Background
Dietary changes in overall structure have been clearly shown to be associated with the development of diabetes [
1]. Aside from obviously increased intake of caloric and amounts of dietary fat, both of which have been demonstrated to be important to the development of prediabetes [
2], the changes also toward increased sweetening of the diet, food additives intake, by-products during food processing or storing and other important elements [
3]. Artificial sweeteners (e.g., saccharin) have been found to implicate in the development of obesity and obesity-related metabolic syndrome, associated with the alterations in composition and function of the intestinal microbiota [
4]. It is worth noting that 1,2-dicarbonyl compounds [
5‐
7] and advanced glycation end products (AGEs) [
8,
9], both of which are easily formed from carbohydrates in caramelization course and Maillard reactions in food, have been reported to increase the risk of type 2 diabetes mellitus (T2DM) and its complications. Based on an investigation of the content of 1,2-dicarbonyl compound in a great variety of commonly consumed foods, 3-deoxyglucosone (3DG) was proved to be the predominant 1,2-dicarbonyl compound [
10]. In addition to intensively investigate as a precursor for AGEs, 3DG itself has certain biological activities [
11‐
13], specifically on the ability to induce insulin resistance in vitro [
13]. The further reports in clinical and animal research indicated that 3DG had been linked to an impaired glucose tolerance [
6,
14,
15], thereby constituting an independent factor for the development of prediabetes.
The term “enteroinsular axis” refers to the signaling pathways between the gut and pancreatic islets that regulate blood glucose homeostasis [
16]. The pathogenesis of T2DM is associated with a defect in this enteroinsular axis [
17,
18]. The signaling pathways in gut related to regulation of glucose homeostasis are mediated by gut hormone, microbiota or immune system and those have gradually been a therapeutic target for diabetes. Glucagon-like peptide-1 (GLP-1) is an important gut hormone that can act via the enteroinsular axis to potentiate insulin secretion from pancreatic islets β-cell, known as the incretin effect [
19]. Owing to the incretin effect, analogs of GLP-1, GLP-1 receptor agonists and dipeptidyl peptidase-IV (DPP-IV) inhibitors are available as treatments for T2DM [
20]. GLP-1 secretagogues also represent a potential approach to enhance incretin action in T2DM. Actually, increasing endogenous GLP-1 secretion by dietary non-digestible ingredient (e.g., resistant maltodextrin and oligofructose), has been shown to improve glucose tolerance [
21,
22]. Reduced plasma GLP-1 concentrations were sometimes observed in T2DM [
23‐
25] even prediabetes [
26] stages, which may provide an explanation to the markedly impaired incretin effect in patients with T2DM [
27] in addition to the deficient in the β-cell response to GLP-1 after meal ingestion [
20]. Impairment of GLP-1 action caused by a blunted secretion of L-cells was also observed in early states of T2DM [
28]. Impairment of GLP-1 secretion, therefore, has been also proposed to be associated with a reduced glucose-stimulated insulin secretion and an impaired glucose tolerance [
29].
Considering the significance of the incretin effect of GLP-1, the factors related to harmful effects towards endogenous GLP-1 secretion become very important. To our knowledge, some endogenous or exogenous events that may decrease GLP-1 secretion have been investigated involving of the direct regulation of GLP-1-secreting cell, but the studies tend to be few. Stimulated hyperlipidemia and a high fat diet given to mice induce a reduction of the number of GLP-1-secreting cells in vitro and in vivo [
30]. In one more in vitro study, lipopolysaccharide, a gut bacterial product, was found to induce the apoptosis in intestinal endocrine cell line STC-1 in a dose-dependent manner [
31]. Thus, continuing to seek other factors that potentially harm GLP-1 secretion would help to restore physiological GLP-1 secretion and deserve to be explored. In an earlier study, 3DG was absorbed into the systemic circulation at a percentage of about 1‰ 2 h after single oral administration of 3DG [
32], suggesting the absorption rate of 3DG from foodstuffs is very slow. This result raises the possibility that 3DG has the ability to affect GLP-1 secretion. We therefore investigated if 3DG is capable of accumulating in intestinal tissue where it may have a role in GLP-1 axis after continuous oral administration of 3DG.
In the current study, 3DG was administered by gastric gavage to Sprague–Dawley (SD) rats for 2 weeks to investigate the distribution of 3DG in intestinal tissues. We also examined the effects of intragastric administration of 3DG on plasma levels of GLP-1, insulin and glucagon, and glucose regulation. Furthermore, the expressions of the sweet receptor subunits (TAS1R2, TAS1R3) and its downstream molecule TRPM5 in duodenum and colon tissues of rats, which is related to GLP-1 secretion, were investigated. In addition, we used the STC-1 L-cell model to investigate the direct effect of 3DG on GLP-1 secretion.
Methods
Synthesis of 3DG
According to the method of Kato et al. [
32], 3DG was synthesized from glucose as previously described [
13].
Determination of appropriate doses of intragastric administration of 3DG
Previous reports have estimated an average dietary 3DG intake of about 50 mg/day based on the 3DG content in commonly consumed foods [
10]. In order to achieve the equivocal effect of a potential 3DG intake of 50 mg per day, we calculated a dose based on body surface area (4.5 mg/kg for rats). Previously, we have reported that intragastric administration of 5 mg/kg 3DG for 2 weeks lightly increased plasma glucose level under oral glucose tolerance tests in mice. Therefore, we gave 5, 20 or 50 mg/kg 3DG by gastric gavage.
Animals
11-week-old SD rats were purchased from Matt Albert Technology Co. Ltd (Suzhou, China) and housed in a temperature-controlled room (23 °C) and 12 h light/12 h dark cycle. All of animal experimental procedures were conducted in compliance with Guide for care and use of laboratory animals (Eighth edition, 2011). The study was approved by the local ethic committee of Suzhou Hospital of Traditional Chinese Medicine. The rats had free access to a standard rodent chow diet (Shuangshi Laboratory Animal Feed Science Co. Ltd, Suzhou, China) and water. The diet contained water (≤10%), crude proteins (≥20.5%), crude fat (≥4%), crude fiber (≤5%), crude ash (≤8%) and mixture of vitamins and micronutrients. After 1 week of acclimatization, the rats were randomly divided into four groups with similar fasting glucose concentration, and each group consisted of six rats. Vehicle (control), 5 mg/kg 3DG, 20 and 50 mg/kg 3DG were given by gastric gavage daily with an administrated period of 2 weeks. Body weight was measured daily. The rats were fasted overnight before the experiments.
STC-1 cells culture
STC-1 cells, an enteroendocrine intestinal cell line, were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 15% (v/v) horse serum, 2.5% (v/v) fetal bovine serum (FBS; Zhejiang Tianhang Biological Technology Co., Ltd., Huzhou, China), and 25 mmol/L glucose at 37 °C in a 5% CO2 humidified atmosphere. The cells were grown to 70–80% confluence for the experiments.
Oral glucose tolerance test (OGTT)
After fasting overnight, a basal blood sample was collected from a tail vein for the measurement of fasting glucose levels using a glucose meter (ACCU-CHEK, Roche, US). Then, the rats were fed with glucose by gastric gavage (2.5 g/kg). And additional blood samples were collected from tail vein at 0, 30, 60, 90, 120 and 180 min following the glucose load, and glucose concentration was determined with a glucose meter. The area under the glycaemic curves (AUC) were calculated for each group of rats.
Measurements of GLP-1, GIP, insulin and glucagon in plasma
Blood samples from aorta abdominalis were collected at 15 and 180 min points following the glucose load for the measurements of insulin, glucagon, GLP-1 (total). Plasma levels of insulin and glucagon were assayed with the corresponding radioimmunoassay kits (Beijing North Institute of Biological Technology, Beijing, China). Plasma GLP-1 concentration was measured using the ELISA kits (Millipore, MA, USA). Total GLP-1 includes both intact [GLP-1-(7–36) amide and GLP-1-(7–37)] and inactivated forms of GLP-1 (GLP-19–36 amide and GLP-1 9–37 degraded by DPP-4).
Measurement of plasma dipeptidyl peptidase-4 (DPP-4) activity
According to the method of Pederson et al. [
33], plasma DPP-4 activity was determined by a colorimetric assay, using H-Gly-Pro-
p-nitroanilide (Sigma, St Louis, MO, USA) as a substrate.
Distribution of 3DG in intestinal tissues after treatment with exogenous 3DG
After 2 weeks of intragastric administration of 3DG, the rats were then killed and intestinal tissues were collected for the measurement of 3DG contents by HPLC. Before the measurement, the content of gastrointestinal tract was completely removed.
Western blot analysis
In rats treated with 50 mg/kg 3DG, the duodenum and colon tissues were collected 2 weeks after intragastric administration 3DG. Methods for quantification of whole protein content and western blot have been described previously [
13]. Antibodies against TAS1R2, TAS1R3 and TRPM5 were obtained from Cell Signaling Tech (Massachusetts, USA).
GLP-1 secretion assay in vitro
STC-1 cells were seeded into six-well plates at a density of 2 × 105 cells/well for 48 h; the cells were then incubated with L-DMEM (5.6 mmol/L glucose) containing 10% FBS. After 3 h, the medium was subsequently removed, and the cells were incubated with or without 3DG at final concentrations of 80, 300 and 1000 ng/mL in 0.2% BSA H-DMEM (25 mmol/L) containing 5 × 10−7 M insulin for 6 h. After the incubation, the medium was collected and centrifuged at 12,000×g for 5 min at 4 °C to remove any floating cells. GLP-1 concentration in the supernatant was measured by ELISA (Millipore, MA, USA).
Statistical analysis
Results of the experimental studies are expressed as mean ± SD. Statistical significance of differences was analyzed by the Student’s t test or One-way analysis of variance. All p values ≤0.05 were considered statistically significant.
Discussion
The objective of this study was to investigate whether 3DG is capable of accumulating in intestinal tissue of Sprague–Dawley (SD) rats after 2-week administration of 3DG by gastric gavage and if so, the effects of intragastric administration of 3DG on plasma levels of GLP-1, insulin and glucagon, and glucose regulation are further investigated. We demonstrated for the first time that intragastric administration of 3DG to rats for 2 weeks led to an obvious increase in 3DG content of the upper small intestine, lower small intestine, ileum and colon, and reduced plasma total GLP-1 and insulin concentrations in a similar manner, in conjunction with increased fasting blood glucose concentration and reduced oral glucose tolerance. The reduced plasma GLP-1 levels occurred in conjunction with reduced expressions of TAS1R2, TAS1R3 and TRPM5 in duodenum and colon and plasma dipeptidyl peptidase-4 activity was not altered, which suggested a reduced GLP-1 secretion in 3DG-treated rats. Moreover, non-cytotoxic concentrations of 3DG directly attenuated GLP-1 secretion in STC-1 cells. From this study, we also observed that 3DG-treated rats displayed obviously pancreatic islet cell dysfunction characterized by decreased plasma insulin level and elevated plasma glucagon level, associated with the development of impaired glucose regulation (IGR). Body weight was equivalent between 3DG-treated rats and vehicle-treated rats (data not shown).
A recent study on the influence of dietary on metabolism of 3DG in healthy volunteers experimented by Julia Degen et al. [
34]., who speculated that orally ingested 3DG remained in the content of gastrointestinal tract to a major degree. As previous reports have indicated that the absorption rate of 3DG from foodstuffs is very slow in a single administration study [
32]. This idea is further strengthened by the results that 3DG content of intestinal tissues was significantly higher in rats 2 weeks after intragastric administration of a high dosage (50 mg/kg) of 3DG than control rats, especially in colon section (Fig.
1a). Furthermore, increased 3DG content in colon section was also observed in rats after administration of a lower dosage (20 mg/kg) of 3DG (Fig.
1b). Similar with the distribution of 3DG in intestinal tissue, it has been reported that GLP-1 is secreted postprandially by intestinal L-cells that increase in density along the intestine and are found in highest amount in the colon [
35]. In the present study, plasma GLP-1 concentrations decreased after intragastric administration of 3DG (Fig.
2a). Furthermore, at concentrations similar to those obtained from intestinal tissues contents in 3DG-treated rats, 3DG directly reduced GLP-1 secretion in the STC-1 cells in a dose-dependent manner (Fig.
2c) together with the unaltered plasma DPP-4 activity in 3DG-treated rats (Fig.
2b), indicating that accumulation of 3DG in intestinal tissue could reduce GLP-1 secretion in rats. Additional, although no effect was observed in response to 5 mg/kg of 3DG on any of the parameters, the 5 mg/kg 3DG group had similar GLP-1 content in colon section compared with the control group, and further support for the notion that decreased GLP-1 secretion was the result of increased 3DG content in intestinal tissues. This idea was also supported by the results that 3DG-treated rats displayed reduced expressions of TAS1R2, TAS1R3 and TRPM5 in duodenum and colon (Fig.
3). Several lines of evidence have demonstrated that sweet taste receptors in intestine regulate GLP-1 secretion following sugar ingestion [
36,
37]. Furthermore, disruption of sweet taste receptors action in animal experiments and L-cell model, using antagonists or genetic manipulation, displayed significantly reduced glucose-stimulated GLP-1 secretion [
36‐
38]. Therefore, the attenuated GLP-1 secretion in 3DG-treated rats could be responsible for the decreased plasma GLP-1 concentrations. In addition, there was no significant difference in AGEs levels in the colon section between 20 mg/kg 3DG-treated group and the corresponding control group (Additional file
1: Figure S1). These results clearly indicates that 3DG was capable of accumulating in intestinal tissue of rats 2 weeks after administration of 3DG, which led to reduced GLP-1 secretion independently from AGEs action. Additionally, reduced expressions of TAS1R2, TAS1R3 and TRPM5 in duodenum and colon (Fig.
3) also provide an explanation for the results that 3DG treated decreased GLP-1 secretion in vitro and in vivo. Research for the confirmation of this mechanism is in progress. Although treatment of STC-1 cells with 3DG at concentrations similar to those obtained from intestinal tissues contents in 3DG-treated rats failed to alter cell viability (Fig.
2d), whether intragastric administration of 3DG for 2 weeks also could result in increased apoptosis of GLP-1-secreting cells in vivo remains unknown and deserves to be further investigated.
IGR, sometimes referred to as prediabetes including isolated impaired glucose tolerance (IGT), isolated impaired fasting glucose (IFG) or combined IGT/IFG, is a high risk state for developing diabetes [
39]. It has been reported that intragastric administration of 3DG for 2 weeks increased plasma glucose level under oral glucose tolerance tests (OGTT) in normal mice [
15]. Such an effect was also observed in our present study. The intragastric administration of 3DG for 2 weeks caused normal SD rats to develop IFG (Fig.
5a) in conjunction with IGT (Fig.
5b) and increased AUC (Fig.
5c) in dose-dependent manner. Furthermore, treatment with 3DG resulted in reduction of GLP-1 secretion (Fig.
2) and sweet taste receptors expression in duodenum and colon (Fig.
3). In support of the observations by other studies, (i) disruption of the GLP-1 receptor action in mice caused IFG and IGT [
40]; (ii) reduced glucose tolerance was observed in the TAS1R3
−/− mice [
41]. Additionally, we also observed the elevated plasma glucagon levels in 3DG-treated in addition to decreased plasma insulin levels (Fig.
4). The main pathophysiological feature of T2DM is pancreatic islet cell dysfunction which manifests as both insufficient insulin secretion from β cells and inappropriately elevated glucagon secretion from α-cell [
42]. Therefore, we obtained a conclusion that 3DG-treated SD rats displayed typical pancreatic islet cell dysfunction, suggesting the pancreatic islet cell dysfunction occurred prior to the development of T2DM.
It is now generally accepted that GLP-1 has a broad role in glucose homeostasis, in great part through stimulation of nutrient-induced insulin secretion from pancreatic β-cells [
35]. In healthy individuals, insulinotropic effects of GLP-1 accounted for 50–70% of prandial insulin secretion from pancreatic β-cells [
43]. As previous reports have documented that administration of GLP-1 to T2DM significantly enhanced and may even restore to normal glucose-induced insulin secretion [
44,
45]. In T2DM, reduced postprandial GLP-1 concentrations in T2DM have been suggested to result in an impaired insulin secretion [
23]. This hypothesis is supported by the results that intragastric administration of 3DG for 2 weeks decreased plasma GLP-1 concentrations at fasting and 15 and 180 min points during an oral glucose load in rats (Fig.
2a) and at the same points with insulin (Fig.
4a). In addition, disruption of GLP-1 action in animal experiments, using antagonists or genetic manipulation, displayed significantly reduced insulin secretion [
46,
47]. From above, it was concluded that the decreased plasma GLP-1 concentrations in rats induced by intragastric administration of 3DG resulting from a decreased GLP-1 secretion led to reduced plasma insulin concentrations and thereby resulted in IGR. Thus, the reduced GLP-1 secretion sometimes observed may explain part of impaired incretin effect in T2DM. Additionally, (i) GLP-1 is known to induce the β-cells proliferation, and GLP-1R
−/− mice exhibit increased susceptibility to β-cell apoptosis injury [
48]; (ii) GLP-1 also reduces glucagon secretion, and the GLP-1 secretion in present study was accompanied by an increased in plasma glucagon concentrations (Fig.
4). These observations also support the above suspection. Thus, a decrease in the biological function of GLP-1 from reduced GLP-1 secretion could result in an impaired insulin secretion but may not the only cause. For example, whether intragastric administration of 3DG for 2 weeks increases plasma 3DG levels in rats is unknown. And if so, whether increased plasma 3DG directly affects insulin secretion from β-cells will be investigated.
Authors’ contributions
All authors contributed to the study concept and design, and the interpretation of the data. LZ, XS, LZ, GL, HX, FW, FH and GJ acquired and analyzed the data. GJ, LZ and XS drafted the manuscript. GJ and LZ reviewed the manuscript for important intellectual content. All authors revised the article and approved the final version for publication. GJ is responsible for the integrity of the work as a whole. All authors read and approved the final manuscript.