Observing exocrine pancreas metabolism using a novel pancreas perfusion technique in combination with hyperpolarized [1-13 C]pyruvate.

In a clinical setting, ex vivo perfusions are routinely used to maintain and assess organ viability prior to transplants. Organ perfusions are also a model system to examine metabolic flux while retaining the local physiological structure, with significant success using hyperpolarized (HP) 13 C NMR in this context. We use a novel exocrine pancreas perfusion technique via the common bile duct to assess acinar cell metabolism with HP [1-13 C]pyruvate. The exocrine component of the pancreas produces digestive enzymes through the ductal system and is often neglected in research on the pancreas. Real-time production of [1-13 C]lactate, [1-13 C]alanine, [1-13 C]malate, [4-13 C]malate, [1-13 C]aspartate, and H13 CO3 - was detected. The appearance of these resonances indicates flux through both pyruvate dehydrogenase and pyruvate carboxylase. We studied excised pancreata from C57BL/6J mice and NOD.Rag1-/- .AI4α/ß mice, a commonly used model of Type 1 Diabetes (T1D). Pancreata from the T1D mice displayed increased lactate to alanine ratio without changes in oxygen consumption, signifying increased cytosolic NADH levels. The mass isotopologue analysis of the extracted pancreas tissue using gas chromatography-mass spectrometry revealed confirmatory 13 C enrichment in multiple TCA cycle metabolites that are products of pyruvate carboxylation. The methodology presented here has the potential to provide insight into mechanisms underlying several pancreatic diseases, such as diabetes, pancreatitis, and pancreatic cancer.

Synergistic Effect of ß-Lapachone and Aminooxyacetic Acid on Central Metabolism in Breast Cancer.

The compound ß-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of ß-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of ß-lapachone action through NAD+-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD+/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of ß-lapachone metabolic perturbation in NQO1+ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1+, MDA-MB-231 NQO1-, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-13C]glucose tracer. We show that ß-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1+ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.

Detecting de novo Hepatic Ketogenesis Using Hyperpolarized [2-13C] Pyruvate.

The role of ketones in metabolic health has progressed over the past two decades, moving from what was perceived as a simple byproduct of fatty acid oxidation to a central player in a multiplicity of disease states. Previous work with hyperpolarized (HP) 13C has shown that ketone production can be detected when using precursors that labeled acetyl-CoA at the C1 position, often in tissues that are not normally recognized as ketogenic. Here, we assay metabolism of HP [2-13C]pyruvate in the perfused mouse liver, a classic metabolic testbed where nutritional conditions can be precisely controlled. Livers perfused with long-chain fatty acids or the medium-chain fatty acid octanoate showed no evidence of ketogenesis in the 13C spectrum. In contrast, addition of dichloroacetate, a potent inhibitor of pyruvate dehydrogenase kinase, resulted in significant production of both acetoacetate and 3-hydroxybutyrate from the pyruvate precursor. This result indicates that ketones are readily produced from carbohydrates, but only in the case where pyruvate dehydrogenase activity is upregulated.

Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State.

Type II diabetes and pre-diabetes are widely prevalent among adults. Elevated serum glucose levels are commonly treated by targeting hepatic gluconeogenesis for downregulation. However, direct measurement of hepatic gluconeogenic capacity is accomplished only via tracer metabolism approaches that rely on multiple assumptions, and are clinically intractable due to expense and time needed for the studies. We previously introduced hyperpolarized (HP) [2-13C]dihydroxyacetone (DHA) as a sensitive detector of gluconeogenic potential, and showed that feeding and fasting produced robust changes in the ratio of detected hexoses (6C) to trioses (3C) in the perfused liver. To confirm that this ratio is robust in the setting of treatment and hormonal control, we used ex vivo perfused mouse livers from BLKS mice (glucagon treated and metformin treated), and db/db mice. We confirm that the ratio of signal intensities of 6C to 3C in 13C nuclear magnetic resonance spectra post HP DHA administration is sensitive to hepatic gluconeogenic state. This method is directly applicable in vivo and can be implemented with existing technologies without the need for substantial modifications.

Zinc finger protein 593 is upregulated during skeletal muscle atrophy and modulates muscle cell differentiation.

Skeletal muscle atrophy is a debilitating condition that can arise due to aging, cancer, corticosteroid use, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study analyzed gene expression patterns in gastrocnemius muscle following sciatic nerve transection and found for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is induced in response to denervation. Quantitative PCR and Western blot analyses confirmed that Zfp593 is expressed in both proliferating myoblasts and differentiated myotubes. To assess sub-cellular location, GFP-tagged Zfp593 was expressed in C2C12 cells and found to localize to the nucleus. The Zfp593 protein possesses a putative zinc finger domain and is believed to function as a modulator of the Oct-2 transcription factor. Interestingly, ectopic expression of Zfp593 did not affect the ability of Oct-1 or Oct-2 to inhibit an Oct reporter gene in muscle cells. Finally, Zfp593 overexpression in cultured muscle cells resulted in significant repression of muscle cell differentiation and attenuation of ERK1/2 and p38 phosphorylation, but did not vitiate protein synthesis. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that it is induced in response to neurogenic atrophy furthers our understanding of the molecular genetic events of muscle wasting.