BZD9L1 benzimidazole analogue hampers colorectal tumor progression by impeding angiogenesis.

BACKGROUND: The development of new vasculatures (angiogenesis) is indispensable in supplying oxygen and nutrients to fuel tumor growth. Epigenetic dysregulation in the tumor vasculature is critical to colorectal cancer (CRC) progression. Sirtuin (SIRT) enzymes are highly expressed in blood vessels. BZD9L1 benzimidazole analogue is a SIRT 1 and 2 inhibitor with reported anticancer activities in CRC. However, its role has yet to be explored in CRC tumor angiogenesis. AIM: To investigate the anti-angiogenic potential of BZD9L1 on endothelial cells (EC) in vitro, ex vivo and in HCT116 CRC xenograft in vivo models. METHODS: EA.hy926 EC were treated with half inhibitory concentration (IC50) (2.5 µM), IC50 (5.0 µM), and double IC50 (10.0 µM) of BZD9L1 and assessed for cell proliferation, adhesion and SIRT 1 and 2 protein expression. Next, 2.5 µM and 5.0 µM of BZD9L1 were employed in downstream in vitro assays, including cell cycle, cell death and sprouting in EC. The effect of BZD9L1 on cell adhesion molecules and SIRT 1 and 2 were assessed via real-time quantitative polymerase chain reaction (qPCR). The growth factors secreted by EC post-treatment were evaluated using the Quantibody Human Angiogenesis Array. Indirect co-culture with HCT116 CRC cells was performed to investigate the impact of growth factors modulated by BZD9L1-treated EC on CRC. The effect of BZD9L1 on sprouting impediment and vessel regression was determined using mouse choroids. HCT116 cells were also injected subcutaneously into nude mice and analyzed for the outcome of BZD9L1 on tumor necrosis, Ki67 protein expression indicative of proliferation, cluster of differentiation 31 (CD31) and CD34 EC markers, and SIRT 1 and 2 genes via hematoxylin and eosin, immunohistochemistry and qPCR, respectively. RESULTS: BZD9L1 impeded EC proliferation, adhesion, and spheroid sprouting through the downregulation of intercellular adhesion molecule 1, vascular endothelial cadherin, integrin-alpha V, SIRT1 and SIRT2 genes. The compound also arrested the cells at G1 phase and induced apoptosis in the EC. In mouse choroids, BZD9L1 inhibited sprouting and regressed sprouting vessels compared to the negative control. Compared to the negative control, the compound also reduced the protein levels of angiogenin, basic fibroblast growth factor, platelet-derived growth factor and placental growth factor, which then inhibited HCT116 CRC spheroid invasion in co-culture. In addition, a significant reduction in CRC tumor growth was noted alongside the downregulation of human SIRT1 (hSIRT1), hSIRT2, CD31, and CD34 EC markers and murine SIRT2 gene, while the murine SIRT1 gene remained unaffected, compared to vehicle control. Histology analyses revealed that BZD9L1 at low (50 mg/kg) and high (250 mg/kg) doses reduced Ki-67 protein expression, while BZD9L1 at the high dose diminished tumor necrosis compared to vehicle control. CONCLUSION: These results highlighted the anti-angiogenic potential of BZD9L1 to reduce CRC tumor progression. Furthermore, together with previous anticancer findings, this study provides valuable insights into the potential of BZD9L1 to co-target CRC tumor vasculatures and cancer cells via SIRT1 and/or SIRT2 down-regulation to improve the therapeutic outcome.

System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy.

Rationale: Diabetic retinopathy (DR) is a major complication of diabetes mellitus causing significant vision loss. DR is a multifactorial disease involving changes in retinal microvasculature and neuronal layers, and aberrations in vascular endothelial growth factors (VEGF) and inflammatory pathways. Despite the success of anti-VEGF therapy, many DR patients do not respond well to the treatment, emphasizing the involvement of other molecular players in neuronal and vascular aberrations in DR. Methods: We employed advanced mass spectrometry-based proteome profiling to obtain a global snapshot of altered protein abundances in vitreous humor from patients with proliferative DR (PDR) in comparison to individuals with epiretinal membrane without active DR or other retinal vascular complications. Global proteome correlation map and protein-protein interaction networks were used to probe into the functional inclination of proteins and aberrated molecular networks in PDR vitreous. In addition, peptide-centric analysis of the proteome data was carried out to identify proteolytic processing, primarily ectodomain shedding events in PDR vitreous. Functional validation experiments were performed using preclinical models of ocular angiogenesis. Results: The vitreous proteome landscape revealed distinct dysregulations in several metabolic, signaling, and immune networks in PDR. Systematic analysis of altered proteins uncovered specific impairment in ectodomain shedding of several transmembrane proteins playing critical roles in neurodegeneration and angiogenesis, pointing to defects in their regulating sheddases, particularly ADAM10, which emerged as the predominant sheddase. We confirmed that ADAM10 protease activity was reduced in animal models of ocular angiogenesis and established that activation of ADAM10 can suppress endothelial cell activation and angiogenesis. Furthermore, we identified the impaired ADAM10-AXL axis as a driver of retinal angiogenesis. Conclusion: We demonstrate restoration of aberrant ectodomain shedding as an effective strategy for treating PDR and propose ADAM10 as an attractive therapeutic target. In all, our study uncovered impaired ectodomain shedding as a prominent feature of PDR, opening new possibilities for advancement in the DR therapeutic space.

HTRA1 Regulates Subclinical Inflammation and Activates Proangiogenic Response in the Retina and Choroid.

High-temperature requirement A1 (HtrA1) has been identified as a disease-susceptibility gene for age-related macular degeneration (AMD) including polypoidal choroidal neovasculopathy (PCV). We characterized the underlying phenotypic changes of transgenic (Tg) mice expressing ubiquitous CAG promoter (CAG-HtrA1 Tg). In vivo imaging modalities and histopathology were performed to investigate the possible neovascularization, drusen formation, and infiltration of macrophages. Subretinal white material deposition and scattered white-yellowish retinal foci were detected on CFP [(Tg­33% (20/60) and wild-type (WT)­7% (1/15), p < 0.05]. In 40% (4/10) of the CAG-HtrA1 Tg retina, ICGA showed punctate hyperfluorescent spots. There was no leakage on FFA and OCTA failed to confirm vascular flow signals from the subretinal materials. Increased macrophages and RPE cell migrations were noted from histopathological sections. Monocyte subpopulations were increased in peripheral blood in the CAG-HtrA1 Tg mice (p < 0.05). Laser induced CNV in the CAG-HtrA1 Tg mice and showed increased leakage from CNV compared to WT mice (p < 0.05). Finally, choroidal explants of the old CAG-HtrA1 Tg mice demonstrated an increased area of sprouting (p < 0.05). Signs of subclinical inflammation was observed in CAG-HtrA1 Tg mice. Such subclinical inflammation may have resulted in increased RPE cell activation and angiogenic potential.

Complement Factor B Mediates Ocular Angiogenesis through Regulating the VEGF Signaling Pathway.

Complement factor B (CFB), a 95-kDa protein, is a crucial catalytic element of the alternative pathway (AP) of complement. After binding of CFB to C3b, activation of the AP depends on the proteolytic cleavage of CFB by factor D to generate the C3 convertase (C3bBb). The C3 convertase contains the catalytic subunit of CFB (Bb), the enzymatic site for the cleavage of a new molecule of C3 into C3b. In addition to its role in activating the AP, CFB has been implicated in pathological ocular neovascularization, a common feature of several blinding eye diseases, however, with somewhat conflicting results. The focus of this study was to investigate the direct impact of CFB on ocular neovascularization in a tightly controlled environment. Using mouse models of laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), our study demonstrated an increase in CFB expression during pathological angiogenesis. Results from several in vitro and ex vivo functionality assays indicated a promoting effect of CFB in angiogenesis. Mechanistically, CFB exerts this pro-angiogenic effect by mediating the vascular endothelial growth factor (VEGF) signaling pathway. In summary, we demonstrate compelling evidence for the role of CFB in driving ocular angiogenesis in a VEGF-dependent manner. This work provides a framework for a more in-depth exploration of CFB-mediated effects in ocular angiogenesis in the future.

Largazole Inhibits Ocular Angiogenesis by Modulating the Expression of VEGFR2 and p21.

Ocular angiogenic diseases, characterized by abnormal blood vessel formation in the eye, are the leading cause of blindness. Although Anti-VEGF therapy is the first-line treatment in the market, a substantial number of patients are refractory to it or may develop resistance over time. As uncontrolled proliferation of vascular endothelial cells is one of the characteristic features of pathological neovascularization, we aimed to investigate the role of the class I histone deacetylase (HDAC) inhibitor Largazole, a cyclodepsipeptide from a marine cyanobacterium, in ocular angiogenesis. Our study showed that Largazole strongly inhibits retinal vascular endothelial cell viability, proliferation, and the ability to form tube-like structures. Largazole strongly inhibits the vessel outgrowth from choroidal explants in choroid sprouting assay while it does not affect the quiescent choroidal vasculature. Largazole also inhibits vessel outgrowth from metatarsal bones in metatarsal sprouting assay without affecting pericytes coverage. We further demonstrated a cooperative effect between Largazole and an approved anti-VEGF drug, Alflibercept. Mechanistically, Largazole strongly inhibits the expression of VEGFR2 and leads to an increased expression of cell cycle inhibitor, p21. Taken together, our study provides compelling evidence on the anti-angiogenic role of Largazole that exerts its function through mediating different signaling pathways.

Leucine-Rich α-2-Glycoprotein 1 Suppresses Endothelial Cell Activation Through ADAM10-Mediated Shedding of TNF-α Receptor.

Elevated serum concentrations of leucine-rich α-2-glycoprotein (LRG1) have been reported in patients with inflammatory, autoimmune, and cardiovascular diseases. This study aims to investigate the role of LRG1 in endothelial activation. LRG1 in endothelial cells (ECs) of arteries and serum of patients with critical limb ischemia (CLI) was assessed by immunohistochemistry and ELISA, respectively. LRG1 expression in sheared and tumor necrosis factor-α (TNF-α)-treated ECs was analyzed. The mechanistic role of LRG1 in endothelial activation was studied in vitro. Plasma of 37-week-old Lrg1 -/- mice was used to investigate causality between LRG1 and tumor necrosis factor receptor 1 (TNFR1) shedding. LRG1 was highly expressed in ECs of stenotic but not normal arteries. LRG1 concentrations in serum of patients with CLI were elevated compared to healthy controls. LRG1 expression was shear dependent. It could be induced by TNF-α, and the induction of its expression was mediated by NF-κB activation. LRG1 inhibited TNF-α-induced activation of NF-κB signaling, expression of VCAM-1 and ICAM-1, and monocyte capture, firm adhesion, and transendothelial migration. Mechanistically, LRG1 exerted its function by causing the shedding of TNFR1 via the ALK5-SMAD2 pathway and the subsequent activation of ADAM10. Consistent with this mechanism, LRG1 and sTNFR1 concentrations were correlated in the serum of CLI patients. Causality between LRG1 and TNFR1 shedding was established by showing that Lrg1 -/- mice had lower plasma sTNFR1 concentrations than wild type mice. Our results demonstrate a novel role for LRG1 in endothelial activation and its potential therapeutic role in inflammatory diseases should be investigated further.

Investigating the Role of PPARß/δ in Retinal Vascular Remodeling Using Pparß/δ-Deficient Mice.

Peroxisome proliferator-activated receptor (PPAR)ß/δ is a member of the nuclear receptor superfamily of transcription factors, which plays fundamental roles in cell proliferation and differentiation, inflammation, adipogenesis, and energy homeostasis. Previous studies demonstrated a reduced choroidal neovascularization (CNV) in Pparß/δ-deficient mice. However, PPARß/δ's role in physiological blood vessel formation and vessel remodeling in the retina has yet to be established. Our study showed that PPARß/δ is specifically required for disordered blood vessel formation in the retina. We further demonstrated an increased arteriovenous crossover and wider venous caliber in Pparß/δ-haplodeficient mice. In summary, these results indicated a critical role of PPARß/δ in pathological angiogenesis and blood vessel remodeling in the retina.

Collaborative Regulation of LRG1 by TGF-ß1 and PPAR-ß/δ Modulates Chronic Pressure Overload-Induced Cardiac Fibrosis.

BACKGROUND: Despite its established significance in fibrotic cardiac remodeling, clinical benefits of global inhibition of TGF (transforming growth factor)-ß1 signaling remain controversial. LRG1 (leucine-rich-α2 glycoprotein 1) is known to regulate endothelial TGFß signaling. This study evaluated the role of LRG1 in cardiac fibrosis and its transcriptional regulatory network in cardiac fibroblasts. METHODS: Pressure overload-induced heart failure was established by transverse aortic constriction. Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunohistochemistry were used to evaluate the expression level and pattern of interested targets or pathology during fibrotic cardiac remodeling. Cardiac function was assessed by pressure-volume loop analysis. RESULTS: LRG1 expression was significantly suppressed in left ventricle of mice with transverse aortic constriction-induced fibrotic cardiac remodeling (mean difference, -0.00085 [95% CI, -0.0013 to -0.00043]; P=0.005) and of patients with end-stage ischemic-dilated cardiomyopathy (mean difference, 0.13 [95% CI, 0.012-0.25]; P=0.032). More profound cardiac fibrosis (mean difference, -0.014% [95% CI, -0.029% to -0.00012%]; P=0.048 for interstitial fibrosis; mean difference, -1.3 [95% CI, -2.5 to -0.2]; P=0.016 for perivascular fibrosis), worse cardiac dysfunction (mean difference, -2.5 ms [95% CI, -4.5 to -0.4 ms]; P=0.016 for Tau-g; mean difference, 13% [95% CI, 2%-24%]; P=0.016 for ejection fraction), and hyperactive TGFß signaling in transverse aortic constriction-operated Lrg1-deficient mice (mean difference, -0.27 [95% CI, -0.47 to -0.07]; P<0.001), which could be reversed by cardiac-specific Lrg1 delivery mediated by adeno-associated virus 9. Mechanistically, LRG1 inhibits cardiac fibroblast activation by competing with TGFß1 for receptor binding, while PPAR (peroxisome proliferator-activated receptor)-ß/δ and TGFß1 collaboratively regulate LRG1 expression via SMRT (silencing mediator for retinoid and thyroid hormone receptor). We further demonstrated functional interactions between LRG1 and PPARß/δ in cardiac fibroblast activation. CONCLUSIONS: Our results established a highly complex molecular network involving LRG1, TGFß1, PPARß/δ, and SMRT in regulating cardiac fibroblast activation and cardiac fibrosis. Targeting LRG1 or PPARß/δ represents a promising strategy to control pathological cardiac remodeling in response to chronic pressure overload.

Sustained delivery of anti-VEGFs from thermogel depots inhibits angiogenesis without the need for multiple injections.

Anti-vascular endothelial growth factor (anti-VEGF) proteins are the gold-standard treatment for posterior eye segment proliferative vascular diseases such as Age-Related Macular Degeneration (AMD) and Diabetic Retinopathy (DR). However, the standard of care requires inconvenient monthly intravitreal injections. This underlies an unmet clinical need to develop alternative solutions for sustained delivery of biologics. In this paper, we demonstrated that anti-VEGFs can be encapsulated by a simple mild process into our polyurethane thermogel depots. By changing the hydrophilic-hydrophobic balance in the copolymer, anti-VEGF release rates can be modulated. The antibody in the thermogel partitions into protein domains which vary in size corresponding to the hydrophilicity balance of the polymer. Anti-VEGFs can be released in a relatively linear manner from the thermogel for up to 40 days in vitro. The encapsulated anti-VEGFs demonstrate anti-angiogenic bioactivity by inhibiting vessel outgrowth in rat ex vivo choroidal explants, and reducing vascular leakage in a VEGF-driven neovascularization rabbit model. In conclusion, we show that these thermogels can be tuned in terms of hydrophilicity and used for sustained delivery of bioactive anti-VEGFs. Physically cross-linked polyurethane thermoresponsive hydrogels could be a promising platform for sustained delivery of biologically active therapeutic proteins.

Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases.

Purpose: Abnormal blood vessel formation is a defining feature of many blinding eye diseases. Targeting abnormal angiogenesis by inhibiting VEGF has revolutionized the treatment of many ocular angiogenic diseases over the last decade. However, a substantial number of patients are refractory to anti-VEGF treatment or may develop resistance over time. The objective of this study was to determine the efficacy and the mechanism of action of Apratoxin S4 in ocular angiogenesis. Methods: Retinal vascular cell proliferation, migration, and the ability to form tube-like structure were studied in vitro. Ex vivo aortic ring, choroid, and metatarsal assays were used to study Apratoxin S4's impact on vessel outgrowth in a multicellular environment. Apratoxin S4 was also tested in mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), and in a rabbit model of persistent retinal neovascularization (PRNV). Western blot and ELISA were used to determine the expression of key angiogenic regulators after Apratoxin S4 treatment. Results: Apratoxin S4 strongly inhibits retinal vascular cell activation by suppressing multiple angiogenic pathways. VEGF-activated vascular cells and angiogenic vessels are more susceptible to Apratoxin S4 treatment than quiescent vascular cells and vessels. Both intraperitoneal and intravitreal delivery of Apratoxin S4 are able to impede ocular neovascularization in vivo. Apratoxin S4 specifically attenuates pathological ocular angiogenesis and exhibits a combinatorial inhibitory effect with standard-of-care VEGF inhibitor drug (aflibercept). Conclusions: Apratoxin S4 is a potent antiangiogenic drug that inhibits the activation of retinal endothelial cells and pericytes through mediating multiple angiogenic pathways.

A Role of Agrin in Maintaining the Stability of Vascular Endothelial Growth Factor Receptor-2 during Tumor Angiogenesis.

Endothelial cell (EC) recruitment is central to the vascularization of tumors. Although several proteoglycans have been implicated in cancer and angiogenesis, their roles in EC recruitment and vascularization during tumorigenesis remain poorly understood. Here, we reveal that Agrin, which is secreted in liver cancer, promotes angiogenesis by recruiting ECs within tumors and metastatic lesions and facilitates adhesion of cancer cells to ECs. In ECs, Agrin-induced angiogenesis and adherence to cancer cells are mediated by Integrin-ß1, Lrp4-MuSK pathways involving focal adhesion kinase. Mechanistically, we uncover that Agrin regulates VEGFR2 levels that sustain the angiogenic property of ECs and adherence to cancer cells. Agrin attributes an ECM stiffness-based stabilization of VEGFR2 by enhancing interactions with Integrin-ß1-Lrp4 and additionally stimulates endothelial nitric-oxide synthase (e-NOS) signaling. Therefore, we propose that cross-talk between Agrin-expressing cancer and ECs favor angiogenesis by sustaining the VEGFR2 pathway.

Molecular Beacon Gold Nanosensors for Leucine-Rich Alpha-2-Glycoprotein-1 Detection in Pathological Angiogenesis.

Leucine-rich alpha-2-glycoprotein-1 (Lrg1) is an emerging biomarker for angiogenesis. Its expression in ocular tissues is up-regulated in both human patients with proliferative diabetic retinopathy and rodent models of pathological angiogenesis. However, there is no existing sensor that allows visualization and monitoring of Lrg1 expression noninvasively and in real time. Herein, we report a nucleic acid-gold nanorod-based nanosensor for the noninvasive monitoring of cellular Lrg1 expression in angiogenesis. Specifically, this platform is constructed by covalently conjugating molecular beacons onto gold nanorods, which prequench the fluorophores on the molecular beacons. Upon intracellular entry and endosomal escape, the complexes interact with cellular Lrg1 mRNA through hybridization of the loop area of the molecular beacons. This complexation distances the fluorophores from nanorod and restores the prequenched fluorescence. The reliability of this platform is confirmed by examining the increased Lrg1 expression in migrating keratinocytes and the Lrg1 gene changes in different postnatal stages of mouse retinal vasculature growth in the mouse retina model.

Absence of NUCKS augments paracrine effects of mesenchymal stem cells-mediated cardiac protection.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) contribute to myocardial repair after myocardial infarction (MI) by secreting a panel of growth factors and cytokines. This study was to investigate the potential mechanisms of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) in regulation of the profiles of BM-MSCs secretion and compare the therapeutic efficacy of NUCKS-/-- and wide type-BM-MSCs (WT-BM-MSCs) on MI. The secretion profiles between NUCKS-/-- and WT-BM-MSCs under hypoxia (1%O2) were analyzed. Gene function analysis showed that compared with WT-BM-MSCs-conditioned medium (CdM), some genes over-presented in NUCKS-/--BM-MSCs-CdM were closely associated with inflammatory response, regulation of cell proliferation, death, migration and secretion. Notably, VEGFa in NUCKS-/--BM-MSCs-CdM was higher than that of WT-BM-MSCs-CdM. WT-BM-MSCs and NUCKS-/--BM-MSCs were transplanted into the peri-infarct region in mice of MI. At 4 weeks after cell transplantation, NUCKS-/-- or WT-BM-MSCs group significantly improved heart function and vessels density and reduced infarction size and apoptosis of cardiomyocytes. Furthermore, NUCKS-/--BM-MSCs provided better cardioprotective effects than WT-BM-MSCs against MI. Our study demonstrates that depletion of NUCKS enhances the therapeutic efficacy of BM-MSCs for MI via regulating the secretion.

Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn.

Corneal wound healing involves a complex cascade of cytokine-controlled cellular events, including inflammatory and angiogenesis responses that are regulated by transcriptional chromatin remodeling. Nuclear Ubiquitous Casein and cyclin-dependent Kinase Substrate (NUCKS) is a key chromatin modifier and transcriptional regulator of metabolic signaling. In this study, we investigated the role of NUCKS in corneal wound healing by comparing its effects on corneal alkali burn in NUCKS knockout (NKO) and NUCKS wild-type (NWT) mice. Our data showed that following alkali-injury, inhibition of NUCKS (NKO) accelerated ocular resurfacing and suppressed neovascularization; the cytokine profile of alkali burned corneas in NKO mice showed suppressed expression of inflammation cytokines (IL1A &IL1B); upregulated expression of antiangiogenic factor (Pigment Epithelium-derived Factor; PEDF); and downregulated expression of angiogenic factor (Vascular Endothelial Growth Factor, VEGF); in vitro, following LPS-induced NFκB activation, NKO corneal cells showed reduced expression of IL6, IP10 and TNFα. In vitro, corneal epithelial cells showed reduced NF-κb activation on silencing of NUCKS and corresponding NFκB-mediated cytokine expression was reduced. Here, we illustrate that inhibition of NUCKS played a role in cytokine modulation and facilitated corneal recovery. This reveals a potential new effective strategy for ocular burn treatment.

Phosphoprotein network analysis of white adipose tissues unveils deregulated pathways in response to high-fat diet.

Despite efforts in the last decade, signaling aberrations associated with obesity remain poorly understood. To dissect molecular mechanisms that define this complex metabolic disorder, we carried out global phosphoproteomic analysis of white adipose tissue (WAT) from mice fed on low-fat diet (LFD) and high-fat diet (HFD). We quantified phosphorylation levels on 7696 peptides, and found significant differential phosphorylation levels in 282 phosphosites from 191 proteins, including various insulin-responsive proteins and metabolic enzymes involved in lipid homeostasis in response to high-fat feeding. Kinase-substrate prediction and integrated network analysis of the altered phosphoproteins revealed underlying signaling modulations during HFD-induced obesity, and suggested deregulation of lipogenic and lipolytic pathways. Mutation of the differentially-regulated novel phosphosite on cytoplasmic acetyl-coA forming enzyme ACSS2 (S263A) upon HFD-induced obesity led to accumulation of serum triglycerides and reduced insulin-responsive AKT phosphorylation as compared to wild type ACSS2, thus highlighting its role in obesity. Altogether, our study presents a comprehensive map of adipose tissue phosphoproteome in obesity and reveals many previously unknown candidate phosphorylation sites for future functional investigation.

Hypothalamic NUCKS regulates peripheral glucose homoeostasis.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate (NUCKS) is highly expressed in the brain and peripheral metabolic organs, and regulates transcription of a number of genes involved in insulin signalling. Whole-body depletion of NUCKS (NKO) in mice leads to obesity, glucose intolerance and insulin resistance. However, a tissue-specific contribution of NUCKS to the observed phenotypes remains unknown. Considering the pivotal roles of insulin signalling in the brain, especially in the hypothalamus, we examined the functions of hypothalamic NUCKS in the regulation of peripheral glucose metabolism. Insulin signalling in the hypothalamus was impaired in the NKO mice when insulin was delivered through intracerebroventricular injection. To validate the hypothalamic specificity, we crossed transgenic mice expressing Cre-recombinase under the Nkx2.1 promoter with floxed NUCKS mice to generate mice with hypothalamus-specific deletion of NUCKS (HNKO). We fed the HNKO and littermate control mice with a normal chow diet (NCD) and a high-fat diet (HFD), and assessed glucose tolerance, insulin tolerance and metabolic parameters. HNKO mice showed mild glucose intolerance under an NCD, but exacerbated obesity and insulin resistance phenotypes under an HFD. In addition, NUCKS regulated levels of insulin receptor in the brain. Unlike HNKO mice, mice with immune-cell-specific deletion of NUCKS (VNKO) did not develop obesity or insulin-resistant phenotypes under an HFD. These studies indicate that hypothalamic NUCKS plays an essential role in regulating glucose homoeostasis and insulin signalling in vivo.

NUCKS: a potential biomarker in cancer and metabolic disease.

Nuclear, casein kinase and cyclin-dependent kinase substrate (NUCKS), a protein similar to the HMG (high-mobility group) protein family, is one of the most modified proteins in the mammalian proteome. Although very little is known about the biological roles of NUCKS, emerging clinical evidence suggests that this protein can be a biomarker and therapeutic target in various human ailments, including several types of cancer. An inverse correlation between NUCKS protein levels and body mass index in humans has also been observed. Depletion of NUCKS in mice has been reported to lead to obesity and impaired glucose homoeostasis. Genome-wide genomic and proteomic approaches have revealed that NUCKS is a chromatin regulator that affects transcription. The time is now ripe for further understanding of the role of this novel biomarker of cancer and the metabolic syndrome, and how its sundry modifications can affect its function. Such studies could reveal how NUCKS could be a link between physiological cues and human ailments.

NUCKS is a positive transcriptional regulator of insulin signaling.

Although much is known about the molecular players in insulin signaling, there is scant information about transcriptional regulation of its key components. We now find that NUCKS is a transcriptional regulator of the insulin signaling components, including the insulin receptor (IR). Knockdown of NUCKS leads to impaired insulin signaling in endocrine cells. NUCKS knockout mice exhibit decreased insulin signaling and increased body weight/fat mass along with impaired glucose tolerance and reduced insulin sensitivity, all of which are further exacerbated by a high-fat diet (HFD). Genome-wide ChIP-seq identifies metabolism and insulin signaling as NUCKS targets. Importantly, NUCKS is downregulated in individuals with a high body mass index and in HFD-fed mice, and conversely, its levels increase upon starvation. Altogether, NUCKS is a physiological regulator of energy homeostasis and glucose metabolism that works by regulating chromatin accessibility and RNA polymerase II recruitment to the promoters of IR and other insulin pathway modulators.

Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice.

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome.