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Research Article CODEN: IJPRNK IMPACT FACTOR: 4.278 ISSN: 2277-8713 Sai Sandeep Y, IJPRBS, 2014; Volume 3(6): 250-257 IJPRBS

Available Online at www.ijprbs.com 250

INVESTIGATIONS ON EFFECT OF HYPERINSULIN ON BLOOD GLUCOSE AND

LIVER TISSUES OF RATTUS NORWEGICUS

JAYALAKSHMI ANNAPRAGADA1, SARITHA. A2, SUNITHA REDDY. G3, SAI SANDEEP.Y4,

JAYASHREE V. HANCHINALMATH5, KIRAN. B6

1. Research Trainee, Department of Biotechnology Research Centre, Tierra seed science Pvt. Ltd., Plot N0.198, Khajaguda, Goloconda Post,

Hyderabad-500 008, India.

2. Department of Biotechnology, Veerashaiva College, Cantonment, Bellary-583 104, India.

3. HR Executive, Department of Human Resource Management, JSW Severfield Structures Ltd., Cantonment, Bellary-583 104, India.

4. Research Scholar, Department of Botany, Sikkim Central University, Gangtok – 737 102, Sikkim, India.

5. Department of Biotechnology, Gulbarga University, Gulbarga – 585 106, Karnataka, India

6. Assistant Professor, Department of Botany, Sikkim Central University, Gangtok – 737 102, Sikkim, India

Accepted Date: 01/12/2014; Published Date: 27/12/2014

Abstract: The albino rats, Rattus norwegicus were injected with insulin of 0.5 ml per rat (five group each) at an interval of 24, 48, 72 and 96 hours respectively and the rate of heart beat was monitored three times in a day chronologically. The blood serum was collected for the estimation of glucose following the standard procedure. The effect of hyperinsulin was also studied in liver tissues by taking the cross section and the difference in specific cells was identified compared to control.

Keywords: Rattus norwegicus, Glucose, Insulin, Liver.

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Sai Sandeep Y, IJPRBS, 2014; Volume 3(6): 250-257



INTRODUCTION

Diabetes mellitus is a disease characterized by the inability to regulate blood glucose levels,

resulting in chronically increased blood glucose levels, or ‘hyperglycemia’ Saltiel et al (2001).

Diabetes is chronic metabolic disorder characterized by hyperglycaemia, glycosuria,

hyperlipaemia and sometime ketonaemia; in the endocrine system (Vineeta and Janeshwer,

2014). Multiple types of diabetes mellitus can be distinguished on the basis of the cause of the

hyperglycemia, which either result from insufficient or even absence of insulin secretion by the

β-cells, in combination with a suboptimal response of peripheral target tissues to insulin, a

phenomenon referred to as insulin resistance. In type 1 diabetes, dysregulation of the immune

system results in immunological intolerance towards the insulin-producing β-cells. This leads to

inflammation of the islets of Langerhans and selective destruction of the β-cells. (Atkinson et al

2001). Insulin synthesis and secretion are also affected in Maturity-onset diabetes of the young

(MODY) and Maternally inherited Diabetes and Deafness (MIDD), due to genetic factors

impacting on β-cell development and mitochondrial function (Brownlee and Cerami, 1981).

Liver failure is increasing dramatically and some estimate it will triple in the next 20 years. The

Centre for Disease Control and Prevention estimates that 30 million people in the USA suffer

from a liver disorder and 27,000 per year die from liver disease. Currently, the most common

causes of chronic liver failure are viral hepatitis and alcohol. In Canada, liver disease is the

fourth leading cause of death (Khan et al 2012). The most common cause is a condition known

as fatty liver disease but Hepatitis B and C are also major causes of chronic liver disease. It is

estimated that more than two million Canadians - regardless of age, sex, ethnic origin or

lifestyle - will be affected by a liver or biliary tract disease in their lifetime, and upwards of 400

liver transplant operations are performed every year. Considering the effect of hyperinsulin, in

the present study, the effect of insulin on heart beat and glucose level of blood and the

variation in specific cells in tissues of liver both in treated and control rats were evaluated at

different hours of duration.

MATERIALS AND METHODS:

Test Animal: The albino rat, Rattus norwegicus of 38 days aged were collected from GYAN

research foundation, Bangalore, and housed in polypropylene cages under hygienic conditions

and feedings were done using rat pellet diet (Hindustan Lever Limited) and water ad libitum.

Chemicals: Bouins fluid, 70% Alcohol, 80% Alcohol, 90% Alcohol, Absolute Alcohol, Xylene,

Paraffin wax, Hematoxylin, Eosin, DPX, were obtained from Hi-Media laboratories Pvt. Ltd.,

India, Merck India, Pvt. Ltd. Which were of Analytical grade.

Injection of Insulin: The albino rat, Rattus norwegicus of 38 days aged were collected and the

rats were grouped into 1st and 2nd group of 5 each. One group is meant for treatment and

another group act as control without any treatment. The 1st group of rats was made with



subcutaneous injections of exogenous 0.5ml of insulin to each rat at an interval of 24,48, 72 and

96 hours. The 2nd group of rats was injected with saline solutions at an interval of 24,48, 72 and

96 hours which acts as control (Liliane et al 2011).

Protein Precipitant: 10g of Na2 WO4.2H2O; 10gram Na2HPO4; 9 gram NaCL dissolved in 800ml of

water. Add about 125ml of NHCL to bring the pH to 3.0. Add I gram of phenol and make the

volume to 1 liter with distilled water (Trinder , 1969).

Colour reagent: 75ml of 4% Na2HPO4, 215ml of water, 5ml of fermcozyme AM glucose oxidase,

0.5ml of 0.1% peroxidase, 300mg of sodium azide and 100mg of 4-amino phenazone were

mixed and kept for 8 weeks at 40C.(Trinder , 1969).

Determination of heart beat and estimation of Glucose: The heart beat rate was recorded

three times in a day chronologically at 24, 48, 72 and 96 hours respectively. The animals were

vivisected after 24, 48, 72 and 96 hour. The blood was collected and 0.1ml of blood was taken

and mixed to 2.9ml of protein precipitate. The obtained mixture was centrifuged. I ml of

supernatant was transferred to test tube and labeled as test tube 1, Take 1ml of standard

(200mg/100ml) and 2.9ml of protein in test tube and label it as test tube 2. To all three tubes,

add 3ml of colour reagent and incubate for 370C for 10 minutes. The OD of all the tubes were

measured at 515nm using 10mm cells using the formulae blood glucose mg/100ml=OD of test

tube 1/ OD of test tube 2 X 200 (Trinder , 1969).

Collection and fixation of Kidney tissues: the kidney tissue was removed carefully and

preserved in different fixatives for histological studies. The fixed liver tissue were dehydrated in

different grades alcohol and embedded in paraffin wax (58-60 C). The sections were cut at 7µ

thick and stained with Ehrlich's Haemotoxylin Eosin. Sections were examined and photographs

were taken for further histological studies.

RESULT AND DISCUSSION:

Determination of heart beat and estimation of Glucose: The results indicated that the rate of

heartbeat found to be 62 beat / minute in control rats. Whereas, the rats treated with insulin

showed increased heart beat rate initially during 24 and 48 hours (62 and 92/minute). The

value decreased to 74 beat/minute in 72 hours. Further, the rate of heartbeat increased to 82

beats/min in the rats treated for 96 hours (Figure 1). The blood glucose level found to be 388

mg/dl in control rats. The value decreased to 200 mg/dl in the rats treated with 24 hours with

insulin. Further the glucose level increased to 157mg/dl in 48 hours and 195 mg/dl in 72 hours

treatment. Whereas, the value decreased to 134 mg/dl in the rats treated for 96 hours (Figure

2).

Study of Histology: The liver is an important organ as it performs so many functions directly or

indirectly related to varied metabolic processes. Much information liver of Rattus norwegicus is



multi lobed structure extending across the anterior region of the stomach on its ventral side.

The surface of the liver is covered with serous membrane and some connective tissues extend

inward into parenchyma. It is composed of parenchyma cells (hepatis cells) and lattice fibers. In

the sections of control rat, the hepatocytes form a cord-like pattern. These cords are arranged

around tributaries of the hepatic vein. The liver cells are large in size, polygonal in shape with

homogenous eosinophilic cytoplasm and centrally located nuclei. A large number of blood

sinusoids were observed and separates the hepatic cords one from the other. The Islets of

Langerhans (Hepatopancreas) are seen scattered in the hepatic tissue in association with the

blood vessels (Figure 3).

The liver, after 24 hours of treatment, the hepatic cells show feebly stained cytoplasm (Figure

4). The depletion of secretary material in the cells was noticed in the tissue treated with insulin.

The hepatic cells become smaller and show sparse granulation around the nucleus. A few

degranulated cells were also observed. Besides these changes, nuclear hypotrophy and

pycnosis was also observed. Blood sinusoids are noticed in the intercellular spaces. The islets of

langerhans exhibited both granulated and degranulated cells. The cells were spherical/ oval

with indistinct cell boundaries containing large nuclei. The histomorphological changes were

more pronounced in the animals treated for prolonged duration (96 hours). More number of

vacuoles found in between the hepatic cord with sparsely granulated cells. The blood sinusoids

were more prominent with degranulated Islets of langerhans (Figure 5).

The liver is an important organ, which performs many vital functions directly or indirectly

related to large number of metabolic processes. The liver may also be considered as a target

organ for environmental imbalance. In the present study the effect of insulin on liver shows

alteration in structure of the cells. Proliferation of ducted cells and small spaces appear in

between the hepatic cell is due to the effect of chemicals. The liver of few vertebrates have

already been reported (Bucke, 1971; Anderson and Mitchum, 1974; Chapman, 1981). Similar

type of hepatic cells has been reported in fishes like Channa punctatus (Dubale and Shah, 1979)

and Clarius batrachus (Mandal and Kulshreshtha, 1980). In the present investigation the liver

cells shows not much alteration in rats. However the insulin affect on endocrine and exocrine

part of the liver which causes vaculisation, necrosis and autolysis of cells. The changes in the

tissues like pycnosis of hepatocyte cell nuclei and cytoplasmic vacuolation in several animals,

were observed after treatment with chemicals (Amminikutty, 1975; Amminikutty and Rege,

1977). The present study also revealed that the liver showed cytological changes with

degranulation and vacuolization in the cytoplasm and conspicuous intercellular spaces. The

intercellular disturbances in the hepatocytes is directly related to the concentration of insulin

treated. Similar observation were made (Buchanan, and McCarroll, 1971).



CONCLUSION:

From the above experiment it can be concluded that, the overdose of insulin shows direct

effect on glucose metabolism and also the cells in the tissues of liver which involves directly in

synthesis of glucose. A further research is necessary to identify the mode of action of specific

cells to hyperinsulin in liver tissues.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. P. Srinivas, PES College, Hanumanth Nagar, Bangalore, Gyan

Research Foundation, Bangalore for providing laboratory facilities and Dr. F. C. Hosur and Sri. G.

Monahar, Veerashaiva College, Bellary for their valuable guidance.

Figure 1: The rate of heart beat in the control and treated rats at different hours

Figure 2: The rate of blood glucose in the control and treated rats at different hours

0

20

40

60

80

100

Control 24 hr 48 hr 72 hr 96 hr

Heart beat (mean)

Blood glucose (mg/dl)

0

100

200

300

400

500

Control 24 hr 48 hr 72 hr 96 hr



Figure 3: The cross section of control liver treated with saline.

Figure 4: The cross section of liver treated with insulin for 24 hours

Figure 5: The cross section of liver treated with insulin for 96 hours



REFERENCES:

1. Amminikutty CK and Rege: Effects of acute and chronic exposure to pesticides, Thiodon, E.C.

35 & Agallol '3' on the liver of window tetra Gymnocorymbus ternetzi (Boulenger). Ind. J. of

Expt. Biol. 1977; 15: 197-200

2. Amminikutty CK: M.Sc. Thesis, University of Bombay1975: 22-24

3. Anderson BG and Mitchum DL: Atlas of trout histology, Laramie Wyoming Game Fish

Department. 1974: 120-123.

4. Bucke D: The Anatomy and Histology of the alimentary tract on the carnivorous fish the pike

Esox lucius C. J. Fish. Biol. 1971; 3: 421-431.

5. Buchanan KD and McCarroll AM: Radioimmuno-assay methods. 1st ed. Kirkham, K.E.,

Hunter, W.M.,. Edinburgh: E. and S. Livingstone. 1971: 136–142.

6. Chapman GB: Ultra structure of the liver of the fingerling rainbow trout Salmo gairdneri. J.

of Fish Biol. 1981; 18: 553-567.

7. Dubale MS and Shah P: Histopathological lesions induced by malathion in the liver of Channa

punctatus. Indian J. Exp. Biol. 1979; 17: 693-797.

8. Mandal PK and Kulshreshtha AK: Histopathological changes induced by sublethal sumithion

in Clarius batracus(linn). I.J.Expt. Biol. 1980; 18: 547-558.

9. Saltiel AR and Kahn CR: Insulin Signaling and the Regulation of Glucose and Lipid Metabolism.

Nature, 2001; 41(4); 799-806.

10. Khan V, Najmi AK, Akhtar M, Mohd A and Pillai KK: A pharmacological appraisal of

medicinal plants with antidiabetic potential. Journal of Pharmacy And Bio-allied Sciences. 2012;

1(4):27-42.

11. Brownlee M and Cerami A. The biochemistry of the complications of diabetes mellitus.

Annu Rev Biochem. 1981;50: 385–432.

12. Liliane SP, Anderson DM and Ana Eliza A: Protocol of Insulin Therapy For Streptozotocin-

Diabetic Rats Based on a Study of Food Ingestion and Glycemic Variation. Scand. J. Lab. Anim.

Sci. 2011; 38(2): 117-127.

13. Trinder P: Determination of glucose in blood using glucose oxidase with an alternative

oxygen acceptor. Ann.Clin.Biochem, 1969; 6: 24.



14. Atkinson MA, Eisenbarth GS: Type 1 diabetes, new perspectives on disease pathogenesis

and treatment. Lancet 2001; 35(8): 221-229.

15. Vineeta T and Janeshwer V: Current Updates of Indian Antidiabetic Medicinal Plants.

International Journal of Research In Pharmacy And Chemistry 2014; 4(1): 114-118.

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