الفهرس 
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Abstract
The present study was conducted to investigate the effect of dietary manipulation in the form of reversion to regular rat chow diet or alternate day fasting as well as their combination on hepatic steatosis in an experimental model of metabolic syndrome in adult male rats.
The study was carried out on 66 local strain male rats, initially weighing 120-180 grams (age of rats was ≃ 6-10 months).
Animals were distributed randomly into the following two groups:
group I; Control rat group (C, n=12).
Rats in this group were fed the control diet and were given (4 ml distilled water / rat /day) introduced daily by gavage for 8 weeks. At the end of the 8th week, two rats were scarified for histopathological study of the liver.
On the 9th week of the study, the other 10 rats were continued on the control diet and were given (4 ml distilled water / rat /day) introduced daily by gavage for additional 4 weeks.
group II; Metabolic syndrome with liver steatosis rat group (M, n=54).
Metabolic syndrome was induced by high fructose diet and rats were given additional fructose solution (4g fructose dissolved in 4 ml distilled water / rat /day) introduced daily by gavage. The high fructose diet and solution was given for 8 weeks. At the end of the 8th week, three rats were scarified for histopathological study of the liver.
On the 9th week of the study, the proven metabolic syndrome rats (n=51) were randomly allocated into the following four experimental groups:
A. Untreated Metabolic syndrome group (MS, n=14).
Rats in this group were continued on high fructose diet and the fructose solution by gavage similar to M group for additional 4 weeks, which is the end of the study period.
B. Metabolic syndrome group reverted to regular rat chow (MSRD, n=12).
Rats in this group were reverted to control diet and were given (4 ml distilled water / rat /day) introduced daily by gavage for 4 weeks.
C. Metabolic syndrome group on alternate-day fasting (MSF, n=13) according to Varady et al, 2010.
Rats in this group underwent alternate-day fasting (only for food) regimen in the form of alternating 24-h periods of ad libitum feeding and fasting (100% calorie restriction on fast day, ad libitum high fructose diet on feed day with the fructose solution by gavage similar to the M group for 4 weeks.
D. Metabolic syndrome group reverted to regular rat chow and on alternate-day fasting (MSRDF, n=12).
Rats in this group underwent alternate-day fasting (only for food) regimen in the form of alternating 24-h periods of ad libitum feeding and fasting (100% calorie restriction on fast day, ad libitum control diet on feed day) and were given (4 ml distilled water / rat /day) introduced daily by gavage for 4 weeks.
At the end of the accommodation period, all rats were subjected to the following as a basal assessment.
• Measurement of body weight (BW), body mass index (BMI), waist circumference (WC),
• Fasting blood glucose level (FBG), intraperitoneal glucose tolerance test (IPGTT),
• Retro-orbital blood samples for determination of serum high density lipoprotein cholesterol (HDL-C) and serum triglycerides (TGs).
Throughout the experiment, all rats were subjected to weekly assessment of body weight (BW).
After 8 weeks, rats were subjected to estimation of the following parameters; body weight (BW), body mass index (BMI), waist circumference (WC), fasting blood glucose level (FBG), intraperitoneal glucose tolerance test (IPGTT), serum high density lipoprotein cholesterol (HDL-C), serum triglycerides (TGs) and histopathological study of liver tissue.
At the end of the study on the 12th week, all rats were subjected to the following measurements:
1. Body weight (BW).
2. Body mass index (BMI).
3. Waist circumference (WC).
4. Fasting blood glucose level (FBG).
5. Intraperitoneal glucose tolerance test (IPGTT).
6. Absolute and relative liver weights.
7. Absolute and relative visceral fat weights.
8. Glycosylated hemoglobin (HbA1C).
9. Determination of serum levels of:
a) High-density lipoprotein cholesterol (HDL-C)
b) Triglycerides (TGs)
c) Total cholesterol (TC) and Calculation of low-density lipoprotein cholesterol (LDL-C)
d) Fasting insulin and the calculation of homeostasis model assessment of insulin resistance (HOMA-IR).
e) C-reactive protein as proinflammatory marker.
f) Albumin.
g) Total bilirubin.
h) Alanine transferase (ALT)
10. Determination of a marker of oxidative stress malondialdehyde (MDA) in liver tissue.
11. Histopathological study of liver tissue.
The 8th week values of BW, BMI, and WC were significantly higher than their corresponding initial values in both C and M groups; 8th week values of the blood glucose levels during IPGTT at 60 min., 90 min., and 120 min., and AUC were significantly increased in C rats compared to their corresponding initial values. For M group, the 8th week values of FBG; blood glucose levels during IPGTT at 30 min., 60min., 90 min., and 120 min.; PG and AUC were significantly increased compared to their corresponding initial values; both C and M groups presented a significantly higher serum level of TGs along with a significantly lower HDL-C in the 8th week compared to their corresponding initial values.
The initial values of BW, BMI, WC, FBG, IPGTT, PG, AUC, serum levels of TGs & HDL-C were insignificantly different between C and M groups. After 8 weeks of high fructose diet, M rats exhibited a significantly higher values of WC; blood glucose levels during IPGTT at 30 min., and 120 min.; PG, AUC associated with a significant lower serum levels of HDL-C in comparison to C group.
The final BW and BMI were not significantly changed, whereas WC was significantly increased in the C and MS groups in comparison to their corresponding 8th week’s values. BW and WC were significantly increased in MSRD group compared to their corresponding 8th week’s values. However, BW, BMI, and WC were significantly decreased in MSF and MSRDF groups versus their corresponding 8th week’s values.
Compared to 8th week values, final FBG; blood glucose levels during IPGTT at 30 min., 60 min., 90 min., and 120 min.; PG; and AUC; all were not significantly changed in the C group. The MS group exhibited a significantly lower final levels of blood glucose during IPGTT at 60 min., 90 min., and 120 min., along with a significant decrease in AUC versus their corresponding 8th week’s values. Although, alternate day fasting alone significantly decreased the final FBG only in MSF rats; reversion to regular rat chow, whether alone or in combination with alternate day fasting, resulted in a significant decrease in FBG and blood glucose levels at 30 min., 60 min., 90 min., and 120 min.; PG; and AUC in both MSRD and MSRDF groups compared to their corresponding 8th week’s values.
Compared to 8th week values, the final levels of serum TGs were significantly decreased in C group, but significantly increased in MS group, but showed significant decrease in MSF group and insignificant change in MSRD and MSRDF groups. On the other hand, HDL-C showed significant increase in C, MSRD, MSF, and MSRDF groups with insignificant change in MS group.
MS group demonstrated non-significant changes in the final values of BW, BMI, WC, VFW and VFW/BW compared to those of the C group, similarly MSRD group had no significant difference in these parameters compared to MS and C rat groups. On the other hand, these parameters were significantly decreased in both MSF and MSRDF groups compared to MS, MSRD groups, as well as C group.
Final values of FBG and blood glucose during IPGTT; PG; and AUC; Hb A1C, all were insignificantly changed in MS group compared to those of the C rats, while the serum fasting insulin level and HOMA-IR were significantly increased in MS group compared to the C group. MSRD group showed non-significant changes in the final values of FBG and blood glucose during IPGTT; PG; and AUC in addition to Hb A1C when compared to those of the MS or C groups. MSRD group showed significant decrease in serum fasting insulin level and HOMA-IR compared to MS group, and significant increase compared to the C group. MSF group showed significant increase in the levels of blood glucose during IPGTT at 90 min., 120 min., non-significant change in PG and AUC but significant decrease in serum fasting insulin level, HOMA-IR, and Hb A1C compared to MS rats. Compared to MSRD group, MSF group presented a significantly elevated blood glucose levels during IPGTT at 60 min. and 90 min., along with non-significant changes in PG, AUC, serum fasting insulin level, HOMA-IR, and Hb A1C. In comparison to the C group, all the measured glucose homeostasis parameters were not significantly different in MSF rats except for a significantly higher serum fasting insulin and HOMA-IR. MSRDF group exhibited a significant lower level of FBG when compared to MSRD; MSF; C and MS rat groups. The blood glucose levels during IPGTT at 60 min., 90 min., and 120 min., were decreased in MSRDF group compared with MSF group. Compared to the C group, the levels of blood glucose measured during IPGTT in MSRDF group were not significantly different except for a significantly lower value at 90 min. PG was not significantly changed in MSRDF group compared to the other four groups. AUC was significantly decreased in MSRDF group in comparison to MSF and control groups. Serum fasting insulin and HOMA-IR were significantly decreased in MSRDF group versus MS, MSRD, and MSF groups. HbA1C was decreased in MSRDF group compared with MSRD, MSF and MS groups, being significant in MS group only. In comparison to C rats, serum fasting insulin was still significantly higher in MSRDF group, while HOMA-IR and HbA1C were not significantly different.
MS group showed a significantly higher final serum levels of TGs, TC, and LDL-C associated with a significantly lower HDL-C compared to the C group. MSRD and MSF groups demonstrated a significantly reduced final serum levels of TGs, TC, and LDL-C associated with a significantly elevated HDL-C compared to the MS group, however, TGs, TC, and LDL-C were still significantly higher, and HDL-C was significantly lower compared to the C group. On comparing the parameters of lipid profile between the MSRD and MSF groups, non-significant changes were observed. MSRDF group exhibited a significant decrease of the final serum levels of TGs, TC, and LDL-C, along with a rise in HDL-C compared to the MS group as well as MSRD group as regards serum levels of TGs, TC and LDL-C with insignificant change in HDL-C. Furthermore, in comparison to the MSF group, MSRDF group showed a significant decrease in the final serum levels TC and LDL-C with no significant changes in TGs or HDL-C. Moreover, the final serum levels of TC and LDL-C in MSRDF group attained those of the controls, yet TGs was still significantly higher and the HDL-C was significantly lower than those of the C group.
MS group showed a significant increase in the final LW and LW/BW versus the C group. MSRD and MSF groups presented a significant decrease in LW and LW/BW compared to MS group. Also, LW was significantly decreased in MSF group versus MSRD group, while the LW/BW was not significantly changed. In comparison to the control rats, LW/BW was still significantly higher in MSRD and in MSF groups, but the LW was not significantly different. MRSDF group showed a significant decrease in LW compared to MS, MSRD, and MSF groups as well as LW/BW when compared to the MS and MSF groups only with the LW in MSRDF group becoming significantly lower than that of the control rats and with the LW/BW insignificantly different from control value.
MS group demonstrated a significant increase in the serum level of ALT associated with a significantly lower level of serum albumin compared to the C group. Although the levels of serum ALT were significantly decreased associated with a significant increase in serum albumin in MSRD and MSF groups compared to the MS group, yet these parameters were not normalized when compared to control rats. In comparison to the MSRD group, serum level of albumin was not significantly changed in MSF group, but the serum level of ALT was significantly lower. MSRDF rats showed significant decrease of ALT and non-significant changes of albumin compared to MSRD group, but when compared to MSF group, no significant difference was observed in either ALT or albumin. However, MSRDF group showed a significant decrease in serum ALT together with a significant increase in serum albumin compared to MS group. In comparison to the C group, the serum ALT was not significant, while the serum albumin was significantly lower in MSRDF group. Concerning the serum bilirubin levels, non-significant changes were recorded among the five study groups.
MDA was significantly increased in MS group versus C group, and was significantly decreased in MSRD, MSF, and MSRDF groups in comparison to MS group, though it was still significantly higher than those of the control rats in these three groups. MSF and MSRDF presented a significantly lower liver levels of MDA in comparison to the MSRD rats. The liver level of MDA was not significantly different between MSF and MSRDF groups. Serum level of CRP was significantly increased in MS group versus C group. MSRD, MSF, and MSRDF groups showed a significant decrease in serum CRP compared to MS group, becoming not significantly different from the level of control rats. The serum level of CRP was not significantly different among MSRD, MSF, and MSRDF groups.
Histopathological study of liver tissue was used to detect % of steatosis and grading of NAFLD that revealed the following:
At the end of 8th week; C group showed 0% steatosis, FC score (1); and M group 69% steatosis, FC score (4).
Four weeks later at the end of the study; C and MSRDF groups showed 0% steatosis, FC score (1); MS group 82% steatosis, FC score (4); MSRD and MSF groups 42% steatosis, FC score (3).
At the end of 8th week; M group was classified as Grade (1) (PF: 0 and AS: 2).
Four weeks later at the end of the study; MS, MSRD and MSF groups were classified as Grade (1), MS (PF: 0 and AS: 4), MSRD (PF: 0 and AS: 3) and MSF (PF: 0 and AS: 1).
Conclusion and recommendations:
It can be concluded that high fructose intake for 8 weeks induced metabolic syndrome associated - grade 1 NAFLD, reversion to either regular diet or alternate day fasting was associated with partial improvement in some measured parameters as well as some histopathological features with no improvement in NAFLD -grading.
On the other hand, metabolic syndrome associated - grade 1 NAFLD was cured by lifestyle intervention in the form of stop of fructose intake and adoption of alternate day fasting which resolved all pathogenic criteria of MetS and NAFLD.
It could be recommended that individuals on high fructose intake as well as patients with metabolic syndrome should be investigated for liver involvement and that dietary habits of patients with grade -1 NAFLD, should be investigated and corrected with recommendation of alternate day fasting if feasible.