Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement.

Despite the important breakthroughs of immune checkpoint inhibitors in recent years, the objective response rates remain limited. Here, we synthesize programmed cell death protein-1 (PD-1) antibody-iRGD cyclic peptide conjugate (αPD-1-(iRGD)2) through glycoengineering methods. In addition to enhancing tissue penetration, αPD-1-(iRGD)2 simultaneously engages tumor cells and PD-1+ T cells via dual targeting, thus mediating tumor-specific T cell activation and proliferation with mild effects on non-specific T cells. In multiple syngeneic mouse models, αPD-1-(iRGD)2 effectively reduces tumor growth with satisfactory biosafety. Moreover, results of flow cytometry and single-cell RNA-seq reveal that αPD-1-(iRGD)2 remodels the tumor microenvironment and expands a population of "better effector" CD8+ tumor infiltrating T cells expressing stem- and memory-associated genes, including Tcf7, Il7r, Lef1, and Bach2. Conclusively, αPD-1-(iRGD)2 is a promising antibody conjugate therapeutic beyond antibody-drug conjugate for cancer immunotherapy.

Reconstruction of Metabolic-Protein Interaction Integrated Network of Eriocheir sinensis and Analysis of Ecdysone Synthesis.

Integrated networks have become a new interest in genome-scale network research due to their ability to comprehensively reflect and analyze the molecular processes in cells. Currently, none of the integrated networks have been reported for higher organisms. Eriocheir sinensis is a typical aquatic animal that grows through ecdysis. Ecdysone has been identified to be a crucial regulator of ecdysis, but the influence factors and regulatory mechanisms of ecdysone synthesis in E. sinensis are still unclear. In this work, the genome-scale metabolic network and protein-protein interaction network of E. sinensis were integrated to reconstruct a metabolic-protein interaction integrated network (MPIN). The MPIN was used to analyze the influence factors of ecdysone synthesis through flux variation analysis. In total, 236 integrated reactions (IRs) were found to influence the ecdysone synthesis of which 16 IRs had a significant impact. These IRs constitute three ecdysone synthesis routes. It is found that there might be alternative pathways to obtain cholesterol for ecdysone synthesis in E. sinensis instead of absorbing it directly from the feeds. The MPIN reconstructed in this work is the first integrated network for higher organisms. The analysis based on the MPIN supplies important information for the mechanism analysis of ecdysone synthesis in E. sinensis.

Signaling Transduction Pathways and G-Protein-Coupled Receptors in Different Stages of the Embryonic Diapause Termination Process in Artemia.

Artemia is a widely distributed small aquatic crustacean, renowned for its ability to enter a state of embryonic diapause. The embryonic diapause termination (EDT) is closely linked to environmental cues, but the precise underlying mechanisms remain elusive. In this study, ATAC-seq and RNA-seq sequencing techniques were employed to explore the gene expression profiles in Artemia cysts 30 min after EDT. These profiles were compared with those during diapause and 5 h after EDT. The regulatory mechanisms governing the EDT process were analyzed through Gene Ontology (GO) enrichment analysis of differentially expressed genes. Furthermore, the active G-protein-coupled receptors (GPCRs) were identified through structural analysis. The results unveiled that the signaling transduction during EDT primarily hinges on GPCRs and the cell surface receptor signaling pathway, but distinct genes are involved across different stages. Hormone-mediated signaling pathways and the tachykinin receptor signaling pathway exhibited heightened activity in the '0-30 min' group, whereas the Wnt signaling pathway manifested its function solely in the '30 min-5 h' group. These results imply a complete divergence in the mechanisms of signal regulation during these two stages. Moreover, through structural analysis, five GPCRs operating at different stages of EDT were identified. These findings provide valuable insights into the signal regulation mechanisms governing Artemia diapause.

High-plex spatial transcriptomic profiling reveals distinct immune components and the HLA class I/DNMT3A/CD8 modulatory axis in mismatch repair-deficient endometrial cancer.

PURPOSE: Tumors bearing mismatch repair deficiency (MMRd) are characterized by a high load of neoantigens and are believed to trigger immunogenic reactions upon immune checkpoint blockade treatment such as anti-PD-1/PD-L1 therapy. However, the mechanisms are still ill-defined, as multiple cancers with MMRd exhibit variable responses to immune checkpoint inhibitors (ICIs). In endometrial cancer (EC), a distinct tumor microenvironment (TME) exists that may correspond to treatment-related efficacies. We aimed to characterize EC patients with aberrant MMR pathways to identify molecular subtypes predisposed to respond to ICI therapies. METHODS: We applied digital spatial profiling, a high-plex spatial transcriptomic approach covering over 1,800 genes, to obtain a highly resolved TME landscape in 45 MMRd-EC patients. We cross-validated multiple biomarkers identified using immunohistochemistry and multiplexed immunofluorescence using in-study and independent cohorts totaling 123 MMRd-EC patients and validated our findings using external TCGA data from microsatellite instability endometrial cancer (MSI-EC) patients. RESULTS: High-plex spatial profiling identified a 14-gene signature in the MMRd tumor-enriched regions stratifying tumors into "hot", "intermediate" and "cold" groups according to their distinct immune profiles, a finding highly consistent with the corresponding CD8 + T-cell infiltration status. Our validation studies further corroborated an existing coregulatory network involving HLA class I and DNMT3A potentially bridged through dynamic crosstalk incorporating CCL5. CONCLUSION: Our study confirmed the heterogeneous TME status within MMRd-ECs and showed that these ECs can be stratified based on potential biomarkers such as HLA class I, DNMT3A and CD8 in pathological settings for improved ICI therapeutic efficacy in this subset of patients.

Trimming Crystallizable Fragment (Fc) Glycans Enables the Direct Enzymatic Transfer of Biomacromolecules to Antibodies as Therapeutics.

Glycoengineering has provided powerful tools to construct site-specific antibody conjugates. However, only small-molecule payloads can be directly transferred to native or engineered antibodies using existing glycoengineering strategies. Herein, we demonstrate that reducing the complexity of crystallizable fragment (Fc) glycans could dramatically boost the chemoenzymatic modification of immunoglobulin G (IgG) via an engineered fucosyltransferase. In this platform, antibodies with Fc glycans engineered to a simple N-acetyllactosamine (LacNAc) disaccharide are successfully conjugated to biomacromolecules, such as oligonucleotides and nanobodies, in a single step within hours. Accordingly, we synthesized an antibody-conjugate-based anti-human epidermal growth factor receptor 2 (HER2)/ cluster of differentiation 3 (CD3) bispecific antibody and used it to selectively destroy patient-derived cancer organoids by reactivating endogenous T lymphocyte cells (T cells) inside the organoid. Our results highlight that this platform is a general approach to construct antibody-biomacromolecule conjugates with translational values.

Sublethal exposure to spinetoram impacts life history traits and dengue virus replication in Aedes aegypti.

Insecticides are anthropogenic environmental stressors and also a common stressor for mosquito vectors. However, the use of insecticides is often guided by short-term efficacy, and the sublethal effect on their target or nontarget species has long been ignored. Here, we analyzed how sublethal exposure of the promising vector-control bioinsecticide spinetoram to Aedes aegypti larvae alter adult performance and susceptibility to dengue virus (DENV) infection. We found that the surviving adult mosquitoes were significantly smaller and exhibited weaker blood-feeding capacity than control females, apart from the extended immature development period. In terms of reproductive potential, although the F0 generation produced a similar number of eggs and offspring during the first gonotrophic cycle, the survival rates of the F1 generations were significantly lower as compared to the control group, suggesting transgenerational sublethal effects on the F1 generation. Notably, surviving adult females had higher DENV-2 viral loads than the control group after spinetoram sublethal exposure. Mechanistically, transcriptomic analysis showed that inhibition of oxidative phosphorylation may function in stimulating DENV production in adult Ae. aegypti. In Aag2 cells, significant accumulation of apoptosis, mitochondrial reactive oxygen species production, and DENV-2 replication by spinetoram exposure consistently support our conclusion. Our study highlights the threat of sublethal spinetoram exposure on outbreaks of mosquito-borne viruses.

A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens.

Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization approach by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen (FF) and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability. By screening 106 phospho-antibodies using RPPA, we demonstrated that AP treatment could serve as an independent factor to be adopted for rapid phospho-antibody selection. We also showed desirable reproducibility and specificity in clincical specimens indicating its potential for tissue-based phospho-protein profiling. Of further clinical significance, using the same approach, based on melanoma and lung cancer FFPE samples, we showed great interexperimental reproducibility and significant correlation with pathological markers in both tissues generating meaningful data that match clinical features. Our findings set a benchmark of an efficient workflow for phospho-antibody characterization that is compatible with high-plex clinical proteomics in precison oncology.

Tumor Microenvironment Profiles Reveal Distinct Therapy-Oriented Proteogenomic Characteristics in Colorectal Cancer.

Advances in immunotherapy have made an unprecedented leap in treating colorectal cancer (CRC). However, more effective therapeutic regimes need a deeper understanding of molecular architectures for precise patient stratification and therapeutic improvement. We profiled patients receiving neoadjuvant chemotherapy alone or in combination with immunotherapy (PD-1 checkpoint inhibitor) using Digital Spatial Profiler (DSP), a high-plex spatial proteogenomic technology. Compartmentalization-based high-plex profiling of both proteins and mRNAs revealed pronounced immune infiltration at tumor regions associated with immunotherapy treatment. The protein and the corresponding mRNA levels within the same selected regions of those patient samples correlate, indicating an overall concordance between the transcriptional and translational levels. An elevated expression of PD-L1 at both protein and the mRNA levels was discovered in the tumor compartment of immunotherapy-treated patients compared with chemo-treated patients, indicating potential prognostic biomarkers are explorable in a spatial manner at the local tumor microenvironment (TME). An elevated expression of PD-L1 was verified by immunohistochemistry. Other compartment-specific biomarkers were also differentially expressed between the tumor and stromal regions, indicating a dynamic interplay that can potentiate novel biomarker discovery from the TME perspectives. Simultaneously, a high-plex spatial profiling of protein and mRNA in the tumor microenvironment of colorectal cancer was performed.

Effective cancer immunotherapy by Ganoderma lucidum polysaccharide-gold nanocomposites through dendritic cell activation and memory T cell response.

Polysaccharides purified from natural herbs possess immunoregulatory functions, while the efficacy of natural polysaccharides on cancer treatment remains unreliable, likely due to their low prescribed doses and fast clearances in clinical settings. In this study, gold nanocomposites containing Ganoderma lucidum polysaccharide (GLP-Au) efficiently induced dendritic cell (DC) activation, evident by the increase of CD80/CD86/CD40/MHCII, decrease of phagocytic ability and acid phosphatase activity, and increased cytokine transcription. GLP-Au significantly promoted the proliferation of CD4+ and CD8+ T cells in splenocytes. DC/T cell co-culture study proved that GLP-Au activation on DC directly resulted in T cell proliferation. GLP-Au exhibited strong inhibitory effects on 4T1 tumor growth and pulmonary metastasis when combined with doxorubicin. GLP-Au recovered body weight loss by doxorubicin and increased the percentage of CD4+/CD44+ memory T cells. This work suggests that polysaccharides from natural herbs can be incorporated into nanocomposites with immunoregulatory characteristics for enhanced efficacy on tumor therapy.

Enhanced anticancer effect of MAP30-S3 by cyclosproin A through endosomal escape.

Cyclosporin A (CsA) is a calcium antagonist and can enhance the efficacy of some protein drugs, but its mechanism remains unknown. In this study, MAP30, a ribosome-inactivating protein reported to have apoptotic effects on cancer cells, was fused with S3, an epidermal growth factor receptor (EGFR)-targeting peptide. In addition, CsA was used to investigate whether it can further promote the apoptotic effects of S3 fused MAP30 (MAP30-S3). Our result showed that the internalization of FITC-labeled MAP30-S3 was increased significantly by S3 in HeLa cells. Unexpectedly, MAP30-S3 only showed a minor decrease in the viability of EGFR-overexpressing cancer cells, including HeLa, SMMC-7721, and MGC803 (IC50>5 µmol/l). However, 2 µmol/l CsA significantly increased the cytotoxicity of MAP30-S3, especially for HeLa cells (IC50=40.3 nmol/l). In comparison, CsA did not further decrease the cytotoxicity of MAP30-S3 on MRC-5, an EGFR low-expressing cell line from normal lung tissue, indicating that CsA did not affect the cancer-targeting specificity of MAP30-S3. Our results also showed that CsA further increased the apoptotic activity of MAP30-S3 in HeLa cells. CsA could promote the endosomal escape of FITC-MAP30-S3 with a diffused pattern in the cytoplasm. Five endocytic inhibitors were used to investigate the cellular uptake mechanism of MAP30-S3, and the results showed that the endosomal escape-enhancing effect of CsA on MAP30-S3 may be associated with the clathrin-dependent endocytic pathways. Our study suggested that CsA could be a novel endosomal escape enhancer to potentiate the intracellular release of anticancer protein drugs, resulting in their improved therapeutic efficacy.

The protective role of autophagy in nephrotoxicity induced by bismuth nanoparticles through AMPK/mTOR pathway.

Bismuth is widely used in metallurgy, cosmetic industry, and medical diagnosis and recently, bismuth nanoparticles (NPs) (BiNP) have been made and proved to be excellent CT imaging agents. Previously, we have synthesized bovine serum albumin based BiNP for imaging purpose but we found a temporary kidney injury by BiNP. Due to the reported adverse events of bismuth on human health, we extended our studies on the mechanisms for BiNP induced nephrotoxicity. Blood biochemical analysis indicated the increase in creatinine (CREA) and blood urea nitrogen (BUN), and intraluminal cast formation with cell apoptosis/necrosis was evident in proximal convoluted tubules (PCTs) of mice. BiNP induced acute kidney injury (AKI) was associated with an increase in LC3II, while the autophagic flux indicator p62 remained unchanged. Chloroquine and rapamycin were used to evaluate the role of autophagy in AKI caused by BiNP. Results showed that BiNP induced AKI was further attenuated by rapamycin, while AKI became severe when chloroquine was applied. In vitro studies further proved BiNP induced autophagy in human embryonic kidney cells 293, presented as autophagic vacuole (AV) formation along with increased levels of autophagy-related proteins including LC3II, Beclin1, and Atg12. Specifically, reactive oxygen species (ROS) generated by BiNP could be the major inducer of autophagy, because ROS blockage attenuated autophagy. Autophagy induced by BiNP was primarily regulated by AMPK/mTOR signal pathway and partially regulated by Akt/mTOR. Our study provides fundamental theory to better understand bismuth induced nephrotoxicity for better clinical application of bismuth related compounds.

Detection of nanocarrier potentiation on drug induced phospholipidosis in cultured cells and primary hepatocyte spheroids by high content imaging and analysis.

Considerable effort has been made to develop nanocarriers for controlled drug delivery over the last decade, while it remains unclear how the strength of adverse drug effect will be altered when a drug is loaded on the nanocarrier. Drug-induced phospholipidosis (DIP) is characterized with excessive accumulation of phospholipids in cells and is common for cationic amphiphilic drugs (CAD). Previously, we have reported that PEGylated graphene oxide (PEG-GO) loaded with several CAD can potentiate DIP. In current study, we extended our study on newly identified phospholipidosis (PLD) inducers that had been identified from the Library of Pharmacologically Active Compounds (LOPAC), to investigate if PEO-GO loaded with these CAD can alter DIP. Twenty-two CAD were respectively loaded on PEG-GO and incubated with RAW264.7, a macrophage cell line. The results showed that when a CAD was loaded on PEG-GO, its strength of PLD induction can be enhanced, unchanged or attenuated. PEG-GO loaded with Ifenprodil exhibited the highest PEG-GO potentiation effect compared to Ifenprodil treatment alone in RAW264.7 cells, and this effect was confirmed in human hepatocellular carcinoma HepG2, another cell line model for PLD induction. Primary hepatocyte culture and spheroids mimicking in vivo conditions were used to further validate nanocarrier potentiation on DIP by Ifenprodil. Stronger phospholipid accumulation was found in PEG-GO/Ifenprodil treated hepatocytes or spheroids than Ifenprodil treatment alone. Therefore, evidences were provided by us that nanocarriers may increase the adverse drug effects and guidance by regulatory agencies need to be drafted for the safe use of nanotechnology in drug delivery.

Feed additive production by fermentation of herb Polygonum hydropiper L. and cassava pulp with simultaneous flavonoid dissolution.

Fermentation of herb Polygonum hydropiper L. (PHL) and cassava pulp (CP) for feed additive production with simultaneous flavonoid dissolution was investigated, and a two-stage response surface methodology (RSM) based on Plackett-Burman factorial design (PB design) was used to optimize the flavonoid dissolution and protein content. Using the screening function of PB design, four different significant factors for the two response variables were acquired: factors A (CP) and B (PHL) for the flavonoid dissolution versus factors G (inoculum size) and H (fermentation time) for protein content. Then, two RSMs were used sequentially to improve the values of the two response variables separately. The mutual corroboration of the experimental results in the present study confirmed the validity of the associated experimental design. The validation experiment showed a flavonoid dissolution rate of 94.00%, and a protein content of 18.20%, gaining an increase in 21.20% and 199.10% over the control, respectively. The present study confirms the feasibility of feed additive production by Saccharomyces cerevisiae with CP and PHL and simultaneous optimization of flavonoid dissolution and protein content using a two-stage RSM.

Continuous ethanol production from sugarcane molasses using a newly designed combined bioreactor system by immobilized Saccharomyces cerevisiae.

Continuous ethanol fermentation using polyvinyl alcohol (PVA), immobilized yeast, and sugarcane molasses (22 and 35°Bx) with 8 g/L urea was run in a combined bioreactor system consisting of three-stage tubular bioreactors in series. The effect of the dilution rate (D) at 0.0037, 0.0075, 0.0117, 0.0145, 0.018, and 0.0282 H(-1) on continuous ethanol fermentation was investigated in this study. The results showed that D had a significant effect on fermentation efficiency, sugar-utilized rate, ethanol yield, and ethanol productivity in this designed continuous fermentation system. The D had a linear relationship with residual sugar and ethanol production under certain conditions. The highest fermentation efficiency of 83.26%, ethanol yield of 0.44 g/g, and the lowest residual sugar content of 6.50 g/L were achieved at 0.0037 H(-1) in the fermentation of 22°Bx molasses, indicating that the immobilization of cells using PVA, sugarcane pieces, and cotton towel is feasible and the established continuous system performs well.

Rhesus macaques develop metabolic syndrome with reversible vascular dysfunction responsive to pioglitazone.

BACKGROUND: The metabolic syndrome (MetS) is a constellation of clinical features that include central obesity, hypertension, atherogenic dyslipidemia, and insulin resistance. However, the concept remains controversial; it has been debated whether MetS represents nothing more than simultaneous co-occurrence of individual risk factors or whether there are common shared pathophysiological mechanisms that link the individual components. METHODS AND RESULTS: To investigate the emergence of metabolic and cardiovascular components during the development of MetS, we identified MetS-predisposed animals (n=35) in a large population of rhesus macaques (Macaca mulatta, 12.7±2.9 years old, n=408), acclimated them to standardized conditions, and monitored the progression of individual component features over 18 months. In 18 MetS animals with recently developed fasting hyperinsulinemia, central obesity, hypertension, and atherogenic dyslipidemia, we found that individual metabolic and cardiovascular components track together during the transition from pre-MetS to onset of MetS; MetS was associated with a 60% impairment of flow-mediated dilation, establishing the mechanistic link with vascular dysfunction. Pioglitazone treatment (3 mg/kg body weight/d for 6 weeks), a peroxisome proliferator-activated receptor γ agonist, reversibly improved atherogenic dyslipidemia and insulin resistance and fully restored flow-mediated dilation with persistent benefits. CONCLUSIONS: Coemergence of metabolic and cardiovascular components during MetS progression and complete normalization of vascular dysfunction with peroxisome proliferator-activated receptor γ agonists suggest shared underlying mechanisms rather than separate processes, arguing for the benefit of early intervention of MetS components. Predictive nonhuman primate (NHP) models of MetS should be highly valuable in mechanistic and translational studies on the pathogenesis of MetS in relation to cardiovascular disease and diabetes mellitus.

Hypothalamic nitric oxide in hypoglycemia detection and counterregulation: a two-edged sword.

Hypoglycemia is the main complication for patients with type 1 diabetes mellitus receiving intensive insulin therapy. In addition to the obvious deleterious effects of acute hypoglycemia on brain function, recurrent episodes of hypoglycemia (RH) have an even more insidious effect. RH impairs the ability of the brain to detect and initiate an appropriate counterregulatory response (CRR) to restore euglycemia in response to subsequent hypoglycemia. Knowledge of mechanisms involved in hypoglycemia detection and counterregulation has significantly improved over the past 20 years. Glucose sensitive neurons (GSNs) in the ventromedial hypothalamus (VMH) may play a key role in the CRR. VMH nitric oxide (NO) production has recently been shown to be critical for both the CRR and glucose sensing by glucose-inhibited neurons. Interestingly, downstream effects of NO may also contribute to the impaired CRR after RH. In this review, we will discuss current literature regarding the molecular mechanisms by which VMH GSNs sense glucose. Putative roles of GSNs in the detection and initiation of the CRR will then be described. Finally, hypothetical mechanisms by which VMH NO production may both facilitate and subsequently impair the CRR will be discussed.

Brain insulin action regulates hypothalamic glucose sensing and the counterregulatory response to hypoglycemia.

OBJECTIVE: An impaired ability to sense and appropriately respond to insulin-induced hypoglycemia is a common and serious complication faced by insulin-treated diabetic patients. This study tests the hypothesis that insulin acts directly in the brain to regulate critical glucose-sensing neurons in the hypothalamus to mediate the counterregulatory response to hypoglycemia. RESEARCH DESIGN AND METHODS: To delineate insulin actions in the brain, neuron-specific insulin receptor knockout (NIRKO) mice and littermate controls were subjected to graded hypoglycemic (100, 70, 50, and 30 mg/dl) hyperinsulinemic (20 mU/kg/min) clamps and nonhypoglycemic stressors (e.g., restraint, heat). Subsequently, counterregulatory responses, hypothalamic neuronal activation (with transcriptional marker c-fos), and regional brain glucose uptake (via (14)C-2deoxyglucose autoradiography) were measured. Additionally, electrophysiological activity of individual glucose-inhibited neurons and hypothalamic glucose sensing protein expression (GLUTs, glucokinase) were measured. RESULTS: NIRKO mice revealed a glycemia-dependent impairment in the sympathoadrenal response to hypoglycemia and demonstrated markedly reduced (3-fold) hypothalamic c-fos activation in response to hypoglycemia but not other stressors. Glucose-inhibited neurons in the ventromedial hypothalamus of NIRKO mice displayed significantly blunted glucose responsiveness (membrane potential and input resistance responses were blunted 66 and 80%, respectively). Further, hypothalamic expression of the insulin-responsive GLUT 4, but not glucokinase, was reduced by 30% in NIRKO mice while regional brain glucose uptake remained unaltered. CONCLUSIONS: Chronically, insulin acts in the brain to regulate the counterregulatory response to hypoglycemia by directly altering glucose sensing in hypothalamic neurons and shifting the glycemic levels necessary to elicit a normal sympathoadrenal response.

Ventromedial hypothalamic nitric oxide production is necessary for hypoglycemia detection and counterregulation.

OBJECTIVE: The response of ventromedial hypothalamic (VMH) glucose-inhibited neurons to decreased glucose is impaired under conditions where the counterregulatory response (CRR) to hypoglycemia is impaired (e.g., recurrent hypoglycemia). This suggests a role for glucose-inhibited neurons in the CRR. We recently showed that decreased glucose increases nitric oxide (NO) production in cultured VMH glucose-inhibited neurons. These in vitro data led us to hypothesize that NO release from VMH glucose-inhibited neurons is critical for the CRR. RESEARCH DESIGN AND METHODS: The CRR was evaluated in rats and mice in response to acute insulin-induced hypoglycemia and hypoglycemic clamps after modulation of brain NO signaling. The glucose sensitivity of ventromedial nucleus glucose-inhibited neurons was also assessed. RESULTS: Hypoglycemia increased hypothalamic constitutive NO synthase (NOS) activity and neuronal NOS (nNOS) but not endothelial NOS (eNOS) phosphorylation in rats. Intracerebroventricular and VMH injection of the nonselective NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) slowed the recovery to euglycemia after hypoglycemia. VMH l-NMMA injection also increased the glucose infusion rate (GIR) and decreased epinephrine secretion during hyperinsulinemic/hypoglycemic clamp in rats. The GIR required to maintain the hypoglycemic plateau was higher in nNOS knockout than wild-type or eNOS knockout mice. Finally, VMH glucose-inhibited neurons were virtually absent in nNOS knockout mice. CONCLUSIONS: We conclude that VMH NO production is necessary for glucose sensing in glucose-inhibited neurons and full generation of the CRR to hypoglycemia. These data suggest that potentiating NO signaling may improve the defective CRR resulting from recurrent hypoglycemia in patients using intensive insulin therapy.

AMP-activated protein kinase and nitric oxide regulate the glucose sensitivity of ventromedial hypothalamic glucose-inhibited neurons.

The mechanisms by which glucose regulates the activity of glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH) are largely unknown. We have previously shown that AMP-activated protein kinase (AMPK) increases nitric oxide (NO) production in VMH GI neurons. We hypothesized that AMPK-mediated NO signaling is required for depolarization of VMH GI neurons in response to decreased glucose. In support of our hypothesis, inhibition of neuronal nitric oxide synthase (nNOS) or the NO receptor soluble guanylyl cyclase (sGC) blocked depolarization of GI neurons to decreased glucose from 2.5 to 0.7 mM or to AMPK activation. Conversely, activation of sGC or the cell-permeable analog of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), enhanced the response of GI neurons to decreased glucose, suggesting that stimulation of NO-sGC-cGMP signaling by AMPK is required for glucose sensing in GI neurons. Interestingly, the AMPK inhibitor compound C completely blocked the effect of sGC activation or 8-Br-cGMP, and 8-Br-cGMP increased VMH AMPKalpha2 phosphorylation. These data suggest that NO, in turn, amplifies AMPK activation in GI neurons. Finally, inhibition of the cystic fibrosis transmembrane regulator (CFTR) Cl(-) conductance blocked depolarization of GI neurons to decreased glucose or AMPK activation, whereas decreased glucose, AMPK activation, and 8-Br-cGMP increased VMH CFTR phosphorylation. We conclude that decreased glucose triggers the following sequence of events leading to depolarization in VMH GI neurons: AMPK activation, nNOS phosphorylation, NO production, and stimulation of sGC-cGMP signaling, which amplifies AMPK activation and leads to closure of the CFTR.

Characterization of glucosensing neuron subpopulations in the arcuate nucleus: integration in neuropeptide Y and pro-opio melanocortin networks?

Four types of responses to glucose changes have been described in the arcuate nucleus (ARC): excitation or inhibition by low glucose concentrations <5 mmol/l (glucose-excited and -inhibited neurons) and by high glucose concentrations >5 mmol/l (high glucose-excited and -inhibited neurons). However, the ability of the same ARC neuron to detect low and high glucose concentrations has never been investigated. Moreover, the mechanism involved in mediating glucose sensitivity in glucose-inhibited neurons and the neurotransmitter identity (neuropeptide Y [NPY] or pro-opio melanocortin [POMC]) of glucosensing neurons has remained controversial. Using patch-clamp recordings on acute mouse brain slices, successive extracellular glucose changes greater than and less than 5 mmol/l show that glucose-excited, high glucose-excited, glucose-inhibited, and high glucose-inhibited neurons are different glucosensing cell subpopulations. Glucose-inhibited neurons directly detect decreased glucose via closure of a chloride channel. Using transgenic NPY-green fluorescent protein (GFP) and POMC-GFP mice, we show that 40% of NPY neurons are glucose-inhibited neurons. In contrast, <5% of POMC neurons responded to changes in extracellular glucose >5 mmol/l. In vivo results confirm the lack of glucose sensitivity of POMC neurons. Taken together, hypo- and hyperglycemia are detected by distinct populations of glucosensing neurons, and POMC and NPY neurons are not solely responsible for ARC glucosensing.