INTRODUCTION:

Obesity is reaching pandemic proportions globally resulting in increased health care burden and decreased life expectancy^.
1–4. Fasting with modifications has been used as therapeutic tool in controlling obesity and in weight reduction^{5- 6}.

Body weight, caloric intake, and oxidative stress has been linked in various pathophysiological mechanisms^7-8. Fasting with modifications has been used as therapeutic tool mainly in controlling obesity and weight loss ⁹. Therefore, it becomes very essential to search for a simple fasting regimen which could be helpful in diminution of the pathophysiology leading to metabolic syndrome. Obese rat model closely mimics the human obesity syndrome. The present study aims to observe the effect of complete and intermittent fasting in rats with high fat high sugar diet.

MATERIAL AND METHODS:

Ethical approval:

All the procedures of this experiments were reviewed and approved by the Institutional Animal Ethical (IAEC)Kasturba Medical College, Mangalore (KMC/MNG/IAEC/14-2019). All the study procedures were conducted and maintained according to guidelines proposed by the Committee for Control and supervision of experimentation on animals (CPCSEA), Government of India.

Study period and location:

Laboratory animals used in the study: Adult Wistar albino rats weighing 200-250 grams were used for the present experimentation procedures. The animals were procured from the central animal house of our institution. All the animals were maintained at 25±2°C temperature with 12 h of light and dark cycles with adlibtum access to laboratory food (commercial rat pellets from VRK nutritional solutions, India) and water.

High Fat, High Sugar (HFHS) Diet:

High fat diet was prepared using Vanaspati ghee-coconut oil in the ratio 3:1 in volume mixed with rat feed (3 ml/kg body Wt/day). High sugar diet made of 25%, weight to volume, fructose water orally by replacing it for plain water for 6 weeks ^10,11.

Intermittent fasting (IF):

Rats were subjected to fasting for 16 hours every day, the standard pellet diet was removed from the cage every evening at 5:00 PM and reintroduced the next morning at 9:00 AM. Plain water intake was unrestricted.

Complete fasting (CF):

Rats were subjected to 24 hours fasting once every 4 day, the standard pellet diet was removed from the cage at 9 AM every fourth day and reintroduced next day at 9 AM. Plain water intake was unrestricted.

Grouping of Animals:

The animals were divided in four groups of 6 animals each. The control group (Group I ) rats were fed with standard rat chow and plain water . The obese group (Group II ) animals were fed with HFHS diet for 8 weeks. The intermittent fasting group (Group III) of animals were fed with HFHS diet for 6 weeks and after 6 weeks, in continuation with HFHS diet, the rats were subjected to intermittent fasting for 2 weeks. Group IV animals were fed with HFHS diet for 6 weeks. After 6 weeks, in continuation with HFHS diet, the rats were completely fasted for 2 weeks (complete fasting groups)

Calculation of the BMI:

The body weight of the animal was recorded using animal weighing balance and the fractions of weights was expressed to the nearest gram unit. The length of the animal was measured from nose tip to the base of the tail using measuring tape and was expressed in centimeters. The measurements were done on day 1, on day 42, that is at the end of 6 weeks, and on day 56, that is the last day of the experiment. Using the length and weight, BMI was calculated. After 8 weeks, The animals were anesthetized using ketamine (100mg/kg body weight) The blood sample were collected by cardiac puncture for the estimations of biochemical parameters which included blood glucose, serum lipid profile, and liver function test . Serum lipid profile was determined using diagnostic kits purchased from Diatek healthcare private limited using spectrophotometer. The estimations of the liver enzymes such as ALT, AST were estimated by UV kinetic methods^12-13. Blood glucose level was estimated using the Accu- Chek® Active-glucometer and Accu- Chek® Active-strips through tail puncture

Histology of liver and Pancreas:

The excised liver and pancreatic tissue was stored in 10% formalin for 48 hours for the process of fixation. Once the fixation has been done the paraffin blocks were made. Sections of liver and pancreatic tissue was done using rotary microtome of thickness about 6-7μ thickness were made and the sections were stained with crystal violet stain

Statistical analysis:

The data entered on to Statistical Package for Social Sciences (SPSS 16) version 16. The result was expressed as proportional and mean using appropriate tables and figures. Results in tables are expressed as median with interquartile range in bracket. Kruskal-Wallis test and Mann-Whitney U test used. Significance level was set at p<0.05

RESULTS:

Effect of HFHS diet and fasting on BMI:

At the end of 6 weeks, the rats fed with HFHS diet showed significant increase (p<0.05 ) . BMI measured did not show any significance across all the groups. after the two diet regimen at the end of eighth week (Table 1)

Table. 1. Changes in the BMI in the different experimental groups

Parameter

Control

Obese

P value

BMI on day 0

(in gm/cm²)

0.539

(0.505-0.566)

0.617

(0.566-0.676)

0.575

(0.487-0.648)

0.575

(0.531-0.633)

0.109

BMI on day 45

(in gm/cm²)

0.578

(0.538-0.633)

0.60

(0.555-0.683)

0.508^b

(0.493-0.571)

0.589 ^c

(0.554-0.674)

0.021^a

BMI on day 60

(in gm/cm²)

0.588

(0.493-0.634)

0.610

(0.609-0.655)

0.634

(0.555-0.686)

0.619

(0.583-0.681)

0.214

a p<0.05, across all the groups; b p<0.05, obese vs IF; ^cP<0.01, IF vs CF.

Effect of fasting on blood glucose, cholesterol, liver weight and liver enzymes:

The IF group had a significant rise (p<0.05). in blood glucose with respect to the obese group. Serum cholesterol level significantly increased (p<0.05). in both IF and CF when compared to control group. Comparatively, IF group had a higher level of cholesterol when compared to CF group. The AST level significantly decreased in obese as compared IF group (p<0.01) The ALP level significantly decreased in the IF group when compared CF group(Table 2)

Table. 2.The Blood Glucose, Cholesterol, Liver Weight and Liver Enzymes in the different experimental groups

Parameter

Control

Obese

P value

BG (mg/dl)

140

(131.25-157.75)

106.5 ^b

(102-115)

139.5 ^d

(131.5-226)

131

(105.25-147.75)

0.02^a

Cholesterol(mg/dl)

85 (79-88.5)

(78.75-105.75)

106.5 ^c

(88-122.75)

89^c

(82-113)

0.045^a

LW(g)

(5.75-10)

(4.75-10)

(7-10)

(6-10)

0.73

AST(IU/L)

(2.5-11.5)

10.5

(8.25-15)

4^d

(3-6.75)

(3-11)

0.129

ALT(IU/L)

(37-57.5)

42.5

(28-48)

32.5

(28.75-40.75)

(29-46)

0.253

ALP(IU/L)

(51-99)

66.5

(43-102.25)

103^e

(56.25-117.5)

(46-94)

0.161

a; p<0.05, across all groups; b; p<0.05, control vs obese; c;pP<0.05, control vs IF, control vs CF; d; p<0.01, obese vs IF; e; p<0.05,obese vs IF

Table. 3. Variations of MDA and GSH Level in the pancreas and liver in the different experimental groups

Parameter	Control	Obese	IF	CF	P Value
MDA-P (µmol)	0.1165 (0.1035-0.122)	0.224 (0.2115-0.228)	0.07950 (0.0735-0.0857)	0.0595 (0.049-0.0605)	0.000 ^a
GSH-P (µmol)	0.1655 (0.14375-0.185)	0.1365 (0.1255-0.145)	0.3075 (0.2752-0.3335)	0.2485 (0.24-0.2702)	0.000 ^b
MDA-L (µmol)	0.1765 (0.16-0.1847)	0.3205 (0.316-0.330)	0.14600 (0.1410-0.15375)	0.121 (0.1162-0.125)	0.000 ^c
GSH-L (µmol)	0.135 (0.12975-0.1495)	0.1885 (0.182-0.194)	0.1985 (0.1927-0.2147)	0.3055 (0.289-0.317)	0.000 ^d

a; p<0.001across all groups, a; p<0.05 control vs obese, control vs IF, control vs CF; a; p<0.01 obese vs IF obese vs CF; a; p<0.01, IF vs CF; b; p<0.001 across all groups. b; p<0.05 control vs IF, control vs CF; b; p<0.01 obese vs IF, obese vs CF; b; p<0.05 IF vs CF; c;p<0.001across all groups; c; p<0.01 control vs obese, control vs CF; c; p<0.05 control vs IF; c; p<0.01, obese vs IF, obese vs CF; c; p<0.01, IF vs CF; d; p<0.001 across all groups; d; p<0.01, control vs obese, control vs IF, control vs CF; d; p<0.05obese vs IF; d; p<0.01obese vs CF; d; p<0.01 IF vs CF.

The MDA level in the pancreatic tissue significantly decreased (p<0.001), Whereas the GSH level significantly increased (p<0.001 in both the fasting groups when compared to the control groups. The observed results was more significant in the complete fasting group (p<0.001). when compared to intermittent fasting group. In the liver, MDA level significantly decreased (p<0.001). in both the fasting groups . More sigfnicant decline (p<0.001). in MDA level and more significant increase in the GSH level (p<0.001). of the liver was observed in the complete fasting group when compared to the intermittent fasting groups (Table 3)

Histology of the liver and the Pancreas:

In the pancreas, the acini were enlarged in obese rats compared to the control group. Architecture of acinar cells were distorted in obese rats. Furthermore, we observed multiple vacuoles in the acinar cells in obese rats. The histological observation of the liver tissue in the obese rats showed a vesiculation (Fig 1 , Fig 2)

Figure 1: Histology of the Liver

Figure 2: Histology of the Pancreas

DISCUSSION:

Different fasting regimens varying in duration of calorie restriction have been experimented previously with similar outcomes ¹⁴. The present study differs from them in comparing and contrasting two different calorie restricting diet regimens in rats being fed with HFHS diet. By the end of study, there was no significant change in BMI among different groups but after the intervention of 2 different diet regimens into the respective fasting group for 2 weeks, both the fasting groups showed an increase in BMI. Previous studies have also shown the development of consuming behavior in mice subjected to calorie restriction ¹⁵. Over-compensatory feeding developed during the fasting period might have resulted in the increased BMI in fasting group. In the present study , the blood glucose level increased in the fasting group when compared to obese group . Reduced insulin sensitivity due to caloric restriction might be the cause of higher blood glucose.

Liver secretes larger quantity of certain enzymes when it is inflamed or damaged. Deposition of fat in the liver causes mild degree inflammation which leads to metabolic dysfunction and releases liver enzymes¹⁶. In our study, there were no significant changes in the liver enzymes . This is in accordance to the study done by Megan et al where fasting for small duration from 0 to 24hrs did not affect the liver enzyme values ¹⁷. The IF group had a higher level of cholesterol in comparison to CF group. This finding is in support of studies previously conducted on animal models and human subjects¹⁸. The increase in cholesterol during fasting state can be attributed to the tissue mobilization of cholesterol during food deprivation ¹⁹.

Reactive oxygen species (ROS) are free radicals derived from oxygen leading to cell injury. They are produced physiologically during mitochondrial respiration. ROS are scavenged by antioxidants and radical scavenging enzymes such as glutathione peroxidase. Increased production or decreased scavenging of ROS leads to oxidative stress resulting in peroxidation of lipids²⁰. Malondialdehyde is a biochemical marker for lipid peroxidation due to oxidative damage . Glutathione is an antioxidant. We observed that there was significant rise in oxidative stress in the obese group. Higher MDA level and lower GSH level was observed in the pancreatic tissue in the HFHS diet induced obese groups , indicating maximum oxidative damage and the poorest protective mechanism in pancreas. Increased endogenous peroxides inducing the pathological complications of obesity further supports our findings. ROS activate redox sensitive inflammatory transcription factors, nuclear factor kb and activator protein-1might be responsible for triggering the release of ROS, establishing a vicious cycle ^{20, 21}

It is interesting to note that in our study the obese group showed increased oxidative stress despite of not having any significant increase in BMI, this finding supports the study conducted by warolin et al where they suggested that overall adiposity is associated with oxidative stress rather than the BMI ²². Simultaneous decline in anti-oxidant level was also observed to be significant in the obese group. This is in line with the study conducted by Christina et al, which also showed altered antioxidant defense mechanisms in obese individuals ²³. In our study, fasting significantly decreased the oxidative stress and also produced an improved antioxidant profile both in the liver and pancreas. suggesting an improved anti oxidative mechanism. The present results are in consistent with the previous reports ^{22, 23}.

Steatohepatitis is one of the consistent histologic features associated with metabolic syndrome. Steatohepatitis is also likely to progress to severe liver disease. Accumulation of substances results in oxidative stress overwhelming the liver’s capacity to metabolize energy substrates . In the present study the histological observation of the liver tissue in the obese rats showed a vesiculation, suggestive of steatosis indicating the onset of liver dysfunction.. In the pancreas, the acini were enlarged in obese rats compared to the control group. Architecture of acinar cells were distorted in obese rats. Furthermore, we observed multiple vacuoles in the acinar cells in obese rats which might be due to fat deposition . Similar findings was observed by Akiko Matsuda et al in the pancreatic tissue of the rats fed on long term high-fat diet ²⁴. The pancreas and liver histology in obese group reflects the oxidative damage produced by increased free radicals and reduced anti-oxidant defense mechanisms ^25. Both the groups subjected to 2 weeks of fasting after 6 weeks of HFHS diet displayed a much-improved liver and pancreas histology compared to rats fed with HFHS diet for 8 weeks. This suggests that oxidative damage produced by HFHS diet can be reversed with fasting. Rats subjected to IF showed significantly higher oxidative damage in pancreas as compared to CF group. This finding is in accordance with the histology, which shows better pancreatic architecture with greater regenerative changes in CF group. In liver, it was again observed that there was a significantly greater oxidative damage in rats treated with IF in comparison to CF. CF group also showed a significantly better anti-oxidant profile than IF even though histologically, there were lesser vacuoles and better architecture was appreciated in IF compared to CF group. In the present study Complete fasting showed significantly lower oxidative damage and better antioxidant profile compared to that of intermittent fasting. Histology examination of rats treated with complete fasting displayed greater regenerative changes in pancreas compared to intermittent fasting further proving that complete fasting is a more effective than intermittent fasting at reversing the damage done due to HFHS diet. The present study is limited to animal models and was time bound. Further studies should be done to explore the long-term effects of these dietary regimens for a more comprehensive analysis. Adverse effects associated with these dietary regimens over a longer period of time must be investigated. Outcomes associated with antioxidant supplementation and fasting should be compared and contrasted. The study can further be expanded to clinical studies/trials and be explored as a treatment option to manage diseases where oxidative stress is implicated to play a key role.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

All the authors thank the Manipal Academy of Higher Education for providing all the support and the facilities needed for this research work. A seed money of Rs.10,000 was sanctioned by Manipal Academy of Higher education

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Received on 06.08.2021 Modified on 17.09.2021

Research J. Pharm. and Tech 2022; 15(11):5094-5098.

DOI: 10.52711/0974-360X.2022.00856