Data_Sheet_1_Extracellular enzyme stoichiometry reveals carbon and nitrogen limitations closely linked to bacterial communities in China’s largest saline lake.docx

Saline lakes possess substantial carbon storage and play essential roles in global carbon cycling. Benthic microorganisms mine and decompose sediment organic matter via extracellular enzymes to acquire limiting nutrients and thus meet their element budgets, which ultimately causes variations in sediment carbon storage. However, current knowledge about microbial nutrient limitation and the associated organic carbon changes especially in saline lake remains elusive. Therefore, we took Qinghai Lake, the largest saline lake of China, as an example to identify the patterns and drivers of microbial metabolic limitations quantified by the vector analyses of extracellular enzyme stoichiometry. Benthic microorganisms were dominantly colimited by carbon (C) and nitrogen (N). Such microbial C limitation was aggravated upon the increases in water salinity and sediment total phosphorus, which suggests that sediment C loss would be elevated when the lake water is concentrated (increasing salinity) and phosphorus becomes enriched under climate change and nutrient pollution, respectively. Microbial N limitation was predominantly intensified by water total nitrogen and inhibited by C limitation. Among the microbial drivers of extracellular enzyme investments, bacterial community structure consistently exerted significant effects on the C, N, and P cycles and microbial C and N limitations, while fungi only altered the P cycle through species richness. These findings advance our knowledge of microbial metabolic limitation in saline lakes, which will provide insights towards a better understanding of global sediment C storage dynamics under climate warming and intensified human activity.</p

Image_3_Anti-inflammatory Effects of Curcumin in Microglial Cells.JPEG

<p>Lipoteichoic acid (LTA) induces neuroinflammatory molecules, contributing to the pathogenesis of neurodegenerative diseases. Therefore, suppression of neuroinflammatory molecules could be developed as a therapeutic method. Although previous data supports an immune-modulating effect of curcumin, the underlying signaling pathways are largely unidentified. Here, we investigated curcumin’s anti-neuroinflammatory properties in LTA-stimulated BV-2 microglial cells. Inflammatory cytokine tumor necrosis factor-α [TNF-α, prostaglandin E2 (PGE2), and Nitric Oxide (NO] secretion in LTA-induced microglial cells were inhibited by curcumin. Curcumin also inhibited LTA-induced inducible NO synthases (iNOS) and cyclooxygenase-2 (COX-2) expression. Subsequently, our mechanistic studies revealed that curcumin inhibited LTA-induced phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, Akt and translocation of NF-κB. Furthermore, curcumin induced hemeoxygenase (HO)-1HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf-2) expression in microglial cells. Inhibition of HO-1 reversed the inhibition effect of HO-1 on inflammatory mediators release in LTA-stimulated microglial cells. Taken together, our results suggest that curcumin could be a potential therapeutic agent for the treatment of neurodegenerative disorders via suppressing neuroinflammatory responses.</p

Ghrelin Inhibits the Differentiation of T Helper 17 Cells through mTOR/STAT3 Signaling Pathway

<div><p>Enhanced activity of interleukin 17 (IL-17) producing T helper 17 (Th17) cells plays an important role in autoimmune and inflammatory diseases. Significant loss of body weight and appetite is associated with chronic inflammation and immune activation, suggesting the cross talk between immune and neuroendocrine systems. Ghrelin has been shown to regulate the organism immune function. However, the effects of ghrelin on the differentiation of Th17 cells remain elusive. In the present study, we observed the enhanced differentiation of Th17 cells in spleens of growth hormone secretagogue receptor 1a (GHSR1a)^-/- mice. Treatment of ghrelin repressed Th17 cell differentiation in a time- and concentration-dependent manner. Phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) was increased in the spleens of GHSR1a^-/- mice. Activation of mTOR signaling by injection of Cre-expressiong adenovirus into tuberous sclerosis complex 1 (TSC1) ^loxp/loxp mice increased the differentiation of Th17 cells in spleen, which was associated with an increment in the phosphorylation of STAT3. Activation of mTOR signaling by leucine or overexpression of p70 ribosome protein subunit 6 kinase 1 (S6K1) activated mTOR signaling in isolated T cells, while reversed the ghrelin-induced inhibition of iTh17 cell differentiation. In conclusion, mTOR mediates the inhibitory effect of ghrelin on the differentiation of Th17 cells by interacting with STAT3.</p></div

Peripheral Effects of Nesfatin-1 on Glucose Homeostasis

<div><p>Aims/hypothesis</p><p>The actions of peripherally administered nesfatin-1 on glucose homeostasis remain controversial. The aim of this study was to characterize the mechanisms by which peripheral nesfatin-1 regulates glucose metabolism.</p><p>Methods</p><p>The effects of nesfatin-1 on glucose metabolism were examined in mice by continuous infusion of the peptide via osmotic pumps. Changes in AKT phosphorylation and Glut4 were investigated by Western blotting and immnuofluorescent staining. Primary myocytes, adipocytes and hepatocytes were isolated from male mice.</p><p>Results</p><p>Continuous peripheral infusion of nesfatin-1 altered glucose tolerance and insulin sensitivity in mice fed either normal or high fat diet<b>,</b> while central administration of nesfatin-1 demonstrated no effect. Nesfatin-1 increases insulin secretion in vivo, and in vitro in cultured min6 cells. In addition, nesfatin-1 up-regulates the phosphorylation of AKT in pancreas and min6 islet cells. In mice fed normal diet, peripheral nesfatin-1 significantly increased insulin-stimulated phosphorylation of AKT in skeletal muscle, adipose tissue and liver; similar effects were observed in skeletal muscle and adipose tissue in mice fed high fat diet. At basal conditions and after insulin stimulation, peripheral nesfatin-1 markedly increased GLUT4 membrane translocation in skeletal muscle and adipose tissue in mice fed either diet. In vitro studies showed that nesfatin-1 increased both basal and insulin-stimulated levels of AKT phosphorylation in cells derived from skeletal muscle, adipose tissue and liver.</p><p>Conclusions</p><p>Our studies demonstrate that nesfatin-1 alters glucose metabolism by mechanisms which increase insulin secretion and insulin sensitivity via altering AKT phosphorylation and GLUT 4 membrane translocation in the skeletal muscle, adipose tissue and liver.</p></div

Ghrelin inhibited the differentiation of Th17 cells <i>in vitro</i>.

<p>(A&B) Total T cells were isolated from mouse spleens and induced to differentiate into Th17 cells with TGF-β (5 ng/ml) and IL-6 (20 ng/ml), then treated with ghrelin with final concentration (A) and time (B) as indicated. The mRNA level of RORγt was analyzed with RT-PCR. (C) Differentiated Th17 cells were treated with ghrelin (10^–8 M). The mRNA level of IL-17A was analyzed with RT-PCR. (D)The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) Cells were stimulated with PMA, Ionomycin and Brefeldin A for 4–6 hours. The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

Effects of peripheral nesfatin-1 infusion on phosphorylation of AKT in mice fed high fat diet.

<p>Mice were fed high fat diet for 8 weeks, and then received subcutaneous infusion of saline (control) or nesfatin-1 for 2 weeks. Insulin at a dose of 2 IU/kg was injected intraperitoneally 10 min before animals were sacrificed. Phospho AKT (ser473) and AKT were detected by Western blotting using specific antibodies, in which AKT was used as internal controls. Shown are representative results from saline (control) and nesfatin-1 infusion mice with or without insulin injection. Levels of AKT phosphorylation were examined in skeletal muscle (A), adipose tissue (B) and liver (C) in mice fed high fat diet. Intensity of phosphor-AKT signals were quantified by NIH Image J software and expressed as mean±SEM. *<i>P</i><0.05 vs. insulin stimulation without nesfatin-1 treatment. Six samples were examined for each condition.</p

Effects of peripheral nesfatin-1 infusion on GLUT4 in mice fed high fat diet.

<p>Immunofluorescent staining was performed using specific antibody against GLUT4 (red) in skeletal muscle and adipose tissue derived from mice consuming high fat diet and either saline (control) or nesfatin-1. Nuclei were stained with Hoechst dye. Controls include substituting primary antibodies with mouse IgG. Shown were representative results from six individual experiments. Images depict immunofluorescent staining for GLUT4 (red) in skeletal muscle (A) and adipose tissue (B). Data are expressed as mean±SEM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071513#pone-0071513-g004" target="_blank">Figure 4</a>. *<i>P</i><0.05 vs. control, # <i>P</i><0.05 vs. insulin stimulation without nesfatin-1 treatment. Shown were representative results from six individual experiments.</p

Enhanced differentiation of Th17 cells in GHSR1a^-/- mice.

<p>8 to 10-wk-old GHSR1a^WT (n = 5) and GHSR1a^-/- (n = 6) male mice were fed standard chow. (A) Total T cells were isolated from the spleen of GHSR1a^WT and GHSR1a^-/- mice. The mRNA level of GHSR1a was evaluated to verify the deficiency of GHSR1a. (B&C) The mRNA levels of RORγt and IL-17A in splenic total T cells and CD4⁺ T cells were analyzed with RT-PCR. (D) The concentration of IL-17A in serum was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus wild type control.</p

Leucine rescued the inhibitory effect of ghrelin on Th17 cells.

<p>Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells. (A) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine (1 mM). The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B&C) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine with final concentration as indicated. The mRNA levels of RORγt (B) and IL-17A (C) were analyzed with RT-PCR. (D) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine (1 mM). The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

mTOR signaling pathway mediated the inhibitory effect of ghrelin on Th17 cells.

<p>(A) Total T cells were isolated from spleens of GHSR1a ^WT and GHSR1a^-/- mice. The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B) 8 to 10-wk-old TSC1^loxp/loxp mice were injected intravenously through caudal vein with GFP or Cre virus for 4 weeks. The location of Ad-GFP in spleen was observed using immunoluorescence microscopy. (C) Total T cells were isolated from the spleens of mice injected with Ad-GFP or Ad-Cre. The expression of TSC1 and the phosphorylation of S6 were analyzed with Western Blot. Relative protein signal intensity was quantified. (D) Total T cells were isolated from the spleens of mice injected with Ad-GFP and Ad-Cre. The mRNA levels of RORγt and IL-17A were analyzed with RT-PCR. (E) The concentration of IL-17A in mice serum was examined with ELISA. *<i>P</i><0.05 versus Ad-GFP injected group.</p