The formation of aggregated chromatin/chromosomes in mouse oocytes treated with high concentration of IBMX as a model for a chromosome transfer in human.

The presence of cyclic adenosine monophosphate (cAMP) has been considered to be a fundamental factor in ensuring meiotic arrest prior to ovulation. cAMP is regarded as a key molecule in the regulation of oocyte maturation. However, it has been reported that increased levels of intracellular cAMP can result in abnormal cytokinesis, with some MI oocytes leading to symmetrically cleaved 2-cell MII oocytes. Consequently, we aimed to investigate the effects of elevated intracellular cAMP levels on abnormal cytokinesis and oocyte maturation during the meiosis of mouse oocytes. This study found that a high concentration of isobutylmethylxanthine (IBMX) also caused chromatin/chromosomes aggregation (AC) after the first meiosis. The rates of AC increased the greater the concentration of IBMX. In addition, AC formation was found to be reversible, showing that the re-formation of the spindle chromosome complex was possible after the IBMX was removed. In human oocytes, the chromosomes aggregate after the germinal vesicle breakdown and following the first and second polar body extrusions (the AC phase), while mouse oocytes do not have this AC phase. The results of our current study may indicate that the AC phase in human oocytes could be related to elevated levels of intracytoplasmic cAMP.

Analyses of the onset mechanisms of cardio-stimulatory action by aciclovir.

Cardio-stimulatory actions of aciclovir have been considered to primarily depend on the sympathetically-mediated reflex resulting from its hypotensive effect. To further clarify onset mechanisms of the cardio-stimulatory actions, we initially studied them using isoflurane-anesthetized dogs under thorough ß1-adrenoceptor blockade with atenolol (1 mg/kg, i.v.) (n = 4). Aciclovir (20 mg/kg/10 min, i.v.) decreased mean arterial blood pressure by 10 mmHg, whereas it increased heart rate by 10 bpm and maximum upstroke velocity of ventricular pressure by 928 mmHg/s, and shortened AH interval by 2 ms, indicating that cardio-stimulatory actions were not totally abolished by ß1-adrenoceptor blockade. Then, unknown mechanisms of cardio-stimulatory action were explored. Since aciclovir has a similar chemical structure to theophylline, in silico molecular docking simulation was performed, indicating aciclovir as well as theophylline possesses strong likelihood of interactions with phosphodiesterase 1A, 1C and 3A. Indeed, aciclovir inhibited phosphodiesterase 1A derived from the bovine heart (n = 4), moreover it exerted positive chronotropic action on the atrial tissue preparation of rats along with an increase of tissue cyclic AMP concentration (n = 4). These results indicate that cardio-stimulatory actions of aciclovir could result from not only hypotension-induced, reflex-mediated increase of sympathetic tone but also its inhibitory effects on phosphodiesterase in the heart.

Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, and inverse agonists) of G-protein coupled receptor ligands. Here, we describe the cyclic AMP assay that is carried out with commercially available nonradioligand ready-to-use kits and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

Cyclic adenosine monophosphate (cAMP) analog and phosphodiesterase inhibitor (IBMX) ameliorate human sperm capacitation and motility.

OBJECTIVES: Human sperm quality is decreasing progressively. One of the foremost reasons for infertility is the failure in sperm capacitation. We examined the influence of a cAMP (cyclic-adenosine mono phosphate analog)+IBMX (3-isobutyl-1-methylxanthine) on the motility and capacitation rate of human sperm over time. MATERIAL AND METHODS: Samples were gotten from 20 asthenozoospermic infertile patients referring to the Academic Center for Education, Culture and Research unit of the infertility research center, Qom, Iran. Samples were processed with a Density Gradient Centrifuging. Spermatozoa were divided into 4 groups: control, experimental 1, 2 and 3 (E1, E2, E3) based on the dose/time schedules (cAMP 5mmol+IBMX 0.2mmol/2, 4, and 6h, respectively). The computer-assisted sperm analysis and chlortetracycline assays were used to measure sperm motility and capacitation. RESULTS: After incubation with a cAMP analog and IBMX, the levels of progressive motile sperms considerably improved in all experimental groups compared to the control group (E1=18.89±7.1, E2=30±9.7, E3=26.3±9.6 vs Control=10.28±6.2, P<0.05) especially in E2 group (P<0.05), indicating a greater effect of db cAMP (5mmol) and IBMX (0.2mmol) for 4h compared to the same doses at 2 and 6h. Also, non-progressive motile sperms significantly decreased in E2 group compared to the other groups (P<0.05). Moreover, both patterns C and B were substantially improved in all experimental groups especially in E2 group (P<0.05). CONCLUSION: Our findings support that the supplementation of sperm with db cAMP+IBMX specially for 4h, could be useful for men with asthenozoospermia to improve the success of assisted reproductive technology.

Increase in Ca2+-Activated cAMP/PKA Signaling Prevents Hydroxychloroquine-Induced Bradycardia of the Cardiac Pacemaker.

Bradycardia or tachycardia are known side effects of drugs that limit their clinical use. The heart pacemaker function which control the heart rate under normal conditions is determined by coupled clock system. Thus, interfering with specific clock mechanism will affect other clock mechanisms through changes in interconnected signaling and can lead to rhythm disturbance. However, upregulation of a different clock components can compensate for this change. We focus here on hydroxychloroquine (HCQ), which has been shown effective in treating COVID-19 patients, however its bradycardic side effect limits its clinical use. We aim to decipher the mechanisms underlying the effect of HCQ on pacemaker automaticity, to identify a potential drug that will eliminate the bradycardia. We used isolated rabbit sinoatrial node (SAN) cells, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and mouse SAN cells residing in SAN tissue. Further, we employed SAN cell computational model to suggest mechanistic insights of the effect of HCQ on pacemaker function. HCQ increased mean spontaneous beat interval and variability in all three models in parallel to slower intracellular kinetics. The computational model suggested that HCQ affects the pacemaker (funny) current (If), L-type Ca2+ current (ICa,L), transient outward potassium (Ito) and due to changes in Ca2+ kinetics, the sodium-calcium exchanger current (INCX). Co-application of 3'-isobutylmethylxanthine (IBMX) and HCQ prevented the increase in beat interval and variability in all three experimental models. The HCQ-induced increase in rabbit and mice SAN cell and hiPSC-CM spontaneous beat interval, can be prevented by a phosphodiester inhibitor that restores automaticity due to slower intracellular Ca2+ kinetics.

Tyrosinase suppresses vasculogenic mimicry in human melanoma cells.

Melanoma is a type of skin cancer that derives from melanocytes; this tumor is highly metastatic and causes poor clinical outcomes in patients. Vasculogenic mimicry (VM), a vascular-like network that is formed by tumor cells instead of endothelial cells, promotes the growth and metastasis of tumors by providing tumors with oxygen- and nutrient-containing blood. VM correlates with a poor prognosis in patients with melanoma, but the melanoma-specific mechanisms of VM are unknown. The present study revealed that treatment with the melanogenesis stimulators 3-isobutyl 1-methylxanthine (IBMX) and α-melanocyte-stimulating hormone (α-MSH) significantly inhibited VM in MNT-1 human pigmented melanoma cells. Tyrosinase (TYR), an essential enzyme in melanin production, was upregulated on treatment with α-MSH and IBMX, prompting an examination of the association between TYR and VM. A TYR inhibitor, arbutin, promoted VM in melanoma cells. Furthermore, CRISPR/Cas9-mediated knockout (KO) of TYR increased VM by melanoma cells. Notably, even in non-pigmented melanoma cells, TYR attenuated VM. Although re-expression of wild-type TYR suppressed VM in TYR-KO cells, T373K TYR, a frequently detected mutation in individuals with albinism, failed to inhibit VM. Overall, these results demonstrated that TYR negatively regulates VM, providing novel insights into the antioncogenic function of TYR in melanomas.

Effect of selected natural and synthetic substances on rabbit reproduction-A mini review.

Numerous natural and synthetic substances have effects on reproduction through several mechanisms. This review aims to summarize the impact of green tea (GT), yucca schidigera (YS) extract, curcuma longa (CL), adenosine 3',5'-cyclic monophosphate (cAMP) and isobutyl-1-methyl-xanthine (IBMX) stimulators on rabbit reproduction performance. To obtain a comprehensive overview of this topic, the keywords "reproduction," "substances," "spermatogenesis," "embryogenesis,"hormonal profil", "green tea", "yucca schidigera" were searched in such databases as WOS and PubMed to obtain relevant information. Spermatozoa profile was positively effected by the GT and YS, however, cAMP inhibitors stimulated spermatozoa motility resulted in positive or negative effects depending on the doses. Similarly, embryogenesis and hormonal profile were positively influenced by the GT, YS, cAMP and IBMX in a proper administration dose. Further research is needed to improve current knowledge about these substances to identify potential effects on the other reproduction parameters. Furthermore, future studies should combine GT, YS and CL with different plant extracts to determine their effects on spermatozoa status, embryogenesis as well as hormonal profile as key outcomes. This review summarizes current knowledge about effect of natural and synthetic substances on rabbit reproduction.

cAMP Modulators before In Vitro Maturation Decrease DNA Damage and Boost Developmental Potential of Sheep Oocytes.

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased (p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

The SPOM-adapted IVM system improves in vitro production of bovine embryos.

This study aimed to test the effects of an IVM SPOM adaptation (SPOM-adapted IVM) on the production, total number of cells (TNC), apoptosis, and cryotolerance (post-warming survival and cytoskeleton actin integrity) of bovine IVP embryos. Two experiments were conducted with two experimental groups based on IVM treatment: A control group (TCM 199 without FCS) and an SPOM-adapted group (TCM 199 with forskolin and IBMX in pre-IVM and IVM with cilostamide). The first experiment evaluated embryo in vitro production, TNC, and apoptosis rate on D9 of development. In the second experiment, embryos were vitrified/warmed at D7 (control fresh and vitrified; SPOM-adapted fresh and vitrified) and assessed regarding post-warming survival rates and cytoskeleton actin integrity. Statistical analysis was performed using GraphPad INSTAT software at a significance level of 5%. An increase (p < 0.05) in blastocyst production was observed in the SPOM-adapted group comparing to the control group. There was no difference (p > 0.05) in the TNC or apoptosis rate between the groups. Regarding cryopreservation, no differences were found (p > 0.05) in actin integrity or post-warming survival rates between the vitrified groups. In both vitrified groups, we observed a significantly lower uninjured pattern of actin integrity compared to the fresh groups (p < 0.05). We conclude that the SPOM-adapted IVM system is beneficial for blastocyst production and does not affect the quality and cryotolerance of the produced embryos.

The Phosphodiesterase Inhibitor, Isobutyl-1-Methylxanthine Prevents the Sudden Drop in Cyclic Adenosine Monophosphate Concentration and Modulates Glucose Metabolism of Equine Cumulus-Oocyte Complexes Matured in Vitro.

Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus-oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus-oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus-oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro-matured oocytes causing poor developmental competence.

Molecular Mechanisms Involved in Neural Substructure Development during Phosphodiesterase Inhibitor Treatment of Mesenchymal Stem Cells.

Stem cells are highly important in biology due to their unique innate ability to self-renew and differentiate into other specialised cells. In a neurological context, treating major injuries such as traumatic brain injury, spinal cord injury and stroke is a strong basis for research in this area. Mesenchymal stem cells (MSC) are a strong candidate because of their accessibility, compatibility if autologous, high yield and multipotency with a potential to generate neural cells. With the use of small-molecule chemicals, the neural induction of stem cells may occur within minutes or hours. Isobutylmethyl xanthine (IBMX) has been widely used in cocktails to induce neural differentiation. However, the key molecular mechanisms it instigates in the process are largely unknown. In this study we showed that IBMX-treated mesenchymal stem cells induced differentiation within 24 h with the unique expression of several key proteins such as Adapter protein crk, hypoxanthine-guanine phosphoribosyltransferase, DNA topoisomerase 2-beta and Cell division protein kinase 5 (CDK5), vital in linking signalling pathways. Furthermore, the increased expression of basic fibroblast growth factor in treated cells promotes phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) cascades and GTPase-Hras interactions. Bioinformatic and pathway analyses revealed upregulation in expression and an increase in the number of proteins with biological ontologies related to neural development and substructure formation. These findings enhance the understanding of the utility of IBMX in MSC neural differentiation and its involvement in neurite substructure development.

Different regulation of lipogenesis in sebocytes and subcutaneous preadipocytes in hamsters in vitro.

Sebaceous gland cells (sebocytes) differentiate to intracellularly accumulate lipid droplets - a phenomenon similar to that found in adipocytes. In the present study, we examined whether the regulation of lipogenesis in sebocytes is the same as that in preadipocytes. When sebocytes and preadipocytes, prepared from auricle and subcutaneous adipose tissues from the inguinal region of hamsters, respectively, were treated with a common differentiation inducer, insulin, intracellular lipid-droplet formation and triacyglycerol (TG) production were dose- and time-dependently augmented in both. Insulin increased the production of perilipin, a differentiation marker in both sebocytes and adipocytes. Insulin-like growth factor 1 (IGF-1) augmented the intracellular level of TG in sebocytes and preadipocytes. In addition, the action of 1α,25-dihydroxyvitamin D3 [1,25(OH2)D3] on TG production was the opposite between sebocytes and preadipocytes. Furthermore, 5α-dihydrotestosterone (5α-DHT) augmented the TG level in sebocytes, whereas it did not alter TG production in preadipocytes. Moreover, insulin-augmented TG production in sebocytes was enhanced by IGF-1 and 5α-DHT, while diminished by 1,25(OH2)D3. In preadipocytes, the insulin-augmented production of TG was decreased by IGF-1, 1,25(OH2)D3, and 5α-DHT. These results suggest that sebocytic lipogenesis is partially similar to but substantially different from adipocyte lipogenesis due to the forementioned hormones and growth factors in the skin under physiological conditions.

Effect of the Stromal Vascular Fraction on Changes in Melanin Formation in B16 Cells Treated by IBMX.

OBJECTIVE: To investigate the effect of the stromal vascular fraction (SVF) on changes in melanin formation and tyrosinase activity in B16 cells treated by 3-isobutyl-1 methylxanthine (IBMX) and to explore the mechanism of SVF-mediated inhibition of pigmentation. METHODS: We co-cultured extracted SVFs and B16 cells treated with IBMX in a certain proportion, and the marker molecule HMB-45 was detected by immunochemistry. Melanin content was determined by NaOH lysis. Activity of tyrosinase was measured by the DOPA oxidation method. RESULTS: HMB-45 was commonly expressed in B16 cells induced by IBMX. After the addition of SVFs, the expression of HMB-45 decreased significantly and positively correlated with increases in SVFs. After the induction of B16 cells by IBMX, melanin content increased significantly. However, melanin decreased after SVF and B16 co-culturing; the effect was more substantial with the increase and decrease in SVFs, and the activity of tyrosinase decreased. CONCLUSION: SVFs inhibit the production of melanin and reduce the activity of tyrosinase, possibly providing a new breakthrough for the treatment of pigment disorders. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Effect of pre-maturation with C-type natriuretic peptide and 3-isobutyl-1-methylxanthine on cumulus-oocyte communication and oocyte developmental competence in cattle.

In vitro embryo production depends on oocyte competence, which is acquired during folliculogenesis, involving cytoplasmic and nuclear processes. In vitro maturation (IVM) induces spontaneous resumption of meiosis, preventing full competence acquisition. The incorporation of a pre-IVM phase with supplementation with C-type natriuretic peptide (CNP) and 3-Isobutyl-1-methylxanthine (IBMX) was used with the aim of improving developmental competence of cattle oocytes. In a preliminary experiment, COCs were cultured with increasing CNP concentrations and nuclear stage assessment was performed. Supplementation with both 100 and 200 nM CNP resulted in more germinal vesicle (GV) arrest at 6 h of culture than those in the control group (79.3%, 76.4% and 59.2%, respectively). In a second experiment, use of 100 nM CNP plus 500 µM IBMX resulted in retention of more oocytes in the GV stage (92.0%) at 6 h of culture compared to supplementation with either CNP or IBMX alone (74.8% and 86.7%, respectively). A subsequent assessment of the effect of the pre-IVM system (6-h of culture with CNP plus IBMX), followed by 20-h of IVM, with comparison to the control at 24-h of IVM was performed. Blastocyst development rate was greater after the pre-IVM phase (45.1% compared with 34.5%). The inclusion of the pre-IVM phase also resulted in an enhanced mitochondrial activity in matured oocytes and sustained integrity of transzonal projections for longer after IVM. In conclusion, CNP and IBMX function synergistically to arrest meiosis in cattle oocytes during a pre-IVM phase, which improves cumulus-oocyte communication and embryo development.

Circulating miR-143-3p inhibition protects against insulin resistance in Metabolic Syndrome via targeting of the insulin-like growth factor 2 receptor.

Metabolic syndrome (MetS) is characterized by a cluster of metabolic disorders including obesity, dyslipidemia, hyperglycemia, and hypertension. Here, we report that 27 microRNAs were found to be expressed differently in serum and urine samples of MetS patients compared to control subjects on microarray analysis. Further qualitative real time- polymerase chain reaction analyses confirmed that circulating levels of miR-143-3p were significantly elevated in MetS patients compared with controls, both in serum and urine samples. After accounting for confounding factors, high levels of miR-143-3p remained an independent risk factor for insulin resistance. Inhibition of miR-143-3p expression in mice protected against development of obesity-associated insulin resistance. Furthermore, we demonstrated that insulin-like growth factor 2 receptor (IGF2R) was among the target genes of miR-143-3p by searching 3 widely used bioinformatics databases and preliminary validation. Our experiments suggest that knockdown of circulating miR-143-3p may protect against insulin resistance in the setting of MetS via targeting of IGF2R and activation of the insulin signaling pathway. Our results characterize the miR-143-3p-IGF2R pathway as a potential target for the treatment of obesity-associated insulin resistance.

Fsk and IBMX inhibit proliferation and proapoptotic of glioma stem cells via activation of cAMP signaling pathway.

OBJECTIVE: We aimed to find out the underlying mechanism of forskolin (Fsk) and 3-isobutyl-1-methylxanthine (IBMX) on glioma stem cells (GSCs). METHODS: The expression of cAMP-related protein CREB and pCREB as well as apoptosis-related proteins were detected through Western blot analysis. The level of proliferation and growth rate of human GSCs was measured through thiazolyl blue tetrazolium bromide assay and stem cells forming sphere assay. The apoptosis-related gene expression was measured through reverse transcription-polymerase chain reaction. RESULTS: cAMP signaling pathway was activated in GSCs with Fsk-IBMX administration. Fsk-IBMX could inhibit the proliferation as well as invasion and promote the apoptosis of U87 cells. Besides, U0126 could inhibit MAPK signaling pathway to increase the sensitivity of GSCs to cAMP signaling pathway. As a result, Fsk-IBMX combined with U0126 had more negative effect on GSCs. CONCLUSIONS: The relationship of cAMP and MAPK signaling pathway in GSCs may provide a potential therapeutic strategy in glioma.

A dose-dependent effect of dimethyl sulfoxide on lipid content, cell viability and oxidative stress in 3T3-L1 adipocytes.

Dimethyl sulfoxide (DMSO) is an effective solvent and cytoprotectant agent that can induce diverse actions in experimental settings, ranging from metabolic stress to cytotoxic effects depending on the concentration used. Therefore, for the quality of experiments and reproducibility of results it is essential to establish a precise and non-toxic dose of DMSO within a specific cell system. 3T3-L1 adipocytes, represent a well-established in vitro cell model used to assess the anti-obesity potential of extracts and compounds. Although DMSO is commonly used as a solvent for these experiments, there is limited data available on the compounding effects of using DMSO. The purpose of this study was to assess a concentration-dependent effect of DMSO on lipid content, cell viability and oxidative damage in 3T3-L1 adipocytes. Results showed that DMSO at doses ≥ 0.1% increased mitochondrial membrane potential as measured by JC-1 fluorescent staining, while doses ≥ 10% reduced the lipid content in matured adipocytes. Consistently, higher doses significantly reduced cell viability, elevated reactive oxygen species levels, depleted intracellular glutathione levels, and accelerated apoptosis and cell necrosis. An interesting finding was that a DMSO dose of 0.01% improved glutathione content of 3T3-L1 adipocytes and had minimal effects on cell viability, apoptosis or and necrosis, supporting its antioxidant effect. Therefore, this study provides compelling evidence that precaution should be taken when assessing compounds dissolved in DMSO, particularly doses ≥1% that were shown to induce oxidative stress in 3T3-L1 adipocytes.

cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPß Induction in 3T3-L1 Preadipocytes.

Distal-less homeobox 5 (Dlx5) is a negative regulator of adipogenesis. Dlx5 expression is decreased by adipogenic stimuli, but the mechanisms of Dlx5 downregulation by adipogenic stimuli have not yet been determined. Here, we tested the impact of cAMP/PKA (protein kinase A) signaling induced by 3-isobutyl-1 methyl xanthine (IBMX), forskolin, and 8-CPT-cAMP on the expression of Dlx5 in 3T3-L1 preadipocytes. Significant downregulation of Dlx5 mRNA expression and protein production levels were observed via cAMP/PKA-dependent signaling. Forced expression of cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein ß (C/EBPß) was sufficient for downregulation of Dlx5 expression and revealed that CREB functions upstream of C/EBPß. In addition, C/EBPß knockdown by siRNA rescued Dlx5 expression in IBMX-treated 3T3-L1 preadipocytes. Luciferase assays using a Dlx5-luc-2935 reporter construct demonstrated the requirement of the Dlx5 promoter region, ranging from -774 to -95 bp that contains two putative C/EBPß binding elements (site-1: -517 to -510 bp and site-2: -164 to -157 bp), in the suppression of Dlx5 transcription. Consequently, chromatin immunoprecipitation analysis confirmed the importance of site-1, but not site-2, in C/EBPß binding and transcriptional suppression of Dlx5. In conclusion, we elucidated the underling mechanism of Dlx5 downregulation in IBMX-induced adipogenesis. IBMX activated cAMP/PKA/CREB signaling and subsequently upregulated C/EBPß, which binds to the Dlx5 promoter to suppress Dlx5 transcription.

Stress Odorant Sensory Response Dysfunction in Drosophila Fragile X Syndrome Mutants.

Sensory processing dysfunction (SPD) is present in most patients with intellectual disability (ID) and autism spectrum disorder (ASD). Silencing expression of the Fragile X mental retardation 1 (FMR1) gene leads to Fragile X syndrome (FXS), the most common single gene cause of ID and ASD. Drosophila have a highly conserved FMR1 ortholog, dfmr1. dfmr1 mutants display cognitive and social defects reminiscent of symptoms seen in individuals with FXS. We utilized a robust behavioral assay for sensory processing of the Drosophila stress odorant (dSO) to gain a better understanding of the molecular basis of SPD in FXS. Here, we show that dfmr1 mutant flies present significant defects in dSO response. We found that dfmr1 expression in mushroom bodies is required for dSO processing. We also show that cyclic adenosine monophosphate (cAMP) signaling via PKA is activated after exposure to dSO and that several drugs regulating both cAMP and cyclic guanosine monophosphate (cGMP) levels significantly improved defects in dSO processing in dfmr1 mutant flies.