Hsueh-Ling Cheng

A Cell-Based Screening Identifies Compounds from the Stem of Momordica charantia that Overcome Insulin Resistance and Activate AMP-Activated Protein Kinase

Treatment of insulin resistance is a critical strategy in the prevention and management of type 2 diabetes. The crude extracts from all parts of Momordica charantia L. have been reported by many studies for the effective treatment of diabetes and related complications. However, the exact ingredients responsible for the hypoglycemic effect and the underlying mechanism of their actions have not been well characterized because of the lack of a proper assay and screening system. A new cell-based, nonradioactive, and nonfluorescent screening method was demonstrated in this study to screen for natural products from the stem of M. charantia, aiming to identify hypoglycemic components that can overcome cellular insulin resistance. The results suggest triterpenoids being potential hypoglycemic components of the plant and the mechanism underlying their action involving AMP-activated protein kinase.

Cucurbitane-type triterpenoids from the stems of momordica charantia.

Four new cucurbitane-type triterpenes, cucurbita-5,23(E)-diene-3beta,7beta,25-triol (1), 3beta-acetoxy-7beta-methoxycucurbita-5,23(E)-dien-25-ol (2), cucurbita-5(10),6,23(E)-triene-3beta,25-diol (5), and cucurbita-5,24-diene-3,7,23-trione (6), together with four known triterpenes, 3beta,25-dihydroxy-7beta-methoxycucurbita-5,23(E)-diene (3), 3beta-hydroxy-7beta,25-dimethoxycucurbita-5,23(E)-diene (4), 3beta,7beta,25-trihydroxycucurbita-5,23(E)-dien-19-al (7), and 25-methoxy-3beta,7beta-dihydroxycucurbita-5,23(E)-dien-19-al (8), were isolated from the methyl alcohol extract of the stems of Momordica charantia. The structures of the new compounds were elucidated by spectroscopic methods.

EMCD, a hypoglycemic triterpene isolated from Momordica charantia wild variant, attenuates TNF-α-induced inflammation in FL83B cells in an AMP-activated protein kinase-independent manner.

Insulin resistance is a causative factor for type 2 diabetes, whereas the development of insulin resistance is closely related to chronic inflammation induced by factors such as tumor necrosis factor-α (TNF-α). Momordica charantia, also known as bitter melon, has been used as an herbal medicine and reported to ameliorate inflammation and hyperglycemia. Previously, a triterpene 5β,19-epoxy-25-methoxy-cucurbita-6,23-diene-3β,19-diol (EMCD), purified from M. charantia L. wild variant WB24, was found to activate AMP-activated protein kinase (AMPK) and have a hypoglycaemic effect in TNF-α-treated FL83B cells. AMPK has been a target for developing anti-diabetic medicine and suggested to play a role in anti-inflammation. The current study aims to investigate if EMCD might repress TNF-α-induced inflammation via AMPK. TNF-α-induced inflammation in FL83B cells was characterized using Western blotting and reverse transcriptase-polymerase chain reaction. Consequently, the expression of inflammatory markers including inducible nitric oxide synthase (iNOS), the p65 subunit of nuclear factor-κB (NF-κB), protein-tyrosine phosphatase-1B, TNF-α and interleukin-1β were significantly elevated by TNF-α in the cell, and EMCD obviously suppressed the TNF-α-induced expression of these markers. When the effect of EMCD was tested simultaneously with epigallocatechin-3-gallate (EGCG), a catechin from green tea reported to be anti-inflammatory, EMCD showed a more obvious anti-inflammatory activity than EGCG did. Investigation of the underlying mechanism suggested that EMCD inhibited the activation of the IκB kinase (IKK) complex and the NF-κB pathway, and the effect was likely independent of AMPK. Collectively, the multiple functions of EMCD suggest it to be a potential agent in treating diabetic complications and other inflammation-related disorders.

Activation of PI 3-kinase/Akt/NF-κB and Stat3 signaling by avian reovirus S1133 in the early stages of infection results in an inflammatory response and delayed apoptosis.

Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1beta and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-kappaB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-kappaB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response.

PI 3-kinase/Akt and STAT3 are required for the prevention of TGF-β-induced Hep3B cell apoptosis by autocrine motility factor/phosphoglucose isomerase

We established Hep3B cells stably-expressing wild-type and mutated AMF/PGI with differing enzymatic activities in order to investigate how AMF/PGI affects TGF-beta-induced apoptosis, and demonstrated that AMF/PGI against TGF-beta-induced apoptosis was correlated with its enzymatic activity. AMF/PGI did not alter TGF-beta-receptor expression nor affect TGF-beta-induced PAI-1 gene promoter or Smad3/4 activity. AMF/PGI induced PI 3-kinase activity, IRS and Akt phosphorylation, which can further regulate BAD phosphorylation. Constitutively-active p110 enhanced AMF/PGI-mediated anti-apoptosis activity, and dominant negative Akt alleviated anti-TGF-beta-induced apoptosis. We also demonstrated that STAT3 is a weak anti-apoptotic agent but has an increased anti-apoptotic effect in cooperation with PI 3-kinase/Akt.

Characterization of two truncated forms of xylanase recombinantly expressed by Lactobacillus reuteri with an introduced rumen fungal xylanase gene

The xylanase R8 gene (xynR8) from uncultured rumen fungi was cloned and successfully expressed in Lactobacillus reuteri. A xylanase activity of 132.1 U/mL was found in the broth of L. reuteri R8, the transformant containing pNZ3004 vector with xynR8 gene insertion. Two distinct forms of recombinant xylanase with different hydrophobicities and molecular weights were found in the broth after purification. According to the results of Western blotting, only the T7-tag, fused in the N-terminus of XynR8, could be bound to the expressed proteins, which indicated that the C-terminus of XynR8 had been truncated. These results, combined with tryptic digestion and mass spectrometry analyses, allow us to attribute the two xylanase forms to an optional cleavage of C-terminal sequences, and XynR8A, a 13 amino acid residues truncated form, and XynR8B, a 22 amino acid residues truncated form, were the main products in the extracellular fraction of L. reuteri R8. The specific activities of XynR8A and R8B were 1028 and 395 U/mg protein. Both forms of recombinant xylanase displayed a typical endoxylanase activity when they were reacted with xylan, but XynR8A demonstrated a better specific activity, catalytic efficiency and thermostability than XynR8B according to the results of enzyme characterization. These changes in enzyme properties were highly possibly caused by the present of the β-sheet in the C-terminal undeleted fragment of XynR8A. This study demonstrates that modified forms with different enzyme properties could be produced when a gene was recombinantly expressed by a L. reuteri transformant.