Extract of Torbangun ( Coleus amboinicus Lour) Leaves Inhibits the Expression of PEPCK and G6Pase in Streptozotocin-Induced Diabetic Rats

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INTRODUCTION
The liver plays an important role in blood glucose homeostasis by maintaining a balance between glucose absorption and storage through glycogenesis and glucose release through glycogenolysis and gluconeogenesis.The level of gluconeogenesis is controlled primarily by the activity of several key enzymes, including phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase). 1PCK is an enzyme that controls the rate of hepatic gluconeogenesis and plays an important role as a centre for regulating glucose hemostasis.PEPCK is the first step in gluconeogenesis in the liver. 1,2Besides PEPCK, there are also G6Pase enzymes in the liver which are found in the endoplasmic reticulum.This enzyme plays a role in the formation of free glucose from glucose-6phosphate (G6P) in the final step of gluconeogenesis and glycogenolysis. 3e rate of transcription of the PEPCK gene is enhanced by glucagon (via cAMP) and glucocorticoids and inhibited by insulin.In addition, under certain conditions, glucose is also able to increase the expression of PEPCK genes. 4,5upplement that usually PEPCK expression is induced by glucagon, catecholamines and glucocorticoids during the fasting period and response to stress, but are predominantly inhibited by increases in glucose induced insulin secretion in food. 2 Meanwhile, the catalytic subunit of G6Pase is coded by one of three genes, namely G6PC1, G6PC2 and G6PC3. 6At certain conditions, the expression of the G6PC1 gene is directly regulated by insulin and glucagon. 7ronic hyperglycemia will affect the gene expression of regulating hepatic glucose output and gluconeogenesis.This is explained by Shao et  al (2005) that sustained hyperglycemia can reduce the effects of glucose and insulin inhibition on PEPCK gene expression and increase the expression of the PEPCK gene hormone stimulant, as well as liver glucose production. 1 Furthermore, G6Pase gene expression is enhanced by high glucose concentrations.This shows that hyperglycemia can increase G6Pase activity. 8ntinuous hyperglycemia causes an increase in free radical production called ROS (Reactive Oxygen Species).Increased ROS is associated with the development of diabetes mellitus (DM) because it can cause a decrease in enzymatic antioxidant activity, causing tissue susceptibility to oxidative stress. 9e way to control DM is through nutraceutical consumption or functional food.There have been more than 1,050 anti-DM plants that have been studied. 10[18] Several studies have reported the ability of Torbangun leaves has been used in DM therapy.A study conducted by Viswanathaswamy et al reported that ethanol extract of Torbangun leaves improved pancreatic function and insulinotropic effect. 18thanol extract of Torbangun leaves contains flavonoids as bioactive compounds at a dose of 620 mg/kg BW for 14 consecutive days significantly reduced blood glucose and free radical levels, increased the glucokinase enzyme, and protected cells pancreatic-β cells from STZinduced rats. 16One type of flavonoids, namely quercetin, was able to stimulate glucose uptake in an insulin-dependent mechanism involving AMPK (protein kinase activated by adenosine monophosphate).In the liver, AMPK reduces glucose production through the reduction of key enzymes of gluconeogenesis, PEPCK, and G6Pase. 19 far, there is no information about the effect of Torbangun leaves extract (TLE) on glucose homeostasis key genes expression in the liver, namely PEPCK and GP6ase.Therefore, it is necessary to do a study to explain the mechanism of TLE in influencing the expression of these genes.The results of this study will make a fundamental contribution to find molecular theory at the genetic level about the mechanism of TLE as a potential anti-DM agent and will provide stronger scientific evidence about the application of complementary therapies for DM patients.Therefore, the purpose of this study is to analyze the antihyperglycemic activity of TLE in hyperglycemic rats by observing the effect of TLE on blood glucose levels and PEPCK and G6Pase genes expression.

Main materials
Torbangun samples have been authenticated by the Botanical Gardens Conservation Center, Indonesian Institute of Sciences, Bogor, West Java, Indonesia (No B-2096/IPH.3/KS/VII/2017.The Torbangun seeds were planted in a total area of ±160 m 2 with a spacing distance of 40 cm 20 in Cibeureum, Bogor City, West Java, Indonesia, at an altitude of 298 asl.Torbangun leaves are harvested at the age of 8 weeks, taken about 15 cm from the top of the plant. 21e experimental animals used were 25 male Sprague-Dawley rats aged 8 weeks, with the minimum body weight of 140 g (148-192 g) from PT. Indoanilab Bogor, Indonesia.Standard rat feed was obtained from PT Indonesia Formula Feed.
STZ from Sigma-Aldrich has been used a single dose of 40mg/kg BW through intraperitonial. 22DM drugs for the control group are metformin hydrochloride dose 62.5 mg/kg BW 23 from Dexa Medica, Indonesia.The anesthetics used are ketamine and xylazine. 24Other laboratory materials are 96% ethanol, insulin level analysis kits from the Bioassay Technology Laboratory, China Qiagen Rneasy Insulation Mini Kit, and ReverTra Ace qPCR RT Master Mix with gDNA Toyobo Remover.

Extraction
Fresh Torbangun leaves was washed, dried at 40 o C for three days using a fresh dryer and mashed with a grinder then sifted to a size of 60 mesh. 16Torbangun leaves powder was extracted following the steps of Suryowati which were modified by adding a sonicator tool. 16e extract was made by mixing 25 grams of simplicia powder with Torbangun leaves as much as 25 grams into 250 ml ethanol 96% and then sonicated with Powersonic sonicator 505, 40 kHz for 40 minutes at room temperature (25 o C).The extract was filtered with Whatman filter paper. 25Sonication was repeated twice with the addition of 100 ml ethanol each repetition.Solvent evaporation in extracts was carried out with a rotary evaporator temperature of 60 o C until semi-solid liquid was achieved. 18The extract obtained was stored in an air tight container. 16

Animal intervention
Twenty-five rats were kept in cages, separated individually by standard feeding.Seven rats were separated as normal (N) and the remaining rats were injected with STZ with a single dose of 40 mg/kg BW. 22 Verification of the hyperglycemia is characterized by a fasting blood glucose (FBG) above 126 mg/dL. 26,27 ll rats that received STZ induction experienced hyperglycemic on the third day after injection.Rats confirmed hyperglycemia were divided into groups: NG (control, hyperglycemic rats, 6 rats), H-IM (control of metformin drugs 62.5 mg/kg BW, 6 rats), and H-IT (TLE 620 mg/kg BW, 6 rats).The dose of Torbangun leaves extract is based on Suryowati et al. 16 The administration of TLE and metformin were carried out with the sonde on the same schedule every day for 14 days.TLE was dissolved with NaCMC 0.3% w/v and metformin was dissolved with distilled water to facilitate administration through a sonde. 16All treatments received a standard ration containing 40% protein, 4% fat, 5% crude fiber, 8% ash, 1% Ca mineral, 0.8% mineral P, with 12% moisture content.FBG was taken on days 0, 4 th , 7 th , 11 th and 14 th , measured using glucometers.The rat's blood is taken from the tip of their tail and dropped on a glucometer strip.
At day 15 after the intervention, all rats were treated with a mixture of ketamine: 90 mg and xylazine: 10 mg dissolved in distilled water.Necropsy is done by cutting the skin and abdominal muscles until the abdominal cavity opened.The soleus muscle is stored in later RNA and stored at -20 o C until the sample is ready for analysis.

Gene expression analysis
Liver was collected for gene expression analysis and stored in later Ribonucleid acid (RNA) liquids at -20 o C until samples was ready for analysis.The expression of the gene was carried out by the reverse transcriptase polymerase chain reaction (RT-PCR) technique.The liver was frozen in liquid nitrogen and then stored at -70°C for the determination of the transcription gene.Qiagen Rneasy Mini Isolation Kit was used for RNA purification and Ace qPCR Rever-Tra RT Master Mix with Toyobo's gDNA Remover for reverse transcriptase.The tool used is Analytic Jena's q-TOWER with Thunderbird qPCR Green Sybr Master Mix Toyobo.][30] Primers design used National Center for Biotechnology Information (NCBI) Pubmed software blast primer.RT-PCR was carried out under the following conditions: 40 cycles for 1 minute at 95°C for predenaturation, 15 seconds at 95°C for denaturation, and 1 minute at 60°C for the annealing process.The primers of each gene are presented in Table 1.
Gene expression was calculated based on the approach of the relative number of mRNAs from the target gene with gene control (18SRNA) using the Comparative Ct method (ΔCt), expression between target genes and control genes can be compared with 2-ΔCt equations, with ΔCt = Ct-gen Ct control target gene (housekeeping genes). 31

Statistical analysis
Data was recorded and initial processing was performed using MSExcel software.Differences in blood glucose levels before and after the intervention were analyzed by Paired T-Test Samples.Differences in insulin levels were analyzed by variance (ANOVA) at a 99% confidence interval to see whether there was an effect of treatment, and gene expression was analyzed descriptively.

Ethical considerations
The research protocol implemented has been approved by Ethical Treatment No. 77-2017 from the Animal Ethics Commission, Institute for Research and Community Service, IPB University, Indonesia.

RESULT
Table 2 gives the effect of giving TLE to the blood levels of hyperglycemic rats.A higher FBG level in the STZ-induced group at the start of the comparison showed that the object of the study was increased hyperglycemia as one of the symptoms of DM.The FBG level of the group of rats that were intervened with TLE and the metformin drug control group showed a decline towards normal conditions at the end of the study.Meanwhile, the NG group still hyperglycemia, FBG > 126 mg/dl).The results of the statistical analysis revealed a significant difference between initial and final FBG on the HI-IM group (p = 0.002, α = 0.01) and the H-IT group (p = 0.005, α = 0.01).
This study shows that the administration of TLE in hyperglycemic rats has an effect on the key gene expression of gluconeogenesis in the liver (PEPCK and G6Pase).The group of rats given the TLE intervention (H-IT) showed a decrease in PEPCK gene expression 0.80 times compared to the group of hyperglycemic rats (NG).TLE also decreased G6Pase gene expression by 0.65 times.Although the decrease in value was not as large as the N and H-IM groups, it was seen that the intervention of TLE was able to reduce the expression of PEPCK and G6Pase genes, thereby reducing the rate of gluconeogenesis and reducing glucose levels in the blood (Figure 1).

DISCUSSION
The pathogenesis of DM is characterized by circulation, namely a decrease in peripheral tissue response to insulin. 32Damage to tissues occurs due to an increase in free radicals in the body.Also, the ability of insulin to increase compatibility. 33e results of the statistical analysis revealed a significant difference between the initial and final FBG interventions in the H-IT and H-IM groups.This shows the effect of Torbangun's extract in reducing blood levels in hyperglycemic rats, having the same complexity as metformin.According to, 18 a decrease in blood glucose levels in rats given TLE was carried out through the restoration of pancreatic tissue function and insulinotropic effects.This happens because the antioxidants contained in TLE can trigger free radicals that are formed due to hyperglycemia.Decreased levels of free radicals cause insulin receptors to work properly, thus, carrying can be transported into cells, producing glycogenesis and reducing the rate of gluconeogenesis.TLE contains flavonoids with potential antioxidant effects. 34The results of this study are consistent with Viswanathaswamy et al who reported that TLE can improve the function of pancreatic tissue and insulinotropic effects (production, secretion, and insulin activity). 18e liver plays a major role in blood glucose homeostasis by maintaining a balance between glucose uptake and storage through glycogenesis and glucose release through glycogenolysis and gluconeogenesis.Glucose homeostasis in the body is highly dependent on genes that regulate the formation of enzymes that play a role in glycogenolysis and gluconeogenesis.The key liver genes that govern the process are PEPCK and G6Pase. 36The bioactive component in plants can influence insulin signaling, which can change the expression of the key gene for glucose homeostasis. 37ronic hyperglycemia leading to DM reduces the ability of glucose and insulin to inhibit PEPCK gene expression.9][40][41][42][43][44][45] Metformin plays a role in increasing the secretion of endorphins from the adrenal gland to stimulate receptor-opioid relationships, causing down-regulating of PEPCK gene expression in the liver. 23e process of decreasing PEPCK and G6Pase gene expression in the liver in the H-IT group can be explained by the following two mechanisms.First, it is related to the improvement of pancreatic-β cells, so that the production and effectiveness of insulin improves.The pancreas of DM rats that were treated with the extract of the leaves of Torbangun experienced regeneration changes to normal.
Giving TLE can protect endocrine cells due to STZ induction so that insulin secretion can be increased. 16PEPCK expression up-regulation is influenced by insulin. 39Insulin and glucagon regulate protein expression and activity to maintain blood glucose levels.The main target is PEPCK, an enzyme that is the first catalyst in the process of gluconeogenesis.The regulation of the PEPCK gene is achieved by modulating the transcription of tightly regulated genes by cAMP (glucagon and catecholamine mediators), glucocorticoids, and insulin. 2 Second, there is a stimulation of the flavonoids contained in TLE against AMPK (adenosine monophosphate-activated protein kinase).As stated by Eid et al that AMPK decreases glucose production primarily through the regulation of key enzymes of gluconeogenesis. 19uryowati suspects that the antioxidant quercetin contained in the leaves extract of Torbangun is one of the antioxidants that play a role in stimulating glucose uptake involving AMPK. 16

CONCLUSION
The findings suggested the administration of TLE in STZ-induced hyperglycemic rats was able to reduce blood sugar levels and was involved in inhibiting the expression of key genes of gluconeogenesis (PEPCK and G6Pase) in the liver.

FUNDING
This study has been funded by Ministry of Health, Republic of Indonesia.

Table 2 : Rats' FBG levels before and after the intervention.
Andrestian MD, et al.Extract of Torbangun (Coleus amboinicus Lour) Leaves Inhibits the Expression of PEPCK and G6Pase in Streptozotocin-Induced Diabetic Rats