OBJECTIVE: Type 2 diabetes (T2D), the most common type of diabetes characterized by high blood glucose and insulin resistance, results from both genetic and environmental factors. Our lab has proposed that chronic excess nutrients induce insulin hypersecretion from the pancreatic ß-cell, contributing to hyperinsulinemia, a prequel to T2D. Normal glucose-stimulated insulin secretion (GSIS) is oscillatory, a feature that is lost in patients with T2D. In this thesis we examine the oscillatory secretion profiles of clonal pancreatic ß-cells cultured in normal and excess nutrients that mimic conditions of T2D. We also begin to examine oscillations in protein phosphorylation that may contribute to normal ß-cell metabolism and GSIS, but if altered might potentially lead to impaired insulin secretion.
METHODS: Nutrient regulation of oscillatory insulin release was studied in clonal pancreatic β-cells (INS-1) cultured in multiwell plates in both low (4 mM) and high (11 mM) glucose. Insulin secretion was stimulated in cells from multiwell plates one well at a time at 30 sec intervals and sampled simultaneously at the end of the timecourse. Insulin secretion and insulin content were measured using a homogenous time-resolved fluorescence (HTRF) insulin kit (Cisbio). Protein was extracted from these same cells for analysis of time-dependent phosphorylation by western blot using specific antibodies. Protein phosphorylation was detected using SuperSignal West Femto chemiluminescence reagent (ThermoFisher) and imaged on an iBright Imaging System (Invitrogen).
RESULTS: Insulin secretion from INS-1 cells grown in separate plates and in 4 mM glucose oscillated with a period of 8.2 0.5 min compared to 5.0 0.5 min in cells cultured at 11 mM glucose. The amplitude of oscillations was 40.4 11.5 and 14.6 1.5 for cells cultured in 4 and 11 mM glucose respectively. Oscillations in secretion from cells cultured in 4 and 11 mM glucose in the same plate were not different in period but different in amplitude due in part to reduced insulin content. Oscillation in the phosphorylation patterns of acetyl-CoA carboxylase (ACC) and myristoylated alanine rich C kinase substrate (MARCKS) were measured in cells cultured in 4 mM glucose and both exhibited a peak in phosphorylation that occurred at the nadir of the insulin oscillation between peaks of insulin release.
CONCLUSION: Insulin secretion from pancreatic ß-cells is affected by nutrient status as excess nutrients decrease the amplitude of oscillations in insulin release. The period of oscillations can also be affected. Oscillations in protein phosphorylation are consistent with both ACC and MARCKS contributing to normal GSIS. These initial studies provide evidence of the suitability of this model system to correlate oscillations in protein activity to exocytosis. Future studies focused on the effects of low and high glucose will potentially reveal new important therapeutic targets that may help prevent/reverse/ameliorate insulin hypersecretion leading to insulin resistance and T2D.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/44011 |
| Date | 10 March 2022 |
| Creators | Narmuratova, Gulzhan |
| Contributors | Deeney, Jude T. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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