Investigations into the Signaling Pathways Involving Glucose-Stimulated Zinc Secretion (GSZS) from Prostate Epithelial Cells In Vitro and In Vivo.

PURPOSE: Recently, we reported that exposure of prostate cells in vitro or the in vivo prostate to high glucose results in release of Zn2+ ions, a process now referred to as glucose-stimulated zinc secretion (GSZS). To our knowledge, the metabolic event(s) that trigger GSZS remain largely unknown. Here, we explore several signaling pathways both in vitro using a prostate epithelial cell line and in vivo from the rat prostate. METHODS: PNT1A cells grown to confluence were washed and tagged with ZIMIR to monitor zinc secretion by optical methods. The expression levels of GLUT1, GLUT4, and Akt in cells cultured in either zinc-rich or zinc-poor media and after exposure to high versus low glucose were determined. Zinc secretion from the rat prostate in vivo as detected by MRI was compared in control animals after injection of glucose, deoxyglucose, or pyruvate to initiate zinc secretion and in animals pre-treated with WZB-117 (a GLUT1 inhibitor) or S961 (a peripheral insulin receptor inhibitor). RESULTS: PNT1A cells exposed to high levels of glucose secrete zinc whereas cells exposed to an equivalent amount of deoxyglucose or pyruvate do not. Expression of Akt was dramatically altered by zinc supplementation of the culture media but not after exposure to glucose while GLUT1 and GLUT4 levels were less affected. Rats pre-treated with WZB-117 prior to imaging showed a reduction in GSZS from the prostate compared to controls whereas rats pre-treated with S961 showed no difference. Interestingly, in comparison to PNT1A cells, pyruvate and deoxyglucose also stimulate zinc secretion in vivo likely through indirect mechanisms. CONCLUSIONS: GSZS requires metabolism of glucose both in vitro (PNT1A cells) and in vivo (rat prostate). Pyruvate also stimulates zinc secretion in vivo but likely via an indirect pathway involving rapid production of glucose via gluconeogenesis. These combined results support the conclusion that glycolytic flux is required to trigger GSZS in vivo.

Detection of glucose-derived D- and L-lactate in cancer cells by the use of a chiral NMR shift reagent.

BACKGROUND: Excessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to L-lactate. Although D-lactate can also be produced from glucose via the methylglyoxal pathway in small amounts, less is known about the amount of D-lactate produced in cancer cells. Since the stereoisomers of lactate cannot be distinguished by conventional 1H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1H NMR resonances of D- and L-lactate. METHODS: The production of L-lactate from glucose and D-lactate from methylglyoxal was first demonstrated in freshly isolated red blood cells using the chiral NMR shift reagent, YbDO3A-trisamide. Then, two different cell lines with high GLO1 expression (H1648 and H 1395) were selected from a panel of over 80 well-characterized human NSCLC cell lines, grown to confluence in standard tissue culture media, washed with phosphate-buffered saline, and exposed to glucose in a buffer for 4 h. After 4 h, a small volume of extracellular fluid was collected and mixed with YbDO3A-trisamide for analysis by 1H NMR spectroscopy. RESULTS: A suspension of freshly isolated red blood cells exposed to 5mM glucose produced L-lactate as expected but very little D-lactate. To evaluate the utility of the chiral NMR shift reagent, methylglyoxal was then added to red cells along with glucose to stimulate the production of D-lactate via the glyoxalate pathway. In this case, both D-lactate and L-lactate were produced and their NMR chemical shifts assigned. NSCLC cell lines with different expression levels of GLO1 produced both L- and D-lactate after incubation with glucose and glutamine alone. A GLO1-deleted parental cell line (3553T3) showed no production of D-lactate from glucose while re-expression of GLO1 resulted in higher production of D-lactate. CONCLUSIONS: The shift-reagent-aided NMR technique demonstrates that D-lactate is produced from glucose in NSCLC cells via the methylglyoxal pathway. The biological role of D-lactate is uncertain but a convenient method for monitoring D-lactate production could provide new insights into the biological roles of D- versus L-lactate in cancer metabolism.

Hyperpolarized 15N-labeled, deuterated tris (2-pyridylmethyl)amine as an MRI sensor of freely available Zn2.

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.

In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo 13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Probing carbohydrate metabolism using hyperpolarized 13 C-labeled molecules.

Glycolysis is a fundamental metabolic process in all organisms. Anomalies in glucose metabolism are linked to various pathological conditions. In particular, elevated aerobic glycolysis is a characteristic feature of rapidly growing cells. Glycolysis and the closely related pentose phosphate pathway can be monitored in real time by hyperpolarized 13 C-labeled metabolic substrates such as 13 C-enriched, deuterated D-glucose derivatives, [2-13 C]-D-fructose, [2-13 C] dihydroxyacetone, [1-13 C]-D-glycerate, [1-13 C]-D-glucono-δ-lactone and [1-13 C] pyruvate in healthy and diseased tissues. Elevated glycolysis in tumors (the Warburg effect) was also successfully imaged using hyperpolarized [U-13 C6 , U-2 H7 ]-D-glucose, while the size of the preexisting lactate pool can be measured by 13 C MRS and/or MRI with hyperpolarized [1-13 C]pyruvate. This review summarizes the application of various hyperpolarized 13 C-labeled metabolites to the real-time monitoring of glycolysis and related metabolic processes in normal and diseased tissues.

Improved Efficacy of Synthesizing *MIII-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio- and Physicochemical Study.

Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [68Ga]Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H2O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M(DOTA)]- (MIII= GaIII, CeIII, EuIII, YIII, and LuIII) complexes were investigated in H2O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H2O/EtOH mixtures (0-70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K1H and log K2H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M(DOTA)]- complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M(H2DOTA)]+ intermediates, which are in equilibrium with the kinetically active monoprotonated [M(HDOTA)] intermediates. The rate-controlling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through H2O (*M(HL) kH2O) and OH- (*M(HL) kOH) assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the M(HL) kH2O values increase from CeIII to LuIII. However, the log KM(HL)H protonation constants, analogous to the log KH2 value, decrease with increasing [EtOH], which increases the concentration of the monoprotonated M(HDOTA) intermediate and accelerates formation of the final complexes. The overall rates of complex formation calculated by the obtained rate constants at different EtOH concentrations show a trend similar to that of the complexation rates determined with the use of radioactive isotopes.