Differential Expression of Gluconeogenic Enzymes in Early- and Late- Stage Diabetes: The Effect of Citrullus Colocynthis Seed Extract on Hyperglycemia and Hyperlipidemia

Background: The extent of metabolic disruption and the usefulness of drugs and supplements, such as medicinal plants, in patients with diabetes may be impacted by the severity of the disease. Method: Nicotinamide + STZ together and STZ alone were used to induce early- and late-stage diabetes type 2 (ET2D and LT2D, respectively) in rats. Glucose tolerance test (GTT) was performed because the stage of disease was considered compatible with the magnitude of deviation from normal glucose tolerance test (GTT), as well as the level of FBS. Three main groups of the study were nondiabetic, early-stage diabetic, and late-stage diabetic rats. Each group was divided into two sub-groups, one of which received Citrullus colocynthis seed aqueous extract (CCAE, 90mg/kg BW) for 28 days. Weekly FBS and body weights were recorded during the study. At the end of 28 days, the serum levels of ALT, AST, ALP, TG, urea, creatinine, uric acid, cholesterol, HDL, LDL, c-peptide, and HbA1c were measured; the hepatic mRNA expression of several enzymes of glucose and fat metabolic pathways were also determined by Real-Time PCR. The accumulation of fat in the hepatic tissue was visualized by measuring the TG content and by H&E and Oil-Red staining and the degree of oxidative stress was measured by protein carbonyl content (PCC). Results: The LT2D rats showed maximal deviations from normal GTT. GTT for control and ET2D rats were similar, yet the area under curve (AUC) for ET2D rats was signicantly higher. Urea, ALT, and ALP levels were high in diabetic rats compared to control and signicantly different from each other.Serum TG dropped and ALT, ALP, HDL and LDL signicantly improved after treatment with CCAE. Different patterns of G6Pase and PEPCK expression in ET2D and LT2D suggested their similarity to short- and long-term fasting states, respectively. While the reduction of FBS levels in ET2D could be explained by an inhibition of G6Pase expression and therefore glycogenolysis, the level of PEPCK expression was not signicantly lowered by CCAE in LT2D. H&E staining of liver tissue showed steatoses of varying morphology in both ET2D and LT2D rats. CCAE led to up-regulated PPARα and down-regulated CPT1 expressions. Conclusion: As PEPCK activity is necessary for the continuation of the TG/FA cycle during fasting, it is possible that in LT2D, CCAE directed the PEPCK activity more towards glyceroneogenesis than gluconeogenesis to ensure the persistence of the TG/FA cycle. The enhanced glyceroneogenesis together with an up-regulated PPARa expression and down-regulated CPT1 expression probably led to lower blood and hepatic TG. More research is needed to establish the effect of CCAE on PEPCK expression and its course of activity.


Introduction
Diabetes type 2 (T2D) is the most prevalent type of diabetes worldwide. Patients are diagnosed with T2D when their blood glucose levelsincreaseafter a mealorwhenhungry. All major classes of approved pharmaceuticals currently used to treat diabetes are puri ed compounds [1]. Every so often, patients and the professionals turn to traditional medicines not only to avoid the drug-related side effects,such ashypoglycemia, nausea, vomiting, bloating, diarrhea or constipation but also to take advantage of their low costs and easy availability. Due to having several active components in naturally balanced proportions all in one pot, medicinal plants act simultaneously on multiple metabolic pathways to intelligently reestablish their lost harmony. Medicinal plants with sugar lowering properties are presumed to exert their antidiabetic activity via a variety of mechanisms, such as inhibition of intestinal glucose absorption, suppression of hepatic glucose production, induction of pancreatic insulin secretion, promotion of peripheral tissue (adipose and muscle) glucose uptake, and/or amelioration of oxidative stress [2] but the capacity of medicinal plants to regulate gene expression have remained largely unexplored.
STZ is commonly used to induce late-stage T2D (analogous to uncontrolled T1D) in experimental animals. When STZ is combined with niacinamide (NIA), a stable non-obese version of T2D is created in which the pancreatic b-cells are only moderately destroyed [9,10]. These distinct types of STZ-and STZ/NIA-induced diabetes models display different degrees of hyperglycemia, dyslipidemia, and insulin resistance; and coping with them may require different treatment modalities.
As T2D is characterized by a dysfunction of enzymes involved in hepatic glucose and fat metabolism, we created mild and severe models of diabetes in rats, which we called early-(ET2D) and late-stage (LT2D) diabetes. We aimed to investigate the effects of the aqueous seed extract of C. colocynthis (which we called CCAE) on the mRNA expression of some of the rate-limiting enzymes in sugar metabolism, (gluconeogenesis pathway) including glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), and lipid metabolism, including insulin-dependent sterol regulatory elementbinding protein-1c (SREBP-1c, a regulator of hepatic DNL), acetyl-CoA carboxylase (ACC, the regulator of FA synthesis and degradation), fatty acid synthase (FAS, the regulator of FA synthesis), peroxisome proliferator-activated receptor alpha (PPARa, the regulator of b-oxidation) and carnitine palmitoyltransferase I (CPT1, a regulator of b-oxidation).
The differential expression levels of PEPCK and G6Pase in the mild and advanced stages of diabetes suggested that early-stage diabetes is similar to short-term fasting; late-stage diabetes resembles longterm fasting. The aqueous seed extract of C. colocynthis may regulate gene expression and determine the course or preference of enzyme action.

Materials And Methods
Preparation of aqueous extract of C. colocynthis seed (CCAE) Bitter apple fruitwas collectedfrom the Saleh Abadregionof Ilam province, Iran, and authenticated by Faculty of Pharmacy; Tehran University of Medical Sciences (TUMS), with a voucher number PMP-648. The powdered seeds were soaked in distilled water (100 g/l) and re uxed for 20 min in a water bath (80°C).The cooled, vacuum ltered extract was concentrated by rotary evaporator before lyophilization (EYELA Freeze dryer FD-1, Japan).

Animals
Seven weeks old Male Wistar-Albino rats were maintained in an animal room (22 ± 2°C), with 12 h lightdark cycle and fed with normal rat chow diet and water ad libitum and cared for according to the guidelines for use of laboratory animals. All the animal experiments were approved by the ethical committee of Tehran University of Medical Sciences.

Diabetes induction
After an overnight (8 h) fasting, the rats destined for LT2D and ET2D groups were injected with STZ or STZ + niacinamide (NIA), respectively (Sigma-Aldrich Co.; St. Louis, MO). The dose of STZ was 55 mg/kg body weight (B.W.) in Experiment-1; it was 65 mg/kg b.w. in the Experiment-2, as explained below. Both STZ and NIA were freshly prepared in 0.3 ml cold normal saline and i.p. injected with insulin needles. NIA (200 mg/kg B.W.) was injected 15 min after STZ administration. Diabetes was con rmed by measuring glucose levels in peripheral blood obtained from the tail vein at days 4 and 10 after diabetes induction (GlucoSure STAR, ApexBio, Taiwan). Normal saline was injected into rats of the control group. STZinjected rats with fasting blood sugar (FBS) above 300 mg/dl on both occasions (day 4 and day 10) were considered as LT2D rats, and NIA/STZ-injected rats with stable FBS ranging between 140 and 220mg/dl were considered as ET2D [11]. Experiment 1. Short-term experimental process(120 min IPGTT) for selection of CCAE dosage LT2D and ET2D were induced in twenty rats (average mass of 100 to 160 g), with 10 rats in each group.
Only, 3 of the STZ-injected rats and 7 of the NIA/STZ-injected rats developed the corresponding types of diabetes, respectively. After measuring FBS in the overnight fasted rats, IPGTT was performed in two representative diabetic rats from each group. Glucose (2 g/kg b.w.) was coadministered into separate rats, with CCAE (the dose was either 90 mg/kg b.w. or 110 mg/kg b.w.) or metformin (100 mg/kg b.w.). Bloodsugar was recordedat 30 min intervals in the tail blood. The purpose of this experiment was to select CCAE dosage but it also showed the short-term hypoglycemic effects of CCAE. Experiment 2. Long-term (28-day) experimental process to investigate the effects of CCAE on sugar and fat metabolism After the induction of ET2D and LT2D as above, 14 of 40(35%) STZ-injected rats, and16 of 20 (80%) NIA/STZ-injected ratsdeveloped LT2D and ET2D, respectively. Rats that did not become diabetic (n=28), received a second injection of STZ (55mg/kgb.w.) of which 6diedand8 did not develop diabetes, and the remaining14rats that developed LT2D (FBS above 300 mg/dl on day 4 and 10)were placed in the STZ (6) andCCAE-STZ(8) groups (Supplement 1, Fig. 1A).
Evaluation of impaired glucose tolerance (IGT) using IPGTT To evaluate the status of glucose tolerance, we performed IPGTT on all the rats with ET2D and only 3 representative rats from LT2D in the same manner as above using only glucose without the drugs. The purpose was to prove that despite causing moderate increases in FBS, early-stage diabetes can give rise to signi cant IGT. Even so, 4 NIA/STZ-injected (ET2D) rats that had near-normal GTTwere excluded from the study. It took about 5 days to perform IPGTT on all the ET2D rats since the procedure required overnight fasted. This time plus the prior 10 days spent for the induction of diabetes and its approval made a total of 15 days.

Treatment with CCAE (study groups)
Fifteen days after induction of diabetes, we started the CCAE treatment with the selected dose of CCAE for half of the rats in each group. Finally, we ended up with 6 groups of rats including: Con (n=4), CCAE-Con (n=4), NIA/STZ (n=8), CCAE-NIA/STZ (n=8), STZ (n=12), CCAE-STZ (n=14, were alive on day 28) (Supplement 1, Fig. 1A). The treatment lasted for 28 days. At the end ofeach week,FBS and body weightsweremeasured. The amount of CCAE for the rats' daily (90 mg/kg) injections was determined according to their weekly body weights.
Blood and liver tissue collection After 28 days of treatment,blood (4-5 ml) was collected from the heart under anesthesia by ketamineandxylazine. A sample (2.5 ml) of the collected blood from each animal was transferred into tubes containing ethylenediaminetetraacetic acid (EDTA, 3 mg/ml) to be used for HbA1cdetermination. Sera were prepared from the remaining blood samples. After dissection, liver tissues were frozenin liquid nitrogen. All samples were stored at -80°C until analysis.

Blood biochemistry
The concentration of AST, ALT, ALP, urea, creatinine, TG, cholesterol, LDL, and HDL was measured in the Diabetes Center of Shariati Hospital, Tehran, Iran, using the respective kits (Human diagnostics). The cpeptide concentration and percent HbA1cwere determined using a c-peptide kit (Biosystem, Spain) and Human/Rat/Mice c-peptide ELISA kit (BioVision, America), respectively.

Real-time PCR
Total RNA was extracted from frozen liver tissue and puri ed using Hybrid-R Blood RNA (GeneAll, South Korea) and RNA Riboclear (GeneAll, South Korea) kit, respectively. The quantity and quality of extracted RNA were inspected by spectroscopic absorption at 260 nm and electrophoresis, respectively. Reverse transcription (RT) was performed with the Revert Aid First Strand cDNA Synthesis kit (Takara, Japan).
Real-time PCR was performed with Rotor-Gene Q (Qiagen, Germany) using SYBR Premix Ex Taq (Takara, Japan). Primer sequences used in this study are listed in Table1. The relative changes in gene expression were determined using the ΔC t method and normalized against the housekeeping b-actin expression.To be able to compare the expression of any certain gene in all groups relative to each other, the simultaneous RT-PCR for every gene and the reference actin was performed in groups of two or three that are: Con and CCAE-Con; Con, NIA/STZ, and STZ; Con, NIA/STZ, and CCAE-NIA/STZ; and Con, STZ, and CCAE-STZ. The data were merged together in one graph. Table 1 Primer sequences Protein and PCC determination A sample of the frozen liver (300mg) was homogenized in ice-cold PBS buffer (3 ml, pH 7.4) containing PMSF (5 mM) and anti-protease cocktail. After centrifugation at 1000 g at 4 °C to remove cell debris, the supernatants were collected, aliquoted and stored at -80 °C until used to measure protein concentration by BCA Protein Quanti cation kit (Pars-Tous, Iran) [12]and protein carbonyl content (PCC) by 2,4dinitrophenylhydrazine (DNPH) reagent [13].

Hepatic TG measurement
A small piece of frozen liver (~150 mg) was homogenized in 3 ml of chloroform/methanol (2:1). The hepatic lipids were extracted, using the method suggested by [14]. TG content was measured by colorimetric-enzymatic kit (Parsazmun, Iran) and expressed as mg/g tissue.

Liver histology
For hematoxylin and eosin (H&E) staining, two samples from each frozen liver tissue were xed in 10% formalin and embedded in para n. After cutting into 5 mm thickness, they were stained with H&E. For Oil Red O staining, two other samples from each group were cryostat sectioned. After immersing in 60% isopropanol for 5 min, sections were stained with freshly ltered Oil Red O solution for 45 min at room temperature. The sections were washed with 60% isopropanol and then distilled water. The cryosections were then stained with hematoxylin. All samples were examined under an optical microscope.

Statistical analyses
Statistical analyses were performed using the GraphPad Prism software, version 8.2.1. Results are presented as mean ± SEM unless otherwise stated. Statistical signi cance was de ned as p< 0.05.

Results
Aqueous extract of the seeds from C. colocynthis (CCAE) We used the seeds because they are believed to be less toxic [15] and most effective in lowering blood sugar levels [16]. The aqueous extract may be assumed to keep the constituent compounds in their most natural shapes and has been reported to have better hypoglycemic effects than chloroform, ethanol, glycoside, and alkaloid extracts [17,18]. HPLC pro le of CCAE has been shown in Fig. 1.Further analysis of the extract components has not been performed.

Selection of CCAE dosage using IPGTT
According to the results, CCAE had an acute or short-term hypoglycemic effect in ET2D (Supplement1, Fig. 1B) consistent with Lahfa et al. [18]. Similar to Mojaz Dalfardi et al. [19], we noted that the hypoglycemic potency for 90 mg/kg of CCAE was comparable to 100 mg/kg of metformin. We selected this dosage (90 mg/kg of CCAE) for the daily treatment of rats during the long-term study.

Evaluation of IGT by IPGTT
To show the presence of impaired glucose tolerance, both models were exposed to IPGTT. As the presence of IGT in severe diabetes was predictable, fewer LT2D rats were exposed to IPGTT. FBS levels were not much higher than normal in rats with ET2D, and all seemed well enough to undergo IPGTT, which surprisingly revealed the presence of signi cant IGT in the early stages of diabetes.

FBS and body weights during a long-term experiment
The average FBS levels were increased in both diabetic models (NIA/STZ group, 133.8 ± 7.9 mg/dl, p=0.004; STZ group, 453.0 ± 41.7 mg/dl, p=0.001; compared to Con, 95 mg/dl ± 5.5); and then lowered to near normal levels by CCAE in both groups (p>0.05 vs. Con) (Fig. 2).The extract did not change the FBS in nondiabetic control (CCAE-Con) consistent with Lahfa et al. [18] but against Abdel-Hassan et al. [20] who reported the glucose-lowering effect of C. colocynthis in normal non-diabetic rabbits.

C-peptide levels in serum
Similar to a previous study [11], in the ET2D model the lower than normal levels of c-peptide indicated limited destruction of pancreatic beta cells; a complete lack of c-peptide in LT2D (STZ groups) showed a total absence of insulin in those rats. In contrast to several other studies [22,23], our results suggested that treatment with CCAE for 28 days was not enough to cause an insulinotropic effect (Fig. 3).

HbA1c levels in the blood
The magnitude of elevation in blood HbA1c levels is a function of both duration of exposure to high glucose levels and the actual concentration of blood glucose. Although it has been shown previously that the dried C. colocynthis fruit powder given to T2D patients can signi cantly reduce the HbA1c levels [24,25], the decrease of HbA1c percentage in the CCAE-treated groups compared to their untreated counterparts was insigni cant in the present study, perhaps due to the short treatment time (28 days) with the extract (Fig. 3).

Blood biochemistry
The major biochemical effect of CCAE was to lower serum TG levels in both diabetic models. TG levels rose in the sera of diabetic rats in agreement some reports [26,27]; then signi cantly decreased towards normal in the treated groups (CCAE-NIA/STZ and CCAE-STZ groups vs. NIA/STZ and STZ groups: p = 0.0006 and p=0.0335, respectively) consistent with other reports [28][29][30][31][32]. Oral administration of 70% alcohol extract of the pulp (1.2 g/kg) in atherogenic rabbits was reported to signi cantly lower total cholesterol, triglycerides, and phospholipids in their liver, heart, and serum [33]. Concerning cholesterol levels in the blood, our results showed no signi cant difference between all groups (p>0.9). Urea, creatinine, uric acid, and AST levels in blood did not change in diabetic groups, nor after treatment. Treatment with CCAE caused an increase in LDL levels in ET2D (p=0.011) and a decrease in HDL levels in LT2D (p<0.001). ALP activityincreased in the LT2D rats (STZ vs. Con, p<0.01) ( Table 2). Treatment with CCAE has been shown, in previous studies, to improve some of the biomarkers [34,35]. Hepatic PEPCK and G6Pase gene expression ET2D induction led to a ~4.5-fold increase in the mRNA levels of G6Pase in NIA/STZ-group with a slight increase in PEPCK. On the contrary, LT2D induction was accompanied by a signi cant ~4.5-fold increase in the mRNA levels of PEPCK in the STZ-group (p=0.007 vs. Con) with onlya slight increase in G6Pase levels (Fig. 4). Treatment with CCAE led to signi cant suppression of G6Pase expression in ET2D (p=0.0055). But the suppression of PEPCK expression by CCAE in LT2D was not statistically meaningful (p=0.6).A previous study has reported the suppression of hepatic G6Pase and fructose 1, 6bisphosphatase enzymatic activities in alloxan-induced rats, by a leaf suspension of C. colocynthis [36] but inhibition of PEPCK activity or its mRNA expression has not been reported.
Hepatic fatty acid synthesis and oxidation pathways On day 28, the mRNA expression levels of SREBP-1c, ACC, FAS, and PPARa were signi cantly low in diabetic rats (STZ and NIA/STZ groups vs. Con, p<0.05) (Fig. 5) consistent with previous reports [37,38]. Low ACC activity was detected by [30] in the sera of alloxan-induced diabetes which, in contrast to our results, increased after treatment with C. colocynthis.
Diabetes induction caused a reduction in PPARa levels in contrast to some [39] but in line with other reports that related it to such circumstances as a mitochondrial failure and oxidative stress [38], hyperglycemia [40] and low lipoprotein lipase (LpL) activity [41]. After treatment with the extract, the mRNA levels of PPARα increased to near-normal levels in both CCAE-STZ and CCAE-NIA/STZ groups. Activation of hepatic expression of PPARa has been shown to ameliorate markers of diabetes, such as hepatic insulin resistance and steatosis [42,43].
Despite the presence of a direct relationship between PPARa and CPT1 expressions [44], decreased expression of PPARα was accompanied by an increase in the mRNA levels of CPT1 in diabetic groups (1.5 and 2.0 folds in ET2D and LT2D, respectively) in agreement with other reports [45][46][47][48]. This upregulation of CPT1 expression, in diabetes, stemmed from the diminished concentration of malonyl-CoA and lifting of its inhibitory effect on CPT1 [26,48,49]. The function of CPT1 is to mediate the transfer of FAs into mitochondria from cytosol; higher levels of CPT1 in STZ-rats implied a greater level of hepatic boxidation compared to NIA/STZ-rats. Treatment with CCAE lowered CPT1 mRNA levels back to near normal values (p<0.001).
Hepatic protein carbonylation (PCC) and TG concentration PCC levels did not increase signi cantly after diabetes induction (Fig. 6), in disagreement with [50]. Yet, upon treatment with CCAE, the decrease in PCC levels was signi cant. This antioxidant activity was in line with research that measured other markers of oxidative stress. For example, diabetic rats treated with various C. colocynthis extracts manifested decreased levels of TBARS, low expression of TNF-α and IL-6, and increased enzymatic activities of SOD and CAT [51,52].
The measurement of hepatic TG concentrations (Fig. 6) as well as H&E and Oil-Red staining of the liver tissue samples (Fig. 7), showed high levels of fat in the untreated diabetic groups relative to Con which paralleled TG levels in serum (Fig. 6, Table 2). Unlike our results, some researchers noted a reduction of TG in the liver of STZ-induced rats which they attributed to increased hepatic b-oxidation [41] or declined rates of lipogenesis [37,48]; yet others reported no change in the hepatic lipid content [49]. TG decreased signi cantly, in both serum and liver, after treatment with CCAE (Fig. 6, Table 2) in agreement with the reported effects of this plant against fatty liver [29].

Liver histology
Harmful changes in the structure of the liver, such as steatosis and brosis, are common ndings in diabetes. Despite the high concentration of hepatic TG in both STZ and NIA/STZ rats ( Fig. 6 and 7, Oil-Red O staining), the absence of large lipid droplets (LDs) in the H&E micrographs of liver from the STZ rats (in Fig. 7) implied differing architectures of LDs in different stages of diabetes. Congested central vein, dilatation of hepatic sinusoids (Fig. 7), brosis (Supplement 2), and excessive glycogen accumulation (Supplement 3) were other complications detected in livers of LT2D rats. Treatment with CCAE led to a reduction of hepatic lipid load in CCAE-NIA/STZ and CCAE-STZ groups ( Fig. 6 and Fig. 7) in agreement with [53] but in contrast to [54] did not seem to alleviate the adverse changes in liver histology in CCAE-STZ.

Discussion
Previous studies have suggested that the hypoglycemic property of various C. colocynthis raw preparations stems from their ability to stimulate pancreatic insulin secretion [22,23,36,55,56]. In the present study, the CCAE extract effectively lowered the FBS and serum TG in both ET2D and LT2D rats ( Fig. 2) even though the insulin levels did not seem to change during 28-day treatment.
Although the results of Oil-RedO staining and the estimation of hepatic TG con rmed the presence of high levels of fat in the liver tissues of both ET2D and LT2D ( Fig. 6 and 7), large LDs were evident only in the liver tissues of ET2D rats. Therefore, the types of fatty liver in ET2D and LT2D may be classi ed as macrovesicular steatosis (the formation of large LDs that displace the nucleus) and microvesicular steatosis (accumulation of small LDs with preserved cellular architecture), respectively [57].
Macrovesicular LDs grow during enhanced local TG synthesis [58]. In ET2D rats, the increased uptake of glucose into hepatocytes due to hyperglycemia-induced GLUT2 expression, and the activation of SREBP-1c by insulin (although insulin concentration was only about 40% of normal) would give rise to DNL [59].
While the newly synthesized hepatic fat and fat absorbed from food, called "new fat", is expected to act as a PPARa agonist and promote FA b-oxidation leading to the prevention of fat accumulation [60], the ongoing production of malonyl CoA by ACC (Fig. 5) and its inhibition of CPT1 may lead to the accumulation of macrovesicular type LDs in the liver of ET2D rats [61,62] (Fig. 7).
In LT2D there is a ow of "old fat" into hepatocytes, due to peripheral lipolysis. Unable to activate PPARa, and therefore mitochondrial b-oxidation [41,60]"old fat" would accumulate in the liver. Meanwhile, the increased ow of FA into the mitochondria of hepatocytes, due to low ACC activity (Fig. 5), overwhelms the mitochondrial b-oxidation and activates the peroxisomal b-oxidation and cytochrome P450dependent w-oxidation in the ER leading to ROS formation, lipid peroxidation, oxidative stress, in ammation, broblast accumulation, brosis, and liver injury, as seen in the liver of LT2D rats (Fig. 7) [ 38,63]. The arrangement of microvesicular LDs in the liver of LT2D rats may be the net effect of the high in ow of old-fat and disproportionate presence of oxidative stress [58] because despite the suppression of hepatic lipogenesis (DNL) in both diabetic models (Fig. 5), the ratio of b-oxidation to lipogenesis (taken as CPT1: SREBP1c ratio) was >2.5 times greater in LT2D (STZ group) compared to ET2D (NIA/STZ group) (Fig. 5) and also ROS level was high in LT2D livers (Fig. 6).
Autophagy is another relevant process that may affect the LD morphology. The presence of large LDs may be a sign of less damaged liver that is better capable of recovery. Larger LDs may be more readily available for fusion with lysosomes into autophagosomes, which are degraded to release the FAs that are then catabolized via b-oxidation [64][65][66]. In the insulin-resistant state, autophagy is greatly diminished. As we did not measure the markers of autophagy in the present study, we could not comment on it further.
In addition to FAs, glycerol-3-phosphate (G3P) is also required for the synthesis of TG within the hepatocytes. In the fed state, G3P is provided by glucose via glycolysis in both liver and adipose tissue.
During the short-term fast, up to several hours, as in between meals, hepatic glycogenolysis maintains stable glucose levels in the blood [67]. The presence of low PEPCK expression and high G6Pase expression in the liver of ET2D rats (Fig. 4) along with relatively low glycogen in the liver of ET2D rats compared to LT2D rats (Supplement 3) suggest that early-stage diabetes is comparable to the short-term fasting state. Bandsma et al observed higher glycogen levels in liver upon 15 or 24 hr fasting [68]in mice with PPARα de ciency. During the rst hours of fasting, therefore, glucose released from glycogen stores, can continue to provide G3P via glycolysis.
During long periods of fasting (>12-14 h), exercise or advanced stages of diabetes, glucose homeostasis is achieved through the gluconeogenesis pathway and FFAs are released into the blood by lipolysis in WAT [69,70]. The majority of FFAs that are produced during lipolysis are re-esteri ed back to TGs in WAT without ever leaving the tissue. Some of the FFAs in blood serve as fuel for tissues such as the central nervous system and cardiac and skeletal muscles, as such or after conversion to ketone bodies in the liver. The leftover FFAs are re-esteri ed with G3P to TGs which normally leave the liver toward WAT in the form of VLDL but may accumulate in the liver to cause fatty liver [64]. The interconversion between FAs and TGs via "FAs + G3P ® TG ® FAs + Glycerol" is known as triglyceride/fatty acid (TG/FA) cycle [71]. It has been found recently that the needed G3P is provided by glyceroneogenesis which is a truncated form of gluconeogenesis pathway for the synthesis of G3P from non-carbohydrate precursors. WAT lacks glycerol kinase activity and depends on the glyceroneogenesis pathway for the de novo synthesis of G3P.Glyceroneogenesis is also the predominant source of hepatic G3P for VLDL synthesis despite the existence of signi cant glycerol kinase activity in the liver [72].
Interestingly, the rate controlling-step in both gluconeogenesis and glyceroneogenesis pathways is catalyzed by the cytoplasmic PEPCK (PEPCK-C) which catalyzes the decarboxylation and then phosphorylation of oxaloacetate to form phosphoenolpyruvate (PEP). PEP is then converted to dihydroxyacetone phosphate which can be partitioned between the paths toward the synthesis of glucose and conversion to G3P [73]. The expression of PEPCK-C is enhanced during long term fasting and carbohydrate-free diet (lack of glucose) to provide a source of carbon for gluconeogenesis and G3P for TG synthesis [74].
While high levels of PEPCK expression are associated with hyperglycemia, obesity, insulin resistance and even cancer [75,76], the tissue-speci c and whole-body knockout of PEPCK or its hypoactivity may lead to type 2 diabetes, insulin resistance, obesity, fatty liver, aging, lipodystrophy, and death [77]. Therefore, a well-balanced PEPCK activity is needed for appropriate serum sugar concentrations and a continuous intracellular (within WAT) and systemic (between WAT and liver) TG/FA cycle during fasting. The high expression of PEPCK in the liver of LT2D rats (Fig. 4) implied that the advanced-stage of diabetes (LT2D) was analogous to the long-term fasting state, and the presence of low levels of G6Pase may imply that most of the remaining PEPCK activity was directed more toward glyceroneogenesis than gluconeogenesis due to higher demand for TG/FA cycle under fasting (diabetic) condition. The TG/FA cycle creates a balance between lipolysis and re-esteri cation and to prevent an overt release of FA into the blood via increasing the uptake of TG into the adipose tissue [78]. It is therefore possible that CCAE spared a truly signi cantly suppression of PEPCK activity, in the present study, to allow the continuous operation of TG/FA cycle, which could partially accountfor the signi cant reduction of TG levels in the sera of LT2D rats (CCAE-STZ group) (Fig. 6).
CCAE's protection against hyperglycemia and hepatic lipid accumulation, in both ET2D and LT2D, can also be attributed to the activation of PPARa expression and the inhibition of the expression of CPT1 in the liver (Fig. 5).PPARa agonists are used as remedies for both hyperglycemia and steatohepatitis [60,67,79].

Conclusion
The glucose tolerance was signi cantly impaired in both early-and late-stages of diabetes. C. colocynthis seed extract (CCAE) showed acute (in ET2D) and chronic (in both ET2D and LT2D) hypoglycemic effects. ET2D and LT2D seemed to be analogous to short-and long-term fasting states, respectively. CCAE affected both the sugar and fat metabolic pathways. It inhibited the expression of G6Pase, and therefore glycogenolysis, in the liver of ET2D rats, but its glucose-lowering activity in LT2D rats had little to do with the reduction of PEPCK expression; CCAE seemed to lower the rate of gluconeogenesis pathway mainly by rerouting PEPCK activity toward glyceroneogenesis. Low blood and hepatic TG could be the outcomes of the enhanced hepatic FA utilization, due to an up-regulated PPARa expression and down-regulated CPT1 expression, as well as the enhanced intracellular (within WAT) and systemic (between WAT and liver) TG/FA recycling due to an improved glyceroneogenesis.More study is needed to establish the effect of CCAE on PEPCK and the glyceroneogenic pathway.  Figure 1 Typical HPLC chromatogram of CCAE.

Figure 2
Weekly FBS and body weights. After diabetes induction (i.e., on day zero), FBS increased 0. 44   C-peptide and HbA1c levels on day 28. C-peptide levels indicate that pancreatic β-cells were totally or partially destroyed in STZ-and NIA/STZ-rat groups, respectively, and that CCAE did not seem to increase insulin secretion from the remaining β-cells. HbA1C levels increased in diabetic groups; 28 days of treatment with CCAE did not produce any change in HbA1C levels of treated diabetic groups relative to untreated counterparts. Con: non-diabetic control, CCAE-Con: non-diabetic control treated with CCAE, NIA/STZ: early-stage diabetic group, CCAE-NIA/STZ: the early-stage diabetic group treated with CCAE, STZ: late-stage diabetic group, CCAE-STZ: the late-stage diabetic group treated with CCAE. *, p<0.05; †, p=0.001.   Effect of CCAE on hepatic protein carbonylation and triglyceride levels. Increased levels of PCC (LT2D and ET2D vs Con, p=0.73 and p=0.19, respectively) and TG were observed in the liver tissue of both diabetic groups compared to Con. Treatment with CCAE led to a signi cant reduction of both variables in all treated groups compared to their untreated controls. Con: non-diabetic control, CCAE-Con: non-diabetic  Effect of CCAE on liver histology. Liver histology was studied using H&E and Oil Red O staining.
According to H&E, large macrovesicular LDs accumulated in the liver from early-stage T2D, which were eliminated by CCAE treatment. Lipid in LT2D liver was less notable perhaps due to its different (microvesicular) morphology. Fibrosis of the portal area and broblast accumulation in the liver were other remarkable manifestations in the late-stage diabetic liver tissues which seemed not to improve by CCAE treatment. Oil Red O staining approved that in diabetic livers, fat accumulated in greater extent and treatment with CCAE tended to decrease the hepatic fat.

Figure 8
Schematic presentation of sugar and fat metabolism and the effect of CCAE. Low FAoxidation/disposal may give rise to the fatty liver when accompanied by increased DNL, as it may occur in ET2D. Enhanced -oxidation of FAs should prevent fatty liver, unless the rate of FA entry into the liver overwhelms the capacity of the mitochondria, as it may occur in LT2D, when it may lead to mitochondrial dysfunction. The attenuation of free radical scavenging mechanisms would then lead to fatty liver, oxidative stress, brosis, and liver damage. Citrullus colocynthis aqueous seed extract (CCAE) was able to ght against hyperglycemia and fatty liver in many frontlines, including free radical scavenging and lowering ROS levels, inhibiting CPT1, activating the expression of PPARα, and directing PEPCK activity more towards glyceroneogenesis than towards gluconeogenesis. In addition to gluconeogenesis and glyceroneogenesis, another important function of PEPCK is to carry out cataplerosis, the removal of citric acid cycle intermediates to prevent their accumulation in mitochondrial matrix, which has not been shown [74].

Supplementary Files
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