Plcb1f/f; Pdx1-CreERT2 mice fed a high-fat diet developed more severe glucose intolerance due to a defect in insulin secretion. Herein, we investigated the effects of zafirlukast on insulin secretion and the possible underlying mechanisms.
Background
Diabetes mellitus – Type II diabetes
Mechanism of insulin secretion
Glucose-stimulated insulin secretion (GSIS) in the pancreatic β-cell is mainly regulated by glucose-stimulated inducing and amplifying pathways by a variety of factors [ 5 ]. In pancreatic islets, innervation and vascularization are important mechanisms in the regulation of insulin secretion.
PLCβ-mediated regulation of GSIS
Overview of G q PCRs which potentiates GSIS through PLCβ signaling
However, whether activation of the ERK1/2 pathway correlates with induction of insulin secretion remains controversial [76]. PLCβ signaling has been shown to be involved in the Ang II-mediated regulation of GSIS.
High-throughput luminescent reporter of insulin secretion
Reduced selective GqPCR-induced insulin release in Plcb1f/f; Pdx1-CreERt2 islets (A) Selective increase of insulin secretion in pancreatic islets. Overall, we have identified a novel function of zafirlukast as a regulator of insulin secretion in pancreatic β-cells.
Phospholipase Cβ1 potentiates glucose-stimulated insulin secretion
Introduction
Therefore, recent studies have focused on improving β-cell function by describing new secretagogues in insulin secretion and the molecular mechanisms underlying these effects [ 4 ]. The sequence of events that occur as part of the triggering pathway is well established, viz. glucose metabolism, closure of ATP-sensitive K+ channels, membrane depolarization and Ca2+ entry through voltage-gated Ca2+ channels. Increased intracellular Ca2+ levels induce exocytosis of insulin vesicles by activation of the exocytotic mechanism [6].
However, the precise mechanism underlying the enhancing effect has remained elusive and further studies are needed to identify additional targets and mechanisms involved in the regulation of insulin secretion. Emerging evidence has shown that PLCβ are considered key effectors in GPCR-mediated signal transduction to potentiate insulin secretion. However, previous studies have not focused on the role of specific PLCβ isoenzymes and have only shown a putative role of PLCβ in pancreatic β cells, suggesting that blockade with non-selective PLC inhibitors, PKC inhibitors or Ca2+ chelators results in reduced insulin release [18, 19].
Taken together, the data presented here demonstrate that PLCβ1 is an important factor in the regulation of insulin secretion.
Materials and methods
The inflated pancreas was then dissected and incubated at 37°C for 15 min with manual shaking every 5 min. Islets were picked from the resulting layers and washed at least three times with ice-cold modified HBSS. The whole pancreas was placed in an acid-ethanol solution (1.5% HCl in 70% EtOH) and incubated overnight at -20°C.
Tissues were then embedded in paraffin, and 2 µm sections were cut and mounted on glass slides. The sections were stained with hematoxylin and eosin (H&E) using standard procedures or immunostained with PLCβ1 (ab185724; Abcam), insulin antibody (ab6995; Abcam), glucagon antibody (G2654; Sigma), Somatostatin antibody American Research Products Inc., Waltham, MA) and Ki-67 antibody (ab15580; Abcam). Free-floating sections were rinsed with PBS and permeabilized in PBS containing 0.2% Triton X-100 for 1 h at room temperature (RT).
For investigation of PLCβ1 expression, brain sections were incubated with anti-PLCβ1 primary antibody (1:200, sc-5291; Santa Cruz Biotechnology) for 12 h at 4°C followed by incubation for 1 h at room temperature with goat anti-mouse Alexa -488 conjugated IgG (1:500, Invitrogen).
Results
Plcb1f/f plasma insulin levels; Pdx1-CreERt2 mice were lower than Plcb1f/f mice, leading to increased plasma glucose levels (Figure 2.4A, B). This abnormal Ca2+ response in Plcb1f/f; Pdx1-CreERt2 mouse islets may have resulted in impaired GSIS. Also, PLCβ1 mRNA levels were reduced in Plcb1f/f; Pdx1-CreERt2 islets relative to Plcb1f/f islets (Figure 2.5B).
Additionally, pancreatic β cells from Plcb1f/f and Plcb1f/f; Pdx1- CreERt2 mice had similar areas (Figure 2.5E). Furthermore, we performed intraperitoneal injections of bethanechol, a non-selective muscarinic agonist, in Plcb1f/f and Plcb1f/f; Pdx1- CreERt2 mice. To test these effects, Plcb1f/f and Plcb1f/f; Pdx1-CreERt2 mice were challenged with a HFD that mimicked human status.
We then performed a glucose tolerance assay to analyze glucose homeostasis in Plcb1f/f; Pdx1-CreERt2 mice during HFD intake.
Conclusion and Discussion
This pronounced deficit in glucose tolerance was correlated with decreased plasma insulin levels, leading to increased glucose levels in Plcb1f/f; Pdx1-CreERt2 mice (Figure 2.7E). Accordingly, the GSIS assay in cultured islets of Plcb1f/f and Plcb1f/f; Pdx1-CreERt2 mice fed a HFD showed that the stimulatory effect of high glucose was lower in Plcb1f/f; Pdx1-CreERt2 islets than in that of Plcb1f/f islets (Figure 2.7F). Although PLCβ1 expression was reduced in hypothalamus, Plcb1f/f; Pdx1-CreERT2 mice showed no change in food intake or body weight (Figure 2.12A, B).
The reduced insulin secretion in Plcb1f/f; Pdx1-CreERt2 mice were attributed to an intrinsic defect in β cells, as isolated islets from these mice showed lower GSIS than those from Plcb1f/f mice. In male adults Plcb1f/f; Pdx1-CreERt2 mice, there was no notable change in β-cell proliferation and β-cell turnover (Figure 2.13A, B). In this study, changes to specific ligands, such as CCK, OXO-M, and OA, did not induce any difference in insulin secretion between pancreatic islets of Plcb1f/f; Pdx1-CreERt2 and Plcb1f/f mice.
PLCβ1 protein expression in α-cell and δ-cells in pancreatic islets from control and Plcb1f/f; Pdx1-CreERt2 islets.
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Ohara-Imaizumi et al., “Serotonin regulates glucose-stimulated insulin secretion from pancreatic β cells during pregnancy,” Proc Natl Acad Sci U S A , vol. Schraenen et al., “Placental lactogens induce serotonin biosynthesis in a subset of rat beta cells during pregnancy,” Diabetologia , vol. Ning et al., “Different signaling downstream of CCK1 receptors regulates distinct functions of CCK in pancreatic β cells,” Br J Pharmacol , vol.
Shang et al., “Effect of high dietary fat on insulin secretion in genetically diabetic Goto-Kakizaki rats,” Pancreas , vol.
Zafirlukast promotes insulin secretion by increasing calcium influx through L-type
Introduction
Materials and methods
The cells were then incubated with dimethyl sulfoxide to dissolve the formazan by shaking for 5 min and the absorbance was measured. After treatment with zafirlukast, pranlukast, and montelukast, MIN6 cells were washed in PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (#89900; Thermo Fisher Scientific). After MIN6 cells were grown on glass coverslips coated with poly-L-lysine for 2 days, the cells were washed twice and loaded with fura-2-acetoxymyl ester (Fura-2AM; final concentration 2 μM) in KRB for 1 h.
The collagenase-injected pancreas was incubated for 15 min in a 37°C water bath and shaken by hand every 5 min. MIN6 cells treated with leukotriene receptor antagonists were lysed in RIPA buffer (Thermo Fisher Scientific), which included a cocktail of protease and phosphatase inhibitors according to the manufacturer's instructions. The membrane was blocked with 5% (w/v) nonfat milk and incubated overnight at 4°C with primary antibodies.
After washing with Tris-buffered saline with Tween 20 buffer, the membrane was incubated with horseradish peroxidase-labeled secondary antibodies and proteins were visualized with enhanced chemiluminescence reagents (GE Healthcare Life Sciences).
Results
Zafirlukast increased insulin secretion in a dose-dependent manner under both low (2.8 mM) and high (16.7 mM) glucose conditions. However, the level of secreted insulin did not change after treatment with pranlukast, and montelukast markedly inhibited glucose-stimulated insulin secretion (GSIS) (Figure 3.2B). Zafirlukast produced a greater increase in insulin secretion at low (2.8 mM) compared with high (16.7 mM) glucose, indicating that zafirlukast is directly involved in the regulation of insulin secretion in the absence of glucose stimulation.
Next, to confirm the effect of zafirlukast on insulin secretion ex vivo, we performed an insulin secretion assay using primary islets isolated from 8-week-old C57BL/6 mice. Interestingly, treatment with pranlukast slightly induced the ERK pathway but not the CaMKII and AKT pathways, resulting in no change in insulin secretion. Consistent with this, nifedipine strongly reduced zafirlukast-dependent insulin secretion in both low (2.8 mM) and high (16.7 mM) glucose conditions (Figure 3.6B).
These findings suggest that zafirlukast induces Ca2+ influx via L-VDCCs, leading to increased insulin secretion in pancreatic β-cells.
Conclusion and Discussion
We demonstrated that zafirlukast potentiates insulin secretion in MIN6 cells and primary pancreatic islets in both basal (2.8 mM) and high glucose (16.7 mM) conditions. The increase in insulin secretion is primarily elicited by extracellular Ca2+ influx through L-VDCC, resulting in increased phosphorylation of CaMKII, AKT and ERK. Interestingly, zafirlukast was able to stimulate insulin secretion by inducing Ca2+ entry and CaMKII, AKT and ERK phosphorylation.
Indeed, treatment with nifedipine, a DHP L-VDCC antagonist, reduced the effect of zafirlukast on both [Ca2+]i and insulin secretion. Our evidence showed that zafirlukast has a greater effect on insulin secretion in low levels of glucose. On the other hand, zafirlukast also affects insulin secretion under high glucose conditions (16.7 mM), indicating the possible use of zafirlukast in controlling blood glucose levels.
We have shown that zafirlukast can stimulate insulin secretion by promoting Ca2+. entry via L-VDCCs, resulting in increased phosphorylation of CaMKII, AKT and ERK.
이 글을 통해 감사의 마음을 전하고 싶습니다. 또한, 학부 연구에 참여하면서 많은 실험기법을 가르쳐주신 박찬영 교수님께 감사의 말씀을 전하고 싶습니다. 어려울 때마다 전화주시고 조언해주시고 도와주시는 용렬님 감사합니다.
제가 박사학위를 무사히 마칠 수 있도록 누구보다 바라주신 부모님, 아버지, 어머니께 진심으로 감사드립니다. 그리고 늘 진심으로 응원해주시는 형님들과 누나들에게도 감사하다는 말씀 전하고 싶습니다. 또한 제가 박사학위를 취득한 것을 매우 기뻐해 주신 형, 어머니, 아버지께도 감사의 말씀을 전하고 싶습니다.
진심으로 감사하고 평생 사랑합니다.
