Measuring the Metabolic State of Tissue-Resident Macrophages via SCENITH.

Functional reprograming of cells is linked to a process of metabolic rewiring that is adapted for such new functions or microenvironment. Macrophages are present in all tissues and exposed to different microenvironments throughout our body. Profiling energetic metabolism of tissue resident and other heterogeneous populations of macrophages in vitro and ex vivo is technologically very challenging. We have recently developed a method to functionally profile energetic metabolism with single-cell resolution, named SCENITH. This method can be performed rapidly ex vivo and does not require specialized equipment. In this book chapter, we will summarize the tissue processing, the procedure and methods, the analysis and example of results, and a series of frequently asked questions.

Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity.

Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.

Circ_0003028 enhances the proliferation and glycolytic capacity and suppresses apoptosis in non-small cell lung cancer cells via the miR-1305/miR-1322-SLC5A1 axis.

Background: Circular RNA (circRNA), a unique RNA molecule with a circular structure, is relevant to the process of non-small cell lung cancer (NSCLC). However, the role and possible mechanisms of circ_0003028 in NSCLC are not completely clear. Here, we investigated the role of circ_0003028 in NSCLC progression. Methods: We first confirmed the stability and head-to-tail junction sequences of circ_000302. Circ_0003028 expression was identified with quantitative reverse transcription polymerase chain reaction (qRT-PCR) in NSCLC tissues, and the survival probability and prognosis were analyzed using Kaplan-Meier survival and receiver operating characteristic (ROC) analyses. Functionally, the proliferation, apoptosis, and glycolytic capacity were examined using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) staining, a flow cytometer, commercial kits [glucose, lactate, and adenosine triphosphate (ATP)], and a Seahorse XF extracellular flux analyzer. Moreover, the potential microRNAs (miRNAs) of circ_0003028 were predicted and identified, and the target gene of miRNA (miR)-1322 and miR-1305 were also screened using DIANA-microT and TargetScan. Results: We first determined the head-to-tail junction sequences of circ_0003028 and its stability. Circ_0003028 was also confirmed to be upregulated in NSCLC tissues. Meanwhile, circ_0003028 had poor overall survival and high diagnostic potential in NSCLC patients. Furthermore, we found that overexpression of circ_0003028 could increase the proliferation and glycolytic capacity and restrain the apoptosis of NSCLC cells, and circ_0003028 silencing played the opposite role to circ_0003028 overexpression. Moreover, circ_0003028 might regulate miR-1305 and miR-1322, which might further regulate solute carrier family 5 member 1 (SLC5A1). Conclusions: Circ_0003028 could accelerate the malignant behaviors and glycolytic capacity of NSCLC cells via a mechanism that may be related to miR-1305 or the miR-1322/SLC5A1 axis. Therefore, the findings of the current study provide a preliminary theoretical basis for NSCLC therapy and diagnosis.

Moderate-intensity training in hypoxia improves exercise performance and glycolytic capacity of skeletal muscle in horses.

We investigated whether moderate-intensity training of horses in moderate hypoxia for 4 weeks elicits greater adaptations in exercise performance, aerobic capacity, and glycolytic/oxidative metabolism in skeletal muscle compared to normoxic training. In a randomized crossover study design, seven untrained Thoroughbred horses (5.9 ± 1.1 years, 508 ± 9 kg) completed 4 weeks (3 sessions/week) of two training protocols consisting of 3-min cantering at 70% of maximal oxygen consumption ( V˙O2max ) in hypoxia (HYP; FI O2  = 14.7%) and normoxia (NOR; FI O2  = 21.0%) with a 4-month washout period. Normoxic incremental exercise tests (IET) were conducted before and after training. Biopsy samples were obtained from the middle gluteal muscle before IET and monocarboxylate transporter (MCT) protein expression and glycolytic/mitochondrial enzyme activities were analyzed. Data were analyzed using mixed models (p < 0.05). Running speed was 7.9 ± 0.2 m/s in both groups and arterial oxygen saturation during training in NOR and HYP were 92.9 ± 0.9% and 75.7 ± 3.9%, respectively. Run time in HYP (+9.7%) and V˙O2max in both groups (NOR, +6.4%; HYP, +4.3%) at IET increased after 4 weeks of training. However, cardiac output, arterial-mixed venous O2 difference, and hemoglobin concentration at exhaustion were unchanged in both conditions. While MCT1 protein and citrate synthase activity did not increase in both conditions after training, MCT4 protein (+13%), and phosphofructokinase activity (+42%) increased only in HYP. In conclusion, 4 weeks of moderate-intensity hypoxic training improves exercise performance and glycolytic capacity of skeletal muscle in horses.

Remote ischemic preconditioning increases accumulated oxygen deficit in middle-distance runners.

The mediators underlying the putative benefits of remote ischemic preconditioning (IPC) on dynamic whole body exercise performance have not been widely investigated. Our objective was to test the hypothesis that remote IPC improves supramaximal exercise performance in National Collegiate Athletic Association (NCAA) Division I middle-distance runners by increasing accumulated oxygen deficit (AOD), an indicator of glycolytic capacity. A randomized sham-controlled crossover study was employed. Ten NCAA Division I middle-distance athletes [age: 21 ± 1 yr; maximal oxygen uptake (V̇o2max): 65 ± 7 ml·kg-1·min-1] completed three supramaximal running trials (baseline, after mock IPC, and with remote IPC) at 110% V̇o2max to exhaustion. Remote IPC was induced in the right arm with 4 × 5 min cycles of brachial artery ischemia with 5 min of reperfusion. Supramaximal AOD (ml/kg) was calculated as the difference between the theoretical oxygen demand required for the supramaximal running bout (linear regression extrapolated from ~12 × 5 min submaximal running stages) and the actual oxygen demand for these bouts. Remote IPC [122 ± 38 s, 95% confidence interval (CI): 94-150] increased (P < 0.001) time to exhaustion 22% compared with baseline (99 ± 23 s, 95% CI: 82-116, P = 0.014) and sham (101 ± 30 s, 95% CI: 80-123, P = 0.001). In the presence of IPC, AOD was 47 ± 36 ml/kg (95% CI: 20.8-73.9), a 29% increase compared with baseline (36 ± 28 ml/kg, 95% CI: 16.3-56.9, P = 0.008) and sham (38 ± 32 ml/kg, 95% CI: 16.2-63.0, P = 0.024). Remote IPC considerably improved supramaximal exercise performance in NCAA Division I middle-distance athletes. Greater glycolytic capacity, as estimated by increased AOD, is a potential mediator for these performance improvements. NEW & NOTEWORTHY Our novel findings indicate that ischemic preconditioning enhanced glycolytic exercise capacity, enabling National Collegiate Athletic Association (NCAA) middle-distance track athletes to run ~22 s longer before exhaustion compared with baseline and mock ischemic preconditioning. The increase in "all-out" performance appears to be due to increased accumulated oxygen deficit, an index of better supramaximal capacity. Of note, enhanced exercise performance was demonstrated in a specific group of in-competition NCAA elite athletes that has already undergone substantial training of the glycolytic energy systems.

Excess glycogen does not resolve high ultimate pH of oxidative muscle.

Skeletal muscle glycogen content can impact the extent of postmortem pH decline. Compared to glycolytic muscles, oxidative muscles contain lower glycogen levels antemortem which may contribute to the higher ultimate pH. In an effort to explore further the participation of glycogen in postmortem metabolism, we postulated that increasing the availability of glycogen would drive additional pH decline in oxidative muscles to equivalent pH values similar to the ultimate pH of glycolytic muscles. Glycolysis and pH declines were compared in porcine longissimus lumborum (glycolytic) and masseter (oxidative) muscles using an in vitro system in the presence of excess glycogen. The ultimate pH of the system containing longissimus lumborum reached a value similar to that observed in intact muscle. The pH decline of the system containing masseter samples stopped prematurely resulting in a higher ultimate pH which was similar to that of intact masseter muscle. To investigate further, we titrated powdered longissimus lumborum and masseter samples in the reaction buffer. As the percentage of glycolytic sample increased, the ultimate pH decreased. These data show that oxidative muscle produces meat with a high ultimate pH regardless of glycogen content and suggest that inherent muscle factors associated with glycolytic muscle control the extent of pH decline in pig muscles.

Resveratrol supplementation does not augment performance adaptations or fibre-type-specific responses to high-intensity interval training in humans.

The present study examined the effect of concurrent exercise training and daily resveratrol (RSV) supplementation (150 mg) on training-induced adaptations following low-dose high-intensity interval training (HIIT). Sixteen recreationally active (∼22 years, ∼51 mL·kg(-1)·min(-1)) men were randomly assigned in a double-blind fashion to either the RSV or placebo group with both groups performing 4 weeks of HIIT 3 days per week. Before and after training, participants had a resting muscle biopsy taken, completed a peak oxygen uptake test, a Wingate test, and a submaximal exercise test. A main effect of training (p < 0.05) and interaction effect (p < 0.05) on peak aerobic power was observed; post hoc pairwise comparisons revealed that a significant (p < 0.05) increase occurred in the placebo group only. Main effects of training (p < 0.05) were observed for both peak oxygen uptake (placebo - pretraining: 51.3 ± 1.8, post-training: 54.5 ± 1.5 mL·kg(-1)·min(-1), effect size (ES) = 0.93; RSV - pretraining: 49.6 ± 2.2, post-training: 52.3 ± 2.5 mL·kg(-1)·min(-1), ES = 0.50) and Wingate peak power (placebo: pretraining: 747 ± 39, post-training: 809 ± 31 W, ES = 0.84; RSV - pretraining: 679 ± 39, post-training: 691 ± 43 W, ES = 0.12). Fibre-type distribution was unchanged, while a main effect of training (p < 0.05) was observed for succinate dehydrogenase activity and glycogen content, but not α-glycerophosphate dehydrogenase activity or intramuscular lipids in type I and IIA fibres. The fold change in PGC-1α, SIRT1, and SOD2 gene expression following training was significantly (p < 0.05) lower in the RSV group than placebo. These results suggest that concurrent exercise training and RSV supplementation may alter the normal training response induced by low-volume HIIT.