Fatty acids stimulate cholecystokinin secretion via an acyl chain length-specific, Ca2+-dependent mechanism in the enteroendocrine cell line STC-1

Abstract

1. The present study has investigated whether fatty acids directly influence peptide release from enteroendocrine cells using STC-1, a mouse intestinal endocrine tumour cell line, previously shown to release cholecystokinin (CCK) in response to other physiological stimuli. 2. Fatty acids elicited a chain length- and dose-dependent stimulation of CCK secretion. Dodecanoic acid (C12) was most effective, producing up to a 5-fold increase in CCK secretion. Fatty acids with less than ten carbon atoms did not increase secretion. The chain length dependence of these effects mimics closely fatty acid-induced CCK secretion previously observed in humans in vivo. 3. Esterification of C12 abolished CCK secretion, indicating a critical role for a free carboxyl group in eliciting secretion. In contrast, modification of the methyl terminus had no effect on C12-induced secretion. The non-metabolizable C12 analogue 2-bromododecanoic acid was equally effective. 4. C12 elicited a marked increase in intracellular calcium levels (200-300 nM) in STC-1 cells which was abolished by the L-type Ca2+ channel antagonist nicardipine. In contrast, C8 produced a smaller and more transient Ca2+ response. C12-induced CCK secretion was also blocked by nicardipine. 5. These data suggest that fatty acids can interact directly with enteroendocrine cells to stimulate CCK secretion via increases in intracellular calcium mediated primarily by L-type Ca2+ channels.

Figure 1. Time course of FA-induced CCK secretion in STC-1 cells

Cells were challenged with 500 μm C12 or HBSS and medium sampled at the time points indicated to 120 min. CCK secretion is presented cumulatively and expressed in relation to the total protein content in representative wells. Data are the means ± s.e.m. of 3 separate experiments.

Figure 2. Dose and chain length dependence of FA-induced CCK secretion

Cells were challenged with increasing concentrations of C6 (n = 4), C8 (n = 4), C10 (n = 5) and C12 (n = 5) saturated fatty acids over 15 min. Results are expressed as a percentage of CCK secretion in control (untreated) cells over the same period. Data are means ± s.e.m. *P < 0.01, **P < 0.001.

Figure 3. Relationship between C12-induced CCK secretion and LDH release

STC-1 cells were exposed to varying concentrations (0–10 mm) of C12 FA over 15 min after which the medium was assayed for CCK and LDH activity. CCK results are expressed as a percentage of secretion in the absence of C12. LDH is expressed as the percentage of total cell-associated activity. Results are means + s.e.m. from n = 5 experiments in each group except 10 mm C12 where n = 3.

Figure 4. CCK secretion following repeat challenge with C12 FA

Cells were challenged with 500 μm C12 for 15 min, after which the FA was removed and cells bathed in control medium for varying times (15–240 min) prior to a second challenge with C12. Results show CCK secretion resulting from the second exposure to C12 expressed as a percentage of the initial response. Data are means + s.e.m. for 9 observations in each group.

Figure 5. Influence of C8 and C12 on intracellular Ca²⁺ levels in STC-1

C8 (500 μm) and C12 (500 μm) added sequentially to fura-2-loaded STC-1 cells as indicated by the bars. The figure shows a single experiment with averaged data from 11 cells. Mean data from several experiments are given in the text.

Figure 6. Effect of L-type Ca²⁺ channel blockade on C12-induced increases in intracellular Ca²⁺

Upper trace, Ca²⁺ response to sequential addition of 500 μm C12 in STC-1 cells. Lower trace, response to sequential addition of C12 where cells have been pretreated with 5 μm nicardipine for 5 min following the initial C12 challenge. Addition of C12 is shown by the bars. The break in each trace represents 5 min during which the cells were perfused with buffer (upper) or buffer + nicardipine (lower) but no fluorescence images or Ca^{2 +}measurements were taken. Figures show results of single experiments with averaged data from 12 (upper trace) and 36 (lower trace) cells. Mean data from several experiments are given in the text.

Figure 7. Inhibition of C12-induced CCK secretion by nicardipine

Effects of 500 μm C12 on CCK secretion in cells pretreated with 5 μm nicardipine () or vehicle (▪) for 5 min. Data are shown as the percentage of CCK released in the absence of C12 and are means + s.e.m. of 9 observations in each group. **P < 0.01.