Shinji Takamatsu

Dietary and genetic control of glucose transporter 2 glycosylation promotes insulin secretion in suppressing diabetes.

Pancreatic beta cell-surface expression of glucose transporter 2 (Glut-2) is essential for glucose-stimulated insulin secretion, thereby controlling blood glucose homeostasis in response to dietary intake. We show that the murine GlcNAcT-IVa glycosyltransferase is required for Glut-2 residency on the beta cell surface by constructing a cell-type- and glycoprotein-specific N-glycan ligand for pancreatic lectin receptors. Loss of GlcNAcT-IVa, or the addition of glycan-ligand mimetics, attenuates Glut-2 cell-surface half-life, provoking endocytosis with redistribution into endosomes and lysosomes. The ensuing impairment of glucose-stimulated insulin secretion leads to metabolic dysfunction diagnostic of type 2 diabetes. Remarkably, the induction of diabetes by chronic ingestion of a high-fat diet is associated with reduced GlcNAcT-IV expression and attenuated Glut-2 glycosylation coincident with Glut-2 endocytosis. We infer that beta cell glucose-transporter glycosylation mediates a link between diet and insulin production that typically suppresses the pathogenesis of type 2 diabetes.

Intra-tumoral distribution of 64Cu-ATSM: A comparison study with FDG

(64)Cu-labeled diacetyl-bis(N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising agent for internal radiation therapy and imaging of hypoxic tissues. In the present study, the intra-tumoral distribution of (64)Cu-ATSM was investigated by comparing it to that of [(18)F]FDG and histological findings. VX2 tumors were implanted into Japanese white rabbits subcutaneously. (64)Cu-ATSM and [(18)F]FDG were co-injected intravenously and the tumor was dissected and cut into 1 mm thick slices 1 h after the injection. The uptake of (64)Cu-ATSM and [(18)F]FDG was measured using a dual-tracer autoradiographic technique. Histological cell biology was estimated from the optical microscopy of tumor sections. The major accumulation of (64)Cu-ATSM was observed around the outer rim of the tumor masses which consisted mainly of active cells and expected to be hypoxic. [(18)F]FDG was distributed more widely with highest levels in the inner regions where pre-necrotic cells were mainly observed. (64)Cu-ATSM appears to be useful for the detection of hypoxic but active tumor cell regions in vivo.

Hypoxic regulation of glycosylation via the N-acetylglucosamine cycle

Glucose is an energy substrate, as well as the primary source of nucleotide sugars, which are utilized as donor substrates in protein glycosylation. Appropriate glycosylation is necessary to maintain the stability of protein, and is also important in the localization and trafficking of proteins. The dysregulation of glycosylation results in the development of a variety of disorders, such as cancer, diabetes mellitus and emphysema. Glycosylation is kinetically regulated by dynamically changing the portfolio of glycosyltransferases, nucleotide sugars, and nucleotide sugar transporters, which together form a part of what is currently referred to as the ”Glycan cycle”. An excess or a deficiency in the expression of glycosyltransferases has been shown to alter the glycosylation pattern, which subsequently leads to the onset, progression and exacerbation of a number of diseases. Furthermore, alterations in intracellular nucleotide sugar levels can also modulate glycosylation patterns. It is observed that pathological hypoxic microenvironments frequently occur in solid cancers and inflammatory foci. Hypoxic conditions dramatically change gene expression profiles, by activating hypoxia-inducible factor-1, which mediates adaptive cellular responses. Hypoxia-induced glycosyltransferases and nucleotide sugar transporters have been shown to modulate glycosylation patterns that are part of the mechanism associated with cancer metastasis. Hypoxia-inducible factor-1 also induces the expression of glucose transporters and various types of glycolytic enzymes, leading to shifts in glucose metabolic patterns. This fact strongly suggests that hypoxic conditions are an important factor in modulating various nucleotide sugar biosynthetic pathways. This review discusses some of the current thinking of how hypoxia alters glucose metabolic fluxes that can modulate cellular glycosylation patterns and consequently modify cellular functions, particularly from the standpoint of the N-acetylglucosamine cycle, a part of the ”Glycan cycle”.

Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice

N-Acetylglucosaminyltransferase-IV (GnT-IV) has two isoenzymes, GnT-IVa and GnT-IVb, which initiate the GlcNAcbeta1-4 branch synthesis on the Manalpha1-3 arm of the N-glycan core thereby increasing N-glycan branch complexity and conferring endogenous lectin binding epitopes. To elucidate the physiological significance of GnT-IV, we engineered and characterized GnT-IVb-deficient mice and further generated GnT-IVa/-IVb double deficient mice. In wild-type mice, GnT-IVa expression is restricted to gastrointestinal tissues, whereas GnT-IVb is broadly expressed among organs. GnT-IVb deficiency induced aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern that might be attributed to increased Ets-1, which conceivably activates the Mgat4a promoter, and thereafter preserved apparent GnT-IV activity. The compensative GnT-IVa expression might contribute to amelioration of the GnT-IVb-deficient phenotype. GnT-IVb deficiency showed mild phenotypic alterations in hematopoietic cell populations and hemostasis. GnT-IVa/-IVb double deficiency completely abolished GnT-IV activity that resulted in the disappearance of the GlcNAcbeta1-4 branch on the Manalpha1-3 arm that was confirmed by MALDI-TOF MS and GC-MS linkage analyses. Comprehensive glycomic analyses revealed that the abundance of terminal moieties was preserved in GnT-IVa/-IVb double deficiency that was due to the elevated expression of glycosyltransferases regarding synthesis of terminal moieties. Thereby, this may maintain the expression of glycan ligands for endogenous lectins and prevent cellular dysfunctions. The fact that the phenotype of GnT-IVa/-IVb double deficiency largely overlapped that of GnT-IVa single deficiency can be attributed to the induced glycomic compensation. This is the first report that mammalian organs have highly organized glycomic compensation systems to preserve N-glycan branch complexity.

A novel second isoenzyme of the human UDP-N-acetylglucosamine:alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase family: cDNA cloning, expression, and chromosomal assignment.

We isolated a novel cDNA encoding a second isoenzyme of UDP-N-acetylglucosamine:α1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GnT-IV; EC 2.4.1.145). The nucleotide and deduced amino acid sequences of the cDNA were homologous to those of the previously cloned human GnT-IV cDNA (63% and 62% identity, respectively). The new cDNA is also confirmed to express GnT-IV activity, suggesting that two isoenzymes of human GnT-IV exist. Although genomic Southern analysis suggested that both genes exist in many mammalian species and the chicken, northern analysis revealed that both genes are expressed in different ways in human tissues. This is the first report concerning the gene family of an N-acetylglucosaminyltransferase in mammals.

Neuroprotective effect of chronic lithium treatment against hypoxia in specific brain regions with upregulation of cAMP response element binding protein and brain-derived neurotrophic factor but not nerve growth factor: comparison with acute lithium treatment.

OBJECTIVES We evaluated the neuroprotective effect of chronically or acutely administered lithium against hypoxia in several brain regions. Furthermore, we investigated the contribution of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and cAMP response element binding protein (CREB) to the neuroprotective effect of lithium. METHODS Brain slices were prepared from rats that had been treated chronically or acutely with lithium. The cerebral glucose metabolic rate (CMRglc) before and after hypoxia loading to brain slices was measured using the dynamic positron autoradiography technique with [(18)F]2-fluoro-2-deoxy-D-glucose. The changes of expression of proteins were investigated using Western blot analysis. RESULTS Before hypoxia loading, the CMRglc did not differ between the lithium-treated and untreated groups. After hypoxia loading, the CMRglc of the untreated group was significantly lower than that before hypoxia loading. However, the CMRglc of the chronic lithium treatment group recovered in the frontal cortex, caudate putamen, hippocampus and cerebellum, but not in the thalamus. In contrast, the CMRglc of the acute lithium treatment group did not recover in any analyzed brain regions. After chronic lithium treatment, the levels of expression of BDNF and phospho-CREB were higher than those of untreated rats in the frontal cortex, but not in the thalamus. However, the expression of NGF did not change in the frontal cortex and thalamus. CONCLUSIONS These results demonstrated that lithium was neuroprotective against hypoxia only after chronic treatment and only in specific brain regions, and that CREB and BDNF might contribute to this effect.

Integrated approach toward the discovery of glyco-biomarkers of inflammation-Related diseases

Glycobiology has contributed tremendously to the discovery and characterization of cancer‐related biomarkers containing glycans (i.e., glyco‐biomarkers) and a more detailed understanding of cancer biology. It is now recognized that most chronic diseases involve some elements of chronic inflammation; these include cancer, Alzheimers disease, and metabolic syndrome (including consequential diabetes mellitus and cardiovascular diseases). By extending the knowledge and experience of the glycobiology community regarding cancer biomarker discovery, we should be able to contribute to the discovery of diagnostic/prognostic glyco‐biomarkers of other chronic diseases that involve chronic inflammation. Future integration of large‐scale “omics”‐type data (e.g., genomics, epigenomics, transcriptomics, proteomics, and glycomics) with computational model building, or a systems glycobiology approach, will facilitate such efforts.

The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytes/macrophages through the DAP12–Syk pathway

We previously demonstrated that Siglec-15, a member of the Siglec family of glycan-recognition proteins, is expressed on a subset of macrophages and preferentially recognizes the sialyl-Tn (sTn) antigen, a tumor-associated glycan structure. In this study, we report on the biological significance of the Siglec-15-mediated interaction between monocytes/macrophages and cancer cells. Siglec-15 is expressed on tumor-associated macrophages (TAMs) in various human tumor tissues. We further demonstrated that its expression is substantially elevated in macrophage colony-stimulating factor-induced M2-like macrophages, which produced more transforming growth factor-β (TGF-β) in response to sTn-positive cells than to negative cells. We designed a co-culture model of THP-1 (human monocytic leukemia) cells and H157 (human lung carcinoma) cells mimicking the interaction between monocytes/macrophages and cancer cells that recapitulated the enhanced TGF-β production in Siglec-15 expressing THP-1 cells by the cellular interaction with sTn expressing H157 cells. The enhanced TGF-β production required a direct interaction between the two cell lines through sialic acids. Siglec-15 associates with adaptor protein DNAX activation protein of 12 kDa (DAP12) at the binding determinant Lys(274) in the transmembrane domain and transduces a signal to spleen tyrosine kinase (Syk). The enhanced TGF-β secretion was significantly attenuated by Syk inhibitor treatment of THP-1 cells or by substitution of the Siglec-15 Lys(274) to Ala, which disrupts the molecular interaction between Siglec15 and DAP12. These findings indicate that Siglec-15 recognizes the tumoral sTn antigen and transduces a signal for enhanced TGF-β secretion in TAMs and further suggest that Siglec-15 on macrophages may contribute to tumor progression by the TGF-β-mediated modulation of intratumoral microenvironments.

Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: Implication of aberrant transforming growth factor-β signaling and matrix metalloproteinase gene expression

Background: Fut8−/− mice show emphysematous lesions, the major risk factor for which is exposure to cigarette smoke (CS). Results: Fut8+/− mice developed CS-induced emphysematous lesions, which are associated with an aberrant Smad7-Smad2-matrix metalloproteinase signaling pathway. Conclusion: Genetic ablation of Fut8 increases sensitivity to CS-induced emphysema. Significance: Core fucosylation appears to be involved in the development of chronic obstructive pulmonary disease. We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-β1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8+/−) that were exposed to cigarette smoke (CS). The Fut8+/− mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8+/− mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-β-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.

A novel noninvasive diagnostic method for nonalcoholic steatohepatitis using two glycobiomarkers.

Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem; thus, discriminating nonalcoholic steatohepatitis (NASH) from NAFLD is of great clinical significance. For the diagnosis of NASH, liver biopsy‐proven histological examination is the current gold standard, and noninvasive and reliable biomarkers are greatly needed. Recently, we found that two glycobiomarkers, fucosylated haptoglobin (Fuc‐Hpt) and Mac‐2 binding protein (Mac2bp), are useful independently for NASH diagnosis. In this study, we confirmed that serum Fuc‐Hpt is suitable for the prediction of ballooning hepatocytes and that serum Mac2bp is suitable for the prediction of liver fibrosis severity in 124 biopsy‐proven NAFLD patients (training cohort). In addition, we found that the combination of serum Fuc‐Hpt and Mac2bp levels was an excellent tool for NASH diagnosis. Using receiver operating characteristic analyses, the area under the receiver operating characteristic curve, sensitivity, and specificity of the combination of these two glycobiomarkers were 0.854, 81.1%, and 79.3%, respectively. We established a prediction model for NASH diagnosis using logistic regression analysis: logit (p) = −2.700 + 0.00242 × Fuc‐Hpt + 1.225 × Mac2bp. To validate the prediction model, another 382 biopsy‐proven NAFLD patients were enrolled (validation cohort). In the validation cohort, the area under the receiver operating characteristic curve of this model for NASH diagnosis was 0.844, with 71.4% and 82.3% sensitivity and specificity, respectively. In addition, we investigated the significance of our developed NASH diagnosis model in ultrasound‐diagnosed NAFLD subjects who received medical health checkups (n = 803). Our model also could predict NAFLD disease severity in this larger population. Conclusion: The combination of serum Fuc‐Hpt and Mac2bp can distinguish NASH from NAFLD patients. Our noninvasive model using two serum glycobiomarkers contributes to a novel NASH diagnostic methodology that could replace liver biopsy. (Hepatology 2015;62:1433–1443)