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Research Notes: MannoseSee also: Biochemistry. 2005 Jan 11. Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose. J Biol Chem. 2004 Mar 12. The biguanide drug metformin stimulates AMP-activated protein kinase, a master regulator of cellular energy metabolism, and has antihyperglycemic activity due to attenuation of gluconeogenesis in hepatocytes and 2-fold stimulation of glucose transport by skeletal muscle. Here we identify a metformin-stimulated d-mannose transport (MSMT) activity in dermal fibroblasts. MSMT increased mannose uptake 1.8-fold and had greater affinity for mannose than basal mannose transport activity. It was attributed to robust stimulation of a transporter expressed weakly in untreated cells. MSMT was not explained by greater glucose transporter activity because metformin unexpectedly decreased transport of 2-deoxy-d-glucose and 3-O-methyl-d-glucose by fibroblasts. Effective inhibitors of MSMT retained specificity for the 3-, 4-, and 6-OH groups of the mannose ring but not the 2-OH group. Thus, MSMT could be strongly inhibited by glucose and 2-deoxy-d-glucose even though the latter was not a good transport substrate. MSMT was significant because in the presence of 2.5 microm mannose, metformin corrected experimentally induced deficiencies in the synthesis of glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol and N-linked glycosylation. MSMT was also identified in congenital disorder of glycosylation types Ia and Ib fibroblasts, and metformin acted synergistically with 100 microm mannose to correct lipid-linked oligosaccharide synthesis and N-glycosylation in the Ia cells. In conclusion, metformin activates a novel fibroblast mannose-selective transport system. This suggests that AMP-activated protein kinase may be a regulator of mannose metabolism and implies a therapy for congenital disorders of glycosylation-Ia. From the full text article: D-Mannose is a component of many eukaryotic glycoconjugates, such as asparagine (N)-linked glycans and their precursor lipid-linked oligosaccharides (LLO),1 glycosylphosphatidylinositols, glycosylphosphatidylinositol-anchored proteins, mannose-containing serine/threonine (O)-linked glycans, and C-mannosyltryptophan (1). By metabolic conversion, extracellularly derived D-glucose can be a major source of D-mannosyl residues. This is illustrated by the human genetic disease congenital disorder of glycosylation (CDG) type Ib, which is characterized by defective phosphomannose isomerase and deficient conversion of the glucose metabolite Fru-6-P to Man-6-P (2). Man-6-P is converted to Man-1-P by phosphomannomutase, which generates GDP-mannose. GDP-mannose is used to form mannose-P-dolichol, and both are required for synthesis of complete LLOs. Thus, CDG-Ib cells have diminished LLO production and incomplete N-linked glycosylation (2). Extracellularly derived mannose itself is also a significant physiological precursor of mannosyl residues. It is well known that [3H]mannose can be incorporated into glycoproteins when added to eukaryotic cell cultures. In dermal fibroblasts, this process is selective over glucose (3). Furthermore, [3H]mannose is incorporated into glycoproteins when injected into mice (4), and a mannose-selective transporter has been identified in human dermal fibroblasts (5). Extracellular mannose can counteract abrogated LLO synthesis caused by culturing normal fibroblasts in glucose-deficient medium (6), and it can correct the LLO defects in cultured CDG-Ia (phosphomannomutase-deficient) and CDG-Ib cells (2, 7, 8). Paradoxically, disease in CDG-Ia patients is not corrected by dietary mannose therapy, and CDG-Ia transferrin remains underglycosylated (9). CDG-Ib is successfully treated with dietary mannose, but excess quantities of mannose can cause side effects such as bloating and kidney damage (10). Thus, in both cases mannose therapy might be more effective if its uptake by cells could be improved. Given the importance of mannose transport activity in normal physiology, as well as the potential for treatment of glycosylation disorders by mannose supplementation, we sought to identify agents that would enhance mannose transport. Metformin is a biguanide drug used to treat type II diabetes and has antihyperglycemic properties (11, 12) due to its ability to stimulate glucose transport activity in skeletal muscle about 2-fold (13–15) and decrease hepatic gluconeogenesis (16). Metformin acts by stimulating AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis in liver, skeletal muscle, adipocytes, and pancreatic islets (14, 17). AMPK is normally activated when cellular AMP concentrations rise relative to ATP, such as during vigorous exercise, and triggers a set of responses that help restore the AMP/ATP ratio to normal. Although an activator, metformin does not appear to interact with AMPK directly (18). In this study we investigated the effects of metformin on mannose transport and protein glycosylation. Because these processes are not well characterized in skeletal muscle, experiments were performed instead with dermal fibroblasts from both normal and CDG type I patients. A metformin-stimulated mannose transport (MSMT) activity was identified. The stimulation of mannose transport, although only roughly 2-fold, is comparable with the 2-fold effect of metformin on glucose transport by skeletal muscle. This was not due to the doubling of activity of a single transporter but rather an increase in the activity of a novel transporter that has little contribution in untreated cells. Unexpectedly, metformin decreased glucose transport activity in fibroblasts, a distinction with MSMT. Finally, we show that the effects of metformin can contribute a major fraction of the hexose necessary to assemble lipid-linked oligosaccharides and that metformin has the potential to counteract the glycosylation defect in CDG-Ia cells. ... MSMT in Congenital Disorder of Glycosylation Type Ia and Ib Cells — LLO synthesis is abrogated in type I CDG (9, 25). Type Ia (phosphomannomutase-deficient) and Ib (phosphomannose isomerase-deficient) CDG each result in diminished GDP-mannose production (8). However, the effects on the LLO pools differ. Type Ia cells accumulate LLO intermediates, resulting in a pattern with predominantly Man2–5GlcNAc2-P-P-dolichol. Thus, a key hallmark of the CDG-Ia phenotype is a relatively high percentage of undermannosylated glycans found on newly synthesized glycoproteins. Because undermannosylated LLOs are poor donor substrates for oligosaccharyltransferase, CDG-Ia glycoproteins also tend to be underglycosylated. By comparison, type Ib cells appear to contain fewer LLO molecules, but they are extended efficiently to Glc3Man9GlcNAc2-P-P-dolichol. As mentioned earlier, CDG-Ib patients can be treated effectively with dietary mannose (2), but curiously, therapy with dietary mannose is not effective for CDG-Ia patients (9). Metformin increased mannose uptake in CDG-Ia (Fig. 10, panel a) and CDG-Ib (panel b) cells. The potential effects of metformin on LLO synthesis in CDG-Ib cells were difficult to assess because extension was already efficient, but the abnormal LLO pool in CDG-Ia cells was more amenable to analysis. Although proper LLO synthesis in normal cells incubated with 0.5 mM glucose was achieved by adding 0.1 mM mannose (Fig. 7), 0.1 mM mannose supplementation had only a modest effect on glycosylation in CDG-Ia cells (Fig. 11, panels a, c, e, and g). By itself, metformin did not affect CDG-Ia LLO synthesis (Fig. 11, panels a and b) or the types of oligosaccharides transferred to protein (panels e and f). However, metformin acted synergistically with mannose. By combining 2 mM metformin treatment with 0.1 mM mannose supplementation, extension of LLO intermediates to Glc3Man9GlcNAc2-P-P-dolichol increased from 5 (panel c) to 19% (panel d) of the LLO pool. The fraction of highly mannosylated glycans on protein increased from 46 (panel g) to 69% (panel h), and the resulting HPLC profiles of N-glycans resembled those from normal cells labeled for the same period under optimal conditions with 0.1 mM mannose (data not shown). Similar data for CDG-Ia cells were observed with 0.5 and 5.0 mM metformin. As indicated by Fig. 8, these results can be attributed to the dual effects of metformin on fibroblast LLO synthesis, one of which is due to increased mannose uptake. The CDG-Ia phenotype may be tissue-dependent (26), and it is difficult to predict how the results obtained here with dermal fibroblasts apply to cells of other tissues. However, in CDG-Ia patients, metformin may be a useful adjuvant for dietary mannose therapy, which by itself is ineffective. Similarly, metformin may allow CDG-Ib patients to be treated with lower quantities of mannose. Implications for AMPK as a Regulator of Mannose Metabolism and Protein Glycosylation in Dermal Fibroblasts — The only known target of metformin is AMPK (18), and its role in mannose transport has not been reported. In skeletal muscle AMPK activation results in fusion of intracellular vesicles containing glucose transporters with the cell surface (14). However, because conventional glucose transport activity was not increased in metformin-treated fibroblasts, this is not a likely cause of the enhanced mannose uptake. Taken together, these data support the possibility that a population of transporters in skin fibroblasts with a selectivity for mannose, distinct from those already described (5), may respond to metformin treatment. Increased mannose uptake due to activation of AMPK may also explain earlier paradoxical results (27) in which the LLO extension defect in CDG-Ia cells was corrected by 12 h of glucose deprivation. This result was surprising, because withdrawal of a source of hexose precursors for LLO extension would be expected to make the problem worse. It is important to understand the mechanism of this effect because, as discussed above, there is currently no effective treatment for CDG-Ia. Extended glucose deprivation would be expected to activate AMPK. As shown with metformin, mannose uptake should then be increased. Because mannose is a much more efficient precursor for LLO synthesis than glucose (3, 4), even a small increase of mannose transport might be effective for correction of the CDG-Ia defect. Possible Biological Function of MSMT — Two aspects of this study suggest that the function of the transport process stimulated by metformin may be specialized. First, MSMT is completely inhibited by physiological concentrations of glucose in the blood (typically 5 mM). Second, with physiological glucose, LLO synthesis in fibroblasts is highly efficient and does not require additional mannose (24). In this regard, the biological role of the dermal fibroblast should be considered. Typically quiescent in healthy skin, fibroblasts aid in the repair of damaged skin by replicating to form granulation tissue and by producing extracellular components. In the absence of a well formed dermal microvasculature, the supply of precursors for extracellular components such as glucose and mannose may be variable. Indeed, direct microprobe measurements of glucose concentrations at sites of skin inflammation were four times lower than blood glucose concentrations (28). It is therefore plausible that signals within the granulation tissue, perhaps associated with compromised access to glucose, activate AMPK to stimulate processes such as mannose transport. Activation of AMPK may promote uptake of mannose as an alternative energy source to compensate for the lack of glucose, as well as an alternative for glucose as a precursor for glycan synthesis. MSMT has an EC50 for mannose of 500 µM (Fig. 2) and was easily detected with both 0.6 and 100 µM mannose (Fig. 4). As shown in Fig. 8, the potential contribution of MSMT to glycan synthesis was significant with 2.5 µM mannose. Because blood contains 20–50 µM mannose (4), even a small fraction of mannose originating from the circulation would be effective. J Biol Chem. 1997 Sep 12. Mannose in N-linked oligosaccharides is assumed to be derived primarily from glucose through phosphomannose isomerase (PMI). The discovery of mammalian mannose-specific transporters that function at physiological concentrations suggested that mannose might directly contribute to oligosaccharide synthesis. To determine the relative contribution of glucose and mannose, human fibroblasts were labeled with either [2-3H]mannose or [1,5,6-3H]glucose at the same specific activity, and the N-linked chains were released by PNGase F digestion. Most of the trichloroacetic acid-precipitable [3H]mannose label was released by this digestion, but only about 10% of the trichloroacetic acid-precipitable material was released from cells labeled with [1,5,6-3H]glucose. Both sugars labeled a similar array of oligosaccharides, and acid hydrolysis of these chains showed that [2-3H]mannose contributed 65-75% of the [3H]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose. Mannose consumption and [2-3H]mannose utilization were within the range of rates expected for mannose transport via the mannose-specific transporter. About 7-14% of the [2-3H]mannose is used for glycosylation, while the rest (86-93%) is catabolized to 3H2O via PMI. Increasing the exogenous mannose concentration beyond mannose transporter saturation results in the conversion of >99% of [2-3H]mannose into 3H2O. Long term labeling of cells with [2-3H]mannose showed that the specific activity of mannose in glycoproteins reached 77% of the specific activity of [2-3H]mannose added to the medium. These results show that when fibroblasts are provided with physiological concentrations of mannose, they use the mannose-specific transporter to supply the majority of mannose needed for glycoprotein synthesis. PMI may normally be used to catabolize excess mannose rather than to primarily supply Man-6-P for glycoprotein synthesis. From the full text article: Eukaryotic cells contain mannose primarily in N-linked oligosaccharides and glycophospholipid anchors (1, 2). The only known pathway providing mannose for these molecules requires the conversion of Man-6-P Man-1-P GDP-Man and dolichyl-P-Man (3, 4). Man-6-P can be formed in two ways; either directly from mannose using hexokinase or derived from the pathway of Glc Glc-6-P Fru-6-P Man-6-P (Fig. 1). Phosphomannose isomerase (PMI)1 (Fru-6-P Man-6-P) plays a pivotal role in the second route, and it has long been assumed that most, if not all, of the mannose in macromolecules is derived from glucose. This assumption is based on the universal distribution of PMI (5), and the fact that this enzyme is essential for the survival of yeast grown in the absence of mannose (6). [2-3H]Mannose is practically the only isotope used to specifically label newly synthesized N-linked oligosaccharides (3), since its diversion into any other metabolic pathway begins with PMI, which generates 3H2O. This reaction is irreversible in terms of metabolic labeling, since the product is immediately diluted into 55.5 M water (3). In contrast, [3H]glucose can be catabolized through glycolysis and the tricarboxylic acid cycle to generate energy or used to synthesize a wide variety of metabolic intermediates including sugars, amino acids, and fatty acids (7, 8). The conspicuous lack of selective incorporation into sugar chains explains why [3H]glucose is rarely used to label them. Normal blood levels of mannose have been measured at 50-100 µM in a few mammals (9-12), but its potential contribution to glycoprotein synthesis has not been investigated. This is probably due to the assumption that its entry into cells via the common glucose (GLUT) transporters is competed by the 100-fold higher levels of blood glucose (13). However, we recently identified a mannose-specific transporter with a Kuptake of 35-70 µM that can supply mannose for glycoprotein synthesis under physiological conditions of 5.0 mM glucose and 50 µM mannose (14). This finding prompted us to ask which of these sugars is preferred as a source of mannose for glycoprotein biosynthesis when both are present at their physiological levels. We find that mannose is highly preferred over the 100-fold greater concentration of glucose for N-linked oligosaccharide synthesis, underscoring the functional significance of the mannose-specific transporter. ... Identifying the source of mannose for oligosaccharide synthesis became a vital question when we found that mannose, but not glucose, corrected glycosylation abnormalities in fibroblasts from children with carbohydrate-deficient glycoprotein syndrome (CDGS) type 1 (15). These CDGS cells made a high proportion of truncated lipid-linked oligosaccharides and underglycosylated their proteins by severalfold (15). This finding raised the possibility that if mannose were directly used for glycoprotein synthesis, it might be a potential therapy for these patients. The case in favor of direct mannose involvement for glycosylation increased when we (14), and others (21-23) also identified a mannose-specific transporter in a variety of mammalian cells that had a Kuptake near the concentrations of mannose found in the blood of several mammalian species (9-12). Since mannose entry via this transporter was not significantly inhibited by glucose at physiological concentration, extracellular mannose might be directly used as a precursor for sugar chain biosynthesis. The results presented here show that the N-glycosylation pathway preferentially uses free mannose over free glucose to synthesize the same set of sugar chains. The long held assumption that glucose is the major source of mannose for glycoprotein synthesis does not appear to be based on published experiments. There have been no direct comparative studies of the incorporation of mannose and glucose into glycoprotein-bound mannose in mammalian cells. This is undoubtedly due to the inherent difficulties of labeling cells with glucose. Since it is mostly catabolized, it is an unsuitable choice for labeling oligosaccharides. By contrast, [2-3H]mannose is an ideal specific label for newly synthesized N-linked oligosaccharides, since it can only be catabolized through PMI to generate 3H2O or be converted to [2-3H]Man-6-P and then to other glycosylation intermediates and finally to macromolecular products (3). These decisive advantages make it practically the universal choice for specific labeling of N-linked chains. Since the facilitated glucose transporters can also transport mannose at high concentrations (13, 24, 25), it was assumed that high glucose concentrations would reduce [3H]mannose uptake. Therefore, reducing the glucose concentration should improve mannose labeling efficiency. However, reducing the glucose concentration led to the synthesis of truncated lipid-linked precursor and underglycosylation of many key proteins (26-35), much like that recently seen in CDGS type I fibroblasts (15). The glycosylation problems associated with the "glucose starvation effect" were reversed by adding either glucose or mannose. However, glucose was always the preferred choice, since it could clearly be converted into the required precursors, while not decreasing the specific activity of [3H]mannose used for labeling. The interpretation of our results on the relative utilization of [3H]mannose versus [3H]glucose in the analysis of N-linked chains relies on several assumptions. The first is that there is a single pool of Man-6-P that is freely accessible to both Man and Fru-6-P and that subsequent conversions of Man-6-P to Man-1-P and GDP-Man cannot discriminate the origin of Man-6-P. As far as we are aware, there is no evidence for separate pools of these intermediates. Second, the specific activity of the Fru-6-P and Man-6-P pools must quickly attain that of exogenous [3H]mannose and [3H]glucose labels. The Man-1-P, Man-6-P, and GDP-Man pools are very small in cultured cells, rat brain, liver, and kidney (36-40) and probably equilibrate within a few minutes of adding [3H]mannose (14). The Fru-6-P and Glc-6-P pools have been measured at 0.1-1.0 nmol/mg of protein in various rat organs (38-40). If these figures are comparable for fibroblasts, all of the relevant precursor pools should equilibrate with exogenous glucose and mannose within a few minutes (14). Given our measured rate of utilization of both labeled mannose and glucose by the cells, it is likely that the internal pools equilibrate with the exogenous radiolabeled medium within seconds or, at most, a few minutes. This is especially important for the 1-h incubations. Continuous labeling of cells with mannose and glucose for 6 and 16 h further ensured equilibration of the label. The data in Table IV show that mannose consumption measured by direct enzymatic assay and that estimated by accounting for 3H2O and [3H]mannose in the glycosylation pathway closely agree with each other, and mannose uptake rates calculated are comparable with the predicted estimates for mannose transporter (14). As mannose is consumed from the medium, its relative contribution to glycosylation could decrease, but some of this loss is undoubtedly due to N-linked oligosaccharide processing. As long as exogenous mannose remains at physiological levels of 50 µM, it contributes the bulk of mannose to glycoproteins synthesized in fibroblasts. Long term labeling experiments require daily renewal of mannose. The direct demonstration that the specific activity of mannose incorporated into glycoproteins can reach 77% of the specific activity of mannose added to the medium shows that under these physiological conditions, fibroblasts rely primarily on mannose for glycosylation rather than converting glucose into mannose via the well known phosphomannose isomerase-based pathway. The relatively long time needed to achieve maximal labeling with [3H]mannose could reflect a preferential reutilization of mannose salvaged from glycoprotein catabolism. Alternatively, the radiolabel might be self-diluting. A single conversion of [3H]mannose-6-P fructose-6-P + 3H2O by PMI is sufficient to produce a nonlabeled molecule, but the energetically neutral reaction (Keq = 1.03) could sometimes be reversed to generate an unlabeled mannose-6-P that could participate in glycosylation reactions. The 23% contributed by other sources appears to be primarily from glucose, but a small amount could arise from gluconeogenesis. Including pyruvate in the medium did not affect the ultimate specific activity of [3H]mannose in glycoconjugates, but the calculated time required to reach this level was longer than in its absence. At present we cannot tell whether this is due to culture conditions or other factors. It is important to point out that the relative contribution of [3H]mannose to glycoprotein synthesis is nearly the same whether measured by isotope dilution analysis or by counting samples labeled with each precursor in short term labelings. The origin of mannose in the blood is unknown. Some is probably derived from the diet; however, neither the content nor the bioavailability of mannose in foods has been investigated. Mannose may also be derived from normal oligosaccharide processing or from glycoprotein-bound or free oligosaccharide degradation (1, 41). Clearly, glucose can be converted into mannose, but the amount may be cell type- or tissue-dependent. If the mannose transporters are major suppliers of mannose for glycoprotein synthesis in mammalian systems, we would expect to find mannose in the blood of all species. Regardless of their specific diets, mammals should also have an intestinal transport system that delivers mannose to the blood. A key factor for supplying mannose is likely to be the efficiency of mannose-specific transporters. The identification of mannose as a major source for glycosylation in fibroblasts has physiological, nutritional, and medical implications beyond a potential therapy for CDGS type 1 (15, 42-44). In addition to CDGS patients, sera from chronic alcoholics have underglycosylated glycoproteins and glucose-starved cultured hepatoma cells make underglycosylated glycoproteins that lack entire carbohydrate chains (26, 45-48). Several reports have underscored the problem of underglycosylation of important glycoproteins (49-51). Based on the results presented here, mannose may promote more efficient glycosylation of proteins than glucose. Clin Chem. 1997 Mar. We describe a new and improved enzymatic assay for determining the concentration of D-mannose in sera. Serum D-glucose is selectively converted to glucose-6 phosphate with the highly specific thermostable glucokinase (EC 2.7.1.2) from Bacillus stearothermophilus. The anionic reaction products and excess substrates are removed by a rapid and simple anion-exchange chromatography step in microcentrifuge spin columns. D-Mannose in the glucose-depleted sample is then assayed spectrophotometrically by using coupled enzymatic reactions. The quantitative elimination of glucose from the serum samples allowed the accurate and reproducible assay of serum mannose in the 0-200 mumol/L range. Recovery of mannose added to serum (5-200 mumol/L) was 94% +/- 4.4%. The intraassay CV was 6.7% at 40 mumol/L mannose (n = 5; 39.6 +/- 1.6 mumol/L) and 4.4% at 80 mumol/L (n = 11; 75.0 +/- 1.8 mumol/L); the interassay CV at these concentrations was 12.2% (n = 7; 36.9 +/- 2.1 mumol/L) and 9.8% (n = 7; 74.2 +/- 2.7 mumol/L), respectively. Sera from 11 healthy human volunteers contained an average of 54.1 +/- 11.9 mumol/L mannose (range 36-81 mumol/L). J Biol Chem. 1996 Apr 19. The concentration of D-mannose in serum is 20-50 micron, but its physiological significance for glycoprotein synthesis is unknown. Here, we show that the uptake of D-mannose by different mammalian cell lines involves a mannose-specific transporter(s) with a K(uptake) of about 30-70 micron and a V(max) which is probably sufficient to account for the bulk of mannose needed for glycoprotein synthesis. Mannose uptake appears to be through a facilitated transport process since it is not inhibited by cyanide. Phloretin completely inhibits mannose uptake, but phloridzin inhibits only 25-30%. Both of these inhibitors can block 2-deoxyglucose uptake in fibroblasts which occurs through the typical glucose transporters. None of 9 other sugars tested inhibited mannose transport. Most importantly, 5 mM D-glucose only inhibits mannose uptake by 50% showing that it is not an efficient competitor. These results suggest that this transporter(s) may use serum mannose for glycoprotein synthesis. |