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.

Mitochondrial adaptation during non-alcoholic fatty liver disease (NAFLD) include remodeling of ketogenic flux and sustained tricarboxylic acid (TCA) cycle activity, which are concurrent to onset of oxidative stress. Over 70% of obese humans have NAFLD and ketogenic diets are common weight loss strategies. However, the effectiveness of ketogenic diets toward alleviating NAFLD remains unclear. We hypothesized that chronic ketogenesis will worsen metabolic dysfunction and oxidative stress during NAFLD. Mice (C57BL/6) were kept (for 16-wks) on either a low-fat, highfat, or high-fat diet supplemented with 1.5X branched chain amino acids (BCAAs) by replacing carbohydrate calories (ketogenic). The ketogenic diet induced hepatic lipid oxidation and ketogenesis, and produced multifaceted changes in flux through the individual steps of the TCA cycle. Higher rates of hepatic oxidative fluxes fueled by the ketogenic diet paralleled lower rates of de novo lipogenesis. Interestingly, this metabolic remodeling did not improve insulin resistance, but induced fibrogenic genes and inflammation in the liver. Under a chronic "ketogenic environment," the hepatocyte diverted more acetyl-CoA away from lipogenesis toward ketogenesis and TCA cycle, a milieu which can hasten oxidative stress and inflammation. In summary, chronic exposure to ketogenic environment during obesity and NAFLD has the potential to aggravate hepatic mitochondrial dysfunction.

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.

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The compound β-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 β-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 β-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 β-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 β-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.

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) 13C NMR in this context. We use a novel exocrine pancreas perfusion technique via the common bile duct to assess acinar cell metabolism with HP [1‐13C]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 [1‐13C]lactate, [1‐13C]alanine, [1‐13C]malate, [4‐13C]malate, [1‐13C]aspartate, and H13CO3− 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/β 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 13C 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.

In this study, untargeted metabolic profiling by Gas Chromatography Mass Spectrometry (GC‐MS) was leveraged to identify differences between Perchloric acid extraction (PCA) and 3:3:2 acetonitrile/isopropanol/water (IPA) extraction for liver metabolomics research. Perchloric acid extraction (PCA), a common means of precipitating excess protein through acidification, has been used in physiology literature for decades and is considered as the standard method for extracting water soluble metabolites. However, PCA extraction commonly requires tedious pH neutralization with potassium hydroxide (KOH) and subsequent desalting. While IPA extraction can also precipitate protein it is pH neutral and bypasses the need for neutralization and is more amenable for high throughput sampling. Several studies have demonstrated that IPA has a high extraction efficiency amongst other organic solvent based extractions such as 50:50 acetonitrile water and 80% methanol water in mammalian fecal extracts [Yang, Y., Yin, Y., Chen, X. et al Scientific Reports. 2019, 9(12017)] and plasma extracts [Jiye, A. et al. 2005 Anal. Chem. 77, 8086–8094] for GC‐MS. Similarly, plasma extractions for Nuclear Magnetic Resonances (NMR) have demonstrated that 50:50 acetonitrile water extractions, a variant of organic extraction similar to IPA, are reproducible and are capable of eliminating excess protein from extracts while preserving low concentration metabolites [Daykin et al. Analytical Biochemistry 2002, 304, 220 –230]. In this study we expand the current knowledge of IPA versus PCA extractions in the field of liver metabolomics. Liver is a potential target for study, due to the increasing prevalence of Non‐Alcoholic Fatty liver disease (NAFLD) and Non‐alcoholic Steatohepatitis (NASH), which now affects a combined 80‐100 million Americans [Perumpail et al. World J Gastroenterology. 2017, 23(47), 8263]. Due to the multifaceted nature of NAFLD approaches including NMR, mass spectrometry and various extraction variants have aimed to identify metabolic changes that would shed light on the mechanism of pathogenesis. To achieve a relevant background for analyzing the suitability of PCA or IPA for liver metabolomics in NAFLD, we extracted ex vivo C57/BLKS perfused mouse livers under fed, fasted and pooled conditions. We utilize C57/BLKS mice as they are prone to type 2 diabetes, atherosclerosis and obesity, all of which are significant cofactors for NAFLD development. Through our untargeted metabolomics panel several previously unidentified metabolites were assessed by principle components analysis and determined to be similar (Figure I). From our targeted metabolomics panel of extracted standards we also observed that several metabolites are largely similar in extraction efficiency with only the ketone 3‐hydroxybutyrate having a higher extraction efficiency by PCA rather than IPA (Figure I). In conclusion, the extraction efficiency and statistical comparison of PCA and IPA extractions for GC‐MS metabolomics assessments demonstrate their suitability and equi...

Skeletal muscle atrophy is a debilitating medical condition which can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C2C12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with MyoD1 or myogenin expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 has revealed four conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Furthermore, two predicted putative nuclear localization signal sequences have been identified in the Zfp593 protein sequence, further suggesting that this protein is localized to the nucleus. Therefore, in order to determine the sub‐cellular localization of Zfp593 in muscle cells, the Zfp593 cDNA was isolated from C2C12 cells, cloned into a green fluorescent protein (GFP) expression plasmid and transfected into proliferating C2C12 cells. Transfected cells were visualized by confocal microscopy revealing that Zfp593 is localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was supported by University of North Florida Transformational Learning grants to D.W.