BACKGROUND: Intracerebral haemorrhage (ICH) is a devastating disease that leads to severe neurological deficits. Microglia are the first line of defence in the brain and play a crucial role in neurological recovery after ICH, whose activities are primarily driven by glucose metabolism. However, little is known regarding the status of glucose metabolism in microglia and its interactions with inflammatory responses after ICH.
OBJECTIVE: This study investigated microglial glycolysis and its mechanistic effects on microglial inflammation after ICH.
METHODS: We explored the status of glucose metabolism in the ipsilateral region and in fluorescence-activated-cell-sorting-isolated (FACS-isolated) microglia via 2-deoxy-[18F]fluoro-D-glucose positron emission tomography (FDG-PET) analyses and gamma emission, respectively. Energy-related targeted metabolomics, along with 13C-glucose isotope tracing, was utilised to analyse glycolytic products in microglia. Mitochondrial membrane potential and mitochondrial reactive oxygen species (MitoROS) accumulation was assessed by flow cytometry. Behavioural, western blotting, gene regulation, and enzymatic activity analyses were conducted with a focus on microglia.
RESULTS: Neurological dysfunction was strongly correlated with decreased FDG-PET signals in the perihaematomal region, where microglial uptake of FDG was reduced. The decreased quantity of glucose-6-phosphate (G-6-P) in microglia was attributed to the downregulation of glucose transporter 1 (GLUT1) and hexokinase 2 (HK2). Enhanced inflammatory responses were driven by HK2 suppression via decreased mitochondrial membrane potential, which could be rescued by MitoROS scavengers. HK inhibitors aggravated neurological injury by suppressing FDG uptake and enhancing microglial inflammation in ICH mice.
CONCLUSIONS: These findings indicate an unexpected metabolic status in pro-inflammatory microglia after ICH, consisting of glycolysis impairment caused by the downregulation of GLUT1 and HK2. Additionally, HK2 suppression promotes inflammatory responses by disrupting mitochondrial function, providing insight into the mechanisms by which inflammation may be facilitated after ICH and indicating that metabolic enzymes as potential targets for ICH treatment.

Neuroinflammation is a prominent feature of Alzheimer\'s disease (AD). Activated microglia undergo a reprogramming of cellular metabolism necessary to power their cellular activities during disease. Thus, selective targeting of microglial immunometabolism might be of therapeutic benefit for treating AD. In the AD brain, the levels of microglial hexokinase 2 (HK2), an enzyme that supports inflammatory responses by promoting glycolysis, are significantly increased. In addition, HK2 displays non-metabolic activities that extend its inflammatory role beyond glycolysis. The antagonism of HK2 affects microglial phenotypes and disease progression in a gene-dose-dependent manner. HK2 complete loss fails to improve pathology by exacerbating inflammation, while its haploinsufficiency reduces pathology in 5xFAD mice. We propose that the partial antagonism of HK2 is effective in slowing disease progression by modulating NF-κB signaling through its cytosolic target, IKBα. The complete loss of HK2 affects additional inflammatory mechanisms related to mitochondrial dysfunction.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise both sporadically and in patients with the genetic syndrome Neurofibromatosis type 1 (NF1). Prognosis is dismal, as large dimensions, risk of relapse, and anatomical localization make surgery poorly effective, and no therapy is known. Hence, the identification of MPNST molecular features that could be hit in an efficient and selective way is mandatory to envision treatment options. Here, we find that MPNSTs express high levels of the glycolytic enzyme Hexokinase 2 (HK2), which is known to shield cancer cells from noxious stimuli when it localizes at MAMs (mitochondria-associated membranes), contact sites between mitochondria and endoplasmic reticulum. A HK2-targeting peptide that dislodges HK2 from MAMs rapidly induces a massive death of MPNST cells. After identifying different matrix metalloproteases (MMPs) expressed in the MPNST microenvironment, we have designed HK2-targeting peptide variants that harbor cleavage sites for these MMPs, making such peptides activatable in the proximity of cancer cells. We find that the peptide carrying the MMP2/9 cleavage site is the most effective, both in inhibiting the in vitro tumorigenicity of MPNST cells and in hampering their growth in mice. Our data indicate that detaching HK2 from MAMs could pave the way for a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression features of the tumor microenvironment.

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor-stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and response to therapy, making them attractive targets for anti-cancer treatment. Unraveling the mechanisms involved in CAFs activation and maintenance is therefore crucial for the improvement of therapy efficacy. Here, we report that CAFs phenoconversion relies on the glucose-dependent inhibition of autophagy. We show that ovarian cancer cell-conditioning medium induces a metabolic reprogramming towards the CAF-phenotype that requires the autophagy-dependent glycolytic shift. In fact, 2-deoxy-D-glucose (2DG) strongly hampers such phenoconversion and, most importantly, induces the phenoreversion of CAFs into quiescent fibroblasts. Moreover, pharmacological inhibition (by proline) or autophagy gene knockdown (by siBECN1 or siATG7) promotes, while autophagy induction (by either 2DG or rapamycin) counteracts, the metabolic rewiring induced by the ovarian cancer cell secretome. Notably, the nutraceutical resveratrol (RV), known to inhibit glucose metabolism and to induce autophagy, promotes the phenoreversion of CAFs into normal fibroblasts even in the presence of ovarian cancer cell-conditioning medium. Overall, our data support the view of testing autophagy inducers for targeting the tumor-promoting stroma as an adjuvant strategy to improve therapy success rates, especially for tumors with a highly desmoplastic stroma, like ovarian cancer.

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells\' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.

Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.

Acute lung injury (ALI) is an acute inflammatory lung disease associated with both innate and adaptive immune responses. Hexokinase 2 (HK2) is specifically highly expressed in numerous types of inflammation‑related diseases and models. In the present study in vitro and in vivo effects of targeted degradation of HK2 on ALI were explored. The degradation of HK2 by the targeting peptide TAT (transactivator of transcription protein of HIV‑1)‑ataxin 1 (ATXN1)‑chaperone‑mediated autophagy‑targeting motif (CTM) was demonstrated by ELISA and western blotting in vitro and in vivo. The inhibitory effects of TAT‑ATXN1‑CTM on lipopolysaccharide (LPS)‑induced inflammatory responses were examined using ELISAs. The therapeutic effects of TAT‑ATXN1‑CTM on LPS‑induced ALI were examined via histological examination and ELISAs in mice. 10 µM TAT‑ATXN1‑CTM administration decreased HK2 protein expression and the secretion of proinflammatory cytokines (TNF‑α and IL‑1β) without altering HK2 mRNA expression in LPS‑treated both in vitro and in vivo, while pathological lung tissue damage and the accumulation of leukocytes, neutrophils, macrophages and lymphocytes in ALI were also significantly suppressed by 10 µM TAT‑ATXN1‑CTM treatment. TAT‑ATXN1‑CTM exhibited anti‑inflammatory activity in vitro and decreased the severity of ALI in vivo. HK2 degradation may represent a novel therapeutic approach for ALI.

Schiff bases attract research interest due to their applications in chemical synthesis and medicinal chemistry. In recent years, benitrobenrazide and benserazide containing imine moiety have been synthesized and characterized as promising inhibitors of hexokinase 2 (HK2), an enzyme overexpressed in most cancer cells. Benserazide and benitrobenrazide possess a common structural fragment, a 2,3,4-trihydroxybenzaldehyde moiety connected through a hydrazone or hydrazine linker acylated on an N\' nitrogen atom by serine or a 4-nitrobenzoic acid fragment. To avoid the presence of a toxicophoric nitro group in the benitrobenrazide molecule, we introduced common pharmacophores such as 4-fluorophenyl or 4-aminophenyl substituents. Modification of benserazide requires the introduction of other endogenous amino acids instead of serine. Herein, we report the synthesis of benitrobenrazide and benserazide analogues and preliminary results of inhibitory activity against HK2 evoked by these structural changes. The derivatives contain a fluorine atom or amino group instead of a nitro group in BNB and exhibit the most potent inhibitory effects against HK2 at a concentration of 1 µM, with HK2 inhibition rates of 60% and 54%, respectively.

Microgliosis and neuroinflammation are prominent features of Alzheimer\'s disease (AD). Disease-responsive microglia meet their increased energy demand by reprogramming metabolism, specifically, switching to favor glycolysis over oxidative phosphorylation. Thus, targeting of microglial immunometabolism might be of therapeutic benefit for treating AD, providing novel and often well understood immune pathways and their newly recognized actions in AD. We report that in the brains of 5xFAD mice and postmortem brains of AD patients, we found a significant increase in the levels of Hexokinase 2 (HK2), an enzyme that supports inflammatory responses by rapidly increasing glycolysis. Moreover, binding of HK2 to mitochondria has been reported to regulate inflammation by preventing mitochondrial dysfunction and NLRP3 inflammasome activation, suggesting that its inflammatory role extends beyond its glycolytic activity. Here we report, that HK2 antagonism selectively affects microglial phenotypes and disease progression in a gene-dose dependent manner. Paradoxically, complete loss of HK2 fails to improve AD progression by exacerbating inflammasome activity while its haploinsufficiency results in reduced pathology and improved cognition in the 5XFAD mice. We propose that the partial antagonism of HK2, is effective in slowed disease progression and inflammation through a non-metabolic mechanism associated with the modulation of NFKβ signaling, through its cytosolic target IKBα. The complete loss of HK2 affects additional inflammatory mechanisms associated to mitochondrial dysfunction.

Hexokinase 2 (HK2) is a crucial enzyme involved in glycolysis, which converts glucose into glucose-6-phosphate and plays a significant role in glucose metabolism. HK2 can mediate glycolysis, which is linked to the release of inflammatory factors. The over-expression of HK2 increases the production of pro-inflammatory cytokines, exacerbating the inflammatory reaction. Consequently, HK2 is closely linked to various inflammatory-related diseases affecting multiple systems, including the digestive, nervous, circulatory, respiratory, reproductive systems, as well as rheumatoid arthritis. HK2 is regarded as a novel therapeutic target for inflammatory-related diseases, and this article provides a comprehensive review of its roles in these conditions. Furthermore, the development of potent HK2 inhibitors has garnered significant attention in recent years. Therefore, this review also presents a summary of potential HK2 inhibitors, offering promising prospects for the treatment of inflammatory-related diseases in the future.