Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized ¹³C metabolic imaging in humans, where measurement of the input function can be problematic.

Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na⁺/H⁺ exchanger 1 (NHE1), causing a reduction in intracellular [Na⁺] ([Na⁺]_i). However, at resting intracellular pH (pH_i), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na⁺]_i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pH_i recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na⁺]_i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na⁺]_i rise upon superfusion of Na⁺-depleted cells with Na⁺-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na⁺]_i at baseline nor pH_i recovery following acute acidosis, as measured by ²³Na triple quantum filtered NMR and ³¹P NMR, respectively. Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na⁺]_i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na⁺].

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out.

Background: Dichloroacetate (DCA) has been found to have antitumour properties. Methods: We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, ¹H and hyperpolarised ¹³C-magnetic resonance spectroscopy. Results: Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-¹³C] pyruvate-to-lactate exchange rate (k_PL) were decreased in DCA-treated cells, along with increased NAD⁺/NADH ratios and NAD⁺. Steady-state lactate excretion and k_PL returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD⁺ and NADH. Reduced k_PL with DCA treatment was found in HT29 tumour xenografts in vivo. Conclusions: DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced k_PL and increased NAD⁺/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.