Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.

Practical Guidelines for Diagnosing and Treating Thyroid Disease Based on the WOMED Metabolic Model of Disease Focusing on Glycolysis and Coenzyme Q10 Deficiency—A Clinical Alternative to the 2021 Retired Clinical Practice Guidelines of the Endocrine Society

This review aims to provide a functional, metabolic view of the pathogenesis of benign thyroid disease. Here, we summarize the features of our previous publications on the “WOMED model of benign thyroid disease”. As of 2021, the current state of art indicates that the basic alteration in benign thyroid disease is a metabolic switch to glycolysis, which can be recognized using 3D-power Doppler ultrasound. A specific perfusion pattern showing enlarged vessels can be found using this technology. This switch originates from an altered function of Complex I due to acquired coenzyme Q10 deficiency, which leads to a glycolytic state of metabolism together with increased angiogenesis. Implementing a combined supplementation strategy that includes magnesium, selenium, and CoQ10, the morphological and perfusion changes of the thyroid can be reverted, i.e., the metabolic state returns to oxidative phosphorylation. Normalization of iron levels when ferritin is lower than 50 ng/mL is also imperative. We propose that a modern investigation of probable thyroid disease requires the use of 3D-power Doppler sonography to recognize the true metabolic situation of the gland. Blood levels of magnesium, selenium, CoQ10, and ferritin should be monitored. Thyroid function tests are complementary so that hypo- or hyperthyroidism can be recognized. Single TSH determinations do not reflect the glycolytic state.

The Energy Sensor AMPKα1 Is Critical in Rapamycin-Inhibition of mTORC1-S6K-Induced T-cell Memory

Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa)-inhibition of mTORC1 (RIM) promotes T-cell memory. However, the underlying pathway and the role of AMPKα1 in Rapa-induced T-cell memory remain elusive. Using genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice in vivo post Listeria monocytogenesis rLmOVA infection and in vitro transition of effector T (TE) to memory T (TM) cells. IL-2- and IL-2+Rapa-stimulated T [IL-2/T and IL-2(Rapa+)/T] cells, when transferred into mice, differentiate into short-term IL-7R−CD62L−KLRG1+ TE and long-lived IL-7R+CD62L+KLRG1− TM cells, respectively. To assess the underlying pathways, we performed Western blotting, confocal microscopy and Seahorse-assay analyses using IL-2/T and IL-2(Rapa+)/T cells. We determined that IL-2(Rapa+)/T cells activate transcription FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555) and ATG7 molecules and promote mitochondrial biogenesis and fatty-acid oxidation (FAO). We found that rapamycin-treated AMPKα-deficient AMPKα1-KO IL-2(Rapa+)/TM cells up-regulate transcription factor HIF-1α and induce a metabolic switch from FAO to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Taken together, our data indicate that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis, and that AMPKα1 plays a critical role in RIM-induced T-cell memory.

Metabolic Reprogramming of Liver Fibrosis

Liver fibrosis is an excessive and imbalanced deposition of fibrous extracellular matrix (ECM) that is associated with the hepatic wound-healing response. It is also the common mechanism that contributes to the impairment of the liver function that is observed in many chronic liver diseases (CLD). Despite the efforts, no effective therapy against fibrosis exists yet. Worryingly, due to the growing obesity pandemic, fibrosis incidence is on the rise. Here, we aim to summarize the main components and mechanisms involved in the progression of liver fibrosis, with special focus on the metabolic regulation of key effectors of fibrogenesis, hepatic stellate cells (HSCs), and their role in the disease progression. Hepatic cells that undergo metabolic reprogramming require a tightly controlled, fine-tuned cellular response, allowing them to meet their energetic demands without affecting cellular integrity. Here, we aim to discuss the role of ribonucleic acid (RNA)-binding proteins (RBPs), whose dynamic nature being context- and stimuli-dependent make them very suitable for the fibrotic situation. Thus, we will not only summarize the up-to-date literature on the metabolic regulation of HSCs in liver fibrosis, but also on the RBP-dependent post-transcriptional regulation of this metabolic switch that results in such important consequences for the progression of fibrosis and CLD.

In Silico-guided Metabolic Engineering of Bacillus Subtilis for Efficient Biosynthesis of Purine Nucleosides by Blocking the Key Backflow Nodes

Background: Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproductions of purine nucleosides by de novo metabolic engineering have been a great challenge due to their strict and complex regulatory machinery involved in the biosynthetic pathways. Results: In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on the genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes Drm (Purine nucleotides toward PPP) and YwjH (PPP-EMP) for resolving the competitive relationship between biomass and purine nucleotides synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides and greatly increased the inosine production to 13.98–14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of purine operon by promoter replacement further enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, deleting the second backflow node YwjH and overexpressing Zwf were combined to increase the inosine production to 22.01±1.18 g/L by enhancing the metabolic flow of PPP. Through switching on the flux node of the glucose-6- phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81±1.23 g/L of inosine by a pgi-based metabolic switch in shake-flask cultivation, suggesting the highest yield in de novo engineered inosine bacteria. Under the guidance of the in silico-designed strategy, a general chassis bacterium was generated for the first time to efficiently synthesize inosine, adenosine, guanosine, IMP, and GMP, providing the sufficient precursor for the synthesis of various purine intermediates. Conclusions: Overall, in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring the efficient targets, representing a superior strategy for efficient biosynthesis of the biotechnological products.

Modulation of PKM1/2 levels by steric blocking morpholinos alters the metabolic and pluripotent state of murine pluripotent stem cells

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described formative pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 mRNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the formative state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression towards a primed pluripotent state.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA–induced metabolic stress in reconstructed human skin

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Chronobiology and Metabolism: Is Ketogenic Diet Able to Influence Circadian Rhythm?

Circadian rhythms underpin most physiological processes, including energy metabolism. The core circadian clock consists of a transcription-translation negative feedback loop, and is synchronized to light-dark cycles by virtue of light input from the retina, to the central clock in the suprachiasmatic nucleus in the hypothalamus. All cells in the body have circadian oscillators which are entrained to the central clock by neural and humoral signals. In addition to light entrainment of the central clock in the brain, it now emerges that other stimuli can drive circadian clock function in peripheral tissues, the major one being food. This can then drive the liver clock to be misaligned with the central brain clock, a situation of internal misalignment with metabolic disease consequences. Such misalignment is prevalent, with shift workers making up 20% of the working population. The effects of diet composition on the clock are not completely clarified yet. High-fat diet and fasting influence circadian expression of clock genes, inducing phase-advance and phase-delay in animal models. Ketogenic diet (KD) is able to induce a metabolic switch from carbohydrate to fatty acid oxidation, miming a fasting state. In recent years, some animal studies have been conducted to investigate the ability of the KD to modify circadian gene expression, and demonstrated that the KD alters circadian rhythm and induces a rearrangement of metabolic gene expression. These findings may lead to new approaches to obesity and metabolic pathologies treatment.