A global view of T cell metabolism in systemic lupus erythematosus.

Impaired metabolism is recognized as an important contributor to pathogenicity of T cells in Systemic Lupus Erythematosus (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.

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.

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.

Disease network data for the pesticide fipronil in rat dopamine cells.

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.

Mitochondrial and transcriptome responses in rat dopaminergic neuronal cells following exposure to the insecticide fipronil.

The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 µM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 µM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 µM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 µM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 µM fipronil treatments, but there was a ∼40-60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 µM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 µM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 µM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson's disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents.

Dieldrin-induced neurotoxicity involves impaired mitochondrial bioenergetics and an endoplasmic reticulum stress response in rat dopaminergic cells.

Mitochondria are sensitive targets of environmental chemicals. Dieldrin (DLD) is an organochlorine pesticide that remains a human health concern due to high lipid bioaccumulation, and it has been epidemiologically associated to an increased risk for Parkinson's disease (PD). As mitochondrial dysfunction is involved in the etiology of PD, this study aimed to determine whether DLD impaired mitochondrial bioenergetics in dopaminergic cells. Rat immortalized dopaminergic N27 cells were treated for 24 or 48h with one dose of either a solvent control, 2.5, 25, or 250µM DLD. Dopaminergic cells treated with 250µM DLD showed increased Casp3/7 activity at 24 and 48h. DLD also caused a dose dependent reduction in cell viability of ∼25-30% over 24h. No significant effects on cell viability, apoptosis, nor cytotoxicity were detected at 24 or 48h with 2.5µM DLD. Following a 24h exposure to 2.5 and 25µM DLD, viable cells were subjected to a mitochondrial stress test using the Seahorse XFe24 Extracellular Flux Analyzer. Following three independent experiments conducted for rigor, dopaminergic cells that were treated with 2.5 and 25µM DLD consistently showed a reduction in maximum respiration and spare capacity compared to the control group. Molecular responses were measured to determine mechanisms of DLD-induced mitochondrial dysfunction. There were no changes in transcripts associated with mitochondrial membrane potential and permeability (e.g. Ant, Hk1, Tspo, Vdac), nor PI3 K/Akt/mTor signaling or mitochondrial-associated apoptotic factors (Bax, Bcl2, Casp3). However, transcript levels for Chop/Gadd153 (DNA Damage Inducible Transcript 3), an apoptotic gene activated following endoplasmic reticulum (ER) stress, were 3-fold higher in N27 cells treated with DLD, suggesting that DLD-induced mitochondrial dysfunction is related to ER stress. Dopamine cells were also assessed for changes in tyrosine hydroxylase (TH) protein, which did not differ among treatments. This study demonstrates that DLD impairs oxidative respiration in dopamine cells, and ER stress is hypothesized to be associated with the DLD-induced mitochondrial dysfunction. This is important as ER stress is also linked to PD. This study presents mechanistic insight into pesticide-induced mitochondrial dysfunction using a chemical that is reported to be associated to a higher risk for neurodegenerative disease.