Type 2 diabetes patients usually develop abnormal lipid metabolism concomitantly, and blood glucose control alone often fails to diminish the complications in the major blood vessels such as coronary artery in these patients. Thus, control of blood lipid is required in the glucose lowering therapy. A meta-analysis published in JAMA showed, when compared with moderate dose statins, intensive high-dose statin treatment may increase the risk for new diabetes [
A total of 40 male db/db mice aged 8 weeks (specific pathogen free) and weighing 35~48 g) were randomly assigned into 2 groups (
The body weight and blood glucose were measure weekly. Before treatment and after 6-week treatment, the fasting blood glucose, fasting TC, LDL-C, TG, and fasting serum insulin (FSI) were determined. The plasma insulin was measured with a mouse ELISA kit for insulin (Morinaga, Yokohama, Japan). The insulin sensitivity index (ISI) was calculated as follows: ISI = ln 1/(FBS × FSI). HbA1c percentage was determined using the fully automated, high-pressure liquid chromatography Tosoh HLC-723 G8 analyzer (Japan). Plasma no-esterified fatty acid (NEFA, FFA) was measured by enzyme-like immunosorbent assay (ELISA) kit (Westang, Shanghai, China).
After 6-week treatment, 10 mice were randomly selected from each group and received intraperitoneal glucose tolerance test (IPGTT). The area under curve (AUC) insulin was used to evaluate the insulin secretion, and
The remaining 10 mice in each group were anesthetized with phenobarbital. The opening of the common bile duct duodenal papilla was clamped and common bile duct puncture was done followed by injection of 1 mg/mL type IV collagenase (2 mL). Then, the pancreas was rapidly collected and placed in Hank’s solution containing type IV collagenase for digestion for 40 min. After washing under shaking, the pancreatic islets were separated. The in vitro islet perfusion system developed by our department was employed for perfusion of pancreatic islets. The perfusate was collected and the insulin concentration was detected to delineate the curve of first phase insulin secretion.
Animals were anesthetized with sodium phenobarbital and the pancreas was collected, fixed in 4% paraformaldehyde for 4~6 h and embedded in paraffin. ABC method was employed for the detection of insulin. The
Image-Pro Plus 5.0.1 was used to measure the area of pancreas and that of
Statistical analysis was done with SPSS version 11.0 and data were expressed as mean ± standard deviation. Means among groups were compared with one way analysis of variance, and those between two groups were compared with LSD test. Data before and after experiment were compared with paired test. A value of
At baseline, there were no marked differences in the FBG, HbA1c, FSI, TC LDL-C, TG, and FFA between ezetimibe group and control group. The db/db mice aged 8 weeks had the FBG of >15 mmol/L. After treatment with ezetimibe, the development of hyperglycemia was alleviated, and the blood glucose and HbA1c at the end of treatment in the db/db mice was markedly lower than that in the untreated db/db mice
Body weight, FBG, FSI, TC, LDL-C, and ISI in different groups at baseline and end of experiment (mean ± SEM).
Group |
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Body weight (g) | FBG (mmol/L) | FSI (ug/L) | TC (mmol/L) | LDL-C (mmol/L) | ISI |
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db/m | Baseline 20 | 29.10 ± 0.33* | 5.51 ± 0.53* | 2.43 ± 0.23* | 2.65 ± 0.41* | 1.65 ± 0.22* |
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Terminal 20 | 35.80 ± 0.54* | 5.71 ± 0.63* | 2.63 ± 0.31* | 2.55 ± 0.53* | 1.79 ± 0.27* |
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db/db | Baseline 20 | 41.20 ± 3.18 | 17.30 ± 3.99 | 8.51 ± 1.26 | 5.14 ± 0.80 | 3.88 ± 0.44 |
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Terminal 20 | 50.20 ± 4.32 | 25.9 ± 2.00 | 9.55 ± 1.20 | 5.86 ± 0.85 | 4.91 ± 0.37 |
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Ezetimibe | Baseline 20 | 40.92 ± 2.84 | 18.37 ± 4.25 | 9.01 ± 1.37 | 5.30 ± 0.77 | 3.69 ± 0.74 |
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Terminal 20 | 45.40 ± 3.81* | 18.82 ± 3.99* | 7.58 ± 0.67* | 4.21 ± 0.63* | 2.78 ± 0.62* |
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Note: db/m: db/m mice without diabetes; db/db: untreated db/db mice with diabetes; ezetimibe: ezetimibe-treated db/db mice with diabetes; *
Group |
|
TG (mmol/L) | FFA (mmol/L) | HbA1c (%) |
---|---|---|---|---|
db/m | Baseline 20 | 1.03 ± 0.22* | 2.52 ± 0.39* | 5.18 ± 1.21* |
Terminal 20 | 0.93 ± 0.27* | 2.58 ± 0.48* | 4.99 ± 1.03* | |
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db/db | Baseline 20 | 1.99 ± 0.74 | 3.23 ± 0.87 | 8.80 ± 2.54 |
Terminal 20 | 2.28 ± 0.49 | 3.74 ± 0.89 | 11.46 ± 2.87 | |
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Ezetimibe | Baseline 20 | 2.18 ± 0.45 | 3.35 ± 0.84 | 9.08 ± 2.67 |
Terminal 20 | 1.50 ± 0.74* | 2.88 ± 0.53* | 9.77 ± 2.60* |
Note: db/m: db/m mice without diabetes; db/db: untreated db/db mice with diabetes; ezetimibe: ezetimibe-treated db/db mice with diabetes; *
At 120 min after glucose tolerance test, the blood glucose in the ezetimibe group was markedly reduced
Effect of on IPGTT in db/db mice with diabetes. Compared with db/db, ezetimibe provided an improvement of glucose tolerance and first-phase insulin response. *
When compared with untreated db/db mice, the insulin secretion was not significantly increased in the ezetimibe-treated mice in the perfusion of pancreatic islets with low glucose solution, but the insulin secretion was markedly elevated at 1 min after perfusion with 16.7 mM glucose solution. This suggests that ezetimibe can improve the first phase insulin secretion, which, however, was still lower than that in the db/m group (Figure
In vitro perfusion of pancreatic islets in different groups. (a), db/db; (b), db/m; (c), ezetimibe. Ezetimibe improved first-phase insulin response when compared with db/db, but the insulin response was still lower than db/m.
After ezetimibe treatment for 6 weeks, quantitative analysis showed the content of
Comparison of
Group |
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db/m control ( |
14.23 ± 2.45* | 100* |
db/db control ( |
8.36 ± 1.53 | 64.21 ± 7.21 |
Ezetimibe group ( |
12.10 ± 2.55* | 83.36 ± 8.45* |
Versus db/db control mice, *
Immunohistochemistry for insulin in the pancreatic islets of three groups. Representative immunostaining for insulin performed with pancreatic tissue sections derived from db/m mice, placebo-treated db/db mice, and ezetimibe-treated db/db mice.
Content of
Ezetimibe is the first agent of a novel class of selective cholesterol absorption inhibitors and acts on the brush borders of rat small intestinal mucosal cells. Ezetimibe can inhibit the Niemann-Pick C1 Like 1 Protein (NPC1Ll) activity and selectively suppress the transportation of cholesterol in the diet and bile across the small intestine into the liver. Thus, the cholesterol stored in the liver is reduced which leads to reduction of synthesis of LDL receptor in the liver, promotion of LDL metabolism, and decrease in plasma LDL-C [
To date, studies have confirmed the NPC1Ll expression in not only the small intestine but the liver, pancreas, gallbladder, testis, and stomach [
Although there was no significant difference in the blood glucose level of db/db diabetic mice before and after ezetimibe treatment, the fasting glucose level, glucose tolerance, and glycosylated hemoglobin were all significantly lowered as compared with the control group. The abnormality of first phase insulin secretion is an early manifestation of dysfunction of
In our study, after ezetimibe treatment for 6 weeks, quantitative analysis showed ezetimibe could significantly reduce the
The mechanism underlying the ezetimibe induced improvement of glucose metabolism under diabetic status is still poorly understood. Studies show that this might be related to the improvement of peripheral insulin resistance and regulation of signaling pathways. The persistent increase in plasma free fat acid (FFA) may elicit the insulin resistance in the muscles and liver, elevate the gluconeogenesis, and reduce the glucose uptake in the muscles. On the contrary, persistent reduction in FFA may improve the glucose tolerance and elevate the peripheral insulin sensitivity [
Of course, there may be other mechanisms related to the ezetimibe improvement of insulin secretion. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1) are produced by the intestine and are released into the circulation in response to ingestion of macronutrients. Yang et al. had found that ezetimibe significantly active glucagon-like peptide-1 [
Taken together, our results demonstrate that ezetimibe not only can reduce the serum lipids, but also can improve the first phase insulin secretion in
The paper received a Grant from Natural Science Foundation of Jiangsu Province (BK2011661).