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American Journal of Physiology. Cell... Nov 2023Glomerular angiogenesis is a characteristic feature of diabetic nephropathy (DN). Enhanced glycolysis plays a crucial role in angiogenesis. The present study was...
Glomerular angiogenesis is a characteristic feature of diabetic nephropathy (DN). Enhanced glycolysis plays a crucial role in angiogenesis. The present study was designed to investigate the role of glycolysis in glomerular endothelial cells (GECs) in a mouse model of DN. Mouse renal cortex and isolated glomerular cells were collected for single-cell and RNA sequencing. Cultured GECs were exposed to high glucose in the presence (proangiogenic) and absence of a vascular sprouting regimen. MicroRNA-590-3p was delivered by lipofectamine in vivo and in vitro. In the present study, a subgroup of GECs with proangiogenic features was identified in diabetic kidneys by using sequencing analyses. In cultured proangiogenic GECs, high glucose increased glycolysis and phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) protein expression, which were inhibited by overexpressing miRNA-590-3p. Mimics of miRNA-590-3p also increased receptor for sphingosine 1-phosphate (S1pR1) expression, an angiogenesis regulator, in proangiogenic GECs challenged with high glucose. Inhibition of PFKFB3 by pharmacological and genetic approaches upregulated S1pR1 protein in vitro. Mimics of miRNA-590-3p significantly reduced migration and angiogenic potential in proangiogenic GECs challenged with high glucose. Ten-week-old type 2 diabetic mice had elevated urinary albumin levels, reduced renal cortex miRNA-590-3p expression, and disarrangement of glomerular endothelial cell fenestration. Overexpressing miRNA-590-3p via perirenal adipose tissue injection restored endothelial cell fenestration and reduced urinary albumin levels in diabetic mice. Therefore, the present study identifies a subgroup of GECs with proangiogenic features in mice with DN. Local administration of miRNA-590-3p mimics reduces glycolytic rate and upregulates S1pR1 protein expression in proangiogenic GECs. The protective effects of miRNA-590-3p provide therapeutic potential in DN treatment. Proangiogenetic glomerular endothelial cells (GECs) are activated in diabetic nephropathy. High glucose upregulates glycolytic enzyme phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) in proangiogenetic cells. PFKFB3 protects the glomerular filtration barrier by targeting endothelial S1pR1. MiRNA-590-3p restores endothelial cell function and mitigates diabetic nephropathy.
Topics: Mice; Animals; Diabetic Nephropathies; Endothelial Cells; Fructose-Bisphosphatase; Phosphofructokinases; Diabetes Mellitus, Experimental; Sphingosine-1-Phosphate Receptors; Phosphofructokinase-1; Glucose; MicroRNAs; Albumins; Glycolysis
PubMed: 37781737
DOI: 10.1152/ajpcell.00261.2023 -
BioRxiv : the Preprint Server For... Sep 2023Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and...
Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition and respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.
PubMed: 37781627
DOI: 10.1101/2023.09.19.558525 -
Scientific Reports Sep 2023Colon adenocarcinoma (COAD) is a common malignant tumor, and the role of the protein PFKFB4 in glycolysis and pentose phosphate pathways is crucial. Researchers...
Colon adenocarcinoma (COAD) is a common malignant tumor, and the role of the protein PFKFB4 in glycolysis and pentose phosphate pathways is crucial. Researchers investigated the clinical significance of PFKFB4 in COAD by studying its expression in 79 tissue samples using immunohistochemistry. We found that PFKFB4 expression was significantly higher in COAD patients, particularly in the sigmoid colon. Interestingly, high PFKFB4 expression was associated with both improved overall survival (OS) and worse progression-free survival (PPS) in COAD patients. Further analysis revealed that genes associated with PFKFB4 were linked to various metabolic pathways, including amino acid biosynthesis, glycolysis, gluconeogenesis, glucose metabolism, and inflammatory response. PFKFB4 expression also showed correlations with the infiltration of different immune cell types in COAD patients, such as CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), macrophages, neutrophils, dendritic cells, active mast cells, and resting NK cells. Overall, the relationship between PFKFB4 expression and the prognosis of COAD is complex and diverse, possibly playing different roles at different stages of the disease. Moreover, its mechanism might involve interactions with various metabolic pathways and immune infiltration in the tumor microenvironment. These findings provide valuable insights into the potential role of PFKFB4 as a biomarker or therapeutic target in COAD.
Topics: Humans; Colonic Neoplasms; Adenocarcinoma; Colon, Sigmoid; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Prognosis; Tumor Microenvironment; Phosphofructokinase-2
PubMed: 37770581
DOI: 10.1038/s41598-023-43619-4 -
International Journal of Molecular... Sep 2023Postharvest abnormal chilling injury (CI) behavior in papaya ( L.) fruit is a rare phenomenon that may be associated with respiratory metabolism. This study thus aimed...
Postharvest abnormal chilling injury (CI) behavior in papaya ( L.) fruit is a rare phenomenon that may be associated with respiratory metabolism. This study thus aimed to investigate the impacts of storage temperatures (1 and 6 °C) on the respiratory metabolism of postharvest papaya and its impact on CI development. Results demonstrated that 1 °C storage reduced the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), citrate synthase (CS), and α-ketoglutarate dehydrogenase (α-KGDH) and regulated the expression of corresponding enzymes in the Embden-Meyerhof-Parnas (EMP) pathway and tricarboxylic acid (TCA) cycle compared with 6 °C storage, resulting in a lower respiration rate of the EMP-TCA pathway and mitigating the development of CI. Meanwhile, lower contents of nicotinamide adenine dinucleotide (hydrogen) (NAD(H)) were observed in papaya fruit stored at 1 °C. Notably, papaya fruit stored at 1 °C maintained higher activity and transcriptional levels of SDH and IDH during the whole storage period. These findings suggest that 1 °C storage reduced the respiration rate of the EMP-TCA pathway by reducing the expression level and activity of related enzymes, which is conducive to the reduction of respiration substrate consumption and finally alleviating the occurrence of CI.
PubMed: 37762201
DOI: 10.3390/ijms241813898 -
Synthetic and Systems Biotechnology Sep 2023Owing to the feature of strong α-glucosidase inhibitory activity, 1-deoxynojirimycin (1-DNJ) has broad application prospects in areas of functional food, biomedicine,...
Owing to the feature of strong α-glucosidase inhibitory activity, 1-deoxynojirimycin (1-DNJ) has broad application prospects in areas of functional food, biomedicine, etc., and this research wants to construct an efficient strain for 1-DNJ production, basing on HZ-12. Firstly, using the temperature-sensitive shuttle plasmid T2 (2)-Ori, gene in phosphotransferase system (PTS) was weakened by homologous recombination, and non-PTS pathway was strengthened by deleting its repressor gene , and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold, reached 110.72 mg/L. Then, to increase precursor fructose-6-phosphate (F-6-P) supply, phosphofructokinase was weaken, fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement, moreover, regulator gene was deleted, 1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold. Subsequently, promoter of 1-DNJ synthetase cluster was optimized, as well as 5'-UTRs of downstream genes in synthetase cluster, and 1-DNJ produced by the final strain reached 478.62 mg/L. Last but not the least, 1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter, which was the highest yield of 1-DNJ reported to date. Taken together, our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis, this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.
PubMed: 37692204
DOI: 10.1016/j.synbio.2023.05.002 -
Cell & Bioscience Sep 2023The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of...
BACKGROUND
The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism.
RESULTS
Based on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement.
CONCLUSION
Our study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction.
PubMed: 37684702
DOI: 10.1186/s13578-023-01114-3 -
Cell Death & Disease Sep 2023Intracellular Ca signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca is intracellular BAPTA (BAPTA),...
Intracellular Ca signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca is intracellular BAPTA (BAPTA), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTA enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTA enhances cell death in B-cell cancers. In this study, we discovered that BAPTA alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTA provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTA diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTA. Notably, these effects were also induced by a BAPTA analog with low affinity for Ca. Consequently, our findings uncover PFKFB3 inhibition as an Ca-independent mechanism through which BAPTA impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTA are not necessarily related to Ca signaling. Our data support the need for a reassessment of the role of Ca in cellular processes when findings were based on the use of BAPTA.
Topics: Myeloid Cell Leukemia Sequence 1 Protein; Phosphoric Monoester Hydrolases; Egtazic Acid; Phosphofructokinase-2; Neoplasms
PubMed: 37684238
DOI: 10.1038/s41419-023-06120-4 -
BMC Medicine Sep 2023T2D is of high prevalence in the middle east and thus studying its mechanisms is of a significant importance. Using 1026 Qatar BioBank samples, epigenetics, whole genome...
BACKGROUND
T2D is of high prevalence in the middle east and thus studying its mechanisms is of a significant importance. Using 1026 Qatar BioBank samples, epigenetics, whole genome sequencing and metabolomics were combined to further elucidate the biological mechanisms of T2D in a population with a high prevalence of T2D.
METHODS
An epigenome-wide association study (EWAS) with T2D was performed using the Infinium 850K EPIC array, followed by whole genome-wide sequencing SNP-CpG association analysis (> 5.5 million SNPs) and a methylome-metabolome (CpG-metabolite) analysis of the identified T2D sites.
RESULTS
A total of 66 T2D-CpG associations were identified, including 63 novel sites in pathways of fructose and mannose metabolism, insulin signaling, galactose, starch and sucrose metabolism, and carbohydrate absorption and digestion. Whole genome SNP associations with the 66 CpGs resulted in 688 significant CpG-SNP associations comprising 22 unique CpGs (33% of the 66 CPGs) and included 181 novel pairs or pairs in novel loci. Fourteen of the loci overlapped published GWAS loci for diabetes related traits and were used to identify causal associations of HK1 and PFKFB2 with HbA1c. Methylome-metabolome analysis identified 66 significant CpG-metabolite pairs among which 61 pairs were novel. Using the identified methylome-metabolome associations, methylation QTLs, and metabolic networks, a multi-omics network was constructed which suggested a number of metabolic mechanisms underlying T2D methylated genes. 1-palmitoyl-2-oleoyl-GPE (16:0/18:1) - a triglyceride-associated metabolite, shared a common network with 13 methylated CpGs, including TXNIP, PFKFB2, OCIAD1, and BLCAP. Mannonate - a food component/plant shared a common network with 6 methylated genes, including TXNIP, BLCAP, THBS4 and PEF1, pointing to a common possible cause of methylation in those genes. A subnetwork with alanine, glutamine, urea cycle (citrulline, arginine), and 1-carboxyethylvaline linked to PFKFB2 and TXNIP revealed associations with kidney function, hypertension and triglyceride metabolism. The pathway containing STYXL1-POR was associated with a sphingosine-ceramides subnetwork associated with HDL-C and LDL-C and point to steroid perturbations in T2D.
CONCLUSIONS
This study revealed several novel methylated genes in T2D, with their genomic variants and associated metabolic pathways with several implications for future clinical use of multi-omics associations in disease and for studying therapeutic targets.
Topics: Humans; Diabetes Mellitus, Type 2; Epigenome; Metabolome; Multiomics; Phosphofructokinase-2; Middle Eastern People
PubMed: 37679740
DOI: 10.1186/s12916-023-03027-x -
Clinical and Translational Medicine Sep 2023The imbalance between osteoblasts and osteoclasts may lead to osteoporosis. Osteoblasts and osteoclasts have different energy requirements, with aerobic glycolysis being...
BACKGROUND
The imbalance between osteoblasts and osteoclasts may lead to osteoporosis. Osteoblasts and osteoclasts have different energy requirements, with aerobic glycolysis being the prominent metabolic feature of osteoblasts, while osteoclast differentiation and fusion are driven by oxidative phosphorylation.
METHODS
By polymerase chain reaction as well as Western blotting, we assayed coactivator-associated arginine methyltransferase 1 (CARM1) expression in bone tissue, the mouse precranial osteoblast cell line MC3T3-E1 and the mouse monocyte macrophage leukaemia cell line RAW264.7, and expression of related genes during osteogenic differentiation and osteoclast differentiation. Using gene overexpression (lentivirus) and loss-of-function approach (CRISPR/Cas9-mediated knockout) in vitro, we examined whether CARM1 regulates osteogenic differentiation and osteoblast differentiation by metabolic regulation. Transcriptomic assays and metabolomic assays were used to find the mechanism of action of CARM1. Furthermore, in vitro methylation assays were applied to clarify the arginine methylation site of PPP1CA by CARM1.
RESULTS
We discovered that CARM1 reprogrammed glucose metabolism in osteoblasts and osteoclasts from oxidative phosphorylation to aerobic glycolysis, thereby promoting osteogenic differentiation and inhibiting osteoclastic differentiation. In vivo experiments revealed that CARM1 significantly decreased bone loss in osteoporosis model mice. Mechanistically, CARM1 methylated R23 of PPP1CA, affected the dephosphorylation of AKT-T450 and AMPK-T172, and increased the activities of phosphofructokinase-1 and pructose-2,6-biphosphatase3, causing an up-regulation of glycolytic flux. At the same time, as a transcriptional coactivator, CARM1 regulated the expression of pyruvate dehydrogenase kinase 3, which resulted in the inhibition of pyruvate dehydrogenase activity and inhibition of the tricarboxylic acid cycle, leading to a subsequent decrease in the flux of oxidative phosphorylation.
CONCLUSIONS
These findings reveal for the first time the mechanism by which CARM1 affects both osteogenesis and osteoclast differentiation through metabolic regulation, which may represent a new feasible treatment strategy for osteoporosis.
Topics: Animals; Mice; Osteogenesis; Methylation; Cell Differentiation; Arginine; Glucose
PubMed: 37649137
DOI: 10.1002/ctm2.1369 -
Animal Nutrition (Zhongguo Xu Mu Shou... Sep 2023Citrate is an essential substrate for energy metabolism that plays critical roles in regulating glucose and lipid metabolic homeostasis. However, the action of citrate...
Citrate is an essential substrate for energy metabolism that plays critical roles in regulating glucose and lipid metabolic homeostasis. However, the action of citrate in regulating nutrient metabolism in fish remains poorly understood. Here, we investigated the effects of dietary sodium citrate on growth performance and systematic energy metabolism in juvenile Nile tilapia (). A total of 270 Nile tilapia (2.81 ± 0.01 g) were randomly divided into three groups (3 replicates per group, 30 fish per replicate) and fed with control diet (35% protein and 6% lipid), 2% and 4% sodium citrate diets, respectively, for 8 weeks. The results showed that sodium citrate exhibited no effect on growth performance ( > 0.05). The whole-body crude protein, serum triglyceride and hepatic glycogen contents were significantly increased in the 4% sodium citrate group ( 0.05), but not in the 2% sodium citrate group ( > 0.05). The 4% sodium citrate treatment significantly increased the serum glucose and insulin levels at the end of feeding trial and also in the glucose tolerance test ( 0.05). The 4% sodium citrate significantly enhanced the hepatic phosphofructokinase activity and inhibited the expression of pyruvate dehydrogenase kinase isozyme 2 and phosphor-pyruvate dehydrogenase E1 component subunit alpha proteins ( 0.05). Additionally, the 4% sodium citrate significantly increased hepatic triglyceride and acetyl-CoA levels, while the expressions of carnitine palmitoyl transferase 1a protein were significantly down-regulated by the 4% sodium citrate ( 0.05). Besides, the 4% sodium citrate induced crude protein deposition in muscle by activating mTOR signaling and inhibiting AMPK signaling ( 0.05). Furthermore, the 4% sodium citrate significantly suppressed serum aspartate aminotransferase and alanine aminotransferase activities, along with the lowered expression of pro-inflammatory genes, such as , and ( 0.05). Although the 4% sodium citrate significantly increased phosphor-nuclear factor-kB p65 protein expression ( 0.05), no significant tissue damage or inflammation occurred. Taken together, dietary supplementation of sodium citrate could exhibit a double-edged effect in Nile tilapia, with the positive aspect in promoting nutrient deposition and the negative aspect in causing hyperglycemia and insulin resistance.
PubMed: 37635932
DOI: 10.1016/j.aninu.2023.06.005