3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 μM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 μM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.

Aims/hypothesis The aim of this work was to determine whether placental endoplasmic reticulum (ER) stress may contribute to the pathophysiology of gestational diabetes mellitus (GDM) and to test the efficacy of chemical chaperones and antioxidant vitamins in ameliorating that stress in a trophoblast-like cell line in vitro. Methods Placental samples were obtained from women suffering from GDM and from normoglycaemic controls and were frozen immediately. Women with GDM had 2 h serum glucose levels > 9.0 mmol/l following a 75 g oral glucose tolerance test and were treated with diet and insulin when necessary. Western blotting was used to assess markers of ER stress. To test the effects of hyperglycaemia on the generation of ER stress, a new trophoblast-like cell line, BeWo-NG, was generated by culturing in a physiological glucose concentration of 5.5 mmol/l (over 20 passages) before challenging with 10 or 20 mmol/l glucose. Results All GDM patients were well-controlled (HbA_1c 5.86 ± 0.55% or 40.64 ± 5.85 mmol/mol, n = 11). Low-grade ER stress was observed in the placental samples, with dilation of ER cisternae and increased phosphorylation of eukaryotic initiation factor 2 subunit α. Challenge of BeWo-NG with high glucose activated the same pathways, but this was as a result of acidosis of the culture medium rather than the glucose concentration per se. Addition of chemical chaperones 4-phenylbutyrate and tauroursodeoxycholic acid and vitamins C and E ameliorated the ER stress. Conclusions/interpretation This is the first report of placental ER stress in GDM patients. Chemical chaperones and antioxidant vitamins represent potential therapeutic interventions for GDM.

Aim: The current study investigated the impact of maternal obesity on placental phenotype in relation to fetal growth and sex.Methods: Female C57BL6/J mice were fed either a diet high in fat and sugar or a standard chow diet, for 6 weeks prior to, and during, pregnancy. At day 19 of gestation, placental morphology and mitochondrial respiration and dynamics were assessed using high‐resolution respirometry, stereology, and molecular analyses.Results: Diet‐induced maternal obesity increased the rate of small for gestational age fetuses in both sexes, and increased blood glucose concentrations in offspring. Placental weight, surface area, and maternal blood spaces were decreased in both sexes, with reductions in placental trophoblast volume, oxygen diffusing capacity, and an increased barrier to transfer in males only. Despite these morphological changes, placental mitochondrial respiration was unaffected by maternal obesity, although the influence of fetal sex on placental respiratory capacity varied between dietary groups. Moreover, in males, but not females, maternal obesity increased mitochondrial complexes (II and ATP synthase) and fission protein DRP1 abundance. It also reduced phosphorylated AMPK and capacity for lipid synthesis, while increasing indices of oxidative stress, specifically in males. In females only, placental mitochondrial biogenesis and capacity for lipid synthesis, were both enhanced. The abundance of uncoupling protein‐2 was decreased by maternal obesity in both fetal sexes.Conclusion: Maternal obesity exerts sex‐dependent changes in placental phenotype in association with alterations in fetal growth and substrate supply. These findings may inform the design of personalized lifestyle interventions or therapies for obese pregnant women.

Glutamine is a conditionally essential amino acid that is an important metabolic resource for proliferating tissues by acting as a proteinogenic amino acid, a nitrogen donor for biosynthetic reactions and as a substrate for the citric acid or tricarboxylic acid cycle. The human immunodeficiency virus type 1 (HIV-1) productively infects activated CD4⁺ T cells that are known to require glutamine for proliferation and for carrying out effector functions. As a virus, HIV-1 is furthermore entirely dependent on host metabolism to support its replication. In this study, we compared HIV-1 infected with uninfected activated primary human CD4⁺ T cells with regard to glutamine metabolism. We report that glutamine concentrations are elevated in HIV-1-infected cells and that glutamine is important to support HIV-1 replication, although the latter is closely linked to the glutamine dependency of cell survival. Metabolic tracer experiments showed that entry of glutamine-derived carbon into the citric acid cycle is unaffected by HIV-1 infection, but that there is an increase in the secretion of glutamine-derived glutamic acid from HIV-1-infected cells. Western blotting of key enzymes that metabolize glutamine revealed marked differences in the expression of glutaminase isoforms, KGA and CAG, as well as the PPAT enzyme that targets glutamine-derived nitrogen toward nucleotide synthesis. Altogether, this demonstrates that infection of CD4⁺ T cells with HIV-1 leads to considerable changes in the cellular glutamine metabolism.