Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation

Abstract

While serotonin (5-HT) co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+)-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

Figure 1. Pancreata of Tph1−/− mice have a normal morphology but lack 5-HT, which results in hyperglycemia and insulin resistance.

(A) 5-HT contents in Tph1−/− and wt mice show, that Tph1−/− pancreata contain only around 10% of the wt mean. *p<0.05; n = 6. (B) Combined stacks of confocal images of pancreas slices spanning 10 µm with anti-insulin-stained islets (green) and anti-S100-stained ductal structures (red) at postnatal day 3 (P3) reveal no difference between the genotypes; scale bar = 70 µm. (C) Individual islets do not differ in morphometric analysis (Tph1−/−: n = 29; wt: n = 30). (D) Basal blood glucose concentration in freely feeding Tph1−/− mice are significantly reduced at P3 (Tph1−/−: n = 20; wt: n = 11) and elevated at P14 (n = 5) and in adulthood (n = 6) compared to wt littermates. *p<0.05. (E) Glucose tolerance tests with fasted wt and Tph1−/− mice show impaired glucose tolerance of the latter. *p<0.05; n = 6. (F) Insulin tolerance tests with Tph1−/− and wt mice. *p<0.05; n = 6. For (A) and (C–F), data are means ± SEM.

Figure 2. Intra- and extracellular 5-HT modulates insulin secretion in opposing directions.

(A–C) Diabetes mellitus and β-cell dysfunction in Tph1−/− mice aged 64 to 70 wk and rescue with 5-HTP. Glucose tolerance test with simultaneous blood glucose (A) and serum insulin (B) measurements. Glucose to insulin ratios (C) are significantly increased in Tph1−/− without 5-HTP treatment before and after the glucose load. *p<0.05; n = 8 (wt and Tph1−/− + 5-HTP) and n = 6 (Tph1−/−). (D) Pargyline induces hyperglycemia in wt mice. After a meal of 60 min, mice were treated with pargyline (75 mg kg⁻¹) and blood glucose was measured immediately and after additional 60 min. *p<0.05; n = 6. (E) Pargyline treatment substantially elevated insulin secretion in wt mice. Mice were treated as described in (D). *p<0.05; n = 6. (F) Glucose tolerance test of Tph1−/− and wt mice with systemic 5-HT pre-treatment. *p<0.05; n = 6. (G) Extracellular 5-HT inhibits insulin secretion in MIN-6, INS-1, and RINm5F insulinoma cells. Cells were treated with 5-HT (500 µM) at the beginning of a 60 min secretion period. Insulin secretion is normalized to glucose-induced control cells. *p<0.05; n = 4. (H) Insulin secretion of RINm5F cells with or without glucose and 5-HTP (500 µM; 3 h) or pargyline (20 µM; 3 h). *p<0.05; n = 3. All data are presented as means ± SEM.

Figure 3. Impaired exocytosis of Tph1−/− β-cells and rescue with 5-HT.

(A) A train of 50 depolarizing pulses from −80 to +10 mV for 40 ms at 10 Hz induced changes in the membrane capacitance of wt (n = 24) and Tph1−/− (n = 19) β-cells. Data are shown as means ± SEM. (B) Representative current-clamp recordings of the electrical activity of wt and Tph1−/− β-cells in pancreas tissue slices. The slices were perfused with solutions containing different glucose concentrations as indicated in the bar below the traces. (C) Representative membrane capacitance response of isolated wt and Tph1−/− β-cells (top panel) stimulated by ramp [Ca²⁺]_i change induced by slow photo-release of caged Ca²⁺ (bottom panel). The impaired component exocytosis has been partially rescued by extracellular 5-HTP (500 µM, 24 h) and completely restored by pipette intracellular dialysis with 5-HT.

Figure 4. Inhibition of protein serotonylation reduces insulin secretion.

(A) Scheme of protein serotonylation of glutamine residues by TGase. Cysteamine (CTA) and monodansylcadaverin (MDC) are potent inhibitors of TGases. (B and C) Uptake and protein incorporation of [³H]-5-HT in β-TC3 (B) and RINm5F cells (C) in the presence of CTA (500 µM; 3 h). *p<0.05; n = 3. (D) Insulin secretion from MIN-6 cells in the presence of CTA (500 µM; 3 h). *p<0.05; n = 4. (E) Proteasomal degradation of serotonylated proteins in RINm5F cells at different glucose concentrations in the presence or absence of the proteasomal inhibitor LLnL (50 µM; 3 h).*p<0.05; n = 4. (F) Immunoblot of serotonylated (5-HT) and total (5xHis) 6xH-Rab3a (white arrowhead) and 6xH-Rab27a (black arrowhead) prepared from glucose-stimulated RINm5F cells treated with 100 µM MDC, 200 µM CTA, or vehicle. Shown is one representative experiment out of three repetitions. (G) Quantification by Ni-NTA pull-downs of 6xH-Rab3a and 6xH-Rab27a from glucose-stimulated RINm5F cells incubated with [³H]-5-HT and either 50 µM LLnL or vehicle. *p<0.05; n = 3. Vector, vector-transfected control. For (B–E) and (G), data are means ± SEM.

Figure 5. Proposed model of 5-HT-induced exocytosis of β-granules from glucose-stimulated β-cells.

The classic sequel of glucose transport, glucose oxidation and ATP generation, ATP-gated potassium channel closure, membrane depolarisation, and Ca²⁺ influx via voltage-dependent calcium channels is represented in the upper part of the scheme. Amongst other functions, Ca²⁺ activates TGases that serotonylate a variety of proteins. In the β-cell, Rab3a, and Rab27a, which are crucially involved in the exocytosis of insulin, are target signaling proteins that become activated by this mechanism. Insulin and 5-HT co-secretion is the consequence. While insulin then exerts its endocrine function, 5-HT acts via an autocrine/paracrine loop. Initially, high extracellular 5-HT concentrations [5-HT]_e attenuate further insulin secretion through 5-HT_1A receptors, with progressive weakening of this effect by the clearance of [5-HT]_e via uptake through the plasma membrane SERT. Eventually, [5-HT]_i reaches much higher levels than [5-HT]_e, promoting another event of insulin secretion by serotonylation. This 5-HT-dependent cycling between inhibition and induction might contribute to the well-known oscillating nature of insulin exocytosis from glucose-stimulated β-cells.