Herramientas manuales

It appears that SX-fraction is capable of significantly lowering the blood glucose levels in animals and humans, implying its potential prevention and treatment of diabetes. However, the hypoglycemic mechanism of the SX-fraction or how it would work has not been yet fully understood. We then hypothesized that the SX-fraction might enhance insulin sensitization by acting on the insulin signal transduction pathway.

Currently, how or why insulin-targeted tissues/organs would become insulin-resistant remains unknown, but it has been postulated that the insulin receptor (IR) might have a primary clue. The IR is a heterotetrameric glycoprotein consisting of two -subunits and two

-subunits [96]. The binding of insulin to the -subunits of the IR induces a conformational change that leads to trans-autophosphorylation of tyrosine residues on the -subunits, activating their tyrosine kinase activity [96, 97]. One of such specific phosphorylated tyrosine residues, i.e., tyrosine 972, serves as a binding site for the phospho-tyrosine binding domains of insulin receptor substrate 1 (IRS-1) whose tyrosine residues are then phosphorylated [96, 98]. This tyrosine-phosphorylated IRS-1 acts as a docking site/molecule that binds to and activates phosphatidylinositol 3-kinase (PI3K), which in turn activates serine/threonine kinase Akt (protein kinase B) [96, 97, 99,100]. Activated Akt induces the translocation of glucose transporter 4 (GLUT4) to the plasma membrane, ultimately promoting glucose-uptake by insulin responsive cells [97, 99, 101]. This is rather a simplified scheme of the insulin signal transduction pathway, which is triggered by activation of the IR (with insulin) and undergoes a cascade of biochemical events described above. As two other insulin signal pathways have

also been postulated [98, 99], it is indeed a complex biochemical process and more studies are required for further elaboration. Nonetheless, insulin resistance (of the IR) developed by the undefined (acquired and inherited) causes would block the signal pathway, resulting in an accumulation of glucose in the circulation. This prolonged hyperglycemic milieu may further keep inactivating or insensitizing the IR/IRS-1, thereby developing into the chronic hyperglycemic state known as diabetes. Thus, one rational approach to overcome such insulin resistance would be to (re)activate the IR/IRS-1 to successfully execute the entire signal pathway.

Accordingly, we investigated the effects of glucose (Glc) on the insulin signal transduction pathway (ISTP) to learn if the hypoglycemic action of the SX-fraction would actually target this pathway. We focused on the effects of Glc and/or SX-fraction on the

phosphorylation status of IR, IRS-1, and Akt, which would adequately indicate their activation status. In addition, the Glc uptake study [101, 102] was performed to assess the

potential improvement in such Glc uptake with SX-fraction. Although this study has been published elsewhere [93], the key findings are briefly described herein.

The skeletal muscle L6 cells were used as an excellent in vitro model in this study [102] and possible effects of the SX-fraction were examined on some key biochemical parameters involved in the ISTP. Such biochemical alterations were then analyzed on ELISA (enzyme- linked immunosorbent assay). First of all, L6 cells must be differentiated to myotubes, which can then express IR, IRS-1 and other biochemical parameters [102]. In other words, L6 cells cannot simply be used for experiments, due to a lack of key parameters to be studied. Cells were allowed to grow until they reached at ~90% confluence (usually took 3 days). After discarding spent medium, fresh medium (with low serum concentration) was added to cells, which were incubated until they were differentiated to myotubes expressing IR, IRS-1, and other parameters. This differentiation process usually completes in 7-10 days.

Myotubes or differentiated L6 cells were first treated with Glc (35 mM) for 24 h and then treated with SX-fraction (300 µg/ml) or insulin (100 nM) for 15 min. All harvested cells were then subjected to ELISA to assess activities of several biochemical parameters indicated by the phosphorylation state of specific amino acid residues (tyrosine or serine). Compared to control cells (no exposure to Glc), this high Glc was indeed capable of reducing the IR activity by ~15%. However, after 24-h Glc treatment, when cells were exposed to SX-fraction for 15 min, the reduced IR activity went up to ~10% higher than controls or ~30% greater than the Glc-suppressed cell group (Figure 6A). Nearly the same IR activation was also attained with insulin, which was run as a positive control. Although Glc also inactivated IRS- 1 by ~12%, SX-fraction was capable of re-activating or reversing such Glc-suppressed IRS-1 as seen with insulin. Similarly, Akt was considerably (~42%) inactivated by Glc but its reduced activity was then significantly (~2-fold) augmented by SX-fraction as well as insulin. In addition, the reduced (~25%) Glc uptake with Glc exposure was facilitated or increased by ~45% or it was even ~20% higher than that in control cells (Figure 6B). This stimulated Glc uptake was also demonstrated by insulin (slightly better than SX-fraction). As it is known that the amount/rate of Glc uptake reflects the outcome of the ISTP, the increased Glc uptake indicates the successful completion of the ISTP while the decreased uptake indicates the interrupted, incomplete ISTP. Taken all together, these results suggest that SX-fraction may re-activate Glc-inactivated IR, triggering the ISTP to consequently facilitate Glc uptake by the cells. In other words, high Glc alone can inactivate the IR/IRS-1, interrupt the ISTP by inactivating or inhibiting other parameters, and will result in a decreased Glc uptake.

However, SX-fraction (or insulin) re-activates the Glc-suppressed IR/IRS-1, carries out the ISTP by activating or stimulating those parameters, and leads to an increased Glc uptake. Therefore, the SX-fraction appears to act on the insulin signal transduction pathway (ISTP), particularly, overcoming insulin resistance by activating IR/IRS-1 to trigger the pathway and implement the subsequent signaling events. This may then account for the hypoglycemic action of SX-fraction.

Figure 6. Effects of SXF or INS on IR phosphorylation or glucose uptake under high Glc (35 mM). (A) After a 24-h Glc treatment, followed by a 15-min exposure of cells to SXF (300 µg/ml) or INS (100 nM), the IR phosphorylation status was assessed by ELISA. All data are mean  SD from three independent experiments (*p <0.04 versus Glc-treated or **p <0.05 versus control). (B) Following a 24-h Glc treatment and a 15-min SXF/INS exposure, glucose uptake was measured using a radioactive ligand and expressed by the % relative to control (100%). The data are mean  SD from three separate experiments (*p <0.03 versus Glc-treated or **p <0.05 versus control).

Effects of Maitake Extract/SX-Fraction on Hypertension