Consumption of Terpenoids-Rich Padina pavonia Extract Attenuates Hyperglycemia, Insulin Resistance and Oxidative Stress, and Upregulates PPARγ in a Rat Model of Type 2 Diabetes

Seaweeds are rich in structurally diverse bioactive compounds with promising therapeutic effects. This study aimed to isolate and identify terpenes from the brown alga Padina pavonia and to investigate its antidiabetic activity, pointing to the possible involvement of peroxisome proliferator-activated receptor (PPAR)γ. Type 2 diabetes was induced by feeding rats a high fat diet (HFD) for 4 weeks followed by injection of 35 mg/kg streptozotocin (STZ). The diabetic rats received P. pavonia extract (PPE; 50, 100 and 200 mg/kg) for 4 weeks and samples were collected for analyses. HFD/STZ-induced rats showed hyperglycemia, dyslipidemia, impaired glucose tolerance, decreased insulin, and increased HbA1c and HOMA-IR. PPE ameliorated hyperglycemia and dyslipidemia, and improved glucose tolerance and insulin sensitivity in diabetic rats. Treatment with PPE increased hepatic hexokinase activity and glycogen, suppressed glucose-6-phosphatase, fructose-1,6-biphosphatase, and glycogen phosphorylase, and attenuated oxidative stress, inflammation, and liver injury and lipid infiltration in HFD/STZ-induced rats. In addition, PPE boosted antioxidants and upregulated PPARγ gene and protein expression in the liver of diabetic rats. Phytochemical investigation resulted in the isolation of six terpenes from PPE and in silico analysis revealed their binding affinity toward PPARγ. In conclusion, P. pavonia-derived terpenes attenuated hyperglycemia, dyslipidemia, oxidative stress, and inflammation, and improved insulin sensitivity and carbohydrate metabolism in type 2 diabetic rats. These beneficial effects are mediated via PPARγ activation. However, further studies to explore the exact mechanisms underlying the antidiabetic effect of PPE are recommended.


Introduction
Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia resulting from impaired insulin secretion and/or action. It is a major global health concern rising to an alarming

General Experimental Procedures
1 H (400 MHz) and 13 C (100 MHz) NMR were recorded in CDCl3 on a Varian Unity Inova instrument using TMS as the internal standard. Chemical shifts (δ) were expressed in ppm and coupling constants (J) were reported in Hz. Optical rotations were measured using Perkin-Elmer 341 polarimeter and Shimadzu UV-vis 160i spectrophotometer was used for UV measurements. HREIMS and EIMS mass spectroscopic analysis were measured on a Finnigan MAT TSQ 700 mass spectrometer. IR spectra were recorded on KBr pellets on a Shimadzu FTIR-8400 instrument.

Animals and Treatments
Male Wistar rats (160-180 g) obtained from Vacsera (Giza, Egypt) were included in this investigation. The animals were housed under standard conditions (23 ± 2 • C and 50-60% humidity) and were given a free access to food and water. The experimental protocol and treatments were approved by the Animal Care and Use Committee.
The rats were allocated into seven groups, two groups were fed normal chow diet and five groups received high fat diet (HFD; 58% fat, 25% protein, and 17% carbohydrate). After 4 weeks, HFD-fed rat received a single intraperitoneal (i.p.) injection of streptozotocin (STZ; 35 mg/kg; Sigma, St. Louis, MO, USA) dissolved in citrate buffer (pH 4.5), and the control rats received i.p. injection of citrate buffer. Seven days after STZ injection, overnight fasted rats received glucose (3 g/kg) orally and blood glucose levels were determined after 2 h. Rats with blood glucose levels ≥ 250 mg/dL were selected to investigate the anti-diabetic effect of the EtOAc fraction of P. pavonia.
After the treatment period, overnight fasted rats were sacrificed under anesthesia. A blood sample from each rat was collected in a tube containing EDTA for glycated hemoglobin (HBA1c) estimation, whereas other blood was collected to prepare serum. The animals were immediately dissected, and the liver was removed, weighed, and a 10% w/v homogenate was prepared in cold phosphate buffered saline (PBS). Following centrifugation, the clear supernatant was collected for the assay of ROS, LPO, nitric oxide (NO), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Other samples were collected on 10% neutral buffered formalin for histological investigation, whereas others were stored at −80 • C.

Oral Glucose Tolerance Test (OGTT)
To investigate the effect of PPE on glucose tolerance, blood samples were collected from the lateral tail vein of overnight fasted rats. Other blood samples were collected at 30, 60, 90, and 120 min following the oral administration of 3 g/kg glucose solution. Glucose was assayed using Spinreact reagent kit (Girona, Spain) according to the method of Trinder [35].

Determination of HBA1c, Insulin, and HOMA-IR
HbA1c and insulin were determined using Biosystems (Barcelona, Spain) and RayBiotech (Peachtree Corners, GA, USA) assay kits, respectively. Homeostasis model of insulin resistance (HOMA-IR) [36] was calculated using the following equation:

Determination of Serum and Liver Lipids
Serum triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were assayed using Spinreact (Girona, Spain) kits according to the manufacturer's instructions. Very low-density lipoprotein cholesterol (vLDL-C) and LDL-C were calculated using the following equations: Atherogenic index of plasma (AIP) was calculated as follows [43]: To determine the TC and TG levels in the liver, total lipids were extracted by chloroform:methanol mixture (2:1, v/v) following the method of Folch et al. [44]. Briefly, 100 mg liver was homogenized in 500 µL PBS and the homogenate was centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a new tube and mixed with 1 mL chloroform/methanol followed by sonication for 30 min. The chloroform phase was collected, dried, and 1 mL isopropanol was added for reconstitution. TG and TC were determined using Spinreact (Girona, Spain) kits.

Histology
Samples from the liver were fixed for 24 h and then processed for dehydration and embedding in paraffin wax. Five micrometer sections were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Thereafter, the sections were examined using a light microscope.

PPARγ Expression
The effect of PPE and HFD/STZ on liver PPARγ was determined by qRT-PCR and Western blotting as previously reported [26,51]. Briefly, RNA was isolated using TRIzol reagent (Invitrogen, Waltham, MA, USA) and treated with RNase-free DNase (Qiagen, Germany). The purified RNA was quantified on a nanodrop and samples with A260/A280 nm >1.7 were selected for reverse transcription.
cDNA was amplified using SYBR Green master mix and the following primers; PPARγ forward: 5 -GGACGCTGAAGAAGAGACCTG-3 , PPARγ reverse: 5 -CCGGGTCCTGTCTGAGTATG-3 , β-actin forward: 5 -AGGAGTACGATGAGTCCGGC-3 and β-actin reverse: 5 -CGCAGCTCAGTAACAGTCCG-3 . The obtained data were analyzed by the 2 −∆∆Ct method [52]. For Western blotting, liver samples were homogenized in RIPA buffer with proteinase inhibitors and protein content was estimated using Bradford reagent [53]. Forty microgram protein was electrophoresed on 10% SDS/PAGE and transferred to nitrocellulose membrane. After blocking in 5% skimmed milk in tris buffered saline/tween 20 (TBST) for 1 h at room temperature (RT), the membrane was probed with anti-PPARγ and anti-β-actin (Novus Biologicals, USA) overnight at 4 • C. The blots were washed with TBST and incubated with the secondary antibodies for 1 h at RT. After washing with TBST, the membrane was developed using enhanced chemiluminescence detection kit (BIO-RAD, Philadelphia, PA, USA). The obtained bands were scanned, and their intensity was determined using ImageJ (NIH, Bethesda, MD, USA).

Molecular Docking
The 3D crystal structure of PPARγ was obtained from Protein Data Bank, (http://www.rcsb.org/pdb) (PDB, ID: 2PRG). Macromolecules were viewed and separated from ligand, solvent and unneeded residues by using UCSF Chimera [54]. Isolated protein receptor was prepared for docking by optimization by means of Autodock Tools (ADT) v1.5.6. This optimization comprises the addition of polar hydrogens and setting the grid box according to the best configuration of the active site amino acid residues [55]. The size of grid box was set at 30 x 36 x 26 (x,y,z). The center of the grid box was located at 59.388 × −6.279 × 36.212 (x,y,z). The 2D structure of the isolated compounds (1-6) was drawn using ChemBioDraw software, then converted to .pdb 3D structures using UCSF Chimera [54]. Ligands were optimized by using ADT v1.5.6. Molecular docking analysis for the isolated compounds (1-6) was carried out by AutoDock Vina (http://vina.scripps.edu/), and the obtained results were analyzed and visualized using PyMOL v2.3.2.

Statistical Analysis
The results were presented as mean ± SEM (standard error of mean). All statistical comparisons were made by one-way ANOVA followed by Tukey's test and the differences were considered statistically significant at P < 0.05. The statistical analysis was carried out using GraphPad Prism 7 (La Jolla, CA, USA).

PPE Ameliorates Hyperglycemia and IR in HFD/STZ-Induced Rats
Diabetic rats showed a significant hyperglycemia throughout the experiment ( Figure 2A). OGTT of the diabetic rats revealed significant increase in blood glucose levels at 0, 30, 60, 90, and 120 min ( Figure 2C). Area under curve analysis of the blood glucose during the treatment period ( Figure 2B) and OGTT ( Figure 2D) revealed significant hyperglycemia in HFD/STZ-induced rats and the antihyperglycemic effect of PPE and PIO. PPE exerted a dose-dependent anti-hyperglycemic effect in diabetic rats, with no effect in normal rats. Hyperglycemia in HFD/STZ-induced rats was confirmed by the significantly elevated HBA1c% (P < 0.001), an effect that was remarkably attenuated by PPE and PIO ( Figure 2E).
Serum insulin was decreased in HFD/STZ-induced rats ( Figure 2F), and treatment with 50 and 100 mg/kg PPE failed to increase insulin significantly in diabetic rats, whereas the 200 mg/kg (P < 0.05) and PIO (P < 0.01) significantly improved serum insulin levels. Given that the assay of glucose or insulin can poorly detect impaired insulin sensitivity [62], IR was evaluated by determining HOMA-IR which was significantly increased in diabetic rats (P < 0.001; Figure 2G). Treatment with 50, 100, and 200 mg/kg PPE and PIO decreased HOMA-IR significantly.

PPE Ameliorates Hyperglycemia and IR in HFD/STZ-Induced Rats
Diabetic rats showed a significant hyperglycemia throughout the experiment (Figure 2A). OGTT of the diabetic rats revealed significant increase in blood glucose levels at 0, 30, 60, 90, and 120 min ( Figure 2C). Area under curve analysis of the blood glucose during the treatment period ( Figure 2B) and OGTT ( Figure 2D) revealed significant hyperglycemia in HFD/STZ-induced rats and the anti-hyperglycemic effect of PPE and PIO. PPE exerted a dose-dependent anti-hyperglycemic effect in diabetic rats, with no effect in normal rats. Hyperglycemia in HFD/STZ-induced rats was confirmed by the significantly elevated HBA1c% (P < 0.001), an effect that was remarkably attenuated by PPE and PIO ( Figure 2E).
Serum insulin was decreased in HFD/STZ-induced rats ( Figure 2F), and treatment with 50 and 100 mg/kg PPE failed to increase insulin significantly in diabetic rats, whereas the 200 mg/kg (P < 0.05) and PIO (P < 0.01) significantly improved serum insulin levels. Given that the assay of glucose or insulin can poorly detect impaired insulin sensitivity [62], IR was evaluated by determining HOMA-IR which was significantly increased in diabetic rats (P < 0.001; Figure 2G). Treatment with 50, 100, and 200 mg/kg PPE and PIO decreased HOMA-IR significantly. Data are mean ± SEM, n = 6. *** P < 0.001 versus control and # P < 0.05, ## P < 0.01 and ### P < 0.001 versus diabetic.

PPE Improves Liver Glucose Metabolizing Enzymes and Glycogen in HFD/STZ-Induced Rats
To explore the anti-hyperglycemic mechanism of PPE in diabetic rats, glucose-metabolizing enzymes and glycogen content were determined. Hexokinase activity was noticeably decreased in the liver of HFD/STZ rats (P < 0.001; Figure 3A). In contrast, G-6-Pase ( Figure 3B), F-1,6-BPase ( Figure 3C), and glycogen phosphorylase ( Figure 3D) were increased in the diabetic rats. Liver glycogen content was diminished in the diabetic group when compared with the control rats (P < 0.001; Figure 3E). Oral supplementation of PPE and PIO increased hepatic hexokinase activity and glycogen, and suppressed G-6-Pase, F-1,6-BPase, and glycogen phosphorylase HFD/STZ-induced rats. Of note, PPE decreased the activity of G-6-Pase and F-1,6-BPase dose-dependently in diabetic rats, whereas exerted no effect in normal rats.

PPE Attenuates Hyperlipidemia in HFD/STZ-Induced Rats
To evaluate the anti-hyperlipidemic effect of PPE in diabetic rats, serum lipids were determined. HFD/STZ-induced rats exhibited significant hypertriglyceridemia ( Figure 4A) and hypercholesterolemia ( Figure 4B) when compared with the control group (P < 0.001). LDL-C ( Figure 4C), vLDL-C ( Figure 4D) and AIP ( Figure 4F) were increased in HFD/STZ-induced rats, whereas HDL-C was significantly declined ( Figure 4E). Treatment of the diabetic rats with PPE (50, 100, and 200 mg/kg) as well as PIO reduced serum TG, TG, LDL-C, vLDL-C, and AIP. The 200 mg/kg dose of PPE increased HDL-C levels in diabetic rats and exerted no effect on lipid profile in normal rats.

PPE Prevents Liver Injury and Lipid Accumulation in HFD/STZ-Induced Rats
The protective effect of PPE on diabetes-induced liver dysfunction was evaluated through the assessment of serum transaminases, liver cholesterol and TG, and histopathological changes. HFD/STZ-induced rats exhibited significantly elevated serum ALT ( Figure 5A) and AST ( Figure 5B), and hepatic cholesterol ( Figure 5C) and TG ( Figure 5D). These biochemical findings were confirmed by the histological investigation where the HFD/STZ-induced rats showed centrilobular hepatic vacuolation of round border and clear vacuoles consistent with fatty changes (Figure 6).
Oral administration of PPE (50, 100, and 200 mg/kg) and PIO reduced serum ALT and AST, and liver cholesterol and TG ( Figure 5) and prevented hepatic steatosis ( Figure 6) in diabetic rats. Rats received 200 mg/kg PPE for 4 weeks showed normal liver function ( Figure 5) and histological architecture ( Figure 6).

PPE Prevents Liver Injury and Lipid Accumulation in HFD/STZ-Induced Rats
The protective effect of PPE on diabetes-induced liver dysfunction was evaluated through the assessment of serum transaminases, liver cholesterol and TG, and histopathological changes. HFD/STZ-induced rats exhibited significantly elevated serum ALT ( Figure 5A) and AST ( Figure 5B), and hepatic cholesterol ( Figure 5C) and TG ( Figure 5D). These biochemical findings were confirmed by the histological investigation where the HFD/STZ-induced rats showed centrilobular hepatic vacuolation of round border and clear vacuoles consistent with fatty changes (Figure 6).
Oral administration of PPE (50, 100, and 200 mg/kg) and PIO reduced serum ALT and AST, and liver cholesterol and TG ( Figure 5) and prevented hepatic steatosis ( Figure 6) in diabetic rats. Rats received 200 mg/kg PPE for 4 weeks showed normal liver function ( Figure 5) and histological architecture ( Figure 6).

PPE Attenuates Oxidative Stress and Enhances Antioxidants in HFD/STZ-Induced Rats
The protective effect of PPE on oxidative stress in diabetic animals was assessed through the assessment of hepatic ROS, LPO, and NO, and antioxidant defenses (Figure 7). Rats received HFD and STZ showed hepatic oxidative stress manifested by the significantly increased ROS, LPO, and NO when compared with the control animals (P < 0.001; Figure 7A-C). Additionally, hepatic GSH ( Figure 7D), SOD ( Figure 7E), and CAT ( Figure 7F) were markedly reduced in HFD/STZ-induced rats (P < 0.001). Oral administration of PPE or PIO suppressed ROS, LPO, and NO and enhanced hepatic GSH, SOD, and CAT (Figure 7). Normal rats received 200 mg/kg PPE for 4 weeks showed no changes in hepatic ROS, LPO, NO, and antioxidants.

PPE Attenuates Oxidative Stress and Enhances Antioxidants in HFD/STZ-Induced Rats
The protective effect of PPE on oxidative stress in diabetic animals was assessed through the assessment of hepatic ROS, LPO, and NO, and antioxidant defenses (Figure 7). Rats received HFD and STZ showed hepatic oxidative stress manifested by the significantly increased ROS, LPO, and NO when compared with the control animals (P < 0.001; Figure 7A-C). Additionally, hepatic GSH ( Figure 7D), SOD ( Figure 7E), and CAT ( Figure 7F) were markedly reduced in HFD/STZ-induced rats (P < 0.001). Oral administration of PPE or PIO suppressed ROS, LPO, and NO and enhanced hepatic GSH, SOD, and CAT (Figure 7). Normal rats received 200 mg/kg PPE for 4 weeks showed no changes in hepatic ROS, LPO, NO, and antioxidants.

PPE Suppresses Inflammation in HFD/STZ-Induced Rats
The anti-inflammatory effect of PPE in diabetic rats was evaluated by determining serum TNF-α ( Figure 8A) and IL-6 ( Figure 8B). The circulating levels of TNF-α and IL-6 were increased significantly in diabetic rats (P < 0.001) and treatment with PPE or PIO ameliorated these cytokines. Oral supplementation of 200 mg/kg PPE did not alter serum TNF-α and IL-6 in normal rats.

PPE Suppresses Inflammation in HFD/STZ-Induced Rats
The anti-inflammatory effect of PPE in diabetic rats was evaluated by determining serum TNFα ( Figure 8A) and IL-6 ( Figure 8B). The circulating levels of TNF-α and IL-6 were increased significantly in diabetic rats (P < 0.001) and treatment with PPE or PIO ameliorated these cytokines. Oral supplementation of 200 mg/kg PPE did not alter serum TNF-α and IL-6 in normal rats.

PPE Upregulates Hepatic PPARγ in HFD/STZ-Induced Rats
To investigate the role of PPARγ in mediating the beneficial effects of PPE in diabetic rats, we determined its gene and protein expression, and conducted a molecular docking study to assess its binding with the isolated compounds. There was a significant downregulation of hepatic PPARγ mRNA ( Figure 9A) and protein expression ( Figure 9B) in HFD/STZ diabetic rats. PPE and PIO administration significantly increased PPARγ mRNA and protein expression in diabetic rats.
The binding affinity of the isolated compounds (1-6) toward PPARγ was studied using molecular docking. The compounds showed binding in the range of −6.2 (kcal/mol) to −7.4 (kcal/mol) ( Table 3). Compound 5 showed the lowest binding energy toward PPARγ, representing the most stable complex ( Figure 9C). Docking analysis of the compounds 1, 5, and 6 revealed binding affinities toward Arg288 and Glu343, whereas compounds 2 and 3 showed affinities toward Arg288 and compound 4 binds Arg288 and Glu295 ( Figure 9C).

PPE Upregulates Hepatic PPARγ in HFD/STZ-Induced Rats
To investigate the role of PPARγ in mediating the beneficial effects of PPE in diabetic rats, we determined its gene and protein expression, and conducted a molecular docking study to assess its binding with the isolated compounds. There was a significant downregulation of hepatic PPARγ mRNA ( Figure 9A) and protein expression ( Figure 9B) in HFD/STZ diabetic rats. PPE and PIO administration significantly increased PPARγ mRNA and protein expression in diabetic rats.
The binding affinity of the isolated compounds (1-6) toward PPARγ was studied using molecular docking. The compounds showed binding in the range of −6.2 (kcal/mol) to −7.4 (kcal/mol) ( Table 3). Compound 5 showed the lowest binding energy toward PPARγ, representing the most stable complex ( Figure 9C). Docking analysis of the compounds 1, 5, and 6 revealed binding affinities toward Arg288 and Glu343, whereas compounds 2 and 3 showed affinities toward Arg288 and compound 4 binds Arg288 and Glu295 ( Figure 9C).

Discussion
T2DM is characterized by IR accompanied with insufficient insulin secretion, resulting in hyperglycemia that can promote organ damage and serious complications [3]. Therefore, management of IR and hyperglycemia is critical for the treatment of DM and prevention of its complications. Herein, we investigated the potential of a terpenoid rich fraction of the marine alga P. pavonia to ameliorate hyperglycemia, IR, hyperlipidemia, and oxidative stress in HFD/STZ-induced diabetic rats, pointing to the possible involvement of PPARγ.
HFD/STZ-induced diabetes has been a widely accepted model that mimic human T2DM. Feeding a HFD to rodents induces IR and metabolic derangements [63], and STZ was evidenced to Figure 9. PPE upregulates hepatic PPARγ in HFD/STZ-induced rats. PPE and PIO increased mRNA abundance (A) and protein expression (B) levels of PPARγ in the liver of diabetic rats. Data are mean ± SEM, n = 6. *** P < 0.001 versus control and ### P < 0.001 versus diabetic. (C) Molecular docking analysis showing the binding between P. pavonia-derived terpenes (Compounds 1-6) and PPARγ. Compounds 1, 5, and 6 interact with Arg288 and Glu343, compounds 2 and 3 interact with Arg288 and compound 4 binds Arg288 and Glu295 in PPARγ.

Discussion
T2DM is characterized by IR accompanied with insufficient insulin secretion, resulting in hyperglycemia that can promote organ damage and serious complications [3]. Therefore, management of IR and hyperglycemia is critical for the treatment of DM and prevention of its complications. Herein, we investigated the potential of a terpenoid rich fraction of the marine alga P. pavonia to ameliorate hyperglycemia, IR, hyperlipidemia, and oxidative stress in HFD/STZ-induced diabetic rats, pointing to the possible involvement of PPARγ.
HFD/STZ-induced diabetes has been a widely accepted model that mimic human T2DM. Feeding a HFD to rodents induces IR and metabolic derangements [63], and STZ was evidenced to damage pancreatic β-cells resulting in decreased insulin release [64]. Therefore, the T2DM model induced by HFD and STZ is used frequently to understand the pathogenesis of DM and to evaluate the beneficial effects of new therapeutic agents. Accordingly, HFD/STZ-induced rats in this study exhibited hyperglycemia, and impaired glucose tolerance and insulin secretion. In addition, HbA1c was significantly elevated in HFD/STZ-induced rats, demonstrating the development of hyperglycemia and diabetes. HbA1c is a valuable indicator for the diagnosis and management of hyperglycemia in diabetes and levels lower than 7% indicate good glycemic control [65]. Previous work from our lab [15] as well as others [66,67] have shown hyperglycemia, elevated HbA1c% and altered insulin secretion, and sensitivity in HFD/STZ-induced rodents. In addition, the values of HOMA-IR suggested the development of IR in HFD/STZ-induced rats. Interestingly, treatment of the diabetic rats with different doses of PPE ameliorated hyperglycemia, glucose intolerance, and HbA1c%, indicating its potent anti-hyperglycemic efficacy. PPE increased insulin release and improved insulin sensitivity as demonstrated by the significant reduction of HOMA-IR. The anti-diabetic effect of P. pavonia hydroethanolic extract has been previously reported in STZ/nicotinamide (NA) diabetic rats [68]. We have reported significant improvement of glucose tolerance and insulin sensitivity following treatment of the STZ/NA-induced rats with 100 mg/kg P. pavonia hydroethanolic extract [68]. Here, we introduced new information that a terpenoid rich fraction of P. pavonia can effectively ameliorate hyperglycemia and IR in type 2 diabetic rats.
Liver plays a key role in glucose homeostasis and uncontrolled hepatic gluconeogenesis and glycogenolysis are centrally implicated in hyperglycemia [69]. In addition, impaired insulin release and/or action causes hyperglycemia through increasing hepatic glucose output and decreasing glucose utilization by peripheral tissues [69]. The enzymes regulating hepatic glucose production are potential targets for the control of glucose homeostasis in diabetes. Therefore, we determined the effect of PPE on hepatic glucose-metabolizing enzymes and glycogen in diabetic rats. Hexokinase activity was decreased whereas G-6-Pase and F-1,6-BPase were significantly increased in the liver of HFD/STZ-induced rats as previously reported [70,71]. Hexokinase is an insulin-dependent enzyme involved in the oxidation of glucose and its activity is decreased in insulin deficiency and IR [72,73]. The declined hepatic hexokinase activity in diabetes results in decreased glucose oxidation rats via glycolysis, leading to hyperglycemia as shown in the current study. In contrast, G-6-Pase and F-1,6-BPase were increased in diabetic rats. Both enzymes are involved in the regulation of glucose homeostasis through eliciting gluconeogenesis. F-1,6-BPase catalyzes the dephosphorylation of fructose-1,6-bisphosphate to fructose-6-phosphate [74] and G-6-Pase completes the ending step in gluconeogenesis and glycogenolysis by catalyzing the dephosphorylation of glucose-6-phosphate to glucose [75]. In diabetes, the increase in hepatic glucose production is directly associated with impaired suppression of G-6-Pase [76]. The increased activity of both G-6-Pase and F-1,6-BPase is directly attributed to impaired insulin release. Accordingly, hexokinase was diminished and G-6-Pase and F-1,6-BPase were activated in the liver of diabetic rats exhibiting insulin insufficiency [70,71,73]. Consequently, hepatic glycogen phosphorylase activity was increased, and glycogen content was declined in HFD/STZ diabetic rats, demonstrating exaggerated glycogenolysis and gluconeogenesis. Glycogen is the storage form of glucose in liver and skeletal muscle and plays an important role in glucose homeostasis. Given that insulin stimulates glycogenesis by stimulating glycogen synthase, and suppressing glycogen phosphorylase and G-6-Pase, decreased glycogen levels reflect insulin insufficiency and/or resistance [77,78]. PPE significantly improved hexokinase activity and suppressed F-1,6-BPase, G-6-Pase, and glycogen phosphorylase, resulting in restoration of the hepatic glycogen content. Hence, it is noteworthy assuming that activation of hexokinase secondary to increased insulin secretion and sensitivity enhanced glucose oxidation and glycogenesis in the liver of HFD/STZ-induced rats. In addition, amelioration of fasting blood glucose in diabetic rats treated with PPE is a direct result of increased hexokinase activity and consequently glucose oxidation.
Dyslipidemia is one of the most common complications seen in diabetes irrespective of insulin deficiency or IR [7]. Diabetic dyslipidemia is featured with increased serum TG, TC, and LDL-C and decreased HDL-C, with hypertriglyceridemia representing the most common abnormality [79]. HFD/STZ diabetic rats in the present investigation exhibited atherogenic lipid profile manifested by increased TG, TC, LDL-C, and vLDL-C, and decreased HDL-C as previously demonstrated [80]. Administration of PPE significantly decreased serum TG, TC, LDL-C, vLDL-C, and AIP, a frequent predictor of atherosclerosis, and the high dose markedly increased serum HDL-C. Given the role of insulin in promoting the storage of lipids, the anti-hyperlipidemic effect of PPE could be mediated, at least in part, via increasing insulin sensitivity and PPARγ activation. This notion is supported by the improvement of lipid profile in PIO-treated diabetic rats. PIO is PPARγ agonist that ameliorates IR and improves TG by enhancing vLDL-TG removal from the circulation and increased HDL-C levels [81,82].
Diabetes can accelerate the progression of liver injury, thereby aggravating abnormal glucose and lipid metabolism [4]. In addition, the accumulation of lipids in the liver may worsen IR, leading to severe metabolic alterations. Excessive accumulation of hepatic lipids and hyperglycemia can provoke hepatocyte injury and liver dysfunction [83]. Here, HFD/STZ-induced diabetic rats showed liver injury evidenced by the elevated serum ALT and AST, the indicators of liver function. Increased serum transaminases has been associated with hepatic IR [84] and rats received HFD and/or STZ showed elevated serum ALT and AST [14]. In addition, hepatic TG and TC were increased in HFD/STZ-induced rats in the present investigation. These biochemical findings were supported by the histological examination which showed centrilobular hepatic vacuolation of round border and clear vacuoles consistent with fatty changes. Hepatic lipid infiltration occurs as a consequence to IR-related lipolysis which promote dyslipidemia and increase fatty acids taken up by the liver as an energy source. Accumulated fatty acids increases lipogenesis and disturbs mitochondrial β-oxidation, resulting in further lipid infiltration in the liver [85].
Hyperglycemia and hyperlipidemia were associated with increased generation of ROS, oxidative stress, and inflammation [15,86]. The role of ROS in liver injury has been established by studies showing that reduction of ROS can protect against hepatocyte damage induced by several insults [87][88][89]. HFD/STZ diabetic rats in this study showed excessive production of ROS associated with increased MDA and NO. Increased ROS can promote oxidative damage to lipids, proteins, and DNA [90,91]. Although the liver is equipped with antioxidant defenses, including GSH, SOD, and CAT, hyperglycemia-induced ROS generation was associated with diminished levels of these antioxidants as previously demonstrated [14,15]. Excess ROS can also activate NF-κB, a transcription factor eliciting the expression of TNF-α, IL-6, and inducible NO synthase (iNOS). These ROS-mediated effects explain the elevated hepatic MDA and NO levels as well as the pro-inflammatory cytokines in diabetic rats. In this context, circulating levels of TNF-α and IL-6, and liver NF-κB expression have been reported to increase in HFD/STZ-induced rats [14,15]. In patients with T2DM, increased circulating levels of TNF-α and IL-6 was reported [13]. Increased IL-6 is associated with impaired glucose tolerance [16] as well as IR in hepatocytes [17]. IL-6 reduces insulin receptor substrate (IRS)-1 tyrosine phosphorylation and inhibits insulin-dependent activation of protein kinase B/Akt in hepatocytes [17]. TNF-α impairs the ability of insulin to stimulate peripheral glucose uptake and to suppress hepatic glucose production [18], increases lipolysis in adipocytes [19], and impairs insulin signaling in muscle cells [20]. Therefore, counteracting oxidative stress and inflammation can result in improved insulin sensitivity.
PPE attenuated diabetes-associated liver injury and lipid accumulation in HFD/STZ-induced rats. All doses of PPE reduced serum transaminases and hepatic TC and TG levels and prevented liver injury in HFD/STZ-induced rats. In addition, PPE suppressed ROS generation, LPO and pro-inflammatory cytokines and boosted GSH and antioxidant enzymes in the liver of HFD/STZ-induced rats. Attenuation of ROS and inflammation significantly contributed to the improvement of insulin sensitivity and suppression of hepatic steatosis. We have previously demonstrated the hepatoprotective efficacy of P. pavonia ethanolic extract in azoxymethane-induced mice [32]. P. pavonia ameliorated liver function, prevented histological alterations, attenuated oxidative stress and NF-κB and increased the expression of PPARγ in liver [32] and colon [31] of azoxymethane-induced mice. Therefore, we thought that PPARγ mediated, at least in part, the beneficial effects of P. pavonia-derived terpenes in diabetic rats. To explore the possible involvement of PPARγ, we determined the effect of PPE on its gene and protein expression and conduced a molecular docking to study its binding with the isolated terpenes. Interestingly, PPE increased both mRNA abundance and protein expression levels of hepatic PPARγ in diabetic rats. PPARγ agonists such as PIO function as insulin sensitizers that enhance insulin action, ameliorate hyperglycemia in T2DM, lower plasma free fatty acids and TG, modulate the expression of inflammatory cytokines and prevent excessive lipid accumulation in liver and other peripheral tissues [22]. Hence, it is noteworthy assuming that the antihyperglycemic, insulin-sensitizing and anti-hyperlipidemic effects of PPE were mediated via PPARγ activation. The ability of PPE to activate PPARγ was supported by the molecular docking which revealed that compounds 1, 3, 5, and 6 possess binding affinity toward Arg288 and Glu343, compound 2 showed binding affinity toward Arg288 and compound 4 bound to Arg288 and Glu295. In addition to the metabolic effects, PPARγ activation is associated with suppressed inflammation and increased expression of antioxidant enzymes. PPARγ prevents NF-κB-dependent inflammatory genes transcription by inhibiting IκBα degradation, suppressing p65 nuclear translocation [92], and reducing NF-κB-dependent transcriptional control [93]. Moreover, activation of PPARγ can directly promote the expression of antioxidant enzymes [94] and suppress NADPH oxidase-dependent ROS production [95]. Besides binding PPARγ, P. pavonia-derived terpenes may exert free radical-scavenging and anti-inflammatory activities. However, studies scrutinizing the antioxidant and anti-inflammatory activities of the isolated terpenes are scarce. For instance, loliolide has been recently reported to suppress oxidative stress and increase the expression of heme oxygenase-1 in H 2 O 2 -induced HaCaT cells [96].

Conclusions
This study confers new information on the antidiabetic potential of P. pavonia-derived terpenes. Treatment of T2DM rats with PPE ameliorated hyperglycemia, glucose intolerance, and IR. PPE increased hepatic hexokinase activity and glycogen, and suppressed G-6-Pase, F-1,6-BPase, and glycogen phosphorylase, suggesting its efficacy to improve carbohydrate metabolism and reduce hepatic glucose production. In addition, PPE prevented hyperlipidemia and inflammation, and protected the liver against injury, lipid accumulation, and oxidative stress. These beneficial effects of PPE are associated with PPARγ activation and in silico investigations showed the binding affinity of the isolated terpenes toward PPARγ. The antidiabetic effects of P. pavonia-derived terpenes are summarized in Figure 10. Therefore, P. pavonia-derived terpenes represent promising leads in the development of effective anti-diabetic agents, pending further studies to explore the underlying mechanisms. effect of PPE on its gene and protein expression and conduced a molecular docking to study its binding with the isolated terpenes. Interestingly, PPE increased both mRNA abundance and protein expression levels of hepatic PPARγ in diabetic rats. PPARγ agonists such as PIO function as insulin sensitizers that enhance insulin action, ameliorate hyperglycemia in T2DM, lower plasma free fatty acids and TG, modulate the expression of inflammatory cytokines and prevent excessive lipid accumulation in liver and other peripheral tissues [22]. Hence, it is noteworthy assuming that the antihyperglycemic, insulin-sensitizing and anti-hyperlipidemic effects of PPE were mediated via PPARγ activation. The ability of PPE to activate PPARγ was supported by the molecular docking which revealed that compounds 1, 3, 5, and 6 possess binding affinity toward Arg288 and Glu343, compound 2 showed binding affinity toward Arg288 and compound 4 bound to Arg288 and Glu295. In addition to the metabolic effects, PPARγ activation is associated with suppressed inflammation and increased expression of antioxidant enzymes. PPARγ prevents NF-κB-dependent inflammatory genes transcription by inhibiting IκBα degradation, suppressing p65 nuclear translocation [92], and reducing NF-κB-dependent transcriptional control [93]. Moreover, activation of PPARγ can directly promote the expression of antioxidant enzymes [94] and suppress NADPH oxidase-dependent ROS production [95]. Besides binding PPARγ, P. pavonia-derived terpenes may exert free radicalscavenging and anti-inflammatory activities. However, studies scrutinizing the antioxidant and antiinflammatory activities of the isolated terpenes are scarce. For instance, loliolide has been recently reported to suppress oxidative stress and increase the expression of heme oxygenase-1 in H2O2induced HaCaT cells [96].

Conclusions
This study confers new information on the antidiabetic potential of P. pavonia-derived terpenes. Treatment of T2DM rats with PPE ameliorated hyperglycemia, glucose intolerance, and IR. PPE increased hepatic hexokinase activity and glycogen, and suppressed G-6-Pase, F-1,6-BPase, and glycogen phosphorylase, suggesting its efficacy to improve carbohydrate metabolism and reduce hepatic glucose production. In addition, PPE prevented hyperlipidemia and inflammation, and protected the liver against injury, lipid accumulation, and oxidative stress. These beneficial effects of PPE are associated with PPARγ activation and in silico investigations showed the binding affinity of the isolated terpenes toward PPARγ. The antidiabetic effects of P. pavonia-derived terpenes are summarized in Figure 10. Therefore, P. pavonia-derived terpenes represent promising leads in the development of effective anti-diabetic agents, pending further studies to explore the underlying mechanisms.