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*Correspondence Author:
I Nyoman Suarsana, Laboratory of Biochemistry, Faculty of Veterinary Medicine, Udayana University, Bali-Indonesia
[email protected]; [email protected]
Received: 2017-07-03 Accepted: 2017-07-15 Published: 2017-07-17
ABSTRACT
Background: Under normal physiological conditions, the glucose uptake into the cell is regulated by glucose transporter (GLUT4) which is stimulated by insulin to be afterward metabolized into energy and/ or stored as glycogen. The research aimed to determine the expression of glucose transporter (GLUT4), glycogen levels on cells liver and muscle tissues in hyperglycemic and normoglycemic conditions on rats.
Method: A total of 20 rats of Spraque Dawley strain were grouped into two treatment groups. The normal group and the hyperglycemia group. The observations were performed on blood glucose level by glucose oxidase biosensor method by using the Blood Glucose Test Meter of GlucoDr™, liver and muscle glycogen levels by spectrophotometry and GLUT4 analysis on cells of liver and muscles tissues by using the immunohistochemical technique.
Results: The results showed that 80% of sucrose administration had caused hyperglycemic rats with the blood glucose levels of
131.2 g/dl. In hyperglycemic conditions, the levels of liver glycogen (9.4 µg/mg) and muscle (8.68 µg/ mg) were significantly higher (P <0.05) than in the liver (8.54 µg / mg) and muscle (7.87 µg / mg) in normoglycemic. Meanwhile, the liver glycogen levels were significantly higher (P <0.05) when compared with the muscle glycogen levels in both hyperglycemic and normoglycemic conditions. The expression of GLUT4 in hyperglycemia was significantly higher (P <0.05) in both liver (64.05%) and muscle (56.15%) when compared with the liver (53.21%) and the muscle (48.67% ) in normoglycemic. Between the liver and muscle tissues, the expression of GLUT4 was significantly higher (P <0.05) in the liver when compared with muscle tissue in both hyperglycemic and normoglycemic conditions.
Conclusion: Expression of GLUT4 and levels of glycogen in the hyperglycemic liver is higher compared to the muscles.
Keywords: glucose, glycogen, hyperglycemia, GLUT4
Cite This Article: Suarsana, I.N., Utama, I.H., Arjentinia, I.P.G.Y., Kardena, I.M., Adi, A.A.A.M. 2017. The differential expression of GLUT4 and glycogen levels on cells of liver and muscle tissues in hyperglycemic and normoglycemic conditions. Bali Medical Journal 3(3): S84-S89. DOI:10.15562/bmj.v3i3.728
INTRODUCTION
Carbohydrates play a fundamental role in life, not only as the primary source of energy for living things but also as compounds that store chemical energy. The glucose uptake into the cell is regulated by a transporter and distributed to various tissues in the body. The distribution process involves a family of transport proteins known as GLUT. Glucose transporter (GLUT) works as a carrier to move glucose through the cell membrane.1 Glucose
that enters the cell undergoes a series of metabolic processes converted into energy or stored as energy reserves in the form of glycogen.2
Glucose is a very important universal biomol-ecule as a biological fuel for mammalian cells. Correspondingly, every cell in the body expresses at least one type of glucose transporter.3 According to
Thorens and Mueckler,4 there is 14 GLUT proteins
work as carriers. However, the ones that have been known and well known as the glucose transport-ers are the forms of GLUT 1 to GLUT 4. Glucose
transporters of 1 to 4 have been known for differ-ent regulatory and kinetic properties in glucose homeostasis in the body.
Based on biochemical properties, GLUT 1-4 is distributed in various tissues.5 GLUT 1 to GLUT 4
are the glucose transporters for glucose. GLUT 1 is spread in the brain and erythrocytes, GLUT 2 is located in the liver cell membranes, pancreas, intes-tine and kidneys, GLUT 3 is located in the brain, and GLUT 4 is a common transporter of glucose in the skeletal muscle tissue, brain, heart, and adipose tissue.
GLUT 4 is a specific protein that facilitates glucose transport. GLUT 4 is a glucose transporter that is responsive to insulin in muscle and adipose tissue in both humans and rodents. GLUT4 trans-locates to the plasma membrane in response to insulin. GLUT4 is known as a major glucose trans-porter and can regulate insulin-stimulated glucose secreted by beta cells as a glucose sensor.6
1Laboratory of Biochemistry,
Faculty of Veterinary Medicine, Udayana University, Jln. P.B. Sudirman Denpasar, Bali, Indonesia 80233
2Laboratory of Internal Disease
Veteriner, Faculty of Veterinary Medicine, Udayana University, Jln. P.B. Sudirman Denpasar, Bali, Indonesia 80233
3Laboratory of Phatology
Veteriner, Faculty of Veterinary Medicine, Udayana University, Jln. P.B. Sudirman Denpasar, Bali, Indonesia 80233
Volume No.: 3
Issue: 3
First page No.: S84
P-ISSN.2089-1180
E-ISSN.2302-2914
Doi: http://dx.doi.org/10.15562/bmj.v3i3.728
IBL Conference 2017 - Proceedings
The differential expression of GLUT4 and glycogen
levels on cells of liver and muscle tissues in
hyperglycemic and normoglycemic conditions
GLUT 4 is located at the Golgi apparatus and serves for uptake of glucose-stimulated insulin or insulin sensitive. Meanwhile, GLUT1 and GLUT3 in normal physiological conditions have been asso-ciated with the plasma membrane so that the mech-anism of action of glucose uptake is not dependent on insulin.5
According to Watson and Pessin,7 there are five
forms of the glucose transporter, namely GLUT1 to GLUT5. GLUT1 to GLUT4 serves as the glucose transporters whereas GLUT5 is a fructose trans-porter. GLUT1 to GLUT5 are spread across multi-ple tissues. In muscle cells and adipose tissues, insulin stimulates the delivery of GLUT4 glucose transporter from the intracellular site to the cell surface, where GLUT4 facilitates plasma glucose uptake into cells.1 The primary function of insulin
is to stimulate the transport of glucose into cells, especially muscle and adipose cells which are then used as energy sources or stored in glycogen.
This study aims to determine the levels of glucose, glycogen levels of liver and muscle as well as profiles of glucose transporter (GLUT4) in vari-ous organs (brain, heart, muscle, liver, kidney, and pancreas) of the hyperglycemic rats.
METHOD
Preparation of experimental animals
A total of 20 rats of Spraque Dawley strain with body weight between 200-225 g were used in this study. The rats were divided into two treatment groups with each group consisted of 10 rats, i.e. the group of normal rats and the group of hyperglyce-mic rats. Before treatments, the experimental rats were adapted to laboratory conditions for seven days with commercial ration and drinking water ad libitum. Treatment of hyperglycemia was by the administration of 80% sucrose solution (b/v) 2 cc orally twice daily for three weeks. Blood glucose levels were analyzed at the beginning and at the end of treatment. At the end of the study, all rats were sacrificed by anesthesia with ketamine-HCl. Rats were dissected, then the gastrocnemius muscles and liver tissue were taken for analysis of GLUT4 and glycogen levels.
Analysis of blood glucose levels
Measurement of blood glucose levels on day 0 (initial data base) and at the end of treatment was carried out. Blood glucose levels were determined by the glucose oxidase biosensor method, using the Blood glucose Test Meter GlucoDr™ of AGM-2100 Model (manufactured by Allmedicus Co Ltd., Korea). Blood was taken from the tip of the tails of the rats that had previously been cleaned with 70% alcohol, then rubbed slowly before the tail tip
was punctured with a small needle.8 The blood that
came out was then placed on a glucometer strip. Blood glucose levels will be read on GlucoDr™ screen after 11 seconds, and blood glucose levels are expressed in mg/dl.
Analysis of glycogen levels of liver and muscle
Glycogen analysis was conducted by using the method according to Peungvicha et al.9 Each 5 g
of liver and muscle (M. gastrocnemius) were taken and then dried in an oven at 50°C for one night, then crushed into flour. Each sample was taken 25 mg and extracted with one mL of 30% KOH solution and incubated in a boiling water bath for 20 minutes, then cooled. It was added 1.5 ml of cold ethanol 95% to the sample tubes and stored at 4°C for 30 minutes. To separate the glycogen depos-its, the sample was centrifuged at 2500 rpm for 20 minutes. The sample deposition was diluted with 1 ml of aquadest. Into the filled tubes of each 100 μl muscle sample and 100 μl liver sample was added 3 ml of 0.2% (w/v) anthrone-sulfuric acid. The green color that appears indicates that the sample of solu-tion contained glycogen. The absorbance value was measured at a wavelength of 620 nm. The glycogen levels were calculated by comparing with standard glycogen levels.
Immunohistochemical analysis on glucose transporter 4 (GLUT4)
Glucose transporter GLUT4 was immunohisto-chemically analyzed by using methods according to Beesley.10 The tissue was fixed for 24 hours in 10%
formalin solution and then processed by a standard method using paraffin. The tissue was incubated with H2O2 in methanol for 15 minutes. Further, it was dropped with a 10% bovine serum albumin (BSA) for 45 minutes at 37° C. After washing; the tissue was spilled with antibody primary monoclo-nal anti-GLUT 4 and left at room temperature for 1 hour. Furthermore, the tissue was dropped with biotinylated IgG for 30 minutes at room tempera-ture. The next stage was spilled with avidin biotin HRP for 30 minutes. The antigen-antibody reaction results were visualized by using diamino benzidine (DAB) at room temperature for 5 minutes, then counterstained with HE. In each new treatment, the tissue was washed with PBS. The observa-tions were performed under a light microscope of 20 time-magnifications. The results were positively showed GLUT4 if the tissue indicated brown color.
Calculating GLUT4 expression
depends on the concentration of GLUT4. How to calculate GLUT4 expression on cells using H-score IHC. The IHC score was calculated using a modi-fication of cancer cell count according to Cohen.11
The intensity of the color expression is calculated in five different fields of view with 400X magnification. Color intensity with score: 1+: weak; 2+: medium; and 3+: strong. The number of cell expression in percentage (%) is calculated by the formula: [(1 x %cell 1+) + (2 x %cell 2+) + (3 x %cell 3+)]/3.
Experiment design and data analysis This study used unpaired design (independent) with the treatment I was a normal rat and treatment II of the hyperglycemic rat. The data of glycogen levels and GLUT4 expression obtained were analyzed by using unpaired t-test.
RESULTS
Analysis of blood glucose levels
The findings of blood glucose level analysis in rats for three weeks of treatment are shown in Figure 1.
The average blood glucose level in the normal group on day 0 was 93.6 mg/dl and on the 21st day was 95.1 mg/dl. In the treatment of hypergly-cemia, the average initial glucose level (day 0) was 91.5 mg/dl and on the 21st day after the treatment, the average of blood glucose level was 131.2 mg/dl. The initial average of blood glucose level (day 0) in both the normal group and the hyperglycemic
group was not significantly different (P> 0.05), in other words, it was still within the normal range.
Analysis of glycogen levels
The findings of glycogen level analysis on the liver and the muscle of rats during the 21 days of treat-ment are presented in Table 1.
The average levels of liver glycogen of hypergly-cemia group reached 9.40 µg/mg, was significantly higher (P<0.05) compared with the average of the normal group glycogen levels of 8.54 µg/mg. The glycogen levels in muscle tissues in hyperglycemia group amounted to 8.68 µg/mg was significantly higher (P<0.05) compared with the average of the control group glycogen levels of 7.87 µg/mg. The levels of liver glycogen in both normoglycemic and hyperglycemic states were significantly higher (P<0.05) compared with muscle (Table 1, Figure 2 and Figure 3)
The glycogen levels between the liver and the muscle tissues also show different levels. Levels of liver glycogen in the normal group were signifi-cantly higher (P <0.05) when compared with glycogen levels of muscle tissues. The same thing happened in the treatment group of hyperglycemia. Levels of liver glycogen in the treatment of hyper-glycemia were significantly higher (P <0.05) when compared with glycogen levels of muscle tissues.
The results of immunohistochemical staining of GLUT4 in liver and muscle
The glucose transporter is a family of proteins embedded in cell membranes that serve to mediate the absorption of glucose from the surrounding environment into cells. The results of immuno-histochemical staining and percentage of GLUT4 expression in liver and muscle in hyperglycemic and normoglycemic conditions are presented in Table 2 and Figure 4.
Immunohistochemical staining results show that the intensity of GLUT4 expression is stronger in hyperglycemic liver and muscles compared to the normoglycemic. Qualitatively, the expression of GLUT4 in hyperglycemic conditions is indicated by the strong brown intensity in the liver and muscle cells (Figure 4).
Based on quantitative results, the average GLUT4 expression in hyperglycemic liver was significantly higher (P <0.05) than with normoglycemic liver. Similarly, the expression of GLUT4 in hypergly-cemic muscles are also significantly higher than that of normoglycemic muscles. GLUT4 expres-sion in the liver was significantly higher (P <0.05) compared to the muscles in both hyperglycemic and normoglycemic conditions (Table 2).
The results immunohistochemical staining of GLUT4 expression on cells of liver and muscles Figure 1 Profile of blood glucose levels of rats during 21 days of treatment
tissues in normoglycemic and hyperglycemic conditions are presented in Figure 4.
GLUT4 is expressed and spread to the liver, brain, muscle, heart, kidneys, and pancreas with varying intensity in each organ. Expressions with strong intensity are seen in the organs of the heart, brain, muscles. Then on the liver and kidneys. Descriptively, GLUT 4 expression in hyperglycemic conditions showed stronger intensity in all organs or tissues compared to the normal conditions (Figure 4).
In the liver, GLUT4 is expressed in the cell nucleus, cytoplasm, and plasma membrane. On the brain, it is seen in the Purkinje layer, the granular cell layer, the pyramidal cell layer, and the myelin fibers. In the heart muscle, it is spread on the cyto-plasm and nucleus. On the muscle, it is seen in the cytoplasm and nucleus of the cell. In the kidneys,
it is spread on glomerular cells, and on the distal tubules. In Pancreas, it is expressed in endocrine cells and on the islet of Langerhans.
DISCUSSION
Some researchers reported that the normal blood glucose levels of rats are 74.35 to 84.85 mg/dl,12 and
between 99 to 127 mg/dl.13 Blood glucose levels of
normal rats vary widely depending on the physiolog-ical conditions, sex, feed, and metabolic processes. The group of hyperglycemic treatment until day 21 showed significantly higher glucose levels (P <0.05) than on day 0, and this indicated that experimental rats had experienced hyperglycemia.
Glucose concentrations in the blood will stimu-late the secretion of the insulin hormone to induce glucose transport into the cell. Liver and muscle cells play an important role as a buffering of post-prandial hyperglycemia by involving mechanisms of energy synthesis and converted into energy stores in the form of glycogen.
The ability to take up glucose at the cellular level are traits possessed by most organisms. Most mammalian cells enter glucose through a facili-tated diffusion process that is mediated by GLUT members (glucose transporter) which are a group of membrane transport proteins. Physiologically, there are 14 GLUTs, but half of them are well known. Glucose transporter 4 (GLUT 4) is the primary insulin-responsive glucose transporter in skeletal muscle and adipose tissue in both humans and rodents.6
Table 1 Results of the analysis of glycogen levels in liver and muscle tissues in normoglycemic and hyperglycemic conditions
Treatments
Glycogen levels (µg/mg)
Liver Muscle
Normal group 8.54 + 0.57aA 7.87 + 0.59aB
Hyperglycemia group 9.40 + 0.43bA 8.68 + 0.66bB
Description: The numbers followed by different (lower) letters towards the columns show significantly different (P <0.05), and the numbers followed by different (upper) letters to the rows indicate significantly different (P <0.05)
Table 2 Findings of the analysis percentage of GLUT4 expression on liver and muscle tissues in normoglycemic and hyperglycemic conditions
Treatments
GLUT4 expression (% cells)
Liver Muscle
Normal group 53.21 + 3.16aA 48.67 + 2.91aB
Hyperglycemia group 64.05 + 4.16bA 56.15 + 3.84bB
Description: The numbers followed by different (lower) letters towards the columns show significantly different (P <0.05), and the numbers followed by different (upper) letters to the rows indicate significantly different (P <0.05)
Figure 3 Glycogen levels of rats liver during 21 days of treatment
Figure 4 Photo micrograph immunohistochemical staining GLUT4 on liver tissues (L), and
muscles (M) in normoglycemic and
Glucose can regulate gene transcription, enzyme activity, hormone secretion, and activity of
gluco-regulatory neurons,4 and possibly can regulate
GLUT 4 expression. Oleszczak et al.,14 reported that
hyperglycemia and hypoglycemia affect the glucose transport and expression of glucose transporters in B and T lymphocytes in humans. Many agents regulate the transport of glucose in lymphocytes. The research findings on lymphocyte of hyperglyce-mia condition showed GLUT 4 was expressed with higher intensity compared with GLUT 1 and GLUT 3. This study demonstrated that changes in glucose concentration significantly affected the expression of glucose transporter in B and T lymphocytes.
Study by Klip et al. in rats with both IDDM
(insulin dependent diabetes mellitus) and non-insu-lin dependent diabetes mellitus (NIDDM), in which the blood glucose levels are higher, but lower insulin levels, showed the total of GLUT 4 levels and the mRNA levels of GLUT 4 in adipose tissue and the skeletal muscle were decreased.15 This may be due to
insulin induced the GLUT 4 translocation from the intracellular membrane compartment to the surface of the fat cell membrane and the skeletal muscle (skeleton) to incorporate glucose into the cell.16
Vannucci et al.,17 reported that GLUT 4
expres-sion in rat brains of cerebral increased in diabetic conditions, as well as hyperinsulinemia and its expression, is higher in female rats compared with male ones. In addition, Choeiri et al.,18 in his
research of glucose transporters in rat brain immu-nohistochemically reported that GLUT 1, GLUT 3, and GLUT 4 expression were found to be higher than GLUT 2, GLUT 5, and GLUT 8. It is specifi-cally reported that GLUT 4 expression in the cells of Golgi apparatus and cell layers of Granular of the cerebellum. GLUT 1 is expressed in the front of the motor cortex of the brain, while GLUT 3 is expressed in the body of the Purkinje cell near the axon hill. The presence of GLUT4 in the area strongly supports the role in providing the energy needed for motoric activity control.
In basal conditions, GLUT 4 is mostly located in intracellular organelles, and GLUT 1 is primarily in plasma membranes.15 Therefore, in order to move to
the surface of the cell membrane, GLUT 4 requires signals from insulin. According to Kobayashi et al., the expression of some forms of glucose transporter on the surface of the cell plays a role in glucose uptake, signal generation, and metabolism to main-tain the body’s cellular metabolic integrity.6
In all cells, the insulin-responsive GLUT 4 pool is localized to the membrane vesicles, i.e. insulin
responsive vesicles.19,20 GLUT 4 is expressed in
insulin-sensitive tissues, e.g. in the fat tissue, skel-etal muscle, and some neurons, the only glucose transporter responsible for the effects of insulin on
the use of postprandial blood glucose.21 Similarly,
Huang et al.,22 suggests that adipose cells, skeletal
muscle cells, and some neurons respond to insulin stimulation by transferring GLUT4 to the plasma membrane of the set.
The results of this study indicate that the hyper-glycemic conditions of GLUT4 expression are higher than normoglycemic conditions in both liver and muscles. The increase of GLUT4 expres-sion correlates with the increased levels of glyco-gen in both the liver and muscle in hyperglycemic conditions. GLUT4 plays an active role in inserting glucose into cells. Inside the cells, glucose undergoes metabolism converted into energy and also glyco-gen. The resulting glycogen is stored as a reservoir of energy in the cell, both liver cells, and muscle cells.
CONCLUSION
Expression of GLUT4 and levels of glycogen in hyperglycemic liver is higher compared to the muscles
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to the Directorate General of Higher Education of the Ministry of National Education and the Rector of Udayana University who has provided the research funds from the RM Udayana University Fund with the Letter of Research Appointment Agreement Number: 104.12 / UN14.2 / PNL.01.03.00 / 2014, dated March 5, 2014.
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