Changes in Metabolic Markers in Insulin-Producing β-Cells during Hypoxia-Induced Cell Death As Studied by NMR Metabolomics

Abstract

This study was designed to investigate changes in the metabolites in the intracellular fluid of the pancreatic β-cell line INS-1 to identify potential early and late biomarkers for predicting hypoxia-induced cell death. INS-1 cells were incubated under normoxic conditions (95% air, 5% CO₂) or hypoxic conditions (1% O₂, 5% CO₂, 95% N₂) for 2, 4, 6, 12, or 24 h. The biological changes indicating the process of cell death were analyzed using the MTT assay, flow cytometry, Western blotting, and immunostaining. Changes in the metabolic profiles from cell lysates were identified using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy, and the spectra were analyzed by the multivariate model Orthogonal Projections to Latent Structure-Discriminant Analysis. Cell viability decreased approximately 40% after 12-24 h of hypoxia, coincident with a high level of cleaved caspase-3. A high level of HIF-1α was detected in the 12-24 h hypoxic conditions. The metabolite profiles were altered according to the degree of exposure to hypoxia. A spectral analysis showed significant differences in creatine-containing compounds at the early stage (2-6 h) and taurine-containing compounds at the late stage (12-24 h), with the detection of HIF-1α and cleaved caspase-3 in cells exposed to hypoxia compared to normoxia. Glycerophosphocholine decreased during the early stage hypoxia. The change in taurine- and creatine-containing compounds and choline species could be involved in the β-cell death process as inhibitors or activators of cell death. Our results imply that assessment by ¹H NMR spectroscopy would be a useful tool to predict the cell death process and to identify molecules regulating hypoxia-induced cell death mechanisms.

Metabolomics Study of the Effects of Inflammation, Hypoxia, and High Glucose on Isolated Human Pancreatic Islets

“…Further, polyamine metabolites were decreased, albeit only in the media, as a result of the oxidative stress 75 . The effects of (severe) hypoxia (1% O 2 ) for different durations (2, 4, 6, 12, and 24 h) in INS-1 cells were investigated relatively recently by NMR metabolomics by Tian and co-workers 76 . Cell viability decreased considerably after 12 to 24 h of hypoxia (by approximately 15% and 40% at 5% and 1% O 2 , respectively) even if these were single cell cultures and not islet spheroids, where hypoxia in the core is likely to be much more severe 15 .…”

Metabolomics applied to the pancreatic islet

“…Choline and phosphocholine concentrations peaked during the first 2–6 h of hypoxic exposure, while glycerophosphocholine levels dipped. These observations correlate well with cell viability, indicating these markers could be exploited to monitor β-cell health in a noninvasive, nondestructive fashion via imaging techniques [100]. …”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…PCr/Cr pools have been detected in insulinoma cell lines (67,128,337,402,450,451,515,536), as well as primary rodent islets (146,177,271). The question is whether a significant PCr/Cr shuttle is operative in ␤-cells, whether it can be exploited to monitor their cytosolic ATP/ADP ratios, and what the metabolic consequences of such a shuttle might be for adenine nucleotide signaling to the K ATP channel.…”