2,4,5,6-Tetraoxypyrimidine (alloxan) was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Metformine was purchased from Shanxi Baozhilin Pharmaceuticals Co., Ltd. (Shanxi, China). Compound K16, a cucurbitane-type triterpenoid isolated from M. charantia was provided by Shenyang Pharmaceutical University (Shenyang, China) (10), and its structure is presented in Fig. 1. Antibodies against insulin receptor (IR; cat. no. 3025s; Cell Signaling Technology, Inc., Danvers, MA, USA), and phosphorylated IR (cat. no. 3024s; Cell Signaling Technology, Inc.), IRS1 (phospho-Ser636; cat. no. D120888; Bio Basic Canada, Inc., Markham, ON, Canada), and glycogen synthase kinase 3β (GSK3β; phospho-Tyr216; cat. no. D155143; Bio Basic Canada, Inc.) were used. Antibody against phosphorylated Akt (cat. no. 13038P) was obtained from Cell Signaling Tachnology, Inc. Antibody against α-tubulin (cat. no. Ab102) was purchased from Vazyme (Piscataway, NJ, USA).

Male C57BL/6J mice (5 weeks old; 18–20 g) obtained from Liao Ning Chang Sheng Biotechnology Co., Ltd. (Benxi, China) were maintained in a 12 h light/dark cycle, and provided with water and food ad libitum. The animals were housed in cages maintained at 21±2.0°C with 50±5% humidity. The study was approved by the ethics committee of Liaoning Traditional Chinese Medicine University (Shenyang, China).

After 1 week of acclimation, male mice (6 weeks old) were randomly divided into 2 groups: Control group (n=10) and model group (n=40). Mice in the control group mice were administered 0.9% saline solution, whereas those in the model group received alloxan (50 mg/kg body weight). The saline solution and alloxan were administered via intraperitoneal injection, and a total of three injections were given, at 48 h intervals. Subsequently, blood samples were collected from the tails of the animals and the glucose levels in the blood samples were measured with a glucometer (Roche Diagnostics, Basel, Switzerland). The animals were subjected to a 16-h fasting period prior to the blood glucose test. Successful establishment of this model was based on mice exhibiting a blood glucose concentration between 10 and 14 mM, and exhibiting behavioral changes, including polydipsia, polyphagia and polyuria (11). Only these animals were used in subsequent experiments.

The alloxan-induced hyperglycemic mice were randomly sorted into 4 groups (n=10 per group), with each group having approximately the equivalent average body weight. One group was administered 0.9% saline solution (alloxan group). Another group was administered metformin (10 mg/kg; metformine group) as a positive control as metformin improves insulin resistance. The remaining two groups were each administered compound K16 (25 or 50 mg/kg body weight). Administration of the drugs was performed by oral gavage, and the treatment lasted for 4 weeks, with three treatments per week. Simultaneously, mice in the control group, (without administration of alloxan) also received 0.9% saline solution via oral gavage for the equivalent duration and frequency. These mice were considered as healthy control mice.

Blood glucose levels were measured weekly. Blood samples were collected from the tail of the animals following a fasting period of 16 h. The glucose levels in the samples were measured with a glucometer. Glucose tolerance was determined by the intraperitoneal glucose tolerance test (IPGTT), which was performed at week 4 of treatment. Again, the animals were subjected to a fasting period of 16 h, and blood samples were then collected from the tail before (0 time) and at 5, 15, 30, 60 and 120 min after intraperitoneal injection of glucose (1 mg/kg body weight). The glucose levels in the blood samples were measured by a glucometer. Area under the curve of glucose concentration vs. time plot was calculated using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

At the end of week 4, blood samples were collected by eyeball extirpating and the animals were sacrificed by cervical dislocation. Serum was isolated from the blood samples by centrifugation at 5,000 × g min-1 for 10 min and the levels of cholesterol and triglycerides in the serum were then measured with a cholesterol (CHO) and triglyceride (TG) kit (Beijing BHKT Clinical Reagent Co., Ltd., Beijing, China). The liver, kidney, spleen and skeletal muscle were removed from the animals. They were weighed, snap-frozen in liquid nitrogen and stored immediately at −80°C. The organ index was calculated as: Organ index = organ weight/body weight.

RT-qPCR was performed to measure the expression levels of Glut4 and Ampkα1 in the liver of mice from different treatment groups. Total RNA was extracted from the liver tissue of three mice from each group. Liver tissue (20 mg) was ground in liquid nitrogen in a mortar and the total RNA was extracted from the tissue homogenate using TRIzol (Beijing Kang Century Biotechnology Co., Ltd., Beijing, China). RT-qPCR analysis was performed as described previously (12) using the ABI Prism 7900-HT Real-Time PCR System (Applied Biosystem; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Primers used in the RT-qPCR were as follow: Glut4, 5′-CTTGGCTCCCTTCAGTTTGG-3′ (forward), 5′-CTACCCAGCCACGTTGCATT-3′ (reverse); Ampkα1, 5′-AAGCCGACCCAATGACATCA-3′ (forward) and 5′-CTTCCTTCGTACACGCAAAT-3′ (reverse); β-actin, 5′-AGGCAAACCGTGAAAAGATG-3′ (forward) and 5′-AGGCAAACCGTGAAAAGATG-3′ (reverse). β-actin was used as an internal control and expression levels were calculated using the ΔΔCq method (13).

The expression levels of several key proteins involved in the insulin signaling pathway in the liver were analyzed by western blotting. Liver tissue was extracted in radioimmunoprecipitation assay cell lysis buffer (Cell Signaling Technology, Inc.) for 15 min on ice (12). Subsequently, the extract was briefly sonicated and then centrifuged at 12,280 × g for 15 min at 4°C. The supernatant of the sample was retained and the protein concentration in the supernatant was measured by bicinchoninic acid assay. Aliquots of the supernatant containing total protein (40 µg) was resolved in 10% SDS-polyacrylamide gel and the protein bands were then transferred to a nitrocellulose membrane and blocked with 5% bovine serum albumin for 3 h at room temperature. Then the membranes were probed with the appropriate primary antibody overnight at 4°C followed by horseradish peroxidase conjugated secondary antibody (1:5,000; cat. nos. BL003A and BL001A; Biosharp Inc., Hefei, China) at room temperature for 2 h. Primary antibodies were used at the following dilutions: IR (1:1,000); phosphorylated IR (1:1,000); IRS1 (1:1,000); GSK3β (1:1,000); p-GSK3β (1:500); phosphorylated Akt (1:1,000); and α-tubulin (1:10,000). Positive signals of the blot were detected by an enhanced chemiluminescence assay (GE Healthcare Life Sciences, Chalfont, UK). The relative density of proteins was analyzed using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Data were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test performed with the SPSS software (version 16; SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard error and P<0.05 was considered to indicate a statistically significant difference.

The effect of compound K16 on the blood glucose levels of alloxan-induced diabetic mice was investigated by administering mice with compound K16 over a period of 4 weeks and monitoring the changes in the blood glucose levels each week. The blood glucose level of healthy control group was approximately one-third of the level of the alloxan-induced diabetic mice (alloxan group) and remained almost unchanged over the entire four weeks (P<0.05; Fig. 2A). The blood glucose level of the metformine group was higher than that of alloxan group on the first week, but then decreased steadily to ~70% of the level of the alloxan group by week 4 (Fig. 2B). The blood glucose levels of the two K16 groups were similar to that of the alloxan group week 1, however, the levels decreased over the following 3 weeks Although with the lower dosage of K16 the blood glucose level appeared to increase slightly at week 4, the high dosage of K16 reduced the blood glucose level to ~50% the level of the alloxan group (Fig. 2C and D). These results demonstrated the positive antidiabetic activity of K16 regarding its ability to reduce blood glucose levels in drug-induced diabetic mice, although the extent of reduction did not reach the blood glucose level of healthy mice.

The effect of compound K16 on the blood glucose tolerance of diabetic mice was investigated by measuring the blood glucose level of the animal prior to and following administration of glucose at week 4, and subsequently monitoring the changes in blood glucose levels after 5, 10, 30, 60 and 120 min. The blood glucose level of control mice was approximately one third the levels of alloxan-induced diabetic mice (P<0.05), and peaked at about 13 mmol/l after 15 min, and then decreased to baseline level after 120 min (Fig. 3A). The blood glucose level of alloxan group followed essentially the same pattern, peaking at 15 min, with a concentration of about 36 mmol/l and decreased to baseline levels after 120 min. The blood glucose levels the metformine and two K16 groups over the entire 120 min were fairly similar; lower than the levels of the alloxan group (Fig. 3B-D). To compare the absolute change in the blood glucose over the entire 120 min period, the IPGTT AUC was calculated to demonstrate the total change in glucose. The results demonstrated that no significant difference in glucose tolerance was observed between the metformine treatment and the no treatment group (alloxan group; Fig. 3E). However, the AUC of the K16 groups was significantly reduced compared with the alloxan group (P<0.05), indicating that compound K16 improved the glucose tolerance in drug-induced diabetic mice by eliminating exogenously administered glucose from the blood.

Alloxan-induced diabetic mice treated with compound K16 over a period of 4 weeks were sacrificed at the end of the week 4. The TG levels of alloxan-induced diabetic mice were ~50% higher than the level of control mice (P<0.01; Fig. 4A). The levels of serum TG in the two K16 groups were significantly decreased compared with the alloxan group (P<0.01), to levels that were even lower than the healthy control mice. As for serum CHO level, the alloxan group exhibited levels ~30% higher than the healthy control group (P<0.01), with the metformine group yielding even higher levels (Fig. 4B), whereas the K16 groups exhibited reduced serum cholesterol levels compared with the alloxan group. However, only the high dosage (50 mg/kg) of compound K16 resulted in a significant reduction relative to the alloxan group (P<0.01; Fig. 4B). These results indicated that compound K16 reduces the serum TG and CHO levels of drug-induced diabetic mice.

The effect of compound K16 on the organs of the mice was evaluated by determining the organ indexes (organ weight/body weight) of the spleen, liver and kidney following sacrifice of the animals at the end of the 4-week treatment. Overall, there was no significant difference in the organ indexes among all the different groups (Fig. 5). This suggested that compound K16 exhibited no toxic effect on the animals.

The aforementioned experimental results indicated that compound K16 indeed exerted antidiabetic activity, as demonstrated by its ability to lower blood glucose level and to increase glucose tolerance in diabetic mice. Thus, to investigate the mechanisms associated with the antidiabetic activity of compound K16, the effect of the compound on the expression of genes or the activation of proteins that are involved in glycometabolism was analyzed. The expression/phosphorylation of IR, IRS1, GSK-3β, Akt, Glut4 and Ampkα1 were determined in the current study (14,15). Compared with control mice, the phosphorylated protein levels of IR and IRS1 were significantly reduced in the alloxan group, however this reduction was significantly abolished in the K16 groups, at the low and high dosages (Fig. 6A). At the high dosage, compound K16 also significantly increased the levels of phosphorylated GSK-3β and Akt, compared with the levels in the alloxan group. Furthermore, RT-qPCR revealed that the mRNA levels of Glut4 and Ampkα1 were significantly increased when the mice were treated with metformine or compound K16 compared with the alloxan group (Fig. 6B). These results provided evidence that compound K16 mediated upregulation of certain genes involved in glycometabolism, and suggested that this may be part of the mechanism by which compound K16 exerts its antidiabetic activity.