Dopamine synthesis and D3 receptor activation in pancreatic β-cells regulates insulin secretion and intracellular [Ca(2+)] oscillations

Abstract

Pancreatic islets are critical for glucose homeostasis via the regulated secretion of insulin and other hormones. We propose a novel mechanism that regulates insulin secretion from β-cells within mouse pancreatic islets: a dopaminergic negative feedback acting on insulin secretion. We show that islets are a site of dopamine synthesis and accumulation outside the central nervous system. We show that both dopamine and its precursor l-dopa inhibit glucose-stimulated insulin secretion, and this inhibition correlates with a reduction in frequency of the intracellular [Ca(2+)] oscillations. We further show that the effects of dopamine are abolished by a specific antagonist of the dopamine receptor D3. Because the dopamine transporter and dopamine receptors are expressed in the islets, we propose that cosecretion of dopamine with insulin activates receptors on the β-cell surface. D3 receptor activation results in changes in intracellular [Ca(2+)] dynamics, which, in turn, lead to lowered insulin secretion. Because blocking dopaminergic negative feedback increases insulin secretion, expanding the knowledge of this pathway in β-cells might offer a potential new target for the treatment of type 2 diabetes.

Fig. 1.

The effects of l-dopa on pancreatic islet dopamine content in vitro and in vivo. A and B, The dopamine content of pancreatic islets was measured immediately after isolation and after incubation in presence (■) (A) (n = 4–7) or absence (□) (B) (n = 3–4) of 10 μm l-dopa for different time intervals. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. incubation time = 0 h. C, The dopamine content of pancreatic islets was measured after a 30-min incubation in presence of 50 μm benserazide, 10 μm l-dopa, and a combination of 50 μm benserazide + 10 μm l-dopa (n = 2–3). **, P < 0.01 vs. 50 μm benserazide; ^#, P < 0.05 vs. 10 μm l-dopa. D, The dopamine content of pancreatic islets was measured immediately after isolation from mice that have received an ip injection of saline solution or 50 mg/kg of l-dopa 30 min before surgery (n = 2). ***, P < 0.001 vs. saline.

Fig. 2.

The effects of l-dopa-induced dopamine accumulation and of dopamine receptor antagonists on glucose-stimulated insulin secretion. A, Insulin secretion measured at 2.8 mm glucose, 16.7 mm glucose, and 16.7 mm glucose plus increasing concentrations of l-dopa as indicated (n = 5–13). B, Sigmoidal dose-response curve fit of insulin secretion stimulated by 16.7 mm glucose in presence of 0.1 μm, 1 μm, 3 μm, 10 μm, and 100 μm l-dopa; R² = 0.97; gray line indicates, best-fit EC₅₀ = 4.4 μm (n = 5–6). C, Insulin secretion measured at 2.8 mm glucose, and 2.8 mm glucose + 0.1 μm, 1 μm, 10 μm l-dopa, and 10 μm quinpirole (n = 4–12). D, Insulin secretion measured at 2.8 mm glucose, 16.7 mm glucose, 16.7 mm glucose after pretreatment with 10 nm, 100 nm and 10 μm l-dopa, 16.7 mm glucose + 10 μm quinpirole (n = 4–11). E, Insulin secretion measured at 2.8 mm glucose (white bar), 16.7 mm glucose (black bar), and 16.7 mm glucose + 10 μm dopamine (gray bar). The selective antagonist for dopamine receptor D2 (L-741,626), or for dopamine receptor D3 (GR 103691) was added to the three stimuli as indicated (n = 6–8). F, Dopamine secretion and insulin secretion from pancreatic islets measured at 2.8 mm glucose, 16.7 mm glucose, 16.7 mm glucose + 50 μm forskolin (n = 4–5). ##, P < 0.01; ###, P < 0.0001 vs. 2.8 mm glucose; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 vs. 16.7 mm glucose; +, P < 0.05; +++, P < 0.001 vs. the respective untreated control.

Fig. 3.

The effects of l-dopa and dopamine on the redox state of pancreatic islets. A, NAD(P)H autofluorescence from isolated islets was measured at 8 mm glucose and at 16.7 mm glucose and with 10 μm dopamine or 10 μm l-dopa as indicated; results are normalized to the maximum signal obtained with 3 mm sodium cyanide (n =2–4). B, Islets were incubated with or without 10 μm l-dopa for 30 min before the experiment after which NAD(P)H autofluorescence was measured at 8 mm and 16.7 mm glucose (n = 3–4).

Fig. 4.

The effects of dopamine and l-dopa on [Ca²⁺]_i oscillation frequency in isolated islets. A, Representative patterns of [Ca²⁺]_i oscillations from a single islet before and after dopamine stimulus. The typical oscillation pattern, stimulated by increasing glucose concentration from 2 mm to 8 mm, is shown in in the first 300 sec; the pattern changed after the addition of dopamine at the indicated concentration. Black arrows indicate the time when dopamine was added; the three plots are offset for an easier comparison. B, Representative patterns of [Ca²⁺]_i oscillations from a single islet before and after l-dopa stimulus. The oscillations were triggered by increasing glucose concentration from 2 mm to 8 mm; black arrows indicate the time when l-dopa was added. C, Sigmoidal dose-response curve fit of [Ca²⁺]_i oscillation frequency in response to treatment with dopamine (▴) or l-dopa (■) in conjunction with 8 mm glucose stimulus; gray line indicates, best-fit EC₅₀ = 3.8 μm, R² = 0.78 (n = 4–7); black line indicates, best-fit EC₅₀ = 7.2 μm, R² = 0.89 (n = 5–14); The difference between EC₅₀ values is statistically significant with a P < 0.05. D, Plot of [Ca²⁺]_i oscillation frequency vs. insulin secretion from two independents sets of experiments; the gray line represents the linear fit of the data (R² = 0.999); Pearson's correlation coefficient r = 1 with P < 0.0001. E, The [Ca²⁺]_i oscillation frequency of control islets vs. islets with elevated dopamine content resulting from a pretreatment with l-dopa as indicated (n = 8); ***, P < 0.001. F, The [Ca²⁺]_i oscillation frequency of control islets vs. islets treated with D3 antagonist (GR 103691) or a mixture of D3 antagonist and dopamine as indicated (n = 9–14); ***, P < 0.001. DA, Dopamine; s, second.

Fig. 5.

Immunodetection of dopamine receptors and DAT in pancreatic islets. A, Immunoblot for DRD2: lane 1, mouse brain extract (25 μg total protein); lane 2, pancreatic islets lysate (54 μg total protein); lane 3, pancreatic islets lysate (45 μg total protein); the white box overlay highlights the 58-kDa band present in all lanes. B, Control immunoblot for DRD2 using the blocking peptide for the primary antibody: lane 1, mouse brain extract (20 μg total protein); lane 2, empty; lanes 3–5, pancreatic islets lysate (20 μg total protein); the white box overlay highlights the 58-kDa band missing in all lanes. C, Immunoblot for DRD3: lane 1, islet membrane fraction (18 μg); lane 2, islet cytosolic fraction (18 μg); lane 3, islet nuclear fraction (18 μg); white box overlay highlights the 48-kDa band present in the islet membrane fraction. D, Control immunoblot for DRD3: lane 1, islet membrane fraction (18 μg); lane 2, islet cytosolic fraction (18 μg); lane 3, islet nuclear fraction (18 μg): white box indicates the absence of the 48-kDa band in the islet membrane fraction. E, Immunoblot for DAT: lane 1, mouse brain extract (10 μg total protein); lanes 2–5, pancreatic islets lysate (76, 73, 46, and 24 islets, respectively); lane 6, empty.

Fig. 6.

Live subcellular localization of DRD2_L-mVenus and quantification of dopamine secretion. A, Sum intensity projection of 47 confocal images of an islet expressing DRD2-mVenus (representative image of 33 cells from seven islets); white line indicates the islet outline; scale bar, 20 μm. B, Confocal image of a single cell from the islet shown in panel A; scale bar, 5 μm. C, Wide-field fluorescence image of a MIN6 expressing DRD2_L-mVenus; scale bar, 5 μm. D, TIRF image of the same field of view shown in panel C; scale bar, 5 μm.