Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino ...acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.
In this study, approximately half the patients with red-cell pyruvate kinase deficiency who were treated with mitapivat had an improvement in their hemoglobin level and decreased hemolysis that was ...sustained for nearly 3 years. Patients who had missense mutations that allowed for synthesis of a hypofunctioning enzyme were most likely to have a response.
The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. ...Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2−/− mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.
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•Pyruvate kinase activators (PKa) amplify insulin release in preclinical T2DM models•PKa amplify insulin release via the phosphoenolpyruvate cycle in vivo•PKa acutely short-circuit gluconeogenesis and accelerate red blood cell glycolysis•PKa improve insulin sensitivity, steatosis, and dyslipidemia in obese rats
Abulizi et al. show that small molecule activation of mitochondrial PEPCK (pck2)-dependent phosphoenolpyruvate (PEP) cycling amplifies glucose-stimulated insulin secretion without evidence of islet injury and improves insulin sensitivity in vivo. These are accompanied by decreased gluconeogenesis, increased red blood cell glycolysis, and reduced hepatic steatosis in preclinical rodent models of diabetes.
Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red cell (RBC) glycolysis, causing changes in metabolism including a deficiency in ATP. This affects red cell homeostasis, ...promoting premature removal of RBCs from the circulation. In this study we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBCs and erythroid precursors from PK-deficient patients. In 15 patients ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patient cells after 24 hours (mean increase 1.8-fold, range 1.2-3.4). ATP levels increased (mean increase 1.5-fold, range 1.0-2.2) similar to control cells (mean increase 1.6-fold, range, 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBCs. Ex vivo treatment with AG-348 increased residual activity 1.4 to >10-fold than residual activity of vehicle-treated samples. Protein analyses suggests that a sufficient level of PK protein is required for cells to respond to AG-348 treatment ex-vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG-348 effectively upregulates PK enzymatic activity and increases stability in PK-deficient RBCs over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).
The spindle checkpoint ensures accurate chromosome segregation by delaying cell-cycle progression until all sister kinetochores capture microtubules from opposite poles and come under tension (for ...reviews, see refs 1, 2). Although the checkpoint is activated by either the lack of kinetochore-microtubule attachments or defects in the tension exerted by microtubule-generated forces, it is not clear whether these signals are linked. We investigated the connection between tension and attachment by studying the conserved budding yeast Ipl1Aurora protein kinase that is required for checkpoint activation in the absence of tension but not attachment. Here, we show that spindle-checkpoint activation in kinetochore mutants that seem to have unattached kinetochores depends on Ipl1 activity. When Ipl1 function was impaired in these kinetochore mutants, the attachments were restored and the checkpoint was turned off. These data indicate that Ipl1 activates the checkpoint in response to tension defects by creating unattached kinetochores. Moreover, although the Dam1 kinetochore complex has been implicated as a key downstream target, we found the existence of unidentified Ipl1 sites on Dam1 or additional important substrates that regulate both microtuble detachment and the checkpoint.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ...ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 alleleâalcohol interaction may be an even greater human public health hazard than previously appreciated.
About 40% of East Asians and over 500 million people worldwide carry a specific polymorphism, ALDH2*2, and exhibit âAsian flushâ after alcohol drinking. We generated a mouse strain with this engineered polymorphism and demonstrated its resemblance to human carriers in terms of defective alcohol metabolism. With this model, we show that murine ALDH2*2 increases ALDH2 protein turnover and promotes chemical-induced liver tumor development. Importantly, ALDH2 is unstable in ALDH2*2 human liver samples and is significantly down-regulated in human liver tumors. Data from our mouse and clinical studies suggest that ALDH2 is a liver tumor suppressor and the ALDH2*2 polymorphism is a risk factor for liver cancer.
Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the ...neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.
Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with ...insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.
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•High-fat diet induces, whereas PKCε-knockdown rescues hepatic PKL S113 phosphorylation•PKL phosphorylation at S113 by CDKs or S12 by PKA increases gluconeogenesis•Neither S12 or S113 phosphorylation alters PKL kinetics or structure•CDK1-mediated phosphorylation of PKL S113 traps PKL in the nucleus
Gassaway et al. identify a diet-induced, cyclin-dependent kinase-regulated phosphorylation site at S113 on pyruvate kinase. Although they determine that neither phosphorylation of this site nor the canonical PKA-regulated S12 site directly impacts enzyme kinetics, they demonstrate that S113 phosphorylation alters pyruvate kinase subcellular localization and influences glucose production.
The process of gene transcription requires the recruitment of a hypophosphorylated form of RNA polymerase II (Pol II) to a gene promoter. The TFIIH-associated kinase Cdk7/Kin28 hyperphosphorylates ...the promoter-bound polymerase; this event is thought to play a crucial role in transcription initiation and promoter clearance. Studies using temperature-sensitive mutants of Kin28 have provided the most compelling evidence for an essential role of its kinase activity in global mRNA synthesis. In contrast, using a small molecule inhibitor that specifically inhibits Kin28 in vivo, we find that the kinase activity is not essential for global transcription. Unlike the temperature-sensitive alleles, the small-molecule inhibitor does not perturb protein-protein interactions nor does it provoke the disassociation of TFIIH from gene promoters. These results lead us to conclude that other functions of TFIIH, rather than the kinase activity, are critical for global gene transcription.
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