Serotonin- and Dopamine-Related Gene Expression in db/db Mice Islets and in MIN6 β -Cells Treated with Palmitate and Oleate

© 2016 L. R. Cataldo et al. High circulating nonesterified fatty acids (NEFAs) concentration, often reported in diabetes, leads to impaired glucose-stimulated insulin secretion (GSIS) through not yet well-defined mechanisms. Serotonin and dopamine might contribute to NEFA-dependent β-cell dysfunction, since extracellular signal of these monoamines decreases GSIS. Moreover, palmitate-treated β-cells may enhance the expression of the serotonin receptor Htr2c, affecting insulin secretion. Additionally, the expression of monoamine-oxidase type B (Maob) seems to be lower in islets from humans and mice with diabetes compared to nondiabetic islets, which may lead to increased monoamine concentrations. We assessed the expression of serotonin- and dopamine-related genes in islets from db/db and wild-type (WT) mice. In addition, the effect of palmitate and oleate on the expression of such genes, 5HT content, and GSIS in MIN6 β-cell was determined. Lower Maob expression was found in islets from

Prolonged Activation of the Htr2b Serotonin Receptor Impairs Glucose Stimulated Insulin Secretion and Mitochondrial Function in MIN6 Cells

<div><p>Aims</p><p>Pancreatic β-cells synthesize and release serotonin (5 hydroxytryptamine, 5HT); however, the role of 5HT receptors on glucose stimulated insulin secretion (GSIS) and the mechanisms mediating this function is not fully understood. The aims of this study were to determine the expression profile of 5HT receptors in murine MIN6 β-cells and to examine the effects of pharmacological activation of 5HT receptor Htr2b on GSIS and mitochondrial function.</p><p>Materials and Methods</p><p>mRNA levels of 5HT receptors in MIN6 cells were quantified by RT qPCR. GSIS was assessed in MIN6 cells in response to global serotonergic activation with 5HT and pharmacological Htr2b activation or inhibition with BW723C86 or SB204741, respectively. In response to Htr2b activation also was evaluated the mRNA and protein levels of PGC1α and PPARy by RT-qPCR and western blotting and mitochondrial function by oxygen consumption rate (OCR) and ATP cellular content.</p><p>Results</p><p>We found that mRNA levels of most 5HT receptors were either very low or undetectable in MIN6 cells. By contrast, Htr2b mRNA was present at moderate levels in these cells. Preincubation (6 h) of MIN6 cells with 5HT or BW723C86 reduced GSIS and the effect of 5HT was prevented by SB204741. Preincubation with BW723C86 increased PGC1α and PPARy mRNA and protein levels and decreased mitochondrial respiration and ATP content in MIN6 cells.</p><p>Conclusions</p><p>Our results indicate that prolonged Htr2b activation in murine β-cells decreases glucose-stimulated insulin secretion and mitochondrial activity by mechanisms likely dependent on enhanced PGC1α/PPARy expression.</p></div

Prolonged activation of Htr2b reduces GSIS in MIN6 cells.

<p>(A) MIN6 cells were treated with vehicle or increasing concentrations (10 nM– 10 μM) of the selective Htr2b agonist BW723C86 for 6 hours and then subjected to GSIS procedure. (B) MIN6 cells were treated with vehicle, 50 μM 5HT or co-treated with 5HT (50 μM) plus a selective Htr2b antagonist SB204741 (100 nM) for 6 hours and then subjected to GSIS procedure. The GSIS assays were carry out with KRH buffer 0 (white bars) or 20 mM glucose (black bars). Concentration of insulin accumulated over 1 hour was measured by ELISA. The bars represent mean ± SEM of three independent experiments in triplicate. Number over the bars represents the stimulation index (SI). ** p<0.01, *** p<0.001 and ns, non-significant. One-way ANOVA analysis.</p

5HT receptors mRNA levels in MIN6 cells.

<p>Total RNA was extracted from MIN6 cells and 5HT receptors mRNA levels assessed by RT-qPCR. The mRNA levels was expressed as fold-change to Insulin receptor (Insr) (2^{−ΔCt 5HT receptors}/2^{−ΔCt Insr}). Bars correspond to mean ± SEM of three independent experiments. Numbers over the bars correspond to mean Ct values.</p

Prolonged Htr2b activation increases PGC1α and PPARy levels in MIN6 cells and the effect of PGC1α1 overexpression on GSIS.

<p>(A) MIN6 cells were treated for 6 hours with the Htr2b selective agonist BW723C86 (10 μM) and then the mRNA levels of PGC1α and PPARy were quantified by RT-qPCR. Relative mRNA levels are expressed as fold-change of 2^−ΔCt in treated vs. control cells (2^{−ΔCt treated}/2^{−ΔCt control}). Numbers over bars correspond to mean Ct values. (B) MIN6 cells were treated for 24 hours with BW723C86 (10 μM) and then, PGC1α and Gapdh protein expression was evaluated by immunoblot. (C and D) MIN6 cells were transfected with a pcDNA3.1-PGC1α1 or an empty vector (mock) for 48 hours. (C) Protein levels of PGC1α1 and (D) insulin secretion were measured by immunoblot and GSIS procedure, respectively. Graph bars represent means ± SEM of three independent experiments in triplicate. ** p<0.01 and *** p<0.001 in one-way ANOVA analysis (A and D) or Student’s t test (B and C).</p

5HTP increases extracellular 5HT levels in MIN6 cells and 5HT reduces GSIS.

<p>(A) MIN6 cells were incubated with 5HT precursor 5HTP (500 μM) (black bars) or vehicle (white bars) for 6 hours and then 5HT concentration was quantify in the conditioned medium by HPLC. (B) MIN6 cells were incubated with 5HT (50 μM) or vehicle during 6 hours in DMEM without FBS and then subjected to GSIS procedure with 0 (white bars) or 20 mM glucose (black bars) for 1 hour. Insulin concentration in the KRH buffer was quantified by ELISA. Graph bars represent mean ± SEM of measurements of three independent experiments in triplicate. The symbol *** denotes p<0.001 in Student’s t (A) tests and in one-way ANOVA (B).</p

Proposed model for the inhibitory effect of prolonged Htr2b activation on GSIS in MIN6 cells.

<p>MIN6 cells constitute a functional microserotoninergic system, with the ability to synthesis 5HT from its precursors (tryptophan or 5HTP), storage, degrade and release this monoamine. The extracellular 5HT may decrease GSIS through the activation of the Htr2b receptor, the most abundantly 5HT receptor expressed in MIN6 cells. Prolonged Htr2b activation is accompanied with calcium release from endoplasmic reticulum and its inhibitory effects on GSIS is likely the result of enhanced PGC1α/PPARy expression and mitochondrial dysfunction with decreased glucose-stimulated ATP synthesis.</p