IF1 Promotes Cellular Proliferation and Inhibits Oxidative Phosphorylation in Mouse Embryonic Fibroblasts under Normoxia and Hypoxia.

ATP synthase inhibitory factor subunit 1 (IF1) is an inhibitory subunit of mitochondrial ATP synthase, playing a crucial role in regulating mitochondrial respiration and energetics. It is well-established that IF1 interacts with the F1 sector of ATP synthase to inhibit the reversal rotation and, thus, ATP hydrolysis. Recent evidence supports that IF1 also inhibits forward rotation or the ATP synthesis activity. Adding to the complexity, IF1 may also facilitate mitophagy and cristae formation. The implications of these complex actions of IF1 for cellular function remain obscure. In the present study, we found that IF1 expression was markedly upregulated in hypoxic MEFs relative to normoxic MEFs. We investigate how IF1 affects cellular growth and function in cultured mouse embryonic fibroblasts derived from mouse lines with systemic IF1 overexpression and knockout under normoxia and hypoxia. Cell survival and proliferation analyses revealed that IF1 overexpression exerted limited effects on cellular viability but substantially increased proliferation under normoxia, whereas it facilitated both cellular viability and proliferation under hypoxia. The absence of IF1 may have a pro-survival effect but not a proliferative one in both normoxia and hypoxia. Cellular bioenergetic analyses revealed that IF1 suppressed cellular respiration when subjected to normoxia and was even more pronounced when subjected to hypoxia with increased mitochondrial ATP production. In contrast, IF1 knockout MEFs showed markedly increased cellular respiration under both normoxia and hypoxia with little change in mitochondrial ATP. Glycolytic stress assay revealed that IF1 overexpression modestly increased glycolysis in normoxia and hypoxia. Interestingly, the absence of IF1 in MEFs led to substantial increases in glycolysis. Therefore, we conclude that IF1 mainly inhibits cellular respiration and enhances cellular glycolysis to preserve mitochondrial ATP. On the other hand, IF1 deletion can significantly facilitate cellular respiration and glycolysis without leading to mitochondrial ATP deficit.

Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.

Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.

Association of fruits, vegetables, and fiber intake with COVID-19 severity and symptoms in hospitalized patients: A cross-sectional study.

Background and aims: Fruits and vegetables are rich in fiber and a good source of anti-inflammatory and immune-boosting vitamins, minerals, and antioxidants. We investigated the association between fruits, vegetables, and fiber intake and severity of COVID-19 and related symptoms in hospitalized patients. Methods: A total of 250 COVID-19 hospitalized patients aged 18 to 65 years were recruited for this cross-sectional study in Kashan, Iran, between June and September of 2021. Dietary intakes were assessed using an online validated 168-item food frequency questionnaire (FFQ). COVID-19 severity and symptoms were evaluated using the National Institutes of Health (NIH) COVID-19 Treatment Guidelines. Moreover, we examined COVID-19 symptoms, inflammatory biomarkers, and additional factors. Results: The mean age of participants was 44.2 ± 12.1 years, and 46% had severe COVID-19. Patients with higher consumption of fruits (OR: 0.28; 95% CI: 0.14-0.58, P-trend <0.001), vegetables (OR: 0.33; 95% CI: 0.16-0.69, P-trend <0.001), and dietary fiber (OR: 0.25; 95% CI: 0.12-0.53, P-trend <0.001) had lower odds of having severe COVID-19. In addition, they had shorter hospitalization and convalescence periods, lower serum C-reactive protein (CRP), and a reduced risk of developing COVID-19 symptoms such as sore throat, nausea and vomiting, dyspnea, myalgia, cough, weakness, fever, and chills. Conclusion: Higher consumption of fruits, vegetables, and fiber was inversely linked with COVID-19 severity, clinical symptoms, hospitalization and convalescence duration, and CRP concentrations. The results should be interpreted with caution in light of the limitations, and prospective cohort studies are required to further evaluate these findings.

The effects of acute sumatriptan treatment on renal ischemia/reperfusion injury in rat and the possible involvement of nitric oxide.

Renal ischemia/reperfusion (I/R) injury is a common pathological condition. Studies reported renal toxicity following administration of triptans, which are commonly used for treating migraine headaches. To investigate the effects of sumatriptan and the molecular mechanisms involved in renal I/R injury in rats, ischemia was induced by bilateral clamping of renal pedicles followed by 24 h of reperfusion. Sumatriptan was administered in three different doses (5, 10, and 20 mg/kg) before I/R injury induction. Biochemical and histopathological changes were evaluated. The contribution of nitric oxide in modulating the effects of sumatriptan was determined by administrating aminoguanidine at 50 mg/kg 60 min before I/R injury. The tissue level of nitrite, superoxide dismutase (SOD), and malondialdehyde (MDA) were measured. Sumatriptan at 10 and 20 mg/kg increased the serum level of creatinine (Cr) and blood urea nitrogen (BUN) significantly. There was also a significant increase in nitrite level of animals that received 10 mg/kg sumatriptan. Co-administration of sumatriptan with aminoguanidine significantly decreased the BUN and Cr. Depletion of SOD level (P < 0.05) and elevation of serum levels of MDA (P < 0.001) indicated the involvement of oxidative stress in sumatriptan adverse effects. Overall, the administration of sumatriptan intensified renal I/R injury through activation of inducible nitric oxide synthase and oxidative responses in rats.

Glatiramer acetate attenuates renal ischemia reperfusion injury in rat model.

Chronic renal failure can ultimately lead to kidney transplantation. Renal transplantation is associated with ischemia-reperfusion injury (I/R).2 The subsequent processes of kidney I/R can lead to irreversible damages to the kidney tissue. Glatiramer acetate is an immunomodulatory drug for the treatment of multiple sclerosis (MS) and the anti-inflammatory effects of this drug have already been proven in some inflammatory models. The purpose of this study was to evaluate the protective effects of Glatiramer on reducing the damages arising from kidney ischemia-reperfusion. In this study, 35 Wistar rats were used which divided into 5 groups: sham, control (I/R), I/R + Glatiramer 0.5 mg/kg, I/R + Glatiramer 1 mg/kg, I/R + Glatiramer 2 mg/kg. Renal arteries were clamped bilaterally for 45 min, then the clamps were removed and the reperfusion process continued to 24 h. In the following, serum and kidneys were separated for analysis. In the control group, serum levels of LDH, inflammatory factor TNF-α and renal functional markers such as BUN and Creatinine were remarkably increased, but in the treatment groups, especially in Glatiramer 2 mg/kg received group, a significant decrease in these factors was observed. Tissue concentration of MDA was reduced following Glatiramer treatment. Besides, Glatiramer attenuated the increased kidney level of NF-κB protein using immunohistochemical assay. NFkB migration to the nucleolus increases inflammatory cytokines production. The anti-inflammatory factor, IL-10, in serum was significantly increased in the treatment group of Glatiramer 2 mg/kg. Furthermore, Glatiramer decreased renal tissue injury score according to the histopathological study. These results demonstrate that Glatiramer may play protective effects in kidney ischemia-reperfusion injury by reducing inflammatory and oxidative damages.