2Unité de Biochimie et de Biologie Moléculaire, Laboratory of Biochemistry and Bioactives Natural Substances, Faculty of Science and Technology, University of Abomey-calavi 01 BP: 4521 Cotonou, Benin
3Laboratoire de Biologie et de Typage Moléculaire en Microbiologie, Faculty of Science and Technology 05 BP 1604 Cotonou, University of Abomey-calavi, Benin
4Laboratoire de Neurosciences, Unité de Formation Biosciences 22 BP 582 Abidjan 22 Université de Cocody-Abidjan (Rép. de Côte-d’Ivoire)
5INSERM U866, Physiologie de la Nutrition & Toxicologie. Université de Bourgogne. 6, Boulevard Gabriel, U.F.R Sciences de la Vie, Dijon 21000, France
Phyllanthus amarus also modulated the activities of carbohydrate-metabolizing enzymes by significantly increasing the activity of hexokinase and pyruvate kinase (p< 0.05) and significantly reducing the activity of glucose-6-phosphatase, fructose-1,6-diphosphatase and glycogen phosphorylase (p< 0.05). Phyllanthus amarus administration up-regulated mrna expression of Glucose Transporter-2 (GLUT-2), and increased lipolysis and cholesterol metabolism through up-regulation of lipoprotein lipase (LPL), Sterol Responsible Element Binding Protein-1a (STREBP-1a expression. FAS expression was down regulated. The Phyllanthus amarus induced increase in serum insulin level, glucokinase (GK), aldolase, pyruvate kinase (PK), succinate dehydrogenase (SDH), and glycogen synthase activities in addition to a higher expression of insulin receptor A (IRA), GK, SDH.
Keywords: P. amarus; Diabetes II; Gene expression ; Gene regulation ; Insulin ; Carbohydrate metabolic enzymes; lipid metabolism
Phyllanthus amarus Schumach. & Thonn is from the branch of the Magnoliophyta and the large family of Euphorbiaceae, APG 2009. Euphorbiaceae is one of the largest plant families in the world (326 genus, 7750 species). P. Amarus is used traditionally to treat hepatitis [2]. Plants of the genus Phyllanthus are widely distributed in most tropical and subtropical countries and are generally used in traditional medicine to treat chronic liver disease [3]. Still called bitter Phyllanthus (French); Henlenwe (fon); Ashasha (yoruba, nagot); Sobaru (bariba); Banna banna biriku (dendi) [4], P. Amarus is an erect grass up to 50 cm tall, glabrous. The stem and leaves are light green; the flowers and fruits are small and hanging at the tips of the twigs. This species flowers and then fructifies between March and October. P. Amarus is a pan tropical species found in forest galleries, fallows, and roadside. This plant is used as a diuretic, astringent [5] also in the treatment of diabetes [6]. The phytochemical analysis of the P. Amarus extract confirmed the presence of tannins, saponins, flavonoids and alkaloids. The plant extract contains high levels of saponins, tannins, flavonoids and alkaloids [7, 8].
Diabetes mellitus is a chronic, hereditary disease characterized by an abnormally elevated level of blood glucose (hyperglycemia) and by the excretion of the excess of glucose in the urine (glycosuria). The basic defect appears to be an absolute or relative lack of insulin or decrease in insulin receptors on the membrane of the target cells, which lead to abnormalities in carbohydrate metabolism as well as in lipid and protein metabolism [9].
Diabetes mellitus, a serious chronic metabolic disorder, is identified by hyperglycemia resulting from deficiency in insulin secretion and/or decreased reaction of the organs to insulin [10, 11].
Diabetes mellitus has been and will probably continue to be classified as growth or juvenile onset and maturity or adult onset. In both the 1980 and 1985 reports, other classes of diabetes included other types [12]. The cause for type 1 diabetes is an absolute deficiency of insulin secretion, whereas type 2 diabetes is a combination of resistance to insulin action and inadequate compensatory insulin secretory response.
The world prevalence of diabetes among adults (aged 20 - 79 years) was 6.4%, affecting 285 million adults, in 2010, and will increase to 7.7%, and 439 million adults by 2030 [13].
Epidemiological studies have suggested that dyslipidaemia is a risk factor for diabetic neuropathy [14]. In diabetic subjects overproduction of FFA and impaired lipoprotein metabolism induces an increase in plasma lipid components [15]. Type 1 and Type 2 diabetes mellitus are associated with metabolic syndrome and a marked increase in the risk of coronary heart disease [16]. Adipose tissue is now recognized as an endocrine organ that contributes to the physiopathology of type 2 diabetes. Diabetes mellitus has also been associated with an increased risk for developing premature atherosclerosis due to an increase in triglycerides (TG) and low-density lipoproteins (LDL), and decrease in high density lipoprotein levels (HDL) [17].
Abnormalities of lipoprotein metabolism cause various hypoor hyperlipoproteinemias. The most common of these is diabetes mellitus, where insulin deficiency causes excessive mobilization of FFA and underutilization of chylomicrons and VLDL, leading to hypertriacylglycerolemia. Most other pathologic conditions affecting lipid transport are due primarily to inherited defects, some of which cause hypercholesterolemia, and premature atherosclerosis.
Obesity particularly abdominal obesity is a risk factor for increased mortality, hypertension, type 2 diabetes mellitus, hyperlipidemia, hyperglycemia, and various endocrine dysfunctions [18]. In addition to the established major risk factors, atherosclerosis in type 2 diabetes is also related to alterations in lipid and lipoprotein profile [19]. Many epidemiological studies have demonstrated that type 2 diabetes mellitus is a wellknown risk factor for the development of cardiovascular disease, cerebrovascular disease, and peripheral vascular diseases [20, 21].
Many of the drugs currently used in Diabetes mellitus (DM) are expensive and side effects are of serious concern [22]. Hence, natural products with perceived cost-effectiveness and no longterm side effects but which elicit better antidiabetic activities are highly desired. The health-promoting properties of some herbal teas or Chinese herbal medicines reported to be rich in antioxidants, particularly phenolics and antioxidant vitamins, can exert inhibitory capacities against α-glucosidase and exert hypoglycemic effects [23, 24, 25].
A number of studies have shown that diabetes mellitus is associated with oxidative stress, leading to an increased production of reactive oxygen species. Antioxidant research is an important topic in the medical field as well as in the food industry. Recent research with important bioactive compounds in many plant and food materials have received much attention. The oxidation induced by ROS can result in cell membrane disintegration, membrane protein damage and DNA mutation, which can further initiate or propagate the development of many diseases, such as cancer, liver injury and cardiovascular disease [26].. Although the body possesses such defense mechanisms, as enzymes and antioxidant nutrients, which arrest the damaging properties of ROS [27]., continuous exposure to chemicals and contaminants may lead to an increase in the amount of free radicals in the body beyond its capacity to control them, and cause irreversible oxidative damage [28]. Therefore, antioxidants with free radical scavenging activities may have great relevance in the prevention and therapeutics of diseases in which oxidants or free radicals are implicated [29]. In this respect, polyphenolic compounds, like flavonoids and phenolic acids, commonly found in plants have been reported to have multiple biological effects, including antioxidant activity [30, 31, 32, 33]. Currently, the possible toxicity of synthetic antioxidants has been criticized. It is generally assumed that frequent consumption of plantderived phytochemicals from vegetables, fruit, tea, and herbs may contribute to shift the balance toward an adequate antioxidant status [34]. Thus interest in natural antioxidant, especially of plant origin, has greatly increased in recent years [35].
The relationship between Phyllanthus amarus Schumach. & Thonn and streptozotocin-induced diabetic rats with regard to carbohydrate and lipid metabolism is not fully examined. Thus, this study was conducted to test the hypothesis that streptozotocin-induced diabetic rats induces systemic alteration in antioxidant activity and lipid profiles may be ameliorated by Phyllanthus amarus Schumach. & Thonn. Administration through modulating expression of genes responsible for carbohydrate and lipid metabolism.
Total phenolics of each extract were estimated by Folinciocalteu reagent method [38]. This method is based on the reduction in alkaline media of phosphotungstic mixture (WO42- ) phosphomolybdic (moo42-) of Folin reagent by the oxidizable group of phenolic compounds, leading to the formation of blue reduction products. Latter have a maximum absorption at 765 nm whose intensity is proportional to the amount of polyphenols present in the sample. Then, 200 μl of diluted sample were added to 1 ml of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 800 μl of saturated sodium carbonate (75 g/l) was added. After 2 h of incubation at room temperature, the absorbance at 765 nm was measured. The standard calibration curve was plotted using gallic acid (y =0,043x – 0,051; R2 = 0,994). The mean of three readings was used and the results expressed as mg of Gallic Acid Equivalents (GAE)/100 mg of extract.
Condensed tannins: Condensed tannins were estimated using the method of author [40] modified by author [41] Vanillin reagent was prepared by mixing equal volume: 8%, methanol at 37% and 4% of vanillin in methanol. The mixture was maintained at 30° C before the assay. Two hundred (200) μl of each extract to be analyzed were added to 1000 μl of reagent of vanillin; the mixture was stirred and incubated in darkness at 30°C for 20 min. The absorbance was measured at 500 nm by a spectrophotometer UV (Perkin Elmer) against white consisting of a mixture of methanol (37%) and HCl (8%) with equal volume.
The glycemias were taken with a SD CHECK glucometer. The extracts were administered in the morning for 21 days. The general guidelines for the care and use of laboratory animals, recommended by the Doctoral School (Life Sciences) of the Faculty of Science and Technology (FAST) at the University of Abomey Calavi (UAC) under the number (UAC/FAST/EDSV/1112107), were followed and the protocol was approved by the Regional Ethical Committee.
The fasting serum glucose measured by using commercially available kits (Agappe Diagnostics, Ernakulam, India). Activity of glycolytic enzymes was assayed: hexokinase was estimated by the method of Crane and Sols [44]; pyruvate kinase was estimated by the method of Bucher and Pfleiderer [45]. Hepatic glycogen content was estimated by the method of Carroll et al. [46]. Gluconeogenic enzyme activities in the liver were assayed using the following procedures: glucose-6-phosphatase was estimated by the method described by Koide and Oda [47], fructose-1,6- diphosphatase was estimated by the method of Pontremoli [48], and the activity of glycogen phosphorylase was assayed by the procedure described by Singh et al. [49].
Other parts from the liver tissues were also frozen in on liquid nitrogen used for molecular analysis. One gram of each liver tissue samples were homogenized in 9 ml of the homogenizing buffer at ph 7.4 for preparation of tissue homogenate.13 This homogenate was used to assay the activities of the hepatic enzymes. The hepatic glucokinase (GK) activity was determined by measuring the glucose-6-phosphate (G6P) formed by GK by formation of nicotinamide-adenine dinucleotide phosphate in the presence of glucose-6-phosphate dehydrogenase enzyme. The activity of succinate dehydrogenase (SDH) was measured by monitoring the reduction of 2, 6-dichlorophenolindophenol at 600 nm.14 Hepatic aldolase activity was assayed by spectrophotometry at 340 nm using Aldolase Activity Colorimetric Assay kit (Biovision, Cat. No. K665-100, Milpitas, CA, USA), where one unit of aldolase activity is defined as the amount of enzyme that produced one μmol of NADH per minute at 25°C.15 Hepatic pyruvate kinase (PK) activity was determined in the liver homogenate with 50 mm glycylglycine, 15 mm ethylene diamine tetraacetic acid (EDTA), and 5 mm potassium phosphate. The total PK activity was determined in the supernatants as described.16 Glycogen synthase (GS) activity was measured in homogenates in the presence of 6.6 mm G6P, and the enzyme activity was expressed as mu/mg protein.17 Hepatic glycogen content was determined as previously described.
Hepatic homogenate protein concentration was measured using a Bio-Rad assay reagent. Total RNA was extracted from the liver tissues using rneasy mini kit (Cat. No. 74104, Qiagen, China) following the manufacturer instructions. The amount of extracted RNA was quantified and qualified using nanodrop ND- 1000 Spectrophotometer (nanodrop Technologies, Wilmington, Delaware, USA). The purity of RNA was checked and it ranged between 1.8 and 2.1 demonstrating the high quality of the RNA. First strand cdna was produced using a specific kit that was supplied by Fermentas (Pittsburgh, PA, USA).
The polymerase chain reaction (PCR) was carried out by (2×) PCR Master Mix (Fermentas Inc., Pittsburgh, PA, USA). A 2720 thermocycler (Applied Biosystems, Foster City, CA, USA) was used in performing the PCR reactions. We used 10 pmol/ μl of each forward and reverse primer for the measured genes. The housekeeping gene β-actin was used as a constitutive control for normalization.. All primers were provided by Sigma Aldrich (Chemie gmbh, Steinheim, Germany) as shown in (Table 1). Amplified PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide in 1x Tris acetate EDTA buffer (TAE) (ph: 8.3-8.5). The electrophoretic picture wasvisualized and analyzed by gel documentation system (Bio Doc Analyze, Biometra, Göttingen, Germany).
mRNA expression |
Forward primer |
Reverse primer |
PK (229 bp) |
5ʹ-ATTGCTGTGACTGGATCTGC-3ʹ |
5ʹ-CCCGCATGATGTTGGTATAG-3ʹ |
PEPCK (236 bp) |
5ʹ-TTTACTGGGAAGGCATCGAT-3ʹ |
5ʹ-TCGTAGACAAGGGGGCAC-3ʹ |
GLUT-2 (330 bp) |
5ʹ-AAGGATCAAAGCCATGTTGG-3ʹ |
5ʹ-GGAGACCTTCTGCTCAGTGG-3ʹ |
SDH (249 bp) |
5’-TGGCTTTCACTTCTCTGTTGG-3’ |
5’-ATCTCCAGTTGTCCTCTT CCA-3’ |
FAS (345 bp) |
5ʹ- CCAGAGCCCAGACAGAGAAG-3ʹ |
5ʹ-GACGCCAGTGTTCGTTCC-3ʹ |
LPL (269 bp ) |
5ʹ-CCTGATGACGCTGATTTTGT-3ʹ |
5ʹ-TATGCTTTGCTGGGGTTTTC-3ʹ |
SREBP-1a (290 bp ) |
5ʹ-ACACAGCGGTTTTGAACGACATC-3ʹ |
5ʹ-ACGGACGGGTACATCTTTACAG-3ʹ |
INSULIN RECEPTOR A (222bp) |
5’-TTCATTCAGGAAGACCTTCGA-3’ |
5’-AGGCCAGAGATGACAAGTGAC-3’ |
BETA-ACTIN (309 bp) |
5’-TCACTATCGGCAATGTGCGG-3’ |
5- GCTCAGGAGGAGCAATGATG-3’ |
Chemical compound |
Ethanolic extract |
Coumarin |
+ |
Flavonoid |
+ |
Naphtoquinone |
+ |
Alkaloid |
+ |
anthracene derivative |
+ |
saponin |
+ |
lignan |
+ |
triterpene |
+ |
Tanins |
+ |
triterpene |
+ |
Ethanolic extract |
Total Phenolic (a) compound |
Flavonoids (b) |
Condensed tannins(c) |
Phyllanthus amarus Schum. & Thonn. |
1,395 ± 0,21 |
11,815 ± 0,118 |
5,432 ± 0,05 |
(b)mg equivalent of rutin/g of extract;
(c)mg equivalent of catechin/mg of extract.
Effect of repeated dose administration of P. amarus extract on oral glucose tolerance test in streptozotocin induced diabetic rats
The STZ induced diabetic group had low serum insulin levels. The STZ induced diabetic rats had significant decrease in the mRNA expression of hepatic insulin receptor A (IRA). While animals administrated with Phyllanthus amarus Schumach. & Thonn showed a increase the levels of serum insulin (Fig 6 (a)) and hepatic IRA relative gene expression when compared with the diabetic rats (Fig 6 (b)). The Phyllanthus amarus Schumach. & Thonn action was dose dependent where the highest effects observed in rats treated with a dose of 500 mg/kg bwt, and less effect with a low dose 200 mg/kg bwt.
Hepatic glycogen content in diabetic rats was found to be significantly reduced (p< 0.05) compared with the normal control. Treatment with P. amarus enhanced the glycogen storage efficiency of liver of diabetic rats compared with diabetic control animals (Fig 7).
groups (p>0.05). The activities of pyruvate kinase (Fig 9) and hexokinase (Fig 10) were significantly diminished (p< 0.05) in STZ-induced diabetic rats as compared with normal control animals. However, P. amarus treatment significantly increased (p< 0.05) the activities of pyruvate kinase and hexokinase in liver tissues of diabetic rats.
The STZ induced diabetic rats had significant decrease in the levels of GK, SDH relative gene expression compared with control rats (p< 0.05). While animals administered with P. amarus showed an increase in the levels of hepatic, GK, SDH relative gene expression when compared with the diabetic rats (Fig 14, 15). While animals administrated with P. amarus induced the activities of hepatic aldolase, and GS, when compared with the diabetic rats (Fig 16, 17). The P. amarus action was dose dependent where the highest effects observed in rats treated with a dose of 500 mg/kg bwt, and less effect with a low dose 200 mg/kg bwt.
The main goal of this study is to determine the free radical scavenging properties screened for in vitro plant extraction of Phyllanthus amarus Schum. & Thonn. Free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The result of the present study showed that the ethanol extract of Phyllanthus amarus Schum. & Thonn., contains highest amount of phenolic compounds. Phenolic compounds are known as high-level antioxidants because of their ability to scavenge free radicals and active oxygen species, such as singlet oxygen, superoxide free radicals and hydroxyl radicals [57]. The radical-scavenging activity is attributed to replacement of hydroxyl groups in the aromatic ring systems of the phenolic compounds as a result of their hydrogen donating ability [58].
The possible mechanism of the extract may in part be attributed to its antioxidant activities. Complementing our findings, earlier studies have reported that the extract may have antioxidant activity. Phyllanthus amarus have been reported to be rich in phenolic compounds (Martin-Nizard et al. 2003) and these compounds were previously served as free radical scavengers [59]. Hyperglycemia generates reactive oxygen species (ROS), which in turn cause lipid peroxidation and membrane damage [60].
The possible mechanism of the extract may in part be attributed to its antioxidant activities. Complementing our findings, earlier studies have reported that the extract may have antioxidant activity. Phyllanthus amarus have been reported to be rich in phenolic compounds [61] and these compounds were previously served as free radical scavengers [59]. Hyperglycemia generates reactive oxygen species (ROS), which in turn cause lipid peroxidation and membrane damage [60].
The experimentally induced diabetic rats showed severe hyperglycemia interrelated with a decrease in endogenous insulin secretion and release. Rats treated with Phyllanthus amarus extract showed a significant decrease in the level of blood glucose and an increase in the level of serum insulin. These results indicated that Phyllanthus amarus produced anti-hyperglycemic activity and the glucose-lowering activity of Phyllanthus amarus may be attributed to pancreatic-enhancement of insulin secretion. Increment in plasma insulin by Phyllanthus amarus is probably due to the presence of derivatives which possess insulin secretagogue activity. It may also be due to the regeneration of pancreatic b cells which are destroyed by STZ [62].
According to previous reports, Diabetes mellitus (DM) was presented with alterations in glucose homeostasis that contribute to persistent hyperglycemia and liver plays a major role in the regulation of glucose metabolism [63]. The experimentally induced diabetic rats showed severe hyperglycemia interrelated with a decrease in endogenous insulin secretion and release. Rats treated with Phyllanthus amarus extract showed a significant decrease in the level of blood glucose and an increase in the level of serum insulin.
The phytochemical screening of Phyllanthus amarus showed the presence of constituents which were characterized and identified for the first time in this work and were having medicinal properties such as antioxidant [64]. Anti-inflammatory [65], anticancer [66], and hypoglycemic activities [67]. Hypoglycaemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid. The antidiabetic activity of Phyllanthus amarus may be attributed to the cumulative effect of these major compounds present in it.
Oral glucose tolerance test (OGTT) outcomes from the present study could lead us to believe that Phyllanthus amarus Schum. & Thonn. May repair pancreas beta cells and enhance insulin secretion that then decrease blood glucose levels. Furthermore, treatment with Phyllanthus amarus Schum. & Thonn. Improves glucose tolerance in diabetic rats, suggesting an enhanced insulin secretion. The ogttevoked hyperglycaemia is normalized after 2 h in control animals; however, the same phenomenon is decreased, but not normalized, in diabetic animals.
This study was designed to investigate the effect of the oral administration of Phyllanthus amarus Schum. & Thonn. Extract on plasma glucose, triglycerides, LDL, HDL, VLDL total cholesterol levels in normal and diabetic rats.
Our results show that Phyllanthus amarus Schum. & Thonn. Administration could decrease triglycerides, cholesterol, VLDL and LDL cholesterol and also increase HDL cholesterol. The decrease in serum triglycerides may be associated with the change in total serum Mg concentration. There is increasing evidence for the role of magnesium in the modulation of serum lipids and lipid uptake in macrophages [68].
Our hypothesis is Phyllanthus amarus induces insulin production and tissues insulin sensitivity, leading to an increase in the tissues glucose uptake, storage, and oxidation. In this study, we demonstrated the anti-hyperglycemic action of Phyllanthus amarus in STZ-induced diabetic rats. It decreased the blood glucose level in male STZ-induced diabetic rats particularly with the high dose (500 mg/kg bwt). The anti-hyperglycemic effect of Phyllanthus amarus was dose dependent. The Phyllanthus amarus extracts decreased the serum glucose levels in diabetic rats. The anti- hyperglycemic effect of Phyllanthus amarus might be exerted by increasing insulin synthesis, and release by the beta cells of the islet of Langerhans and inducing the sensitivity of cell receptors to insulin. This appears through the induction of expression of the insulin gene in pancreatic cells and IRA in hepatic cells, and increasing the serum insulin levels consequently increased glucose uptake through induction of Glut 2 gene expression. The hypoglycemic effect of Phyllanthus amarus was exerted through potentiation of insulin synthesis and release from the existing beta cells, as well as increasing the tissues sensitivity of insulin to glucose uptake [69].
The STZ induces a selective destruction of pancreatic β-cells leading to poor glucose utilization inducing hyperglycemia, but leaving many of the surviving beta cells, which can be regenerated [70]. Such regeneration is enhanced by the administration of Phyllanthus amarus, and results in stimulating insulin release through increasing the level of gene expression, and so increasing its level in the blood, which can improve glucose metabolism.
Insulin receptors are expressed with different ranges in all tissues that are sensitive to insulin [71]. This enforce our results, which showed high hepatic IRA gene expression levels in the groups that were administrated high doses of Phyllanthus amarus. That possess hypoglycemic, as well as antioxidant properties. Some flavonoids have hypoglycemic properties because they improve altered glucose and oxidative metabolisms of the diabetic states. They also exert a stimulatory effect on insulin secretion by changing Ca++ concentration [72].
The activity of enzymes like hexokinase, pyruvate kinase, glucose-6-phosphatase, and fructose-1,6-diphosphatase was markedly altered, resulting in hyperglycemia, which leads to the pathogenesis of diabetic complications [73]. The altered the activity of hexokinase and pyruvate kinase, key enzymes in the catabolism of glucose, diminishing the metabolism of glucose and ATP production in diabetic conditions. The reduction in the activities of these enzymes in the liver tissues of diabetic rats is an indication of reduced glycolysis and amplified gluconeogenesis signifying that these two pathways are distorted in diabetes. In agreement with the above reports, the activities of hexokinase and pyruvate kinase were significantly decreased in the STZinduced DM group. The PK is a glycolytic enzyme playing a central role in hepatic glucose and lipid metabolism. It is regulated by phosphorylation, allosteric modification, hormones, and nutrients. Excess glucose utilization by tissue induces glycolysis, L-Pyruvate kinase (L-PK) activity, glycogenesis, de novo-synthesis of fatty acids, and lipid storage. The L-PK gene transcription is induced by insulin stimulated glucose metabolism [74]. Treatment with 200 mg/kg and 500 mg/kg bwt of Phyllanthus amarus causes an increase in the serum insulin level (p< 0.05). The elevation in the serum insulin could result in activation of L-PK gene expression, and the enzyme activity that presented in our results, which refer to increase in the glycolytic pathway by treatment with 200 and 500 mg/kg bwt of Phyllanthus amarus, whereas treatment with 200 mg/kg bwt failed to produce this effect. The PK activity decreases as the result of diabetes and increases by the administration of insulin to diabetic rats in the liver tissues [75]. The increase in activity of PK in the liver tissue of rats is the cause of the increase in glycolysis and the decrease in gluconeogenesis as indicated by PEPCK mrna expression [76].
Administration of Phyllanthus amarus to diabetic rats significantly elevated these enzyme activities in liver. The activities of regulatory enzymes in gluconeogenesis, like glucose- 6-phosphatase and fructose-1,6-diphosphatase, are elevated in Diabetes mellitus [77] and increased activities of these enzymes in STZ-induced diabetic rats may be due to insulin insufficiency [78]. Glucose-6-phosphatase and fructose-1,6-diphosphatase are dephosphorylating enzymes which impair hepatic glucose utilization. Our results showed that the activities of glucose-6- phosphatase and fructose-1,6-diphosphatase were significantly decreased by the administration of Phyllanthus amarus. Glycogen is the primary intracellular storage form of glucose and its quantity in various tissues is a direct manifestation of insulin activity as insulin supports intracellular glycogen deposition [79]. The reduced glycogen store in diabetic rats has been attributed to the loss of glycogen synthase-activating system and/or the increased activity of glycogen phosphorylase [80]. In the present study, there was a decrease in the hepatic glycogen content of diabetic rats which suggests the increased glucose output during insulin deficiency. Here diabetic animals showed increased glycogen phosphorylase activity when compared with normal control animals. Treatment with Phyllanthus amarus restored the levels of glycogen, probably by means of decreasing the activity of glycogen phosphorylase.
Hepatic glucose utilization was induced possibly due to the induction of gene expression of the Glut 2 gene.
The latter is a membrane bound glucose transporter present mainly in the liver, and not dependent on insulin. It has a high glucose Michaelis constant (Km) and so the transporting of glucose into hepatic tissue is unlimited [81]. Glucose that is transported to the liver is either oxidized, or stored as glycogen. The Phyllanthus amarus improved the activity and gene expression of hepatic glucose catabolic enzymes GK, aldolase and SDH in harmony by increasing the level of gene expression of insulin gene and serum insulin levels. The GK activity and gene expression was increased in the Phyllanthus amarus treated rats when compared with diabetic non-treated rats.
The gene expression of GK is correlated with enzyme activity. The GK enzyme catalyzes the first hepatic glycolysis reaction, it has a low blood glucose affinity with high Km. So it has a high sensitivity to blood glucose level change [81]. The GK activity and expression levels are decreased in diabetic patients [82]. The GK is a strong diabetic therapy marker because it enhances the hepatic glucose uptake and insulin secretion from pancreatic tissue [83]. Aldolase is a bi-functional enzyme in both glycolysis and gluconeogenesis; it is closely related to PFK-1 (phosphofructokinase- 1) in its action as it cleaves its product (F1, 6 bisphosphate) into glyceraldhyde-3-phosphate and dihydroxy acetone phosphate. Its level was reported lower in the diabetic models [84], the increase in its activity after Phyllanthus amarus treatment may refer to improvement of glucose oxidation. The SDH is an oxidative mitochondrial enzyme that controls transcription of metabolism-related genes in mitochondria, and promotes glucose and lipid metabolism. The SDH mrna expression levels were reduced in diabetic animals [85]. Our study showed that treatment with 200 and 500 mg/kg bwt of Phyllanthus amarus can increase the SDH mrna levels and activity that improves the oxidative status in diabetic rats [86]. Glycogenesis is another pathway for glucose utilization in the liver that is directly affected by insulin; the decrease in insulin level in diabetic rats results in the decrease of GS activity, and so decrease in liver glycogen content [87]. Such decrease in the activity was reversed by Phyllanthus amarus treatment with doses of 200 and 500 mg/kg bwt by the correction of insulin level in the blood. The hypoglycemic action of Phyllanthus amarus was accompanied by the activation of GS, and increases the hepatic glycogen content [88]. The STZ induces a selective destruction of pancreatic β-cells, which may be regenerated giving false results.
It could be predicted that glycogen levels in tissues (muscle and liver) decreased as the influx of glucose in liver, thus, inhibited in the absence of insulin and recovered on insulin treatment [89]. Our findings showed that the administration of Phyllanthus amarus to streptozotocin-induced diabetic wistar rats significant increase in PK mrna expression without changes in PEPCK mrna expression. Moreover, Glut 2 expression increased after Phyllanthus amarus administration. As known, Glut 2 is a trans-membrane carrier protein, which enables passive glucose movement across cell membranes. Glut 2 is the principal transporter for the transfer of glucose between liver and blood, and for renal glucose reabsorption [90].
Elevated TG, cholesterol and LDL levels, and reduced HDL are the key abnormalities that constitute dyslipidemia [91]. Here, our results show that Phyllanthus amarus administration normalized the changes induced in lipid profiles suggesting an improvement in insulin sensitivity through up-regulation in LPL, STREBP-1a expression, while FAS expression was down regulated. All together shows the importance of Phyllanthus amarus as nutrient molecules which help in preventing the changes in lipid profiles and regulated the gene expression of carbohydrate, lipid metabolism and with their antioxidant activities in streptozotocin-induced diabetic rats.
Our results support the hypothesis that Phyllanthus amarus has a potential role in the management of diabetes and in the prevention of some complications in STZ-induced diabetic rats and that it may be useful in the treatment of hyperlipidemia in diabetes. Further studies will be needed to confirm the role of Phyllanthus amarus in lipid metabolism control.
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