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Rahmawati Aisyah　Seminar

 

New roles for dopamine D2 and D3 receptors in pancreatic beta cell insulin secretion

Zachary J. Farino Travis J. Morgenstern Antonella Maffei Matthias Quick Alain J. De Solis Pattama Wiriyasermkul Robin J . Freyberg Despoina Aslanoglou Denise Sorisio Benjamin P. Inbar R. Benjamin Free Prashant Donthamsetti Eugene V. Mosharov Christoph Kellendonk Gary J. Schwartz David R. Sibley Claudia Schmauss Lori M. Zeltser Holly Moore Paul E. Harris Jonathan A. Javitch Zachary Freyberg 

 

Molecular Psychiatry, 2019

 

Introduction

Antipsychotic drugs (APDs) used for psychotropic medications cause metabolic disturbances, particularly on glycemic control. Although the mechanisms behind APD-induced metabolic dysfunction are still unclear, all clinically effective APDs have a single unifying characteristic that is their action on dopamine (DA) D2-like receptors (D2, D3, and D4 receptors).

D2-like receptors are expressed in brain regions that regulate feeding behaviors and appetite. Recent studies have shown that D2 (D2R) and D3 (D3R) receptors are also found in peripheral tissues essential for metabolic regulation, including insulin-secreting pancreatic beta cells. Previous studies suggest that DA and possibly D2R/D3R signaling possess significant roles in modulating glucose-stimulated insulin secretion (GSIS). However, the individual roles of each receptor in GSIS regulation are still poorly understood as most agonists and blockers share affinities for both. The use of global D2R and D3R KO mice produced ambiguous results as the receptors are expressed in several central nervous systems (CNS) and peripheral tissues related to metabolic regulation.

Pancreatic beta cells have the ability to synthesize and catabolize DA, comparable to CNS DA neurons.  Beta cells express aromatic amino-acid decarboxylase (AADC), which coverts L-DOPA (DA precursor) to DA, monoamine oxidases A and B (MAO-A and MAO-B), which catabolize mono-amine, and vesicular monoamine transporter (VMAT2), which is needed to load DA into vesicles.  However, the exact roles of beta cell DA synthesis/utilization in metabolism and the role of D2R and D3R in these processes remain unclear.

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Figure 1. Insulin-secreting INS-1E cells require the dopamine precursor L-DOPA for dopamine biosynthesis and release.

  1. HPLC traces of secreted DA and its metabolites from INS-1E cells in the absence of precursor L-DOPA supplementation. No DA or DA metabolites were detectable.

  2. HPLC traces of secreted DA and its metabolites from INS-1E with precursor L-DOPA supplementation (30μM, 90 min, 37 °C). DA and DA metabolites were detected.

HPLC analysis of glucose-stimulated DA secretion. Glucose stimulation (20μM, 90 min, 37 °C) increased secreted DA by 70% compared with the unstimulated condition (P=0.013)

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Figure 2. Glucose stimulation enhances L-DOPA uptake in INS-1E cells.

  1. Comparative qPCR analysis of L-type amino acid transporter 1 (LAT1) mRNA transcripts in INS-1E cells, wildtype C57BL/6J mouse pancreatic islets, hypothalamus, and striatum. LAT1 mRNA levels were comparable between all groups.

  2. Comparative qPCR analysis of L-type amino acid transporter 2 (LAT2) mRNA transcripts in INS-1E cells, wildtype C57BL/6J mouse pancreatic islets, hypothalamus, and striatum. LAT2 mRNA was present in all groups, with the highest in INS-1E cells (P = 0.0062).

  3. Time course of [3H]L-DOPA (2 μM) uptake in the presence (blue square) or absence (black circle) of 20 mM glucose stimulation. Glucose stimulation caused a 40% increase in [3H]L-DOPA uptake within 30 min relative to the unstimulated condition (P=0.0005). The AADC inhibitor benserazide (5 μM, red triangle) inhibited a decrease in [3H]L-DOPA accumulation at later timepoints (60–120 min).

Area under the curve (AUC) analysis of [3H]L-DOPA uptake for the three conditions shown in c. Glucose stimulation caused a significant overall AUC change [F(2,5) = 42.72, P = 0.001]. There was a 2.5-fold increase in the glucose-stimulated condition compared with the unstimulated condition (P = 0.002). Benserazide (5 μM) addition further enhanced this AUC increase (P = 0.001)

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Figure 3. Effects of L-DOPA and D2R/D3R blockade on glucose-stimulated insulin secretion. Homogenous time resolve fluorescence (HTRF) based measurement of secreted insulin in INS-1E cells in response to 20 mM glucose stimulation (90 min, 37 °C).

  1. Increasing concentrations of L-DOPA caused dose-dependent inhibition of GSIS, which was best fit to a sigmoidal curve (in black; IC50= 38.3 μM, R2 = 0.84). AADC inhibition by benserazide (10 μM) abolished L-DOPA’s inhibition of GSIS (in red).

  2. Treatment with the D2R/D3R agonist quinpirole produced a dose-dependent inhibition of GSIS (in black; IC50 = 10.3 μM, R2 = 0.90).

Concurrent blockade of D2R and D3R by increasing concentrations of raclopride in the presence of 100 μM L-DOPA attenuated L-DOPA’s inhibitory effects on GSIS.

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Figure 4. Effects of L-DOPA and dopamine on glucose-stimulated insulin secretion in pancreatic islets of D2R or D3R knockout mice.

  1. L-DOPA treatment significantly inhibited glucose-stimulated insulin secretion (GSIS) in pancreatic islets from wildtype (WT) C57BL/6J mice in a concentration-dependent manner [F(2,67) = 12.32, P < 0.0001]. GSIS was reduced both with 10 μM L-DOPA (P = 0.006), and 30 μM L-DOPA (P < 0.0001) compared with stimulation with 20mM glucose alone (n = 20 for all groups).

  2. Inhibitory effects of L-DOPA on GSIS were attenuated in pancreatic islets from homozygous global D3R KO mice at both 10 μM and 30 μM L-DOPA concentrations [F (2,42) = 1.12, P > 0.05, n = 15 for all groups].

  3. Although no significant GSIS inhibition was evident at 10 μM L-DOPA (P > 0.05), and largely attenuated at 30 μM L-DOPA (P = 0.05; n = 16 for all groups) in islets from β-cell-selective D2R KO mice.

  4. DA treatment of pancreatic islets from WT mice significantly inhibited GSIS in a dose-dependent manner compared with stimulation with 20mM glucose alone [F(3,74) = 13.11, P < 0.001] (100 nM: P = 0.026; 1 μM: P < 0.0001; 10 μM: P < 0.0001; n = 35 for all groups).

  5. Pancreatic islets from global D3R KO mice exhibited significant DA-induced GSIS inhibition [F(3,56) = 5.17, P = 0.003]. Though D3R KO islets did not significantly respond to 100 nM DA, both 1 μM DA (P = 0.03), and 10 μM DA treatment significantly inhibited GSIS (P = 0.001; n = 15 for all groups).

  6. Pancreatic islets from β-cell-specific D2R KO mice responded to DA treatment [F(3,60) = 6.60, P = 0.001]. D2R KO islets exhibited GSIS inhibition at higher DA concentrations: 1 μM (P < 0.0001) and 10 μM DA (P = 0.0003), but not at 100 nM DA (P > 0.05; n = 16 for all groups). Results are represented as % maximal insulin secretion based on mean HTRF values from n ≥ 6 replicate samples in age-matched mice. Assays were conducted on n ≥ 3 independent experimental days. All bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

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Figure S5. Glucose-stimulated DA secretion is reduced in D2R and D3R KO pancreatic islets.

  1. Pancreatic islets isolated from homozygous global D3R KO mice secreted significantly less DA (32% reduction) compared to wildtype (WT) littermate controls in response to stimulation with 20 mM glucose and 30 μM L-DOPA (P=0.012; n=6 D3R KO, n=8 WT).

Pancreatic islets from homozygous β-cell-specific D2R KO mice secreted 55% less DA compared to WT littermate controls (P<0.0001; n=5 for D2R KO and WT)

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Figure 5. D2R KO in beta cells disrupts postprandial insulin regulation in vivo

  1. Postprandial elevation in serum insulin levels was threefold higher in homozygous pancreatic β-cell-selective D2R KO mice (n = 12) compared with WT littermate controls (n = 9; P = 0.038).

  2. There were no significant differences in either pre-meal fasting or postprandial glucose levels between homozygous pancreatic β-cell-selective D2R KO mice (n = 12) compared with WT littermate controls (n = 9; P > 0.05).

  3. Intraperitoneal glucose tolerance test (ipGTT, 2 g/kg). There were no significant differences in glucose tolerance between homozygous pancreatic β-cell-selective D2R KO mice (n = 6) compared with WT littermate controls (n = 10; P > 0.05).

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