Chronic fructose renders pancreatic beta-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP
BARTLEY, Clarissa,et al
Fructose is widely used as a sweetener in processed food and since then is associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular regarding the pancreatic beta-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human beta-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels, without affecting mitochondrial function, induced AMPK activation and favored ATP release from the beta-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both pannexin1 channels and P2Y1 receptors in beta-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve beta-cells potentiated insulin [...]
BARTLEY, Clarissa,et al
. Chronic fructose renders pancreatic beta-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.American Journal of Physiology. Endocrinology and Metabolism
, 2019, vol. 317, no. 1, p. E25-E41
PMID : 30912960
DOI : 10.1152/ajpendo.00456.2018
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Chronic fructose renders pancreatic beta-cells hyper-responsive through extracellular ATP signaling
by Bartley et al.
SI Table S1-S2
SI Figures and Legends 1-5
2 Supplementary Table S1. Clinical data of organ donors and characteristics of corresponding islet batches.
Abbreviations: F: Female, M: Male; BMI: Body Mass Index. Purity, measured by dithizone staining, indicates the percentage of endocrine cells in the human islet preparations.
Supplementary Table S2. Primers used for quantitative RT-PCR analysis
4 Supplementary Fig. 1. Chronic fructose exposure potentiates GSIS in human islets, whereas aspartame does not stimulate GSIS in INS-1E ß-cells. A: For acute tests, control CM INS-1E ß-cells were exposed for 1h to G8.3 + 5.5 mM aspartame (acute aspartame) and 100 µM ARL67156. B: Cells were also exposed for 4 days to aspartame. Results are expressed as means ± SEM of at least 3 independent experiments. #P<
0.05 CM+F cells versus CM cells; ***P<0.005 versus CM G2.5 control values; $P<0.05, $$$P<0.005 versus corresponding basal cells. C and D: Modified INS-1E ß-cells expressing Gaussia luciferase were cultured with either 5.5 mM fructose (CM+F) or control medium only (CM) for 4 days and then challenged online for 25 min with basal G2.5 followed by 30 mM KCl stimulation. C: Representative native traces of basal secretion at G2.5 in untreated and fructose-treated cells. D: Results are expressed as relative luminescence units (RLU) normalized to signal of CM cells at G2.5.The first phase is defined as the first 3.2 min of secretion and the second phase as the subsequent 6.5 min of secretion. (n=4). E-H: Isolated human islets from three donors were treated for 4 days with 5.5 mM fructose in complete medium (CMRL+F). E and G:
The insulin secretory responses of 100 hand-picked treated human islets from two additional donors were perifused with G2.8 (basal) and G8.3 (stimulation; dashed line) for the indicated time periods. Values are expressed as means ± SEM of 3 independent perifusion chambers (except for values CMRL+F;
Supplementary fig. 1E, 1F; n=2 chambers). F and H: Means of area under the curve (AUC) of the respective perifusion profiles is shown in the right panel. ###P< 0.005 CMRL+F islets versus CMRL islets; *P< 0.05,
***P< 0.005 versus corresponding basal secretion.
Supplementary Fig. 2. AMPK activation and mitochondrial function in INS-1E ß-cells after chronic fructose exposure. Cells were treated for 4 days with 5.5 mM fructose in complete medium (CM+F). A: Quantitative analysis of pAMPK/AMPK band densities normalized to ACTIN from immunoblot represented in Figure 2A.
Results are expressed as protein levels normalized to CM control values at the end of the 4-day culture period.
B and C: Human islets were treated for 4 days to 5.5 mM fructose (F) in complete CRML-1066 medium (CMRL+F).
B: Representative immunoblotting showing levels of pAMPK, AMPK and ACTIN from treated islets isolated from one donor after 1 h post-culture of glucose lowering at 1 mM (G1, 0 min) followed by glucose stimulation for 10 min at 8.3 mM (G8.3, 10 min). C: Quantitative analysis of pAMPK/AMPK band densities normalized to ACTIN.
Results are expressed as protein levels normalized to CMRL G1 control values. Representative oxygen consumption rate (OCR) profiles from Seahorse measurements in treated INS-1E ß-cells further stimulated with D: G2.5 and E: G15 followed by the sequential addition of oligomycin (Oligo), FCCP and antimycin A/rotenone (Ant/Rot). F: The coupled respiration from 3 independent experiments is indicated as oxygen consumption rate (OCR) and expressed as the percentage of the baseline respiration before stimulation. G: Uncoupled mitochondrial respiration expressed as oxygen consumption rate (pmol O2/min). H: Representative oxygen consumption rate (OCR) profiles in control CM cells acutely stimulated with 5.5 mM fructose after a 2 h preincubation with the indicated glucose concentrations.. (I) ATP/ADP ratio in treated cells right after the 4-day
6 Supplementary Fig. 3. Expression of pannexin1 channels and P2Y1 purinergic receptors in INS-1E ß-cells after chronic fructose exposure. Cells were cultured for 4 days in complete medium (CM) and CM supplemented with 5.5 mM fructose (CM+F). Transcript levels of (A) actin, tubulin, cyclophilin and (B) the transcription factor Pdx1 and the taste receptor T1r3 subunit were quantified in treated cells (n=3) as described in Materials and Methods.
C and D: Associated profiles across single cells (from immunofluorescence images Fig. 3, F and G) show the intensity and location of Panx1 and P2Y1 in INS-1E ß-cells. Additional confocal microcopy images (n>3) to Fig. 3 obtained from treated cells immunolabeled with antibodies against (E) Panx1 (green), DAPI (blue) and insulin (red) and (F) P2Y1 (blue).
Supplementary Fig. 4. Expression of pannexin1 channels and P2Y1 purinergic receptors in mouse islets. A and B: Additional confocal microscopy images (out of 3 mice) to Fig. 5 obtained from both male and female WT mouse pancreas sections immunolabeled with antibodies against insulin (red) and glucagon (blue) combined with either (A) Panx1 or (B) P2Y1 (green). C: Representative images of Panx1 immunofluorescent staining (green) in WT and Panx1-/- mouse pancreas sections (out of 3 mice). Nuclei were stained with DAPI (in blue) and sections were counterstained with Evans Blue (in red). D and E: Immunoblotting and associated densitometry showing levels of Panx1 from isolated control (WT) or Panx1-/- mouse islets (out of 2 mice). F: Insulin content at the end
8 Supplementary Fig. 5. Chronic fructose exposure potentiates GSIS in human islets through extracellular ATP signaling mediated by pannexin1 channels and P2Y1 receptors. Isolated human islets from different donors were cultured for 4 days in complete medium (CMRL) or CMRL supplemented with 5.5 mM fructose (CMRL+F). At the end of the 4-day culture period, islets were pre-incubated for 1 h with G2.8. A: Immunoblotting from 3 donors showing the protein levels of PANX1 and P2Y1 in non-deglycosylated whole islet lysates and quantification of the immunoreactive bands (n=4). B: After preincubation for 1 h at G2.8, islets were stimulated for 1 h with G16.7 (n=4 donors done in triplicate)) and 10 M of the P2Y1 agonist 2MeSADP or the P2Y1 antagonist MRS2179. C:
Isolated islets from the same donor as in Supplementary Fig. 5A were also stimulated with G8.3 in the presence of 10 M 2MeSADP, 100 M ARL67156 and 10 µM Mfq. Stimulation was preceded by 1 h pretreatment with Mfq or the different antagonists. Data are expressed as means ± SEM (n=3 batches per condition). *P< 0.05,
**P<0.01, ***P<0.005 versus CM G2.5 control values; $P<0.05, $$P<0.01, $$$P<0.005 versus corresponding basal islets.