High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

Summary

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

Abstract

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

Introduction

The primary purpose of the pancreatic beta cell is to maintain system normoglycemia through glucose-stimulated insulin secretion. The beta cells sense physiological changes in circulating glucose largely due to the low affinity, high capacity glucose transporter GLUT2 (Glucose Transporter 2, K_m 16.7 mM)¹. As circulatory glucose levels rise, this high-capacity low-affinity transporter facilitates a proportional increase in intracellular glucose within the beta cell. Glucose is metabolized through glycolysis, the TCA cycle (tricarboxylic acid cycle) and mitochondrial respiration resulting in elevated cellular ATP (adenosine triphosphat....

Protocol

1. Cell Culture

1. Culture INS-1 derived 832/13 beta cells in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 10 mM HEPES, 2 mM Glutamine, 1 mM Sodium Pyruvate, and 0.05 mM 2-mercaptoethanol^{9,10,11,12,13}.
2. Remove 832/13 cells from a T75 flask using 2 mL of 0.25% trypsin, incubating at 37 °C for 10 min. Neutralize trypsin by adding 8 mL of complete RPMI 1640 media.
3. Count cells using a hemocytometer and dilute 100 µL of the cell volum....

Results

INS-1 832/13 beta cells that are prepared and harvested as described in the protocol will demonstrate modulation in oxygen consumption based on the various chemical interventions (Figure 1A). An increase in respiration will be observed when the glucose concentration is increased to 16.7 mM Glucose (Figure 1B). Respiration will decrease when the intact cells are treated with Oligomycin A. This respiration is known as LEAK, which is defined as the basal, nonphosph.......

Discussion

The objective of this protocol is to use high-resolution respirometry to measure respiratory rates in intact pancreatic beta cells. This method allows the measurement of the beta cell response to increased glucose levels. The protocol also allows for the pretreatment with various compounds, as demonstrated in this protocol with the naturally occurring monomeric epicatechin or curcumin^4,5. Treatment with various other compounds cou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
O2k Core: Oxygraph-2k	Oroboros Instruments	10000-02	Instrument for high-resolution respirometry
DatLab 6 Program	Oroboros Instruments	27141-01	Computer program for analysing high-reolution respirometry
INS-1 832/13 cell line	Duke University Medical Center	NONE	Beta cell line, gift from Dr. Christopher Newgard
Curcumin	Sigma	C7727	Pre-treatment of beta cells
Cocoa epicatechin monomer	Virginia Polytechnic Institute and State University	NONE	Pre-treatment of beta cells, gift from Dr. Andrew Neilson
Trypsin	Sigma	T4049	For cell culture
RPMI-1640	Sigma	R8758	832/13 beta cell media
Fetal Bovine Serum	Hyclone	SH30071.03	832/13 beta cell media component
Penicillin-streptomycin	Sigma	P4458	832/13 beta cell media component
HEPES	Sigma	H3662	832/13 beta cell media component/ 1x SAB Buffer
Glutamine	Caisson	GLL01	832/13 beta cell media component
Sodium Pyruvate	Sigma	S8636	832/13 beta cell media component
2-Mercaptoethanol	Sigma	M3148	832/13 beta cell media component
NaCl	Fisher	S271	Component of 10x SAB buffer
KCl	Sigma	P9541	Component of 10x SAB buffer
KH2PO4	Sigma	P5655	Component of 10x SAB buffer
MgSO4	Sigma	M2643	Component of 10x SAB buffer
D-(+)-Glucose Solution	Sigma	G8769	Component of 1x SAB buffer, chemical for respiration assay
CaCl2	Sigma	C5670	Component of 1x SAB buffer
35% BSA Solution	Sigma	A7979	Component of 1x SAB buffer
NaHCO3	Sigma	S5761	Component of 1x SAB buffer
200 proof ethanol	Sigma	459844	Washing Oxygraph O2k Chambers
Oligomycin A	Sigma	O4876	Chemicals for respiration assay
FCCP	Sigma	C2920	Chemicals for respiration assay
Anitmycin A	Sigma	A8674	Chemicals for respiration assay
BCA Protein Kit	Thermo Fisher	23225	For determining protein concentration