Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells - PubMed (original) (raw)

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Karel A Erion et al. J Biol Chem. 2015.

Abstract

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the β-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S0.25 = 3.5 mm) and 4 mm glucose with oleate (S0.25 = 4.5 mm) compared with 4 mm glucose alone (S0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r(2) = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca(2+) responses between left- and right-shifted β-cells. However, a left shift in the sensitivity of exocytosis to Ca(2+) was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S0.25 = 1.1 and 1.7 μm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.

Keywords: beta cell (β-cell); calcium imaging; hyperinsulinemia; insulin secretion; lipid; metabolism; type 2 diabetes.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Preserving insulin content during culture in high lipid concentration increases insulin secretion at basal and intermediate glucose in isolated rat islets. Islets were cultured in standard culture medium (11 m

m

glucose) with or without 0.2 m

m

oleate plus or minus 0.4 m

m

DZ for 48 h. A, insulin content following exposure to these conditions. B, islets were perifused at basal (3 m

m

) glucose for 30 min, and a baseline was collected for 10.5 min. Islets were stimulated with 11 m

m

glucose and then brought to 15 m

m

glucose at 34 min. Lines are a two-point running average fit to data points. Error bars are displayed every sixth point for clarity. C, the same insulin secretion as in B but expressed as a percentage of total insulin content secreted. D, -fold stimulation over basal insulin secretion by 11 m

m

glucose derived from B. Basal insulin secretion was the average of 0–12.6 min, and stimulated secretion was the average of 16.1–17.2 min. Data are derived from three independent experiments. Error bars represent ±S.E. (*, p < 0.05; +, p < 0.001). Colors from A correspond to B, C, and D.

FIGURE 2.

FIGURE 2.

Rat islets cultured in a high lipid environment exhibit a left-shifted concentration dependence of GSIS. Whole islets from the same rat were incubated in standard culture medium (11 m

m

glucose) with or without 0.1 m

m

oleate. Following a 48-h incubation, the total insulin content (A) and concentration dependence of GSIS (B) were assessed. Each experiment was normalized to its own content. The S0.25 was derived from the mean dose-response curve of all experiments. Data comprise 11 separate experiments from independent isolations. Error bars represent ±S.E. (*, p < 0.05).

FIGURE 3.

FIGURE 3.

Reduction of culture glucose decreases lipid stores, increases insulin content, and right-shifts the dose-response curve of GSIS in INS-1 cells. Lipid stores were assessed with the dye Nile red, and representative images were taken at 20× magnification are shown for 11G (A) and 4G (B) cells. The total insulin content (C) and acute insulin secretion response (D) to a range of glucose concentrations in 4G and 11G cells are shown. Insulin secretion was normalized to the total insulin content and expressed as a percentage of total content secreted. Data are means from 12 and 13 independent experiments for 4G and 11G cells, respectively (n = 3–4 replicates for each point in a given experiment). Error bars represent ±S.E. A two-tailed Student's t test was used for comparisons between conditions (*, p < 0.05; #, p < 0.01; +, p < 0.001).

FIGURE 4.

FIGURE 4.

INS-1 cells cultured in excess nutrients have a left-shifted insulin secretion glucose-dose response. A, transition of 11G cells to 4 m

m

glucose for 48 h. B, supplementation of 4G cells with 0.15 m

m

oleate (FA) for 24 h. C, incubation of 11G cells with 0.15 m

m

FA for 48 h (eight, six, and four independent experiments for 4G (48 h), 4G FA, and 11G FA, respectively). D, correlation analysis between intracellular lipid and the quarter-saturation level for GSIS derived from Table 1 (♦, 4G cells; ♦, 4 m

m

glucose for 48 h; ♦, 4G cells with 0.15 m

m

oleate; ■, 11G cells; ■, 11G cells with 0.15 m

m

oleate). Data are expressed as the mean from independent experiments. Error bars represent ±S.E. (*, p < 0.05; #, p < 0.01; +, p < 0.001).

FIGURE 5.

FIGURE 5.

Chronic culture in high glucose does not affect GK activity in INS-1 cells. GK activity was determined by the rate of NADPH appearance as described under “Experimental Procedures.” The slope of NADPH fluorescence was determined at different glucose concentrations and was normalized to the protein content of the soluble portion of the extract (n = 6 independent experiments). Data are presented as a mean of independent experiments. Error bars represent ±S.E. with no significant differences detected between 4G and 11G cells as determined by one-way analysis of variance. RFU, relative fluorescence units.

FIGURE 6.

FIGURE 6.

No difference in NAD(P)H concentration in β-cells with left- and right-shifted GSIS. A, the autofluorescence of NAD(P)H was assessed in 4G (black) and 11G (gray) cells both basally (2 m

m

glucose) and in response to glucose stimulation at 4, 6, 8, and 12 m

m

glucose. B, NAD(P)H production in response to glucose was assessed in whole isolated pancreatic islets cultured in 0 m

m

oleate (black) and 0.1 m

m

oleate (gray). Islets were brought from basal (3 m

m

glucose) to the concentrations indicated. NAD(P)H is expressed as a percentage of the full scale as described under “Experimental Procedures.” n = 6–7 and 6 separate experiments for A and B, respectively. Each experiment consisted of four to six islets per condition in B. Data are presented as a mean from independent experiments. Error bars represent ±S.E. One-way analysis of variance was used for comparisons between conditions (#, p < 0.01).

FIGURE 7.

FIGURE 7.

Increased mitochondrial leak in 11G cells but no change in the glucose dose response of O2 consumption. A, increase in oxygen consumption as a percent increase over basal in 4G (black) and 11G (gray) cells. Oxygen consumption traces document the differences between 4G (♦) and 11G (♦) in response to acute stimulation with 4 (B), 6 (C), and 8 m

m

(D) glucose. E, oligomycin-sensitive respiration used to estimate ATP synthesis. F, oligomycin-insensitive respiration used to estimate mitochondrial leak. Figures are averages from four separate experiments. Error bars represent ±S.E. (n = 4 replicates per experiment) (*, p < 0.05).

FIGURE 8.

FIGURE 8.

Increase in [Ca2+]i at 4 mm glucose in both 4G and 11G cells. 4G (A) and 11G (B) cells were brought from 2 m

m

glucose to 4 m

m

glucose at 4 min, and the change in average [Ca2+]i was documented. The same procedure was carried out in response to 8 m

m

glucose in 4G (C) and 11G (D) cells. Experiments consisted of ∼150 cells per trace. Data are expressed as the mean from four independent experiments. Error bars represent ± S.E. Error bars are shown every 10th reading for clarity.

FIGURE 9.

FIGURE 9.

Decreased lag time but no change in [Ca2+]i glucose dose response in whole islets cultured with oleate. Whole islets were brought from a baseline of 3 m

m

glucose to 7 m

m

glucose at 3 min and then to 15 m

m

glucose at 15 min. Average responses from control islets (31 from five rats) cultured without oleate (A) and islets cultured for 48 h with 0.1 m

m

oleate (26 from five rats) (B) are shown. Error bars represent ± S.E. Error bars are shown every 10th measurement for clarity. A three-point running average of a representative islet from control culture (C) and 0.1 m

m

oleate culture (D) is shown.

FIGURE 10.

FIGURE 10.

No change in amplification of insulin secretion by glucose in 4G and 11G cells. Data are presented as a mean from three independent experiments (n = 3–4 wells per glucose concentration). Error bars represent ±S.E. No significant differences were detected by a one-way analysis of variance.

FIGURE 11.

FIGURE 11.

11G cells have a left-shifted sensitivity of exocytosis to Ca2+. Cells were permeabilized and exposed to different Ca2+ concentrations in the presence of an ATP-regenerating system. Each curve is a mean of four independent experiments. Error bars represent ±S.E. (n = 3–4 wells per condition). The S0.25 and S0.5 values were derived from the mean of values obtained from each individual experiment ±S.E. A two-tailed Student's t test was used for comparisons between conditions (*, p < 0.05; #, p < 0.01; +, p < 0.001).

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