Insulin secretion and α-glucosidase inhibitory effects of dicaffeoylquinic acid derivatives

In this study, we investigated the effects of dicaffeoylquinic acid derivatives, including 1,4-di-O-caffeoylquinic acid (1,4-DCQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), 4,5-di-O-caffeoylquinic acid (4,5-DCQA), and 1,5-di-O-caffeoylquinic acid (1,5-DCQA) on glucose-stimulated insulin secretion (GSIS) activity and α-glucosidase activity were compared in rat INS-1 pancreatic β-cells. The α-glucosidase inhibitory activities of dicaffeoylquinic acid derivatives were as follows: 1,4-DCQA > 1,5-DCQA > 3,4-DCQA > 4,5-DCQA > 3,5-DCQA. In INS-1 cells, dicaffeoylquinic acid derivatives showed no cytotoxic effect at any concentration (2.5–10 μM). In addition, the GSIS activities of dicaffeoylquinic acid derivatives were as follows: 4,5-DCQA > 3,4-DCQA > 1,4-DCQA > 3,5-DCQA > 1,5-DCQA. Treatment of INS-1 cells with 4,5-DCQA resulted in a marked increase in protein expression of extracellular signal-regulated protein kinases (ERK), insulin receptor substrate-2 (P-IRS-2), Akt, phosphoinositide 3-kinase (P-PI3K), and pancreatic and duodenal homeobox-1 (PDX-1), which might be related to its GSIS activity in INS-1 cells. These findings indicate that the location of the dicaffeoyl functional group influences the anti-diabetic activity of quinic acid.


Introduction
Diabetes mellitus (DM) is metabolic endocrine disorder in the world associated with abnormal compromised lipid and carbohydrate metabolism. One approach for the treatment of type 2 DM is using α-glucosidase inhibitors as an oral anti-hyperglycemic drug [1]. α-Glucosidase inhibitors has its own mechanism of action that diminish the levels of postprandial blood glucose. It can help in retarding the absorption of carbohydrates by decreasing α-glucosidase activity in the epithelium of small intestine [2]. Acarbose, miglitol, and voglibose are clinically approved as α-glucosidase inhibitors [3]. These three α-glucosidase inhibitors are sugars or its derivatives, which can induce gastrointestinal side effects [3]. A range of chemical compounds isolated from natural products have been reported to be effective in inhibiting the α-glucosidase activity. Most of the chemical compounds reported as α-glucosidase inhibitors in previous studies are secondary metabolites including flavonoids, alkaloids, anthocyanins, terpenoids, and phenolic acids [4].
Caffeoylquinic acid derivatives have been claimed to have various biological effects including neuroprotective activity [5,6], anti-oxidant effect [7,8], anti-inflammatory activity [9,10], anti-viral effect [11,12], anti-cancer activity [13], and anti-hepatotoxic activity [14]. Furthermore, their inhibitory effects on α-glucosidase activity have been scientifically evaluated in the previous many reports [15][16][17]. However, little is known concerning their effect on glucose-stimulated insulin secretion (GSIS). Another approach for the treatment of type 2 DM is an increase in GSIS. GSIS had been considered the exclusive mechanism of insulin regulation [18]. Defective insulin secretion is a characteristic of pancreatic β cell dysfunction, which develops early and gets worse further in T2D [19]. Sulfonylureas known as oral insulinotropic agents to treat T2DM promote insulin secretion by closing K + ATP channels at the plasma membrane, while medicines in this group are known to often lead to hypoglycemia. This is because it continuously stimulates insulin secretion, regardless of plasma glucose levels [20]. Thus, identification of potential compounds that stimulate GSIS is highly desirable. Therefore, in this study, the inhibitory effects of dicaffeoylquinic acid derivatives (Fig. 1) on α-glucosidase inhibitory were compared, and it was also confirmed whether the dicaffeoylquinic acid derivatives enhance insulin secretion in pancreatic β cells using only stimulatory glucose. In addition, the corresponding mechanisms were investigated.

Plant materials and chemiclas
The dried aerial parts of Saussurea grandifolia were extracted with methanol under reflux.

GSIS assay
INS-1 cells plated on 12-well plates for 24 h were used to measure the effects of dicaffeoylquinic acid derivatives on GSIS. To this end, INS-1 cells were kept in Krebs-Ringer bicarbonate HEPES buffer (KRBB) supplemented with 2.8 mM glucose for 2 h. Thereafter the INS-1 cells were incubated for 1 h in the fresh KRBB with the denoted glucose concentrations (2.8 or 16.7 mM glucose) and test agents (gliclazide and dicaffeoylquinic acid derivatives). Glucose stimulated index (GSI) was calculated by dividing the insulin concentration that had accumulated during exposure to 16.7 mM glucose by the insulin accumulated during exposure to 2.8 mM glucose. After incubation a cell culture supernatant was analyzed using a rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Shibukawa, Gunma, Japan) as recommended by the producer to measure the GSIS.

Statistical analysis
All analyses were conducted using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA). Nonparametric comparisons of samples were conducted with the Kruskal-Wallis test to analyze the results. Statistical significance was set at p < 0.05.

Discussion
Inhibitory effect of dicaffeoylquinic acid derivatives on α-glucosidase activity have been scientifically evaluated in the previous many studies [26][27][28].
Little is known about effects of dicaffeoylquinic acid derivatives on insulin secretion compared to their α-glucosidase activities in the in vivo and in vitro models of type 2 DM. Although it has been suggested that Gynura divaricata rich in 4,5-DCQA restore pancreatic function in type 2 DM mice [30], the effect on 4,5-DCQA itself has not been investigated yet. In the present study, we compared the effects of  In addition, treatment with 4,5-DCQA increased protein expressions of ERK, IRS-2, PDX-1, Akt, and PI3K compared to untreated controls in INS-1 cells. These results indicated that GSIS activity of 4,5-DCQA might be partly related to PDX-1 expression via IRS-2/ Akt/PI3K signaling pathway and ERK expression. ERK belongs to the mitogen-activated protein kinases (MAPK) family and plays an essential role in regulating not only cellular apoptosis and proliferation, but also differentiation. Earlier study indicates that the MAPK inhibitor PD98059 inhibit ERK phosphorylation and GSIS in β-TC6 mouse pancreatic cells [31].
Similar results are observed with U0126, a specific MAPK/ERK kinase inhibitor, reduces GSIS in mice pancreatic islets. ERK appears to regulate pancreatic β-cell survival and expression of insulin gene [32]. Many studies have shown that phosphorylated IRS-2 triggers PI3K/ Akt pathway activation, and the participation of IRS-2/ PI3K/Akt signaling in the regulation of maintenance of β-cell mass and normal pancreatic β-cell function is demonstrated [33]. In addition, IRS-2/PI3K/Akt signaling is known as the upstream of PDX-1. It has been reported that administration of Gynura divaricata rich in 4,5-DCQA enhances the PDX-1 expression in the pancreatic tissue of diabetic mice, thus retaining mature β-cell function [30]. PDX-1 is a vital transcription factor in the development of pancreas and transactivates insulin gene. Moreover, impaired GSIS is observed in PDX-1-deficient mice [34,35]. Our current study suggested that treatment with 4,5-DCQA increased the PDX-1 expression via IRS-2/Akt/PI3K signaling pathway and ERK1/2 Based on the results, we reported the potent α-glucosidase inhibitory potential of dicaffeoylquinic acid derivatives and their GSIS effect. All dicaffeoylquinic acid derivatives exerted promising α-glucosidase inhibitory effects. 1,4-DCQA among dicaffeoylquinic acid derivatives exhibited maximum inhibitory effcets. Further, GSIS assay supported potentiation effect on GSIS shown by the dicaffeoylquinic acid derivatives. In addition, GSIS effect of 4,5-DCQA was supported by increased protein expressions of ERK, IRS-2, Akt, PI3K, and PDX-1. Our study provided partial evidence for the applicability of dicaffeoylquinic acid derivatives as candidates in the treatment of diabetes. However, further study including effect in animal models of T2D and in human islets are necessary.