Upregulation of circ-IGF1R increased therapeutic effect of hypoxia-pretreated ADSC-derived extracellular vesicle by regulating miR-503-5p/HK2/VEGFA axis.

Diabetes mellitus is a major cause of blindness and chronic ulcers in the working-age population worldwide. Wound healing is deeply dependent on neovascularization to restore blood flow. Former research has found that differentially expressed circular RNAs (circRNAs) are associated with hyperglycaemia-induced endothelial cell damage, and hypoxia-pretreated adipose-derived stem cells (ADSCs)-extracellular vesicle (HEV) transplants have a more therapeutic effect to enhance wound healing in diabetic mice by delivery circRNA. The current investigation employed high-throughput sequencing to identify circRNAs that are abnormally expressed between EV and HEV. The regulatory mechanism and predicted targets of one differentially expressed circRNA, circ-IGF1R, were investigated utilizing bioinformatics analyses, luciferase reporter assays, angiogenic differentiation assays, flow cytometric apoptosis analysis and RT-qPCR. Circ-IGF1R expression increased in HEV, and downregulation of circ-IGF1R suppressed and reversed the promotion effect of HEV on angiogenesis in ulcerated tissue. Bioinformatics analyses and luciferase reporter assays confirmed that miR-503-5p was the downstream target of circ-IGF1R, and inhibiting miR-503-5p restored the promotion effect of HEV on angiogenesis after circ-IGF1R silence. The study also found that miR-503-5p can interact with 3'-UTR of both HK2 and VEGFA. Overexpression of HK2 or VEGFA restored the promotion effect of HExo on angiogenesis after circ-IGF1R silence. Overexpression miR-503-5p or silence HK2/VEGFA reversed the protective effect of circ-IGF1R to MLMECs angiogenic differentiation. Overexpression of circ-IGF1R increased the protective effect of HEV on the promotion of wound healing in mice with diabetes. Circ-IGF1R promotes HIF-1α expression through miR-503-5p sponging. Our data demonstrate that circ-IGF1R overexpression EVs from ADSCs suppress high glucose-induced endothelial cell damage by regulating miR-503-5p/HK2/VEGFA axis.

AKT1 Promotes Tumorigenesis and Metastasis by Directly Phosphorylating Hexokinases.

The importance of protein kinase B (AKT) in tumorigenesis and development is well established, but its potential regulation of metabolic reprogramming via phosphorylation of the hexokinase (HK) isozymes remains unclear. There are two HK family members (HK1/2) and three AKT family members (AKT1/2/3), with varied distribution of AKTs exhibiting distinct functions in different tissues and cell types. Although AKT is known to phosphorylate HK2 at threonine 473, AKT-mediated phosphorylation of HK1 has not been reported. We examined direct binding and phosphorylation of HK1/2 by AKT1 and identified the phosphorylation modification sites using coimmunoprecipitation, glutathione pull-down, western blotting, and in vitro kinase assays. Regulation of HK activity through phosphorylation by AKT1 was also examined. Uptake of 2-[1,2-3H]-deoxyglucose and production of lactate were investigated to determine whether AKT1 regulates glucose metabolism by phosphorylating HK1/2. Functional assays, immunohistochemistry, and tumor experiments in mice were performed to investigate whether AKT1-mediated regulation of tumor development is dependent on its kinase activity and/or the involvement of HK1/2. AKT interacted with and phosphorylated HK1 and HK2. Serine phosphorylation significantly increased AKT kinase activity, thereby enhancing glycolysis. Mechanistically, the phosphorylation of HK1 at serine 178 (S178) by AKT significantly decreased the Km and enhanced the Vmax by interfering with the formation of HK1 dimers. Mutations in the AKT phosphorylation sites of HK1 or HK2 significantly abrogated the stimulatory characteristics of AKT on glycolysis, tumorigenesis, and cell migration, invasion, proliferation, and metastasis. HK1-S178 phosphorylation levels were significantly correlated with the occurrence and metastasis of different types of clinical tumors. We conclude that AKT not only regulates tumor glucose metabolism by directly phosphorylating HK1 and HK2, but also plays important roles in tumor progression, proliferation, and migration.

Gastric cancer mesenchymal stem cells promote tumor glycolysis and chemoresistance by regulating B7H3 in gastric cancer cells.

Despite surgical treatment combined with multidrug therapy having made some progress, chemotherapy resistance is the main cause of recurrence and death of gastric cancer (GC). Gastric cancer mesenchymal stem cells (GCMSCs) have been reported to be correlated with the limited efficacy of chemotherapy in GC, but the mechanism of GCMSCs regulating GC resistance needs to be further studied. The gene set enrichment analysis (GSEA) was performed to explore the glycolysis-related pathways heterogeneity across different cell subpopulations. Glucose uptake and lactate production assays were used to evaluate the importance of B7H3 expression in GCMSCs-treated GC cells. The therapeutic efficacy of oxaliplatin (OXA) and paclitaxel (PTX) was determined using CCK-8 and colony formation assays. Signaling pathways altered by GCMSCs-CM were revealed by immunoblotting. The expression of TNF-α in GCMSCs and bone marrow mesenchymal stem cells (BMMSCs) was detected by western blot analysis and qPCR. Our results showed that the OXA and PTX resistance of GC cells were significantly enhanced in the GCMSCs-CM treated GC cells. Acquired OXA and PTX resistance was characterized by increased cell viability for OXA and PTX, the formation of cell colonies, and decreased levels of cell apoptosis, which were accompanied by reduced levels of cleaved caspase-3 and Bax expression, and increased levels of Bcl-2, HK2, MDR1, and B7H3 expression. Blocking TNF-α in GCMSCs-CM, B7H3 knockdown or the use of 2-DG, a key enzyme inhibitor of glycolysis in GC cells suppressed the OXA and PTX resistance of GC cells that had been treated with GCMSCs-CM. This study shows that GCMSCs-CM derived TNF-α could upregulate the expression of B7H3 of GC cells to promote tumor chemoresistance. Our results provide a new basis for the treatment of GC.

Hexokinase 2 nonmetabolic function-mediated phosphorylation of IκBα enhances pancreatic ductal adenocarcinoma progression.

Aberrant signaling in tumor cells induces nonmetabolic functions of some metabolic enzymes in many cellular activities. As a key glycolytic enzyme, the nonmetabolic function of hexokinase 2 (HK2) plays a role in tumor immune evasion. However, whether HK2, dependent of its nonmetabolic activity, plays a role in human pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains unclear. Here, we demonstrated that HK2 acts as a protein kinase and phosphorylates IκBα at T291 in PDAC cells, activating NF-κB, which enters the nucleus and promotes the expression of downstream targets under hypoxia. HK2 nonmetabolic activity-promoted activation of NF-κB promotes the proliferation, migration, and invasion of PDAC cells. These findings provide new insights into the multifaceted roles of HK2 in tumor development and underscore the potential of targeting HK2 protein kinase activity for PDAC treatment.

AST-120 alleviates renal ischemia-reperfusion injury by inhibiting HK2-mediated glycolysis.

OBJECTIVE: Renal ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), which is associated with high incidence and mortality. AST-120 is an oral carbonaceous adsorbent that can alleviate kidney damage. This study aimed to explore the effects of AST-120 on renal IRI and the molecular mechanism. METHODS: A renal IRI mouse model was established and administrated AST-120, and differentially expressed genes were screened using RNA sequencing. Renal function and pathology were analyzed in mice. Hypoxia/reoxygenation (H/R) cell model was generated, and glycolysis was evaluated by detecting lactate levels and Seahorse analysis. Histone lactylation was analyzed by western blotting, and its relationship with hexokinase 2 (HK2) was assessed using chromatin immunoprecipitation. RESULTS: The results showed that HK2 expression was increased after IRI, and AST-120 decreased HK2 expression. Knockout of HK2 attenuated renal IRI and inhibits glycolysis. AST-120 inhibited renal IRI in the presence of HK2 rather than HK2 absence. In proximal tubular cells, knockdown of HK2 suppressed glycolysis and H3K18 lactylation caused by H/R. H3K18 lactylation was enriched in HK2 promoter and upregulated HK2 levels. Rescue experiments revealed that lactate reversed IRI that suppressed by HK2 knockdown. CONCLUSIONS: In conclusion, AST-120 alleviates renal IRI via suppressing HK2-mediated glycolysis, which suppresses H3K18 lactylation and further reduces HK2 levels. This study proposes a novel mechanism by which AST-120 alleviates IRI.

Gemigliptin mitigates TGF-ß-induced renal fibrosis through FGF21-mediated inhibition of the TGF-ß/Smad3 signaling pathway.

Fibroblast growth factor 21 (FGF21), a well-known regulator of metabolic disorders, exhibits the potential to prevent renal fibrosis by negatively regulating the transforming growth factor ß (TGF-ß)/Smad3 signaling pathway. Gemigliptin and other dipeptidyl peptidase-4 inhibitors are frequently used for the management of patients with type 2 diabetes. However, the protective effect of gemigliptin against renal fibrosis, particularly its potential to upregulate the expression of FGF21, remains incompletely understood. This study assessed the renoprotective effects of gemigliptin against TGF-ß-induced renal fibrosis by enhancing the expression of FGF21 in the cultured human proximal tubular epithelial cell line HK-2. Treatment with FGF21 effectively prevented TGF-ß-induced renal fibrosis by attenuating the TGF-ß/Smad3 signaling pathway. Similarly, gemigliptin exhibited protective effects against TGF-ß-induced renal fibrosis by mitigating TGF-ß/Smad3 signaling through the upregulation of FGF21 expression. However, the protective effects of gemigliptin were blocked when FGF21 expression was knocked down in TGF-ß-treated HK-2 cells. These results indicate that gemegliptin has the potential to exhibit protective effects against TGF-ß-induced renal fibrosis by elevating FGF21 expression levels in cultured human proximal tubular epithelial cells.

CCT6A facilitates lung adenocarcinoma progression and glycolysis via STAT1/HK2 axis.

BACKGROUND: Chaperonin Containing TCP1 Subunit 6 A (CCT6A) is a prominent protein involved in the folding and stabilization of newly synthesized proteins. However, its roles and underlying mechanisms in lung adenocarcinoma (LUAD), one of the most aggressive cancers, remain elusive. METHODS: Our study utilized in vitro cell phenotype experiments to assess CCT6A's impact on the proliferation and invasion capabilities of LUAD cell lines. To delve into CCT6A's intrinsic mechanisms affecting glycolysis and proliferation in lung adenocarcinoma, we employed transcriptomic sequencing and liquid chromatography-mass spectrometry analysis. Co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) assays were also conducted to substantiate the mechanism. RESULTS: CCT6A was found to be significantly overexpressed in LUAD and associated with a poorer prognosis. The silencing of CCT6A inhibited the proliferation and migration of LUAD cells and elevated apoptosis rates. Mechanistically, CCT6A interacted with STAT1 protein, forming a complex that enhances the stability of STAT1 by protecting it from ubiquitin-mediated degradation. This, in turn, facilitated the transcription of hexokinase 2 (HK2), a critical enzyme in aerobic glycolysis, thereby stimulating LUAD's aerobic glycolysis and progression. CONCLUSION: Our findings reveal that the CCT6A/STAT1/HK2 axis orchestrated a reprogramming of glucose metabolism and thus promoted LUAD progression. These insights position CCT6A as a promising candidate for therapeutic intervention in LUAD treatment.

PDLIM1 interacts with HK2 to promote gastric cancer progression through enhancing the Warburg effect via Wnt/ß-catenin signaling.

PDZ and LIM domain protein 1 (PDLIM1) is a cytoskeletal protein and is associated with the malignant pathological features of several tumors. However, the prognostic value of PDLIM1 and the molecular mechanisms by which it is involved in the metabolism and progression in gastric cancer (GC) are still unclear. The GEPIA database was used to predict the expression and prognosis of PDLIM1 in GC. qRT-PCR and western blot assays were applied to detect the mRNA and protein expression in GC tissues and cells. Loss- and gain-of-function experiments were performed to evaluate the biological role of PDLIM1 in GC cells. The Warburg effect was detected by a battery of glycolytic indicators. The interaction of PDLIM1 and hexokinase 2 (HK2) was determined by a co-immunoprecipitation assay. Furthermore, the modulatory effects of PDLIM1 and HK2 on Wnt/ß-catenin signaling were assessed. The results showed that PDLIM1 expression was upregulated in GC tissues and cells and was associated with a poor prognosis for GC patients. PDLIM1 inhibition reduced GC cell proliferation, migration and invasion and promoted cell apoptosis. In the glucose deprivation (GLU-D) condition, the PDLIM1 level was reduced and PDLIM1 overexpression led to an increase in glycolysis. Besides, mechanistic investigation showed that PDLIM1 interacted with HK2 to mediate biological behaviors and the glycolysis of GC through Wnt/ß-catenin signaling under glucose deprivation. In conclusion, PDLIM1 interacts with HK2 to promote gastric cancer progression by enhancing the Warburg effect via Wnt/ß-catenin signaling.

LncARSR promotes glioma tumor growth by mediating glycolysis through the STAT3/HK2 axis.

BACKGROUND: Glioma represents the predominant malignant brain tumor. This investigation endeavors to elucidate the impact and prospective mechanisms of glycolysis-related lncARSR on glioma. METHODS: LncARSR level was assessed in normal glial cells and glioma cells. Cell proliferation, migration, and invasion measurements were conducted through CCK-8, wound healing, and transwell assay. Flow cytometry was utilized to measure cell apoptosis and cell cycle. Biochemical assay kits and immunoblotting were employed to measure the content of glycolysis-related indicators and protein expression, respectively. We analyzed the impact of both lncARSR knockdown and overexpression of the Signal Transducer and Activator of Transcription 3 (STAT3) on Hexokinase 2 (HK2) using dual luciferase reporter assays and Chromatin Immunoprecipitation (ChIP) experiments. Further assessment of the impact of lncARSR on glioma progression was conducted through animal experiments. RESULTS: LncARSR was expressed at elevated levels in glioma cells compared to normal glial cells. Overexpressing lncARSR enhanced proliferation, migration, invasion, and G2/M phase arrest in glioma cells and also increased glucose, lactate, ATP production, as well as the expression of HK2, PFK1, PKM2, GLUT1, and LDHA. STAT3 binding to the HK2 gene promoter was weakened following the knockdown of lncARSR. Upregulation of STAT3 reversed the suppressed functions of knocking down lncARSR on cell proliferation, migration, invasion, G2/M phase arrest, and glycolysis and counteracted its promotional effect on cell apoptosis. In vivo, knocking down lncARSR inhibits glioma growth and ki67 and PCNA expression. CONCLUSION: LncARSR promotes the development of glioma by enhancing glycolysis through the STAT3-HK2 axis.

ALKBH5 facilitates the progression of infantile hemangioma by increasing FOXF1 expression in a m6A-YTHDF2 dependent manner to activate HK-2 signaling.

Alkylation repair homolog protein 5 (ALKBH5) is reported to participate in infantile hemangioma (IH) progression. However, the underlying mechanism of ALKBH5 in IH remains unclear. Using qRT-PCR and Western blotting, ALKBH5, forkhead box F1 (FOXF1) and hexokinase 2 (HK-2) expressions in IH tissues and IH-derived endothelial cells XPTS-1 were assessed. The Me-RIP assay was used to analyze FOXF1 m6A level. CCK8, colony formation, flow cytometry and transwell assays were employed to determine IH cell viability, proliferation, apoptosis, migration and invasion. The interactions between YTH (YT521-B homology) domain 2 (YTHDF2), FOXF1 and HK-2 were analyzed by RIP, dual luciferase reporter gene assay and/or ChIP assay. The in vivo IH growth was evaluated in immunocompromised mice. FOXF1 was overexpressed in IH tissues, and its silencing inhibited IH cell proliferation, migration and invasion whereas promoting cell apoptosis in vitro. ALKBH5 upregulation facilitated FOXF1 mRNA stability and expression in IH cells in a m6A-YTHDF2-dependent manner. FOXF1 downregulation reversed the impact of ALKBH5 upregulation on IH cellular phenotypes. It also turned out that FOXF1 positively regulated HK-2 expression in IH cells through interacting with the HK-2 promoter. HK-2 upregulation abolished FOXF1 knockdown's inhibition on IH cell aggressive behaviors. ALKBH5 or FOXF1 silencing suppressed IH tumor development via HK-2 signaling in immunocompromised mice. ALKBH5 promoted FOXF1 expression m6A-YTHDF2 dependently, which in turn elevated HK-2 expression, thereby accelerating IH development.