which include all the plastic ware needed to calibrate and perform the assays (sensor cartridges, cell culture plate, and calibrant). Sample types can include primary cells, immortalized cells, iPSC derived human cells, and isolated mitochondria. The XFe96 analyzer utilized by the Core enables high throughput in a 96-well plate format.

Dynamic and Static Culture Systems

A state-of-the-art fully automated perifusion system (Biorep Technologies PERI4-FA-115 perifusion system) is available for real time in vitro analysis of cell or tissue samples in response to a range of stimuli. This system is amenable to almost any cell or tissue type and is ideally suited for studies looking at dynamic changes in the production of secreted analytes or changes in cell viability.

• Hormone release (e.g. insulin, glucagon)
• Inflammatory molecule production
• Neuropeptide release
• Neuropeptide release

The Core also provides static assessment of cell function. This is preferable where a large number of conditions need to be analyzed in parallel, or measures need to be made in the absence of flow. Since it allows for the accumulation of an endproduct over time, measurement of low-abundance analytes is possible. This system is optimized for pancreatic islets, where hormone secretion and content can be quantified.

Pancreatic Islets

Investigators can obtain isolated islets from rats and mice. The Core has experience isolating islets from Fischer, Sprague-Dawley, Wistar and BB rats, and C57BL/6, DBA/2, db/db and other strains and genetically modified mice. Although it is strain-dependent, rodents yield about 700 islets per rat, and 200 islets per mouse. Once prepared, islets may be cultured under desired conditions and/or treatments until being either shipped or further analyzed by methods listed above.

The Core can also assist investigators with acquiring human islets through the NIDDK-funded Integrated Islet Distribution Program and the IsletCore at the Alberta Diabetes Institute in Canada, as well as monkey islets from the Oregon National Primate Research Center.

Laboratory Assays

The Core can perform specific hormone assays on plasma/serum, cell lysates, tissue extracts, cell culture media or urine from both human and animal samples. These include, but are not limited to, ELISAs for insulin, glucagon and GLP-1, as well as a glucose oxidase assay for glucose.

Consultation and Training

Input on study design and data analysis, together with training in the techniques utilized by the Core are offered. Contact the Director, Sakeneh Zraika or Associate Director, Ron Tian , for more details.