IL-6 deficiency in mice neither impairs induction of metabolic genes in the liver nor affects blood glucose levels during fasting and moderately intense exercise.

AIMS/HYPOTHESIS: Fasting and exercise are strong physiological stimuli for hepatic glucose production. IL-6 has been implicated in the regulation of gluconeogenic genes, but the results are contradictory and the relevance of IL-6 for fasting- and exercise-induced hepatic glucose production is not clear. METHODS: Investigations were performed in rat hepatoma cells, and on C57Bl6 and Il6(-/-) mice under the following conditions: IL-6 stimulation/injection, non-exhaustive exercise (60 min run on a treadmill) and fasting for 16 h. Metabolite analysis, quantitative real-time PCR and immunoblotting were performed. RESULTS: IL-6 stimulation of rat hepatoma cells led to higher glucose production. Injection of IL-6 in mice slightly increased hepatic Pepck (also known as Pck1) expression. Fasting of Il6(-/-) mice for 16 h did not alter glucose production compared with wild-type mice, since plasma glucose concentrations were similar and upregulation of phosphoenolpyruvate carboxykinase (PEPCK) and Pgc-1alpha (also known as Ppargc1a) expression was comparable. In the non-fasting state, Il6(-/-) mice showed a mild metabolic alteration including higher plasma glucose and insulin levels, lower NEFA concentrations and slightly increased hepatic PEPCK content. Moderately intense exercise resulted in elevated IL-6 plasma levels in wild-type mice. Despite that, plasma glucose, insulin, NEFA levels and hepatic glycogen content were not different in Il6(-/-) mice immediately after running, while expression of hepatic G6pc, Pgc-1alpha, Irs2 and Igfbp1 mRNA was similarly increased. CONCLUSIONS/INTERPRETATION: These data suggest that in mice IL-6 is not essential for physiologically increased glucose production during fasting or non-exhaustive exercise.

Activation of the mitogen-activated protein kinase (MAPK) signalling pathway in the liver of mice is related to plasma glucose levels after acute exercise.

AIMS/HYPOTHESIS: We aimed to identify, in the liver of mice, signal transduction pathways that show a pronounced regulation by acute exercise. We also aimed to elucidate the role of metabolic stress in this response. METHODS: C57Bl6 mice performed a 60 min run on a treadmill under non-exhaustive conditions. Hepatic RNA and protein lysates were prepared immediately after running and used for whole-genome-expression analysis, quantitative real-time PCR and immunoblotting. A subset of mice recovered for 3 h after the treadmill run. A further group of mice performed the treadmill run after having received a vitamin C- and vitamin E-enriched diet over 4 weeks. RESULTS: The highest number of genes differentially regulated by exercise in the liver was found in the mitogen-activated protein kinase (MAPK) signalling pathway, with a pronounced and transient upregulation of the transcription factors encoded by c-Fos (also known as Fos), c-Jun (also known as Jun), FosB (also known as Fosb) and JunB (also known as Junb) and phosphorylation of hepatic MAPK. Acute exercise also activated the p53 signalling pathway. A major role for oxidative stress is unlikely since the antioxidant-enriched diet did not prevent the activation of the MAPK pathway. In contrast, lower plasma glucose levels after running were related to enhanced levels of MAPK signalling proteins, similar to the upregulation of Igfbp1 and Pgc-1alpha (also known as Ppargc1a). In the working muscle the activation of the MAPK pathway was weak and not related to plasma glucose concentrations. CONCLUSIONS/INTERPRETATION: Metabolic stress evidenced as low plasma glucose levels appears to be an important determinant for the activation of the MAPK signalling pathway and the transcriptional response of the liver to acute exercise.

Selective association of plasma non-esterified fatty acid species with circulating interleukin-8 concentrations in humans.

Elevated plasma concentrations of non-esterified fatty acids (NEFA), due to high fat intake and/or adipose tissue lipolysis, are a hallmark of the metabolic syndrome. We assessed whether certain plasma NEFA species contribute to the chronic low-grade inflammatory state seen in the metabolic syndrome. We determined the fasting plasma NEFA patterns of 75 overweight non-diabetic subjects and analysed their relationship with plasma inflammatory parameters. After adjustment for gender, age, body fat, and waist-hip ratio, no strong correlations of single NEFA species with leukocyte number, C-reactive protein, interleukin-6, tumour necrosis factor-alpha, or monocyte chemoattractant protein-1 were detected. However, oleate was negatively (r=-0.36, p=0.0015) and myristate (r=0.41, p=0.0003) as well as the omega3-polyunsaturated NEFA alpha-linolenate (r=0.37, p=0.0011), eicosapentaenoate (r=0.40, p=0.0003), and docosahexaenoate (r=0.40, p=0.0004) were positively associated with interleukin-8 concentrations. The other NEFA species as well as the total plasma NEFA concentration did not correlate with interleukin-8. The correlations of myristate, oleate, and the sum of all omega3-polyunsaturated NEFA with interleukin-8 were independent of plasma tumour necrosis factor-alpha and overall adiposity. Our data demonstrate close and selective associations of oleate, myristate, and omega3-polyunsaturated NEFA with plasma concentrations of the pro-inflammatory chemokine interleukin-8. Thus, these NEFA species may represent specific determinants of plasma interleukin-8.

The RAGE pathway in inflammatory myopathies and limb girdle muscular dystrophy.

Oxidative stress and nuclear factor-kappaB (NF-kappaB) activation are linked to the pathogenesis of many metabolic, degenerative, and chronic inflammatory diseases. Activation of the receptor for advanced glycation end products (RAGE) by its specific ligand N(epsilon)-carboxymethyllysine (CML) results in the activation of NF-kappaB and the production of proinflammatory cytokines. To determine whether engagement of RAGE contributes to the pathogenesis of inflammatory myopathies, we performed immunohistochemical studies on the presence of CML-modified proteins, RAGE and activated NF-kappaB in muscle biopsies of patients with polymyositis (PM, n=10), dermatomyositis (DM, n=10), limb girdle muscular dystrophy (LGMD, n=10) and in 10 controls with normal muscle biopsy results. In inflammatory myopathies CML, RAGE and NF-kappaB were detected in mononuclear cells and in regenerating muscle fibers. CML, NF-kappaB and, to a lesser extent, RAGE were also found in degenerating muscle fibers, but colocalization of CML, RAGE and NF-kappaB was only seen in infiltrating mononuclear cells and regenerating muscle fibers. Immunofluorescence double labeling demonstrated an expression of CML, RAGE and NF-kappaB in CD4-, CD8-, CD22- and CD68-positive mononuclear cells. Western blot analysis showed an increased immunoreactivity for CML-modified proteins in PM and DM. In LGMD, CML, RAGE and NF-kappaB were found in regenerating muscle fibers and less frequently in degenerating muscle fibers, and with lower staining intensities than in inflammatory myopathies. Our data suggests that the CML-RAGE-NF-kappaB pathway is an evident proinflammatory pathomechanism in mononuclear effector cells in PM and DM. RAGE-mediated NF-kappaB activation may be involved in muscle fiber regeneration in inflammatory myopathies and LGMD.

Glutamine:fructose-6-phosphate aminotransferase enzyme activity is necessary for the induction of TGF-beta1 and fibronectin expression in mesangial cells.

AIMS/HYPOTHESIS: Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate aminotransferase (GFAT) as a rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine TGF-beta1, a key mediator in the development of diabetic nephropathy and possibly other diabetic angiopathies. In this study we investigated the effect of enhanced expression of wild-type GFAT and two enzymatically inactive GFAT mutants on TGF-beta1 synthesis in mesangial cells. METHODS: Mutated human GFAT expression vectors were prepared by PCR-site directed mutagenesis. Wild-type and mutated vectors were transfected into human embryonic kidney 293 cells and mesangial cells and GFAT enzyme activity was assessed by formation of glucosamine-6-phosphate. Production of TGF-beta1 and fibronectin protein was examined by ELISA. RESULTS: Mutation of histidine 577 or lysine 676 to alanine led to a complete loss of GFAT enzyme activity. An increased concentration of wild-type GFAT in mesangial cells enhanced both TGF-beta1 and fibronectin production 1.5-fold, while mesangial cells transfected with the mutated GFAT constructs showed no effect. CONCLUSION/INTERPRETATION: The data indicate that the hexosamine pathway-mediated induction of TGF-beta1 synthesis in mesangial cells is dependent on GFAT enzyme activity. Our results add to previous observations showing that the hexosamine pathway could increase the transcriptional activity of nuclear proteins leading to enhanced cytokine synthesis.

Glucose oversupply increases Delta9-desaturase expression and its metabolites in rat skeletal muscle.

AIM/HYPOTHESIS: Previous studies have shown that prolonged glucose infusion causes insulin resistance and triglyceride accumulation in rat skeletal muscle. In this study, we investigated a possible relationship between insulin resistance and the composition of different accumulated lipid fractions in rat skeletal muscle. METHODS: Continuous glucose infusion was carried out in rats for 7 days. Lipids were extracted from skeletal muscle, separated by thin layer chromatography and fatty acid composition of phospholipids, triglycerides, diglycerides, free fatty acids and cholesterol esters fractions was analysed by gas chromatography. Delta9-Desaturase mRNA was measured by real time polymerase chain reaction. The enzyme activity was measured in the microsomal fractions. RESULTS: Prolonged glucose infusion (5 days) increased the relative content of palmitoleic acid (16:1 N7) several-fold (2.3- to 5.8-fold) in four out of five lipid fractions and enhanced oleic acid (18:1 N9) two-fold in three lipid fractions suggesting increased Delta9-desaturase activity while the content of several polyunsaturated fatty acids was reduced. In parallel, Delta9-Desaturase mRNA contents and enzyme activities in skeletal muscle were increased 10-fold, 75-fold, 2.6-fold and 7.7-fold after 2 and 5 days of glucose infusion, respectively. CONCLUSION/INTERPRETATION: Our results suggest that long-term glucose oversupply induces a rapid increase in Delta9-desaturase expression and enzyme activity in skeletal muscle which leads to fast and specific changes in fatty acid metabolism possibly contributing to the insulin resistance in this animal model.

Angiotensin II induces human TGF-beta 1 promoter activation: similarity to hyperglycaemia.

AIMS/HYPOTHESIS: Activation of the renal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. Because previous in vitro studies demonstrated the angiotensin II (ang II)-mediated up-regulation of the prosclerotic transforming growth factor beta 1 (TGF) we studied the molecular mechanism of ang II-induced TGF-beta 1 gene activation. METHODS: Mesangial cells were stimulated with 100 nmol/l ang II with or without inhibitors of protein kinase C (PKC) and p38 MAPK and the TGF-beta 1 promoter activity was determined by promoter-reporter assays. The effect of ang II on the binding of nuclear proteins to the regulatory AP-1 site B, previously shown to mediate the high glucose-response of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays. RESULTS: Ang II enhanced the activity of the TGF-beta1 promoter fragment -453/+11 approximately 1.6-fold. Mutation of each of two AP-1 binding sites or inhibition of the PKC- and p38 MAPK-dependent pathways blocked the ang II-stimulated activity completely. Furthermore, ang II activated the binding of nuclear proteins to the AP-1 box B of the TGF-beta 1 promoter. These effects were similar to those previously observed with high glucose. Co-incubation with ang II and high glucose had no additive effect on TGF-beta 1 promoter activity, protein binding to the AP-1 box B or activation of p38 MAPK. CONCLUSION/INTERPRETATION: The findings indicate that ang II and hyperglycaemia stimulate the TGF-beta 1 gene activation through the same PKC- and p38 MAPK-dependent pathways by the same regulatory elements of the TGF-beta 1 promoter. Our data could also be relevant for e.g. hypertension-induced glomerulosclerosis.

N(epsilon)-Carboxymethyllysine in diabetic and non-diabetic polyneuropathies.

Increased oxidative stress and advanced glycosylation are important factors in the development of diabetic neuropathy. In non-diabetic neuropathies their influence has not been investigated in detail so far. We studied the localisation of N(epsilon)-carboxymethyllysine (CML) - a biomarker for oxidative stress - by immunohistochemistry in sural nerve biopsies of 31 patients with different polyneuropathies [diabetic polyneuropathy (n=5), alcohol-associated polyneuropathy (n=4), vitamin B12-deficient polyneuropathy (n=6), chronic inflammatory demyelinating polyneuropathy (CIDP) (n=6), vasculitic neuropathy (n=6), Charcot-Marie-Tooth disease type I (CMT I) (n=4)] and 4 normal controls. CML was detected in the perineurium of patients with diabetic, alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. Epineurial, perineurial and endoneurial vessels were CML positive in diabetic, vitamin B12-deficient and vasculitic polyneuropathies. CML was also found in mononuclear inflammatory cells in vasculitic neuropathy. In CIDP and normal controls there was only marginal perineurial CML deposition in 2/6 and 1/4 cases. In CMT I no CML was detected. Immunohistochemical results were confirmed by immunoblot. Our data suggest a role of oxidative stress in the pathogenesis not only of diabetic but also of alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. It may be a minor pathogenetic factor in CIDP and may not be involved in CMT I. Underlying causes for increased oxidative stress may be an elevated production of reactive oxygen species and an impairment of antioxidative defences. Therefore, an antioxidative treatment should be considered in alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathy.

Prolonged glucose infusion into conscious rats inhibits early steps in insulin signalling and induces translocation of GLUT4 and protein kinase C in skeletal muscle.

AIMS/HYPOTHESIS: Previous studies on diabetic patients have shown that hyperglycaemia increases glucose uptake in an apparently insulin-independent manner. However, the molecular mechanism has not been clarified. METHODS: We studied rats receiving continuous glucose infusion to address this question. In this animal model, rats accommodate systemic glucose oversupply and rapidly develop insulin resistance. RESULTS: Glucose infusion increased both plasma glucose and insulin concentrations to peak after one day. In spite of continuous glucose infusion normoglycaemia was reached after 5 days while insulin concentrations remained higher. Focusing our studies in day 2 (hyperglycaemia/hyperinsulinaemia) and day 5 (normoglycaemia/hyperinsulinaemia) we found, particularly in day 5, that the early steps of the insulin signalling cascade in skeletal muscle of glucose-infused rats were not more activated when compared to control animals as assessed by a comparable phosphorylation of the insulin receptor, IRS-1 and PKB and by an unaltered IRS-1-associated Ptd(Ins) 3' kinase activity. Continuous glucose infusion induced GLUT4 protein expression and translocation to the plasma membrane while neither expression nor translocation of GLUT1 was affected. Translocation of PKC- betaI, - betaII (> threefold) and -alpha, -theta (to a lesser extent) to the plasma membrane was significantly induced after 2 days but not after 5 days of glucose infusion when normoglycaemia was reached. CONCLUSIONS/INTERPRETATION: Our data support the hypothesis that continuous glucose infusion induces translocation of GLUT4 while the early steps of the insulin signalling cascade were not increased. These effects could be mediated by activation of PKC.

Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.

Low-molecular-weight heparin prevents high glucose- and phorbol ester-induced TGF-beta 1 gene activation.

BACKGROUND: Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy. Since previous in vivo studies demonstrated a renoprotective effect of low-molecular-weight (LMW) heparin in experimental animals, and recent in vitro data showed an interaction of this drug with the overactivated TGF-beta 1 cascade in high glucose- and phorbol ester-stimulated mesangial cells, we studied the molecular mechanism of these effects on TGF-beta 1 gene expression. METHODS: Mesangial cells were stimulated with 30 mmol/L glucose or with 0.5 micromol/L phorbol ester [phorbol myristate acetate (PMA)] in the absence or presence of LMW heparin. TGF-beta 1 promoter activity was determined in promoter-reporter luciferase assays. The effect of LMW heparin on the binding of nuclear proteins to a regulatory activator protein-1 (AP-1) site, which mediates the high glucose and PMA responsiveness of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays. RESULTS: The presence of LMW heparin completely prevented TGF-beta 1 gene activation in both high glucose- and PMA-stimulated cells. Preincubation of the cells with LMW heparin and subsequent stimulation of the cells with both stimuli yielded the same result. Furthermore, treatment with LMW heparin prevented the enhanced binding of nuclear proteins to the regulatory AP-1 site, while binding to a Sp1 site was unaffected. Basal promoter activity and basal AP-1 binding also was reduced by LMW heparin. The LMW heparin effect on basal promoter activity was abolished by mutation of the regulatory AP-1 box B and by deletion of this AP-1 binding site. CONCLUSIONS: LMW heparin prevents high glucose- and PMA-mediated TGF-beta 1 expression by inhibiting the activation of the TGF-beta 1 promoter and by preventing the enhanced binding of nuclear proteins to the regulatory AP-1 site.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis.

Divergence in distribution and prognostic significance of major basement components in laryngeal carcinomas.

In previous studies, we demonstrated a loss of major basement membrane (BM) components in laryngeal squamous cell carcinomas and provided initial evidence that this was of potential prognostic significance. In our current study, we extended the panel of BM antibodies and enlarged our study group in order to perform a multivariate statistical analysis. We analyzed 26 laryngeal squamous cell carcinomas immunohistochemically for the distribution of the BM-components collagen IV, collagen VII, laminin-1, laminin-5, perlecan and fibronectin. The resulting data were correlated with clinical prognostic factors and statistical correlation coefficients were determined for independent uni- and multivariate analysis. All carcinomas analyzed revealed defects of the peritumoral BM with more extensive loss of collagen VII than collagen IV, laminin-1, perlecan and fibronectin. Laminin-5 in contrast was present even in poorly differentiated tumors showing an enhanced intracytoplasmatic staining in the tumor cells. Furthermore, our statistical analysis did not show independent prognostic significance of any of the BM-components. Our observations indicate a divergence between the loss of several major BM-components (collagens IV, VII, laminin-1, perlecan) and an enhanced deposition of laminin-5. This suggests a severely altered cell-matrix interaction, since laminin-5 links the collagen VII-containing anchoring fibrils to cell receptors of the integrin type.

Overexpression of glutamine:fructose-6-phosphate-amidotransferase induces transforming growth factor-beta1 synthesis in NIH-3T3 fibroblasts.

Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate-amidotransferase (GFAT) as rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine transforming growth factor beta1 (TGF-beta1) in fibrotic complications, particularly in diabetic kidney disease. Here, we investigate in a stable transfection system the effect of overexpression of GFAT on TGF-beta1 synthesis in NIH-3T3 fibroblasts. We demonstrate a 1.8-fold stimulation of TGF-beta1 mRNA and a 1.9-fold increased protein expression, whereas TGF-beta2 production was not upregulated. The 1.5-fold enhanced TGF-beta1 promoter activity suggests a transcriptional regulation. The elevated TGF-beta1 protein is biologically active since GFAT-overexpressing cells exhibit a 2-fold fibronectin production. The results indicate a GFAT-dependent induction of TGF-beta1 synthesis.

Induction of apoptosis by glyoxal in human embryonic lung epithelial cell line L132.

Oxidative stress has been suggested to play a central role in the pathogenesis of lung fibrosis and lung epithelial cell apoptosis is considered to be a key event during fibrogenesis. Studies from various laboratories have indicated that metabolic conditions may initiate oxidative stress, thereby contributing to epithelial cell death. This study was designed to test the hypothesis that glyoxal, an intermediate product in the glycation reaction leading to advanced glycation end products (AGEs), may induce lung epithelial cell apoptosis. We investigated the in vitro effects of glyoxal on fetal human lung epithelial L132 cells. Immunocytochemical analysis of paraffin-embedded cells and fluorescence-activated cell sorter analysis revealed a dose-dependent accumulation of the glycoxidation product (epsilon)N-carboxymethyllysine (CML) in all compartments of the cell. It has been shown that CML modification of proteins may serve as an indicator for oxidative stress. To examine the role of apoptosis in epithelial lung cells we investigated glyoxal-dependent changes in pro- and antiapoptotic mediators bax and activated caspase-3, and galectin-3 and bcl-2, respectively. Increasing concentrations of glyoxal (50 to 400 microM) induced an increase in the number of apoptotic cells. The apoptotic changes were confirmed by transmission electron microscopy. Immunocytochemical analysis of treated cells revealed the presence of other AGEs such as pentosidine as well as products of lipid peroxidation.

Role of the hexosamine biosynthetic pathway in diabetic nephropathy.

The hexosamine biosynthetic pathway has been hypothesized to be involved in the development of insulin resistance and diabetic vascular complications. In particular, it was demonstrated that hyperglycemia-induced production of transforming growth factor-beta (TGF-beta1), a prosclerotic cytokine causally involved in the development of diabetic nephropathy. Several lines of evidence indicate that TGF-beta1 induction is mediated by the hexosamine pathway. In cultured mesangial cells, high glucose levels induce TGF-beta1 production. This effect is eliminated by inhibition of glutamine: fructose-6-phosphate-amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased levels of TGF-beta1 protein, mRNA, and promoter activity. Inasmuch as stimulation or inhibition of GFAT increased or decreased high glucose-stimulated activity of protein kinase C (PKC), respectively, the observed effects appear to be transduced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-alpha and basic fibroblast growth factor [bFGF]) was demonstrated in vascular smooth muscle cells. These studies also revealed a rapid increase in GFAT activity by treatment with agents that elevated levels of cyclic adenosine 3',5' monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immunohistochemistry and in situ hybridization, high expression of GFAT was found in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells. whereas glomerular cells remained essentially unstained. However, significant staining occurred in glomerular cells of patients with diabetic nephropathy. Current data indicate that the flux through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, particularly diabetic nephropathy.

Simultaneous, quantitative analysis of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose and UDP-galactose in human peripheral blood cells, muscle biopsies and cultured mesangial cells by capillary zone electrophoresis.

The aim of this study was to develop and evaluate a capillary zone electrophoretic (CE) procedure for the accurate quantification of the UDP-hexosamines as well as for the corresponding UDP-hexoses in samples from various biological origins. Testing different buffer conditions, voltages, capillary dimensions and temperatures, optimal results were achieved with a 90 mM borate buffer, pH 9.0, at 18 degrees C and 15.5 kV in an uncoated fused-silica capillary of 50 cm x 50 microm and a detection wavelength of lambda = 262 nm. The total procedure, i.e., including variations of the sample preparation, showed coefficients of variation for the peak areas between 4. 1% and 10.4% in mesangial cells (n = 7) and between 7.8 and 10.3% (n = 6) in leukocytes for the components of interest. To improve precision, an internal standard was used for calibration. The limit of detection for all compounds is an absolute amount of 180 fmol, sufficient for the precise analysis of UDP-sugars in a limited amount of biological samples, such as human leukocytes (obtained from a 10 mL blood sample), muscle biopsies (< or = 100 mg), and mesangial kidney cells (ca. 2.5 x 10(5) cells). This reproducible, quantitative analysis of all four UDP-sugars from various biomedically relevant origins by CZE is a definite improvement over the generally used high performance liquid chromatography (HPLC) procedures. The CZE method allows the study of the flux through the hexosamine pathway in diabetes mellitus and other diseases in a simple, quantitative and accurate way.

Molecular mechanism of diabetic nephropathy.

Diabetic nephropathy is one of the main causes of renal end-stage disease. Morphologically, the development of diabetic nephropathy is characterized by progressive thickening of the glomerular basement membrane and by expansion of the mesangial matrix which correlates to glomerular filtration function. In vitro studies with cultured mesangial cells revealed that elevated glucose concentrations increase collagen synthesis similar to the in vivo situation. These studies showed that hyperglycemia may be toxic either by non-enzymatic reaction of glucose with proteins and subsequent formation of advanced glucosylation end products or by increased metabolism leading to increased oxidative stress and activation of protein kinase C resulting in increased production of cytokines. Particularly, de novo synthesis of transforming growth factor beta1 (TGF-beta1) is induced and TGF-beta1 appears also involved since blockage of this prosclerotic factor inhibits high glucose-induced collagen synthesis. Interestingly, it could be demonstrated that angiotensin II also stimulates TGF-beta1 production possibly via the same signal transduction pathway. Besides the classical clinical chemical parameters for evaluation of renal function, the measurement of urinary albumin excretion is now widely used for detection of developing diabetic nephropathy. Since diabetes causes glomerular and tubular changes, tubular marker proteins may be used to detect early renal damage. An increased urinary excretion of matrix proteins (e.g. collagen) and cytokines (e.g. TGF-beta1) was found in early diabetic nephropathy. However, the diagnostic value of these new parameters remains to be established.