Cyclic AMP Levels Research Articles

Spint1 disruption in mouse pancreas leads to glucose intolerance and impaired insulin production involving HEPSIN/MAFA

SPINT1, a membrane-anchored serine protease inhibitor, regulates cascades of pericellular proteolysis while its tissue-specific functions remain incompletely characterized. In this study, we generate Spint1-lacZ knock-in mice and observe Spint1 expression in embryonic pancreatic epithelium. Pancreas-specific Spint1 disruption significantly diminishes islet size and mass, causing glucose intolerance and downregulation of MAFA and insulin. Mechanistically, the serine protease HEPSIN interacts with SPINT1 in β cells, and Hepsin silencing counteracts the downregulation of Mafa and Ins1 caused by Spint1 depletion. Furthermore, we demonstrate a potential interaction between HEPSIN and GLP1R in β cells. Spint1 silencing or Hepsin overexpression reduces GLP1R-related cyclic AMP levels and Mafa expression. Spint1-disrupted mice also exhibit a significant reduction in Exendin-4-induced insulin secretion. Moreover, SPINT1 expression increases in islets of prediabetic humans compared to non-prediabetic groups. The results unveil a role for SPINT1 in β cells, modulating glucose homeostasis and insulin production via HEPSIN/MAFA signaling.

Health Benefits of Epigallocatechin Gallate and Forskolin with a Special Emphasis on Glaucoma and Other Retinal Diseases

This review highlights the therapeutic potential of epigallocatechin gallate (EGCG) and forskolin in managing retinal diseases, with a focus on glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy. EGCG, a potent polyphenol from green tea, exhibits significant antioxidant, anti-inflammatory, and neuroprotective effects, making it a promising candidate for reducing oxidative stress and inflammation in ocular tissues. Forskolin, a diterpene from Coleus forskohlii, increases cyclic AMP (cAMP) levels, which helps lower intraocular pressure (IOP) and provides neuroprotection. Both compounds target critical pathways involved in retinal disease progression, including oxidative stress, mitochondrial dysfunction, and inflammation, offering complementary therapeutic benefits. This review consolidates preclinical and clinical studies, highlighting the potential of EGCG and forskolin as adjunctive or alternative treatments for retinal diseases. Future research should explore the synergistic effects of these compounds, particularly in combination therapies aimed at addressing multiple pathogenic mechanisms in retinal health.

Epidermal Growth Factor Reduces Collecting Duct Water Permeability by Inhibiting Aquaporin-2 Phosphorylation at Ser269

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism of this response. Using the Burg isolated perfused tubule technique in microdissected rat inner medullary collecting ducts (IMCDs), we found that addition of 0.1 μM EGF to the peritubular bath significantly decreased 0.1 nM 1‐deamino‐8‐D‐arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on dDAVP-stimulated water permeability was associated with a reduction in intracellular cyclic AMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces dDAVP-stimulated aquaporin-2 (AQP2) phosphorylation at S264 and S269. This effect was absent when membrane-permeable 8-cpt-cAMP (0.1 mM) was used to stimulate AQP2 phosphorylation, suggesting that EGF’s effect was on a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for S269 phosphorylation, we used Bayesian analysis to integrate multiple -omic datasets. Thirteen top ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at S256, S264, and S269. None of the other kinases phosphorylated S269. Additionally, PKA inhibitors, H-89 and PKI, strongly inhibited dDAVP-stimulated AQP2 phosphorylation at S269. These results indicate that EGF decreases the water permeability of the collecting duct by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at S269, a site previously shown to regulate AQP2 endocytosis. This work was funded by NHLBI project ZIA-HL001285 and ZIA-HL006129, M.A.K. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of glucagon to support postabsorptive glucose flux and glycemia in healthy rats and its attenuation in male Zucker diabetic fatty rats.

Hyperglucagonemia is a hallmark of type 2 diabetes (T2DM), yet the role of elevated plasma glucagon (P-GCG) to promote excessive postabsorptive glucose production and contribute to hyperglycemia in patients with this disease remains debatable. We investigated the acute action of P-GCG to safeguard/support postabsorptive endogenous glucose production (EGP) and euglycemia in healthy Zucker control lean (ZCL) rats. Using male Zucker diabetic fatty (ZDF) rats that exhibit the typical metabolic disorders of human T2DM, such as excessive EGP, hyperglycemia, hyperinsulinemia, and hyperglucagonemia, we examined the ability of hyperglucagonemia to promote greater rates of postabsorptive EGP and hyperglycemia. Euglycemic or hyperglycemic basal insulin (INS-BC) and glucagon (GCG-BC) clamps were performed in the absence or during an acute setting of glucagon deficiency (GCG-DF, ∼10% of basal), either alone or in combination with insulin deficiency (INS-DF, ∼10% of basal). Glucose appearance, disappearance, and cycling rates were measured using [2-3H] and [3-3H]-glucose. In ZCL rats, GCG-DF reduced the levels of hepatic cyclic AMP, EGP, and plasma glucose (PG) by 50%, 32%, and 50%, respectively. EGP fell in the presence GCG-DF and INS-BC, but under GCG-DF and INS-DF, EGP and PG increased two- and threefold, respectively. GCG-DF revealed the hyperglucagonemia present in ZDF rats lacked the ability to regulate hepatic intracellular cyclic AMP levels and glucose flux, since EGP and PG levels fell by only 10%. We conclude that the liver in T2DM suffers from resistance to all three major regulatory factors, glucagon, insulin, and glucose, thus leading to a loss of metabolic flexibility.NEW & NOTEWORTHY In postabsorptive state, basal plasma insulin (P-INS) and plasma glucose (PG) act dominantly to increase hepatic glucose cycling and reduce endogenous glucose production (EGP) and PG in healthy rats, which is only counteracted by the acute action of basal plasma glucagon (P-GCG) to support EGP and euglycemia. Hyperglucagonemia, a hallmark of type 2 diabetes (T2DM) present in Zucker diabetic fatty (ZDF) rats, is not the primary mediator of hyperglycemia and high EGP as commonly thought; instead, the liver is resistant to glucagon as well as insulin and glucose.

Insulin Resistance Develops Due to an Imbalance in the Synthesis of Cyclic AMP and the Natural Cyclic AMP Antagonist Prostaglandylinositol Cyclic Phosphate (Cyclic PIP)

The reasons initiating insulin resistance are not identified. Various metabolic derailments have been characterized. These are the outcome and not the initiation of insulin resistance. In animal models of type 2 diabetes and hypertension, a decreased hormonal stimulation of the synthesis of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP) was determined. The resultant imbalance of the action of cyclic AMP and cyclic PIP shifts metabolic regulation to the dominance of catabolism and a decrease in imperative anabolism. This dominance develops gradually since the more cyclic AMP dominates, the more the synthesis of cyclic PIP will be inhibited. Vanishing actions of cyclic PIP are its 10-fold activation of glucose uptake in adipocytes, its inhibition of insulin release from pancreatic β-cells, its inhibition of PKA and its 7-fold activation of protein ser/thr phosphatase. Reduced synthesis of cyclic PIP results from (a) decreased substrate availability, (b) long-time elevated cyclic AMP levels resulting from stress overloads and (c) aging and the gradual decrease in the synthesis of hormones which likely maintain mechanisms that stimulate cyclic PIP synthesis. The need is to discover which hormones, such as growth hormone, insulin-like growth factor-1, dehydroepiandrosterone, and testosterone, are involved in maintaining the stimulation of cyclic PIP synthesis.

Biochemical differences between high and low dose methadone clients on stable maintenance therapy

The methadone dose required to prevent withdrawal symptoms in chronic methadone users varies dramatically from 5 to 300 mg/day, which could be attributed to a number of biochemical factors, including human µ-opioid receptor (hMOR) density, down-stream desensitization, concurrent drug use or P-glycoprotein (P-gp) expression, or other, possibly genetic factors. Here we describe some biochemical changes of patients taking higher versus lower doses in chronic methadone maintenance therapy (MMT). hMOR density was measured in leukocytes from methadone-maintained patients receiving lower dose (<100 mg/day) or higher dose (≥100 mg/day) and naive subjects by flow cytometry. Desensitization of receptor signaling was examined by measuring in vivo and attenuated cyclic AMP (cAMP) levels in response to increasing methadone concentrations. P-gp expression and the presence of other drugs were measured using validated methods.

Dysregulated Cyclic Nucleotide Metabolism in Alcohol-Associated Steatohepatitis: Implications for Novel Targeted Therapies.

Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking. Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH). Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis. These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.

SAT648 The Role Of Olfactory Marker Protein On Adipogenesis

Abstract Disclosure: Y. Bao: None. Y. Cho: None. E. Lee: None. Olfactory marker protein (OMP) plays a role in regulating the of olfactory transduction but is also expressed in adipose tissue. OMP serves as a regulating buffer for cyclic AMP (cAMP) levels; thus, we hypothesized that Omp may play a role in modulating adipocyte differentiation, a cAMP-dependent process. To determine the role of OMP in adipogenesis, we examined the differences in body weight, adipose tissue mass, and adipogenic or thermogenic gene expression of normal and Omp-knockout (KO) mice fed a high-fat diet. The cAMP production, adipogenic gene expression, and cAMP response element binding protein (CREB) phosphorylation were measured during differentiation in 3T3-L1 preadipocytes and mouse embryonic fibroblasts (MEFs). Finally, RNA sequencing was performed to find out why adipogenesis was reduced when Omp was deleted. We observed a decrease in body weight, adipose tissue mass, and the size of adipocytes in Omp-KO mice. We confirmed low cAMP production and CREB phosphorylation in adipogenesis induced in Omp-/- MEFs, and nuclear factor kappa B activation due to significantly reduced IκBα expression. Collectively, our results suggest that loss of OMP function inhibits adipogenesis through affecting adipocyte differentiation. Presentation: Saturday, June 17, 2023

Fushenmu treatment ameliorates RyR2 with related metabolites in a zebrafish model of barium chloride induced arrhythmia

BackgroundFushenmu (Pini Radix in Poria, FSM) is a folk parasitic herb that has been mainly used for palpitation and amnesiain in traditional Chinese medicine (TCM). Recently, as an individual herb or a component of formulations, Fushenmu exhibits therapeutic potential for the treatment of cardiac arrhythmias. Yet, how specific targets or pathways of Fushenmu inhibit arrhythmia has not yet been reported.MethodsHere, based on clinical functional genomics, metabolomics and molecular biologic technologies, a network construction strategy was adopted to identify FSM therapeutic targets and biomarkers that might explore its functions.ResultsIn this study, it was found that FSM recovered arrhythmia-associated heart failure in barium chloride (BaCl2) induced arrhythmic zebrafish embryos, as was evidenced by the shortened cardiac sinus venosus—bulbus arteriosus (SV-BA) distance, smaller cardiovascular bleeding areas, and reduced cardiomyocyte apoptosis. Moreover, analysis via ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-QTOF-ESI-MS/MS) components identification and network pharmacology prediction showed that 11 main active components of FSM acted on 33 candidate therapeutic targets. Metabolomic analysis also suggested that FSM could rescue 242 abnormal metabolites from arrhythmic zebrafish embryos. Further analysis based on the combination of target prediction and metabolomic results illustrated that FSM down-regulated Ryanodine Receptor 2 (RyR2) expressions, inhibited adrenaline and 3',5'-Cyclic AMP (cAMP) levels in a dose-dependent manner, which was confirmed by metabolites quantification and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay.ConclusionIn summary, this study revealed that FSM mitigated BaCl2 induced cardiac damage caused by arrhythmia by suppressing RyR2 expressions, decreasing adrenaline and cAMP through the adrenergic signalling pathway.

Salt-inducible kinases regulate androgen synthesis in theca cells by enhancing CREB signaling

Ovulation is the pinnacle of folliculogenesis, a process that requires an interplay between the oocyte, the granulosa cells, and the theca cells (TCs). TCs are the only source of ovarian androgens, which play a vital role in female fertility. However, abnormally elevated androgen levels reduce fertility. Therefore, uncovering novel mechanisms regulating androgen synthesis in TCs is of great significance. We have shown that salt-inducible kinases (SIKs) regulate granulosa cell steroidogenesis. Here, we investigated whether SIKs regulate androgen production in TCs. SIK2 and SIK3 were detected in the TCs of mouse ovaries and isolated TCs. Next, TCs in culture were treated with luteinizing hormone (LH) in the presence or absence of a highly specific SIK inhibitor. SIK inhibition enhanced the stimulatory effect of LH on steroidogenic gene expression and androgen production in a concentration-dependent manner. SIK inhibition alone stimulated the expression of steroidogenic genes and increased androgen production. Activation of adenylyl cyclase with forskolin or emulation of increased intracellular cyclic AMP levels stimulated steroidogenesis, an effect that was enhanced by the inhibition of SIK activity. The stimulatory effect of downstream targets of cyclic AMP was also significantly augmented by SIK inhibition, suggesting that SIKs control targets downstream cyclic AMP. Finally, it is shown that SIK2 knockout mice have higher circulating testosterone than controls. This evidence shows that TCs express SIKs and reveal novel roles for SIKs in the regulation of TC function and androgen production. This information could contribute to uncovering therapeutic targets to treat hyperandrogenic diseases.

Impact of the cAMP efflux and extracellular cAMP-adenosine pathway on airway smooth muscle relaxation induced by formoterol and phosphodiesterase inhibitors

β2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by β2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5′-nucleotidases (ecto-5′NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that β2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5′NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by β2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only β2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.

Olfactory marker protein regulates adipogenesis via the cAMP–IκBα pathway

Olfactory marker protein (OMP) regulates olfactory transduction and is also expressed in adipose tissue. Since it serves as a regulatory buffer for cyclic AMP (cAMP) levels, we hypothesized that it plays a role in modulating adipocyte differentiation. To determine the role of OMP in adipogenesis, we examined the differences in body weight, adipose tissue mass, and adipogenic or thermogenic gene expression between high-fat diet-fed control and Omp-knockout (KO) mice. cAMP production, adipogenic gene expression, and cAMP response element binding protein (CREB) phosphorylation were measured during the differentiation of 3T3-L1 preadipocytes and mouse embryonic fibroblasts (MEFs). RNA sequencing was performed to determine the gene expression patterns responsible for the reduction in adipogenesis when Omp was deleted. Body weight, adipose tissue mass, and adipocyte size decreased in Omp-KO mice. Furthermore, cAMP production and CREB phosphorylation reduced during adipogenesis induced in Omp−/− MEFs, and the Nuclear factor kappa B was activated due to significantly reduced expression of its inhibitor. Collectively, our results suggest that loss of OMP function inhibits adipogenesis by affecting adipocyte differentiation.

Association of a single amino acid replacement with dorsal pigmentation in a lizard from the Qinghai-Tibetan Plateau

Reptiles can evolve adaptive colors in different environments, but relatively little is known about the genetic mechanisms. Here, we identified the MC1R gene and its association with intraspecific color variation in the lizard Phrynocephalus erythrurus. Analysis of the MC1R sequence in 143 individuals from dark South Qiangtang Plateau (SQP) and light North Qiangtang plateau (NQP) populations, revealed two amino acid sites that showed significant differences in frequency between two areas. One SNP, corresponding to Glu183Lys residue, was found to be a highly significant outlier and differentially fixed for SQP and NQP populations. This residue is located in an extracellular area in the second small extracellular loop within the secondary structure of MC1R, which represents an “attachment pocket” part of the 3D structure. Cytological expression of MC1R alleles with the Glu183Lys replacement showed a 39 % increase in intracellular agonist-induced cyclic AMP levels and a 23.18 % greater cell surface expression of MC1R protein in the SQP relative to the NQP allele. Further in silico 3D modeling and in vitro binding experiments indicated a higher MC1R-α-MSH binding for the SQP allele, and elevated melanin synthesis. We provide an overview of how a single amino acid replacement leads to fundamental changes in MC1R function, and hence shapes variation in dorsal pigmentation in lizards from different environments.

Relaxin-like Gonad-Stimulating Peptides in Asteroidea.

Starfish relaxin-like gonad-stimulating peptide (RGP) is the first identified peptide hormone with gonadotropin-like activity in invertebrates. RGP is a heterodimeric peptide, comprising A and B chains with disulfide cross-linkages. Although RGP had been named a gonad-stimulating substance (GSS), the purified peptide is a member of relaxin-type peptide family. Thus, GSS was renamed as RGP. The cDNA of RGP encodes not only the A and B chains, but also signal and C-peptides. After the rgp gene is translated as a precursor, mature RGP is produced by eliminating the signal and C-peptides. Hitherto, twenty-four RGP orthologs have been identified or predicted from starfish in the orders Valvatida, Forcipulatida, Paxillosida, Spinulosida, and Velatida. The molecular evolution of the RGP family is in good accordance with the phylogenetic taxonomy in Asteroidea. Recently, another relaxin-like peptide with gonadotropin-like activity, RLP2, was found in starfish. RGP is mainly present in the radial nerve cords and circumoral nerve rings, but also in the arm tips, the gonoducts, and the coelomocytes. RGP acts on ovarian follicle cells and testicular interstitial cells to induce the production of 1-methyladenine (1-MeAde), a starfish maturation-inducing hormone. RGP-induced 1-MeAde production is accompanied by an increase in intracellular cyclic AMP levels. This suggests that the receptor for RGP (RGPR) is a G protein-coupled receptor (GPCR). Two types of GPCRs, RGPR1 and RGPR2, have been postulated as candidates. Furthermore, 1-MeAde produced by RGP not only induces oocyte maturation, but also induces gamete shedding, possibly by stimulating the secretion of acetylcholine in the ovaries and testes. Thus, RGP plays an important role in starfish reproduction, but its secretion mechanism is still unknown. It has also been revealed that RGP is found in the peripheral adhesive papillae of the brachiolaria arms. However, gonads are not developed in the larvae before metamorphosis. It may be possible to discover new physiological functions of RGP other than gonadotropin-like activity.

Loss-of-Function Variants in DRD1 in Infantile Parkinsonism-Dystonia.

The human dopaminergic system is vital for a broad range of neurological processes, including the control of voluntary movement. Here we report a proband presenting with clinical features of dopamine deficiency: severe infantile parkinsonism-dystonia, characterised by frequent oculogyric crises, dysautonomia and global neurodevelopmental impairment. CSF neurotransmitter analysis was unexpectedly normal. Triome whole-genome sequencing revealed a homozygous variant (c.110C>A, (p.T37K)) in DRD1, encoding the most abundant dopamine receptor (D1) in the central nervous system, most highly expressed in the striatum. This variant was absent from gnomAD, with a CADD score of 27.5. Using an in vitro heterologous expression system, we determined that DRD1-T37K results in loss of protein function. Structure-function modelling studies predicted reduced substrate binding, which was confirmed in vitro. Exposure of mutant protein to the selective D1 agonist Chloro APB resulted in significantly reduced cyclic AMP levels. Numerous D1 agonists failed to rescue the cellular defect, reflected clinically in the patient, who had no benefit from dopaminergic therapy. Our study identifies DRD1 as a new disease-associated gene, suggesting a crucial role for the D1 receptor in motor control.

Intracellular Nitric Oxide and cAMP Are Involved in Cellulolytic Enzyme Production in Neurospora crassa.

Although molecular regulation of cellulolytic enzyme production in filamentous fungi has been actively explored, the underlying signaling processes in fungal cells are still not clearly understood. In this study, the molecular signaling mechanism regulating cellulase production in Neurospora crassa was investigated. We found that the transcription and extracellular cellulolytic activity of four cellulolytic enzymes (cbh1, gh6-2, gh5-1, and gh3-4) increased in Avicel (microcrystalline cellulose) medium. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) detected by fluorescent dyes were observed in larger areas of fungal hyphae grown in Avicel medium compared to those grown in glucose medium. The transcription of the four cellulolytic enzyme genes in fungal hyphae grown in Avicel medium was significantly decreased and increased after NO was intracellularly removed and extracellularly added, respectively. Furthermore, we found that the cyclic AMP (cAMP) level in fungal cells was significantly decreased after intracellular NO removal, and the addition of cAMP could enhance cellulolytic enzyme activity. Taken together, our data suggest that the increase in intracellular NO in response to cellulose in media may have promoted the transcription of cellulolytic enzymes and participated in the elevation of intracellular cAMP, eventually leading to improved extracellular cellulolytic enzyme activity.

Small Hepatitis B Virus Surface Antigen Promotes Hepatic Gluconeogenesis via Enhancing Glucagon/cAMP/Protein Kinase A/CREB Signaling.

Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (PEPCK) in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. IMPORTANCE Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both in vivo and in vitro. This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.

ODP117 Pseudohypoparathyroidism Type IB: A Rare Cause of Hypocalcemia

Abstract Introduction Pseudohypoparathyroidism (PHP) describes disorders of hypoparathyroidism that are the result of resistance of target tissues to the actions of parathyroid hormone (PTH). Studies have estimated the prevalence of PHP to be 0.34 in 100,000 in Japan and 1.1 in 100,000 in Denmark. The first biochemical abnormalities to become apparent are elevated serum levels of PTH and elevated serum levels of phosphorus, followed by hypocalcaemia. We present a patient diagnosed with PHP Type 1B (PHP1B), which was presented to us many years after her initial diagnosis, with other major comorbidities now present. Case description This patient is an 81 year old female presenting to the clinic to discuss her previously diagnosed PHP1B. Her past medical history revealed she began having symptoms of intermittent muscle cramping at the age of 22, followed by carpopedal spasms and periorbital paresthesia a year later. She has also had a physical examination significant for a positive Trousseau sign, but no phenotypical signs of Albright hereditary Osteodystrophy (AHO). Lab evidence from 1977 was recovered showing urinary cyclic AMP level <20 nmol/mg after administration of PTH, consistent with PHP1B. The family history in her parents and children has revealed no similar genetic history so far. Other comorbidities she has developed since her diagnosis are hypokalemia, hypothyroidism, osteoporosis with a femur fracture, obesity, chronic kidney disease and nephrolithiasis. She has been continuing treatment throughout her life with calcium, calcitriol and vitamin D supplementation for hypocalcemia. She has been mostly eucalcemic since then, and complains of occasional carpopedal spasms and perioral numbness. Discussion PHP1B is isolated resistance to PTH in the kidney and normal PTH response in bone without features of AHO. PHP1A and 1C have PTH resistance in bone, and both express AHO. These various forms of PHP are due to defects in the GNAS gene that lead to decreased expression of the alpha-subunit of G protein (Gsa). In the absence of molecular analysis, the clinical and biochemical overlap between PHP and related disorders can lead to challenges in diagnostic classification. There are 2 types of PHP1B cases- autosomal dominant (AD) and sporadic. However, the majority of Patients with PHP1B do not have mutations within the GNAS but they have methylation and imprinting defects that result in absence of maternal Gsa. To emphasize, epimutation study rather than sequencing of the coding region in GNAS should be performed for genetic investigation, which has now become standard practice. Although there is no difference in clinical presentation between AD PHP1B and sporadic PHP1B, their differentiation early during the disease onset can help providers give patients more reliable genetic and reproductive counseling and provide optimal medical management of complications such as hypocalcemia. Presentation: No date and time listed

Quantification of cyclic AMP and cyclic GMP levels in Krebs-Henseleit solution by LC-MS/MS: Application in washed platelet aggregation samples

Cyclic Nucleotides are important in regulating platelet function. Increases in 3′5′-cyclic adenosine monophosphate (cAMP) and 3′5′-cyclic guanosine monophosphate (cGMP) inhibit platelet aggregation and are pharmacological targets for antiplatelet therapy. Here we report an improved method for determining cAMP and cGMP concentrations and, for the first time, in washed platelet supernatants by combining high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). Characteristic peaks of the substrates, cGMP or cAMP and their internal standards were identified in negative-ion electrospray ionisation using multiple reaction monitoring. Compared with previously reported methods, the method presented here shows high precision with the lowest lower limit of quantification (LLoQ) to date (10 pg/mL). The effect of a novel catecholamine, 6-nitrodopamine, on cyclic nucleotide levels was quantified. Our results showed that this new method was fast, sensitive, and highly reproducible.

Piezo1 stimulates mitochondrial function via cAMP signaling.

Mechanical signals stimulate mitochondrial function but the molecular mechanisms are not clear. Here, we show that the mechanically sensitive ion channel Piezo1 plays a critical role in mitochondrial adaptation to mechanical stimulation. The activation of Piezo1 induced mitochondrial calcium uptake and oxidative phosphorylation (OXPHOS). In contrast, loss of Piezo1 reduced the mitochondrial oxygen consumption rate (OCR) and adenosine triphosphate (ATP) production in calvarial cells and these changes were associated with increased expression of the phosphodiesterases Pde4a and lower cyclic AMP (cAMP) levels. In addition, Piezo1 increased cAMP production and the activation of a cAMP-responsive transcriptional reporter. Consistent with this, cAMP was sufficient to increase mitochondrial OCR and the inhibition of phosphodiesterases augmented the increase in OCR induced by Piezo1. Moreover, the inhibition of cAMP production or activity of protein kinase A, a kinase activated by cAMP, prevented the increase in OCR induced by Piezo1. These results demonstrate that cAMP signaling contributes to the increase in mitochondrial OXPHOS induced by activation of Piezo1.