Silymarin Induces Insulin Resistance through an Increase of Phosphatase and Tensin Homolog in Wistar Rats

Background and aims

Phosphatase and tensin homolog (PTEN) is a phosphoinositide phosphatase that regulates crucial cellular functions, including insulin signaling, lipid and glucose metabolism, as well as survival and apoptosis. Silymarin is the active ingredient in milk thistle and exerts numerous effects through the activation of PTEN. However, the effect of silymarin on the development of insulin resistance remains unknown.

Methods

Wistar rats fed fructose-rich chow or normal chow were administered oral silymarin to identify the development of insulin resistance using the homeostasis model assessment of insulin resistance and hyperinsulinemic- euglycemic clamping. Changes in PTEN expression in skeletal muscle and liver were compared using western blotting analysis. Further investigation was performed in L6 cells to check the expression of PTEN and insulin-related signals. PTEN deletion in L6 cells was achieved by small interfering ribonucleic acid transfection.

Results

Oral administration of silymarin at a dose of 200 mg/kg once daily induced insulin resistance in normal rats and enhanced insulin resistance in fructose-rich chow-fed rats. An increase of PTEN expression was observed in the skeletal muscle and liver of rats with insulin resistance. A decrease in the phosphorylation of Akt in L6 myotube cells, which was maintained in a high-glucose condition, was also observed. Treatment with silymarin aggravated high-glucose-induced insulin resistance. Deletion of PTEN in L6 cells reversed silymarin-induced impaired insulin signaling and glucose uptake.

Conclusions

Silymarin has the ability to disrupt insulin signaling through increased PTEN expression. Therefore, silymarin should be used carefully in type-2 diabetic patients.     

A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins

We demonstrate the existence of a large endoplasmic reticulum (ER)-localized multiprotein complex that is comprised of the molecular chaperones BiP; GRP94; CaBP1; protein disulfide isomerase (PDI); ERdj3, a recently identified ER Hsp40 cochaperone; cyclophilin B; ERp72; GRP170; UDP-glucosyltransferase; and SDF2-L1. This complex is associated with unassembled, incompletely folded immunoglobulin heavy chains. Except for ERdj3, and to a lesser extent PDI, this complex also forms in the absence of nascent protein synthesis and is found in a variety of cell types. Cross-linking studies reveal that the majority of these chaperones are included in the complex. Our data suggest that this subset of ER chaperones forms an ER network that can bind to unfolded protein substrates instead of existing as free pools that assembled onto substrate proteins. It is noticeable that most of the components of the calnexin/calreticulin system, which include some of the most abundant chaperones inside the ER, are either not detected in this complex or only very poorly represented. This study demonstrates an organization of ER chaperones and folding enzymes that has not been previously appreciated and suggests a spatial separation of the two chaperone systems that may account for the temporal interactions observed in other studies.     

Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

Liver fibrosis is a common characteristic of chronic liver diseases. The activation of hepatic stellate cells (HSCs) plays a key role in fibrogenesis in response to liver injury, yet the mechanism by which damaged hepatocytes modulate the activation of HSCs is poorly understood. Our previous studies have established that liver-specific deletion of O-GlcNAc transferase (OGT)leads to hepatocyte necroptosis and spontaneous fibrosis. Here, we report that OGT-deficient hepatocytes secrete trefoil factor 2 (TFF2) that activates HSCs and contributes to the fibrogenic process. The expression and secretion of TFF2 are induced in OGT-deficient hepatocytes but not in WT hepatocytes. TFF2 activates the platelet-derived growth factor receptor beta signaling pathway that promotes the proliferation and migration of primary HSCs. TFF2 protein expression is elevated in mice with carbon tetrachloride-induced liver injury. These findings identify TFF2 as a novel factor that mediates intercellular signaling between hepatocytes and HSCs and suggest a role of the hepatic OGT–TFF2 axis in the process of fibrogenesis.     

Additional N-glycosylation in the N-terminal region of recombinant human alpha-1 antitrypsin enhances the circulatory half-life in Sprague-Dawley rats

Glycosylation affects the circulatory half-lives of therapeutic proteins. However, the effects of an additional N-glycosylation in the unstructured region or the loop region of alpha-1 antitrypsin (A1AT) on the circulatory half-life of the protein are largely unknown. In this study, we investigated the role of an additional N-glycosylation site (Q4N/D6T, Q9N, D12N/S14T, A70N, G148T, R178N, or V212N) to the three naturally occurring N-glycosylation sites in human A1AT. A single-dose (445 μg/kg) pharmacokinetic study using male Sprague-Dawley rats showed that, among the seven recombinant A1AT (rA1AT) mutants, Q9N and D12N/S14T showed the highest serum concentration and area under the curve values, as well as similar circulatory half-lives that were 2.2-fold higher than plasma-derived A1AT and 1.7-fold higher than wild-type rA1AT. We further characterized the Q9N mutant regarding the N-glycan profile, sialic acid content, protease inhibitory activity, and protein stability. The results indicate that an additional N-glycosylation in the flexible N-terminal region increases the circulatory half-life of rA1AT without altering its protease inhibitory activity. Our study provides novel insight into the use of rA1AT for the treatment of emphysema with an increased injection interval relative to the clinically used plasma-derived A1AT.     

Anti-inflammatory property of the urinary metabolites of nobiletin in mouse

Nobiletin, a major component of polymethoxyflavones in citrus fruits, has a broad spectrum of health beneficial properties including anti-inflammatory and anti-carcinogenic activities. The metabolite identification of nobiletin in mouse urine has concluded that it undergoes mono-demethylation (3'- and 4'-demethylnobiletin) and di-demethylation (3',4'-didemethylnobiletin) metabolic pathway. Biological screening of nobiletin and its metabolites has revealed that the metabolites possess more potent anti-inflammatory activity than their parent compound. Therefore, this letter reports the identification of nobiletin metabolites and their anti-inflammatory activity against LPS-induced NO production and iNOS, COX-2 protein expression in RAW264.7 macrophage.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.     

Induction of endothelial apoptosis by 4-hydroxyhexenal.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.