Microfluidic-enabled quantitative measurements of insulin release dynamics from single islets of Langerhans in response to 5-palmitic acid hydroxy stearic acid.

Proper release of insulin from pancreatic islets of Langerhans is essential for maintaining glucose homeostasis. For full efficacy, both the pattern and the amount of hormone release are critical. It is therefore important to understand how insulin levels are secreted from single islets in both a quantitative fashion and in a manner that resolves temporal dynamics. In this study, we describe a microfluidic analytical system that can both quantitatively monitor insulin secretion from single islets while simultaneously maintaining high temporal sampling to resolve dynamics of release. We have applied this system to determine the acute and chronic effects of a recently-identified lipid, 5-palmitic acid hydroxy stearic acid (5-PAHSA), which is a member of the fatty acid hydroxy fatty acid class of lipids that are upregulated in healthy individuals. Chronic incubation (48 h) with 5-PAHSA significantly increased glucose-stimulated insulin secretion (GSIS) in murine islets compared to chronic incubation without the lipid or in the presence of palmitic acid (PA). The studies were continued in human islets from both healthy donors and donors diagnosed with type 2 diabetes mellitus (T2DM). Total amounts of GSIS were not only augmented in islets that were chronically incubated with 5-PAHSA, but the dynamic insulin release profiles also improved as noted by more pronounced insulin oscillations. With this quantitative microfluidic system, we have corroborated the anti-diabetic effects of 5-PAHSA by demonstrating improved islet function after chronic incubation with this lipid via improved oscillatory dynamics along with higher basal and peak release rates.

Dual Detection System for Simultaneous Measurement of Intracellular Fluorescent Markers and Cellular Secretion.

Glucose-stimulated insulin secretion from pancreatic ß-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with high-speed automated insulin immunoassays. As a demonstration of the system, intracellular [Ca2+] ([Ca2+]i) was measured by imaging Fura-2 fluorescence simultaneously with insulin secretion from islets exposed to elevated glucose levels. Both [Ca2+]i and insulin were oscillatory during application of 10 mM glucose with temporal and quantitative profiles similar to what has been observed elsewhere. In previous work, sinusoidal glucose levels have been used to test the entrainment of islets while monitoring either [Ca2+]i or insulin levels; using this newly developed system, we show unambiguously that oscillations of both [Ca2+]i and insulin release are entrained to oscillatory glucose levels and that the temporal correlation of these are maintained throughout the experiment. It is expected that the developed analytical system can be expanded to investigate a number of other intracellular messengers in islets or other stimulus-secretion pathways in different cells.

The PA domain is crucial for determining optimum substrate length for soybean protease C1: structure and kinetics correlate with molecular function.

A subtilisin-like enzyme, soybean protease C1 (EC 3.4.21.25), initiates the degradation of the ß-conglycinin storage proteins in early seedling growth. Previous kinetic studies revealed a nine-residue (P5-P4') length requirement for substrate peptides to attain optimum cleavage rates. This modeling study used the crystal structure of tomato subtilase (SBT3) as a starting model to explain the length requirement. The study also correlates structure to kinetic studies that elucidated the amino acid preferences of soybean protease C1 for P1, P1' and P4' locations of the cleavage sequence. The interactions of a number of protease C1 residues with P5, P4 and P4' residues of its substrate elucidated by this analysis can explain why the enzyme only hydrolyzes peptide bonds outside of soybean storage protein's core double ß-barrel cupin domains. The findings further correlate with the literature-reported hypothesis for the subtilisin-specific protease-associated (PA) domain to play a critical role. Residues of the SBT3 PA domain also interact with the P2' residue on the substrate's carboxyl side of the scissile bond, while those on protease C1 interact with its substrate's P4' residue. This stands in contrast with the subtilisin BPN' that has no PA domain, and where the enzyme makes stronger interaction with residues on the amino side of the cleaved bond. The variable patterns of interactions between the substrate models and PA domains of tomato SBT3 and soybean protease C1 illustrate a crucial role for the PA domain in molecular recognition of their substrates.