Stored RBC transfusions leads to the systemic inflammatory response syndrome in anemic murine neonates.

OBJECTIVE: RBC transfusions (RBCT) are life-saving treatment for premature and critically ill infants. However, the procedure has been associated with the development of systemic inflammatory response syndrome (SIRS) and potentially multiple organ dysfunction syndrome (MODS) in neonates. The present study aimed to investigate the mechanisms of RBCT-related SIRS in severely anemic murine neonates. METHODS: C57BL/6 (WT), TLR4-/- and myeloid-specific triggered myeloid receptor-1 (trem1)-/- mouse pups were studied in 4 groups (n = 6 each): (1) naïve controls, (2) transfused control, (3) anemic (hematocrit 20-24%) and (4) anemic with RBC transfused using our established murine model of phlebotomy-induced anemia (PIA) and RBC transfusion. Plasma was measured for quantifying inflammatory cytokines (IFN-γ, IL-1ß, TNF-α, IL-6, MIP-1α, MIP-1ß, MIP2 and LIX) using a Luminex assay. In vitro studies included (i) sensitization by exposing the cells to a low level of lipopolysaccharide (LPS; 500 ng/ml) and (ii) trem1-siRNA transfection with/without plasma supernatant from stored RBC to assess the acute inflammatory response through trem1 by qRT-PCR and immunoblotting. RESULTS: Anemic murine pups developed cytokine storm within 2 h of receiving stored RBCs, which increased until 6 h post-transfusion, as compared to non-anemic mice receiving stored RBCTs ("transfusion controls"), in a TLR4-independent fashion. Nonetheless, severely anemic pups had elevated circulating endotoxin levels, thereby sensitizing circulating monocytes to presynthesize proinflammatory cytokines (IFN-γ, IL-1ß, TNF-α, IL-6, MIP-1α, MIP-1ß, MIP2, LIX) and express trem1. Silencing trem1 expression in Raw264.7 cells mitigated both endotoxin-associated presynthesis of proinflammatory cytokines and the RBCT-induced release of inflammatory cytokines. Indeed, myeloid-specific trem1-/- murine pups had significantly reduced evidence of SIRS following RBCTs. CONCLUSION: Severe anemia-associated low-grade inflammation sensitizes monocytes to enhance the synthesis of proinflammatory cytokines and trem1. In this setting, RBCTs further activate these monocytes, thereby inducing SIRS. Inhibiting trem1 in myeloid cells, including monocytes, alleviates the inflammatory response associated with the combined effects of anemia and RBCTs in murine neonates.

Activation of the canonical ER stress IRE1-XBP1 pathway by insulin regulates glucose and lipid metabolism.

Knockout of the transcription factor X-box binding protein (XBP1) is known to decrease liver glucose production and lipogenesis. However, whether insulin can regulate gluconeogenesis and lipogenesis through XBP1 and how insulin activates the inositol-requiring enzyme-XBP1 ER stress pathway remains unexplored. Here, we report that in the fed state, insulin-activated kinase AKT directly phosphorylates inositol-requiring enzyme 1 at S724, which in turn mediates the splicing of XBP1u mRNA, thus favoring the generation of the spliced form, XBP1s, in the liver of mice. Subsequently, XBP1s stimulate the expression of lipogenic genes and upregulates liver lipogenesis as previously reported. Intriguingly, we find that fasting leads to an increase in XBP1u along with a drastic decrease in XBP1s in the liver of mice, and XBP1u, not XBP1s, significantly increases PKA-stimulated CRE reporter activity in cultured hepatocytes. Furthermore, we demonstrate that overexpression of XBP1u significantly increases cAMP-stimulated expression of rate-limiting gluconeogenic genes, G6pc and Pck1, and glucose production in primary hepatocytes. Reexpression of XBP1u in the liver of mice with XBP1 depletion significantly increases fasting blood glucose levels and gluconeogenic gene expression. These data support an important role of XBP1u in upregulating gluconeogenesis in the fasted state. Taken together, we reveal that insulin signaling via AKT controls the expression of XBP1 isoforms and that XBP1u and XBP1s function in different nutritional states to regulate liver gluconeogenesis and lipogenesis, respectively.

The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor.

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRß) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRß's tyrosine kinase activity and directly promotes IRß interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.

Sirtuin 4 Regulates Lipopolysaccharide Mediated Leydig Cell Dysfunction.

Bacterial lipopolysaccharide (LPS) is the most important contributing factor in pathogenesis of bacterial infection in male accessory glands; and it has shown to inhibit testicular steroidogenesis and induce apoptosis. The present study demonstrates that LPS causes mitochondrial dysfunction via suppression of sirtuin 4 (SIRT4); which in turn affects Leydig cell function by modulating steroidogenesis and apoptosis. LC-540 Leydig cells treated with LPS (10 µg/ml) showed impaired steroidogenesis and increased cellular apoptosis. The mRNA and protein expression of SIRT4 were decreased in LPS treated cells when compared to controls. The obtained data suggest that the c-Jun N-terminal kinase (JNK) activation suppresses SIRT4 expression in LPS treated Leydig cells. Furthermore, the overexpression of SIRT4 prevented LPS induced impaired steroidogenesis and cellular apoptosis by improving mitochondrial function. These findings provide valuable information that SIRT4 regulates LPS mediated Leydig cell dysfunction.

HDAC7 modulates TNF-α-mediated suppression of Leydig cell steroidogenesis.

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. In the present study, we demonstrate that TNF-α activates histone deacetylases 7 (HDAC7), which regulates the expression of steroidogenic enzyme genes in Leydig cells. LC-540 Leydig cells were treated with TNF-α (10 ng/ml) for different time intervals. TNF-α treatment significantly suppressed histone H3 acetylation and methylation and, concomitantly, increased the total histone deacetylases activity in LC-540 Leydig cells. RT-PCR and western blot analysis revealed that HDAC7 was up-regulated in TNF-α-treated cells. Our results also demonstrated that an siRNA-mediated knockdown of HDAC7 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells. These findings provide valuable information that TNF-α-mediated suppression of steroidogenesis involves HDAC7 in Leydig cells.

TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1.

OBJECTIVE AND DESIGN: The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. A detailed understanding of the mechanisms by which TNF-α regulates testicular steroidogenesis will be helpful in the design of novel clinical interventions for the treatment and prevention of male reproductive disorders. Here, we report that TNF-α-mediated activation of DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is involved in the inhibition of Leydig cell steroidogenesis. MATERIALS AND METHODS: Rat testis Leydig tumor cells (LC-540) were treated with TNF-α (10 ng/ml) for different time intervals. To elucidate the pathways of intracellular signal transduction that regulate DAX-1 expression, we utilized specific inhibitors. The siRNA transfection of DAX-1 into LC-540 cells was performed by electroporation. The mRNA and protein levels were determined by RT-PCR and Western blotting, respectively. RESULTS: We found that the mRNA and protein levels of DAX-1 were increased by threefold approximately in TNF-α-treated cells when compared to controls. Staurosporine, JNK inhibitor SP600125 and ERK inhibitor PD98059 significantly decreased DAX-1 expression in TNF-α-treated Leydig cells when compared to their respective controls. Further, a siRNA-mediated knockdown of DAX-1 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells. CONCLUSIONS: These findings provide valuable information that TNF-α activates DAX-1 through JNK/ERK MAP kinase pathway which regulates the expression of steroidogenic enzyme genes in Leydig cells.

A Detailed Protocol for the Induction of Anemia and RBC Transfusion-associated Necrotizing Enterocolitis in Neonatal Mice.

Anemia is a common and serious health problem, nearly universally diagnosed in preterm infants, and is associated with increased morbidity and mortality worldwide. Red blood cell (RBC) transfusion is a lifesaving and mainstay therapy; however, it has critical adverse effects. One consequence is necrotizing enterocolitis (NEC), an inflammatory bowel necrosis disease in preterm infants. The murine model of phlebotomy-induced anemia and RBC transfusion-associated NEC enables a detailed study of the molecular mechanisms underlying these morbidities and the evaluation of potential new therapeutic strategies. This protocol describes a detailed procedure for obtaining murine pups with phlebotomy-induced anemia and delivering an RBC transfusion that develops NEC.

Protocol for measuring mitochondrial size in mouse and human liver tissues.

Mitochondrial dynamic process is important for cell viability, metabolic activity, and mitochondria health. Here, we present a protocol for measuring mitochondrial size through immunofluorescence staining, confocal imaging, and analysis in ImageJ. We describe the steps for tissue processing, antigen retrieval, mitochondrial staining using an integrating immunofluorescence assay, and computerized image analysis to measure each mitochondrial size in mouse and human liver tissues. This protocol reduces tissue sample volume and processing time for the preparation of primary cells. For complete details on the use and execution of this protocol, please refer to Pearah et al.1.

Regulation of Liver Glucose and Lipid Metabolism by Transcriptional Factors and Coactivators.

The prevalence of nonalcoholic fatty liver disease (NAFLD) worldwide is on the rise and NAFLD is becoming the most common cause of chronic liver disease. In the USA, NAFLD affects over 30% of the population, with similar occurrence rates reported from Europe and Asia. This is due to the global increase in obesity and type 2 diabetes mellitus (T2DM) because patients with obesity and T2DM commonly have NAFLD, and patients with NAFLD are often obese and have T2DM with insulin resistance and dyslipidemia as well as hypertriglyceridemia. Excessive accumulation of triglycerides is a hallmark of NAFLD and NAFLD is now recognized as the liver disease component of metabolic syndrome. Liver glucose and lipid metabolisms are intertwined and carbon flux can be used to generate glucose or lipids; therefore, in this review we discuss the important transcription factors and coactivators that regulate glucose and lipid metabolism.

Far-western Blotting Detection of the Binding of Insulin Receptor Substrate to the Insulin Receptor.

Far-western blotting, derived from the western blot, has been used to detect interactions between proteins in vitro, such as receptor-ligand interactions. The insulin signaling pathway plays a critical role in the regulation of both metabolism and cell growth. The binding of the insulin receptor substrate (IRS) to the insulin receptor is essential for the propagation of downstream signaling after the activation of the insulin receptor by insulin. Here, we describe a step-by-step far-western blotting protocol for determining the binding of IRS to the insulin receptor.

Blocking AMPKαS496 phosphorylation improves mitochondrial dynamics and hyperglycemia in aging and obesity.

Impaired mitochondrial dynamics causes aging-related or metabolic diseases. Yet, the molecular mechanism responsible for the impairment of mitochondrial dynamics is still not well understood. Here, we report that elevated blood insulin and/or glucagon levels downregulate mitochondrial fission through directly phosphorylating AMPKα at S496 by AKT or PKA, resulting in the impairment of AMPK-MFF-DRP1 signaling and mitochondrial dynamics and activity. Since there are significantly increased AMPKα1 phosphorylation at S496 in the liver of elderly mice, obese mice, and obese patients, we, therefore, designed AMPK-specific targeting peptides (Pa496m and Pa496h) to block AMPKα1S496 phosphorylation and found that these targeting peptides can increase AMPK kinase activity, augment mitochondrial fission and oxidation, and reduce ROS, leading to the rejuvenation of mitochondria. Furthermore, these AMPK targeting peptides robustly suppress liver glucose production in obese mice. Our data suggest these targeting peptides are promising therapeutic agents for improving mitochondrial dynamics and activity and alleviating hyperglycemia in elderly and obese patients.

Development and Functions of Mitochondria in Early Life.

Mitochondria are highly dynamic organelles of bacterial origin in eukaryotic cells. These play a central role in metabolism and adenosine triphosphate (ATP) synthesis and in the production and regulation of reactive oxygen species (ROS). In addition to the generation of energy, mitochondria perform numerous other functions to support key developmental events such as fertilization during reproduction, oocyte maturation, and the development of the embryo. During embryonic and neonatal development, mitochondria may have important effects on metabolic, energetic, and epigenetic regulation, which may have significant short- and long-term effects on embryonic and offspring health. Hence, the environment, epigenome, and early-life regulation are all linked by mitochondrial integrity, communication, and metabolism.

Effect of biogenic silver nanocubes on matrix metalloproteinases 2 and 9 expressions in hyperglycemic skin injury and its impact in early wound healing in streptozotocin-induced diabetic mice.

Microbial contamination along with over expressions of matrix metalloproteinases 2 and 9 impairs wound healing in diabetic patients. Silver-based antimicrobial agents have been successfully used for treating non-healing chronic wounds associated with infection. However, topical application of silver-ion compounds impairs wound healing process. Thus, usage of biogenic silver nanoparticles appears as a new means to reduce the toxicity of silver compounds in the wound care system. Here, following our previous method, AgNPs was synthesized using the culture filtrate of Brevibacillus brevis KN8(2) then characterized by UV-visible spectrophotometry, TEM, SAED, XRD and DLS measurements. The antibacterial activity of AgNPs was evaluated against the most common wound infecting pathogens Pseudomonas aeruginosa and Staphylococcus aureus by well diffusion assay. Further, the wound healing efficacy of biogenic AgNPs was examined in streptozotocin-induced diabetic mice by measuring wound area closure, histopathology, mRNA and protein expressions of MMP-2, MMP-9. Our results demonstrates that besides antimicrobial activity, biogenic AgNPs decreased the mRNA and protein expression of MMP-2 and MMP-9 in wounded granulation tissues leads to early wound healing in diabetic mice. These findings revealed that biogenic AgNPs synthesized from B. brevis KN8(2) could be an effective therapeutic agent in the management of diabetic foot ulcer with/without infection.

Helicobacter pylori pathogen regulates p14ARF tumor suppressor and autophagy in gastric epithelial cells.

Infection with Helicobacter pylori is one of the strongest risk factors for development of gastric cancer. Although these bacteria infect approximately half of the world's population, only a small fraction of infected individuals develops gastric malignancies. Interactions between host and bacterial virulence factors are complex and interrelated, making it difficult to elucidate specific processes associated with H. pylori-induced tumorigenesis. In this study, we found that H. pylori inhibits p14ARF tumor suppressor by inducing its degradation. This effect was found to be strain-specific. Downregulation of p14ARF induced by H. pylori leads to inhibition of autophagy in a p53-independent manner in infected cells. We identified TRIP12 protein as E3 ubiquitin ligase that is upregulated by H. pylori, inducing ubiquitination and subsequent degradation of p14ARF protein. Using isogenic H. pylori mutants, we found that induction of TRIP12 is mediated by bacterial virulence factor CagA. Increased expression of TRIP12 protein was found in infected gastric epithelial cells in vitro and human gastric mucosa of H. pylori-infected individuals. In conclusion, our data demonstrate a new mechanism of ARF inhibition that may affect host-bacteria interactions and facilitate tumorigenic transformation in the stomach.

Bacterial lipopolysaccharide differently modulates steroidogenic enzymes gene expressions in the brain and testis in rats.

Bacterial lipopolysaccharide (LPS) is a major component of the cell wall of gram negative bacteria contributing to the pathogenesis of bacterial infection, in particular in those diseases affecting central nervous system and reproductive tissues. The present work is an attempt to study the regulation of steroidogenic enzymes gene expression in the brain and testis in LPS induced rats. Adult male albino rats were administered LPS (5mg/kg BW) to induce acute inflammation. LPS administration induced severe oxidative damage in the brain and testicular tissue which was evident from decreased activities of enzymic antioxidants and increased lipid peroxidation levels. The mRNA expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and androgen receptor corepressor-19kDa (ARR19) in the brain and testis were determined. The mRNA expression of 3ß-HSD and 17ß-HSD was increased in the brain with significant decrease in the testis at 24h and 48h in LPS treated animals. The results also demonstrated an interesting finding that LPS treatment completely represses ARR19 in the brain, while not in the testis. These findings show ARR19 might play a crucial role in regulation of neuronal and testicular steroidogenesis in inflammatory diseases.

The role of phosphoenolpyruvate carboxykinase in neuronal steroidogenesis under acute inflammation.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme found in many tissues throughout the body including brain. In the present study, we have investigated the effect of bacterial lipopolysaccharide (LPS) on PEPCK and its role in neuronal steroidogenesis. Adult female albino rats were administered LPS (5mg/kg body weight) to induce acute inflammation. LPS administration resulted in a significant increase of PEPCK mRNA expression with concomitant increase in mRNA levels of steroidogenic acute regulatory (StAR) protein and other steroidogenic enzymes including 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and aromatase in brain tissue. Further, the inhibition of PEPCK expression by glipizide significantly decreased the mRNA expression of steroidogenic proteins and concurrently increased the mRNA levels of proinflammatory cytokines under LPS administration. The results of this study suggest a novel finding that PEPCK may have an important role in neuronal steroidogenesis; which serves as an adaptive response under inflammation.