scholarly journals Effect of hypoxia on purine metabolism of human skeletal muscle cells

Biotecnia ◽  
2021 ◽  
Vol 23 (2) ◽  
Author(s):  
Tania Zenteno-Savín ◽  
Crisalejandra Rivera-Pérez ◽  
Ramón Gaxiola-Robles ◽  
Norma Olguín-Monroy ◽  
Orlando Lugo-Lugo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Muscle Cells ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Purine Metabolism ◽  
Inosine Monophosphate Dehydrogenase ◽  
Inosine Monophosphate ◽  
Hypoxic Conditions ◽  
Human Skeletal Muscle Cells

Mammals experience some degree of hypoxia during their lifetime. In response to hypoxic challenge, mammalian cells orchestrate specific responses at transcriptional and posttranslational level which lead to changes in the purine metabolites in order to cope with threatening conditions. The aim of this study was to evaluate the response of the enzymes involved in the purine metabolism of human muscle cells to hypoxic conditions. Muscle cells in culture were exposed to hypoxia and the enzymatic activity of inosine monophosphate dehydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP) and hypoxanthine guanine phosphoribosyl transferase (HGPRT) as well as their transcript expression were quantified under normoxic and hypoxic conditions. Purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD+), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine diphosphate (GDP) and guanosine triphosphate (GTP)) concentrations were also quantified. Significant reduction of IMPDH activity and HX and IMP concentrations (p < 0.05) were observed after hypoxia, suggesting a decrease of de novo synthesis of purines. After hypoxia a global reduction of transcripts was observed, suggesting a reduction of the metabolic machinery of purine metabolism to new steady states that balance ATP demand and ATP supply pathways.

scholarly journals A Narrative Review on the Role of AMPK on De Novo Lipogenesis in Non-Alcoholic Fatty Liver Disease: Evidence from Human Studies

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1822
Author(s):  
Christian von Loeffelholz ◽  
Sina M. Coldewey ◽  
Andreas L. Birkenfeld
Keyword(s):  
Liver Disease ◽  
Fatty Liver ◽  
Fatty Liver Disease ◽  
Mammalian Cells ◽  
De Novo ◽  
Adenosine Monophosphate ◽  
De Novo Lipogenesis ◽  
Alcoholic Fatty Liver ◽  
Alcoholic Fatty Liver Disease

5′AMP-activated protein kinase (AMPK) is known as metabolic sensor in mammalian cells that becomes activated by an increasing adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio. The heterotrimeric AMPK protein comprises three subunits, each of which has multiple phosphorylation sites, playing an important role in the regulation of essential molecular pathways. By phosphorylation of downstream proteins and modulation of gene transcription AMPK functions as a master switch of energy homeostasis in tissues with high metabolic turnover, such as the liver, skeletal muscle, and adipose tissue. Regulation of AMPK under conditions of chronic caloric oversupply emerged as substantial research target to get deeper insight into the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Evidence supporting the role of AMPK in NAFLD is mainly derived from preclinical cell culture and animal studies. Dysbalanced de novo lipogenesis has been identified as one of the key processes in NAFLD pathogenesis. Thus, the scope of this review is to provide an integrative overview of evidence, in particular from clinical studies and human samples, on the role of AMPK in the regulation of primarily de novo lipogenesis in human NAFLD.

scholarly journals Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na+,K+-ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247377
Author(s):  
Vid Jan ◽  
Katarina Miš ◽  
Natasa Nikolic ◽  
Klemen Dolinar ◽  
Metka Petrič ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Human Skeletal Muscle ◽  
De Novo ◽  
Muscle Cells ◽  
Electrical Pulse ◽  
Electrical Pulse Stimulation ◽  
Human Skeletal Muscle Cells ◽  
Pulse Stimulation ◽  
Low Serum

Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7–10 days. A short-term co-culture (10–11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs.

A Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Overcomes Conventional Drug-Resistance in Multiple Mueloma Cells in the Bone Marrow Microenvironment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2468-2468
Author(s):  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Makoto Hamasaki ◽  
Raje Noopur ◽  
Kumar Shaji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
De Novo ◽  
Parp Cleavage ◽  
Inosine Monophosphate Dehydrogenase ◽  
Apoptotic Pathway ◽  
Inosine Monophosphate ◽  
Induced Apoptosis ◽  
Monophosphate Dehydrogenase

Abstract Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA) is a small molecule, selective, uncompetitive novel inhibitor directed against human IMPDH enzyme. IMPDH inhibitors have been demonstrated to induce growth arrest, and extensively investigated as immunosuppressants. Here we show that VX-944 inhibits growth of human multiple myeloma (MM) cell lines, including those resistant to conventional agents, via induction of apoptosis and S phase arrest in vitro. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs), do not protect against VX-944-induced MM cell growth inhibition. We next delineated the molecular mechanism of VX-944-induced MM cell death in the MM.1S human MM cell line. VX-944 induced apoptosis in MM.1S cells, confirmed by PARP cleavage as well as flow cytometric detection of the mitochondrial membrane protein 7A6 and TdT-mediated dUTP nick-end labelling (TUNEL) positive cells, without significant cleavage of caspases 3, 8 and 9. While the pan-caspase inhibitor z-VAD-fmk did not inhibit the VX-944-induced apoptosis and cell death suggesting that VX-944 triggers apoptosis in MM1.S cells primarily via caspase-independent pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin, melphalan and bortezomib, all of which activate caspases in MM cells and induce apoptosis, even in the presence of BMSCs. Taken together, our data demonstrate non-caspase-dependent apoptotic pathway triggered by VX-944 thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional and/or novel agents which trigger caspase activation. Our ongoing studies are delineating the mechanisms whereby VX-944 induces MM cell apoptosis.

scholarly journals Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi

2021 ◽  
Vol 22 (6) ◽  
pp. 2948
Author(s):  
Rene Buchet ◽  
Camille Tribes ◽  
Valentine Rouaix ◽  
Bastien Doumèche ◽  
Michele Fiore ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Atp Hydrolysis ◽  
Muscle Cells ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Extracellular Medium ◽  
Inorganic Pyrophosphate

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.

scholarly journals The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathione-β-synthase domain

2021 ◽  
Author(s):  
Zdeněk Knejzlík ◽  
Michal Doležal ◽  
Klára Herkommerová ◽  
Kamila Clarova ◽  
Martin Klima ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
De Novo ◽  
Gene Knockout ◽  
Guanosine Monophosphate ◽  
Inosine Monophosphate Dehydrogenase ◽  
Purine Salvage ◽  
Inosine Monophosphate ◽  
Salvage Pathways ◽  
Cbs Domains ◽  
Purine Salvage Pathways

Purine metabolism plays a pivotal role in bacterial life cycle, however, regulation of the de novo and purine salvage pathways have not been extensively detailed in mycobacteria. By gene knockout, biochemical and structural analyses, we identified Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) guaB1 gene product as a novel type of guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine salvage pathway and contains cystathione β-synthase (CBS) domains with atypical orientation in the octamer. CBS domains share a much larger interacting area with a conserved catalytic domain in comparison with the only known CBS containing protozoan GMPR and closely related inosine monophosphate dehydrogenase structures. Our results revealed essential effect of pH on allosteric regulation of Msm GMPR activity and oligomerization with adenine and guanosine nucleotides binding to CBS domains.Bioinformatic analysis indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

scholarly journals DRES-09. REGULATORY EFFECTS OF THE CILIARY GTPASE ARL13B ON PURINE METABOLISM IN GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi73-vi73
Author(s):  
Miranda Saathoff ◽  
Jack Shireman ◽  
Eunus Ali ◽  
Cheol Park ◽  
Issam Ben-Sahra ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Specific Activity ◽  
Purine Metabolism ◽  
P Value ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Double Strand Breaks ◽  
Purine Nucleotides ◽  
Regulatory Effects

Abstract Glioblastoma (GBM) is the most common form of adult primary brain cancer. Despite an aggressive treatment regimen – surgical resection, irradiation, and temozolomide (TMZ) chemotherapy – patients’ prognosis is still grim. TMZ acts by methylating purines, specifically at the O6 and N7 positions of guanine, to induce cytotoxic DNA double-strand breaks. We thus wanted to explore how purine metabolism may contribute to TMZ-resistance. In mammalian cells, purine nucleotides can be recycled by the salvage pathway or generated via de novo synthesis. The salvage pathway is energetically inexpensive relative to de novo thus, highly proliferative GBM cells preferentially utilize the salvage pathway. We have shown that salvage synthesis is reduced in response to TMZ (p-value=0.0021), hinting that the cells may utilize de novo to evade therapy induced alkylation of purines. Using immunoprecipitation-mass spectroscopy analysis, we found a novel interaction between the ciliary GTPase ARL13B and IMPDH2, the rate-limiting enzyme in de novo synthesis. We have shown that this interaction, occurring at the C-terminal domain of ARL13B, plays a significant role in the regulation of purine biosynthesis as abolishing it through ARL13B knockdown reduced flux through de novo (p-value< 0.0001) synthesis as measured by the specific activity of IMPDH2. Further, the lentiviral-mediated rescue of ARL13B brings IMPDH2 activity back to basal levels (p< 0.0001). Given its canonical function as a GTPase, we hypothesize that ARL13B acts as a novel regulator of de novo synthesis by sequestering GDP, allowing IMPDH2 to sense and respond to the cytosolic levels of guanine nucleotides. Without ARL13B the de novo pathway is halted, forcing the cells to rely on salvage to replenish nucleotide pools. Reliance on this pathway in the presence of TMZ causes cells to incorporate damaged nucleotides as a result of the drug’s alkylating action leading to the increased therapeutic efficacy of TMZ.

scholarly journals Compositional complexity of rods and rings

2018 ◽  
Vol 29 (19) ◽  
pp. 2303-2316 ◽  
Author(s):  
Cara R. Schiavon ◽  
Maxwell E. Griffin ◽  
Marinella Pirozzi ◽  
Raman Parashuraman ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Glucose Deprivation ◽  
Cell Types ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Nucleotide Synthesis ◽  
Inosine Monophosphate ◽  
Binding Partners ◽  
Rods And Rings

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.

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