To survive extremely different environments, intracellular parasites require highly adaptable physiological and metabolic systems. Leishmania donovani extracellular promastigotes reside in a glucose-rich, slightly alkaline environment in the sand fly vector alimentary tract. On entry into human macrophage phagolysosomes, promastigotes differentiate into intracellular amastigotes. These cope with an acidic milieu, where glucose is scarce while amino acids are abundant. Here, we use an axenic differentiation model and a novel high-coverage, comparative proteomic methodology to analyze in detail protein expression changes throughout the differentiation process. The analysis identified and quantified 21% of the parasite proteome across 7 time points during differentiation. The data reveal a delayed increase in gluconeogenesis enzymes, coinciding with a decrease in glycolytic capacity. At the same time, beta-oxidation, amino acid catabolism, tricarboxylic acid cycle, mitochondrial respiration chain, and oxidative phosphorylation capacities are all up-regulated. The results indicate that the differentiating parasite shifts from glucose to fatty acids and amino acids as its main energy source. Furthermore, glycerol and amino acids are used as precursors for sugar synthesis, compensating for lack of exogenous sugars. These changes occur while promastigotes undergo morphological transformation. Our findings provide new insight into changes occurring in single-cell organisms during a developmental process.

The pathogenic intracellular parasites Leishmania donovani cycle between sand fly gut and the human macrophage phagolysosome, differentiating from extracellular promastigotes to intracellular amastigote forms. Using isobaric tagging for relative and absolute quantifications (iTRAQ/LC-MS/MS) proteomic methodology, we recently described the ordered gene expression changes during this process. While protein abundance changes in Leishmania were documented, little is known about their PTMs. Here we used iTRAQ to detect protein phosphorylation, methylation, acetylation, and glycosylation sites throughout differentiation. We found methylation of arginines, aspartic acids, glutamic acids, asparagines, and histidines. Detected acetylation sites included serines and protein N-terminal acetylations on methionines, serines, alanines, and threonines. Phosphorylations were detected on serines and threonines, but not tyrosines. iTRAQ identified novel fucosylation sites as well as hexosylations. We observed quantity changes in some modifications during differentiation, suggesting a role in L. donovani intracellular development. This study is the first high-throughput analysis of PTM sites dynamics during an intracellular parasitic development.

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Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-γ induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 ± 14 μM, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 ± 1.7 μM, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and α-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.

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Hypoxia, which characterizes ischemia, trauma, inflammation, and solid tumors, recruits monocytes, immobilizes them, and alters their function, leading to an anti-inflammatory and proangiogenic phenotype. Monocyte extravasation from the circulation and their migration in tissues are partially mediated by the balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The mechanisms evoked by hypoxia that regulate monocyte migration and activation are not entirely clear. Specifically, the effect of hypoxia on TIMPs in these cells has hardly been investigated. We show that hypoxia reduces TIMP-2 secretion from human primary monocytes and from the monocyte-like cell lines U937 and THP-1 by three- to fourfold (P < 0.01), by inhibiting TIMP-2 transcription through mechanisms that involve the transcription factor SP-1. Hypoxia also lowers TIMP-2 protein secretion from human endothelial cells (by 2-fold, P < 0.05). TIMP-2 levels do not influence the reduced migration of THP-1 cells in hypoxia; however, low TIMP-2 levels enhance endothelial cell migration/proliferation, their ability to form tubelike structures in vitro, and the appearance of mature blood vessels in a Matrigel plug assay in vivo. Thus we conclude that reduced TIMP-2 levels secreted from both hypoxic monocytes and endothelial cells are proangiogenic.

Glucocorticoids play an important role in the treatment of inflammation and immune disorders, despite side effects, which include metabolic derangements such as central adiposity. These studies examine the role of protein phosphatase 5 (Ppp5) in glucocorticoid receptor (GR) complexes which mediate response to glucocorticoids. Mice homozygous for inactivated Ppp5 (Ppp5 D274A/D274A ) exhibit decreased adipose tissue surrounding the gonads and kidneys compared with wild-type mice. Adipocyte size is smaller, more preadipocytes/stromal cell are present in their gonadal fat tissue and differentiation of preadipocytes to adipocytes is retarded. Glucocorticoid levels are raised and the GR is hyperphosphorylated in adipose tissue of Ppp5 D274A/D274A mice at Ser212 and Ser220 (orthologous to human Ser203 and Ser211) in the absence of glucocorticoids. Preadipocyte cultures from Ppp5 D274A/D274A mice show decreased down regulation of Delta-like protein-1/preadipocyte factor-1, hyperphosphorylation of extra-cellular signal regulated kinase 2 (ERK2) and increased concentration of (sex determining region Y)-box 9 (SOX9), changes in a pathway essential for preadipocyte differentiation, which leads to decreased concentrations of the transcription factors CEBPβ and CEBPα necessary for the later stages of adipogenesis. The data indicate that Ppp5 plays a crucial role in modifying GR-mediated initiation of adipose tissue differentiation, suggesting that inhibition of Ppp5 may potentially be beneficial to prevent obesity during glucocorticoid treatment.

Background Guidelines from 2016 onwards recommend early use of SGLT2i or GLP-1 RA for patients with type 2 diabetes (T2D) and cardiovascular disease (CVD), to reduce CV events and mortality. Many eligible patients are not treated accordingly, although data are lacking for Central and Eastern Europe (CEE). Methods The CORDIALLY non-interventional study evaluated the real-world characteristics, modern antidiabetic treatment patterns, and the prevalence of CVD and chronic kidney disease (CKD) in adults with T2D at nonhospital-based practices in CEE. Data were retrospectively collated by medical chart review for patients initiating empagliflozin, another SGLT2i, DPP4i, or GLP-1 RA in autumn 2018. All data were analysed cross-sectionally, except for discontinuations assessed 1 year ± 2 months after initiation. Results Patients (N = 4055) were enrolled by diabetologists (56.7%), endocrinologists (40.7%), or cardiologists (2.5%). Empagliflozin (48.5%) was the most prescribed medication among SGLT2i, DPP4i, and GLP-1 RA; > 3 times more patients were prescribed empagliflozin than other SGLT2i (10 times more by cardiologists). Overall, 36.6% of patients had diagnosed CVD. Despite guidelines recommending SGLT2i or GLP-1 RA, 26.8% of patients with CVD received DPP4i. Patients initiating DPP4i were older (mean 66.4 years) than with SGLT2i (62.4 years) or GLP-1 RA (58.3 years). CKD prevalence differed by physician assessment (14.5%) or based on eGFR and UACR (27.9%). Many patients with CKD (≥ 41%) received DPP4i, despite guidelines recommending SGLT2is owing to their renal benefits. 1 year ± 2-months after initiation, 10.0% (7.9–12.3%) of patients had discontinued study medication: 23.7–45.0% due to ‘financial burden of co-payment’, 0–1.9% due to adverse events (no patients discontinued DPP4i due to adverse events). Treatment guidelines were ‘highly relevant’ for a greater proportion of cardiologists (79.4%) and endocrinologists (72.9%) than diabetologists (56.9%), and ≤ 20% of physicians consulted other physicians when choosing and discontinuing treatments. Conclusions In CORDIALLY, significant proportions of patients with T2D and CVD/CKD who initiated modern antidiabetic medication in CEE in autumn 2018 were not treated with cardioprotective T2D medications. Use of DPP4i instead of SGLT2i or GLP-1 RA may be related to lack of affordable access, the perceived safety of these medications, lack of adherence to the latest treatment guidelines, and lack of collaboration between physicians. Thus, many patients with T2D and comorbidities may develop preventable complications or die prematurely. Trial registration NCT03807440.