Iron overload by transferrin receptor protein 1 regulation plays an important role in palmitate-induced insulin resistance in human skeletal muscle cells.

Free fatty acid is considered to be one of the major pathogenic factors of inducing insulin resistance. The association between iron disturbances and insulin resistance has recently begun to receive a lot of attention. Although skeletal muscles are a major tissue for iron utilization and storage, the role of iron in palmitate (PA)-induced insulin resistance is unknown. We investigated the molecular mechanism underlying iron dysregulation in PA-induced insulin resistance. Interestingly, we found that PA simultaneously increased intracellular iron and induced insulin resistance. The iron chelator deferoxamine dramatically inhibited PA-induced insulin resistance, and iron donors impaired insulin sensitivity by activating JNK. PA up-regulated transferrin receptor 1 (tfR1), an iron uptake protein, which was modulated by iron-responsive element-binding proteins 2. Knockdown of tfR1 and iron-responsive element-binding proteins 2 prevented PA-induced iron uptake and insulin resistance. PA also translocated the tfR1 by stimulating calcium influx, but the calcium chelator, BAPTA-AM, dramatically reduced iron overload by inhibiting tfR1 translocation and ultimately increased insulin sensitivity. Iron overload may play a critical role in PA-induced insulin resistance. Blocking iron overload may thus be a useful strategy for preventing insulin resistance and diabetes.-Cui, R., Choi, S.-E., Kim, T. H., Lee, H. J., Lee, S. J., Kang, Y., Jeon, J. Y., Kim, H. J., Lee, K.-W. Iron overload by transferrin receptor protein 1 regulation plays an important role in palmitate-induced insulin resistance in human skeletal muscle cells.

GADD45γ regulates TNF-α and IL-6 synthesis in THP-1 cells.

OBJECTIVE AND DESIGN: This study investigated the link between growth arrest and DNA damage 45γ (GADD45γ) expression and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) synthesis. METHODS: We stimulated THP-1 monocyte cells using lipopolysaccharide (LPS). We knocked-down and over-expressed GADD45γ using lentiviral vectors harboring GADD45γ short hairpin RNA and GADD45γ open reading frame, respectively. To inhibit activation of c-Jun-terminal kinase (JNK), we used a specific inhibitor, SP600125. RESULTS: LPS stimulation of THP-1 cells resulted in increased expression of GADD45γ mRNA which reached its peak 2 h after stimulation and gradually diminished thereafter. TNF-α and IL-6 were up-regulated at both the mRNA and protein levels in activated THP-1 cells. Knock-down of GADD45γ reduced TNF-α protein production by up to 75 % and IL-6 protein by up to 60 %. In contrast, over-expression of GADD45γ increased TNF-α production by six-fold and IL-6 protein by 80-fold. There was a discrepancy between TNF-α mRNA and its protein level, whereas IL-6 mRNA and its protein level were correlated. Knock-down of GADD45γ decreased the JNK activity, suggesting that JNK may play the role of a downstream mediator for the pro-inflammatory effects of GADD45γ. CONCLUSIONS: We show evidence that GADD45γ may regulate TNF-α and IL-6 expression in activated THP-1 monocyte cells.

Growth arrest and DNA damage 45γ is required for caspase-dependent renal tubular cell apoptosis.

BACKGROUND: Growth Arrest and DNA Damage 45γ (GADD45γ) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs. METHODS: Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45γ expression was silenced, HRE cells were transduced with a plasmid encoding GADD45γ-specific shRNA. The recombinant adenovirus containing the GADD45γ gene was synthesized to overexpress GADD45γ protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry. RESULTS: Expression of the GADD45γ gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45γ, and significantly augmented by the overexpression of GADD45γ. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45γ, and was augmented by the overexpression of GADD45γ, indicating that caspase activation is dependent on the expression of GADD45γ. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part. CONCLUSIONS: To our knowledge, this is the first study to show that GADD45γ is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

Activin A stimulates IgA expression in mouse B cells.

In this study, a potential role for activin A in mouse immunoglobulin (Ig) regulation was investigated. We observed that activin A increased IgA secretion in B lymphoma cells. In contrast, little effect was observed on IgM and IgG2b secretion. Activin A also significantly increased surface IgA expression and Ig germ-line alpha transcript (GLT(alpha)) levels. In parallel, activin A increased GLT(alpha) and post-switch transcripts alpha (PST(alpha)) expression in normal B cells, which was augmented by IL-5. An increase in IgA production by surface IgA negative B cells by activin A was apparent. Finally, the increase of IgA secretion by activin A was blocked by an activin receptor inhibitor (SB431542). The increase of GLT(alpha) by activin A was augmented by Smad3/4 overexpression and abolished by Smad3 dominant negative overexpression. These results suggest that activin A induces IgA isotype switching via Smad3/4-mediated germ line alpha transcription.

Bitter melon extract ameliorates palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in HepG2 cells and high-fat/high-fructose-diet-induced fatty liver.

BACKGROUND: Bitter melon (BM) improves glucose level, lipid homeostasis, and insulin resistance in vivo. However, the preventive mechanism of BM in nonalcoholic fatty liver disease (NAFLD) has not been elucidated yet. AIM & DESIGN: To determine the protective mechanism of bitter melon extract (BME), we performed experiments in vitro and in vivo. BME were treated palmitate (PA)-administrated HepG2 cells. C57BL/6J mice were divided into two groups: high-fat/high-fructose (HF/HFr) without or with BME supplementation (100 mg/kg body weight). Endoplasmic reticulum (ER) stress, apoptosis, and biochemical markers were then examined by western blot and real-time PCR analyses. RESULTS: BME significantly decreased expression levels of ER-stress markers (including phospho-eIF2α, CHOP, and phospho-JNK [Jun N-terminal kinases]) in PA-treated HepG2 cells. BME also significantly decreased the activity of cleaved caspase-3 (a well known apoptotic-induced molecule) and DNA fragmentation. The effect of BME on ER stress-mediated apoptosis in vitro was similarly observed in HF/HFr-fed mice in vivo. BME significantly reduced HF/HFr-induced hepatic triglyceride (TG) and serum alanine aminotransferase (ALT) as markers of hepatic damage in mice. In addition, BME ameliorated HF/HFr-induced serum TG and serum-free fatty acids. CONCLUSION: These data indicate that BME has protective effects against ER stress mediated apoptosis in HepG2 cells as well as in HF/HFr-induced fatty liver of mouse. Therefore, BME might be useful for preventing and treating NAFLD.

Activin A stimulates mouse macrophages to express APRIL via the Smad3 and ERK/CREB pathways.

A proliferation-inducing ligand (APRIL) is primarily expressed by macrophages and dendritic cells, and stimulates B cell proliferation, differentiation, survival, and Ig production. In the present study, we investigated the role and signaling mechanisms of activin A in APRIL expression by mouse macrophages. Activin A markedly enhanced APRIL expression in mouse macrophages at both the transcriptional and protein levels. Overexpression of dominant-negative (DN)-Smad3 and SB431542 abrogated activin-induced APRIL transcription. Furthermore, activin A induced Smad3 phosphorylation. These results indicate that activin A enhances APRIL expression through both activin receptor-like kinase 4 (ALK4) and Smad3. In a subsequent analysis of activin A signaling, it was found that PD98059, an extracellular signal-related kinase (ERK) inhibitor, eliminated activin A-induced APRIL expression. On the other hand, overexpression of cAMP responsive element-binding protein (CREB), a molecule downstream of ERK, augmented activin A-induced APRIL expression, and this effect could be abolished by PD98059. This finding that activin A induces ERK and CREB phosphorylation suggests that ERK and CREB act as intermediates in APRIL expression. Taken together, these results demonstrate that activin A can enhance APRIL expression through two different pathways, Smad3 and ERK/CREB.

Further Characterization of Activin A-induced IgA Response in Murine B Lymphocytes.

We have recently shown that activin A, a member of TGF-beta superfamily, stimulates mouse B cells to express IgA isotype but other isotypes. In the present study, we further characterized effects of activin A on B cell growth and IgA expression. We found that activin A did not have effect on LPS-stimulated cell viability. In parallel, CFSE staining analysis revealed that activin A did not alter cell division. An increase of IgA secretion by activin A was completely abrogated by anti-activin A Ab but not by anti-TGFbeta1 Ab. In the same conditions, no other isotypes are significantly affected by each antibody treatment. Finally, activin A, as similar to TGF-beta1, increased IgA secretion by mesenteric lymph node cells. These results suggest that activin A can specifically stimulate IgA response, independent of TGF-beta in the gut.