Identification and role of a 6-deoxy-4-keto-hexosamine in the lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3.

The outer core (OC) region of Yersinia enterocolitica serotype O:3 lipopolysaccharide is a hexasaccharide essential for the integrity of the outer membrane. It is involved in resistance against cationic antimicrobial peptides and plays a role in virulence during early phases of infection. We show here that the proximal residue of the OC hexasaccharide is a rarely encountered 4-keto-hexosamine, 2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (Sugp) and that WbcP is a UDP-GlcNAc-4,6-dehydratase enzyme responsible for the biosynthesis of the nucleotide-activated form of this rare sugar converting UDP-2-acetamido-2-deoxy-D-glucopyranose (UDP-D-GlcpNAc) to UDP-2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (UDP- Sugp). In an aqueous environment, the 4-keto group of this sugar was present in the 4-dihydroxy form, due to hydration. Furthermore, evidence is provided that the axial 4-hydroxy group of this dihydroxy function was crucial for the biological role of the OC, that is, in the bacteriophage and enterocoliticin receptor structure and in the epitope of a monoclonal antibody.

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells.

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants. Not surprisingly, changes in O-GlcNAc protein modifications were noted during cell activation with and without GlcN addition. These findings could not be explained by some non-specific change in cell metabolism because ATP concentrations did not significantly change. We speculate that HBP-active compounds may contribute to patient care in certain inflammatory and autoimmune diseases.

The hexosamine signaling pathway: deciphering the "O-GlcNAc code".

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. Like phosphorylation, protein O-GlcNAcylation dramatically alters the posttranslational fate and function of target proteins. Indeed, O-GlcNAcylation may compete with phosphorylation for certain Ser/Thr target sites. Like kinases and phosphatases, the enzymes of O-GlcNAc metabolism are highly compartmentalized and regulated. Yet, O-GlcNAc addition is subject to an additional and unique level of metabolic control. O-GlcNAc transfer is the terminal step in a "hexosamine signaling pathway" (HSP). In the HSP, levels of uridine 5'-diphosphate (UDP)-GlcNAc respond to nutrient excess to activate O-GlcNAcylation. Removal of O-GlcNAc may also be under similar metabolic regulation. Differentially targeted isoforms of the enzymes of O-GlcNAc metabolism allow the participation of O-GlcNAc in diverse intracellular functions. O-GlcNAc addition and removal are key to histone remodeling, transcription, proliferation, apoptosis, and proteasomal degradation. This nutrient-responsive signaling pathway also modulates important cellular pathways, including the insulin signaling cascade in animals and the gibberellin signaling pathway in plants. Alterations in O-GlcNAc metabolism are associated with various human diseases including diabetes mellitus and neurodegeneration. This review will focus on current approaches to deciphering the "O-GlcNAc code" in order to elucidate how O-GlcNAc participates in its diverse functions. This ongoing effort requires analysis of the enzymes of O-GlcNAc metabolism, their many targets, and how the O-GlcNAc modification may be regulated.

Glucose-containing peritoneal dialysis fluids regulate leptin secretion from 3T3-L1 adipocytes.

BACKGROUND: A marked elevation of serum leptin is observed soon after the start of peritoneal dialysis (PD), suggesting that leptin production may be stimulated by this treatment. Glucose metabolism is the major factor regulating leptin. The current study was designed to test if glucose-based PD fluids might regulate leptin production in vitro. METHODS: 3T3-L1 adipocytes were exposed to a 50:50 mixture of dialysis solutions and medium M199 containing 10% serum for

Activation of the hexosamine signaling pathway in adipose tissue results in decreased serum adiponectin and skeletal muscle insulin resistance.

Overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine:fructose-6-phosphate amidotransferase) in muscle and adipose tissue of transgenic mice was previously shown to result in insulin resistance and hyperleptinemia. Explanted muscle from transgenic mice was not insulin resistant in vitro, suggesting that muscle insulin resistance could be mediated by soluble factors from fat tissue. To dissect the relative contributions of muscle and fat to hexosamine-induced insulin resistance, we overexpressed glutamine:fructose-6-phosphate amidotransferase 2.5-fold, specifically in fat under control of the aP2 promoter. Fasting glucose, insulin, and triglycerides were unchanged in the transgenic mice; leptin and beta-hydroxybutyrate levels were 91% and 29% higher, respectively. Fasted transgenic mice have mild glucose intolerance and skeletal muscle insulin resistance in vivo. In fasting transgenic mice, glucose disposal rates with hyperinsulinemia were decreased 27% in females and 10% in males. Uptake of 2-deoxy-D-glucose into muscle was diminished by 45% in female and 21% in male transgenics. Serum adiponectin was also lower in the fasted transgenics, by 37% in females and 22% in males. TNF alpha and resistin mRNA levels in adipose tissue were not altered in the fasted transgenics; levels of mRNA for leptin were increased and peroxisome proliferator-activated receptor gamma decreased. To further explore the relationship between adiponectin and insulin sensitivity, we examined mice that have been refed for 6 h after a 24-h fast. Refeeding wild-type mice resulted in decreased serum adiponectin and increased leptin. In transgenic mice, however, the regulation of these hormones by refeeding was lost for adiponectin and diminished for leptin. Refed transgenic female and male mice no longer exhibited decreased serum adiponectin in the refed state, and they were no longer insulin resistant as by lower or unchanged insulin and glucose levels. We conclude that increased hexosamine levels in fat, mimicking excess nutrient delivery, are sufficient to cause insulin resistance in skeletal muscle. Changes in serum adiponectin correlate with the insulin resistance of the transgenic animals.

Hexosamines as mediators of nutrient sensing and regulation in diabetes.

High concentrations of glucose induce insulin resistance, impair insulin secretion, and affect hepatic glucose production in a manner that mirrors Type 2 diabetes, and hexosamines mimic many of these effects. This has led to the hypothesis that cells use hexosamine flux as a glucose- and satiety-sensing pathway. The hexosamine hypothesis for glucose sensing has been validated by overexpressing the rate-limiting enzyme for hexosamine synthesis, glutamine: fructose-6-phosphate amidotransferase (GFA) in several tissues including muscle, liver, fat, and beta cells. With overexpression of GFA in transgenic animals, skeletal muscle becomes insulin resistant, the liver synthesizes excess fatty acid, and the beta cell secretes excess insulin leading to hyperinsulinemia. Thus, excess hexosamine flux leads to a coordinated response whereby fuel is shunted toward long-term storage, mirroring the "thrifty phenotype." Chronically, however, these same adaptive changes result ultimately in obesity, hyperlipidemia, beta cell failure, and Type 2 diabetes. These results suggest a mechanism by which chronic overnutrition leads to the phenotype of Type 2 diabetes.

Hexosamines regulate leptin production in 3T3-L1 adipocytes through transcriptional mechanisms.

This study was undertaken to examine the regulation of leptin gene (LEP) transcription and leptin release by hexosamines in 3T3-L1 adipocytes. Glucosamine (1 mM), an intermediate in hexosamine biosynthesis, increased leptin release to 117.0 +/- 7.3% (P = 0.0430; n = 9) and 134.6 +/- 6.5% of the control value (P = 0.0367; n = 4) by 48 and 96 h, respectively. With 0.01 mM glucosamine, leptin release was increased to 120.0 +/- 3.0% of the control value (P = 0.0069; n = 4) by 96 h of treatment. Glucose at 5 and 20 mM stimulated leptin release to 759 +/- 227% and 1104 +/- 316% of the control value over the 96-h culture period. Inhibition of hexosamine biosynthesis with 6-diazo-5-oxonorleucine (20 microM) reduced glucose-stimulated leptin release 13 +/- 2.3% and 29.9 +/- 6.6% at 24 and 96 h, respectively (n = 4; P < 0.05). A 24-h incubation in 5 mM glucose significantly increased (163.0 +/- 19.3%; n = 7) the activity of a human LEP promoter electroporated into differentiated 3T3-L1 cells. Glucosamine (1 mM; 48 h) also increased LEP promoter activity 170.0 +/- 13.0% (n = 5). Mutation of the three Sp1 binding sites in the LEP construct significantly reduced promoter activity. However, glucose (5 mM; 24 h) and glucosamine (1 mM; 48 h) increased the activity of the mutated promoter to 165 +/- 40% (n = 8) and 143 +/- 13% of the control value (n = 8). Glucosamine significantly increased O-glycosylation of Sp1 by 16.1 +/- 4.5% (P = 0.0305; n = 3). These data demonstrate that glucose and hexosamines regulate leptin production through transcriptional mechanisms localized to the proximal portion of the LEP promoter. Hexosamine-mediated regulation of LEP gene expression does not depend on Sp1 binding to traditional sites on the promoter.

Hexosamines regulate leptin production in human subcutaneous adipocytes.

The hexosamine biosynthetic pathway has recently been proposed as a mechanism through which cells "sense" nutrient flux to regulate leptin release. This study was undertaken to examine the regulation of leptin production by hexosamines in human adipocytes. Adipose tissue UDP-N-acetylglucosamine, an end product of hexosamine biosynthesis, was elevated 3.2-fold, and ob messenger ribonucleic acid was elevated 2-fold in the sc adipose tissue of 17 obese [body mass index (BMI), 41.3+/-12.0 kg/m2; age, 31+/-5 yr] subjects compared to 14 lean (BMI, 23.4+/-1.6 kg/m2; age, 33+/-11 yr) subjects. Serum leptin was increased 2.7-fold in the obese subjects. A significant positive relationship was found between adipose tissue UDP-N-acetylglucosamine and BMI (Spearman correlation = 0.576; P = 0.0007) and between UDP-N-acetylglucosamine and serum leptin (Spearman correlation = 0.4650; P = 0.0145). Treatment of isolated sc adipocytes with 1 mmol/L glucosamine, an intermediate product in UDP-N-acetylglucosamine biosynthesis, increased leptin release 21.4+/-17.6% (mean +/- SD) over control (P = 0.0365) and 74.5+/-82.8% over control (P = 0.0271) in adipocytes from lean (BMI, 23.2+/-1.6 kg/m2; n = 6) and obese (BMI, 55.4+/-13.0 kg/m2,; n = 9) subjects, respectively, by 48 h of culture. Inhibition of UDP-N-acetylglucosamine biosynthesis with 6-diazo-5-oxo-norleucine reduced glucose-stimulated leptin release from cultured adipocytes 21.8+/-32.4% (P = 0.0395; n = 12) and ob gene expression 19.9+/-18.9% (P = 0.0208; n = 8) by 48 h of treatment. These findings suggest that hexosamine biosynthesis regulates leptin production in human adipose tissue.

Hexosamine regulation of glucose-mediated laminin synthesis in mesangial cells involves protein kinases A and C.

Hyperglycemia leads to alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation. These adverse effects of glucose may be mediated by glucose metabolism through the hexosamine biosynthesis pathway (HBP). The HBP converts fructose-6-phosphate to glucosamine-6-phosphate via the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFA). We have investigated the effects of high glucose (HG, 25 mM) and glucosamine (GlcN, 1.5 mM) on the synthesis of the ECM protein laminin in a SV-40-transformed rat kidney mesangial (MES) cell line. The roles of protein kinases C (PKC) and A (PKA) in mediating laminin accumulation were also investigated. Treatment of MES cells with HG or GlcN for 48 h increased laminin levels in cellular extracts more than twofold compared with 5 mM glucose (low glucose; LG). The presence of the GFA inhibitor diazo-oxo-norleucine (DON, 10 microM) blocked HG but not GlcN-induced laminin synthesis. HG resulted in a time-dependent increase in total PKC and PKA activities, 57+/-11.3 (P < 0.01 vs. LG) and 85+/-17.4% (P < 0.01 vs. LG), respectively. GlcN had no effect on the total PKC activity; however, both glucose and glucosamine increased membrane-associated PKC activity by twofold compared with LG. GlcN stimulated total PKA activity by 47+/-8.4% (P < 0.01 vs. LG). Similarly, membrane- associated PKA activity was also increased by HG and GlcN approximately 1.8 and 1.5-fold, respectively. HG and GlcN increased cellular cAMP levels 2.2- and 3. 4-fold, respectively. Pharmacological downregulation of PKC by long-term incubation of MES cells with 0.5 microM phorbol 12-myristate 13-acetate (PMA) or inhibition of PKA activity by 2 microM H-8 blocked the effects of HG and GlcN on laminin synthesis. These results demonstrate that glucose-induced laminin synthesis in MES cells is mediated by flux through the HBP and that this stimulation involves PKC and PKA signaling pathways.

Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione.

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg x min were 39-90% lower in such transgenic mice, compared with their control littermates (P < or = 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 +/- 17.8 vs. 166.8 +/- 15.1 pmol deoxyglucose/g x min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5'-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Hexosamines and insulin resistance.

Glucose is an important regulator of cell growth and metabolism. Thus, it is likely that some of the adverse effects of hyperglycemia are reflections of normal regulation by abnormal concentrations of glucose. How the cell senses glucose, however, is still incompletely understood. Evidence has been presented that the hexosamine biosynthesis pathway serves this function for regulation of aspects of glucose uptake, glycogen synthesis, glycolysis, and synthesis of growth factors. Excess hexosamine flux causes insulin resistance in cultured cells, tissues, and intact animals. Further evidence for the possible role of this pathway in normal glucose homeostasis and disease is that the level of activity of the rate-limiting enzyme in hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase, is correlated with glucose disposal rates (GDRs) in normal humans and transgenic mice.

Agglutination of Leishmania promastigotes by midgut lectins from various species of phlebotomine sandflies.

Lectins which agglutinate Leishmania promastigotes were demonstrated in gut lysates from laboratory colonies of five Phlebotomus and two Lutzomyia species. In general, the highest agglutination titres were found in P. halepensis and Lu. longipalpis (Jacobina). Marked differences were found in the agglutination of promastigotes of various Leishmania species and strains and high agglutination titres were observed in some natural vector-parasite combinations, such as Phlebotomus argentipes and Le. donovani. Intraspecific variability, in agglutination of Le. major strains, could be related to the varying infectivity of the strains to laboratory animals. Similar carbohydrates, of which the most effective were D-mannosamine and N-acetyl-D-glucosamine, inhibited the agglutination of Le. major and Le. donovani promastigotes by midgut extracts from P. papatasi and Lu. longipalpis. D-Mannosamine and N-acetyl-D-glucosamine inhibited agglutination of promastigotes in all vector-parasite combinations. The results of the carbohydrate-inhibition tests indicate that the lectin specificities in Phlebotomus are similar to those in Lutzomyia.

Glucose regulation of transforming growth factor-alpha expression is mediated by products of the hexosamine biosynthesis pathway.

We have recently shown that glucose and glucosamine regulate the transcription of transforming growth factor-alpha (TGF alpha) in rat aortic smooth muscle (RASM) cells. Based on the increased potency of glucosamine compared to glucose, we hypothesized that stimulation of TGF alpha transcription by glucose is mediated through the hexosamine biosynthesis pathway. The yeast cDNA for the rate-limiting enzyme of this pathway, glutamine:fructose-6-phosphate amidotransferase (GFA), was therefore expressed in RASM cells. GFA-transfected cells showed an increase in GFA activity, exhibiting a 2.2-fold increase in the synthesis of glucosamine-6-phosphate, the first product of the hexosamine biosynthetic pathway. To test the effect of GFA overexpression on TGF alpha transcriptional activity, cells were transiently cotransfected with GFA along with a reporter plasmid containing the firefly luciferase gene under control of the TGF alpha promoter. GFA-transfected cells exhibited a glucose-dependent 2-fold increase in TGF alpha activity compared to control cells. Maximal stimulation of TGF alpha-luciferase activity by glucosamine, however, was equivalent in GFA-and control-transfected cells, confirming that the stimulation observed by both agents operated through the same pathway. This increase in TGF alpha activity was inhibited (85% at 0.5 mM glucose and 69% at 30 mM glucose) by the glutamine analog and inhibitor of GFA, 6-diazo-5-oxonorleucine (10 microM). Control studies confirmed that the increased TGF alpha-luciferase activity in the GFA-expressing cells was not an artifact of altered growth, survival, or transfection efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical behavior of articular cartilage quantitative changes with enzymatic alteration of the proteoglycan fraction.

The in-vitro viscoelastic mechanical response of normal rabbit articular cartilage is strongly dependent on the quantity and integrity of the proteoglycan fraction of the tissue matrix. Experimental results demonstrate that specific functional relationships exist between shear moduli, retardation time spectra, and proteoglycan content. Quantitative enzymolysis of the proteoglycan fraction of the tissue alters the form of these relationships in a fashion consistent with the altered physiochemical make-up of the tissue. The observed changes in mechanical behavior with controlled enzymolysis are similar to those associated with the early stages of osteoarthritis, rheumatoid arthritis, joint sepsis, and synovitis in animal models.