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In-vitro Study of Hexane Fraction of P. Niruri Whole Plant

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Effect of hexane fraction of P. niruri on glucose uptake in L6 cells:

Figure 79 represents the effect of hexane fractions of P. niruri whole plant, insulin and metformin on glucose uptake by L-6 myotubes. It is evident from the results that hexane fraction of P. niruri caused increases glucose uptake in concentration dependent manner in L6 cells. The hexane fraction of P. niruri whole plant increases basal glucose uptake in L6 myotubes to a significant level at a minimum concentration of 10 μg/ml (1.26-fold, p<0.05). Maximum stimulation was observed at 20 μg/ml concentration, calculated to be around 1.52-fold (p<0.01) as compared to basal control cells. Results were compared with the standard antidiabetic drug Insulin and metformin. Insulin alone caused around 1.65-fold (p<0.01) stimulation and metformin caused nearly 1.78 fold (p<0.01) stimulation at 100nM and 500μM concentration, respectively (Fig 79).

Figure 1: Effect of hexane fraction of P. niruri whole plant on 2-deoxyglucose uptake in L6 myotubes. Cells were incubated for 16 h with different concentrations of P. niruri. After incubation myotubes were also stimulated with 500 μM concentration of metformin for 16 h, followed by the determination of 2-DG uptake. Results are expressed as in fold change.

Effect of wortmannin on hexane fraction of P. niruri whole plant stimulated glucose uptake in L6 cells:

To clarify the mechanism of the enhancement of glucose uptake by hexane fraction of P. niruri, we examined whether hexane fraction of P. niruri whole plant induced glucose uptake was reversed by wortmannin, which is a specific inhibitor for PI-3-kinase that blocks the insulin-signaling pathway. Presence of wortmannin (100 nM) completely reversed the insulin-induced glucose uptake, to the basal level (Figure 80). Treatment of cells with hexane fraction of P. niruri at 20 μg/ml conc. for 16 h, in presence of wortmannin inhibited hexane fraction of P. niruri induced glucose uptake. Hexane fraction-mediated potentiation of insulin response to increase glucose uptake was also completely abolished to basal level in presence of wortmannin as shown in Figue 80. These results suggest that the signal transduction leading to glucose uptake by hexane fraction of P. niruri is primarily mediated via PI3K dependent pathway.

Figure 2: Effect of wortmannin on insulin and hexane fraction of P. niruri whole plant induced glucose uptake in L6 myotubes. Cells were incubated in the absence (Cont) or the presence of hexane fraction of P. niruri at 20 μg/ml conc. for 16 h without or with wortmannin (100 nM). After incubation myotubes were left untreated (white bars) or stimulated with 100 nM insulin (black bars) for 20 min, followed by the determination of the glucose uptake. Results are expressed as fold stimulation over control basal.

Effect of hexane fraction of P. niruri whole plant on mRNA expression of insulin signaling pathway genes in L6 cells:

Figure 85 represents the effect of hexane fraction of P. niruri whole plant on mRNA expression of insulin signaling pathway genes in L6 cells. The gene expression profile results showed that hexane fraction of P. niruri whole plant could upregulated the expression of IRS-1(Insulin receptor substrate-1), AKT2 (Protein Kinase-B), HNF-α (Hepatic nuclear factor-α), PPAR-γ (Peroxysome proliferator activated receptor-γ) and GLUT4 gene, while expression of GSK-3β (Glycogen synthase kinase-3β) remain unchanged. These results suggest that hexane fraction of P. niruri whole plant stimulates insulin signaling pathways genes which may account for the antihyperglycemic effect of this fraction.

Figure 3: Effect of the hexane fraction of P. niruri whole plant on the expression of IRS-1, AKT2, GLUT4, PPAR-γ, GSK-3β and HNF-α genes in L6 myotubes. L6 myotubes were treated with 20 μg/ml concentrations of hexane fraction of P. niruri for 16 h and then subjected to Real Time PCR analysis. Experiments are performed in triplicate. Results shown are mean ± SE of three independent experiments. *p< 0.05, **p<0.01, relative to control.

Effect of hexane fraction of P. niruri whole plant on IRS-1, AKT, PI3-kinase and AMPK proteins in L6 cells:

Figure 82 represents the effect of hexane fraction of P. niruri whole plant on IRS-1, PI3-kinase-AKT proteins in L6 cells. The glucose uptake can be mediated by the insulin signaling pathway, which can stimulate the translocation of glucose transporter 4 (GLUT4)-containing vesicles to the plasma membrane. Subsequently, GLUT4 transports glucose across the plasma membrane into cytoplasm. To investigate the mechanistic aspects of the antidiabetic action of hexane fraction of P. niruri whole plant on expression of genes involved in insulin signaling pathway were studied by western blot analysis. As shown in the Figure 82 hexane fraction of P. niruri whole plant increases the proteins expression profile of p-IRS-1, PI3K, p-AKT, and GLUT-4 proteins. Indicating that hexane fraction of P. niruri enhances the insulin signaling pathway protein and thus increasing the glucose metabolism.

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Figure 4: Effect of hexane fraction of P. niruri whole plant on the phosphorylation of IRS-1, PI3K, AKT, and GLUT-4 proteins in L6 myotubes. L6 myotubes were treated with 20 μg/ml concentrations of hexane fraction of P. niruri whole plant for 16 h and then subjected to western blot analysis. Experiments are performed in triplicate. Results are shown in mean ± SE of three independent experiments.

Discussion

The whole plant of P. niruri has been used as natural medicine since the Vedic time. Present study was the investigation of antidiabetic effect of hexane fraction of ethanolic extract of P. niruri whole plant at the dose of 100 mg/kg of b.w in validated models of type 2 diabetes mellitus (T2DM) ie., STZ-induced diabetic rats; high-fructose high-fat diet fed low dosed STZ-induced diabetic rats; n2-STZ-induced diabetic rats and db/db mice.

T2DM is a disorder of dysregulated glucose homeostasis which may due to either alteration in insulin secretion and/or insulin sensitivity, resulting in decreased insulin stimulated glucose uptake, failure to suppress hepatic glucose production, and accumulation of hepatic lipids. Many oral antidiabetic agents are now available for the management of patients with type 2 diabetes mellitus. These agents have different modes of action and have different types of side effects. These oral agents can be used in combination to give better glycemic control than is possible with each alone. The multi-factorial pathogenicity of diabetes demands a multimodal therapeutic approach. Thus future therapeutic strategies require the combination of various types of antidiabetic agents. Therefore, better management of type 2 diabetes is sorely needed with plant based drugs that can enhance the glucose uptake and inhibit PTP1b in the muscles, hepatocytes, and adipose tissues and also inhibits intestinal α-glucosidase, and eye lens aldose reductase activity.

STZ selectively damage β cells by free radical injury of DNA fragmentation and nitric oxide generation induces diabetes in all species animals.   

STZ-induced diabetic animals are most widely used for screening the compounds including natural products for their insulinomimetic, insulinotropic and other hypoglycemic/antihyperglycemic activities (Bates et al., 2000). In diabetes the hyperglycemia also induces the elevation of plasma levels of urea, uric acid and creatinine, which are considered as the significant markers of renal dysfunction and chronic hyperglycemia is involved in the etiology of development of diabetic complications. (Daisy et al., 2009, Mahalingam and Kannabiran 2010).

The present study revealed a significant decrease of basal serum insulin after 10 weeks of STZ injection. This decrease was of percentage change, 45-49% as compared with normal non-diabetic rats. Such results agree with that of (Akhani et al., 2004) and may be ascribed to the diabetogenic effect of STZ which lead to destruction of β-cells and decreased number of insulin-containing secretory granules as indicated in the present study. On the other hand the results obtained for blood glucose profile of STZ-induced diabetic rats showed high levels of blood glucose and impaired glucose tolerance as compared with the normal non-diabetic rats. These results are in accordance with the finding of several authors using STZ-induced diabetic animals (El-Naggar et al., 2005, Helmy et al., 2007, Hassan et al., 2008). As recorded by (Caro et al., 1990) glucose intolerance could arise from either a defect in insulin secretion as in case of insulin dependent diabetes (Type 1) or a defect in insulin resistance (receptor or post-receptor defect) as in case of non insulin dependent diabetes mellitus (Type 2). Our finding on serum insulin concentration confirms the previously mentioned hypothesis.

In the present study, total lipids were increased in serum of STZ-diabetic rats as compared with the normal ones at the end of the experiment. Our results are in accordance with the finding of (Lalhlenmawia et al., 2007, Rawi et al., 1995) who recorded a marked increase of total lipids in serum of STZ-induced diabetic rats. Several investigators, however, recognized that insulin deficiency in STZ -diabetic animals brings about an enhanced breakdown of fat (Sheela et al.,1992, Rawi et al., 1998) increase in mobilization of free fatty acids from the peripheral depots (Kumar et al., 2010, Shirwaiker et al., 2004) and consequence of the uninhibited actions of lipolytic hormones (glucagon and catecholamines) on the fat depots (Ravi et al., 2005). The antihyperglycemic and anti dyslipidemic activity of the hexane fraction of the ethanolic extract was confirmed by multiple dose treatment of hexane fraction of P. niruri whole plant on STZ induced diabetic rats for 28 consecutive days. It has been found that treatment of this plant fraction significantly improved glucose tolerance, markedly elevates the serum insulin level of diabetic rats this indicates that fraction may promote the release of insulin from β cells of pancreas. As shown in the Table 5 the elevation of serum AST and ALT levels (liver function marker enzymes) may reflect the damage of the hepatic cells (Rawi et al., 1995, Kim et al., 2006), concluded that the elevation in AST and ALT levels may be due to the destructive changes in the hepatic cells as a result of toxemia. Hyperglyceamia also induces the elevation of plasma levels of urea, uric acid and creatinine, which are considered as the significant markers of renal dysfunction (Daisy et a; 2009, Mahalingam and Kannabiran, 2010) is significant increase in the serum level of urea, uric acid and creatinine in the diabetic rats when compared with respective normal control rats.

Type 2 diabetes can be produced by combination of diet [high fat (HFD) or high fructose diet] plus low dose of STZ (35 mg/kg, ip) treatment (Srinivasan et al., 2005). Short term HFD feeding produces hyperinsulinaemia and insulin resistance initially followed by treatment with STZ that causes the beta cell damage and frank hyperglycaemia in the presence of almost absolute normal insulin circulating concentrations in rats (Reed et al., 2000, Zhang et al., 2003, Srinivasan et al., 2005). These HFD-fed, low dose STZ-treated rat model, exhibits stable, long lasting hyperglycaemia and the symptoms of type 2 diabetes like polyuria, polydipsia, and polyphagia and diabetic complications such as hypertension. Hence, the treatment of hexane fraction of P. niruri(100 mg/kg) to high-fructose high fat fed low dosed STZ induced diabetic rats, significantly reversed the hyperglycemia, hyperinsulinemia and it also improves the serum lipid profile declining the levels of triglycerides, total cholesterol, LDL-cholesterol along with increasing the level of HDL-cholesterol.

The C57BL/KsJ-db/db mice at 12 weeks of age exhibited most of the human characteristics of type 2 diabetes including hyperglycemia in the fasting and fed states, hyperinsulinemia and insulin resistance (Shafrir et al., 1992). The hexane fraction of P. nirurishowed significant fall in peripheral blood glucose profile and also improved the glucose tolerance of db/db mice. Further hexane fraction of P. niruriwhole plant also showed antidyslipidemic activity in db/db mice that is comparable to rosiglitazone, at 10.0mg/kg b. w.

Therefore in present study the hexane fraction of P. niruriwas further evaluated on glucose uptake in L6 cell lines of rat skeletal muscles. Since skeletal muscle cells are considered a well-established in vitro model to study the regulation of glucose transport, since in skeletal muscle, glucose transporters are the first rate-limiting step for glucose utilization under physiological condition (Clow et al., 2004). Glut 4 exists exclusively in insulin-sensitive tissue mainly skeletal, cardiac muscle and adipose tissue and is thus the major transporter responsible for insulin-mediated whole-body uptake (Eddouks et al., 2009). Impaired glucose transport with reduced Glut-4 translocation and disturbance in insulin signaling cascade are the major defects in insulin resistance and type-2 diabetes mellitus. Results of the present study demonstrated that incubation with hexane fraction of P. niruricaused significant stimulation in glucose uptake in L6 skeletal muscle cells. Incubation with 20 μg/ml concentration of hexane fraction of P. niruristimulated the glucose uptake by around 1.52-fold, which is higher than 500 µM concentration of standard antidiabetic drug metformin which shows 1.78-fold stimulation at 20 μg/ml concentration. Since uptake of glucose is the rate limiting step in its utilization, observed antidiabetic effect of hexane fraction of P. nirurimay be mediated, at least in part, through increased utilization of glucose and skeletal muscle may be the major target of action.

Hexane fraction of P. niruri whole plant treatment to L6 muscle cell lines, stimulated glucose uptake in a concentration dependent manner. Further to investigate the mechanism of stimulation of glucose uptake, the effect of hexane fraction on cellular signaling pathways known to modulate these processes was studied. The observations demonstrate that hexane fraction stimulates glucose uptake by inducing GLUT4 translocation in a PI3K dependent manner. This was confirmed by the inhibitory effect of hexane fraction mediated glucose uptake by wortmannin, an inhibitor of PI3K activation. In order to identify the downstream activators of PI3K, an analysis was carried out on the phosphorylation status of Akt (protein kinase B) the important protein kinases shown to regulate insulin mediated GLUT4 translocation. Like insulin, hexane fraction of P. niruri whoe plant stimulates Akt protein in differentiated myotubes (L6 cells). However, hexane fraction of P. niruri treatment resulted in the activation of Akt similar to insulin, suggesting hexane fraction of P. niruri whole plant stimulate GLUT4 translocation and glucose uptake in an Akt dependent manner. Involvement of PI-3-kinase independent pathways in insulin stimulated GLUT4 translocation has been established (Fryer et al., 2002; Tan et al., 2008).


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