IN-HOUSE VALIDATION OF HIGH FAT DIET INDUCED OBESITY MODELS

INHOUSE VALIDATION OF HIGH FAT DIET INDUCED OBESITY MODELS

Prepared by:-POOJA A. SHARMA

Exam no. 49B.Pharm

Guided by:-Dr. Shrikalp DeshpandeM.Pharm., Ph.D., L.L.B

DEPARTMENT OF PHARMACOLOGYK.B. INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH

GANDHINAGAR

INTRODUCTION

OBJECTIVE

METHODOLOGY

EVALUATION PARAMETERS

RESULT AND DISCUSSION

CONCLUSION

Contents

2

DEFINITION

• Obesity is a multi-factorial disorder of energy

balance, in which

– calorie intake exceeds energy output (1)

– excessively large BMI occurs (1)

– abnormal accumulation of fat deposits (2)

– excessive secretion of adipokines occurs (3)

– insulin resistance occurs (4)

1

Potential Consequences of Obesity

Obesity is associated with a rise in many co-morbid conditions, including:

– Type 2 Diabetes(5)

– Metabolic syndrome X(6).

– Hyperlipidemia(7).

– Hypertension(7).

– Obstructive Sleep Apnea(8)

– Atherosclerosis (9).

– Pulmonary embolism (10)

– Back and lower extremity weight-

bearing degenerative problems(11).

– Cancer (12)

2

• To understand the genetic and environmental basis of obesity,

animal models have proven useful by allowing manipulations

technically or ethically not feasible in humans (13)

• In these models, obesity can be induced by genetic mutations,

pharmacologically, for example by injecting gold thioglucose (14)

or by a variety of dietary manoeuvres.

• These models of obesity have allowed insights into some critical

pathways but their overall relevance is nonetheless questionable

since common obesity cannot be attributed to a single gene or

single pathway. Thus, the polygenic nature of obesity calls for a

more realistic approach to generate animal models of obesity.

• In this respect, diet-induced obesity allows to mimic situations

more closely related to what can be observed in humans.3

HIGH FAT DIEThyperphagia (15)

calorie intake (16)

diet-induced thermogenesis (17)

the adipose tissue (18)

Summary of the effects of high fat diets

the liver (19)

the small intestine (20) 4

• A growing body of literature reviews rodents as models of human

obesity, though there are many confounding factors including species,

strain, gender, age, type of diet, level of fat and duration of diet.

• In order to rationalize the effect of these variables it was necessary to

carry out a validation study regarding the type of the high fat diet

used, the metabolic energy supplied by the diet and the mode of diet

administration. Further it was essential to consider the time factor and

the economic factor while developing a suitable model of obesity as

per the need.

• In this study we have developed and compared in-house high fat diet

induced obesity models and compared them in various strains and

gender, using commercially available high fat diet as standard.

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This dissertation aimed at comparing and validating

different diet induced obesity models utilizing different

high fat diet regimens in different strains and gender.

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Animals :-Sprague Dawley and Wistar rats.

Table 1. Study groups and treatment

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• Group 1 and 2: - were fed with in-house prepared high fat diet

(table no. ) containing 26.39 % fat supplying 52.56 %kcal from

fat and having a metabolic energy of 5261 kcal/kg.

• Groups 3 and 4:- Edible coconut oil and vanaspati ghee were

procured from the market and a mixture of the two was

prepared in a ratio of 2: 3 respectively v/v (21,22,23,24). This

high fat diet, at a dose of 10 ml/kg (8.13 kcal/ml) body weight,

was fed to the animals, per orally, daily in addition to normal

pellet diet (NPD) supplies energy of 3620 kcal/kg. (25).

• Group 5 and 6:- Animals were fed with commercially available

market preparation of high fat diet containing 24 % fat

supplying energy 48%kcal from fat and having a metabolic

energy of 4880 kcal/kg (26).8

Table 2. Nutritional information of In-house prepared HFD

gm % kcal %

Protein 13.745 12.16

Carbohydrate 39.34 34.82

Fat 26.39 52.56

kcal/gm 5.24

Ingredients Weight (gm) kcal

Normal pellet diet (NPD) 200 724

Casein 135 540

Lard 260 2340

Sucrose 245 980

DL-methionine 3 12

Corn starch 150 600

Vitamin and mineral mix 5 20

Salt 1 0

Soyabean oil 5 45

Total 1004 5261 9

4.2 EVALUATION PARAMETERS

Physical parameters

– Daily food intake

– Daily calorie intake

– Daily water intake

– Weekly body weight

Biochemical parameters

– Oral Glucose Tolerance Test(27)

– Insulin Tolerance Test(28)

– Plasma Total Cholesterol

– Plasma Triglyceride

– HDL Cholesterol

– LDL Cholesterol

– VLDL Cholesterol10

1. Food intake

All the values are expressed asMean±SEM

a*** = GP 5 showed significantdifference with GP 6(p<0.05)

b*** = GP 4 showed significantdifference with GP 2 , GP 3 and GP 5 (p<0.05)

c** = GP 2 showed significantdifference with GP 3(p<0.05)

Fig 1. Effect of various High fat diets on weekly food intake (gm/ d/rat) vs. time (days)

Food intake of marketed group was the highest, that of the in-housed prepared HFD comparativelylower and vegetable ghee:coconut oil group ate NPD the lowest. No strain wise difference in foodintake was observed.

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2. Calorie intake

All the values are expressed as Mean±SEM

a*** = GP 4 showed significant difference with GP 1, 2, 3, 5 (p<0.05)

b***= GP 5 showed significant difference with GP 6 (p<0.05)

Fig. 2. Effect of various HFD on Calorie intake (kcal/d/rat) vs. time (days)

Calorie intake of marketed group was the highest, that of the in-housed prepared HFD wasalmost similar to that of vegetable ghee:coconut oil group. SD rats showed higher food intakethan wistar rats. Gender wise no difference was observed.

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3. Water intake


a*** = GP 3 showed significant difference with GP 6 (p<0.05)

b*** = GP 2 showed significant difference with GP 6 (p<0.05)

Fig. 3. Effect of various HFD on water intake (ml/d/rat) vs. time (days)

Water intake of males given vegetable ghee:coconut oil group was comparatively higher than thatof the females given the same diet.

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4. Body weight


$ = GP 1 showed significant difference with GP 2 (p<0.01)

# = GP 1 showed significantdifference with GP 2 (p<0.001)

ɸ = GP 5 showed significant difference with GP 4 (p<0.01)and GP 6 (p<0.05)

Fig. 4. Effect of various HFD on % increase in body weight vs. time (days)

The % increase in body weight of SD strain was higher compared to that of wistar strain given thesame diet. In case of female rats, those on marketed diet showed higher weight gain than v.g:c.odiet. v.g:c.o diet showed more weight gain compared to inhouse HFD.

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a*** = GP 3 showed significantdifference with GP 6 (P<0.05)

b* = GP 4 showed significantdifference with GP 5 (P<0.05)

Fig.5.1 Effect of various HFD on Oral Glucose Tolerance Test (28 day)

5. Oral Glucose Tolerance Test

Gender specific variation in plasma glucose concentration was observed, being higher for malesthan females. No sign of glucose intolerance developed in case of any of the diet manipulationsby 28 days. 15


a* = GP 4 showed significant difference with GP 2 and GP 3.

Fig.5.2 Effect of various HFD on Oral Glucose Tolerance Test (42 day)

Marketed HFD showed significantly higher glucose intolerance by 42 days in comparison to in-house HFD and v.g:c.o diet

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6. Insulin Tolerance Test

Fig. 6.1 Effect of various HFD on insulin tolerance test (15 day)


No signs of insulin resistance were observed by 15 day of dietary manipulation

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a** = GP 4 showed significant difference with GP 3(P<0.05)

Fig.6.2 Effect of various HFD on insulin tolerance test (49 day)

Marketed HFD showed comparatively marked insulin resistance by 49 days of dietarymanipulation. v.g:c.o diet group was the least affected.

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7. Plasma Total Cholesterol


$ = GP 1 showed significant difference with GP 2 (p<0.01)

# = GP 4 showed significant differencewith GP 2(p<0.01) and GP 5 (p<0.001)

&= GP 3 showed significant difference with GP 2 (p<0.05)

ɸ = GP 1 showed significant difference with GP 2 (p<0.05)

α = GP 4 showed significant difference with GP 2 (p<0.05)

Fig. 7. Effect of various HFD on % increase in total cholesterol vs. time (days)

SD strain showed higher increase in total cholesterol than wistar strain. Marketed HFD showedcomparatively marked increase in total cholesterol than other diets. Vegetable ghee:coconut oildiet had more effect compared to in-house HFD.

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8. Plasma Triglyceride


$ = GP 1 showed significantdifference with GP 2 (p<0.05)

# = GP 3 showed significant difference with GP 2 (p<0.05)

& = GP 3 showed significant difference with GP 2 (p<0.05)

Φ = GP 6 showed significant difference with GP 5 (p<0.01)

α = GP 6 showed significant difference with GP 5 (p<0.01)

Fig. 8. Effect of various HFD on % increase in plasma triglyceride vs. time (days)

At the end of 49 day, SD strain showed higher increase triglyceride than wistar strain. At the endof 28 days only, higher plasma triglyceride level was observed in case of vegetable ghee:coconutoil diet compared to inhouse HFD in both gender.

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9. Plasma HDL cholesterol


$ = GP 1 showed significant difference with GP 4 (p< 0.01)

# = GP 1 showed significant difference with GP 4 (p<0.05)

& = GP 2 showed significant difference with GP 4(p<0.01)

@= GP 2 showed significant difference with GP 4 (p<0.05)

Fig.9. Effect of various HFD on % Decrease in plasma HDL-C vs. time (days)

The % decrease in HDL-C was highest in case of inhouse HFD and was lowest in case of marketeddiet indicating the usefulness of marketed diet for screening of anti-obesity drugs utilizing HDL-C as therapeutic target. Vegetable ghee:coconut oil diet showed comparatively less decrease inHDL-C than inhouse HFD.

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10. Plasma LDL cholesterol


$=GP 3 showed significantdifference with GP 2 (p<0.01)GP 4 (p<0.001)& GP 6 (p<0.001)

Fig. 10. Effect of various HFD on % increase in LDL-C vs. time (days)

Male vegetable ghee:coconut oil treated rats showed higher increase in LDL-C compared toinhouse HFD, marketed HFD and females with vegetable ghee:coconut oil diet.

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11. Plasma VLDL cholesterol


# = GP 3 showed significant difference with GP 1 (p<0.05) and GP 2 (p<0.01)

$ = GP 6 showed significantdifference with GP 5 (p<0.05)

Fig.11. Effect of various HFD on % increase in VLDL-C vs. time (days)

Vegetable ghee:coconut oil treated rats showed higher increase in VLDL-C compared to inhouseand marketed HFD in both gender.

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• Overall, the results of the present validation study indicate that there exists gender specific, diet

specific and strain specific variation in obesity models.

• A better achieved lipid profile and body weight gain was observed in case of vegetable

ghee::coconut oil (3:2) diet in a shorter duration of time and it ensures equal amount of fat being

given to the entire group of animals. Moreover it is more feasible in terms of economy also.

• Marketed HFD produced higher body weight gain and better lipid profile than in-house prepared

high fat diet at the end of 4 week but at the end of 7 week almost equal results were obtained.

However marketed diet showed more glucose intolerance and insulin resistance and hence can be

utilized as a model for screening of anti-obesity drugs showing usefulness in diabetes.

• Gender wise not much difference was found, however better lipid achieved lipid profile was

obtained in case of males than females.

• Strain wise Sprague-Dawley rats served as better model than Wistar rats in case of in-house

prepared HFD. The reason for this strain specific variation requires further investigation.

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Plasma Total Cholesterol

Fig. 1 Effect of various HFD on plasma total cholesterol (mg/dl) vs. time (days)

Plasma triglyceride

Fig. 2 Effect of various HFD on plasma triglyceride (mg/dl) vs. time (days)

Plasma HDL cholesterol

Fig.3 Effect of various HFD on plasma HDL-C (mg/dl) vs. time (days)

Plasma LDL cholesterol

Fig. 4 Effect of various HFD on plasma LDL-C (mg/dl) vs. time (days)

Plasma VLDL cholesterol

Fig. 5 Effect of various HFD on plasma VLDL-C (mg/dl) vs. time (days)

Education

IN-HOUSE VALIDATION OF HIGH FAT DIET INDUCED OBESITY MODELS