Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.

Material Basis For Combining Euphrobia Kansui And Licorice Based On Rat Liver Microsomal

Background: The herbal-pair, Kansui and Licorice, belongs to the "eighteen incompatible medicaments" category of traditional Chinese medicine. Kansuiphorin C (KC) is the main toxic component of Kansui. The main component of licorice is glycyrrhizic acid, which is hydrolyzed to glycyrrhetinic acid. Currently, the synergistic mechanism between Kansui and Licorice is unclear. Methods: Rat liver microsomes were used in this experiment, HPLC was used to detect the contents of KC, glycyrrhizic acid, and glycyrrhetinic acid to determine whether these compounds are metabolic substrates of CYP450. A control group with isozyme inhibitors was also employed to reveal the isozyme subtypes involved in compound metabolism. To further explain the induction or inhibitory effect of the above compounds on liver microsomal enzymes, enzyme activity was indirectly revealed based on changes in the contents of known metabolites of CYP2E1, CYP2C9, and CYP3A4. Results: KC and glycyrrhetinic acid were metabolic substrates of CYP450. CYP2E1 and CYP2C9 are mainly involved in the partial metabolism of glycyrrhizic acid in the liver. CYP2E1 and CYP3A4 are mainly involved in the partial metabolism of glycyrrhetinic acid in the liver. CYP2E1, CYP2C9, and CYP3A4 did not play a major role in the metabolism of KC. KC had little effect on the metabolism of glycyrrhizic acid and glycyrrhetinic acid. Glycyrrhizic acid, glycyrrhetinic acid, and KC induced CYP3A4 and inhibit CYP2E1. Both glycyrrhizic acid and glycyrrhetinic acid could inhibit the induction of CYP3A4 after combination with KC. KC with glycyrrhizic acid could synergistically inhibit the activity of CYP2E1, while KC with glycyrrhetinic acid could synergistically induce the activity of CYP2E1 Conclusion: KC and glycyrrhetinic acid were metabolic substrates of CYP450. KC, glycyrrhizic acid and glycyrrhetinic acid have different inducing and inhibiting effects on CYP450 enzyme.

Mapping Consistent, Reproducible, and Transcriptionally Relevant Connectome Hubs of the Human Brain

Macroscopic functional connectomic analyses have identified sets of densely connected regions in the human brain, known as connectome hubs, which play a vital role in understanding network communication, cognitive processing, and brain disorders. However, anatomical locations of functional connectome hubs are largely inconsistent and less reproducible among extant reports, partly due to inadequate sample size and differences in image processing and network analysis. Moreover, the genetic signatures underlying the robust connectome hubs remain unknown. Here, we conduct the first worldwide voxelwise meta-connectomic analysis by pooling resting-state functional MRI data of 5,212 healthy young adults across 61 independent international cohorts with harmonized image processing and network analysis protocols. We identify highly consistent and reproducible functional connectome hubs that are spatially distributed in multiple heteromodal and unimodal regions, with the most robust findings mainly located in lateral parietal regions. These connectome hubs show unique, heterogeneous connectivity profiles and are critical for both intra- and inter-network communications. Using transcriptome data from the Allen Human Brain Atlas and BrainSpan Atlas as well as machine learning, we demonstrate that these robust hubs are significantly associated with a transcriptomic pattern dominated by genes involved in the neuropeptide signaling pathway, neurodevelopmental processes, and cellular metabolic processes. This pattern represents microstructural and metabolic substrates underlying the development and functioning of brain hubs. Together, these results highlight robustness of macroscopic connectome hubs of the human brain and their potential cellular and molecular underpinnings and have implications for understanding how brain hubs support the connectome organization in health and disease.

Mitochondrial H+ Leak and Thermogenesis

Mitochondria of all tissues convert various metabolic substrates into two forms of energy: ATP and heat. Historically, the primary focus of research in mitochondrial bioenergetics was on the mechanisms of ATP production, while mitochondrial thermogenesis received significantly less attention. Nevertheless, mitochondrial heat production is crucial for the maintenance of body temperature, regulation of the pace of metabolism, and prevention of oxidative damage to mitochondria and the cell. In addition, mitochondrial thermogenesis has gained significance as a pharmacological target for treating metabolic disorders. Mitochondria produce heat as the result of H+ leak across their inner membrane. This review provides a critical assessment of the current field of mitochondrial H+ leak and thermogenesis, with a focus on the molecular mechanisms involved in the function and regulation of uncoupling protein 1 and the ADP/ATP carrier, the two proteins that mediate mitochondrial H+ leak. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Osmoregulatory Plasticity of Juvenile Greater Amberjack (Seriola dumerili) to Environmental Salinity

Osmotic costs in teleosts are highly variable, reaching up to 50% of energy expenditure in some. In several species, environmental salinities close to the isosmotic point (~15 psu) minimize energy demand for osmoregulation while enhancing growth. The present study aimed to characterize the physiological status related to osmoregulation in early juveniles of the greater amberjack, Seriola dumerili, acclimated to three salinities (15, 22, and 36 psu). Our results indicate that plasma metabolic substrates were enhanced at the lower salinities, whereas hepatic carbohydrate and energetic lipid substrates decreased. Moreover, osmoregulatory parameters, such as osmolality, muscle water content, gill and intestine Na+-K+-ATPase activities, suggested a great osmoregulatory capacity in this species. Remarkably, electrophysiological parameters, such as short-circuit current (Isc) and transepithelial electric resistance (TER), were enhanced significantly at the posterior intestine. Concomitantly, Isc and TER anterior-to-posterior intestine differences were intensified with increasing environmental salinity. Furthermore, the expression of several adeno-hypophyseal genes was assessed. Expression of prl showed an inverse linear relationship with increasing environmental salinity, while gh mRNA enhanced significantly in the 22 psu-acclimated groups. Overall, these results could explain the better growth observed in S. dumerili juveniles kept at salinities close to isosmotic rather than in seawater.

Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS Glucose : 0.1315±0.012; FS FFA : 0.1428±0.0127; FS 3OHB : 0.1343±0.014; FS MIX : 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS Glucose : 0.088±0.009***, p<0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS FFA : 0.109±0.0127; FS 3OHB : 0.107±0.012; FS MIX : 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.

Astrocyte metabolism of the medium-chain fatty acids octanoic acid and decanoic acid promotes GABA synthesis in neurons via elevated glutamine supply

The medium-chain fatty acids octanoic acid (C8) and decanoic acid (C10) are gaining attention as beneficial brain fuels in several neurological disorders. The protective effects of C8 and C10 have been proposed to be driven by hepatic production of ketone bodies. However, plasma ketone levels correlates poorly with the cerebral effects of C8 and C10, suggesting that additional mechanism are in place. Here we investigated cellular C8 and C10 metabolism in the brain and explored how the protective effects of C8 and C10 may be linked to cellular metabolism. Using dynamic isotope labeling, with [U-13C]C8 and [U-13C]C10 as metabolic substrates, we show that both C8 and C10 are oxidatively metabolized in mouse brain slices. The 13C enrichment from metabolism of [U-13C]C8 and [U-13C]C10 was particularly prominent in glutamine, suggesting that C8 and C10 metabolism primarily occurs in astrocytes. This finding was corroborated in cultured astrocytes in which C8 increased the respiration linked to ATP production, whereas C10 elevated the mitochondrial proton leak. When C8 and C10 were provided together as metabolic substrates in brain slices, metabolism of C10 was predominant over that of C8. Furthermore, metabolism of both [U-13C]C8 and [U-13C]C10 was unaffected by etomoxir indicating that it is independent of carnitine palmitoyltransferase I (CPT-1). Finally, we show that inhibition of glutamine synthesis selectively reduced 13C accumulation in GABA from [U-13C]C8 and [U-13C]C10 metabolism in brain slices, demonstrating that the glutamine generated from astrocyte C8 and C10 metabolism is utilized for neuronal GABA synthesis. Collectively, the results show that cerebral C8 and C10 metabolism is linked to the metabolic coupling of neurons and astrocytes, which may serve as a protective metabolic mechanism of C8 and C10 supplementation in neurological disorders.

The metabolic flexibility of quiescent CSC: implications for chemotherapy resistance

Quiescence has been observed in stem cells (SCs), including adult SCs and cancer SCs (CSCs). Conventional chemotherapies mostly target proliferating cancer cells, while the quiescent state favors CSCs escape to chemotherapeutic drugs, leaving risks for tumor recurrence or metastasis. The tumor microenvironment (TME) provides various signals that maintain resident quiescent CSCs, protect them from immune surveillance, and facilitates their recurrence potential. Since the TME has the potential to support and initiate stem cell-like programs in cancer cells, targeting the TME components may prove to be a powerful modality for the treatment of chemotherapy resistance. In addition, an increasing number of studies have discovered that CSCs exhibit the potential of metabolic flexibility when metabolic substrates are limited, and display increased robustness in response to stress. Accompanied by chemotherapy that targets proliferative cancer cells, treatments that modulate CSC quiescence through the regulation of metabolic pathways also show promise. In this review, we focus on the roles of metabolic flexibility and the TME on CSCs quiescence and further discuss potential treatments of targeting CSCs and the TME to limit chemotherapy resistance.

The Influence of Epigenetic Modifications on Metabolic Changes in White Adipose Tissue and Liver and Their Potential Impact in Exercise

Background: Epigenetic marks are responsive to a wide variety of environmental stimuli and serve as important mediators for gene transcription. A number of chromatin modifying enzymes orchestrate epigenetic responses to environmental stimuli, with a growing body of research examining how changes in metabolic substrates or co-factors alter epigenetic modifications.Scope of Review: Here, we provide a systematic review of existing evidence of metabolism-related epigenetic changes in white adipose tissue (WAT) and the liver and generate secondary hypotheses on how exercise may impact metabolism-related epigenetic marks in these tissues.Major Conclusions: Epigenetic changes contribute to the complex transcriptional responses associated with WAT lipolysis, hepatic de novo lipogenesis, and hepatic gluconeogenesis. While these metabolic responses may hypothetically be altered with acute and chronic exercise, direct testing is needed.

Integrated Analysis of microRNAs and Metabolomics in Rat’s Serum Reveals Multi-action Modes of Qingfei Paidu Decoction for COVID-19 Treatment

Background During the fight against coronavirus disease 2019 (COVID-19) in China, Qingfei Paidu decoction (QFPDD) has been widely applied to treat COVID-19 patients. Retrospective studies showed that QFPDD could improve clinical outcomes of COVID-19. Thus, it is necessary and interesting to explore the action mode of QFPDD for further application and development.MethodsSprague-Dawley (SD) rats were randomly divided into two groups, QFPDD (n=9) and control (n=10) groups. They were parallelly treated for 12 days with QFPDD and warm distilled water, respectively. At the endpoint, the microRNA (miRNA or miR) profiles in serum were detected to identify differently expressed miRNAs (DEMs). Then, the action mode of QFPDD were explored via review of potential roles of DEMs and functional enrichment analysis of their targets (e.g., GO enrichment and KEGG pathway analysis), especially focusing on the aspects of immunity, inflammation, virus infection and pulmonary fibrosis. Core genes were identified based on KEGG pathway analysis. Metabolomics were detected in serum and significantly changed metabolites (SCMs), especially the metabolic substrates and products of enzyme of core gene were identified as biomarkers to validate the regulation of DEMs to enzyme activity of core gene through metabolomic analysis and linear correlation analysis between SCMs and DEMs. Results23 DEMs were identified in the serum between QFPDD and control groups, with 1636 predicted genes. Reported evidence has showed that both the DEMs and their target genes involve regulation of immunity, inflammation, virus infection and pulmonary fibrosis. Phospholipase C, gamma 1 (Plcg1) was identified as a core gene and predicted to be upregulated attributed to downregulation of novel-89-mature. The levels of three SCMs, PC(P-18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/P-18:0) and PC(16:1(9Z)/16:1(9Z)), which were the metabolic substrates of phospholipase C, were significantly reduced in QFPDD group, in addition, PC(P-18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)) and PC(22:5(4Z,7Z,10Z,13Z,16Z)/P-18:0) presented positively linear correlation with the expression level of novel-89-mature. The level of phosphorylcholine, a product of PCs metabolized by phospholipase C, was significantly elevated in QFPDD group. Conclusion QFPDD can induce modification of miRNAs profile, and subsequently multi-regulate the immunity, inflammation, virus infection and pulmonary fibrosis in vivo, playing an important role for the positive outcomes of COVID-19 patients treated by QFPDD in China.