Protective effects of calorie restriction on insulin resistance and islet function in STZ-induced type 2 diabetes rats

Abstract

Background: Caloric restriction (CR) has become increasingly attractive in the treatment of type 2 diabetes mellitus (T2DM) because of the increasingly common high-calorie diet and sedentary lifestyle. This study aimed to evaluate the role of CR in T2DM treatment and further explore its potential molecular mechanisms.

Methods: Sixty male Sprague-Dawley rats were used in this study. The diabetes model was induced by 8 weeks of high-fat diet (HFD) followed by a single dose of streptozotocin injection (30 mg/kg). Subsequently, the diabetic rats were fed HFD at 28 g/day (diabetic control) or 20 g/day (30% CR regimen) for 20 weeks. Meanwhile, normal rats fed a free standard chow diet served as the vehicle control. Body mass, plasma glucose levels, and lipid profiles were monitored. After diabetes-related functional tests were performed, the rats were sacrificed at 10 and 20 weeks, and glucose uptake in fresh muscle was determined. In addition, western blotting and immunofluorescence were used to detect alterations in AKT/AS160/GLUT4 signaling.

Results: We found that 30% CR significantly attenuated hyperglycemia and dyslipidemia, leading to alleviation of glucolipotoxicity and thus protection of islet function. Insulin resistance was also markedly ameliorated, as indicated by notably improved insulin tolerance and homeostatic model assessment for insulin resistance (HOMA-IR). However, the improvement in glucose uptake in skeletal muscle was not significant. The upregulation of AKT/AS160/GLUT4 signaling in muscle induced by 30% CR also attenuated gradually over time. Interestingly, the consecutive decrease in AKT/AS160/GLUT4 signaling in white adipose tissue was significantly reversed by 30% CR.

Conclusion: CR (30%) could protect islet function from hyperglycemia and dyslipidemia, and improve insulin resistance. The mechanism by which these effects occurred is likely related to the upregulation of AKT/AS160/GLUT4 signaling.

Keywords: AKT /AS160/GLUT4; Calorie restriction; Diabetes; Insulin resistance; Streptozotocin.

Fig. 1

Flow chart of animal study design

Fig. 2

Effects of CR on metabolic indices. The diabetic Sprague–Dawley rats model was induced by feeding HFD for 8 weeks, followed by a single STZ injection (30 mg/kg). The rats were either provided HFD ab libitum (HFD + AL + STZ group) or fed HFD with a 30% restriction regimen (HFD + CR + STZ group). Rats given free access to normal chow diet served as a blank control (ND + AL group). All interventions lasted for 20 weeks. a, b body mass; c, d water intake; e, f RBG; g, h FBG; i IPGTT; j AUC of IPGTT; k FFAs. Data were presented as the mean ± standard deviation (SD). For a–h, n = 10–20 for the ND + AL group and n = 6–14 for the other two groups. Six rats from each group were randomly chosen for the IPGTT experiment and lipid profile detection. *p < 0.05 between the two groups; CR, calorie restriction; HFD, high-fat diet; STZ, streptozotocin; RBG, random blood glucose; FBG, fasting blood glucose; IPGTT, intraperitoneal glucose tolerance test; AUC, area under curve; FFAs, free fat acids; ND + AL, normal diet provided ad libitum (BCT group); HFD + AL + STZ, HFD provided ad libitum and STZ injection (MCT group); HFD + CR + STZ, HFD with 30% CR and STZ injection (CR group)

Fig. 3

Effects of CR on insulin resistance and islets function. The diabetic Sprague–Dawley rats model was induced by feeding HFD for 8 weeks, followed by a single STZ injection (30 mg/kg). The rats were either provided HFD ab libitum (HFD + AL + STZ group) or fed HFD with a 30% restriction regimen (HFD + CR + STZ group). Rats given free access to normal chow diet served as a blank control (ND + AL group). All interventions lasted for 20 weeks. a ITT (n = 6); b GSIS (n = 6); c AUC of GSIS; d HOMA-IR (n = 6–10); e glucose uptake in muscle (n = 4); data were presented as the mean ± standard deviation (SD). *p < 0.05 between the two groups; CR, calorie restriction; HFD, high-fat diet; STZ, streptozotocin; ITT, insulin tolerance test; GSIS, glucose simulate insulin secretion; AUC, area under curve; HOMA-IR, homeostasis model assessment of insulin resistance; IOD, integrated optical density; ND + AL, normal diet provided ad libitum (BCT group); HFD + AL + STZ, HFD provided ad libitum and STZ injection (MCT group); HFD + CR + STZ, HFD with 30% CR and STZ injection (CR group)

Fig. 4

Effects of CR on AKT/AS160/GLUT4 signaling. The diabetic Sprague–Dawley rats model was induced by feeding HFD for 8 weeks, followed by a single STZ injection (30 mg/kg). The rats were either provided HFD ab libitum (HFD + AL + STZ group) or fed HFD with a 30% restriction regimen (HFD + CR + STZ group). Rats given free access to normal chow diet served as a blank control (ND + AL group). All interventions lasted for 20 weeks. Effects of CR on the expressions of AKT, p-AKT, AS160, and GLUT4 were determined by western blotting. a Expressions of AKT, p-AKT, AS160 and GLUT4 in skeletal muscle tissue after 10 and 20 weeks of intervention. b, c Densitometric measurements of band intensity were performed using ImageJ 1.52q. d Expressions of AKT, p-AKT, AS160 and GLUT4 in white adipose tissue after 10 and 20 weeks of intervention. e, f Densitometric measurements of band intensity were performed using ImageJ 1.52q. Data were from at least three independent experiments and presented as the mean ± standard deviation (SD). *p < 0.05 between the two groups; CR, calorie restriction; HFD, high-fat diet; STZ, streptozotocin; ND + AL, normal diet provided ad libitum (BCT group); HFD + AL + STZ, HFD provided ad libitum and STZ injection (MCT group); HFD + CR + STZ, HFD with 30% CR and STZ injection (CR group)

Fig. 5

GLUT4 immunofluorescence in skeletal muscle. The diabetic Sprague–Dawley rats model was induced by feeding HFD for 8 weeks, followed by a single STZ injection (30 mg/kg). The rats were either provided HFD ab libitum (HFD + AL + STZ group) or fed HFD with a 30% restriction regimen (HFD + CR + STZ group). Rats given free access to normal chow diet served as a blank control (ND + AL group). All interventions lasted for 20 weeks. a Representative images of GLUT4 immunofluorescence in skeletal muscle (× 100). b Densitometric measurements of the average fluorescence were performed by ImageJ 1.52q. Data were from at least three independent experiments and presented as the mean ± standard deviation (SD). *p < 0.05 between the two groups; CR, calorie restriction; HFD, high-fat diet; STZ, streptozotocin; ND + AL, normal diet provided ad libitum (BCT group); HFD + AL + STZ, HFD provided ad libitum and STZ injection (MCT group); HFD + CR + STZ, HFD with 30% CR and STZ injection (CR group)