Antidiabetic effect and mode of action of cytopiloyne

Abstract

Cytopiloyne was identified as a novel polyacetylenic compound. However, its antidiabetic properties are poorly understood. The aim of the present study was to investigate the anti-diabetic effect and mode of action of cytopiloyne on type 2 diabetes (T2D). We first evaluated the therapeutic effect of cytopiloyne on T2D in db/db mice. We found that one dose of cytopiloyne reduced postprandial glucose levels while increasing blood insulin levels. Accordingly, long-term treatment with cytopiloyne reduced postprandial blood glucose levels, increased blood insulin, improved glucose tolerance, suppressed the level of glycosylated hemoglobin A1c (HbA1c), and protected pancreatic islets in db/db mice. Next, we studied the anti-diabetic mechanism of action of cytopiloyne. We showed that cytopiloyne failed to decrease blood glucose in streptozocin- (STZ-)treated mice whose β cells were already destroyed. Additionally, cytopiloyne dose dependently increased insulin secretion and expression in β cells. The increase of insulin secretion/expression of cytopiloyne was regulated by protein kinase C α (PKC α ) and its activators, calcium, and diacylglycerol (DAG). Overall, our data suggest that cytopiloyne treats T2D via regulation of insulin production involving the calcium/DAG/PKC α cascade in β cells. These data thus identify the molecular mechanism of action of cytopiloyne and prove its therapeutic potential in T2D.

Figure 1

Anti-diabetic effects of cytopiloyne in db/db mice during long-term treatment. (A) Chemical structure of cytopiloyne. (B) Four groups of 6 to 8-week-old diabetic db/db mice were tube-fed with vehicle, cytopiloyne (CP, 0.5 and 2.5 mg/kg/day), or glimepiride (GLM, 2.5 mg/kg/day) from 0 to 6 weeks. Postprandial blood glucose (BG) levels in these mice were measured. (C) Blood insulin levels from the above mice (B). (D) IPGTT was performed in the above db/db mice (B) on weeks 0 and 6 after-treatment, and blood glucose levels were monitored for 3.5 h. (E) The percentage of glycosylated HbA_1c in whole blood from the above mice (B) was determined 0 and 6 weeks after-treatment. (F) Pancreata of 8- and 16-week-old db/db males, which had received the same treatment as described in (B) for 2 (images a–h) and 10 (images i–p) weeks, were stained with hematoxylin and eosin (H&E, images (a), (c), (e), (g), (i), (k), (m), and (o)) or hematoxylin and an antibody against insulin (H&I, images (b), (d), (f), (h), (j), (l), (n), and (p)). Arrowheads indicate pancreatic islets. Scale bars, 200 μm. Results are expressed as mean ± SEM from 3 independent experiments, and P (*) < 0.05 was considered to be statistically significant. The number of mice (n) is indicated in parentheses.

Figure 2

Cytopiloyne-mediated insulin secretion depends on pancreatic β cells. (a) Rat pancreatic islets were incubated with KRB buffer containing vehicle, glimepiride (GLM, 0 to 10 μM), or cytopiloyne (CP, 1.5 to 42 μM) in the absence or presence of 16.7 mM glucose (HG). The insulin levels were determined using an insulin ELISA kit. The data are presented as mean ± SEM of 3 independent experiments. (b) Fed C57BL mice, which had already received an injection of STZ, were administered an oral dose of vehicle, cytopiloyne (CP, 0.1, 0.5, and 2.5 mg/kg), and an intraperitoneal injection of insulin (Ins, 2.5 IU/kg). Postprandial blood sugar levels in the STZ-treated mice were determined using a glucometer. (c) Fed C57BL mice, which had already received STZ, were orally administered a single dose of vehicle, cytopiloyne (CP, 0.5 and 2.5 mg/kg), glimepiride (GLM, 2.5 mg/kg), or metformin (Met, 60 mg/kg), followed by an intraperitoneal injection with insulin (Ins). Postprandial blood sugar levels in the STZ-treated mice were determined using a glucometer. Results are expressed as mean ± SEM from 3 independent experiments, and P < 0.05 was considered to be statistically significant (*). The number of mice (n) is indicated in parentheses.

Figure 3

Increase in insulin mRNA and protein content by cytopiloyne in pancreatic islets. (a) RIN-m5F β cells transfected with phINS-Luc and pRL-TK plasmids were incubated with medium containing 3.3 mM glucose in the presence of vehicle (LG), glimepiride (GLM, 10 μM), and cytopiloyne (7, 14, or 28 μM) or 16.7 mM glucose (HG). Insulin promoter activity expressed as fold change relative to vehicle-treated control was measured using dual luciferase assays. (b) The relative expression level (R.E.L.) of insulin relative to L13 in rat primary pancreatic islets, which were already treated with 3.3 mM glucose in the presence of vehicle (LG), glimepiride (GLM, 10 μM), or cytopiloyne (7, 14, or 28 μM) or 16.7 mM glucose (HG) for 24 h, was determined by real-time RT-PCR. (c) Rat pancreatic islets received the same treatments as the islets in (b) in the presence of brefeldin A for 24 h. After anti-insulin antibody staining, these cells underwent FACS analysis. The percentage of insulin-positive β cells is shown. Results are expressed as mean ± SEM from 3 independent experiments, and P < 0.05 was considered to be statistically significant (*).

Figure 4

Effects of cytopiloyne on calcium mobilization, DAG generation, and PKCα activation. (a) After Fura 2-AM loading, RIN-m5F cells were stimulated with 16.7 mM glucose (HG) and cytopiloyne (CP) at 7, 14, and 28 μM. The level of intracellular calcium, as shown by the 340/380 nm ratio, was detected using a fluorescence spectrophotometer. (b) RIN-m5F cells were stimulated with glucose, cytopiloyne (CP), PMA, and glimepiride (GLM). Total cell lipids and their commercial standards, DAG and cholesterol (CHL), were resolved on a silica thin layer plate. The quantity of DAG and cholesterol in each sample is replotted into histograms. (c) RIN-m5F cells were incubated with vehicle (NS), cytopiloyne (CP, 7, 14, and 28 μM), PMA (1 μM), and 16.7 mM glucose (HG). Membrane proteins of each sample were subjected to Western blot with anti-PKCα and anti-actin antibodies. (d) RIN-m5F cells were incubated with vehicle, cytopiloyne (28 μM), and PMA (1 μM) in the absence or presence of EGTA (10 μM) and nimodipine (Nimo, 1 μM). Total proteins were subjected to Western blot with anti-PKCα (t-PKCα) and anti-phospho-PKCα (p-PKCα) antibodies.

Figure 5

Cytopiloyne-mediated insulin secretion and expression are abolished by a dominant-negative mutant and a PKCα inhibitor. (a) RIN-m5F cells were grown in medium with 16.7 mM glucose (HG) or 3.3 mM glucose (LG) in the presence of PMA (1 μM), GF109203X (GF, 3 μM), and cytopiloyne (CP, 28 μM). The insulin level in the supernatants was determined using an ELISA kit. (b) RIN-m5F cells were transfected with 5 μg of pHACE-PKCα DN (+) or pcDNA3 (−) plasmid and grown in medium supplemented with 16.7 mM (HG) or 3.3 mM glucose (LG) in the presence of PMA and cytopiloyne. The insulin level was determined as described in (a). The expression level of dominant-negative HA-tagged PKCα (DN) and an internal control, actin, in the transfected cells was determined by Western blot using anti-HA and anti-actin antibodies. (c) RIN-m5F cells were transfected with phINS-Luc and pRL-TK plasmids. The cells were grown in medium with 16.7 mM (HG) or 3.3 mM glucose (LG) in the absence and presence of PMA, GF109203X, and cytopiloyne. The activity of the insulin promoter (pINS) in fold was measured using dual luciferase assays. (d) RIN-m5F cells were transfected with phINS-Luc and pRL-TK plus 5 μg of pHACE-PKCα DN (+) or pcDNA3 (−) plasmids. The cells were grown in medium with 16.7 mM (HG) or 3.3 mM glucose (LG) in the absence or presence of PMA and cytopiloyne. Insulin promoter activity expressed as fold change relative to vehicle-treated control was measured using dual luciferase assays. The expression level of dominant-negative HA-tagged PKCα (DN) and actin in the transfected cells was determined using Western blot and anti-HA and anti-actin antibodies. (e) RIN-m5F cells were grown in medium with 3.3 mM glucose (LG) and/or cytopiloyne (CP, 28 μM) in the presence of EGTA (10 μM) or nimodipine (Nimo, 1 μM). The insulin level in the supernatants was determined.

Figure 6

Schematic diagram of the likely mechanism by which cytopiloyne treats T2D in diabetic mouse models. Cytopiloyne shows anti-diabetic effects in diabetic mice, as evidenced by a reduction in the levels of blood sugar and glycosylated HbA_1c, improvement of glucose tolerance, and its regulation of β-cell functions (e.g., insulin secretion, insulin expression, and pancreatic islet protection). The regulation of insulin secretion/expression in β cells by cytopiloyne involves PKCα and its activators, calcium, and DAG.