Antiobesity Effect of N-Acetylneuraminic Acid by Enhancing Antioxidative Capacity in Mice Fed a High-Fat Diet.

The sialic acid N-acetylneuraminic acid (NANA), an essential factor in bioregulation, is a functional food component that is known to have beneficial health effects, but its antiobesity effect has not been clearly understood. Adipocyte dysfunction in obesity involves a decrease in the level of NANA sialylation. In this study, we investigated the antiobesity effect of NANA in mice fed a high-fat diet (HFD) and in 3T3-L1 adipocytes. Male C57BL/6J mice were randomly divided into three groups and administered the following diets: a normal diet, an HFD, and an HFD with 1% NANA supplementation for 12 weeks. NANA supplementation significantly reduced body weight gain; epididymal adipose tissue hypertrophy; and serum lipid, fasting glucose, and aspartate transaminase levels compared with those in HFD mice. The percentage of lipid droplets in hepatic tissue was also decreased by NANA supplementation in HFD mice. The downregulation of Adipoq expression and upregulation of Fabp4 expression induced by HFD in epididymal adipocytes were improved by NANA supplementation. The downregulation of Sod1 expression and increase in malondialdehyde level were induced by HFD, and they were significantly improved in the liver by NANA supplementation, but not in epididymal adipocytes. However, NANA supplementation had no effect on sialylation and antioxidant enzyme levels in mouse epididymal adipocytes and 3T3-L1 adipocytes. Overall, NANA exerts antiobesity and antihypolipidemic effects and may be beneficial in suppressing obesity-related diseases.

The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid is a glucose-dependent potentiator of insulin secretion.

Sialidase cleaves sialic acid residues from a sialoglycoconjugate: oligosaccharides, glycolipids and glycoproteins that contain sialic acid. Histochemical imaging of the mouse pancreas using a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe used to assess sialidase activity, showed that pancreatic islets have intense sialidase activity. The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) remarkably enhances glutamate release from hippocampal neurons. Since there are many similar processes between synaptic vesicle exocytosis and secretory granule exocytosis, we investigated the effect of DANA on insulin release from ß-cells. Insulin release was induced in INS-1D cells by treatment with 8.3 mM glucose, and the release was enhanced by treatment with DANA. In a mouse intraperitoneal glucose tolerance test, the increase in serum insulin levels was enhanced by intravenous injection with DANA. However, under fasting conditions, insulin release was not enhanced by treatment with DANA. Calcium oscillations induced by 8.3 mM glucose treatment of INS-1D cells were not affected by DANA. Blood insulin levels in sialidase isozyme Neu3-deficient mice were significantly higher than those in WT mice under ad libitum feeding conditions, but the levels were not different under fasting conditions. These results indicate that DANA is a glucose-dependent potentiator of insulin secretion. The sialidase inhibitor may be useful for anti-diabetic treatment with a low risk of hypoglycemia.

A mouse model and 19 F NMR approach to investigate the effects of sialic acid supplementation on cognitive development.

Researchers have observed that a sialic acid (Sia)-supplemented neonatal diet leads to improved cognition in weanling piglets. However, whether cognitive improvement appears with different physiological backgrounds and persists into adulthood is not known. Here, we have established a convenient mouse model and used an 19 F NMR approach to address these questions, test the conditionally essential nutrient hypothesis about Sia supplementation, and assess the prospect of measuring Sia metabolism directly in vivo. Indeed, the neonatal mouse brain uptakes more Sia than the adult brain, and Sia supplementation of neonatal mice improves the cognitive performance of adult mice. The non-invasive 19 F NMR approach and viable mouse model opens unique opportunities for clarifying the interplay of nutritional supplementation, metabolism, and cognitive development.

Effects of oligosaccharide-sialic acid (OS) compound on maternal-newborn gut microbiome, glucose metabolism and systematic immunity in pregnancy: protocol for a randomised controlled study.

INTRODUCTION: The gut microbiota participates in multiple human biological processes, including metabolism and immune responses. During pregnancy, the dynamics of gut microbiota is involved in physiological adaptation. The disturbed profile of microbiome is associated with maternal complications, such as gestational diabetes mellitus (GDM), which further transfers to the offspring and influence their metabolic and immunological functions in the long term. Prebiotics targeting the gut microbiota and modulating metabolic and immune functions have been shown to be effective in non-pregnant populations with metabolic syndrome. Hence, we propose the use of a prebiotic supplement, oligosaccharide-sialic acid (OS) from the first trimester until delivery in pregnant women, can benefit maternal/new-born gut microbiome, glucose metabolism and innate immunity. METHODS AND ANALYSIS: In this prospective double-blinded randomised clinical trial, recruited singleton pregnancies will be stratified by body mass index (BMI) and randomly assigned to consume the OS preparation or placebo daily from the first trimester. At seven later time points (before and after recruitment in the first trimester, in the middle and third trimesters, before delivery, at birth and 42 days postpartum), compliance will be evaluated and/or biological samples will be collected. Along with maternal clinical information, questionnaires on lifestyle and infant development will be recorded. The primary outcomes are the effect of OS on the maternal-offspring gut microbiome and GDM incidence. The secondary outcomes are maternal glycolipid biochemical parameters, cytokine profiles, weight gain during pregnancy and infant morbidities, growth and development. The study aims to validate the effects of OS on reducing maternal morbidity within different BMI groups. The multiple dimensional dataset generated from the study includes clinical and lifestyle-related information, various biological markers and associated protective or risk factors for morbidity and prognosis. An extended follow-up through 42 days after birth could further explore the intrauterine influence on the long-term health of offspring. ETHICS AND DISSEMINATION: This protocol has been approved by Peking University First Hospital, National Unit of Clinical Trial Ethics Committee (reference number: 164). The results are expected to be published in scientific manuscripts by 2021. TRIAL REGISTRATION NUMBER: ChiCTR1800017192.

Targeted cancer imaging and photothermal therapy via monosaccharide-imprinted gold nanorods.

Plasmonic nanomaterials have been widely used for photothermal therapy (PTT) of cancer, but their recognition specificity remains challenging. We prepared monosaccharide-imprinted gold nanorods (AuNRs) for targeted cancer PTT, using sialic acid (SA) as a representative monosaccharide. The SA-imprinted AuNRs exhibited good specificity, enabling the killing of cancer cells without damaging healthy cells.

N-Acetylneuraminic Acid Supplementation Prevents High Fat Diet-Induced Insulin Resistance in Rats through Transcriptional and Nontranscriptional Mechanisms.

N-Acetylneuraminic acid (Neu5Ac) is a biomarker of cardiometabolic diseases. In the present study, we tested the hypothesis that dietary Neu5Ac may improve cardiometabolic indices. A high fat diet (HFD) + Neu5Ac (50 or 400 mg/kg BW/day) was fed to rats and compared with HFD + simvastatin (10 mg/kg BW/day) or HFD alone for 12 weeks. Weights and serum biochemicals (lipid profile, oral glucose tolerance test, leptin, adiponectin, and insulin) were measured, and mRNA levels of insulin signaling genes were determined. The results indicated that low and high doses of sialic acid (SA) improved metabolic indices, although only the oral glucose tolerance test, serum triglycerides, leptin, and adiponectin were significantly better than those in the HFD and HFD + simvastatin groups (P < 0.05). Furthermore, the results showed that only high-dose SA significantly affected the transcription of hepatic and adipose tissue insulin signaling genes. The data suggested that SA prevented HFD-induced insulin resistance in rats after 12 weeks of administration through nontranscriptionally mediated biochemical changes that may have differentially sialylated glycoprotein structures at a low dose. At higher doses, SA induced transcriptional regulation of insulin signaling genes. These effects suggest that low and high doses of SA may produce similar metabolic outcomes in relation to insulin sensitivity through multiple mechanisms. These findings are worth studying further.

High fat diet-induced inflammation and oxidative stress are attenuated by N-acetylneuraminic acid in rats.

BACKGROUND: Serum sialic acid levels are positively correlated with coronary artery disease and inflammation. Although sialic acid is a non-specific marker, it is considered sensitive likely due to its influence in sialylation of glycoprotein structures all over the body. OBJECTIVES: We hypothesized that dietary supplementation with N-acetylneuraminic acid (Neu5Ac), a type of sialic acid, will have profound effects on high fat diet- (HFD-) induced inflammation and oxidative stress in view of the widespread incorporation of sialic acid into glycoprotein structures in the body. METHODS: HFD-fed rats with or without simvastatin or Neu5Ac (50 and 400 mg/kg/day) were followed up for 12 weeks. Lipid profiles, and markers of inflammation (C-reactive protein, interleukin-6, and tumor necrosis factor alpha), insulin resistance (serum insulin and adiponectin, oral glucose tolerance test and homeostatic model of insulin resistance) and oxidative stress (total antioxidant status and thiobarbituric acid reactive species) in the serum and liver were determined, while mRNA levels of hepatic antioxidant and inflammation genes were also quantified. Serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase, urea, creatinine and uric acid were also assessed. RESULTS: HFD feeding caused hyperlipidemia and insulin resistance, and worsened liver and kidney functions. HFD feeding also potentiated inflammation and oxidative stress, partly through modulation of hepatic gene expression, while Neu5Ac especially at higher doses and simvastatin attenuated HFD-induced changes, although Neu5Ac showed better outcomes. CONCLUSIONS: Based on the present results, we surmised that Neu5Ac can prevent HFD-induced inflammation and oxidative stress, and may in fact be useful in the prevention of hyperlipidemia-associated inflammation and oxidative stress. However, the translational implications of these findings can only be determined after long-term effects are established. Hence, the use of Neu5Ac on obesity-related diseases requires additional attention.

Sialic acid supplementation ameliorates puromycin aminonucleoside nephrosis in rats.

Defects in sialylation are known to have serious consequences on podocyte function leading to collapse of the glomerular filtration barrier and the development of proteinuria. However, the cellular processes underlying aberrant sialylation in renal disease are inadequately defined. We have shown in cultured human podocytes that puromycin aminonucleoside (PAN) downregulates enzymes involved in sialic acid metabolism and redox homeostasis and these can be rescued by co-treatment with free sialic acid. The aim of the current study was to ascertain whether sialic acid supplementation could improve renal function and attenuate desialylation in an in vivo model of proteinuria (PAN nephrosis) and to delineate the possible mechanisms involved. PAN nephrotic rats were supplemented with free sialic acid, its precursor N-acetyl mannosamine or the NADPH oxidase inhibitor apocynin. Glomeruli, urine, and sera were examined for evidence of kidney injury and therapeutic efficacy. Of the three treatment regimens, sialic acid had the broadest efficacy in attenuating PAN-induced injury. Proteinuria and urinary nephrin loss were reduced. Transmission electron microscopy revealed that podocyte ultrastructure, exhibited less severe foot process effacement. PAN-induced oxidative stress was ameliorated as evidenced by a reduction in glomerular NOX4 expression and a downregulation of urine xanthine oxidase levels. Sialylation dysfunction was improved as indicated by reduced urinary concentrations of free sialic acid, restored electrophoretic mobility of podocalyxin, and improved expression of a sialyltransferase. These data indicate that PAN induces alterations in the expression of enzymes involved in redox control and sialoglycoprotein metabolism, which can be ameliorated by sialic acid supplementation possibly via its properties as both an antioxidant and a substrate for sialylation.

Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes.

Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx-crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid. PAN treatment of podocytes resulted in a loss of sialic acid from podocyte proteins. This was accompanied by a reduction, in the expression of sialyltransferases and a decrease in the key enzyme of sialic acid biosynthesis N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). PAN treatment also attenuated expression of the antioxidant enzyme superoxide dismutase (mSOD) and concomitantly increased the generation of superoxide anions. Sialic acid supplementation rescued podocyte protein sialylation and partially restored expression of sialyltransferases. Sialic acid also restored mSOD mRNA expression and quenched the oxidative burst. These data suggest that PAN-induced aberrant sialylation occurs as a result of modulation of enzymes involved sialic acid metabolism some of which are affected by oxidative stress. These data suggest that sialic acid therapy not only reinstates functionally important negative charge but also acts a source of antioxidant activity.

Preferential lectin binding of cancer cells upon sialic acid treatment under nutrient deprivation.

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.

Evaluation of sialic acid-analogs for the attenuation of amyloid-beta toxicity.

BACKGROUND: Amyloid-beta peptide (Aß) is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease (AD). To that end, agents which either sequester Aß or interfere with Aß interaction/binding to cells have been investigated as a means to reduce the pathological effects of Aß. METHODS: Different structural analogs of sialic acid (N-acetylneuramic acid) were used to decorate a chitosan backbone using EDC chemistry. FTIR and colorimetric assays were used to characterize the complexes. The ability of these complexes to attenuate Aß toxicity was investigated in vitro using a model neuroblastoma cell line SH-SY5Y. RESULTS: Oxygen substitution in ring structure is responsible for the increase in toxicity and increase in protective properties of the complexes. Also, the multi OH tail present in sialic acid is critical to attenuate toxicity. Analogs show no protective properties which reinforces the conclusion that clustering of sugars in cellular membranes play a significant role in Aß binding. CONCLUSIONS: Successfully produced compounds that showed varying degree of efficacy in attenuating Aß toxicity to cells in culture. This work elucidates the impact that certain structures of sialic acid and its analogs can have on Aß binding. It will allow for more specific and detailed improvements in the therapeutic polysaccharide structures that can be developed and modified to overcome other shortcomings of AD therapeutic development, particularly of penetrating the blood-brain barrier. GENERAL SIGNIFICANCE: Oxygen atom plays crucial role on therapeutic effectiveness. This work can help as a general guideline for further therapeutic development.

Sialic acid feeding aged rats rejuvenates stimulated salivation and colon enteric neuron chemotypes.

Old age is linked to numerous changes of body functions such as salivation, gastrointestinal motility, and permeability all linked to central and enteric nervous system decline. Thus, gut motility and barrier functions suffer. Sialic acid plays a key role in the nervous system at large and for many receptor functions specifically. Decreased sialylation in the elderly suggests an endogenous sialic acid deficit. We used a rat model of aging, to ask whether sialic acid feeding would affect (i) stimulated salivation, (ii) gut functions, and (iii) sialic acid levels and neuronal markers in brain and gut. We observed reduced levels of pilocarpine-stimulated salivation in old versus young rats and restored this function by sialic acid feeding. Brain ganglioside bound sialic acid levels were found lower in aged versus young rats, and sialic acid feeding partly restored the levels. The hypothalamic expression of cholinergic and panneuronal markers was reduced in aged rats. The expression of the nitrergic marker nNOS was increased upon sialic acid feeding in aged rats. Neither fecal output nor gut permeability was different between young and aged rats studied here, and sialic acid feeding did not alter these parameters. However, the colonic expression of specific nervous system markers nNOS and Uchl1 and the key enzyme for sialic acid synthesis GNE were differentially affected in young and aged rats by sialic acid feeding indicating that regulatory mechanisms change with age. Investigation of sialic acid supplementation as a functional nutrient in the elderly may help those who suffer from disorders of reduced salivation. Further research is needed to understand the differential effects of sialic acid feeding in young and aged rats.

A novel sialic acid-specific lectin from Phaseolus coccineus seeds with potent antineoplastic and antifungal activities.

A novel lectin (PCL) with specificity towards sialic acid was purified from Phaseolus coccineus L. (P. multiflorus willd) seeds using ion exchange chromatography on CM and DEAE-Sepharose, and gel filtration on Sephacryl S-200 column. PCL was a homodimer consisting of 29,831.265 Da subunits as determined by gel filtration and MS. Also, PCL was a non-metalloprotein and its N-terminal 23-amino acid sequence, ATETSFSFQRLNLANLVLNKESS, was determined. Subsequently, MTT method, cell morphological analysis and LDH activity-based cytotoxicity assays demonstrated that PCL was highly cytotoxic to L929 cells and induced apoptosis in a dose-dependent manner. Using caspase inhibitors, a typical caspase-dependent pathway was confirmed. PCL also showed remarkable antifungal activity towards some plant pathogenic fungi. Furthermore, when sialic acid-specific activity was fully inhibited, cytotoxicity and antifungal activity were abruptly decreased, respectively, suggesting a significant correlation between sialic acid-specific site and its bi-functional bioactivities.

Gating of the shaker potassium channel is modulated differentially by N-glycosylation and sialic acids.

N-linked glycans, including sialic acids, are integral components of ion channel complexes. To determine if N-linked sugars can modulate a rapidly inactivating K+ channel, the glycosylated Drosophila melanogaster Shaker K+ channel (ShB) and the N-glycosylation-deficient mutant (ShNQ), were studied under conditions of full and reduced sialylation. Through an apparent electrostatic mechanism, full sialylation induced uniform and significant hyperpolarizing shifts in all measured voltage-dependent ShB gating parameters compared to those measured under conditions of reduced sialylation. Steady-state gating of ShNQ was unaffected by changes in sialylation and was nearly identical to that observed for ShB under conditions of reduced sialylation, indicating that N-linked sialic acids were wholly responsible for the observed effects of sialic acid on ShB gating. Interestingly, the rates of transition among channel states and the voltage-independent rates of activation and inactivation were significantly slower for ShNQ compared to ShB. Both effects were independent of sialylation, indicating that N-linked sugars other than sialic acids alter ShB gating kinetics but have little to no effect on the steady-state distribution of channels among states. The effect of sialic acids on channel gating, particularly inactivation gating, and the impact of other N-linked sugars on channel gating kinetics are unique to the ShB isoform. Thus, ShB gating is modulated by two complementary but distinct sugar-dependent mechanisms, (1) an N-linked sialic acid-dependent surface charge effect and (2) a sialic acid-independent effect that is consistent with N-linked sugars affecting the stability of ShB among its functional states.

Dietary sialic acid supplementation improves learning and memory in piglets.

BACKGROUND: Sialic acid, a key component of both human milk oligosaccharides and neural tissues, may be a conditional nutrient during periods of rapid brain growth. OBJECTIVE: We tested the hypothesis that variations in the sialic acid content of a formula milk would influence early learning behavior and gene expression of enzymes involved in sialic acid metabolism in piglets. DESIGN: Piglets (n = 54) were allocated to 1 of 4 groups fed sow milk replacer supplemented with increasing amounts of sialic acid as casein glycomacropeptide for 35 d. Learning performance and memory were assessed with the use of easy and difficult visual cues in an 8-arm radial maze. Brain ganglioside and sialoprotein concentrations and mRNA expression of 2 learning-associated genes (ST8SIA4 and GNE) were measured. RESULTS: In both tests, the supplemented groups learned in significantly fewer trials than did the control group, with a dose-response relation for the difficult task (P = 0.018) but not the easy task. In the hippocampus, significant dose-response relations were observed between amount of sialic acid supplementation and mRNA levels of ST8SIA4 (P = 0.002) and GNE (P = 0.004), corresponding with proportionate increases in protein-bound sialic acid concentrations in the frontal cortex. CONCLUSIONS: Feeding a protein-bound source of sialic acid during early development enhanced learning and increased expression of 2 genes associated with learning in developing piglets. Sialic acid in mammalian milks could play a role in cognitive development.