Transport and storage of 5-hydroxytryptamine in pancreatic β-cells

doi:10.1016/0006-2952(72)90062-7

Biochemical Pharmacology

Volume 21, Issue 5, 1 March 1972, Pages 695-706

https://doi.org/10.1016/0006-2952(72)90062-7 Get rights and content

Abstract

To elucidate the role of biogenic amines in insulin secretion, pancreatic islets rich in β-cells were microdissected from obese-hyperglycemic mice and were incubated with ¹⁴C-labelled 5-hydroxytryptamine (5-HT). The saturability of uptake and the fact that 5-HT was accumulated to high levels indicated that the β-cells possess a transport system with great capacity for this amine. The initial uptake was not sensitive to glucose or diazoxide.

Efflux of radioactivity from islets preloaded with ¹⁴C-labelled 5-HT exhibited complex kinetics suggesting incorporation of the amine into some less mobile compartment of the β-cells. This compartment may be the insulin-containing secretory granules, since homogenization and centrifugation of preloaded islets revealed closely parallel sedimentation profiles for insulin and ¹⁴C. The apparent co-sedimentation of insulin and 5-HT probably reflects the ultrastructural organization of the β-cells, as insignificant radioactivities were recovered in the sediments after adding ¹⁴C-labelled 5-HT to homogenized islets. Furthermore, gel filtration of insulin on 5-HT-equilibrated Sephadex G-50 did not indicate any great affinity of insulin for the amine.

Neither glucose nor glibenclamide could be shown to mobilize granule-bound 5-HT from intact β-cells. In these experiments insulin release was slow despite a glucose concentration as high as 20 mM. It seems possible that co-storage of insulin and 5-HT reduces the ability of β-granules to undergo normal emiocytosis.

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Previous studies further indicate that serotonin is produced and secreted from beta cells almost exclusively under conditions of increased metabolic demand. Although the machinery for serotonin uptake and release is present in beta cells of several species (Gylfe, 1978; Hellman et al., 1972; Schäfer et al., 2013), detecting serotonin secretion from rodent beta cells requires islets to be preloaded in vitro with serotonin or its precursors (Barbosa et al., 1998; Rosário et al., 2008; Smith et al., 1995). This suggests that serotonin may not play a paracrine role in the islet in the normal physiological state.
In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT_1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT_1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity.
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The relevance of serotonin and in particular that of 5-HT₃ receptors is unequivocal with respect to emetic/antiemetic effects, but it is controversial with respect to antidiabetic effects. The effects of tropisetron (5-HT₃ receptor antagonist) and various ginger (Zingiber officinale) extracts (known to interact with the 5-HT₃ receptor channel system) were investigated. Serotonin (32 to 500 µM) inhibits insulin release (RIA) from INS-1 cells which is reversed by tropisetron (10 to 100 µM) and two different ginger extracts (spissum and an oily extract). Their effects are obvious even in the absence of serotonin but are more pronounced in its presence (doubled to tripled). Specific 5-HT₃ binding sites are present in INS-1 cells using 0.4 nM [³H] GR65630 in displacement experiments. The in vitro data with respect to ginger are corroborated by in vivo data on glucose-loaded rats showing that blood glucose (Glucoquant^®) is decreased by ~ 35% and plasma insulin (RIA) is increased by ~ 10%. Both the spissum extract and the oily ginger extract are effective in two other models: they inhibit [¹⁴C] guanidinium uptake into N1E-115 cells (model of 5-HT₃ effects) and relax rat ileum both directly and as a serotonin antagonistic effect. Other receptors addressed by ginger are 5-HT₂ receptors as demonstrated by using methysergide and ketanserin. They weakly antagonize the serotonin effect as well. It may be concluded that serotonin and in particular the 5-HT₃ receptor channel system are involved in modulating insulin release and that tropisetron and various ginger extracts can be used to improve a diabetic situation.
<sup>3</sup>H-serotonin as a marker of oscillatory insulin secretion in clonal β-cells (INS-1)
2007, FEBS Letters
Citation Excerpt :
Due to difficulties in making reliable insulin specific electrodes, a more basic serotonin-sensing carbon fiber is often used to measure exocytosis from cells, which have been preincubated with serotonin for 2–24 h [6,7]. Serotonin is taken up into the β-cell, localizes to the insulin secretory granules [8,9] and is then co-secreted with insulin [10]. Serotonin release from single preloaded pancreatic β-cells has been measured using the amperometric technique and has correlated well to insulin exocytosis as measured by the same technique using the specific insulin sensitive carbon fiber electrode [11].
Serotonin release from preloaded pancreatic β-cells has been used as a marker for insulin release in studying exocytosis from single cells using the amperometric technique. We found that single pancreatic β-cells exhibited oscillations in exocytosis with a period of 1–1.5 min as measured amperometrically by serotonin release. We also show that ³H-serotonin can be used to monitor exocytosis from intact and streptolysin-O permeabilized clonal insulin-secreting cells preloaded with labeled serotonin and that serotonin release correlated with insulin secretion in the same cells. The use of ³H-serotonin provides a real-time indicator of exocytosis from populations of clonal insulin-secreting cells.
Are 5-Hydroxytryptamine-preloaded β-Cells an Appropriate Physiologic Model System for Establishing that Insulin Stimulates Insulin Secretion?
2001, Journal of Biological Chemistry
Citation Excerpt :
In conclusion, our studies and several previous reports (29, 30) demonstrate that prior exposure of β-cells to 5HT results in a profound time-dependent suppression of the insulin secretory process. The concept that the secretion of 5HT from preloaded β-cells reflects physiologic secretion is not supported by these or other studies (28-30). It is premature to conclude that insulin, under physiologic conditions, stimulates its own secretion based exclusively on studies using amperometric measurements of 5HT release without substantial corroborating data including actual measurements of insulin secretion from 5HT-preloaded β-cells.
The release and oxidation of 5-hydroxytryptamine from 5-hydroxytryptamine-preloaded β-cells has been used as a surrogate marker for insulin secretion. Findings made using this methodology have been used to support the concept that insulin stimulates its own release. In the present studies, the effects of 5-hydroxytryptamine on stimulated insulin secretion from isolated perifused rat islets was determined. When added together with stimulatory glucose, 5-hydroxytryptamine (0.5 mm) significantly reduced both phases of 8 mm glucose-induced secretion and reduced the first phase of 15 mmglucose-induced release by 60% without any effect on sustained insulin release rates. Preloading of β-cells with 0.5 mm5-hydroxytryptamine for 3 h resulted in a more severe impairment of 15 mm glucose-induced secretion. First and second phase release rates were reduced by 70 and 55%, respectively. In addition, this pretreatment protocol also abolished 200 μmtolbutamide-induced insulin secretion from perifused islets. These findings confirm that 5-hydroxytryptamine is a powerful inhibitor of stimulated insulin secretion. The responses of 5-hydroxytryptamine-preloaded β-cells may not accurately reflect the biochemical events occurring during the physiologic regulation of insulin secretion. The suggestion that insulin stimulates its own secretion based exclusively on amperometric measurements should be reconsidered.
Real time electrochemical detection of 5-HT/insulin secretion from single pancreatic islets: Effect of glucose and K<sup>+</sup> depolarization
1996, Biochemical and Biophysical Research Communications
We report a highly sensitive electrochemical approach suitable for the real time measurement of insulin release from single islets of Langerhans, the functional endocrine units in the pancreas. The method is based on the detection of the insulin surrogate 5-hydroxytryptamine (5-HT) by carbon fibre microelectrodes implanted in the islets. Based on the combination of this novel approach with the simultaneous microfluorometric recording of cytosolic free Ca²⁺concentration ([Ca²⁺]_i), we demonstrate that glucose-stimulated islets secrete 5-HT/insulin in a pulsatile fashion under physiological conditions, and that this activity is encoded by synchronous [Ca²⁺]_ioscillations. The sensitivity to detect variations in minute amounts of secreted materials is partially conferred by the fact that the tracer is released into a relatively confined space (the intraislet interstitial space).
Amperometric detection of quantal secretion from patch-clamped rat pancreatic β-cells
1996, Journal of Biological Chemistry
Serotonin (5-HT) is taken up in insulin granules and co-released with insulin on stimulation of pancreatic islet β-cells. Based on these observations, we have used microcarbon fiber amperometry to examine secretogogue-induced 5-HT release from rat β-cells preloaded for 4-16 h with 5-HT and then exposed to a bath solution containing 10 μM forskolin. In response to local application of KCl (60 mM) or tolbutamide (50-200 μM), we recorded barrages of amperometric events. Each amperometric event consisted of a short pulse of current measurable at electrode voltages that catalyze 5-HT oxidation. With either secretogogue, release was calcium-dependent. On combining amperometry with perforated patch whole-cell recording, we found that barrages of such events were well coupled in time and graded in intensity with depolarization-induced Ca₂₊ currents and well correlated with increases in membrane capacitance. In cell-attached patch recording, amperometric events evoked by application of tolbutamide followed the closure of ATP-sensitive K₊ channels and coincided with the onset of electrical activity. These experiments suggest that amperometry is a useful technique for studying, in real time, the dynamic aspects of stimulus-secretion coupling in β-cells. Their performance was facilitated by the design of a new carbon fiber electrode (ProCFE) described within.

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Transport and storage of 5-hydroxytryptamine in pancreatic β-cells

Abstract

Life Sci.

Biochem. Pharmac.

Biochim. biophys. Acta

J. biol. Chem.

Adv. Pharmac.

J. Ultrastruct. Res.

Biochem. biophys. Res. Commun.

Life Sci.

Acta endocr. (Kbh.)

Endocrinology

Acta Physiol. Scand. Suppl.