Impaired metabolism is recognized as an important contributor to pathogenicity of T cells in
(SLE). Over the last two decades, we have acquired significant knowledge about the signaling and ...transcriptomic programs related to metabolic rewiring in healthy and SLE T cells. However, our understanding of metabolic network activity derives largely from studying metabolic pathways in isolation. Here, we argue that enzymatic activities are necessarily coupled through mass and energy balance constraints with in-built network-wide dependencies and compensation mechanisms. Therefore, metabolic rewiring of T cells in SLE must be understood in the context of the entire network, including changes in metabolic demands such as shifts in biomass composition and cytokine secretion rates as well as changes in uptake/excretion rates of multiple nutrients and waste products. As a way forward, we suggest cell physiology experiments and integration of orthogonal metabolic measurements through computational modeling towards a comprehensive understanding of T cell metabolism in lupus.
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)
C 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-
Cpyruvate 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
C 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.
Transcriptome data were collected in rat dopamine cells exposed to fipronil for 24 h using microarray analysis. Fipronil is a phenylpyrazole pesticide that acts to inhibit gamma-aminobutyric acid ...(GABA), blocking inhibitory synaptic transmission in the central nervous system. Transcriptome data were subjected to pathway analysis and subnetwork enrichment analysis. We report that 25 µM fipronil altered transcriptional networks in dopamine-synthesizing cells that are associated with Alzheimer's Disease, Huntington Disease, and Schizophrenia. Data analysis revealed that nerve fibre degeneration, nervous system malformations, neurofibrillary tangles, and neuroinflammation were all disease processes related to the transcriptome profile observed in the rat neuronal cells. Other disease networks altered by fipronil exposure at the transcript level were associated with the mitochondria, including mitochondrial DNA depletion syndrome and mitochondrial encephalomyopathies. These data, along with those presented in Souders et al. (2021), are significant because they increase understanding into the molecular mechanisms underlying human disease following exposures to neuroactive pesticides. These data can be reused to inform adverse outcome pathways for neurotoxic pesticides.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The exocrine pancreas displays structural and functional abnormalities in type 1 diabetes, such as acinar atrophy and fibrosis. Metabolic dysfunction may occur alongside these changes. Real-time ...exocrine pancreatic metabolism was investigated using nuclear magnetic resonance (NMR) and hyperpolarized (HP) pyruvate. This work used the hyperglycemic NOD.Rag1-/-.AI4α/β mouse model of T1D (n=4), normoglycemic NOD.Rag1-/-.AI4α/β mice (n=3) to model the pre-T1D state, and the NOD.Rag1-/- control (n=3). NMR spectra were collected at 14 T after injection of 4 mM HP 1-13C pyruvate into the common bile duct, and 13C signals from several central carbon metabolites were detected. Pancreata from diabetic mice produced significantly more (p<0.05) fumarate and aspartate compared to control mice, indicating increased pyruvate carboxylase flux. Alanine to lactate and bicarbonate to lactate ratios were significantly altered in pre-diabetic and diabetic mice compared to the control, indicating a detectable shift in tissue redox balance prior to altered glycemia. Separately, immunofluorescence imaging of pancreas slices revealed pyruvate carboxylase levels were increased in T1D mice. Ultimately, these results indicate that the diabetic pancreas displays increased pyruvate carboxylase (PC) flux and altered redox balance, thus providing unique insights into uncharacterized metabolic shifts in the exocrine pancreas in T1D.
The pancreas functions as both an endocrine and exocrine gland. The endocrine component consists of the islets of Langerhans, whereas the exocrine tissue produces digestive enzymes through the ductal ...system. Although the majority of Type I Diabetes research has focused on the islets, the exocrine tissue is likely implicated in T1D as well. This research utilizes a novel method to investigate exocrine pancreas metabolism with a pancreas perfusion system. I hypothesized that dissolution dynamic nuclear polarization (dDNP) combined with the pancreas perfusion system would provide real-time measurements of central carbon metabolic turnover in the exocrine pancreas. DNP is a phenomenon in which a transfer of high spin polarization greatly enhances the signal in the resulting nuclear magnetic resonance (NMR) spectra. This research compares dDNP measurements in the exocrine pancreas in C57BL/6 male mice under varying glucose conditions. It also examines central carbon metabolism using this system in the T1D mouse model NOD.Rag1-/-.AI4α/β and the NOD.Rag1-/- control. After cannulation of the common bile duct, the exocrine pancreas was perfused for 30 minutes with perfusate containing either 3mM or 16.7mM glucose. Tissue viability was confirmed throughout the perfusion with oxygen consumption measurements. dDNP was performed using the trityl radical and 4mM 1-13Cpyruvate. NMR spectra were collected on a 600MHz instrument with a 10mm 13C-optimized cryoprobe and analyzed with Bruker TopSpin. Several metabolites were identified and quantified in all measured spectra, demonstrating the detection of real-time metabolic turnover. Ultimately, this work demonstrates that dissolution dynamic nuclear polarization can be used to evaluate metabolic flux in the pancreas in multiple mouse models. Future will investigate the real-time metabolic response of the exocrine pancreas to insulin concentrations in the T1D disease model.
Disclosure
A.Rushin: None. M.Mcleod: None. M.Ragavan: None. M.Merritt: None.
Funding
National Institutes of Health (T32 DK10876) National Science Foundation (DMR-1644779)
The pancreas functions as both an endocrine and exocrine gland. The endocrine component consists of the islets of Langerhans, which contain several pancreatic cell types that produce hormones. In the ...islets, α‐cells produce glucagon and β‐cells produce insulin, both of which serve to regulate glucose metabolism. The exocrine tissue produces digestive enzymes such as chymotrypsin and is connected through the ductal system. Type I Diabetes (T1D) is an autoimmune disease in which immune cells attack the pancreatic β‐cells, resulting in the pancreas producing little to no insulin. Although the vast majority of research has focused on the islets, the exocrine tissue may be implicated in T1D as well. This research introduces a novel method to investigate exocrine pancreas metabolism specifically using a pancreas perfusion system through the common bile duct. I hypothesized that the exocrine pancreas would demonstrate metabolic activity through uptake and utilization of 2H7glucose during a perfusion.
Using C57BL/6 male mice, the surgical procedure consisted of clamping the ampulla of Vater with a John Hopkins bulldog clamp and cannulation of the common bile duct. The pancreas was then perfused for 20 minutes with perfusate containing Krebs electrolytes, free fatty acids, and 16.7mM of either unlabeled or 2H7glucose. The tissue was flash frozen in liquid nitrogen and subjected to an acetonitrile isopropanol water extraction. The extracts were derivatized using methoxyamine and MTBSTFA before gas chromatography‐mass spectrometry (GC‐MS) analysis.
GC‐MS measurements confirmed the pancreas exocrine tissue metabolized 2H7glucose, as shown by an increased enrichment in lactate mass isotopologues in the tissue perfused with isotopically labeled glucose compared to the unlabeled control. Additional central carbon metabolites also demonstrated increased enrichment, namely malate and fumarate. The observed isotopologue labeling patterns are indicative of anaplerosis. This research demonstrates metabolism specific to the exocrine pancreas can be measured using a pancreas perfusion system and isotopic labeling. Future work will examine real‐time central carbon metabolic flux using dissolution dynamic nuclear polarization (dDNP) combined with the pancreas perfusion system. This research will use the T1D mouse model NOD. Rag1‐/‐.AI4α/β and compare the results to the NOD.Rag1‐/‐ control mouse model to examine how exocrine pancreas central carbon metabolism is altered in T1D models in a more physiologically relevant manner than previously possible.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The exocrine pancreas displays structural and functional abnormalities in type 1 diabetes, such as acinar atrophy and fibrosis. Metabolic dysfunction may occur alongside these changes. We established ...a method to investigate exocrine metabolism using nuclear magnetic resonance (NMR) and hyperpolarized (HP) substrates. This approach conserves the native environment while permitting analysis of metabolic flux in the living pancreas. Here, we compared exocrine pancreas metabolism in fed (n=4) and fasted (n=5) male C57BL/6J mice. Each pancreas was perfused for 30 minutes with oxygenated Krebs-Heinsleit buffer in a 14 T NMR instrument. NMR spectra were collected after injection of 4 mM HP 1-13C pyruvate into the common bile duct. 13C signals from several central carbon metabolites were detected, such as alanine, malate, and fumarate. Pancreata from fasted mice produced less (p<0.05) fumarate and aspartate compared to those from the fed mice. After perfusion, all pancreata were analyzed by gas chromatography - mass spectrometry for metabolite quantification. Malate was significantly enriched (p<0.05) by 13C in the fed pancreas with similar total malate between groups. Overall, these results suggest that the exocrine pancreas displays higher pyruvate carboxylase (PC) flux in the fed state compared to the fasted state. The methodology presented here provides a unique avenue to assess pancreatic metabolism in real-time.
Disclosure
A.Rushin: None. M.Mcleod: None. M.Glanz: None. D.S.Graham: None. M.Merritt: None.
Funding
National Institutes of Health (T32DK10876); National Science Foundation (DMR-1644779)
Insulin resistance is a major contributor to nonalcoholic fatty liver disease (NAFLD) . NAFLD is a progressive disease that leads to impaired hepatic function and liver cirrhosis. Increased ...ketogenesis has been correlated with a protective phenotype in mice and humans. During our study we identified differences in acetyl-CoA partitioning between the oxidative tricarboxylic acid cycle (TCA) , and ketogenesis. 8 week old C57Bl6 mice were fed a high fat or low fat (60% or 10% fat by calories respectively) diet for 16 weeks before performing liver perfusion in 12-14 hours fasted mice with 1 mM D15-octanoate fatty acid tracer. The effluent perfusate was collected and GC-MS analysis was performed to identify the distribution of deuterium label. Enrichments were greater in ketones (˜70%) than the TCA cycle (˜20%) with the unexpected determination that terminal acetyl-CoA units derived from fatty acids were preferentially used for ketogenesis as compared to internal acetyl-CoA units (Figure 1) . These changes were confirmed with fractional enrichment analysis and metabolic modeling of the ketogenic pathway and TCA cycle. This finding elucidates differential handling of acetyl-CoA that could be exploited for therapeutic benefit to NAFLD.
Disclosure
M.Mcleod: None. M.Ragavan: None. A.Rushin: None. M.Merritt: None. A.G.Giacalone: None.
Non‐alcoholic fatty liver disease (NAFLD) is characterized by >5% hepatic fat by mass. As one of the most common liver diseases, with a prevalence of roughly 24% in the U.S. adult population, NAFLD ...related morbidities account for a striking ~300 billion dollars a year in healthcare costs for the U.S. alone Shetty, A., & Syn, W. K. Federal practitioner: for the health care professionals of the VA, DoD, and PHS, (2019), 36(1), 14–19. NAFLD, both in humans and murine models of the disease, has been associated with increased rates of hepatic β‐oxidation and reactive oxygen species production Sunny, N. E., Parks, E. J., Browning, J. D., & Burgess, S. C. Cell metabolism, (2011). 14(6), 804–810. These characteristics are thought to drive a pro‐inflammatory state that causes hepatic damage, and can transition to the much more serious malady nonalcoholic steatohepatitis. To directly quantify hepatic β‐oxidation as a potential biomarker of NAFLD disease development and progression, we are developing deuterium magnetic resonance (DMR) techniques to monitor oxidation of D15‐octanoate. In the perfused mouse liver, we demonstrate that DMR measures of HDO production, as a direct byproduct of β‐oxidation, estimate hepatic β‐oxidation rates within 5% of standard quantitation by mass spectrometry of fatty acid depletion from perfusate. In a dietary model of NAFLD (60% fat by kcal, HFD) we observed increased overall b‐oxidation, but when normalized to the total liver weight, the HFD liver oxidized less than the low‐fat diet control. By coupling DMR spectroscopy, gas chromatography‐mass spectrometry and metabolic modelling, we were able to identify increases in total ketogenesis and FADH2 oxidation in high fat diet perfused livers. When normalizing metabolic flux to liver protein, both octanoate and FADH2 oxidation were significantly lower in high fat diet livers. Increased total fatty acid oxidation and ketogenesis implicate activation of compensatory mechanisms by the liver during NAFLD to address higher levels of circulating lipids. Decreases in efficiency/relative oxidation indicate an accrual of hepatic damage that hinders the efficient clearance of excess lipids. These results demonstrate the utility of DMR as a sensitive measure for detecting changes in metabolic activity during NAFLD progression, and suggests that a clinical imaging paradigm might be feasible.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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 1‐13Cpyruvate. 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 1‐13Clactate, 1‐13Calanine, 1‐13Cmalate, 4‐13Cmalate, 1‐13Caspartate, 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.
Real‐time metabolism of the exocrine pancreas is measured using nuclear magnetic resonance after injection of hyperpolarized 1‐13Cpyruvate into the perfusing pancreas.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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