Agmatine Protects Against the Progression of Sepsis Through the Imidazoline I2 Receptor-Ribosomal S6 Kinase 2-Nuclear Factor-κB Signaling Pathway.

OBJECTIVES: The knowledge that agmatine is found in the human body has existed for several years; however, its role in sepsis has not yet been studied. In the present study, we investigate the role of agmatine in the progression and treatment of sepsis. DESIGN: Clinical/laboratory investigations. SETTING: Medical centers/University-based research laboratory. SUBJECTS: Elective ICU patients with severe sepsis and healthy volunteers; C57BL/6 mice weighing 18-22 g. INTERVENTIONS: Serum agmatine level and its associations with inflammatory markers were assessed in patients with sepsis. Agmatine was administered intraperitoneally to mice before a lipopolysaccharide challenge. Human peripheral blood mononuclear cells and murine macrophages were pretreated with agmatine followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: Serum agmatine levels were significantly decreased in patients with sepsis and lipopolysaccharide-induced mice, and correlated with Acute Physiology and Chronic Health Evaluation II score, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels. In a therapeutic experiment, exogenous agmatine attenuated the cytokine production of peripheral blood mononuclear cells from patients with sepsis and healthy controls. Agmatine also exerted a significant beneficial effect in the inflammatory response and organ damage and reduced the death rate in lipopolysaccharide-induced mice. Imidazoline I2 receptor agonist 2-benzofuran-2-yl blocked the pharmacological action of agmatine; whereas, other imidazoline receptor ligands did not. Furthermore, agmatine significantly impaired the inflammatory response by inactivating nuclear factor-κB, but not protein 38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and inducible nitric oxide synthase signaling in macrophages. Activation of imidazoline I2 receptor or knockdown of ribosomal S6 kinase 2 counteracted the effects of agmatine on phosphorylation and degradation of inhibitor of nuclear factor-κBα. CONCLUSIONS: Endogenous agmatine metabolism correlated with the progression of sepsis. Supplemental exogenous agmatine could ameliorate the lipopolysaccharide-induced systemic inflammatory responses and multiple organ injuries through the imidazoline I2 receptor-ribosomal S6 kinase 2-nuclear factor-κB pathway. Agmatine could be used as both a clinical biomarker and a promising pharmaconutrient in patients with severe sepsis.

Ribosomal S6 kinase (RSK) modulators: a patent review.

INTRODUCTION: The p90 ribosomal S6 kinases (RSK) are a family of Ser/Thr protein kinases that are downstream effectors of MEK1/2-ERK1/2. Increased RSK activation is implicated in the etiology of multiple pathologies, including numerous types of cancers, cardiovascular disease, liver and lung fibrosis, and infections. AREAS COVERED: The review summarizes the patent and scientific literature on small molecule modulators of RSK and their potential use as therapeutics. The patents were identified using World Intellectual Property Organization and United States Patent and Trademark Office databases. The compounds described are predominantly RSK inhibitors, but a RSK activator is also described. The majority of the inhibitors are not RSK-specific. EXPERT OPINION: Based on the overwhelming evidence that RSK is involved in a number of diseases that have high mortalities it seems surprising that there are no RSK modulators that have pharmacokinetic properties suitable for in vivo use. MEK1/2 inhibitors are in the clinic, but the efficacy of these compounds appears to be limited by their side effects. We hypothesize that targeting the downstream effectors of MEK1/2, like RSK, are an untapped source of drug targets and that they will generate less side effects than MEK1/2 inhibitors because they regulate fewer effectors.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 µmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

Differential concentration and time dependent effects of progesterone on kinase activity, hyperactivation and acrosome reaction in human spermatozoa.

Progesterone has been identified to be one of the physiological regulators of sperm hyperactivation and acrosome reaction. However, the high sensitivity of human spermatozoa to progesterone implies that many may undergo premature hyperactivation and acrosome reaction thereby compromising their ability to fertilize. We hypothesized that if a spermatozoon has to preclude the occurrence of these events prematurely, there should be differential dose- and time-dependent effects on motility and acrosome reaction. We observed that low concentrations of progesterone (10 and 100 nm) induce sperm motility and activate tyrosine kinase; higher concentrations (1-10 µm) are required to induce extracellular signal regulated kinases 1/2 (Erk1/2), p90 ribosomal S6 kinase (p90RSK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK1) and AKT phosphorylation, hyperactivation and acrosome reaction. The induction of acrosome reaction and tyrosine phosphorylation in response to higher concentration of progesterone is not absolutely dependent on activation of T-type voltage-gated Ca(2+) channel or CatSper as Mibefradil did not completely abrogate progesterone-mediated effects. These results imply that although the spermatozoa are sensitive to low concentrations of progesterone, they only activate motility and tyrosine kinase activation; higher concentrations are required to induce hyperactivation and acrosome reaction probably by activating multiple kinase pathways including the MAPK and AKT.

5-deoxykaempferol plays a potential therapeutic role by targeting multiple signaling pathways in skin cancer.

Nontoxic small molecules with multitargeting effects are believed to have potential in cancer prevention. Dietary phytochemicals were shown to exhibit cancer-preventive effects attributed to their antioxidant capacities. In this report, we show that the natural compound 5-deoxykaempferol (5-DK) exerts a chemopreventive effect on UVB-induced skin carcinogenesis by targeting multiple signaling molecules. 5-DK suppressed the UVB-induced expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor in mouse skin epidermal JB6 P+ cells. Moreover, 5-DK inhibited phosphorylation of MKK3/6, MKK4, and Akt, but had no effect on phosphorylation of Src, extracellular signal-regulated kinases, or ribosomal S6 kinase (RSK). However, 5-DK affected multiple targets by reducing Src, phosphoinositide 3-kinase (PI3K), and RSK2 activities. In particular, pull-down assays revealed that 5-DK specifically bound to and competed with ATP for binding with Src, PI3K, and RSK2. Exposure to 5-DK significantly suppressed UVB-induced tumorigenesis in mouse skin in a dose-dependent manner, and it inhibited the UVB-induced expression of COX-2, proliferating cell nuclear antigen, vascular endothelial growth factor, and matrix metalloproteinase-9. Our data suggest that 5-DK docks at the ATP-binding site of Src, PI3K, and RSK2. For RSK2, the ATP-binding site is located between the N- and C-lobes of the kinase domain. Taken together, our results indicate that 5-DK holds promise for the treatment of UVB-induced skin cancer by targeting Src, PI3K, and RSK2 signaling.

Chronic high fat feeding attenuates load-induced hypertrophy in mice.

The incidence of obesity and obesity-related conditions, such as metabolic syndrome and insulin resistance, is on the increase. The effect of obesity on skeletal muscle function, especially the regulation of muscle mass, is poorly understood. In this study we investigated the effect of diet-induced obesity on the ability of skeletal muscle to respond to an imposed growth stimulus, such as increased load. Male C57BL/6 mice were randomized into two diet groups: a low fat, high carbohydrate diet (LFD) and a high fat, low carbohydrate diet (HFD) fed ad libitum for 14 weeks. Mice from each diet group were divided into two treatment groups: sedentary control or bilateral functional overload (FO) of the plantaris muscle. Mice were evaluated at 3, 7, 14 or 30 days following FO. By 14 days of FO, there was a 10% reduction (P < 0.05) in absolute growth of the plantaris in response to overload in HFD mice vs. LFD mice. By 30 days the attenuation in growth increased to 16% in HFD mice compared to LFD mice. Following FO, there was a reduction in the formation of polysomes in the HFD mice relative to the LFD mice, suggesting a decrease in protein translation. Further, activation of Akt and S6K1, in response to increased mechanical loading, was significantly attenuated in the HFD mice relative to the LFD mice. In conclusion, chronic high fat feeding impairs the ability of skeletal muscle to hypertrophy in response to increased mechanical load. This failure coincided with a failure to activate key members of the Akt/mTOR signalling pathway and increase protein translation.

Effects of metformin on mammalian target of rapamycin in a mouse model of endometrial hyperplasia.

OBJECTIVE: The effects of metformin on S6K1, which is a crucial effector of mTOR signaling, and on endometrium were studied in a mouse model of endometrial hyperplasia induced by unopposed estradiol or tamoxifen. STUDY DESIGN: Forty-eight oophorectomized Balb/c mice were randomly assigned to receive saline, tamoxifen citrate (4 mg/kg), 17-beta estradiol hemihydrate (4 mg/kg), metformin (50 mg/kg), tamoxifen citrate (4 mg/kg) with metformin (50 mg/kg), or estradiol (4 mg/kg) with metformin (50 mg/kg) for 3 days. Histological markers of uterotrophy, including luminal epithelial cell height and density of endometrial glands were quantified for each slide. Immunohistochemical expression of PCNA and S6K1 was evaluated. H-score was used for S6K1 expression. Statistical analysis was performed using Student's t-test for comparison of two continous variables and one-way ANOVA for comparison of multiple variables. RESULTS: Mice treated either with tamoxifen or estradiol had significantly increased density of endometrial glands and epithelial heights compared to vehicle-only or metformin-only group (p<0.001). Addition of metformin to tamoxifen or estradiol treated mice significantly decreased the density of endometrial glands and epithelial cell heights (p<0.05). Addition of metformin to tamoxifen significantly decreased the H-score of S6K1 (p<0.05) and the immunohistochemical expression of PCNA (p<0.05) in uterine lining epithelium, glandular and stromal cells. Addition of metformin to estradiol significantly decreased the H-score of S6K1 (p<0.05) and the immunohistochemical expression of PCNA (p<0.05) in uterine lining epithelium, glandular and stromal cells. CONCLUSION: Metformin seems to have possible antiproliferative effects on the endometrium of estradiol or tamoxifen treated mice via inhibiting the mTOR mediated S6K1 activation.

Geniposide, a novel agonist for GLP-1 receptor, prevents PC12 cells from oxidative damage via MAP kinase pathway.

Alzheimer's disease (AD) is the most common form of dementia. Glucagon-like peptide-1 (GLP-1) gives a new genre in therapeutic targets for intervention in AD with its neurotrophic and neuroprotective functions. In previous work, we identified that geniposide is a novel agonist for GLP-1 receptor, which shows neurotrophic characteristics to induce the neuronal differentiation of PC12 cells. The aim of this study is to determine whether geniposide prevents neurons from oxidative damage, and to explore its signaling pathways. The results demonstrated that geniposide increased the expression of anti-apoptotic proteins, including Bcl-2 and heme oxygenase-1 (HO-1), to antagonize the oxidative damage in PC12 cells induced by hydrogen peroxide. LY294002 (a PI3K inhibitor) inhibited the effect of geniposide increasing of Bcl-2 level by activation of MAPK, MEK and c-Raf phosphorylation in hydrogen peroxide treated PC12 cells. U0126 (a selective inhibitor of MEK) also attenuated the enhancement of geniposide on Bcl-2 level by inhibiting the phosphorylation of p90RSK in the hydrogen peroxide treated PC12 cells. All these data demonstrate that geniposide, an agonist for GLP-1 receptor, regulates expression of anti-oxidative proteins including HO-1 and Bcl-2 by activating the transcriptor of p90RSK via MAPK signaling pathway in PC12 cells.

Brain-derived neurotrophic factor stimulates the transcriptional and neuroprotective activity of myocyte-enhancer factor 2C through an ERK1/2-RSK2 signaling cascade.

Neurotrophin activation of myocyte-enhancer factor (MEF) 2C is one of the strongest pro-survival signaling pathways in developing neurons. To date, neurotrophin stimulation of MEF2C has been largely attributed to its direct phosphorylation by extracellular signal-regulated kinase (ERK) 5. Because MEF2C is not directly phosphorylated by ERK1/2 in vitro, it is generally assumed that the ERK1/2 signaling cascade does not regulate MEF2C. Surprisingly, we discovered that ERK1/2 are required for both the transcriptional and neuroprotective activity of MEF2C in cortical neurons stimulated by brain-derived neurotrophic factor. ERK1/2 stimulation of MEF2C is mediated by p90 ribosomal S6 kinase 2 (RSK2), a Ser/Thr protein kinase downstream of ERK1/2. RSK2 strongly phosphorylates purified recombinant MEF2C protein in vitro. Furthermore, RSK2 can directly phosphorylate MEF2C on S192, a consensus RSK2-phosphorylation site located in the transactivation domain of MEF2C. Substitution of S192 with a non-phosphorylatable alanine diminishes both the transcriptional and neuroprotective activity of MEF2C to an extent similar to mutation on S387, an established activating phosphorylation site. Together, our data identifies ERK1/2-RSK2 signaling as a novel mechanism by which neurotrophins activate MEF2C and promote neuronal survival.