Phenolipid JE improves metabolic profile and inhibits gluconeogenesis via modulating AKT-mediated insulin signaling in STZ-induced diabetic mice.

Phenolipids are characteristic phytochemicals of Syzygium genus. However, the antidiabetic potential and underlying molecular mechanism of these components are not fully elucidated. Herein, we studied the anti-diabetic effects of jambone E (JE), a phenolipid from S. cumini, with in vitro and in vivo models. Data from current study showed that JE enhanced glucose consumption and uptake, promoted glycogen synthesis, and suppressed gluconeogenesis in insulin resistant (IR)-HepG2 cells and primary mouse hepatocytes. JE also attenuated streptozotocin-induced hyperglycemia and hyperlipidemia in type 1 diabetic (T1D) mice. Eleven metabolites (e.g. trimethylamine n-oxide, 4-pyridoxic acid, phosphatidylinositol 39:4, phenaceturic acid, and hippuric acid) were identified as potential serum biomarkers for JE's antidiabetic effects by an untargeted metabolomics approach. The further molecular mechanistic study revealed that JE up-regulated phosphorylation levels of protein kinase B (AKT), glycogen synthase kinase 3 beta, and forkhead box O1 (FoxO1), promoted nuclear exclusion of FoxO1 whilst decreased gene expression levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha, phosphoenolpyruvate carboxykinase and glucose 6-phosphatase in IR-HepG2 cells and T1D mice. Our data suggested that JE might be a potent activator for AKT-mediated insulin signaling pathway, which was confirmed by the usage of AKT inhibitor and AKT-target siRNA interference, as well as the cellular thermal shift assay. Findings from the current study shed light on the anti-diabetic effects of phenolipids in the Syzygium species, which supports the use of medicinal plants in the Syzygium genus for potential pharmaceutical applications.

Progesterone triggers Rho kinase-cofilin axis during in vitro and in vivo endometrial decidualization.

STUDY QUESTION: Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo? SUMMARY ANSWER: Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium. WHAT IS KNOWN ALREADY: Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls. STUDY DESIGN, SIZE, DURATION: In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 10 for both phases). PARTICIPANTS/MATERIALS, SETTING, METHODS: The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P < 0.05) and are further augmented following the 9-day treatment (P < 0.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P < 0.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation < 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P < 0.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P < 0.001) as well as in vitro decidualized ESCs (P < 0.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P < 0.05). LARGE SCALE DATA: Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243. LIMITATIONS, REASONS FOR CAUTION: The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples. WIDER IMPLICATIONS OF THE FINDINGS: The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies-including the context of decidualization-related subfertility and infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.

Ganoderic acid A is the effective ingredient of Ganoderma triterpenes in retarding renal cyst development in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening monogenetic diseases characterized by progressive enlargement of fluid-filled renal cysts. Our previous study has shown that Ganoderma triterpenes (GT) retards PKD renal cyst development. In the present study we identified the effective ingredient of GT in suppression of kidney cyst development. Using an in vitro MDCK cystogenesis model, we identified ganoderic acid A (GA-A) as the most promising candidate among the 12 ganoderic acid (GA) monomers. We further showed that GA-A (6.25-100 µM) significantly inhibited cyst growth in MDCK cyst model and embryonic kidney cyst model in vitro, and the inhibitory effect was reversible. In kidney-specific Pkd1 knockout (kPKD) mice displaying severe cystic kidney disease, administration of GA-A (50 mg· kg-1 ·d-1, sc) significantly attenuated renal cyst development. In both MDCK cells and kidney of kPKD mice, we revealed that GA-A dose-dependently downregulated the Ras/MAPK signaling pathway. The expression of proliferating cell nuclear antigen (PCNA) was also suppressed, suggesting a possible effect of GA-A on cell proliferation. These experimental data suggest that GA-A may be the main ingredient of GT as a potential therapeutic reagent for treating ADPKD.

Pretreatment with ß-adrenergic receptor agonists facilitates induction of LTP and sharp wave ripple complexes in rodent hippocampus.

Norepinephrine, is involved in the enhancement of learning and memory formation by regulating synaptic mechanisms through its ability to activate pre- and post-synaptic adrenergic receptors. Here we show that ß-agonists of norepinephrine facilitate the induction of both associational LTP and sharp wave ripples (SPW-Rs) in acute slices of rat hippocampus in area CA3. Surprisingly, this facilitating effect persists when slices are only pretreated with ß-receptor agonists followed by wash out and application of the unspecific ß-adrenoreceptor (ßAR) antagonist propranolol. During application of ßAR agonists repeated stimulation resulted in facilitated induction of SPW-Rs. Since SPW-Rs are thought to be involved in memory replay we studied the effects of ßAR-agonists on spontaneous SPW-Rs in murine hippocampus and found that amplitude and incidence of SPW-Rs increased. These effects involve cyclic-AMP and the activation of protein kinase A and suggest a supportive role in memory consolidation. © 2016 Wiley Periodicals, Inc.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 µM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca(2+) was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

cAMP inhibits migration, ruffling and paxillin accumulation in focal adhesions of pancreatic ductal adenocarcinoma cells: effects of PKA and EPAC.

We demonstrated that increasing intracellular cAMP concentrations result in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation is mediated by PKA, whilst EPAC potentiates migration.

A carrier-free, dual-functional hydrogel constructed of antimicrobial peptide Jelleine-1 and 8Br-cAMP for MRSA infected diabetic wound healing.

Bacterial infection and local growth factor deficiency are two of the major causes of the nonunion of diabetic wounds. Antimicrobial peptides (AMPs) are believed to be alternatives to antibiotics against drug-resistant bacterial infections. 8-Bromoadenosine-3', 5'-cyclic monophosphate (8Br-cAMP) can promote cells to secrete growth factors and accelerate cell proliferation. In the present study, we constructed a hydrogel with antimicrobial peptide Jelleine-1 (J-1) and 8Br-cAMP without any other gelators or chemical crosslinking agents. The hydrogel was proved to promote the secretion of transforming growth factor-ß (TGF-ß) and vascular endothelial growth factor-A (VEGFA) in vitro and in vivo. Notably, it exhibited potent potential for wound healing in methicillin-resistant Staphylococcus aureus (MRSA) infected diabetic wounds. This would be attributed to the retention of AMPs and 8Br-cAMP on the wound site by the hydrogel system. In addition, the hydrogel also showed good biodegradability, proper stability, and good biocompatibility. This study would shed light on the development of carrier-free and multifunctional hydrogel for wound healing. STATEMENT OF SIGNIFICANCE: Bacterial infection and local growth factor deficiency are two of the major causes for the nonunion of refractory wounds. In the present study, an injectable carrier-free hydrogel was constructed of a natural antimicrobial peptide J-1 and 8Br-cAMP by eco-friendly physical crosslinking without any other gelators or chemical crosslinking agents. The hydrogel exhibited excellent antimicrobial activity and was proved to promote the secretion of TGF-ß and VEGFA in vitro and in vivo. Correspondingly, the hydrogel showed exceptionally wound healing effects in the wound model of MRSA infected diabetic rats. This study would provide an alternative strategy or a potential hydrogel dressing for the treatment of chronic or refractory wounds.

Angiogenesis and vasculogenic mimicry are inhibited by 8-Br-cAMP through activation of the cAMP/PKA pathway in colorectal cancer.

INTRODUCTION: Vasculogenic mimicry (VM) describes the formation of an epithelial-independent tumor microcirculation system that differs from traditional angiogenesis. Angiogenesis and the formation of VM are closely related through the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and the epithelial-mesenchymal transition (EMT) process. MATERIALS AND METHODS: In this study, 8-Br-cAMP, a cAMP analog and PKA activator, was used to activate the cAMP/PKA pathway to evaluate the effects of cAMP/PKA on angiogenesis and VM in colorectal cancer (CRC) cells. We used a syngeneic model of CRC in BALB/c mice. RESULTS: We discovered that treatment with 8-Br-cAMP significantly reduced tumor number compared to control mice after the 7th, 14th, and 28th days of treatment. VM was evaluated by periodic acid-schiff (PAS)-CD31 staining, and we found that VM was inhibited by 8-Br-cAMP treatment in vivo. Immunohistochemistry confirmed the inhibition of vascular endothelial growth factor (VEGF) and cAMP and the activation of PKA by 8-Br-cAMP; quantitative real-time-PCR (qRT-PCR) demonstrated that 8-Br-cAMP regulated the expression of vascular endothelial (VE)-cadherin, matrix metalloproteinase 2 (MMP2), ephrin type-A receptor 2 (EphA2), and VEGF in vivo. Experiments in vitro revealed that treatment with 8-Br-cAMP and U0126 decreased VEGF expression through PKA-ERK in CT26 cells by qRT-PCR. We further confirmed that tube formation of human umbilical vein endothelial cells was inhibited by 8-Br-cAMP in vitro. DISCUSSION: This study demonstrates that angiogenesis and VM are inhibited by 8-Br-cAMP treatment. Our data indicate that 8-Br-cAMP acts through the cAMP/PKA-ERK pathway and through EMT processes in CRC. These findings provide an insight into mechanisms of CRC and suggest that the cAMP/PKA-ERK pathway is a novel potential therapeutic target for the treatment of CRC.

The role of the cAMP/PKA signalling pathway in the inhibitory influence of melatonin on oxytocin and vasopressin secretion from the rat hypothalamo-neurohypophysial system.

INTRODUCTION: Melatonin was found to inhibit forskolin-stimulated oxytocin (OT) and vasopressin (VP) release in vitro. The purpose of the present investigation was to evaluate the contribution of the cyclic 3',5'-adenosine monophosphate/protein kinase A (cAMP/PKA) signalling pathway in melatonin-dependent inhibition of OT and VP secretion from the rat hypothalamo-neurohypophysial (H-NH) system in vitro. MATERIAL AND METHODS: The H-NH explants were placed in 1 ml of normal Krebs-Ringer (nK-R) buffer and first preincubated for 30 min in control buffer or in the presence of PKA inhibitor, i.e. cAMPS-Rp or H-89. Next, they were incubated in nK-R buffer {fluid F1} and then in buffer as F1 enriched with melatonin (10-9 M or 10-7 M) and/or PKA activator, i.e. cAMP analogue (8-Br-cAMP), or their vehicles {fluid F2}. After 20 min of incubation in fluid F1 and then F2, the media were collected and frozen, to be assayed for OT and VP by the RIA. RESULTS: 8-Br-cAMP increased OT and VP secretion when the H-NH explants were preincubated in control medium, while PKA inhibitors eliminated its stimulatory effect on OT and VP release. Melatonin (10-7 M) diminished basal OT and VP output from the H-NH system, and inhibited (at both concentrations studied) the cAMP analogue-stimulated release of both neurohormones under control conditions. The effect of melatonin on OT and VP release was completely blocked when cAMPS-Rp, but not H-89, was used to disrupt the cAMP/ /PKA pathway. CONCLUSIONS: Melatonin employs the cAMP/PKA signalling pathway to inhibit OT and VP secretion from the rat H-NH system; nonethe-less, other cAMP-mediated mechanisms are not excluded.