Protein Kinase A Protein Research Articles

Glucose inhibits the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA.

Protein kinase A (PKA) plays an important role in cellular life activities. Recently, PKA was found to bind to the inhibitor of nuclear factor-kappaB (IκB), a key protein in the nuclear factor-kappaB (NF-κB) pathway, to form a complex involved in the regulation of inflammatory response. However, the role of PKA in the anti-inflammatory of goose fatty liver is still unclear. A total of 14 healthy 70-d-old male Lander geese were randomly divided into a control group and an overfeeding group. Inflammation level was analyzed by histopathological method in the liver. The mRNA and protein abundance of PKA and tumor necrosis factor-alpha (TNFα), as well as the ubiquitination level of PKA, were detected. Moreover, goose primary hepatocytes were cotreated with glucose, harringtonine, and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132). Finally, the co-immunoprecipitated samples of PKA from the control and overfeeding group were used for protein mass spectrometry. The results showed that no difference in PKA mRNA expression was observed (P > 0.05), while the PKA protein level in the overfed group was significantly reduced (P < 0.05) when compared with the control group. The ubiquitination level of PKA was higher than that of the control group in fatty liver. The mRNA expression of PKA was elevated but protein abundance was reduced in goose primary hepatocytes with 200mmol/L glucose treatment (P < 0.05). The PKA protein abundance was dramatically reduced in hepatocytes treated with harringtonine (P < 0.01) when compared with the glucose-supplemented group. Nevertheless, MG132 tended to alleviate the inhibitory effect of harringtonine on PKA protein abundance (P = 0.081). There was no significant difference in TNFα protein level among glucose-treated groups and control (P > 0.05). Protein mass spectrometry analysis showed that 29 and 76 interacting proteins of PKA were screened in goose normal and fatty liver, respectively. Validation showed that PKA interacted with the E3 ubiquitination ligases ring finger protein 135 (RNF135) and potassium channel modulatory factor 1 (KCMF1). In summary, glucose may inhibit the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA. Additionally, RNF135 and KCMF1 may be involved in the regulation of PKA ubiquitination level as E3 ubiquitination ligases.

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

A mutation in the PRKAR1B gene drives pathological mechanisms of neurodegeneration across species.

Protein Kinase A (PKA) neuronal function is controlled by the interaction of a regulatory (R) subunit dimer to two catalytic (C) subunits. Recently, the L50R variant in the gene encoding the RIβ subunit was identified in individuals with a novel neurodegenerative disease. However, the mechanisms driving the disease phenotype remained unknown. In this study, we generated a mouse model carrying the RIβ-L50R mutation to replicate the human disease phenotype and study its progression with age. We examined postmortem brains of affected individuals as well as live cell cultures. Employing biochemical assays, immunohistochemistry, and behavioral assessments, we investigated the impact of the mutation on PKA complex assembly, protein aggregation and neuronal degeneration. We reveal that RIβ is an aggregation-prone protein that progressively accumulates in wildtype and Alzheimer's mouse models with age, while aggregation is accelerated in the RIβ-L50R mouse model. We define RIβ-L50R as a causal mutation driving an age-dependent behavioral and disease phenotype in human and mouse models. Mechanistically, this mutation disrupts RIβ dimerization, leading to aggregation of its monomers. Intriguingly, interaction with the C-subunit protects the RIβ-L50R from self-aggregating, in a dose-dependent manner. Furthermore, cAMP signaling induces RIβ-L50R aggregation. The pathophysiological mechanism elucidated here for a newly recognized neurodegenerative disease, in which protein aggregation is the result of disrupted homodimerization, sheds light on a remarkably under-appreciated but potentially common mechanism across several neurodegenerative diseases.

Preventive effect of kiwi berry (Actinidia arguta) on loperamide-induced constipation

Constipation is a common intestinal disease. Kiwi berries can effectively prevent constipation. However, studies have yet to be done to determine how kiwi berries prevent constipation. For 2 weeks, mice in this study were continually orally gavaged with kiwi berry, loperamide, or a combination of the two. This study found that the kiwi group's feces had more water than the constipated mice. In addition, kiwi berries can speed up gastrointestinal transit (GI), shorten the time it takes to pass the first dark stool, and dramatically enhance body weight gain. In the interstitial cells of Cajal (ICC) cells and colon tissues, alterations in the protein expression of vasoactive intestinal peptide (VIP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and aquaporin-3 (AQP3) were found. At 3, 6, and 12 h of ICC cells and mouse colon, the kiwi group’s VIP, cAMP, PKA, and AQP3 protein expression levels were lower than those of the constipated mice. The kiwi berry can decrease the Firmicutes to Bacteroidetes ratio and boost the diversity and quantity of gut microbiota. By influencing the gut microbiota and VIP-cAMP-PKA-AQP3 signaling pathway, kiwi berries prevent constipation.

Dibutyl phthalate disrupts [Ca2+]i, reactive oxygen species, [pH]i, protein kinases and mitochondrial activity, impairing sperm function

To explore the mechanism of sperm dysfunction caused by dibutyl phthalate (DBP), the effects of DBP on intracellular [Ca2+] and [pH], reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, phosphorylation of protein kinase A (PKA) substrate proteins and phosphotyrosine (p-Tyr) proteins, sperm motility, spontaneous acrosome reaction, and tail bending were examined in mouse spermatozoa. At 100 µg/mL, DBP significantly increased tail bending and [Ca2+]i. Interestingly, DBP showed biphasic effects on [pH]i. DBP at 10–100 µg/mL significantly decreased sperm motility. Similarly, Ca2+ ionophore A23187 decreased [pH]i sperm motility, suggesting that DBP-induced excessive [Ca2+]i decreased sperm motility. DBP significantly increased ROS and LPO. DBP at 100 µg/mL significantly decreased mPTP closing, MMP, and ATP levels in spermatozoa, as did H2O2, indicative of ROS-mediated mitochondrial dysfunction caused by DBP. DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins. DBP at 1–10 µg/mL significantly increased the spontaneous acrosome reaction, suggesting that DBP can activate sperm capacitation. Altogether, DBP showed a biphasic effect on intracellular signaling in spermatozoa. At concentrations relevant to seminal ortho-phthalate levels, DBP activates [pH]i, protein tyrosine kinases and PKA via physiological levels of ROS generation, potentiating sperm capacitation. DBP at high doses excessively raises [Ca2+]i and ROS and disrupts [pH]i, impairing the mitochondrial function, tail structural integrity, and sperm motility.

Attenuation of propofol-induced hippocampal neuron damage in developing rats by dexmedetomidine

This study explored the mechanism of dexmedetomidine attenuating propofol damage to hippocampal neurons in rats. By constructing rat hippocampal neuron model and carrying out targeted intervention; enzyme linked immunosorbent assay (ELISA) detected IL-1β and IL-18 levels; while Nissl staining observed hippocampus Histopathology; flow cytometry quantitatively analyzed the number of hippocampal neuron cells and apoptosis rate. Moreover, levels of PKA, Caspase-1 and NOD-like receptor thermal protein domain associated protein 3(NLRP3) and corresponding proteins were measured. Results showed that, there were more Nissl bodies in cytoplasm of hippocampal neurons in control group, and distribution in the cytoplasm was relatively uniform. Moreover, IL-1β and IL-18 in model group continued to increase; while dexmedetomidine effectively inhibited apoptosis of hippocampal neurons, which may be related to decreased expression of protein kinase A (PKA). After using PKA inhibitors, apoptosis was significantly inhibited, and when the expression of PKA was reduced, Caspase-1 was down-regulated along with reduced NLRP3 level, which improved the injury of hippocampal neurons. Dexmedetomidine can therefore down-regulate the level of Caspase-1 in hippocampal neurons by inhibiting the PKA signaling pathway, improving apoptosis, reducing the genetic and protein expressions of NLRP3, and slowing down the damage of hippocampal neurons.

Ilex paraguariensis A.St.-Hil. improves lipid metabolism in high-fat diet-fed obese rats and suppresses intracellular lipid accumulation in 3T3-L1 adipocytes via the AMPK-dependent and insulin signaling pathways.

Obesity is closely associated with several chronic diseases, and adipose tissue plays a major role in modulating energy metabolism. This study aimed to determine whether Mate, derived from I. paraguariensis A.St.-Hil., ameliorates lipid metabolism in 3T3-L1 adipocytes and high-fat diet (HFD)-fed obese Sprague-Dawley (SD) rats. 3T3-L1 adipocytes were cultured for 7 days, following which intracellular lipid accumulation and expression levels of lipid metabolism-related factors were examined. Dorsomorphin was used to investigate the potential pathways involved, particularly the adenosine monophosphate-activated protein kinase (AMPK)- dependent pathway. Mate was administered to rat HFD-fed obese SD models for 8 consecutive weeks. The expression of lipid metabolism-related factors in the organs and tissues collected from dissected SD rats was evaluated. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes, increased the protein and gene expression levels of AMPK, hormone sensitive lipase (HSL), calmodulin kinase kinase (CaMKK), liver kinase B1 (LKB1), protein kinase A (PKA), CCAAT/enhancer binding protein β (C/EBPβ), insulin receptor b (IRβ), and insulin receptor substrate 1 (IRS1) (Tyr465), and decreased those of sterol regulatory element binding protein 1C (Srebp1c), fatty acid synthase (FAS), peroxisome-activated receptor γ (PPARγ), and IRS1 (Ser1101). Furthermore, an AMPK inhibitor abolished the effects exerted by Mate on intracellular lipid accumulation and HSL and FAS expression levels. Mate treatment suppressed body weight gain and improved serum cholesterol levels in HFD-fed obese SD rats. Treatment with Mate increased the protein and gene expression levels of AMPK, PKA, Erk1/Erk2 (p44/p42), and uncoupling protein 1 and reduced those of mammalian target of rapamycin, S6 kinase, Srebp1c, ap2, FAS, Il6, Adiponectin, Leptin, and Fabp4 in rat HFD-fed obese SD models. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes and improved lipid metabolism in the epididymal adipose tissue of HFD-fed obese SD rats via the activation of AMPK-dependent and insulin signaling pathways.

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

MHY4571, a novel diarylcyclohexanone derivative, exerts anti-cancer activity by regulating the PKA-cAMP-response element-binding protein pathway in squamous cell lung cancer

BackgroundThe protein kinase A (PKA)/cAMP response element-binding protein (CREB) has been suggested to be related to the inhibition of the proliferation of non-small cell lung cancer (NSCLC) cells. This study aimed to investigate the efficacy of a novel diarylcyclohexanone derivative, MHY4571, in regulating the PKA-CREB pathway and to study its anti-tumor role in squamous NSCLC.MethodsWe designed MHY4571 as a novel PKA inhibitor with acceptable in silico ADME properties and tested it in vitro in lung cancer cell lines and in vivo in xenograft and orthotopic mouse models of squamous cell lung carcinoma.ResultsMHY4571 inhibited PKA activity (> 70% inhibition) and suppressed the expression of p-PKA and p-CREB dose-dependently. MHY4571 treatment reduced lung cancer cell viability and promoted caspase 3-dependent apoptotic cell death. Orally administered MHY4571 significantly suppressed lung tumor growth in xenograft and orthotopic mouse models. PKA catalytic subunit alpha-silencing by siRNA (siPKA) strongly attenuated CREB phosphorylation; siCREB did not alter PKA protein levels or its phosphorylation, suggesting that PKA is an upstream regulator of CREB activity. MHY4571 acted synergistically with cisplatin (on co-treatment) to induce apoptotic cell death in lung cancer cells.ConclusionsOur results imply that MHY4571 may be a potential drug candidate for squamous cell lung cancer treatment.

Melatonin alleviates traumatic brain injury-induced anxiety-like behaviors in rats: Roles of the protein kinase A/cAMP-response element binding signaling pathway.

Melatonin is a hormone produced by the pineal gland. Given its capabilities of neuroprotection and low neurotoxicity, melatonin could be a therapeutic strategy for traumatic brain injury (TBI). The present study was conducted to determine the neuroprotective effects of melatonin on TBI-induced anxiety and the possible molecular mechanism. Rats were randomly divided into seven groups. The rodent model of TBI was established using the weight-drop method. Melatonin was administered by intraperitoneal injection at a dose of 10 mg/kg after TBI. H89 (0.02 mg/kg), a special protein kinase A (PKA) inhibitor, or dibutyryl-cyclic adenosine monophosphate (cAMP; 0.1 mg/kg), an activator of PKA, were administered by stereotactic injection of the brain to evaluate the roles of PKA and cAMP-response element-binding protein (CREB) in melatonin-related mood regulation, respectively. At 30 days post-TBI, the changes in anxiety-like behaviors in rats were measured using the open field and elevated plus maze tests. At 24 h post-TBI, the number of activated astrocytes and neuronal apoptosis were evaluated using immunofluorescence assay. The expression levels of inflammatory cytokines (TNF-α and IL-6) in the amygdala were measured using an enzyme-linked immunosorbent assay. The expression levels of PKA, phosphorylated (p)-PKA, CREB, p-CREB, NF-κB and p-NF-κB in the amygdala were detected using western blotting. It was revealed that melatonin partially reversed TBI-induced anxiety-like behavior in rats, and decreased the number of activated astrocytes and neuronal apoptosis in the amygdala induced by TBI. H89 partially blocked the neuroprotective effects of melatonin; while dibutyryl-cAMP not only reduced the H89-induced emotional disturbance but also enhanced the protective effects of melatonin against TBI. Overall, melatonin can alleviate TBI-induced anxiety-like behaviors in rats. Moreover, the underlying mechanism may be associated with the activation of the PKA/CREB signaling pathway.

Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay

The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation. Therefore, PKA has been viewed increasingly as potential target for variety of drugs and environmental endocrine disruptors. Consequentially, the preparation of PKA protein became an important initial step for the subsequent exploration of PKA’s character in endocrine disrupting effects of pesticides. To investigate PKA protein, which is potential to be the environmental endocrine toxicity target of triazole fungicides, a strategy to heterologously express protein kinase A catalytic alpha subunit of human (hPKAcα) and zebrafish (zPKAcα) in Escherichia coli (E. coli) BL21(DE3) host cells was demonstrated. After optimizing conditions and protein purification, we successfully obtained enzymatically active hPKAcα and zPKAcα. Western blot analysis indicated that the recombinant hPKAcα and zPKAcα still retained their characteristic antigenicity and binding activity, while in vitro kinase activity assays revealed that the recombinant hPKAcα and zPKAcα maintained enzyme activity. By in silico methods including homology modelling and molecular docking, the affinity of ligands and the models of hPKAcα and zPKAcα were further tested. The present study offered a valuable method to achieve the prokaryotic expression of a eukaryotic protein kinase and laid a foundation to facilitate further investigation of toxicological target of triazole pesticides.

Sevoflurane protects against ischemia-reperfusion injury in mice after total knee arthroplasty via facilitating RASD1-mediated protein kinase A pathway activation.

This study aimed to explore effects of Sevoflurane on ischemia-reperfusion (I/R) injury after total knee arthroplasty (TKA). To explore potential molecular mechanism, Ras related dexamethasone induced 1 (RASD1), a Protein kinase A (PKA) activator, frequently associated with various models of I/R injury, was also investigated. In vivo mouse models with I/R injury after TKA and in vitro cell models with I/R injury were induced. Contents of creatinine kinase (CK), lactic dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), serum levels of inflammatory factors, expression of PKA pathway-related genes and cell proliferation and apoptosis were measured. RASD1 was altered and PKA pathway was inhibited in mice and cells to elucidate the involvement of RASD1 and PKA pathway in Sevoflurane treatment on I/R injury. RASD1 was upregulated in I/R injury after TKA. Sevoflurane treatment or silencing RASD1 reduced RASD1 expression, CK, LDH and MDA contents, inflammation, apoptosis, but increased proliferation, SOD content, cAMP expression, and extents of PKA and cAMP responsive element binding protein (CREB) phosphorylation in skeletal muscle cells of I/R injury. Additionally, PKA pathway activation potentiated the therapeutic effect of Sevoflurane on I/R injury after TKA. Altogether, Sevoflurane treatment confines I/R injury after TKA via RASD1-mediated PKA pathway activation.

PKA and AMPK Signaling Pathways Differentially Regulate Luteal Steroidogenesis.

Luteinizing hormone (LH) via protein kinase A (PKA) triggers ovulation and formation of the corpus luteum, which arises from the differentiation of follicular granulosa and theca cells into large and small luteal cells, respectively. The small and large luteal cells produce progesterone, a steroid hormone required for establishment and maintenance of pregnancy. We recently reported on the importance of hormone-sensitive lipase (HSL, also known as LIPE) and lipid droplets for appropriate secretory function of the corpus luteum. These lipid-rich intracellular organelles store cholesteryl esters, which can be hydrolyzed by HSL to provide cholesterol, the main substrate necessary for progesterone synthesis. In the present study, we analyzed dynamic posttranslational modifications of HSL mediated by PKA and AMP-activated protein kinase (AMPK) as well as their effects on steroidogenesis in luteal cells. Our results revealed that AMPK acutely inhibits the stimulatory effects of LH/PKA on progesterone production without reducing levels of STAR, CYP11A1, and HSD3B proteins. Exogenous cholesterol reversed the negative effects of AMPK on LH-stimulated steroidogenesis, suggesting that AMPK regulates cholesterol availability in luteal cells. AMPK evoked inhibitory phosphorylation of HSL (Ser565). In contrast, LH/PKA decreased phosphorylation of AMPK at Thr172, a residue required for its activation. Additionally, LH/PKA increased phosphorylation of HSL at Ser563, which is crucial for enzyme activation, and decreased inhibitory phosphorylation of HSL at Ser565. The findings indicate that LH and AMPK exert opposite posttranslational modifications of HSL, presumptively regulating cholesterol availability for steroidogenesis.

PGE2 upregulates gene expression of dual oxidase in a lepidopteran insect midgut via cAMP signalling pathway.

In insect midgut, prostaglandins (PGs) play a crucial role in defending bacterial and malarial pathogens. However, little is known about the PG signalling pathway in the midgut. A dual oxidase (Se-Duox) with presumed function of catalysing reactive oxygen species (ROS) production in the midgut was identified in beet armyworm, Spodoptera exigua. Se-Duox was expressed in all developmental stages, exhibiting relatively high expression levels in the midgut of late larval instars. Se-Duox expression was upregulated upon bacterial challenge. RNA interference (RNAi) of Se-Duox expression significantly suppressed ROS levels in the midgut lumen. The suppression of ROS levels increased insecticidal activity of Serratia marcescens after oral infection. Interestingly, treatment with a PLA2 inhibitor prevented the induction of Se-Duox expression in response to bacterial challenge. On the other hand, addition of its catalytic product rescued the induction of Se-Duox expression. Especially, PG synthesis inhibitor significantly suppressed Se-Duox expression, while the addition of PGE2 or PGD2 rescued the inhibition. Subsequent PG signals involved cAMP and downstream components because specific inhibitors of cAMP signal components such as adenylate cyclase (AC) and protein kinase A (PKA) significantly inhibited Se-Duox expression. Indeed, addition of a cAMP analogue stimulated Se-Duox expression in the midgut. Furthermore, individual RNAi specific to PGE2 receptor (a trimeric G-protein subunit), AC, PKA or cAMP-responsive element-binding protein resulted in suppression of Se-Duox expression. These results suggest that PGs can activate midgut immunity via cAMP signalling pathway by inducing Se-Duox expression along with increased ROS levels.

PEG‐asparaginase‐induced hepatic steatosis is associated with PKA activation and white adipose tissue lipolysis

BackgroundPEGylated L‐asparaginase (PEG‐ASNase) is an essential chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia (ALL) that depletes L‐asparagine and leads to leukemic cell apoptosis. However, this agent is avoided in adult ALL protocols due to its high risk of liver toxicity. Clinical studies have implied that the mechanism of PEG‐ASNase‐induced liver toxicity is due to defect in hepatic β oxidation; however, no study has investigated the mechanism or the role of β oxidation in PEG‐ASNase‐induced hepatic toxicity. Our study aims to demonstrate that PEG‐ASNase‐induced liver toxicity is not due to hepatic defects in fatty acid oxidation, VLDL secretion, or fatty acid synthesis. Rather, our data suggest that the toxicity may be due to a novel mechanism of fatty liver involving drug‐induced adipose lipolysis.Methods8‐week‐old male Balb/c mice received 1,500 IU/kg of PEG‐ASNase and were sacrifice at 1, 3, 5, or 7 days after drug administration. Blood, liver, and white adipose tissue (WAT) were harvested after sacrifice. Liver lipids were directly quantified and further assessed via Oil red O and H&amp;E staining. Total and direct bilirubin, ALT, AST, and non‐esterified fatty acids (NEFA) were measure in plasma. The mRNA and protein levels of genes involved in hepatic fatty acid oxidation, lipid synthesis, and VLDL secretion were determined. To assess the effect of PEG‐ASNase on WAT lipolysis, the protein levels of Atgl, phosphorylated Hsl, and protein kinase A (PKA) were measured.ResultsPEG‐ASNase treatment in mice led to total body, liver, and WAT weight loss. Mice developed hepatic microvesicular steatosis after 3–7 days of drug administration. Plasma direct and total bilirubin levels were elevated 8‐fold relative to controls, whereas ALT, AST, and NEFA concentrations were elevated 2‐fold 3–7 days after PEG‐ASNase. Furthermore, we found, that the hepatic mRNA and protein levels of Srebp‐1c and Fas, which are involved in fatty acid synthesis, were downregulated suggesting a decrease in lipid synthesis after PEG‐ASNase. Furthermore, the hepatic gene and protein expression of Pparα, Lcad, and Mcad, which are involved in fatty acid oxidation, were upregulated after PEG‐ASNase. In vivo VLDL secretion was also increased 3 days after PEG‐ASNase administration. Taken together, our data indicate that other lipid sources are mediating the drug‐induced fatty liver. Among WAT, we found that PEG‐ASNase elevated ATGL, phosphorylated HSL, and PKA protein levels, consistent with the WAT weight loss and the increased plasma NEFA levels.ConclusionOur data suggest that PEG‐ASNase‐induced WAT lipolysis leads to the development of microvesicular hepatic steatosis and toxicity. Our future studies will determine whether PEG‐ASNase hepatic steatosis can be inhibited via the pharmacological or genetic inhibition of adipose lipolysis and identify the molecular mechanism leading to PKA activation and lipolysis.Support or Funding InformationNIH Grants RO1 CA216815. University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261.

Therapeutic effect and mechanism of polysaccharide from Alpiniae oxyphyllae fructus on urinary incontinence.

The purpose of this paper was to investigate the effects and mechanism of polysaccharide (PAOF) from Alpiniae oxyphyllae fructus on urinary incontinence (UI) in old-age hydruric model rats (OHMR). Results suggested that PAOF can significantly reduce the urination volume, Na+, Cl- emission and increase K+ excretion of OHMR. In addition, PAOF can increase the content of aldosterone (ALD) and antidiuretic hormone (ADH) in blood of OHMR. The coefficients of spleen, thymus and adrenal of OHMR were improved by PAOF. Furthermore, PAOF can not only elevate significantly the expression of β3-adrenoceptor mRNA in bladder detrusor of OHMR, but also increase the content of adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in bladder detrusor of OHMR. Meanwhile, PAOF can elevate significantly the expression of PKA protein in bladder detrusor of rats with polyuria. The data implied that PAOF may offer therapeutic potential against UI.

A3707 Myocardial microRNA-101 downregulates classical B1-adrenoreceptor signing cascade resulting in improving the cardiac function

Objectives: We tested the hypothesis that the miR-101 down-regulates β1-AR expression and signaling resulting in improving the cardiac function. Methods: Bioinformatic analysis andhe dual luciferase assays was used to determine whether miR-101 target β1-AR mRNA. Through miR-101 mimic or inhibitor in H9C2 cell, the expression of β1-AR and protein kinase A (PKA), PKA phosphorylation (p-PKA) were tested by western blot. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine miR-101 expression profiles in the heart of spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) rats (n = 6, respectively). Rats were treated with miR-101 agomiR or antagomiR to build a miR-101-upregulated/ downregulated animal model. Cardiac function and HR were assessed by ultrasound diagnostic instrument, systolic blood pressure (SBP) was determined by non-invasive blood pressure analyzer, the expression of β1-AR, PKA, p-PKA protein were detected. Results: I The dual luciferase assays confomed that β1-AR is a direct target gene of miR-101. In vitro study, the expression level of β1-AR, PKA, p-PKA protein were decreased in H9C2 cell transfected with miR-101 mimic (p < 0.05). Meanwhile the expression level of β1-AR expression, PKA protein, p-PKA protein elevated in both H9C2 cell transfected with miR-101 inhibitor; In vivo study, the level of myocardial miR-101 expression were decreased in SHR rats compared to β1-AR (−/ − ) rats and WKY rats (p < 0.05). Elevated of β1-AR protein, PKA protein, p-PKA protein, cAMP level as well as BP, EF, FS in SHR rats was descresed by miR-101 mimic transfection. (p < 0.05). Conclusion: Myocardial microRNA-101 downregulates classical β1-adrenoreceptor signing cascade resulting in improving the cardiac function

FSH receptor binding inhibitor impacts K-Ras and c-Myc of ovarian cancer and signal pathway.

The present study aimed to investigate FSHreceptor binding inhibitor (FRBI) effects on relative factors (K-Ras, c-Myc and Vascular endothelial growth factor (VEGF)) to ovarian cancer, and expression levels of FSH receptor (FSHR) mRNAs and proteins in the cumulus-oocyte complex (COCs), to determine changes of protein kinase A (PKA) in sheep granulosa cells, further to elucidate signaling pathway of FRBI action. COCs were cultured in vitro for 24h under supplementation of varying concentrations of FRBI (0, 10, 20, 30 and 40μg/mL) or FSH (10IU/mL). Concentrations of K-Ras, c-Myc, VEGF, cAMP and FSH were detected in IVM media fluids, respectively. The results showed that the concentrations of c-Myc, K-Ras and FSH of FRBI groups were gradually reduced with the increase of FRBI doses. VEGF level of the FRBI-4 group was significantly greater than control group (CG). Expression levels FSHR mRNA and protein and PKA of FRBI-3 and FRBI-4 groups were less than that of CG or FSH group (P<0.05 or P<0.01). Inositol trisphosphate (IP3) concentrations of FRBI-3 and FRBI-4 groups were less than FSH group (P<0.05). FRBI administration doses had significant negative correlations to levels or concentrations of K-Ras, c-Myc, VEGF, FSHR mRNA and protein and PKA protein. K-Ras had significant positive correlations with FSHR mRNA and protein and PKA protein. In conclusion, FRBI could promote the production of VEGF of sheep COCs. Higher doses of FRBI (30 and 40μg/mL) suppressed the production of c-Myc and K-Ras, and declined FSH concentrations in the IVM medium fluid, and decreased the expressions of FSHR at the gene and protein levels, additionally attenuated expression of PKA protein in the granulosa cells.

Effect of acute mid-intensity treadmill exercise on the androgen hormone level and uncoupling protein-1 expression in brown fat tissue of mouse.

[Purpose]Brown adipose tissue (BAT) plays an important role in metabolizing different substances, including androgens. The aim of this study was to determine whether a single bout of aerobic exercise would increase the androgen hormone concentration in mouse BAT and whether its increase was associated with uncoupling protein-1 (UCP-1), protein kinase A (PKA)-related mechanism in BAT.[Methods]Twenty, 9-week-old ICR adult male micewere randomly divided into three groups: Control (n=6, CON), Exercise (n=7, EX), and Exercise + SRD5A1A2 inhibitor (n=7, EXIN). SRD5A1A2 is an enzyme needed when free testosterone is metabolized to dihydrotestosterone (DHT). SRD5A1A2 was administered intraperitoneally in the EXIN group, while the CON and EX groups were treated with the vehicle only. One hour later, exercise was performed at 60–70% for 30minutes. The levels of testosterone and DHT in BAT were determined by ELISA, and UCP-1 mRNA level was examined by RT-PCR. UCP-1 and PKA protein levels were determined by western blotting.[Results]After a single period of exercise, testosterone and DHT concentrations in BAT were significantly higher in EX than those in CON, and lower in EXIN than those in EX. The ratio of phosphorylated PKA to total PKA in BAT was significantly higher in EX than that in CON, and lower in EXIN than that in EX. UCP-1 levels in BAT were not different in the three groups.[Conclusion]Aerobic exercise increased bioactive androgen hormone levels in BAT in association with the increase in phosphorylated PKA levels. In contrast, 30minutes of treadmill exercise did not affect UCP-1 expression.

Enriched Chicken Bone Broth as a Dietary Supplement Reduces Nociception and Sensitization Associated with Prolonged Jaw Opening.

To test a commercially available enriched chicken bone broth (ECBB) product for its potential anti-inflammatory properties and to evaluate its ability to reduce nociception and expression of protein kinase A (PKA) in a clinically relevant model of temporomandibular disorder (TMD) caused by prolonged jaw opening in rats. The potential of the ECBB and of a homemade broth was investigated using the Folin-Ciocalteu reagent and percent inhibition of cyclooxygenase-2 (COX-2) activity, which was determined using a commercially available kit. Additionally, the effect of ECBB and homemade broth on nocifensive head withdrawal responses to mechanical stimulation in male Sprague-Dawley rats subjected to prolonged jaw opening was evaluated. Differences were considered significant at P < .025. Changes in PKA expression in the medullary dorsal horn region of the spinal trigeminal nucleus associated with prolonged jaw opening were assessed using immunofluorescence, and these changes were considered significant at P < .05. Behavioral data were analyzed by using multiple nonparametric tests, and immunohistochemistry data were analyzed by using one-way analysis of variance with Games-Howell post hoc tests in SPSS software. ECBB exhibited greater reducing potential and inhibition of COX-2 activity compared to homemade broth. Near maximal jaw opening was sufficient to induce sustained nocifensive responses to mechanical stimuli for 7 days. This increased sensitivity was correlated with elevated levels of the active form of PKA. Importantly, dietary inclusion of ECBB, but not of homemade broth, for 2 weeks prior to jaw opening was sufficient to reduce nocifensive behaviors and PKA expression. Findings from this study provide evidence that ECBB attenuates nociception and expression of the pro-inflammatory protein PKA and thus may be beneficial as a nutraceutical supplement to manage inflammatory pain associated with TMD.