(E)-9-oxooctadec-10-en-12-ynoic acid from Ixora brachiata Roxb. increases glucose uptake in L6 myotubes by activating the PI3K pathway.

In an attempt to discover new scaffolds for anti-diabetic activity from plants, we screened extracts from Ixora brachiata Roxb. for their effect on glucose uptake in L6 myotubes. The petroleum (PE) extract of the plant showed a significant increase in insulin stimulated glucose uptake by L6 myotubes. The bioactivity guided fractionation of the crude extract yielded a compound (E)-9-oxooctadec-10-en-12-ynoic acid (OEA). The compound induced a dose dependent increase in insulin stimulated glucose uptake in L6 myotubes with an EC50 of 22.96µM. OEA also increased the phosphorylation of IRS-1, Akt and AS160 leading to increased GLUT4 translocation to the plasma membrane indicating that it promotes insulin stimulated glucose uptake in L6 myotubes by activating the PI3K pathway.

NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKß are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

NF-κB dependent anti-inflammatory activity of chlorojanerin isolated from Saussurea heteromalla.

Medicinal plants have shown great promise as a source of novel drug compounds for the treatment of inflammatory disorders. In our search for new entities with anti-inflammatory potential, the extracts of the whole plant of Saussurea heteromalla (family-Asteraceae), collected from Himalayas, were evaluated in the high throughput screen for TNF-α and IL-6 inhibitors. The extract blocked TNF-α and IL-6 production in LPS stimulated THP-1 cells (human acute monocyte leukemia cell line) completely at 10 and 30 µg/ml. The plant has been found as a new source of chlorojanerin, a guaianolide type of sesquiterpene lactone. Chlorojanerin was shown to be significantly effective in inhibiting TNF-α and IL-6 production in LPS-stimulated THP-1 cells (IC(50)=2.3±0.2 µM and 1.8±0.7 µM respectively). The compound also blocked TNF-α and IL-6 production from LPS-stimulated human monocytes (IC(50)=1.5±0.4 and 0.7±0.2 µM respectively) and synovial cells from a patient with rheumatoid arthritis (IC(50)<0.03 and 0.5 µM respectively). Transcriptional profiling of the LPS stimulated THP-1 cells revealed that chlorojanerin exerted its anti-inflammatory effect by inhibiting the expression of 8 genes involved in activating the transcription factor - NF-κB. Real time analysis of these genes validated the effect of chlorojanerin on the classical downstream targets of NF-κB. Thus, this study clearly delineated 8 genes which were specifically mitigated due to the effect of chlorojanerin on NF-κB induced signaling at the mRNA level. Further, chlorojanerin at 5 µM also inhibited the binding of NF-κB in a GFP reporter assay system by 55.5% thus validating the microarray gene expression data. This work is a step towards the isolation and characterization of lead anti-inflammatory agents from the extract of Saussurea heteromalla, which can be developed into better therapeutic molecules targeted towards some specific inflammatory diseases.

A preferential p110alpha/gamma PI3K inhibitor attenuates experimental inflammation by suppressing the production of proinflammatory mediators in a NF-kappaB-dependent manner.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the increased production and/or biological activity of proinflammatory cytokines (e.g., TNF-alpha, IL-6). The production of proinflammatory cytokines is controlled at the gene level by the activity of transcription factors, such as NF-kappaB. Phosphatidylinositol 3-kinase (PI3K), a lipid kinase, is known to induce the activation of NF-kappaB. Given this, we hypothesized that inhibitors of PI3K activation would demonstrate anti-inflammatory potential. Accordingly, we studied the effects of a preferential p110alpha/gamma PI3K inhibitor (compound 8C; PIK-75) in inflammation-based assays. Mechanism-based assays utilizing human cells revealed that PIK-75-mediated inhibition of PI3K activation is associated with dramatic suppression of downstream signaling events, including AKT phosphorylation, IKK activation, and NF-kappaB transcription. Cell-based assays revealed that PIK-75 potently and dose dependently inhibits in vitro and in vivo production of TNF-alpha and IL-6, diminishes the induced expression of human endothelial cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1), and blocks human monocyte-endothelial cell adhesion. Most importantly, PIK-75, when administered orally in a therapeutic regimen, significantly suppresses the macroscopic and histological abnormalities associated with dextran sulfate sodium-induced murine colitis. The efficacy of PIK-75 in attenuating experimental inflammation is mediated, at least in part, due to the downregulation of pertinent inflammatory mediators in the colon. Collectively, these results provide first evidence that PIK-75 possesses anti-inflammatory potential. Given that PIK-75 is known to exhibit anti-cancer activity, the findings from this study thus reinforce the cross-therapeutic functionality of potential drugs.