Atomoxetine and Fluoxetine Activate AMPK-ACC-CPT1 Pathway in Human SH-SY5Y and U-87 MG Cells.

OBJECTIVE: Atomoxetine and fluoxetine are psychopharmacologic agents associated with loss of appetite and weight. Adenosine monophosphate-activated protein kinase (AMPK) is the cellular energy sensor that regulate metabolism and energy, being activated by fasting and inhibited by feeding in the hypothalamus. METHODS: Human brain cell lines (SH-SY5Y and U-87 MG cells) were used to study the outcome of atomoxetine and fluoxetine treatment in the activity of AMPK-acetyl-CoA carboxylase (ACC)- carnitine palmitoyl transferase 1 (CPT1) pathway and upstream regulation by calcium/calmodulin-dependent kinase kinase ß (CaMKKß) using immunoblotting and CPT1 enzymatic activity measures. RESULTS: Phosphorylation of AMPK and ACC increased significantly after atomoxetine and fluoxetine treatment in the first 30-60 minutes of treatment in the two cell lines. Activation of AMPK and inhibition of ACC was associated with an increase by 5-fold of mitochondrial CPT1 activity. Although the neuronal isoform CPT1C could be detected by immunoblotting, activity was not changed by the drug treatments. In addition, the increase in phospho-AMPK and phospho-ACC expression induced by atomoxetine was abolished by treatment with STO-609, a CaMKKß inhibitor, indicating that AMPK-ACC-CPT1 pathway is activated through CaMKKß phosphorylation. CONCLUSION: These findings indicate that at the cellular level atomoxetine and fluoxetine treatments may activate AMPK-ACC-CPT1 pathways through CaMKKß in human SH-SY5Y and U-87 MG cells.

Dibutyl phthalate exposure during gestation and lactation in C57BL/6 mice: Maternal behavior and neurodevelopment in pups.

OBJECTIVES: Neurotoxic effects of phthalate during pregnancy on immature brain of the offspring or mature brains of the mothers remain unclear. We examined the effect of dibutyl phthalate (DBP) exposure during gestation and lactation on the maternal behavior of mother mice and neurodevelopment in pups. METHODS: Pregnant mice were treated orally with DBP (0, 50 and 100 mg/kg/day, N = 20 per group) from gestational day 13 to postnatal day (PND) 15. Maternal behavior was measured using pup retrieval and nest shape test at postpartum day 4. For the pups, the neurodevelopment was measured using negative geotaxis, cliff avoidance at PND 7, swimming test and olfactory orientation at PND 14. RNA and protein expressions in the brain cortex of 50 mg/kg/day and control group (0 mg/kg/day) were analyzed using microarray and Western blot analysis. Nissl-stained sections at the coronal level of interaural 2.56 mm, bregma -1,23 mm, were used for counting of dark cortical neurons in mother and pup mice. RESULTS: DBP treated mother mice (50 and 100 mg/kg/day) showed poor maternal behavior, poor nesting and retrieval behavior compared to the control group (0 mg/kg/day). In brain cortex, DBP-treated mothers showed decrease in protein expression of Nr4a3, Egr1, Arc, BDNF and phosphorylation of AKT and CREB, were also decreased in cortex of DBP-treated mothers. Pups exposed to DBP showed significantly decreased scores in negative geotaxis at PND 7 and swimming scores and olfactory orientation tests at PND 14. The cortex of the DBP exposed pups showed increase in expression of dopamine receptor D2 gene. Nissl staining showed that the dark neurons were increased in cortex of DBP treated mothers and DBP exposed pups. Suggesting that phthalate may delay pup development indirectly through inadequate mothering as well as direct phthalate exposure on the brain. CONCLUSION: DBP exposure during gestation and lactation cause impairment in maternal behaviors and downregulation of neuronal plasticity and survival signals. Pups of mothers with exposed to DBP, showed delayed neurodevelopment and dark neurons increase in brain cortex, suggesting that phthalate may delay pup development indirectly through inadequate mothering as well as direct phthalate exposure on the brain.

Rapid securing of reference substances from Peucedanum japonicum Thunberg by recycling preparative high-performance liquid chromatography.

Among the most critical needs of natural product chemistry is a complete library of pure reference substances. Some khellactone-type isomers of pharmacological importance are either still lacking reference substances or references are only available in limited amounts. To address this need, a recycling high-performance liquid chromatography (R-HPLC) strategy was adopted to improve the isomer separation efficiency from Peucedanum japonicum. Under the optimal isolation conditions, we obtained isomerically pure substances, particularly khellactone coumarins with different substituent groups. Isolated compounds attained purities greater than 98% as determined by ultra-performance liquid chromatography-charged aerosol detector (UPLC-CAD) and photodiode array (PDA). The structures of these compounds were identified according to their mass patterns and 2D NMR spectra. The proposed methods of single-column recycling obtained the same amount of product as conventional systems while being simple, increasing efficiency and reducing cost.

Restoration of Cdk5, TrkB and Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor Proteins after Chronic Methylphenidate Treatment in Spontaneous Hypertensive Rats, a Model for Attention-Deficit Hyperactivity Disorder.

OBJECTIVE: Synaptic vesicle mobilization and neurite outgrowth regulation molecules were examined in modulation of effects of methylphenidate (MPH) in Spontaneous Hypertensive Rats (SHRs), a model for attention-deficit hyperactivity disorder (ADHD). METHODS: We compared the changes in the protein expression level of Cyclin dependent kinase 5 (Cdk5) and molecular substrates of Cdk5; tropomyosin receptor kinase B (TrkB), syntaxin 1A (STX1A) and synaptosomal-associated protein 25 (SNAP25). Comparisons were made in prefrontal cortex of vehicle (distilled water i.p. for 7 days)-treated SHRs, vehicle-treated Wistar Kyoto Rats (WKYs) and MPH (2 mg/kg i.p. for 7 days) treated SHRs. RESULTS: The Cdk5 level of vehicle-treated SHRs was significantly decreased compared to the Cdk5 level of vehicle-treated WKY rats, but was restored to the expression level of vehicle-treated WKYs in MPH-treated SHR. The ratio of p25/p35 was significantly decreased in MPH-treated SHR compared to vehicle-treated SHR. Moreover, TrkB, STX1A and SNAP25 of vehicle-treated SHRs were significantly decreased compared to vehicle-treated WKY rats, but were restored to the expression level of vehicle-treated WKYs in MPH-treated SHR. CONCLUSION: The results show that Cdk5, TrkB, STX1A, and SNAP25 were involved in the modulation of MPH effects in prefrontal cortex of SHRs and play important role in treatment of ADHD.