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Oncogenesis Jul 2023MLK4, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, has been implicated in cancer progression. However, its role in lung adenocarcinoma...
MLK4, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, has been implicated in cancer progression. However, its role in lung adenocarcinoma has not been characterized. Here, we showed that MLK4 was overexpressed in a significant subset of lung adenocarcinoma, associated with a worse prognosis, and exerted an oncogenic function in vitro and in vivo. Bioinformatics analyses of clinical datasets identified phosphoenolpyruvate carboxykinase 1 (PCK1) as a novel target of MLK4. We validated that MLK4 regulated PCK1 expression at transcriptional level, by phosphorylating the transcription factor CREB, which in turn mediated PCK1 expression. We further demonstrated that PCK1 is an oncogenic factor in lung adenocarcinoma. Given the importance of PCK1 in the regulation of cellular metabolism, we next deciphered the metabolic effects of MLK4. Metabolic and mass spectrometry analyses showed that MLK4 knockdown led to significant reduction of glycolysis and decreased levels of glycolytic pathway metabolites including phosphoenolpyruvate and lactate. Finally, the promoter analysis of MLK4 unravelled a binding site of transcription factor KLF5, which in turn, positively regulated MLK4 expression in lung adenocarcinoma. In summary, we have revealed a KLF5-MLK4-PCK1 signalling pathway involved in lung tumorigenesis and established an unusual link between MAP3K signalling and cancer metabolism.
PubMed: 37407566
DOI: 10.1038/s41389-023-00478-y -
Proteins Sep 2023Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion...
Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PP -PfPEPCK), which instead of using a nucleotide, utilized PP to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PP -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PP -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PP -PfPEPCK being a Fe activated enzyme in contrast with the Mn activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.
Topics: Phosphoenolpyruvate; Propionibacterium freudenreichii; Phosphoenolpyruvate Carboxykinase (ATP); Oxaloacetic Acid; Guanosine Triphosphate; Nucleotides; Adenosine Triphosphate; Kinetics
PubMed: 37226637
DOI: 10.1002/prot.26513 -
Genes & Diseases Jul 2024Liver cancer stem cells were found to rely on glycolysis as the preferred metabolic program. Phosphoenolpyruvate carboxylase 1 (PCK1), a gluconeogenic metabolic enzyme,...
Liver cancer stem cells were found to rely on glycolysis as the preferred metabolic program. Phosphoenolpyruvate carboxylase 1 (PCK1), a gluconeogenic metabolic enzyme, is down-regulated in hepatocellular carcinoma and is closely related to poor prognosis. The oncogenesis and progression of tumors are closely related to cancer stem cells. It is not completely clear whether the PCK1 deficiency increases the stemness of hepatoma cells and promotes the oncogenesis of hepatocellular carcinoma. Herein, the results showed that PCK1 inhibited the self-renewal property of hepatoma cells, reduced the mRNA level of cancer stem cell markers, and inhibited tumorigenesis. Moreover, PCK1 increased the sensitivity of hepatocellular carcinoma cells to sorafenib. Furthermore, we found that PCK1 activated the Hippo pathway by enhancing the phosphorylation of YAP and inhibiting its nuclear translocation. Verteporfin reduced the stemness of hepatoma cells and promoted the pro-apoptotic effect of sorafenib. Thus, combined treatment with verteporfin and sorafenib may be a potential anti-tumor strategy in hepatocellular carcinoma.
PubMed: 38560500
DOI: 10.1016/j.gendis.2023.101114 -
Nutrients Sep 2023Dark tea has great potential in regulating glycolipid metabolism, and theabrownin (TB) is considered to be the characteristic and bioactive constituent of dark tea. This...
Dark tea has great potential in regulating glycolipid metabolism, and theabrownin (TB) is considered to be the characteristic and bioactive constituent of dark tea. This study evaluated the ability of TB1 (fermented for 7 days) and TB2 (fermented for 14 days) isolated from dark tea to reverse insulin resistance (IR) in HepG2 cells. The results indicated that TB significantly ameliorated oxidative stress by improving mitochondrial function. In addition, TB improved glycogen synthesis and glucose consumption, and inhibited gluconeogenesis and fatty acid synthesis, by regulating GSK3β (Glycogen synthase kinase 3β), G6Pase (Glucose-6-phosphatase), GCK (Glucokinase), PEPCK1 (Phosphoenolpyruvate carboxy kinase 1), SREBP-1C (sterol regulatory element-binding protein 1C), FASN (fatty acid synthase), and ACC (Acetyl-CoA carboxylase). Additionally, the results of Western blot and real-time PCR experiments demonstrated that TB modulated glucolipid metabolism through the IRS-1 (Insulin receptor substrate 1)/PI3K (phosphatidylinositol-3 kinase)/Akt (protein kinase B) signaling pathway. Treatment with the PI3K inhibitor demonstrated a favorable correlation between PI3K activation and TB action on glycolipid metabolism. Notably, we observed that TB2 had a greater effect on improving insulin resistance compared with TB1, which, due to its prolonged fermentation time, increased the degree of oxidative polymerization of TB.
PubMed: 37764646
DOI: 10.3390/nu15183862 -
Biochemical Pharmacology Dec 2023Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors....
Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors. Therefore, molecules that modulate CRYs have the potential to regulate gluconeogenesis. In this study, we discovered a new molecule called TW68 that interacts with the primary pockets of mammalian CRY1/2, leading to reduced ubiquitination levels and increased stability. In cell-based circadian rhythm assays using U2OS Bmal1-dLuc cells, TW68 extended the period length of the circadian rhythm. Additionally, TW68 decreased the transcriptional levels of two genes, Phosphoenolpyruvate carboxykinase 1 (PCK1) and Glucose-6-phosphatase (G6PC), which play crucial roles in glucose biosynthesis during glucagon-induced gluconeogenesis in HepG2 cells. Oral administration of TW68 in mice showed good tolerance, a good pharmacokinetic profile, and remarkable bioavailability. Finally, when administered to fasting diabetic animals from ob/ob and HFD-fed obese mice, TW68 reduced blood glucose levels by enhancing CRY stabilization and subsequently decreasing the transcriptional levels of Pck1 and G6pc. These findings collectively demonstrate the antidiabetic efficacy of TW68 in vivo, suggesting its therapeutic potential for controlling fasting glucose levels in the treatment of type 2 diabetes mellitus.
Topics: Animals; Mice; Cryptochromes; Blood Glucose; Mice, Obese; Glucagon; Diabetes Mellitus, Type 2; Circadian Rhythm; Circadian Clocks; Mammals; Fasting
PubMed: 37898388
DOI: 10.1016/j.bcp.2023.115896 -
Cell Proliferation Oct 2023
Topics: Humans; Liver Neoplasms; Mitochondria; Neoplasm Proteins; Cation Transport Proteins; Cyclic AMP Response Element-Binding Protein; Phosphoenolpyruvate Carboxykinase (GTP); Intracellular Signaling Peptides and Proteins
PubMed: 37435980
DOI: 10.1111/cpr.13527 -
Annals of Botany Nov 2023The relative contributions of C3 photosynthesis and crassulacean acid metabolism (CAM) during the earliest stages of development were investigated to assess how much...
BACKGROUND AND AIMS
The relative contributions of C3 photosynthesis and crassulacean acid metabolism (CAM) during the earliest stages of development were investigated to assess how much each might contribute to cactus pear (Opuntia ficus-indica) productivity.
METHODS
The developmental progression of C3 photosynthesis and CAM was assessed in seedlings and daughter cladodes of mature plants by titratable acidity, δ13C isotopic values and diel gas exchange measurements.
KEY RESULTS
Nocturnal acidification was observed in seedling cladodes and cotyledons at the earliest stages of development and became highly significant by 75 days of development. Seedling cotyledons showed mean δ13C values of -21.4 and -17.1 ‰ at 30 and 100 days of age, respectively. Seedling cladodes showed mean δ13C values of -19.4 and -14.5 ‰ at 30 and 100 days of age, respectively. These values are typical of CAM plants. Net CO2 assimilation was negative, then occurred in both the day and the night, with nighttime fixation becoming predominant once the primary cladode reached 5 cm in size. Emergent daughter cladodes growing on mature plants showed nocturnal titratable acidity at the earliest stages of development, which became significant when daughter cladodes were >2.5-5 cm in height. Emergent daughter cladodes showed mean δ13C values of -14.5 to -15.6 ‰, typical of CAM plants. CO2 assimilation studies revealed that net CO2 uptake was negative in daughter cladodes <12 cm in length, but then exhibited net positive CO2 assimilation in both the day and the night, with net nocturnal CO2 assimilation predominating once the daughter cladode grew larger.
CONCLUSIONS
Developing O. ficus-indica primary and daughter cladodes begin as respiring sink tissues that transition directly to performing CAM once net positive CO2 fixation is observed. Overall, these results demonstrate that CAM is the primary form of photosynthetic carbon assimilation for O. ficus-indica even at the earliest stages of seedling or daughter cladode development.
Topics: Crassulacean Acid Metabolism; Opuntia; Carbon Dioxide; Photosynthesis; Seedlings
PubMed: 37256773
DOI: 10.1093/aob/mcad070 -
Animals : An Open Access Journal From... Nov 2023Palmitic (C16:0), α-linolenic acid (C18:3n-3 ), and propionate regulate bovine pyruvate carboxylase () and phosphoenolpyruvate carboxykinase () expression in vitro. The...
Effects of an Hourly Bolus Postruminal Infusion of Flaxseed Oil or Palm Oil on Circulating Fatty Acid Concentrations and Hepatic Expression of Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Dairy Cattle.
Palmitic (C16:0), α-linolenic acid (C18:3n-3 ), and propionate regulate bovine pyruvate carboxylase () and phosphoenolpyruvate carboxykinase () expression in vitro. The objective of this experiment was to determine the impact of C16:0, C18:3n-3 , propionate, and acetate postruminal infusions on hepatic and expression. We hypothesized that circulating fatty acids alter hepatic and in lactating dairy cows. Acetate, propionate, palm oil, and flaxseed oil were supplied postruminally to lactating cows ( = 4) using two 4 × 4 Latin square studies. For Experiment 1, cows were infused on an hourly basis with either a bolus of propionate, acetate, or the combination of propionate and palm oil, or acetate and palm oil, and Experiment 2 was similar, but flaxseed oil replaced palm oil. Flaxseed infusions increased plasma concentration and the molar percent of C18:3n-3 and decreased C16:0 but did not affect or expression. Palm infusions did not affect blood metabolites or the hepatic expression of or . The lack of responses to short-chain fatty acid infusions and changes in circulating long-chain fatty acids in mature cattle are not suitable models to study the effects of α-linolenic acid and propionate on bovine and expression previously observed in vitro.
PubMed: 38003190
DOI: 10.3390/ani13223572 -
Annals of Botany Nov 2023Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the...
BACKGROUND AND AIMS
Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function.
METHODS
Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction.
KEY RESULTS
The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators.
CONCLUSIONS
The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.
Topics: Amino Acid Sequence; Poaceae; Phosphoenolpyruvate Carboxylase; Biological Evolution; Plants; Serine; Kinetics
PubMed: 37675505
DOI: 10.1093/aob/mcad116 -
International Journal of Biological... Sep 2023Diabetes is one of the major global public health problems. Our previous results found that oat β-D-glucan exhibited ameliorative effects on diabetic mice, but the...
Diabetes is one of the major global public health problems. Our previous results found that oat β-D-glucan exhibited ameliorative effects on diabetic mice, but the underlying mechanism is unclear. The present study indicates that oat β-D-glucan increased glycogen content, decreased glycogen synthase (GS) phosphorylation and increased hepatic glycogen synthase kinase 3β (GSK3β) phosphorylation for glycogen synthesis via PI3K/AKT/GSK3-mediated GS activation. Moreover, oat β-D-glucan inhibited gluconeogenesis through the PI3K/AKT/Foxo1-mediated phosphoenolpyruvate carboxykinase (PEPCK) decrease. In addition, oat β-D-glucan enhanced glucose catabolism through elevated protein levels of COQ9, UQCRC2, COXIV and ATP5F complexes involved in oxidative phosphorylation, as well as that of TFAM, a key regulator of mitochondrial gene expression. Importantly, our results showed that oat β-D-glucan maintained hepatic glucose balance via TLR4-mediated intracellular signal. After TLR4 blocking with anti-TLR4 antibody, oat β-D-glucan had almost no effect on high glucose-induced HepG2 cells. These data revealed that oat β-D-glucan maintains glucose balance by regulating the TLR4/PI3K/AKT signal pathway.
Topics: Mice; Animals; Diabetes Mellitus, Type 2; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Avena; Toll-Like Receptor 4; Glucans; Diabetes Mellitus, Experimental; Glycogen Synthase Kinase 3; Insulin Resistance; Glucose; Glycogen; Glycogen Synthase Kinase 3 beta
PubMed: 37516222
DOI: 10.1016/j.ijbiomac.2023.126039