Regulation of Glucagon Secretion and Trafficking by Proteins in the Glucagon Interactome

Patients with diabetes exhibit hyperglucagonemia, or excess glucagon secretion. The glucagonocentric hypothesis of diabetes states that hyperglucagonemia, rather than hypoinsulinemia, may be the underlying mechanism of hyperglycemia of diabetes. Thus, uncovering mechanisms that regulate glucagon secretion from pancreatic α-cells is crucial for developing treatments for hyperglycemia. One clue to the regulation of glucagon secretion may lie in the proteins that interact with glucagon in α-cell’s secretory pathway, primarily within the secretory granule. The purpose of my work was to identify proteins that interact with glucagon within the secretory granule and characterize a candidate protein within this network that regulates the intracellular trafficking of glucagon to control its secretion. To identify secretory granule proteins that interact with glucagon, I purified secretory granules from α-TC1-6 cells. I then used affinity purification using tagged glucagon to isolate protein complexes that interact with glucagon, and identified these proteins through liquid chromatography/mass spectrometry. In this way, I identified a glucagon “interactome” within the α-cell secretory granule. I found that components of the interactome changed in response to different glucose concentrations, and to treatment with the paracrine inhibitors insulin and GABA. Next, I characterized the function of one interactome protein, the neuronal cytoskeletal protein stathmin-2, in glucagon secretion. Through overexpression and siRNA-mediated silencing of stathmin-2 in α-TC1-6 cells, I showed that stathmin-2 is a tonic inhibitor of glucagon secretion. Using confocal high-resolution immunofluorescence microscopy, I found that stathmin-2 exerts its regulatory role by trafficking of glucagon to the endolysosomal system. Finally, I examined how the trafficking role of stathmin-2 is altered in the hyperglucagonemia of diabetes. Using isolated islets from a mouse model of diabetes, I showed that the increase in cellular glucagon was accompanied by a reduction in stathmin-2 levels. Confocal microscopy analysis indicated that, in diabetes, there is a switch from the anterograde trafficking of glucagon towards the lysosome to retrograde trafficking towards secretory granules, possibility mediated by the endosomal protein Rab7. In summary, my thesis describes the discovery of a regulatory mechanism for glucagon secretion from α-cells that may operate in hyperglucagonemia. These findings have clinical application for treatment of hyperglycemia of diabetes

Role of sperm apoptosis and oxidative stress in male infertility: A narrative review

Activation of caspase, externalization of phosphatidyl serine, change in the mitochondrial membrane potential, and DNA fragmentation are apoptosis markers found in human ejaculated spermatozoa. Also, reactive oxygen species (ROS) play a vital role in the different types of male infertility. In this review, data sources including Google Scholar, Scopus, PubMed, and Science Direct were searched for publications with no particular time restriction to get a holistic and comprehensive view of the research. Apoptosis regulates the male germ cells, correct function and development from the early embryonic stages of gonadal differentiation to fertilization. In addition to maintaining a reasonable ratio between the Sertoli and germ cells, apoptosis is one of the well-known quality control mechanisms in the testis. Also, high ROS levels cause a heightened and dysregulated apoptotic response. Apoptosis is one of the well-known mechanisms of quality control in the testis. Nevertheless, increased apoptosis may have adverse effects on sperm production. Recent studies have shown that ROS and the consequent oxidative stress play a crucial role in apoptosis. This review aims to assimilate and summarize recent findings on the apoptosis in male reproduction and fertility. Also, this review discusses the update on the role of ROS in normal sperm function to guide future research in this area. Key words: Fertility, Spermatogonia, Apoptosis, Reproduction, DNA fragmentation, DNA integrity, ROS

Isoenzymatic pattern of glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase in Iranian Echinococcus granulosus

Morphology and genetic studies of Echinococcus granulosus have indicated that there are 2 different strains of this parasite in Iran, ovine and camel. However, no study has been carried out to date on the phenotypic characterization of this parasite. In the present study the electrophoretic pattern of glucose-6-phosphate dehydrogenase (G6PD) and isocitrate dehydrogenase (ICD) was demonstrated. In this study isolates of ovine and camel hydatid cysts were collected from slaughterhouses across Iran. Hydatid fluid and hydatid sand were separated and collected. The fluid was concentrated and the protoscoleces from the sand were extracted. The amount of total protein in protoscoleces and concentrated hydatid fluid was determined. Electrophoretic pattern of extract was indicated by SDS-PAGE. Non-denaturating electrophoresis was also used for study of electrophoretic pattern of G6PD and ICD; so that major and minor enzyme activities were indicated. Densitometry of electrophoretic pattern indicated 2 major bands for each of these enzymes in camel and sheep with the same pattern in the extract of protoscoleces and hydatid fluid. Based on the fact that the band of enzymes in each of 2 isolates has different molecular patterns; we propose that these represent the 2 different strains (sheep-dog and camel-dog) of this parasite in Iran

Stipagrostis pennata (Trin.) De Winter Artificial Seed Production and Seedlings Multiplication in Temporary Immersion Bioreactors

This study was conducted to develop the protocol for artificial seed production of Stipagrostis pennata (Trin.) De Winter via somatic embryo encapsulation as well as test a temporary bioreactor system for germination and seedling growth. Embryogenic calli were encapsulated using sodium alginate and calcium chloride and then sowed in the Murashige and Skoog (MS) germination medium in in vitro cultures. The experiments were conducted as a factorial based on a completely randomized design with three replications. The treatments include three concentrations of sodium alginate (1.5%, 2.5%, and 3.5%), two ion exchange times (20 and 30 min), and two artificial seed germination media (hormone-free MS and MS supplemented with zeatin riboside and L-proline). Germination percentage and number of days needed until the beginning of germination were studied. The highest percentage of artificial seed germination was obtained when 2.5% sodium alginate was used for 30 min (ion exchange time) and when the seeds were placed on the MS germination medium supplemented with zeatin riboside and L-proline. The results of the analysis of variance in the temporary immersion bioreactor system showed that the main effects observed on the seedling growth were associated with different growth hormones in culture media and the number of feeding cycles. Experimental results also indicated that the total protein analyses of zygotic seedlings and seedlings originating from the synthetic seeds showed no statistically significant differences between these samples

Isoenzymatic pattern of glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase in Iranian Echinococcus granulosus

Morphology and genetic studies of Echinococcus granulosus have indicated that there are 2 different strains of this parasite in Iran, ovine and camel. However, no study has been carried out to date on the phenotypic characterization of this parasite. In the present study the electrophoretic pattern of glucose-6-phosphate dehydrogenase (G6PD) and isocitrate dehydrogenase (ICD) was demonstrated. In this study isolates of ovine and camel hydatid cysts were collected from slaughterhouses across Iran. Hydatid fluid and hydatid sand were separated and collected. The fluid was concentrated and the protoscoleces from the sand were extracted. The amount of total protein in protoscoleces and concentrated hydatid fluid was determined. Electrophoretic pattern of extract was indicated by SDS-PAGE. Non-denaturating electrophoresis was also used for study of electrophoretic pattern of G6PD and ICD; so that major and minor enzyme activities were indicated. Densitometry of electrophoretic pattern indicated 2 major bands for each of these enzymes in camel and sheep with the same pattern in the extract of protoscoleces and hydatid fluid. Based on the fact that the band of enzymes in each of 2 isolates has different molecular patterns; we propose that these represent the 2 different strains (sheep-dog and camel-dog) of this parasite in Iran

Challenges and Pitfalls in the Management of Rhino-Orbital Mucormycosis in Ophthalmology: A Highlighted Problem in the COVID-19 Era

Secondary infections in hospitalized and ill patients with coronavirus disease 2019 (COVID-19) are common. One of these life-threatening infectious diseases is rhino-orbital mucormycosis, which made an outbreak recently. This outbreak was mainly caused by the administration of high-dose corticosteroids in patients with COVID-19, especially those with diabetes mellitus. The increased incidence of rhino-orbital mucormycosis in the COVID-19 era presents different challenges for healthcare providers including ophthalmologists who are directly involved in disease management. We summarized the main challenges and recommendations for ophthalmologists on the management of rhino-orbital mucormycosis

Effect of dietary vitamin E on Eimeria tenella-induced oxidative stress in broiler chickens

An experiment was carried out to investigate the impact of high doses of dietary vitamin E on antioxidant  status in broiler chickens (Ross 308) experimentally infected with Eimeria tenella. One day old chicks were assigned to five groups (25 each) and given basal diet (A and B) or basal diet supplemented with 100, 316 or 562 mg/kg of vitamin E (C to E), respectively. On the 21st day, all chicks except those in group A were  inoculated with E. tenella and monitored for any change in blood vitamin E, malondialdehyde (MDA) and  superoxide dismutase (SOD). Plasma vitamin E decreased by infection, but increased with dietary vitamin E (p<0.05). A significant rise of plasma and erythrocyte MDA was observed in infected birds (p<0.05), however, the chicks fed diet with 316 mg/kg added vitamin E had a lower MDA compared to infected controls (p<0.001). The erythrocyte SOD was not affected by infection (p>0.05), but it was significantly higher in group D than in groups B and E (p<0.05). In conclusion, addition of dietary vitamin E at 316 mg/kg can afford antioxidant protection to chickens infected with E. tenella, but at higher doses it may aggravate the unbalanced  oxidant/antioxidant status.Key words: Eimeria tenella, oxidative stress, broiler chickens, vitamin E, malondialdehyde, superoxide dismutase

Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells

Glucagon is stored within the secretory granules of pancreatic alpha cells until stimuli trigger its release. The alpha cell secretory responses to the stimuli vary widely, possibly due to differences in experimental models or microenvironmental conditions. We hypothesized that the response of the alpha cell to various stimuli could be due to plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. We used tagged glucagon with Fc to pull out glucagon from the enriched preparation of secretory granules in α-TC1-6 cells. Isolation of secretory granules was validated by immunoisolation with Fc-glucagon and immunoblotting for organelle-specific proteins. Isolated enriched secretory granules were then used for affinity purification with Fc-glucagon followed by liquid chromatography/tandem mass spectrometry to identify secretory granule proteins that interact with glucagon. Proteomic analyses revealed a network of proteins containing glucose regulated protein 78 KDa (GRP78) and histone H4. The interaction between glucagon and the ER stress protein GRP78 and histone H4 was confirmed through co-immunoprecipitation of secretory granule lysates, and colocalization immunofluorescence confocal microscopy. Composition of the protein networks was altered at different glucose levels (25 vs. 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. siRNA-mediated silencing of a subset of these proteins revealed their involvement in glucagon secretion in α-TC1-6 cells. Therefore, our results show a novel and dynamic glucagon interactome within α-TC1-6 cell secretory granules. We suggest that variations in the alpha cell secretory response to stimuli may be governed by plasticity in the glucagon “interactome.