The rate of phosphocreatine (PCr) recovery (k ) after exercise, characterizing muscle oxidative capacity, is traditionally assessed with unlocalized P magnetic resonance spectroscopy (MRS) using a single surface coil. However, because of intramuscular variation in fibre type and oxygen supply, k may be non-uniform within muscles. We tested this along the length of the tibialis anterior (TA) muscle in 10 male volunteers. For this purpose, we employed a 3T MR system with a P/ H volume transmit coil combined with a home-built P phased-array receive probe, consisting of five coil elements covering the TA muscle length. Mono-exponential k was determined for all coil elements after 40 s of submaximal isometric dorsiflexion (SUBMAX) and incremental exercise to exhaustion (EXH). In addition, muscle functional MRI ( H mfMRI) was performed using the volume coil after another 40 s of SUBMAX. A strong gradient in k was observed along the TA (P < 0.001), being two times higher proximally vs. distally during SUBMAX and EXH. Statistical analysis showed that this gradient cannot be explained by pH variations. A similar gradient was seen in the slope of the initial post-exercise H mfMRI signal change, which was higher proximally than distally in both the TA and the extensor digitorum longus (P< 0.001) and strongly correlated with k . The pronounced differences along the TA in functional oxidative capacity identify regional variation in the physiological demand of this muscle during everyday activities and have implications for the bio-energetic assessment of interventions to modify its performance and of neuromuscular disorders involving the TA.
We report the effects of Ca(2+) binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. Analysis of the Ca(2+)-induced chemical shifts perturbations yields similar K(D) values of 16-24 microM for the two CBD2-AD Ca(2+)-binding sites, and significant effects are observed up to 20 A away. To quantify the Ca(2+)-induced chemical shift changes, we performed a comparative analysis of eight Ca(2+)-binding proteins that revealed large differences between different protein folds. The CBD2 (15)N relaxation data show the CBD2-AD Ca(2+) coordinating loops to be more rigid in the Ca(2+)-bound state as well as to affect the FG-loop located at the opposite site of the domain. The equivalent loops of the CBD2-BD splice variant do not bind Ca(2+) and are much more dynamic relative to both the Ca(2+)-bound and apo forms of CBD2-AD. A more structured FG-loop in CBD2-BD is suggested by increased S(2) order parameter values relative to both forms of CBD2-AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca(2+)-binding event is felt throughout the molecule. The data suggest that the FG-loop plays an important role in connecting the Ca(2+)-binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG-loop with the first Ca(2+)-binding domain of NCX.
Significance The human prostate accumulates high luminal citrate levels to serve sperm viability. There is only indirect qualitative evidence about metabolic pathways and carbon sources maintaining these levels. Human citrate-secreting prostate cancer cells were supplied with 13 C-labeled substrates, and NMR spectra of extracellular fluid were recorded. We report absolute citrate production rates of prostate cells and direct evidence that glucose is the main carbon source for secreted citrate. Pyruvate carboxylase provides sufficient anaplerotic carbons to support citrate secretion. Glutamine carbons exchange with carbons for secreted citrate but are likely not involved in its net synthesis. Moreover, we developed metabolic models employing the 13 C distribution in extracellular citrate as input to assess intracellular pathways followed by carbons toward citrate.
It is possible to perform prostate (1)H-MRSI at 7T with a SPSP-MRSI sequence while using separate transmit and receive coils. This low-SAR MRSI concept provides the opportunity to increase spatial resolution of MRSI within reasonable scan times.
Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor α-cyano-4-hydroxycinnamic acid.
Background Mutations in isocitrate dehydrogenase 1 ( IDH1 ) occur in various types of cancer and induce metabolic alterations resulting from the neomorphic activity that causes production of D -2-hydroxyglutarate ( D- 2-HG) at the expense of α-ketoglutarate (α-KG) and NADPH. To overcome metabolic stress induced by these alterations, IDH -mutated ( IDH mut ) cancers utilize rescue mechanisms comprising pathways in which glutaminase and glutamate dehydrogenase (GLUD) are involved. We hypothesized that inhibition of glutamate processing with the pleiotropic GLUD-inhibitor epigallocatechin-3-gallate (EGCG) would not only hamper D- 2-HG production, but also decrease NAD(P)H and α-KG synthesis in IDH mut cancers, resulting in increased metabolic stress and increased sensitivity to radiotherapy. Methods We performed 13 C-tracing studies to show that HCT116 colorectal cancer cells with an IDH1 R132H knock-in allele depend more on glutaminolysis than on glycolysis for the production of D -2-HG. We treated HCT116 cells, HCT116- IDH1 R132H cells, and HT1080 cells (carrying an IDH1 R132C mutation) with EGCG and evaluated D- 2-HG production, cell proliferation rates, and sensitivity to radiotherapy. Results Significant amounts of 13 C from glutamate accumulate in D- 2-HG in HCT116- IDH1 wt/R132H but not in HCT116- IDH1 wt/wt . Preventing glutamate processing in HCT116- IDH1 wt/R132H cells with EGCG resulted in reduction of D- 2-HG production. In addition, EGCG treatment decreased proliferation rates of IDH1 mut cells and at high doses sensitized cancer cells to ionizing radiation. Effects of EGCG in IDH-mutated cell lines were diminished by treatment with the IDH1 mut inhibitor AGI-5198. Conclusions This work shows that glutamate can be directly processed into D- 2-HG and that reduction of glutamatolysis may be an effective and promising new treatment option for IDH mut cancers. Electronic supplementary material The online version of this article (10.1186/s40170-019-0198-7) contains supplementary material, which is available to authorized users.
Imaging of hyperpolarized 13 C-labeled substrates has emerged as an important magnetic resonance (MR) technique to study metabolic pathways in real time in vivo . Even though this technique has found its way to clinical trials, in vivo dynamic nuclear polarization is still mostly applied in preclinical models. Its tremendous increase in signal-to-noise ratio (SNR) overcomes the intrinsically low MR sensitivity of the 13 C nucleus and allows real-time metabolic imaging in small structures like the mouse brain. However, applications in brain research are limited as delivery of hyperpolarized compounds is restrained by the blood–brain barrier (BBB). A local noninvasive disruption of the BBB could facilitate delivery of hyperpolarized substrates and create opportunities to study metabolic pathways in the brain that are generally not within reach. In this work, we designed a setup to apply BBB disruption in the mouse brain by MR-guided focused ultrasound (FUS) prior to MR imaging of 13 C-enriched hyperpolarized [1- 13 C]-pyruvate and its conversion to [1- 13 C]-lactate. To overcome partial volume issues, we optimized a fast multigradient-echo imaging method (temporal resolution of 2.4 s) with an in-plane spatial resolution of 1.6 × 1.6 mm 2 , without the need of processing large amounts of spectroscopic data. We demonstrated the feasibility to apply 13 C imaging in less than 1 h after FUS treatment and showed a locally disrupted BBB during the time window of the whole experiment. From detected hyperpolarized pyruvate and lactate signals in both FUS-treated and untreated mice, we conclude that even at high spatial resolution, signals from the blood compartment dominate in the 13 C images, leaving the interpretation of hyperpolarized signals in the mouse brain challenging.
We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).
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