Peptides (v.92, #C)

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways by Xuefei Ma; Wenjian Lin; Zhenyu Lin; Ming Hao; Xinyuan Gao; Yue Zhang; Hongyu Kuang (1-8).
Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H2O2 and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H2O2-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H2O2-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H2O2-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H2O2 induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.
Keywords: Retinal ganglion cells; Autophagy; Liraglutide; Mitochondrial biogenesis; Mitophagy;

Extracellular NAMPT/Visfatin induces proliferation through ERK1/2 and AKT and inhibits apoptosis in breast cancer cells by Zafar Gholinejad; Nejat kheiripour; Mitra Nourbakhsh; Davod Ilbeigi; Kiarash Behroozfar; Zahra Hesari; Abolfazl Golestani; Mohammad Shabani; Nahid Einollahi (9-15).
Visfatin is a novel adipokine and proinflammatory cytokine which is implicated in breast cancer progression. The exact proliferative and anti-apoptotic mechanisms of visfatin are still under debate. In this study, the effect of extracellular visfatin on proliferation and apoptosis of breast cancer cells were investigated considering key regulatory molecules in these procedures.BrdU (Bromodeoxyuridine) experiment was used to assess cell proliferation in response to visfatin treatment. Cell viability and apoptosis were assessed using MTT assay and flowcytometry, respectively. Phosphorylation levels of AKT and ERK1/2 as well as survivin levels and Poly ADP ribose polymerase (PARP) cleavage were investigated by western blot analysis.Visfatin induced proliferation of MCF-7 and MDA-MB-231 cells, an effect that was repressed by using AKT and ERK1/2 inhibitors, indicating involvement of these two signaling pathways in the proliferative effect of visfatin. Similarly, phosphorylation of AKT and ERK1/2 were elevated by visfatin treatment. On the other hand, visfatin improved cell viability and prevented TNF-α-induced apoptosis as well as PARP cleavage. Visfatin also exerted a protective effect on survivin.The results of this study suggest that visfatin induces breast cancer cell proliferation through AKT/PI3K and ERK/MAPK activation and protects against apoptosis in these cells. Thus increased visfatin levels may augment breast cancer development and attenuate treatment efficiency in breast cancer patients.
Keywords: Visfatin; AKT; ERK1/2; Apoptosis; Survivin; Breast cancer;

Long-term effects of angiotensin-(1–7) on lipid metabolism in the adipose tissue and liver by Carolina Campos Lima Moreira; Fabíola Cesário Lourenço; Érica Guilhen Mario; Robson Augusto Souza Santos; Leida Maria Botion; Valéria Ernestânia Chaves (16-22).
The angiotensin (Ang) converting enzyme 2/Ang-(1–7)/Mas axis has been described to have a beneficial role on metabolic disorders. In the present study, the use of a transgenic rat model that chronically overexpresses Ang-(1–7) enabled us to investigate the chronic effects of this peptide on lipid accumulation in the liver and adipose tissue. The transgenic group showed a marked tendency toward increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and decreased lipoprotein lipase (LPL) expression and activity in epididymal adipose tissue. We also showed that Mas receptor-knockout mice had decreased PPARγ expression in adipose tissue, accompanied by an increase in LPL activity. These results confirm the regulation of adipose tissue LPL activity by Ang-(1–7) and suggest that this occurs independent of PPARγ expression. The reduced adiposity index of transgenic rats, due to the effect of Ang-(1–7), was accompanied by a decrease in lipogenesis. These findings suggest a direct effect of Ang-(1–7) on lipogenesis, independent of the stimulatory effect of insulin. Furthermore, the decreased concentration of triacylglycerol in the liver of transgenic rats may result from increased activity of cytosolic lipases and decreased fatty acid uptake from the adipose tissue, determined from fatty acid-binding protein expression, and hepatic de novo fatty acid synthesis, evaluated by fatty acid synthase expression. The data clearly show that Ang-(1–7) regulates lipid metabolism in the adipose tissue and liver.
Keywords: Angiotensin-(1–7); Adipose tissue; Liver; Cytosolic lipases;

In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2 by K. Alagarasu; P.S. Patil; P. Shil; M. Seervi; M.B. Kakade; H. Tillu; A. Salunke (23-30).
Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5 μM–15 μM) or scrambled (Scr) LL-37(5 μM–15 μM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24 h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10–15 μM LL-37 significantly reduced its infectivity as compared to virus control (P < 0.0001). Moreover, pretreatment of virus with 10–15 μM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P < 0.0001). Treatment of virus with 10–15 μM LL-37 resulted in two to three log reduction of mean log10 FFU/ml as compared to virus control (P < 0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P > 0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24 h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.
Keywords: LL-37; Dengue virus; Antimicrobial peptides; Cathelicidin;

Insect selective excitatory β-type sodium channel neurotoxins from scorpion venom (β-NaScTxs) are composed of about 70–76 amino acid residues and share a common scaffold stabilized by four unique disulfide bonds. The phylogenetic analysis of these toxins was hindered by limited sequence data. In our recent study, two new insect selective excitatory β-NaScTxs, LmIT and ImIT, were isolated from Lychas mucronatus and Isometrus maculatus, respectively. With the sequences previously reported, we examined the adaptive molecular evolution of insect selective excitatory β-NaScTxs by estimating the nonsynonymous-to-synonymous rate ratio (ω =  d N/d S). The results revealed 12 positively selected sites in the genes of insect selective excitatory β-NaScTxs. Moreover, these positively selected sites match well with the sites important for interacting with sodium channels, as demonstrated in previous mutagenesis study. These results reveal that adaptive evolution after gene duplication is one of the most important genetic mechanisms of scorpion neurotoxin diversification.
Keywords: Scorpion neurotoxins; Insect selective excitatory β-NaScTxs; Molecular phylogeny; Adaptive evolution;

Spinal activation of the NPY Y1 receptor reduces mechanical and cold allodynia in rats with chronic constriction injury by Mariana Malet; Candelaria Leiguarda; Guillermo Gastón; Carly McCarthy; Pablo Brumovsky (38-45).
Neuropeptide tyrosine (NPY) and its associated receptors Y1R and Y2R have been previously implicated in the spinal modulation of neuropathic pain induced by total or partial sectioning of the sciatic nerve. However, their role in chronic constrictive injuries of the sciatic nerve has not yet been described. In the present study, we analyzed the consequences of pharmacological activation of spinal Y1R, by using the specific Y1R agonist Leu31Pro34-NPY, in rats with chronic constriction injury (CCI). CCI and sham-injury rats were implanted with a permanent intrathecal catheter (at day 7 after injury), and their response to the administration of different doses (2.5, 5, 7, 10 or 20 μg) of Leu31Pro34-NPY (at a volume of 10 μl) through the implanted catheter, recorded 14 days after injury. Mechanical allodynia was tested by means of the up-and-down method, using von Frey filaments. Cold allodynia was tested by application of an acetone drop to the affected hindpaw. Intrathecal Leu31Pro34-NPY induced an increase of mechanical thresholds in rats with CCI, starting at doses of 5 μg and becoming stronger with higher doses. Intrathecal Leu31Pro34 also resulted in reductions in the frequency of withdrawal to cold stimuli, although the effect was somewhat more moderate and mostly observed for doses of 7 μg and higher. We thus show that spinal activation of the Y1R is able to reduce neuropathic pain due to a chronic constrictive injury and, together with other studies, support the use of a spinal Y1R agonist as a therapeutic agent against chronic pain induced by peripheral neuropathy.
Keywords: Allodynia; Inhibition; Neuropathic pain; Neuropeptides; Sciatic nerve; Spinal cord;