Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B

Abstract

Both insulin and insulin-like growth factor 1 (IGF-1) are known to reduce glucose-dependent insulin secretion from the beta cells of pancreatic islets. In this paper we show that the mechanism by which IGF-1 mediates this effect is in large part through activation of a specific cyclic nucleotide phosphodiesterase, phosphodiesterase 3B (PDE3B). More specifically, in both isolated pancreatic islets and insulin-secreting HIT-T15 cells, IGF-1 inhibits insulin secretion that has been increased by glucose and glucagonlike peptide 1 (GLP-1). Moreover, IGF-1 decreases cAMP levels in parallel to the reduction of insulin secretion. Insulin secretion stimulated by cAMP analogs that activate protein kinase A and also are substrates for PDE3B is also inhibited by IGF-1. However, IGF-1 does not inhibit insulin secretion stimulated by nonhydrolyzable cAMP analogs. In addition, selective inhibitors of PDE3B completely block the ability of IGF-1 to inhibit insulin secretion. Finally, PDE3B activity measured in vitro after immunoprecipitation from cells treated with IGF-1 is higher than the activity from control cells. Taken together with the fact that pancreatic beta cells express little or no insulin receptor but large amounts of IGF-1 receptor, these data strongly suggest a new regulatory feedback loop model for the control of insulin secretion. In this model, increased insulin secretion in vivo will stimulate IGF-1 synthesis by the liver, and the secreted IGF-1 in turn feedback inhibits insulin secretion from the beta cells through an IGF-1 receptor-mediated pathway. This pathway is likely to be particularly important when levels of both glucose and secretagogues such as GLP-1 are elevated.

Figure 1

PDE3B expression in the β cells of rat pancreatic islets. (A) The antibody against the C-terminal portion of a GST-PDE3B fusion protein recognizes a single band from a mouse epididymal fat pad extract. This band was competed away by preincubation with 200 ng of GST-PDE3B antigen but not with 200 ng of a GST-PDE1C2 polypeptide of similar length. This serum also specifically detects the PDE3B protein in the extracts of rat epididymal fat pad, rat pancreatic islets, and a hamster pancreatic β cell line, HIT-T15. (B) Confocal images of a rat pancreatic islet double-stained by anti-PDE3B and anti-insulin antibodies. The PDE3B signal was visualized by a fluorescein-conjugated goat anti-rabbit secondary antibody, and the insulin signal by a rhodamine-conjugated donkey anti-mouse secondary antibody. The side-by-side comparison of the two images clearly indicates that PDE3B is expressed in insulin-containing β cells, which is confirmed by the prevailing orange color in the merged image. (×40.) Note: the punctuate staining of insulin reflects insulin-containing secretory vesicles.

Figure 1

PDE3B expression in the β cells of rat pancreatic islets. (A) The antibody against the C-terminal portion of a GST-PDE3B fusion protein recognizes a single band from a mouse epididymal fat pad extract. This band was competed away by preincubation with 200 ng of GST-PDE3B antigen but not with 200 ng of a GST-PDE1C2 polypeptide of similar length. This serum also specifically detects the PDE3B protein in the extracts of rat epididymal fat pad, rat pancreatic islets, and a hamster pancreatic β cell line, HIT-T15. (B) Confocal images of a rat pancreatic islet double-stained by anti-PDE3B and anti-insulin antibodies. The PDE3B signal was visualized by a fluorescein-conjugated goat anti-rabbit secondary antibody, and the insulin signal by a rhodamine-conjugated donkey anti-mouse secondary antibody. The side-by-side comparison of the two images clearly indicates that PDE3B is expressed in insulin-containing β cells, which is confirmed by the prevailing orange color in the merged image. (×40.) Note: the punctuate staining of insulin reflects insulin-containing secretory vesicles.

Figure 2

IGF-1 receptor, but not insulin receptor, is expressed in rat pancreatic islets. (A) Western blot detection of IGF-1 receptor α-subunit expression in rat pancreatic islets (lane I). Although insulin receptor is strongly expressed in rat liver (lane L), no detectable level of expression was seen in pancreatic islets. The solid arrows indicate either the IGF-1 or insulin receptor α subunits. Both antibodies are directed against the N-terminal 20 amino acids of the corresponding receptor α subunits. (B) Confocal image of a rat pancreatic islet. (×20.) The primary antibody is anti-IGF-1 receptor α subunit. The staining indicates that IGF-1 receptor is expressed in the pancreatic β cells.

Figure 2

IGF-1 receptor, but not insulin receptor, is expressed in rat pancreatic islets. (A) Western blot detection of IGF-1 receptor α-subunit expression in rat pancreatic islets (lane I). Although insulin receptor is strongly expressed in rat liver (lane L), no detectable level of expression was seen in pancreatic islets. The solid arrows indicate either the IGF-1 or insulin receptor α subunits. Both antibodies are directed against the N-terminal 20 amino acids of the corresponding receptor α subunits. (B) Confocal image of a rat pancreatic islet. (×20.) The primary antibody is anti-IGF-1 receptor α subunit. The staining indicates that IGF-1 receptor is expressed in the pancreatic β cells.

Figure 3

Effect of IGF-1 on insulin secretion is potentiated by 8-Br-cAMP but not N⁶-benzoyl-cAMP (6-Bnz-cAMP). Newborn rat pancreatic islets in monolayer cell culture were preincubated in low-glucose medium (1.6 mM) for 2 hr before being switched to high glucose (11.1 mM). The cAMP analogs and/or 10 nM IGF-1 were added together with the high-glucose medium. After incubation for 30 min the medium was analyzed for immunoreactive insulin (IRI). Experiments under each set of conditions were carried out in triplicate. IGF-1 suppressed insulin secretion potentiated by 8-Br-cAMP (a relatively good PDE3B substrate) but had little or no effect on the insulin secretion potentiated by N⁶-benzoyl-cAMP, an analog highly resistant to hydrolysis by PDE3B. Twenty-five units of IRI = 1 mg of insulin.

Figure 4

Milrinone prevents the inhibitory effect of IGF-1 on insulin secretion. The newborn rat pancreatic monolayer cells were cultured in low-glucose medium before transfer to high-glucose medium (see the legend of Fig. 2.). Different pharmacological conditions are represented by patterns or stippling. Milrinone, a highly selective inhibitor of PDE3B (IC₅₀ = 0.3 μM), potentiates insulin secretion at low concentration (1 μM), suggesting the presence of PDE3 activity. Although IGF-1 significantly suppressed insulin secretion in the absence of milrinone, it could not inhibit insulin secretion in its presence.

Figure 5

IGF-1 suppresses insulin secretion from HIT-T15 cells. HIT-T15 cells were perifused by Krebs–Ringer buffer containing 0.1% BSA and no glucose until the basal insulin level became stable. The perifused cells were then switched to the same buffer containing high glucose with or without the other agents. The starting point of exposure to high-glucose medium is indicated by the arrow. The results shown here are the average of fractions from duplicate columns. IGF-1 consistently suppressed insulin secretion both with and without 8-Br-cAMP potentiation.

Figure 6

Inhibition of insulin secretion by IGF-1 is accompanied by a decrease in cAMP. HIT-T15 cells were preincubated with Krebs–Ringer buffer containing no glucose for 1 hr before treatment with GLP-1 and/or IGF-1 in the presence of glucose at a high concentration. Different conditions are represented by different stippling patterns. GLP-1, as expected, increased cAMP and potentiated insulin secretion from the HIT-T15. IGF-1 counteracted the GLP-1 effect on insulin secretion and caused a drop in cAMP.

Figure 7

Activation of PDE3B by IGF-1 in HIT-T15 cells. (A) When PDE3B activity was immunoprecipitated from the HIT-T15 extract, the pellet contained only PDE3B, as all of the PDE activity was completely suppressed by milrinone. A significant increase of PDE3B activity was observed when the HIT-T15 cells were treated with IGF-1. As a control, the PDE4 activity remaining in the supernatant did not change under any of the conditions tested. (B) Western blot assay indicating the integrity and relative amount of PDE3B protein present in each assay.

Figure 8

Model for the role of PDE3B in IGF-1 regulation of insulin secretion from pancreatic β cells. In this model increased circulating insulin stimulates the synthesis and release of IGF-1 (thick arrows) from the liver (27, 28). The elevated IGF-1 in the circulation then binds to IGF-1 receptors (IGF-1R) on the pancreatic β cell surface, activating a series of still unidentified kinases that eventually cause activation of PDE3B. As a result, the cAMP levels within the β cells declines, leading to attenuation of insulin release. If the local insulin concentration is too high near the β cells, this regulatory feedback loop may also allow insulin to exert an immediate inhibitory effect on its own secretion through the IGF-1 receptors (as indicated by the thin open arrows). This model does not exclude participation of potential paracrine IGF-1 synthesized locally within the pancreatic islets.