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

Abstract

The transplantation of human pancreatic islets is a therapeutic possibility for a subset of type 1 diabetic patients who experience severe hypoglycemia. Pre- and post-transplantation loss in islet viability and function, however, is a major efficacy-limiting impediment. To investigate the effects of inflammation and hypoxia, the main obstacles hampering the survival and function of isolated, cultured, and transplanted islets, we conducted a comprehensive metabolomics evaluation of human islets in parallel with dynamic glucose-stimulated insulin release (GSIR) perifusion studies for functional evaluation. Metabolomics profiling of media and cell samples identified a total of 241 and 361 biochemicals, respectively. Metabolites that were altered in highly significant manner in both included, for example, kynurenine, kynurenate, citrulline, and mannitol/sorbitol under inflammation (all elevated) plus lactate (elevated) and N-formylmethionine (depressed) for hypoxia. Dynamic GSIR experiments, which capture both first- and second-phase insulin release, found severely depressed insulin-secretion under hypoxia, whereas elevated baseline and stimulated insulin-secretion was measured for islet exposed to the inflammatory cytokine cocktail (IL-1β, IFN-γ, and TNF-α). Because of the uniquely large changes observed in kynurenine and kynurenate, they might serve as potential biomarkers of islet inflammation, and indoleamine-2,3-dioxygenase on the corresponding pathway could be a worthwhile therapeutic target to dampen inflammatory effects.

Keywords: cytokines; human islets; hyperglycemia; hypoxia; insulin secretion; metabolomics; perifusion.

Figure 1

Hierarchical clustering of the present metabolomics data in cell (A) and media samples (B) obtained from human islets (n = 10) cultured under different conditions as indicated (control, hypoxia, and inflammatory cytokines with low or high glucose). Within the clusters, blueish coloring represent elevated metabolite levels, while yellowish coloring represent decreased metabolite levels. Samples are color-coded as indicated by the figure key on the right side.

Figure 2

Box plot data illustrating some of the metabolites significantly altered in both cell and media samples by culture in high glucose conditions (16 vs. 5.5 mM for 24 h). Data (scaled intensity) are shown as median surrounded by a box indicating upper and lower quartiles and bars of minimum and maximum of distribution (mean indicated by a plus symbol, extreme values shown as circles), and are color-coded per condition (control in red, hypoxia in green, and inflammation in blue). LG and HG denote standard (low) and high glucose cultures, respectively during the second 24 h. For glucose-induced changes, compare the darker HG and lighter LG columns within each condition. Values are normalized in terms of raw area counts with each biochemical rescaled to set the median equal to 1.

Figure 3

Box plot data illustrating some of the metabolite significantly altered in both cell and media samples by culture in the presence of inflammatory cytokines (cocktail of IL-1β, IFN-γ, and TNF-α for 24 + 24 h). Notation is the same as in Figure 2. For inflammation-induced changes, compare blue vs. red columns (lighter and darker shades for changes under LG and HG conditions, respectively).

Figure 4

Effect of inflammatory cytokines (IL-1β, IFN-γ, and TNF-α) in standard culture (5.5 mM) on human islet metabolism shown using MetaboLync Pathway Classification Network for both media (top) and cell (bottom) samples for the Lipid and Amino Acid super-families. Within each pathway, the size of the circle correlates with the magnitude of change, and the color indicates significant change vs. control (p < 0.05; red increased, blue decreased).

Figure 5

Brief overview of the tryptophan metabolic pathway with box plots included showing metabolites detected here and significantly altered by culture with inflammatory cytokines (blue rectangles). Metabolites significantly increased are shown in bold red, those significantly decreased in bold green color; the notation of the box plots is the same as used before in Figure 2 or Figure 3. The large changes in kynurenine and kynurenate are associated with significant increases in tryptophan intermediates 3-indoxyl sulfate and indolelactate as well as with concomitant loss of several nicotinamide intermediates suggesting that inflammatory conditions perturb tryptophan metabolism to favor the indole and kynurenine axes.

Figure 6

Box plot data illustrating some of the metabolite significantly altered in both cell and media samples by culture under hypoxic conditions (3% O₂ for 24 + 24 h). Notation is the same as in Figure 2. For hypoxia-induced changes, compare green vs. red columns (lighter and darker shades for changes under LG and HG conditions, respectively).

Figure 7

Effect of hypoxia in standard culture (5.5 mM) on human islet metabolism shown using MetaboLync Pathway Visualization for media samples. Within each pathway, the size of the circle correlates with the magnitude of change, and the color indicates significant change vs. control (p < 0.05; red increased, blue decreased).

Figure 8

Dynamic glucose-stimulated insulin release (GSIR) perifusion data for islets following different incubation conditions from the present study. All data here are from islets cultured with basal glucose for the entire 48 h. The main figure shows the average secreted insulin collected every minute for a low (3 mM) → high (11 mM) → low (3 mM) glucose challenge followed by a KCl step. Data are for n = 4 different islet samples; results of the individual perifusions are shown in the small insets.