Regulation of Intestinal GLP-1 and GLUT2 genes underlie hypoglycemia in Desplatsia subericarpa (Bocq)-Fed Wistar Rats

Desplatsia subericarpa is an understory dense tree growing up to 10 meters tall. The bole is about 4-10 cm in diameter. The glabrous papery leavesare alternate and simple with stipules.Drupe is 6-10cm long and 57.5 cm wide (Burkill, 2000; Keay, 1958). D.Subericarpa has been identified following an ethnopharmacological survey of neglected plants with food and therapeutic uses in Southern Nigeria. Folklorically, the fresh leaves of D. subericarpa are eaten as vegetables while the dried leavesaremade into a decoction useful in managing diabetic conditions yet Journal of Herbal Drug


Introduction
Desplatsia subericarpa is an understory dense tree growing up to 10 meters tall.The bole is about 4-10 cm in diameter.The glabrous papery leavesare alternate and simple with stipules.Drupe is 6-10cm long and 5-without scientific data to validate this claim and explain its mode of action, safety in humans.
Glucagon-like peptide 1 (GLP-1) is a potent incretinhormone produced in the L-cells of thedistal ileum and colon.In the L-cells, tissue-specific posttranslational processing of the proglucagon gene generates GLP-1(Ezcurr et al., 2013;Velasquez et al., 2010), which acts onβ cells GLP-1 receptor, and mediates its glucose responsiveness by promoting insulin exocytosis in GLUT2 (the normally expressed high K m glucose transporter of β cells)-dependent manner (Chen et al., 1990).Indeed, GLP-1/GLP-1R signaling and GLUT2, mediate glucose-stimulated insulin secretion, and their blockage impair glucose sensing with resulting hyperglycemia (MacDonald et al., 2002).TGR5 is a bile acid activated G-protein coupled receptor, which plays key regulatory roles in energy expenditure and diet-induced obesity through intestinal (GLP-1) mechanisms (McKillop et al., 2016;Pols et al., 2010;Wacker et al., 2014).Pharmacological targeting of TGR5 has been identified as a promising incretin-based strategy for managing type 2 diabetes and metabolic disorders such as non-alcoholic fatty liver disease.Therefore, in this study,TGR5/glucagonlike peptide-1 (GLP-1)/insulin pathway 11 along the intestino-hepato-pancreatic axis was investigated in Desplatsia subericarpa Bocq.-fedWistar Rats.Furthermore, we investigated blood glucose regulation in streptozotocin-induced diabetic rats fed Dsubericarpa Bocq-based diet, and finally evaluate nephrotoxicity following sub-chronic exposure.Our results support intestinal GLP-1 up-Regulation and GLUT2 down-regulation in Desplatsia subericarpa Bocq.-fedWistar Rats which may play significant role in its antidiabetic claims.

Plant materials
Desplatsia subericarpa Bocq.leaves were collected in January 2016 from Ugbogiobo village located in Ovia North Local Government Area of Edo State, Nigeria.Leaves were dried in a cool dry place for two weeks and oven died for 30 minutes at 40° C to ensure leaves were crispy.Dried leaves were pulverized using a mechanical mill, sieved and used as feed formulation as described below.A voucher specimen was deposited in the University of Benin Herbarium (UBHm0284).

2.2
Animal studies 6-week-old male inbred Wistar Rats were used with strict compliance with the NIH Guide for the Care and Use of Laboratory Animals.All the protocols were also reviewed and approved by Laboratory Animal Ethical Committee of AdekunleAjasin University, Akungba-Akoko.The Animals (now referred to as subjects) were maintained on a standard rat chow and water ad libitum for 5 days prior to grouping and experimentation.

D. subericarpa-regulated anti-diabetic transcriptional program along intestinal-pancreatichepatic axis
Subjects were housed three (3) per cage under a 12h light/dark condition.Subject randomization and grouping for this study has been presented (fig. 1) for clarity.6-hr prior to sacrifice, food and water were withheld; anesthesia was induced in the subjects using chloroform.The Intestine, pancreas, liver and blood samples (cardiac puncture) were collected for further analysis.

Diabetes study
Diabetes was induced by intraperitoneal injection of single dose of freshly prepared solution of streptozotocin (in 0.1 M sodium citrate buffer, pH 4.5), 45 mg/kg) to overnight fasted subjects.Three days after administration, hyperglycemic subjects were grouped into diabetic control (100% feed/honey) and diabetic treatment groups (10% D. subericarpa leaf powder/90% rat chow mixed in honey), basal group was also fed like diabetic control but without streptozotocin treatment.Blood samples were taken three (3) times over of 9 feeding days for non-fasting blood glucose determination using the glucose oxidase method (Barham & Trinder, 1972).

Blood insulin quantification
Blood samples taken via cardiac puncture were stored in heparinized tubes, centrifuged at 3500 rpm for quantification using insulin ELISA kits (EZRMI-13K) according to the manufacturer's instructions.

2.6
Total RNA isolation, Reverse transcription and PCR amplification RNA samples were extracted from tissues (n=3) using acid guanidiniumthiocyanate-phenol-chloroform extraction method.Genomic DNA contaminant was digested using DNase I (Fermentas, Ontario, Canada) treatment.The reaction mixture contains 2 µL of DNase I, 5 µL of reaction buffer and 23 µL RNA sample.The mixture was incubated at 37°C for 30 minutes.DNase I inactivation was achieved by adding 5 µL EDTA (25 mM) followed by 65°C incubation for 10 minutes 13 .RNA yield and purity were then spectrophotometrically determined.Prior to cDNA conversion, all RNA samples were diluted to 100 ng concentration using nuclease free water.MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (NEB) was used for RNA-to-cDNA conversion according to the manufacturer's instruction.PCR amplification for the determination of genes whose primers (SnapGene software) are listed below was done using the following protocol: PCR amplification was performed in a total of 25 μl volume reaction mixture containing 2 μlcDNA (40 ng), 2 μl primer (100 pmol) 12.5 μl ReadyMix Taq PCR master mix (Sigma Aldrich) and 8.5 μl nuclease-free water.Initial denaturation at 95 °C for 5 minutes was followed by 20 cycles of amplification (denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds and extension at 72 °C for 60 seconds) and ending with final extension at 72 °C for 10 minutes.

Image processing and statistical analysis
In-gel amplicon bands (n=3) were captured using iPhone-5s camera (Noir), image post-processing was done on Keynote platform and band quantification was done using Image-J software.Data presented here expressed as Mean ± Standard Error of Mean (SEM).Statistical analyses were performed using t-test (P < 0.05 vs. basal control) using Graph Pad Prism version.7 (GraphPad Software, Inc. La Jolla, CA, USA) software.

Intestinal GLP1 up-regulation and GLUT2 down-regulation following culinary administration of D. subericarpa
Our understanding of ethnopharmacological uses of D. subericarpa leaves is infantile at this stage and to our knowledge; this study provides the first systematic study at unraveling its antidiabetic activity.First, our choice of administering the whole-leaf in culinary exposure was informed by the consensus that the leaf is traditionally made into vegetable decoction for managing diabetic complications.To mimic this, normal rat chow and the dried leaf powder were mixed together in 90:10 and 70:30 ratios respectively.To aid consumption in animals, 100mL of pure honey was used to formulate the diet.The feed (Fig. 1) was administered ad libitum for 48 hr period.RT-PCR gel electrophoresis of RNA samples isolated from the intestinal crypt of the animals following feeding period showed up-regulated GLP-1 expression and downregulated GLUT2 genes.GLUT2 is the major route for glucose absorption across the brush-border membrane (Kellett & Helliwell, 2000) and its inhibition has been identified as a antidiabetic mechanism in some plants extracts (Nistor Baldea et al., 2010).Uncharacterized components of D. subericarpa leaves interfere with the expression of GLUT2, which appears more robust hypoglycemic mechanism than inhibition or blockage of mobilization to the brush-border.
We propose a plausible mechanism underlying GLUT2 gene downregulation as interference with sterol response element-binding protein (SREBP)-1c, which is known to facilitate glucose-stimulated GLUT2 gene expression (Im et al., 2005).Note that direct inhibition of (SREBP)-1c has been chosen rather than inhibition membrane-bound transcription factor site-1 protease (S1P) (Brown & Goldstein, 1999), which catalyzes its post-translational maturation because direct inhibition of S1P would have resulted in hepatic downregulation of fatty acid synthase and glucose-6phosphate dehydrogenase whose regulation are controlled by (SREBP)-1a (Inoue et al., 2005).Our result show increased expression of hepatic fatty acid synthase and glucose-6-phosphate dehydrogenase (Fig. 3), providing clear evidence against S1P inhibition.
Next, GLP-1 expression pattern was investigated.GLP-1 is an enteroendocrine L-cells hormone produced following accumulating nutrients such as glucose within the gastrointestinal tract.Indeed, repression of GLUT2 should cause glucose accumulation within the gastrointestinal space resulting in increased GLP-1 expression and release, which in turn stimulates insulin and inhibits glucagon release with concomitant hypoglycemic effects (Ezcurra et al., 2013;Velasquez et al., 2010).Additionally, GLP-1 upregulation may proceed though TGR5 activation as naturally occurring TGR5 agonists have been previously shown to modulate GLP-1 biosynthesis and secretion (Jafri et al., 2016).D. subericarpa may contain TGR5 phytochemical agonists; some of these scaffolds have been characterized in plants such as Olea europraea (Sato et al., 2007).

Pancreatic Insulin release not gene expression is regulated by culinary administration of D. Subericarpa
The desired goal in treatment of type-2 diabetes is to improve cell health (Marchetti et al., 2009), increase insulin release, and tissue absorption of blood glucose via GLUT4, which is rapidly translocated to the membrane of target tissue downstream insulin/Insulinreceptor signaling pathway (Leto & Saltiel, 2012).We tested whether D. subericarpa leaves also alter insulin and L-type voltage gated channel transcription in the pancreas (Fig. 2D).While our data showed that none of these genes is significantly upregulated in the test groups (vs.control (p < 0.05), Fig. 2D, ii&iii), blood insulin release is however dose-dependently increased in the treatment groups (Fig. 2D, iv).Increased blood insulin levels following D. subericarpa may have resulted from increased GLP-1 expression as stimulation of insulin secretion from pancreatic β-cells via glucagon-like peptide 1 receptor (GLP-1R)/GLP-1 (or other agonists) is well documented (Meloni et al., 2013) Paradoxically, blunted GLUT2 expression may result in low blood glucose, which should act synergistically with GLP-1 to activate insulin release pathway in pancreatic β-cells (MacDonald et al., 2002), clearly insulin release mediated by D. subericarpa may involve GLP-1 but glucose-independent manner.Interestingly, TGR5 agonists are the scaffolds, which mediate secretion of GLP-1 in the absence of glucose (Lan et al., 2012), whether GLP-1 is sufficient for insulin release in the absence of glucose is yet to be determined.

D. Subericarpa reverses hyperglycemia in experimental animals but exhibit sub-chronic nephrotoxicity
To test the usability of D. subericarpa leaves in experimental diabetic condition, nine (9) animals were subjected to similar initial feeding conditions (normal rat chow with honey base), six (6) of the animals were induced with diabetes using streptozotocin (single dose, 45 mg/kg, i.p.); day-3 post streptozotocin treatment, hyperglycemia was observed in all the treatment groups.Three (3) of the diabetic animals were chosen for treatment (10% D. subericarpa) for 9 days.Blood samples were drawn periodically (once every 3 days) from the tail of the animals for blood glucose determination.Indeed, D. subericarpa leaves in the diet of diabetic rats partially reversed hyperglycemia associated with streptozotocin treatment (Fig. 3A).This result clearly supports the folkloric use of D. subericarpa leaves in diabetic condition.Since diabetes is treated chronically, it should be interesting to know how the kidney functions in a 14-day treatment period.
Here, kidney injury molecule 1 (KIM-1)(Andreucci et al., 2016) (Faubel et al., 2007) patterns were investigated as biomarkers for kidney damage.Surprisingly, both genes were upregulated in both test subjects (Fig. 3B,i-iii).This finding may not be absolutely peculiar to D. subericarpa leaves as consumption of mixed Chinese herbs have resulted in renal tubular acidosis with hypokalaemic paralysis, rhabdomyolysis and subsequent acute renal failure (Lee et al., 1999).
In conclusion, we have demonstrated that D. subericarpa leaves have potent anti-diabetic principles acting via GLUT2 gene downregulation and GLP-1 upregulation mechanisms.We have also shown its applicability in animal model of diabetes.Its application in humans may be limited in whole-leaf form as renal IL-(KIM-1) expression were significantly increased in animals administered D. subericarpa leaves.Thus, reciprocal regulation of GLP-1 and GLUT2 genes by D. subericarpa leaves provides preliminary indication that the leaves contain TGR5 agonists, since TGR5 activation is upstream GLP-1 gene upregulation.Taking a cue from previous studies, we hypothesize that the unidentified compounds may likely be of sterol class especially the triterpenes, which has demonstrated potent TGR5 agonism in vitro and in vivo (Schaap et al., 2014); GLUT2 downregulation in SREBPdependent manner also lends credence to our speculation as sterols likewise regulate the activity of SREBP (Nohturfft et al., 1999).It may be instructive to suggest immediate isolation and characterization of SREBP-1c inhibiting/TGR5 activating principle, which may be different from the nephrotoxic components for anti-diabetes drug development.
Upregulated GLP-1, increased insulin exocytosis but not increased insulin gene expression coupled with GLUT2 down-regulation following culinary exposure to D. Subericarpa leaves solidly support its antidiabetic claims.The anti-diabetic pharmacology may be blunted by nephrotoxicity as KIM-1 and IL-1-β genes were marked up regulated in normal animals exposed (14-day) to D. Subericarpa.We therefore report that the whole leaf indeed D. Subericarpa contains phytochemical principles with potent antidiabetic potency but may be nephrotoxic; for future clinical use, selective fractionation of active components from the toxic components is desirable.

Acknowledgements
The technical assistance of Dr. Elekofehinti of Federal University of Technology Akure, Nigeria is acknowledged.The authors also acknowledged the First Bank of Nigeria for providing Solar-power supply facility to Center for Bio-Computing and Drug Development where the study was conducted.