Increased expression level of ANGPTL8 in white adipose tissue under acute and chronic cold treatment.

BACKGROUND: Angiopoietin-like proteins (ANGPTL), primarily 3, 4, and 8, play a major role in maintaining energy homeostasis by regulating triglyceride metabolism. This study evaluated the level of ANGPTL3, 4, and 8 in the liver, brown adipose tissue (BAT), and subcutaneous white adipose tissue (SAT) of mice maintained under acute and chronic cold conditions. METHODS: C57BL/6J mice were exposed to cold temperature (4 °C) for 10 days with food provided ad libitum. Animal tissues were harvested at Day 0 (Control group, n = 5) and Days 1, 3, 5, and 10 (cold treatment groups, n = 10 per group). The expression levels of various genes were measured in the liver, SAT, and BAT. ANGPTL3, 4, and 8 expressions were measured in the liver. ANGPTL4, 8, and genes involved in browning and lipid metabolism [uncoupling protein 1 (UCP1), lipoprotein lipase (LPL), and adipose triglyceride lipase (ATGL)] were measured in SAT and BAT. Western blotting (WB) analysis and immunohistochemistry (IHC) were performed to confirm ANGPTL8 expression in these tissues. RESULTS: The expressions of ANGPTL3 and 8 mRNA were significantly reduced in mouse liver tissues after cold treatment (P < 0.05); however, the expression of ANGPTL4 was not significantly altered. In BAT, ANGPTL8 expression was unchanged after cold treatment, whereas ANGPTL4 expression was significantly reduced (P < 0.05). ANGPTL4 levels were also significantly reduced in SAT, whereas ANGPTL8 gene expression exhibited over a 5-fold increase. Similarly, UCP1 gene expression was also significantly increased in SAT. The mRNA levels of LPL and ATGL showed an initial increase followed by a gradual decrease with an increase in the days of cold exposure. ANGPTL8 protein overexpression was further confirmed by WB and IHC. CONCLUSIONS: This study shows that exposure to acute and chronic cold treatment results in the differential expression of ANGPTL proteins in the liver and adipose tissues (SAT and BAT). The results show a significant reduction in ANGPTL4 in BAT, which is linked to improved thermogenesis in response to acute cold exposure. ANGPTL8 was activated under acute and chronic cold conditions in SAT, suggesting that it is involved in regulating lipolysis and enhancing SAT browning.

Higher Levels of ANGPTL5 in the Circulation of Subjects With Obesity and Type 2 Diabetes Are Associated With Insulin Resistance.

Objective: The family of angiopoietin-like proteins (ANGPTLs) is composed of eight ANGPTLs members that are involved in regulating various metabolic processes and have been implicated in type 2 diabetes (T2D) and obesity. ANGPTL5 is an understudied member of this family that has been suggested to regulate triglyceride metabolism with a potential role in obesity. This study was designed to investigate the expression levels of ANGPTL5 protein in the circulation of subjects with obesity and T2D. Methods: A total of 204 subjects were enrolled in this cross-sectional study, of which 95 had diagnosed T2D and 109 did not (non-T2D). Within the non-T2D group, 39 subjects were obese (BMI ≥ 30 Kg/m2) and 70 were not (BMI < 30 Kg/m2). Among subjects with T2D, 61 were obese and 34 were non-obese. Circulating ANGPTL5 plasma levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: In this study, we showed that ANGPTL5 levels were higher in the plasma of subjects with T2D [mean ± standard error of the mean (SEM): 5.78 ± 2.70 ng/mL] compared with individuals without T2D (mean ± SEM: 4.42 ± 2.22 ng/mL; P < 0.001). Obese and non-T2D subjects had significantly higher levels of ANGPTL5 (mean ± SEM: 5.115 ± 0.366 ng/mL) compared with non-obese, non-T2D subjects (mean ± SEM: 4.02 ± 0.271 ng/mL; P = 0.003). Similarly, among subjects with diagnosed T2D, those who were obese had higher ANGPTL5 plasma levels than non-obese subjects, although this difference did not reach statistical significance (P = 0.088). Correlation analyses revealed that ANGPTL5 levels positively associated with fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), triglycerides (TGL), and insulin resistance as measured by HOMA-IR. Conclusion: our data shows for the first time that circulating ANGPTL5 levels were higher in obese individuals and those with T2D. Further analysis will be required to better understand the interaction between ANGPTL5 and other metabolic related biomarkers to shed more light on its role in diabetes and obesity.

A novel oxazolidinone derivative PH192 demonstrates anticonvulsant activity in vivo in rats and mice.

The pharmacotherapeutic management of seizure disorders with currently available medications is not optimal due to side effects and failure of some patients to respond to all available medications. As such there is the need to develop new antiseizure drugs by looking at new chemical classes of compounds. We recently screened, in vitro, a new class of compounds, the oxazolidinones, for actions in the brain that may indicate potential for antiseizure activity. A few compounds were identified with such a potential. Here we tested whether one of these lead compounds, PH192, will exhibit in vivo antiseizure activity using chemically- and electrically- induced seizures models in mice and rats. Out of 5 compounds tested, all of them had minimal neurotoxicological effects in mice, with PH192 being the best, with comparable efficacy (ED50) and toxicity (TD50) to only levetiracetam. Intraperitoneal (IP) pretreatment with PH192 produced a dose-dependent protection of mice from seizures induced using the 6 Hz stimulation protocol with an estimated ED50 of 34.5 mg/kg in mice and about 90 mg/kg in rats and a neurotoxic dose >500 mg/kg in mice, yielding a calculated neuro (protective) index of >14.7. When pretreated with 100 mg/kg PH192 for 30 min, about 75% of mice were protected from 6 Hz-induced seizures. When rats were pretreated for 30 min with PH192, 66.6% of rats were protected from seizures induced using the 6 Hz stimulation protocol while 83.3% were protected using the maximal electroshock (MES) stimulation protocol. Pentylenetetrazole (PTZ) injection at 50, and 100 mg/kg produced stage 5 seizures in all rats. Thirty minutes IP pretreatment of rats with 100 mg/kg PH192 protected 80% of rats from the PTZ-induced seizures, a level of protection similar to that obtained with a reference antiepileptic drug (AED) phenytoin (40 mg/kg), that is used clinically for the treatment of various seizure disorders. The results of these studies indicate that PH192 protects against both chemically- and electrically-induced seizures with little central nervous system side effects. This suggests that the oxazolidinone pharmacophore has potential for discovering new antiepileptic drugs with possibly minimal central side effects.

Increased plasma and adipose tissue levels of ANGPTL8/Betatrophin and ANGPTL4 in people with hypertension.

BACKGROUND: Hypertension is a risk factor for both cardiovascular diseases (CVDs) and type 2 diabetes (T2D). Angiopoietin-like proteins (ANGPTLs), mainly ANGPTL3, ANGPTL4 and ANGPTL8, are associated with increased plasma lipid content due to their role in regulating the activity of lipoprotein lipase, a key enzyme in metabolism of the lipoprotein in circulation. Dyslipidaemia is a risk factor for hypertension development; however, the roles of ANGPTL3, ANGPTL4 and ANGPTL8 in subjects with hypertension have not yet been established. This study compared the plasma and adipose tissue levels of ANGPTL3, ANGPTL4 and ANGPTL8 in age- and body mass index-matched subjects with and without hypertension. METHODS: A total of 119 subjects, including 69 hypertensive and 50 non-hypertensive subjects, were enrolled. ANGPTL3, ANGPTL4 and ANGPTL8 plasma levels were measured by ELISA, whereas their levels in adipose tissue were assessed via real-time PCR. RESULTS: We found that ANGPTL4 (202.49 ± 17.44 ng/mL vs. 160.64 ± 10.36 ng/mL, p = 0.04) and ANGPTL8 levels (2310.96 ± 194.88 pg/mL vs. 1583.35 ± 138.27 pg/mL, p = 0.001) were higher in hypertensive subjects than non-hypertensive subjects. However, ANGPTL3 levels were not significantly different between the two populations. Similarly, ANGPTL4 and ANGPTL8 levels were also elevated in subjects with T2D and hypertension than in those with T2D but not hypertension. Additionally, people with highest tertiles of ANGPTL8 had higher odds of having hypertension (odd ratio [OR] = 3.8, 95% confidence interval [CI] = (1.5-9.8), p-Value = 0.005. Similar to its plasma levels, ANGPTL4 and ANGPTL8 were higher in adipose tissue. CONCLUSIONS: In conclusion, our data illustrate that ANGPTL4 and ANGPTL8 levels in both plasma and adipose tissues are increased in subjects with hypertension. The elevated levels of ANGPTL4 and ANGPTL8 in hypertensive subjects highlight their potential involvement, their potential role as biomarkers for hypertension and their therapeutic value in hypertension given their roles in regulating lipid metabolism.

Evaluation of anticonvulsant actions of dibromophenyl enaminones using in vitro and in vivo seizure models.

Epilepsy and other seizure disorders are not adequately managed with currently available drugs. We recently synthesized a series of dibromophenyl enaminones and demonstrated that AK6 and E249 were equipotent to previous analogs but more efficacious in suppressing neuronal excitation. Here we examined the actions of these lead compounds on in vitro and in vivo seizure models. In vitro seizures were induced in the hippocampal slice chemically (zero Mg2+ buffer and picrotoxin) and electrically using patterned high frequency stimulation (HFS) of afferents. In vivo seizures were induced in rats using the 6 Hz and the maximal electroshock models. AK6 (10 µM) and E249 (10 µM) depressed the amplitude of population spikes recorded in area CA1 of the hippocampus by -50.5±4.3% and -40.1±3.1% respectively, with partial recovery after washout. In the zero Mg2+ model, AK6 (10 µM) depressed multiple population spiking (mPS) by -59.3±6.9% and spontaneous bursts (SBs) by -65.9±7.2% and in the picrotoxin-model by -43.3±7.2% and -50.0±8.3%, respectively. Likewise, E249 (10 µM) depressed the zero-Mg2+-induced mPS by -48.8±9.5% and SBs by -55.8±15.5%, and in the picrotoxin model by -37.1±5.5% and -56.5±11.4%, respectively. They both suppressed post-HFS induced afterdischarges and SBs. AK6 and E249 dose-dependently protected rats in maximal electroshock and 6 Hz models of in vivo seizures after 30 min pretreatment. Their level of protection in both models was similar to that obtained with phenytoin Finally, while AK6 had no effect on locomotion in rats, phenytoin significantly decreased locomotion. AK6 and E249, suppressed in vitro and in vivo seizures to a similar extent. Their in vivo activities are comparable with but not superior to phenytoin. The most efficacious, AK6 produced no locomotor suppression while phenytoin did. Thus, AK6 and E249 may be excellent candidates for further investigation as potential agents for the treatment of epilepsy syndromes with possibly less CNS side effects.

Synthesis, neuronal activity and mechanisms of action of halogenated enaminones.

Due to the excellent anticonvulsant activity of previously synthesized halogenated enaminones, more disubstituted analogs were synthesized and evaluated in vitro. The new enaminones either had no effect, depressed, or enhanced population spike (PS) amplitude in the rat hippocampus in a concentration-dependent manner. Structure-activity relationship (SAR) analysis indicated that compounds 21 and 25 (with dibromo substituents) were equipotent, and more potent than compound 2 (with dichloro substituents), with compound 25 being the most efficacious of all tested compounds. Both diiodo derivatives 30 and 31 tested produced no significant effect on PS. For PS depression, phenyl substitution on the cyclohexenone ring produced the most efficacious compound 25. PS depressing analogues also depressed evoked excitatory postsynaptic current (EPSC) and action potential firing frequency. Removal of phenyl or methyl group from position 6 on the cyclohexenone ring of enaminone esters produced compound 28 which exhibited pro-convulsant effects. There was no direct correlation between C log P values and anticonvulsant activity of the halogenated enaminones. The mechanisms of anticonvulsant activity were the indirect suppression of excitatory synaptic transmission by enhancing extracellular GABA, and the direct suppression of action potential firing of the neurons.

The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbit conjunctival epithelial cell layers.

PURPOSE: To delineate the characteristics and mechanisms of uptake of biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles in primary cultured rabbit conjunctival epithelial cells (RCECs). METHODS: Poly(D,L-lactide-co-glycolide) nanoparticles (PLGA 50:50. 101 nm in diameter) containing 6-coumarin (as a fluorescent marker) were used. The effect of size was studied using various particle sizes (100 nm, 800 nm, and 10 microm). The effect of cytochalasin D, nocodazole, and metabolic inhibitors on nanoparticle uptake was investigated. The capability of nanoparticles to enhance the uptake of an encapsulated protein. BSA bound to Texas red (TR-BSA), was evaluated. RESULTS: Maximal uptake of nanoparticles at 37 degrees C occurred at 2 h, and 100-nm particles had the highest uptake in RCECs in comparison with 800-nm and 10-microm particles. Nanoparticle uptake was saturable over the 0.1-4 mg/ml concentration range. Nanoparticle uptake was confirmed by confocal microscopy and was inhibited significantly by coumarin-free nanoparticles (of similar size), by lower incubation temperature, and by the presence of metabolic inhibitors and cytochalasin D. The uptake of encapsulated TR-BSA in RCECs at 4 h was 28% higher than free BSA application. CONCLUSION: Our findings suggest that PLGA nanoparticle uptake in primary cultured rabbit conjunctival epithelial cells occurs most likely by adsorptive-type endocytosis.

Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis.

PURPOSE: The internalization of poly (dl-lactide-co-glycolide, PLGA) nanoparticles in rabbit conjunctival epithelial cells (RCEC) was previously shown to occur by an endocytic process, as evidenced its energy-dependence, inhibition by the vesicle formation blocker cytochalasin D, and by the characteristic display of punctate distribution under confocal microscopy. In addition, clathrin protein was implicated in the endocytosis of these nanoparticles in vascular smooth muscle cells. We sought to examine the expression of clathrin and caveolin-1 in RCECs and to determine whether they play a role in PLGA nanoparticle endocytosis. METHODS: PLGA (50:50) nanoparticles (100 nm in diameter) containing 6-coumarin (fluorescent marker, 0.05% w/v) were used in this study. The effect of pharmacological treatments aimed at disrupting formation of clathrin-coated vesicles (hypertonic challenge and intracellular K+ depletion) and caveolae (nystatin and filipin) on apical uptake of nanoparticles in primary cultured RCEC was investigated. Transferrin was chosen as a marker for clathrin-dependent endocytosis from the basolateral aspect, whereas cholera toxin B subunit was chosen as a marker for caveolae-mediated endocytosis. The staining pattern of nanoparticles in RCECs was compared with that of clathrin heavy chain (HC) and caveolin-1 under fluorescent confocal microscopy to examine possible colocalization using clathrin HC and caveolin-1 mouse monoclonal antibodies (mAb). Two pairs of primers were designed (based on conserved regions of clathrin and caveolin-1 gene in different species) to amplify a 744-bp and 152-bp fragment of clathrin HC and caveolin-1 gene, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for clathrin HC and caveolin-1 was performed using total RNA prepared from freshly isolated RCECs. HEK293 cells were used as positive control for clathrin gene expression, whereas rabbit heart muscle and HEK cells were used as positive control for caveolin-1 gene expression. The RT-PCR products were separated using 2% agarose gel electrophoresis. Western blot analysis was performed to detect the expression of both clathrin and caveolin-1 proteins in RCECs using mouse mAbs. HeLa cells and A431 epidermoid cells were used as positive controls. The effect of transfection of RCECs (using Lipofectamine 2000TM reagent) with specific antisense oligonucleotides designed against the rabbit clathrin isoform on clathrin protein expression and PLGA nanoparticle uptake was investigated. RESULTS: Apical uptake of nanoparticles in primary cultured RCECs was decreased by 45% and 35%, respectively, as a result of K+ depletion and hypertonic media treatments. Likewise, the same treatments significantly decreased the basolateral uptake of FITC-transferrin by 50%. In contrast, nystatin and filipin had no effect on apical uptake of nanoparticles and cholera toxin B subunit in RCECs, suggesting a lack of the involvement of caveolae in the internalization of these two agents. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb, with partial overlap with a nanoparticle staining pattern. RT-PCR confirmed the presence of the clathrin HC gene, but not the caveolin-1 gene, in RCECs as indicated by a 744-bp fragment of the gene. However, caveolin-1 gene was detected in other rabbit tissues such as the epithelium of the cornea and trachea, and heart muscle, as indicated by a 152-bp fragment of the gene. Western blot analysis revealed a clathrin HC band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 protein (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Transfection of RCECs with antisense oligonuceotide directed against clathrin HC resulted in knockdown of the clathrin HC protein in a concentration dependent manner. However, clathrin HC protein knockdown had no effect on apical uptake of nanoparticles in RCECs. CONCLUSIONS: Our findings indicate that endocytosis of nanoparticles in primary cultured RCECs occurs mostly independently of clathrin- and caveolin-1-mediated pathways. In addition, the gene and protein expression of clathrin HC, but not caveolin-1, was identified in rabbit conjunctival epithelial cells.