Possible role of hydrogen sulfide in insulin secretion and in development of insulin resistance.

H(2)S has been proposed as physiological important molecule. It is considered as first endogenous gaseous K(+) channel opener. K(+) ATP channel activity is mainly responsible for insulin secretion. K(+)ATP channel opening of β cells leads to inhibition of insulin secretion and channels closing leads to secretion. H2S is the gaseous K(+) ATP channel opener but it does not have channel specificity. So, H(2)S may have some effect on insulin secretion. H(2)S is high in Zuker diabetic fatty rats. That means H(2)S is high in insulin resistance condition. We tried to find out the role of H(2)S in insulin secretion and in development of insulin resistance. From the result of our study, H(2)S have K(+) ATP channel opening activity on β cells. H(2)S does not have any role in the development of insulin resistance. Decrease in insulin level in Zuker diabetic rat and streptozotocin-induced diabetic rat is due to high H(2)S level.


INTRODUCTION
In recent years, interest has been directed toward other naturally occurring gases, notably H2S. Now, H 2 S has been proposed as physiological important molecule. Recently, high concentration of H 2 S has been observed in brain of rats, humans, and cows. The H 2 S concentration in rat serum has been found to be 46 μM [1] and in brain tissue 50-160 μM. [2] Significant amount of H2S is produced in various tissues. Cystathionine gamma lyase (CSE), cystathionine β synthase (CBS), and 3-mercaptosulfurtransferase are the enzymes responsible for production of H 2 S. [3] The main subtract for these enzymes is L-cysteine. H 2 S is considered as endogenous vasorelaxant factor and cardiovascular function regulator. [4] Zhao showed that H 2 S is the first gaseous K + ATP channel opener. [1] Intravenous injection of That means H 2 S is high in insulin resistance condition. According to Muhammed, experimental diabetes in rats induced by streptozotocin leads to increase expression of the H 2 S producing enzymes. [6] H 2 S may have an inhibitory effect of insulin secretion due to nonspecific K + ATP channel opening activity. On the other side, H 2 S level is high in hyperinsulinemia. So, this study was carried out to investigate the role of H 2 S in insulin secretion and development of insulin resistance.

Animals
Adult albino rats of either sex (Wistar strain) weighing between 200 and 250 g were used for the study. The animals were fed ad libitum with standard pellet diet and had free access to water. All experiments and protocols described in present report were approved by the Institutional Animal Ethics Committee (IAEC).

Experimental protocol
Effect of H 2 S on the K + ATP channel of insulin secreting cell was studied by dividing animals into two groups. Group 1 received NaHS 2 mg/kg i.p. (high rate of mortality on higher dose), and Group 2 received only saline. In the second experiment, animals were divided into two groups. Group 1 received saline only and group 2 received glibenclamide 10 mg/kg, p.o [7] then after NaHS 2 mg/kg i.p. Blood was collected from tail vein at 0, 30, 60, 120 min after administration of drugs up to 5 h and blood glucose and insulin were measured. The blood glucose was measured by the GOD/POD [8] method and insulin was measured by RIA. [9] Protocol to study the role of H 2 S in insulin resistance development The wistar albino rats are divided into two groups (n=6). One group received NaHS 2 mg/kg i.p twice a day for 30 days. Another group received only saline. The development of insulin resistance was studied by performing oral glucose tolerance test (OGTT). Also OGTT was performed after giving insulin by i.m. to measure sensitivity toward exogenous insulin. Insulin and glucose level were also measured before and after 30 days treatment.

Oral Glucose Tolerance Test (OGTT)
After 30 days, 5 g/kg of glucose was administered to fasted animals. Blood samples were collected from tail vein under light anesthesia for 0 min and 15, 30, 60, 90 and 120 min after oral glucose administration. The blood samples were allowed to clot and serum was separated by centrifugation. The samples were measured for glucose level by the GOD/ POD method. [10] Oral Glucose Tolerance Test (OGTT) with Insulin Wistar rats were divided into two groups of six animals. One is treated with NaHS 2 mg/kg for 30 days other is treated with saline. After 30 days animals were orally administered with 5 g/kg of glucose and insulin 1 U/kg i.m. Blood samples were collected from tail vein under light anesthesia before 0 min and 15, 30, 60, 90 and 120 min after oral glucose administration. The samples were allowed to clot and serum was separated by centrifugation. The samples were measured for glucose level by the GOD/ POD method. [10]

Measurement of serum H 2 S level in streptozotocininduced diabetic rat
Type II diabetes was induced in wistar rats by streptozotocine. [11] After induction of diabetes, H 2 S level in serum was measured. Seventy-five microliters of plasma were mixed with 250 µl of 1% w/v zinc acetate and 425 µl distilled water. Then 20 mM N-dimethyl-pphenylenediamine sulfate in 7.2 mM HCl (133 µl) and 30 mM FeCl 3 in 1.2 mM HCl (133 µl) were also added to the test tube for 10-min incubation at room temperature. The protein in the plasma was removed by adding 250 µl of 10% tricholoacetic acid to the reaction mixture and pelleted by centrifugation at 14 000 g (5 min). The absorbance of the resulting solution at 670 nm was measured with a spectrophotometer (TECAN Systems) in a 96-well plate. All samples were assayed in duplicate, and concentration in the solution was calculated against a calibration curve of NaHS (3.125-250 µM). Results show plasma H 2 S concentration in micromolar [12]

Statistical analysis
All the values were expressed as mean+SEM. The statistical analysis was performed using Student's unpaired t-test and by AUC comparison. Value of P less than 5% (P<0.05) was considered statistically significant.

Effect of H 2 S on insulin and glucose level
After administration of H 2 S (2 mg/kg), glucose insulin Role of H 2 S in insulin resistance level were measured. Administration of H 2 S results in an increase in glucose level and a decrease in insulin level which became normal after 5 h [ Table 1]. After administration of glibenclamide 10 mg/kg, p.o H 2 S was given. Insulin and glucose level in animal treated with glibenclamide and H 2 S was not significantly changed compared to salinetreated animals. That means effect of H 2 S was inhibited by glibenclamide [ Table 2].

Effect of H 2 S on insulin resistance
After chronic treatment with H 2 S, oral glucose tolerance test was performed to check the development of insulin resistance. Glucose level was measured after administration of glucose 5 g/kg p.o. There was no significant difference in glucose level compared to normal animals [ Figure 1].
After chronic treatment with H 2 S, oral glucose tolerance test was performed along with administration of insulin 1 U/kg i.m to check the development of insulin sensitivity. Glucose level was measured after administration of glucose 5 g/kg p.o. There was no significant difference in glucose level compared to normal animals [ Figure 2]. Also there was no significant difference in insulin and glucose level before and after chronic administration of H 2 S [ Table 3].

DISCUSSION
The islets of Langerhans contain four main cell types, out of them β cells secrete insulin. The main factor controlling the synthesis and secretion of insulin is the blood glucose concentration. ATP-sensitive K + -channel determines the resting membrane potential in β cells via a membrane transporter called Glut-2 and its subsequent metabolism via glycosidase and glycolysis increases intracellular ATP. This blocks K + ATP causing membrane depolarization and opening of voltage dependant calcium channels, leading to insulin level of PPG-treated ZDF rats were significantly increased in comparison with that of untreated ZDF rats. [15] CONCLUSION From the result of our study, it can be concluded that H 2 S is pancreation K+ ATP channel opener and it inhibit insulin secretion. High H 2 S level in streptozotocin-induced diabetic rat and ZDF rat is responsible for the low level of insulin and it does not have any role in development of insulin resistance. So, H 2 S can be a good target to treat diabetes but further detail investigation of role of H 2 S in insulin resistance is require to study.