Dimethyl [1,4-¹³C₂]Fumarate as a Molecular Sensor for Gluconeogenesis in Renal versus Hepatic Tissues

The TCA cycle intermediate fumarate is converted to malate by fumarase. Because the fumarate dianion enters intact cells slowly but readily penetrates into necrotic cells with compromised membranes, hyperpolarized (HP) [1,4-¹³C₂]fumarate has been established as a molecular imaging probe for detecting cellular necrosis based on its conversion to [1,4-¹³C₂]malate. To broaden its utility to viable cells, we developed HP-¹³C-labeled fumarate esters, assuming that esterification would improve membrane permeability and cellular uptake. Since fumarate is metabolized through malate to oxaloacetate, a key precursor in gluconeogenesis, we investigated dimethyl [1,4-¹³C₂]fumarate as a potential HP probe for imaging gluconeogenic flux. Liquid-state polarization of HP-dimethyl [1,4-¹³C₂]fumarate exceeded 50%. The HP-¹³C NMR spectrum displayed a single resonance at 165.39 ppm, with longitudinal relaxation times (T₁) of 44 s at 3 T and 47 s at 1 T. In vivo spectra demonstrated rapid ester hydrolysis, yielding a predominant fumarate signal (175.4 ppm) along with smaller peaks corresponding to malate and bicarbonate. Isotopomer analysis of tissue extracts showed robust labeling of the phosphoenolpyruvate (PEP) pool and incorporation into newly synthesized glucose. Glucose production was higher in fasted than in fed rats (kidney: 13% vs 6%; liver: 3.7% vs 1%) and was greater in kidney than in liver. Ex vivo experiments confirmed rapid hydrolysis in rat blood but minimal hydrolysis in human blood, consistent with species-specific differences in carboxylesterase activity.