Sinapic Acid Regulates Glucose Homeostasis by Modulating the Activities of Carbohydrate Metabolizing Enzymes in High Fat Diet Fed-Low Dose STZ Induced Experimental Type 2 Diabetes in Rats

Diabetes Mellitus is a chronic metabolic disorder arises due to absolute lack of insulin secretion (T1DM) or its action or both (T2DM). Alterations in glucose metabolism in DM are frequently accompanied by impairment in the activities of enzymes that regulate carbohydrate metabolism. Liver is a vital organ that acts as primary site of endogenous glucose production through gluconeogenesis or glycogenolysis. The enzymes that control glucose metabolism in the liver tissue are considered as potential targets for the maintenance of normal glycemic control in diabetic individuals. Search for new drugs with more effi cacies and without side effects preferably from plant origin continues. Sinapic acid is one such phytochemical which lacks scientifi c validation for its folklore use. It is a naturally occurring carboxylic acid belongs to phenylpropanoid family. It is widely distributed in the various sources such as rye, mustard, berries and vegetables In the present study it was aimed to systematically study the effi cacy of sinapic acid (25mg/kg.b.w./rat for 30 days) in the regulation of glucose homeostasis modulating the activities of carbohydrate metabolizing enzymes in hepatic tissues of high fat diet fed-low dose STZ induced experimental type 2 diabetes in rats. The altered activities of carbohydrate metabolizing enzymes such as glucokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6phosphate dehydrogenase, lactate dehydrogenase in hepatic tissues of diabetic rats were signifi cantly reverted to near normalcy upon oral treatment with sinapic acid. In addition, oral administration of sinapic acid to experimental diabetic groups of rats showed signifi cant reduction in the levels of fasting blood glucose and glycosylated hemoglobin and increased level of plasma insulin and hemoglobin. Thus, the present data demonstrated that the oral administration of sinapic acid to diabetic rats regulates glucose homeostasis by regulating the activities of carbohydrate metabolizing enzymes. Research Article Sinapic Acid Regulates Glucose Homeostasis by Modulating the Activities of Carbohydrate Metabolizing Enzymes in High Fat Diet Fed-Low Dose STZ Induced Experimental Type 2 Diabetes in Rats Ramesh Nithya, Vellai Roshana Devi, Rajendran Selvam and Sorimuthu Pillai Subramanian* Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India Dates: Received: 21 July, 2017; Accepted: 21 August, 2017; Published: 22 August, 2017 *Corresponding author: Sorimuthu Pillai Subramanian, Professor, Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 025, Tamilnadu, India, E-mail:


Introduction
Diabetes Mellitus (DM) is a chronic metabolic disorder arises due to absolute lack of insulin secretion (T1DM) from the -cells of pancreas or its action or both (T2DM) [1]. It is characterized by a persistent elevation in both fasting as well as postprandial blood glucose levels. The prevalence of DM is predicted to rise 642 million by the year 2025 and T2DM accounts for more than 90% of the total diabetic population. Several drugs such as biguanides, sulphonylureas, meglitinides, thiazolidiones, -glucosidase inhibitors, bile acid sequestrants, SGLT2 inhibitors, GLP-1 receptor agonists and insulin with different mode of action are currently used either as monotherapy or combinatorial strategy for the treatment of DM [2]. However, these drugs elicit undesirable side effects and develop resistance after prolonged use. Thus, the maintenance of normoglycemia remains a major task in the treatment of patients with diabetes mellitus and hence the search for new drugs with more effi cacies and without side effects preferably from plant origin continues.
Liver is a one of the major storage organ for glucose reserve in the human body and thus plays a pivotal role in the regulation secondary complications of diabetes mellitus and diminution of hepatic glucose production has certainly been considered as a successful strategy for the treatment of diabetes. It has been reported that the chronic hyperglycemia in diabetes decreases the activities of enzymes in glycolytic and pentose pathways while increases the activity of gluconeogenesis and glycogenolytic pathways [3].
Phytochemicals are ecologically derived plant secondary metabolites which protect them against environmental stress such as UV radiation, pollution, high temperature, extreme cold, drought, fl ood, tissue damage and microbial attacks [4]. Interestingly, these secondary metabolites are known to play a pivotal role in alleviating the primary and secondary complications of dreadful human diseases such as cancer, diabetes and atherosclerosis. More than 60% of the currently available allopathic drugs are originally identifi ed from traditional medicinal plants. The medicinal value of plants lies in some chemical substances that elicit a defi nite physiological action on the human body. The WHO recommends phytotherapy in its health programs and suggested basic procedures for scientifi c validation of phytoingredients for their benefi cial as well as pharmacological properties.
Sinapic acid (3, 5-dimethoxy 4-hydroxy cinnamic acid) is one such phytochemical which lacks scientifi c validation for its folklore use. It is a naturally occurring carboxylic acid belongs to phenylpropanoid family. It is widely distributed in the plant kingdom and is obtained from various sources such as mustard, rye, berries and vegetables [5]. Sinapic acid is known to possess antioxidant [6], anti-infl ammatory [7], anxiolytic [8], peroxynitrite scavenging [9], neuroprotective [10] and antihyperglycemic properties [11]. Serum albumin has been reported to be responsible for the transport of sinapic acid in blood due to its ability to bind with serum albumin through hydrophobic interaction and hydrogen-bonding. Maximum plasma-sinapic acid level has been described as 40nM with a bioavailability of 3% of the total phenolics present in the non-processed cereal meal. Moreover, the small intestine was reported as the major site for absorption of orally administered sinapic acid through active Na + gradient-driven transport [12]. Recently, we have reported the antidiabetic and antioxidant properties of sinapic acid in experimental type 2 diabetes in rats [13,14].
Among the various animal models to study the toxicity and pharmacological screening of newly developed therapeutical agents, high fat diet fed-low dose STZ induced experimental type 2 diabetes in rats is widely accepted as it closely resembles the clinical features of human type 2 diabetes in terms of insulin resistance coupled with insuffi cient insulin secretion. Therefore, in the present study it has been employed as an animal model to systematically study the effi cacy of sinapic acid in the regulation of glucose homeostasis.

Chemicals
Sinapic acid and Streptozotocin were purchased from Sigma-aldrich, St.Louis, USA. Ultra-sensitive ELISA kit for rat insulin was purchased from Crystal Chem Inc., Life Technologies, and India. All other reagents used in the present study were of analytical grade.

Experimental animals
Male albino rats of Wistar strains (160-180 g) were procured from Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai. Rats were housed randomly in spacious polypropylene cages lined with husk under controlled environment (12:12 ± 1 h light/dark cycle; temperature 22°C ± 3°C; relative humidity 55% ± 10%). Before the initiation of the experiments, all animals were acclimatized to standard husbandry conditions for one week to overcome stress. Animals were fed with commercial pellet rat chow (Hindustan Lever Ltd., Bangalore, India), and allowed to have free access to water

High Fat Diet fed streptozotocin induced diabetes
The rats were divided into two dietary regimens by feeding either normal or high fat diet (HFD) for the initial period of two weeks [15]. The ingredients and chemical composition of the HFD was followed as before reported. After two weeks of dietary manipulation, the groups of rats fed with HFD were injected intraperitoneally with a low dose of STZ (35 mg/kg b.w) dissolved in 0.1M cold citrate buffer, pH 4.5). One week after STZ injection, the rats were screened for blood glucose level. Rats having fasting blood glucose (FBG) >250mg/dl that exhibited random hyperglycemia and glycosuria were selected for the experiment. The rats were allowed to continue to feed on the respective diets until the end of the experiments.

Experimental Protocol
The rats were divided into four groups each group comprising six rats. Sinapic acid was dissolved in 0.2% dimethyl sulfoxide and administrated to rats orally using an intragastric tube daily for a period of 30 days.

Oral glucose tolerance test
Overnight fasted rats of all groups were subjected to oral glucose tolerance test on the last week of the experimental period. The blood glucose levels were monitored at 0, 30, 60, 90 and 120 min using One Touch glucometer (Life scan, Johnson and Johnson Company) after oral administration of 2 g/kg b.w.

Sample collection
After 30 days of treatment, the animals were fasted overnight and sacrifi ced by cervical decapitation. Blood was collected with and without anticoagulants for the separation of plasma and serum. The liver and kidney were carefully removed, weighed and washed in ice-cold saline. The liver, kidney and muscle tissues were sliced into pieces and homogenized in an appropriate buffer (pH 7.0). The homogenates were centrifuged at 3000 rpm for 10 min at 0 o C in cold centrifuge. The supernatant was separated and used for various biochemical estimations.
Plasma insulin level was assayed using an ELISA kit (Linco and Alkaline phosphatase (ALP) in serum were assayed [24,25].

Assay on insulin resistance
The insulin resistance developed in the experimental animals was evaluated by a homeostasis model of insulin resistance (HOMA-IR). The HOMA-IR was calculated by the method of Mathews et al. [26], as follows.

   
Fasting insulin level U / ml x Fasting blood glucose mg / dl HOMA IR 405   

Statistical analysis
The results were expressed as mean ± SD of six rats per group and statistical signifi cance was evaluated by one-way analysis of variance (ANOVA) using SPSS (version 16) program followed by LSD. Values were considered statistically signifi cant when p < 0.05.

Results and Discussion
Based on the data obtained through toxicity and dosage fi xation studies, the optimum dose for sinapic acid was fi xed as 25mg/kg.b.w./rat/day for 30 days. The effect of oral administration of sinapic acid on oral glucose tolerance test (OGTT) in control and experimental groups of rats is presented as Figure 1. In control group of rats, the blood glucose level attained the maximum peak at 60 min after an oral glucose load and it was progressively reverted back to physiological level at 120 min indicating the maintenance of normal glucose homeostasis. On the other side, the blood glucose levels in type 2 experimental diabetic rats reached the maximum peak at 60 min and remained unsubsidized over the next 60 min.
Oral treatment with sinapic acid as well as metformin resulted in a signifi cant decrease in both fasting 30 as well as 60 min compared with untreated diabetic rats suggesting the effi cacy of sinapic acid in the maintenance of blood glucose homeostasis during oral glucose load which in turn may be due to its insulin stimulatory and/or insulin mimetic properties.
Epidemiological studies and clinical trials strongly support that chronic hyperglycemia is the principal cause of diabetic complications and an effective glucose control is the key factor in preventing the metabolic disorders. The OGTT signifi es the body's ability to use glucose, the main source of energy. It is a test of immense value instead of using fasting blood glucose concentration alone to facilitate the diagnosis of diabetes. This test can also be used to diagnose pre-diabetes and diabetes.
The glucose lowering effect of sinapic acid was comparable with metformin treated group of rats indicating the signifi cant antidiabetic properties of sinapic acid. Table 1 Table 3 depicts the effect of sinapic acid on AST, ALT and ALP in the serum of control and experimental groups of rats.
Signifi cant elevation of these pathological marker enzymes were observed in diabetic rats. Oral administration with sinapic acid restores the elevated levels of these enzymes to physiological range. The aspartate transaminase and alanine transaminase are well-known cytosolic enzymes used as biomarkers to predict possible toxicity to the hepatic tissues [39]. Elevation of these transaminases was observed in experimental diabetic rats suggested damage to the liver cells as well [40]. Alkaline Table 1: Effect of sinapic acid on the levels of blood glucose, hemoglobin, glycosylated hemoglobin (HbA1c) and plasma insulin, in experimental groups of rats after 30 days experimental period. Values are given as mean ± S.D for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical signifi cance was compared within the groups as follows: a control rats; b diabetic control rats; Values are statistically signifi cant at * p < 0.05. Metformin treated diabetic rats also exhibit similar results. Table 6 shows the level of liver glycogen and the activities of glycogen synthase and glycogen phosphorylase in control and experimental groups of rats. Diabetic rats showed considerable decrease in the liver glycogen content and glycogen synthase activity and a concomitant increase in glycogen phosphorylase activity. Upon oral administration of sinapic acid as well as metformin to diabetic rats restored glycogen level in liver and the activities of glycogen synthase, glycogen phosphorylase was also brought back to physiological levels when compared to control rats.
Being an insulin-dependent enzyme, the hepatic hexokinase activity of diabetic rats is almost entirely inhibited or inactivated due to the absence of insulin [45]. This impairment results in a marked reduction in the rate of glucose oxidation via glycolysis, which ultimately leads to hyperglycemia. In diabetic condition, the activity of this enzyme is inhibited or inactivated due to insulin resistance because it is an insulin-dependent enzyme. Reduced insulin level in HFD-low dose STZ induced type 2 diabetic rats leads to reduced activity of hexokinase which ultimately results in the onset of chronic hyperglycemia.
Upon oral treatment with Sinapic acid, the diabetic rats showed signifi cantly increased activity of hexokinase.
Pyruvate kinase (PK) is a ubiquitously expressed, rate controlling, terminal key glycolytic enzyme that catalyzes the irreversible phosphoryl group transfer from phosphoenol Table 2: Effect of sinapic on the levels of total proteins, blood urea, serum uric acid and serum creatinine of control and experimental groups of rats. Values are given as mean ± S.D for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. The results were compared with a control; b Diabetic control.

Groups Total protein (g/dl) Blood urea (mg/dl) Serum uric acid (mg/dl) Serum creatinine (mg/dl)
Values are statistically signifi cant at *p <0.05. pyruvate to ADP, yielding pyruvate and ATP. Its altered activity during diabetic conditions could be expected to diminish the metabolism of glucose and ATP production. Hence, the observed decline in the activity of PK in the liver of experimental diabetic rats is responsible for the reduced glycolysis and amplifi ed gluconeogenesis signifying that these two pathways are distorted in diabetes [46]. The treatment with sinapic acid to diabetic rats showed a notable increase in plasma insulin that induces a decrease in ATP, a known allosteric inhibitor of PK, thereby increases the PK activity to near normalcy.
Lactate dehydrogenase (LDH) is a cytosolic enzyme that catalyzes the conversion of pyruvate to lactate in anaerobic glycolysis, which is subsequently converted to glucose in gluconeogenic fl ux. In diabetic condition, an increased activity of LDH was observed due to the impairment in glucosestimulated insulin secretion [47]. Thus, increased activity of LDH interferes with normal glucose metabolism and insulin secretion in the pancreatic -cells and it may be directly responsible for insulin secretory defects in diabetes. Oral administration of sinapic acid as well as metformin treated diabetic groups of rats showed a signifi cant reduction in the LDH activity, probably due to the regulation of NAD+/NADH ratio by the oxidation of glucose.
Glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose concentration, is expressed mainly in the liver and kidney and is critical in providing glucose to other organs during diabetes, prolonged fasting or starvation [48].
It catalyzes the dephosphorylation of glucose-6-phosphate to free glucose as the terminal step in gluconeogenesis and glycogenolysis. Fructose-1, 6-bisphosphatase is a key regulatory enzyme of the hepatic gluconeogenesis and appeared as a target for effi cient and safe glycemic control in diabetes [49]. Glucose-6-phosphate dehydrogenase (G6PDH), the fi rst and rate limiting enzyme, catalyzes the oxidation of glucose-6-phosphate to 6-phospho gluconate and at the same time reducing NADP+ to generate NADPH in the pentose phosphate pathway (PPP) that supplies reducing energy to the cells. NADPH is essential to renew reduced glutathione and decreased NADPH production may facilitate oxidative stress [50]. Hyperglycemia can lead to decrease the activity of G6PDH.
G6PDH defi ciency could be a risk factor for the pathogenesis of diabetes.
The activities of glucose-6-phosphatase and fructose-1, 6-bisphosphatase were signifi cantly increased in the liver of the diabetic rat possibly because of insulin defi ciency.
Administration of sinapic acid improved the reversal of high glucose-6-phosphatase and fructose-1, 6-bisphosphatase   activities in high fat diet fed-low dose STZ induced experimental type 2 diabetic rats which in turn revealed its role in the regulation of gluconeogenesis. In the present study, the oral administration of sinapic acid considerably increased the activity of glucose-6-phosphate dehydrogenase indicating the regulatory role of sinapic acid in maintaining normoglycemia in experimental diabetes.
Glycogen is a branched polymer of glucose residues which is synthesized by the enzyme glycogen synthase [51].
Glycogen synthase and glycogen phosphorylase are the ratelimiting enzymes in glycogen metabolism. During diabetic conditions, the glycogen levels, glycogen synthase activity and responsiveness to insulin signaling are diminished and glycogen phosphorylase activity is signifi cantly increased.
The observed decrease in glycogen content and altered activities of glycogen synthase and phosphorylase in diabetic rats were signifi cantly improved to expected range upon oral administration of sinapic acid. Thus it may be concluded that the oral administration of sinapic acid signifi cantly modulates the glycogen metabolism in high fat diet fed-low dose STZ induced experimental type 2 diabetes in rats.

Conclusion
The present study has demonstrated that sinapic acid signifi cantly improved the levels of fasting blood glucose and plasma insulin and thereby modulating the activities of carbohydrate metabolizing enzymes in the hepatic tissues of high fat diet fed-low dose STZ induced experimental type 2 diabetes in rats. The data obtained also evidenced that the effi cacy of sinapic acid at a concentration of 25mg/kg.b.w was comparable with metformin which was administered relatively at a higher concentration (50 mg/kg.b.w.). Therefore, sinapic represents a potential target for the development of an alternative medicine in the treatment and management of diabetes and its secondary complications.