RESEARCH INSULIN

EISSN 2320 6888

Discovery Proteins, Volume 1, Number 2, May 2013

Discovery Proteins RESEARCH INSULIN

ISSN 2320 6640

Proteins

Role of insulin as facilitating transporter for glucose in miniaturized screen printed electrode
Kaur A1, Verma N2
1. Assistant Professor, Department of Biochemistry,Gian Sagar Medical College and Hospital, Ramnagar, Patiala-147 002, Punjab (India) 2. Professor, Department of Biotechnology Punjabi University, Patiala-147 002, Punjab (India)

Correspondence author: Assistant Professor, Department of Biochemistry, Gian Sagar Medical College and Hospital, Ramnagar, Patiala-147002, Punjab (India), Mail: aman_jetty@yahoo.com, Mobile No. +91-9915731079
Received 16 March; accepted 25 April; published online 01 May; printed 16 May 2013

Insulin is a protein hormone. It plays very important role in glucose metabolism. This study was conducted to see the effect of insulin working as facilitating transporter for glucose in two types of cells i.e. a) Red blood cells (RBC) of normal individual were immobilized on the base (screen printed electrode) and b) RBC of IDDM were immobilized on the base (screen printed electrode).The response of both type of cells was seen on the instrument photoflurometer ,which obeyed bioassay principle with the estimation of insulin in terms of deflection. The Red blood cells inherent the enzyme machinery which results in the formation of NADPH. The results of both were significant i.e. p value less than 0.0001. The insulin levels of the screen printed electrode which were immobilized with the RBC of IDDM patients were lower as compared to the RBC of normal individuals which significantly relates the role of insulin as facilitating transporter of glucose. Key words: Insulin, red blood cells, Immobilization, photoflurometer. Abbreviations: SPE-Screen printed electrode, RBC- Red blood corpuscles, IDDM- Insulin dependent diabetic mellitus patients, Non-Insulin dependent diabetes mellitus (NIDDM), PBS- Phosphate buffer saline,NADPH- Nicotinamide dinucleotide phosphatedehydrogenase, WHO-World Health Organization.

ABSTRACT

1. INTRODUCTION

Insulin is a protein hormone secreted from pancreas from cells of islets of Langerhans (Guyton, 2010) as shown in Figure 1. It plays an important role in metabolism causing increased carbohydrates metabolism, glycogensis and glycogen storage, fatty acid synthesis/triglyceride storage and amino acid uptake, protein synthesis increases transmembrane K+ transport and decreases cyclic AMP level in adipose tissue and liver. Von Mering and Minkowski demonstrated the relationship of pancreatic hypo function and diabetic mellitus as early as 1890. Banting & Best finally succeeded in interacting insulin from pancreas in 1922 Talwar et al., (2008).

only about 50 units. 1 mg. of pure crystalline insulin has an activity of 24 units. Increase in the glucose concentration of the blood is a direct stimulus to insulin secretion. The granules move to the periphery of the -cell and are extruded into the blood by a process called emeiocytosis which is the reverse process of pinocytosis. Simultaneous with release of insulin the polypeptide linking the A and B chains the C-peptide is also released into circulation. Glucose can not only stimulate the release of the preformed hormone, but can also stimulate further synthesis of the hormone by the beta cell. Sugars like mannose and fructose also act like glucose in stimulating insulin release (Chang, 2008).

1.1. Bio-chemistry

1.2. Mechanism of Action

Insulin is a heterodimeric protein, has been isolated from pancreas and prepared in the crystalline form. Insulin molecule is composed of two polypeptide chains called Achain and B-chain containing total of 51 amino acids. A chain contains 21 amino acids and B chain contains 30 amino acids. In A chain, N-terminal amino acid is glycine and C-terminal is asparagine. In B-chain, N-terminal amino acid is phenylalanine and C-terminal is threonine. The human pancreas is said to contain about 250 units of insulin at any one time. The daily requirement of the hormone is

Kaur et al. Role of insulin as facilitating transporter for glucose in miniaturized screen printed electrode, Discovery Proteins, 2013, 1(2), 30-33, www.discovery.org.in/dr.htm

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The role of insulin as glucose transporter is a known fact. But this fact was analyzed on the miniaturization technique i.e. immobilization of RBC cells of normal individuals and IDDM patients. The deflection in terms of insulin was low in IDDM patients as compared to the normal individuals which is true to the role of insulin as glucose transporter.

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Insulin

Insulin binds to the -subunit of its receptor, which causes autophosphorylation of the -subunits receptor, which in turn induces tyrosine kinase activity as shown in figure 2. The receptor tyrosine kinase activity begins a cascade of cell phosphorylation that increases or decreases the activity of enzymes that mediate the effects of glucose on glucose, fat and protein metabolism, for e.g. Glucose transporters are moved to the cell membrane to facilitate glucose entry into the cell (Guyton, 2010). Insulin Receptor / Effectors Secondary Messenger Enzyme (Activated / inhibited) (Plasma Membrane) Glucose molecules by themselves are insoluble, so the rate of simple diffusion is too slow. A glucose carrier complex on the other side is soluble and can diffuse throughout it. The overall rate of glucose transport from the blood in the cells is greatly enhanced by the presence of insulin. Insulin facilitates the transport of glucose across the

Discovery Proteins RESEARCH INSULIN

The biochemical actions of insulin may be summarized as follows: Activation of glycogen synthetase and pyruvate dehydorgenase-phosphoprotein phosphatase. 1. Inactivation of c AMP-dependent protein kinase. 2. Inactivation of membrane bound adenylate cyclase, following its stimulation by hormones such as catecholamine and glucagon (thereby inhibiting glycogenolysis) 3. Activation of endoplasmic reticulum insulin-sensitive phosphodiesterase. 4. Activation of cell membrane calcium transport, mitochondrial calcium transport and cell membrane Ca++ Mg ATPase.

1.4. Functions of insulin

1. 2. 3. 4.
Figure 1 Physiologic anatomy of an islet of Langerhans in the pancreas (Guyton, 2010)

Increases activity of glycolytic enzymes Decreases activity of gluconeogenic enzymes Increases glycogen synthesis Increases uptake of glucose by skeletal and cardiac muscle 5. Decreases blood glucose level 6. Increases lipid synthesis 7. Increases transmembrane K+ transport 8. Decreases cyclic AMP level in adipose tissue and liver 9. Enhances tissue uptake of amino acids and accelerates protein synthesis.

1.5. Diabetes Linked to Insulin

Diabetes mellitus is a chronic hyperglycemic condition in which blood glucose level increases above the normal level and there are disturbances in carbohydrates, fats, proteins metabolism associated with absolute or relative deficiency in insulin secretion or insulin action. According to WHO recommendation diabetes mellitus is classified to: Type I : Insulin dependent diabetes mellitus (IDDM) Type II : Non-Insulin dependent diabetes mellitus (NIDDM) Type III : Diabetes associated with pancreatic disease, hormonal diseases, pregnancy, and use of drugs or chemical, abnormalities of insulin or its receptor and genetic syndrome (WHO Diabetes Group, 1985).

2. SCPOE OF STUDY

Figure 2 Schematic of the insulin receptor (Guyton, 2010)

In the present study, role of insulin as facilitating transporter was checked, based on immobilization technique. For this, comparison was done on both the subjects i.e. RBC of normal individual and IDDM patients were immobilized on screen printed electrode and response was seen for the insulin measurement on the instrument photoflurometer. Insulin monitoring is very important for clinical diagnostics. It serves as the predictictor of diabetes, insulinoma (insulin secreting tumour). Serum insulin levels are increased in type 2 diabetes, hyperthyroidism, type IV hyperlipoproteinemia, renal failure, liver cirrhosis, and in generalized acute illnesses. Decreased serum insulin levels are found in type 1 diabetes and are associated with pheochromocytoma, malnutrition and cystic fibrosis.

cell membrane (Chang, 2008). Jones, (2003) reviewed the relationship of the insulin resistance with the cardiovascular disease and added the function of insulin that with increase in glucose level, pancreas releases the insulin which facilitates the transport of glucose. Insulin stimulates intracellular transport of all sugars which have same configuration as glucose in carbon 1, 2 and 3 e.g. arabinose, xylose and galactose except for fructose which differs in C-2 position i.e. has keto group and does not depend on insulin for its transport into the cell (Guyton, 2010).

2.1. Materials
2.1.1. Subjects
The present study comprised of 60 patients from government and private hospital. The consent from the patient was taken who were enrolled for study and approval from ethics committee of the institute.

2.1.2. Blood Sample

The serum sample was used for insulin determination.

Kaur et al. Role of insulin as facilitating transporter for glucose in miniaturized screen printed electrode, Discovery Proteins, 2013, 1(2), 30-33, www.discovery.org.in/dr.htm

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1.3. Biochemical basis

2.1.3. Chemicals / Reagents

Insulin binds to specific receptors on the cell membrane and elicits the release of yet underfined intracellular secondary messengers. The messengers affect intracellular protein synthesis, and bring about changes in enzyme activities, besides producing membrane effects as shown in figure 3.

The screen printed base electrodes were used for immobilization of the red blood cells. The base electrodes have been procured from kind courtesy by Dr. Joseph Wang, New Mexico State University, USA. Reagents used were of analytical grade and were procured from SRL chemical Ltd., Torrent pharmaceuticals Ltd., S.D fine

Discovery Proteins RESEARCH INSULIN

Table 1 Effect of Insulin as Facilitator on Normal individual & IDDM Patient in terms of deflection SPE Immobilized with RBC OF Normal Individual , SPE Immobilized with RBC OF IDDM Patients N = 30 (group-1) N=30 (group-2) Mean(Deflection) 35 29 S.D 0.7 1.4 SEM 0.128 0.256 *p< 0.0001

100 90 80

Deflection

70 60 50 40 0.1 0.5 Insulin Concentration (U) 1

Figure 3 Biochemical Basis of Insulin Action

Figure 4 Standard Curve of Insulin with Photofluorometer

chemicals, Fluka-Germany and Himedia. The 3 ml sized glass cells (i.e. for reaction mixture) were fabricated from Central Scientific Instruments Organization, Chandigarh.

2.2. Methodology

2.2.1. Fabrication of Screen Printed Electrode

normal individual 2) by immobilization with IDDM patient. The results were calculated by formulae: Concentration of unknown sample UB = ------------------X Concentration of standard SB U Deflection of unknown sample S Deflection of standard concentration B Blank Table 1 shows the comparative analysis of effect of insulin as facilitator exploiting the two types of red blood cells i.e. in group -1, the SPE were immobilized with RBC of normal healthy individuals and in group-2, SPE were immobilized with RBC of IDDM patients. The deflection in terms of insulin in group-2 was less as compared to group-1 with significant p value <0.0001.This signifies the role of insulin as glucose transporter.

The red blood cells of normal healthy individuals and IDDM patients were used for immobilization on screen-printed base electrodes. This immobilization has been done with the covalent cross linker, glutaraldehyde. Further for good fixation of biocomponent, silanization with 10% ATS (aminotriethoxysilane) has been done (Kaur and Verma, 2012).

2.2.2. Experimental Part

The experiments were run with the blank, standard and unknown samples. Optimization of various parameters for production of NADPH was studied which included temperature, reaction time, glucose concentration, NADP+ and RBC (for immobilization) (Kaur and Verma 2013).

3. RESULT AND DISCUSSION

3.1. Bioassay Principle
The bioassay principle in electrochemical study is the oxidation of NADPH. The developed electrochemical insulin biosensor inherent red blood cells which contain enzymes hexokinase, glucose-6- phosphate dehydrodegenase, ATP and insulin receptor. This enzymes machinery is used for insulin detection. The insulin facilitates the transport of glucose across rbc membrane by glucose permease transporter (Murray, 2009) which is converted to glucose-6phosphate by hexokinase and further to 6phosphogluconate by glucose-6phosphate dehydrodegenase with the concomitant generation of NADPH. The formation of NADPH was monitored by Photofluorometer by excitation at 360 nm and emission at 470 nm. The fluorescence of NADPH occurred and deflection was observed which was used to monitor insulin level.

2.2.3. Instrument Measuring Procedure

The transducer used in this study was electrochemical biosensor photoflourometer-systronic-151. The response of NADPH was monitored by the oxidation of NADPH. The chemical experiment was conducted using a fabricated SPE in the special fabricated cell of 3ml volume. The SPE inherited both the electrodes (working and reference) was then applied in the cell. The 2 ml reaction mixture was prepared and after optimum time the response i.e 25 mins was observed. The formation of NADPH was monitored by Photofluorometer by excitation at 360 nm and emission at 470 nm. The fluorescence of NADPH occurred and deflection was observed which was used to monitor insulin level.

2.2.4. Insulin standard curve

2.2.5. Calculations

To check the mass transfer efficiency and effect of immobilization on response time, the immobilization on SPE by two types of RBC was done. 1) by immobilization with
Kaur et al. Role of insulin as facilitating transporter for glucose in miniaturized screen printed electrode, Discovery Proteins, 2013, 1(2), 30-33, www.discovery.org.in/dr.htm

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The experiments observed in the study, significantly confirms the role of insulin as transporter across the cell membrane. In IDDM patients, due to deficiency of insulin, the overall rate of glucose transporters is low. This transport

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Insulin standard curve ranging from (0.6-6.0) nM was constructed as shown in Figure 4. This obeyed the bioassay principle.

4. CONCLUSION

Discovery Proteins RESEARCH INSULIN

is enhanced by presence of more insulin. So, insulin plays important role as glucose transporter.

SUMMARY OF RESEARCH

1. This study signifies the role of insulin as facilitating transporter for glucose. 2. The effect on insulin was seen by immobilizing RBC cells of normal individuals and IDDM patients and deflection in terms of insulin was low as compared to normal individuals.

FUTURE ISSUES

From the findings, the further research can be on identification of insulin glucose transporter factors.

DISCLOSURE STATEMENT ACKNOWLEDGEMENTS

The work was carried out in the Department of Biotechnology,Punjabi University,Patiala, with their full support for productive research work. The authors are grateful to the Head, Department of Biotechnology, Punjabi University Patiala.

REFERENCES

1. 2.

3. 4.

Chang R, Physical Chemistry for Biosciences, William College, U.S.A, 2005 Guyton AC, Hall JE. Insulin, glucagon, Diabetes mellitus. In th Textbook of Medical Physiology , 11 ed, W.B Saunders (eds.). A Harcourt publishers International Co. pp. 885-897, 2010 Jones RL. Insulin resistance, diet and cardiovascular disease: a review. Food, Agr. Environ, 2003, 1, 26-29 Kaur A, Verma N. Electrochemical Biosensor for Monitoring Insulin in Normal Individuals and Diabetic Mellitus Patients.

5. 6. 7.

European Journal of Experimental Biology, 2012, 2(2), 389395 Kaur A, Verma N. Miniaturized-Screen printed electrode of Electrochemical Biosensor for Monitoring Insulin in Diabetic Patients. Discovery Science, 2013, 4(10), 9-14 Murray K. Harpers Illustrated Biochemistry. McGrawHill, Inc. New York, 2009 Talwar GP, Srivastva LM, Moudgil KD: Textbook of Biochemistry and Human Biology G.P. Talwar (Eds.), Prentice Hall India, New Delhi. 2008

Kaur et al. Role of insulin as facilitating transporter for glucose in miniaturized screen printed electrode, Discovery Proteins, 2013, 1(2), 30-33, www.discovery.org.in/dr.htm

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