The autonomic nervous system regulates pancreatic β-cell proliferation in adult male rats

Abstract

The pancreatic β-cell responds to changes in the nutrient environment to maintain glucose homeostasis by adapting its function and mass. Nutrients can act directly on the β-cell and also indirectly through the brain via autonomic nerves innervating islets. Despite the importance of the brain-islet axis in insulin secretion, relatively little is known regarding its involvement in β-cell proliferation. We previously demonstrated that prolonged infusions of nutrients in rats provoke a dramatic increase in β-cell proliferation in part because of the direct action of nutrients. Here, we addressed the contribution of the autonomic nervous system. In isolated islets, muscarinic stimulation increased, whereas adrenergic stimulation decreased, glucose-induced β-cell proliferation. Blocking α-adrenergic receptors reversed the effect of epinephrine on glucose + nonesterified fatty acids (NEFA)-induced β-cell proliferation, whereas activation of β-adrenergic receptors was without effect. Infusion of glucose + NEFA toward the brain stimulated β-cell proliferation, and this effect was abrogated following celiac vagotomy. The increase in β-cell proliferation following peripheral infusions of glucose + NEFA was not inhibited by vagotomy or atropine treatment but was blocked by coinfusion of epinephrine. We conclude that β-cell proliferation is stimulated by parasympathetic and inhibited by sympathetic signals. Whereas glucose + NEFA in the brain stimulates β-cell proliferation through the vagus nerve, β-cell proliferation in response to systemic nutrient excess does not involve parasympathetic signals but may be associated with decreased sympathetic tone.

Keywords: autonomic nervous system; nutrients; parasympathetic; sympathetic; β-cell proliferation.

Fig. 1.

Regulation of glucose-induced β-cell proliferation in isolated rat islets by carbachol and epinephrine. Isolated rat islets were cultured in the presence of 5.5, 11.1, or 16.7 mmol/l glucose with or without carbachol (carb; 500 µmol/l) or epinephrine (epi; 1 µmol/l) for 72 h. A–C: representative sections of islets exposed to the indicated glucose concentrations and vehicle (A), carbachol (B), or epinephrine (C) immunostained for insulin (green), Ki-67 (red), and nuclei (blue). Arrows show positive nuclei for Ki-67. D: percentage of Ki-67⁺/insulin⁺ cells over total insulin⁺ cells. E: insulin levels in the islet supernatant. Solid line, vehicle; broken line, carbachol; dotted line, epinephrine. Veh, vehicle. Data are means ± SE (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle, 2-way ANOVA with Dunnett posttest. Scale bar = 50 µm.

Fig. 2.

Regulation of glucose- and nonesterified fatty acid (NEFA)-induced β-cell proliferation in isolated rat islets by α-adrenergic signaling. Isolated rat islets were cultured in the presence of 16.7 mmol/l glucose + NEFA mix (0.5 mmol/l; oleate, linoleate, palmitate; NEFA) with or without epinephrine (epi; 10⁻¹⁰ to 10⁻⁵ mmol/l) (A–C) or in the presence of 5.5 mmol/l glucose, 16.7 mmol/l glucose, or 16.7 mmol/l glucose + NEFA with all conditions exposed to epinephrine with or without yohimbine (yoh; 10 µmol/l) or prazosin (pra; 10 µmol/l) (D–I) for 72 h. A, D, and G: representative sections of islets immunostained for insulin (green), Ki-67 (red), and nuclei (blue). Arrows show positive nuclei for Ki-67. B, E, and H: percentage of Ki-67⁺/insulin⁺ cells over total insulin⁺ cells. C, F, and I: insulin levels in the islet supernatant. Open circles represent individual data points. White bar, 5.5 mmol/l glucose; gray bar, 16.7 mmol/l glucose; black bar, 16.7 mmol/l glucose + NEFA. Veh, vehicle. Data are individual values and means ± SE (n = 4–10). *P < 0.05, **P < 0.01, and ***P < 0.001, 1-way ANOVA with Dunnett posttest. Scale bar = 50 µm.

Fig. 3.

Regulation of glucose- and nonesterified fatty acid (NEFA)-induced β-cell proliferation in isolated rat islets by isoproterenol. Isolated rat islets were cultured in the presence of 16.7 mmol/l glucose + NEFA mix (0.5 mmol/l; oleate, linoleate, palmitate; NEFA) with or without isoproterenol (iso; 10⁻⁸ to 10⁻⁴ mol/l) (A–C) or 5.5 mmol/l glucose, 16.7 mmol/l glucose, or 16.7 mmol/l glucose + NEFA with or without isoproterenol (1 µmol/l) (D–F) for 72 h. A and D: representative sections of islets immunostained for insulin (green), Ki-67 (red), and nuclei (blue). Arrows show positive nuclei for Ki-67. B and E: percentage of Ki-67⁺/insulin⁺ cells over total insulin⁺ cells. C and F: insulin levels in the islet supernatant. Open circles represent individual data points. White bar, 5.5 mmol/l glucose; gray bar, 16.7 mmol/l glucose; black bar, 16.7 mmol/l glucose + NEFA. Data are individual values and means ± SE (n = 6). ***P < 0.001 versus vehicle, 1-way ANOVA with Sidak posttest. Scale bar = 50 µm.

Fig. 4.

Metabolic parameters and β-cell proliferation following central infusion of glucose (GLU) + ClinOleic (CLI). Normal (A–F) and sham-operated (sh) or vagotomized (vgx; G–L) rats were infused with saline (SAL) or GLU + CLI via the carotid artery toward the brain for 72 h. Blood glucose (A and G), plasma insulin (B and H), plasma nonesterified fatty acid (NEFA, C and I), and caloric intake (D and J) during the infusion. E and K: percentage of Ki-67⁺/insulin⁺ cells over total insulin⁺ cells. F and L: representative pancreatic sections immunostained for insulin (green), Ki-67 (red), and nuclei (blue) after 72-h infusions. Arrows show positive nuclei for Ki-67. A–D: black circle, SAL; white circle, GLU + CLI. J: black circle, SAL-sh; white circle, GLU + CLI-sh; black square, SAL-vgx; white square, GLU + CLI-vgx. Open circles represent individual data points. Data are individual values and means ± SE (n = 5–10). *P < 0.05 and **P < 0.01 versus SAL, 1-way ANOVA with Sidak posttest. Scale bar = 50 µm.

Fig. 5.

Metabolic parameters and β-cell proliferation following systemic infusion of glucose (GLU) + ClinOleic (CLI) and effects of celiac vagotomy and atropine. Sham-operated (sh) or vagotomized (vgx) (A–E) or vehicle (veh) or atropine-treated (atr) (F–J) rats were infused with saline (SAL) or GLU + CLI via the jugular vein for 72 h. Blood glucose (A and F), plasma insulin (B and G), and plasma nonesterified fatty acid (NEFA, C and H) during the infusion. D and I: percentage of Ki-67⁺/insulin⁺ cells over total insulin⁺ cells after the 72-h infusion. E and J: representative pancreatic sections immunostained for insulin (green), Ki-67 (red), and nuclei (blue). Arrows show positive nuclei for Ki-67. Open circles represent individual data points. Data are individual values and means ± SE (n = 6). *P < 0.05 and ***P < 0.001 versus SAL, 1-way ANOVA with Sidak posttest. Scale bar = 50 µm.

Fig. 6.

Effects of epinephrine treatment on metabolic parameters and β-cell proliferation in response to systemic infusion of glucose (GLU) + ClinOleic (CLI). Rats were infused with saline (SAL) or GLU + CLI for 72 h and either vehicle (veh) or epinephrine (epi). Blood glucose (A), glucose infusion rate (GIR, B), plasma insulin (C), and plasma nonesterified fatty acid (NEFA, D) during the infusion. E: percentage of Ki-67⁺/insulin⁺ cells over total insulin⁺ cells. F: representative pancreatic sections immunostained for insulin (green), Ki-67 (red), and nuclei (blue). Arrows show nuclei positive for Ki-67. Open circles represent individual data points. Data are individual values and means ± SE (n = 4–6). **P < 0.01 and ***P < 0.001 versus SAL. Scale bar = 50 µm.