Characterization of N-glycosylated type I collagen in streptozotocin-induced diabetes.

The N epsilon-glycosylation of lysine and hydroxylysine residues in collagen from streptozotocin-induced-diabetic rats was confirmed and the stability of the complex shown to be due to an Amadori rearrangement. The studies also demonstrate the relative specificities of glucose, galactose and mannose in their reaction with collagen. The glycosylation of lysine in vitro occurs with glucose and galactose, but not with mannose, whereas only gucose reacts with hydroxylysine to any significant extent. Glycosylation of collagen occurs slowly during normal aging, but in contrast with reports suggesting accelerated aging of collagen in diabetic animals, we clearly demonstrated that the apparent increased stability is not due to an acceleration of the normal maturation process involving the reducible cross-links.     

Apolipoprotein H Is Not Affected by In Vitro Glycosylation

Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstrated in diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H, whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes. Apolipoprotein H was isolated from human plasma and purified through a combination of affinity chromatography and continuous elution electrophoresis. The in vitro glycosylation was performed by incubating purified apolipoprotein H with high concentration of glucose. Our results indicate that the in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoprotein H, despite the fact that this apolipoprotein contains a high number of lysine residues. Since the in vitro concentration of glucose was far higher than the levels normally found in diabetic subjects, it is unlikely for apolipoprotein H to become glycosylated in diabetes.     

Apolipoprotein H Is Not Affected by In Vitro Glycosylation

Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstrated in diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H, whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes. Apolipoprotein H was isolated from human plasma and purified through a combination of affinity chromatography and continuous elution electrophoresis. The in vitro glycosylation was performed by incubating purified apolipoprotein H with high concentration of glucose. Our results indicate that the in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoprotein H, despite the fact that this apolipoprotein contains a high number of lysine residues. Since the in vitro concentration of glucose was far higher than the levels normally found in diabetic subjects, it is unlikely for apolipoprotein H to become glycosylated in diabetes.     

Evidence for glucose-mediated covalent cross-linking of collagen after glycosylation in vitro.

Rabbit forelimb tendons incubated for 15 or 21 days at 35 degrees C in the presence of 8 or 24 mg of glucose/ml were shown to change their chemical, biochemical and mechanical characteristics. The tendons treated with glucose contained up to three times as much hexosyl-lysine and hexosylhydroxylysine as did control tendons as judged by assay of NaB3H4-reduced samples. Measurement of the force generated on thermal contraction showed significant increases in glycosylated tendons compared with controls, indicating the formation of new covalent stabilizing bonds. This conclusion was supported by the decreased solubility of intact tendons and re-formed fibres glycosylated in vitro, and by the evidence from peptide maps of CNBr-digested glucose-incubated tendons. The latter, when compared with peptide maps of control tendons, revealed the presence of additional high-Mr peptide material. These peptides appear to be cross-linked by a new type of covalent bond stable to mild thermal and chemical treatment. This system in vitro provides a readily controlled model for the study of the chemistry of changes brought about in collagen by non-enzymic glycosylation in diabetes.     

Effects of glucose and its modulation by insulin and estradiol on BMSC differentiation into osteoblastic lineages.

It is well known that diabetes affects bone in human and animal models, and leads to osteopenia and osteoporosis. Bone-mineral density and other biochemical markers of bone turnover are very much affected in people with diabetes. Reduced bone mass, occurring with increased frequency in diabetes mellitus, has been attributed to poor glycemic control, but the pathogenic mechanisms remain unknown. High concentrations of glucose (hyperglycemia) in diabetics leads to this complication. Very few in vitro studies using bone-cell lines have been carried out to address this problem. In this study, we examined the effects of different doses of glucose concentration (5.5, 16.5, and 49.4 mmol/L), alone, with insulin (0.6 microg/mL), or with 17beta-estradiol (E2) (10 nmol/L), on rat bone-marrow stromal cells (BMSCs) in the presence of an osteogenic medium. BMSC proliferation and alkaline phosphatase (ALP) were studied after 3 and 7 d of culture, respectively; the area stained for collagen and mineralized nodules was studied after 28 d of culture. With high concentrations of glucose, BMSC proliferation, ALP activity, the number of nodules formed, and the area stained for collagen were greatly reduced. Insulin treatment alone was able to increase [3H]-thymidine uptake or ALP activity, whereas both insulin and estradiol were able to increase the number of mineralized nodules and the area stained for collagen and mineralization. In conclusion, this study suggests that insulin and estradiol are able to contain the deleterious effect of high concentrations of glucose on BMSC-derived osteoblast proliferation and function.     

Influence of monosaccharides on the fibrillogenesis of type I collagen

The in vitro self-assembly of Type I rat tail tendon collagen in the presence of monosaccharides was studied turbidimetrically. D(+)glucose and D(+)mannose delayed fibril formation significantly while D(-)fructose showed no variation. The effect of glucose was concentration dependent. Binding of glucose during fibrillogenesis was shown by the incorporation of [14C]glucose and also by SDS-polyacrylamide slab gel electrophoresis of the product. The results indicate that in vitro nonenzymatic glycosylation can take place in the short duration required for fibril formation.     

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of thrombospondin-1-mediated TGF-��1 on collagen type III synthesis induced by high glucose

Transforming growth factor-β1 (TGF-β1) has been thought to play a major role during cardiac fibrosis in the development of diabetic cardiomyopathy, and cardiac fibrosis mainly as a result of an increase of collagen type III occurs in the human hearts with diabetes. Thrombospondin-1 (TSP-1) has been reported to activate the latent complex of TGF-β1. We examined the effects of TSP-1 on the expression of TGF-β1 and collagen type III by rat cardiac fibroblasts in high ambient glucose. We demonstrated that high glucose induces the mRNA and protein expression of collagen type III, TGF-β1, and TSP-1. Furthermore, the mRNA and protein expression of collagen type III induced by high glucose was downregulated after treatment with TGF-β1 antibody, or TSP-1 siRNA. The expression of TGF-β1 increased by high glucose was also reversed after treatment with TSP-1 siRNA. Our findings suggest that the TSP-1 participates in the upregulation of TGF-β1, collagen type III by high glucose and may provide new therapeutic strategies for diabetic cardiomyopathy.     

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm(2) shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell-matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.     

In vivo glycosylation of dermal and tendon type I collagen

Recent studies show that native collagen fibers in the extracellular space can be subject to nonenzymatic glycosylation and that the extent of such glycosylation increases in clinical hyperglycemia and aging. In the present study, a comparison was made on the extent of glycosylation in rat tail tendon and in the soluble and insoluble fractions of collagen separated from rat skin after in vivo labeling with [14C]glucose. It was observed that nonenzymatic glycosylation occurred maximally in the salt-soluble fraction as measured by the level of ketoamine linked hexose. 14C radioactivity incorporation as well as the number of free amino groups was also increased in this fraction. However, the amounts of O-glycosidically linked sugars did not show much variation between the soluble and insoluble fractions. These findings could be correlated to the enhanced metabolic turnover of newly synthesized collagen in diabetics.     

Release of iron from haemoglobin--a possible source of free radicals in diabetes mellitus

Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes.     

Effect of curcumin on protein glycosylation, lipid peroxidation, and oxygen radical generation in human red blood cells exposed to high glucose levels

Curcumin (diferuloylmethane) is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Experimental studies with diabetic animals demonstrate that curcumin supplementation can suppress cataract development and collagen cross-linking, promote wound healing, and lower blood lipids and glucose levels. The mechanism by which curcumin may cause diabetes-associated vascular damage to regress is not known. Erythrocytes were treated with high levels of glucose (mimicking diabetes) in the presence or absence of curcumin (0-10 muM) in the medium at 37 degrees C for 24 h. This study demonstrates that curcumin prevents protein glycosylation and lipid peroxidation caused by high glucose levels using an erythrocyte cell model. This study also suggests that curcumin may inhibit oxygen radical production caused by high glucose concentrations in a cell-free system, and increase glucose utilization in erythrocytes. This provides evidence for a novel mechanism by which curcumin supplementation may prevent the cellular dysfunction associated with diabetes.     

Effects of diabetes on glucose oxidation in rat aorta after hypophysectomy and adrenalectomy

The influence of diabetes, hypophysectomy and adrenalectomy on glucose oxidation in rat aorta was studied. Diabetes was induced in normal, adrenalectomized and hypophysectomized-cortisone substituted rats by streptozotocin (65 mg/kg body weight). The oxidation of glucose to CO2 was determined during incubation of rat aorta in vitro for 2-3 hours. The aortic glucose oxidation was reduced after hypophysectomy but was unaffected by adrenalectomy. After streptozotocin treatment the rise in blood glucose concentration was similar in normal, adrenalectomized and hypophysectomized-cortisone substituted rats. In shamoperated diabetic rats the aortic glucose oxidation was reduced after a diabetes duration of 4 days. In adrenalectomized diabetic rats the aortic glucose oxidation was not significantly affected after 4 days but was reduced after a diabetes duration of 14 days. When adrenalectomized diabetic rats were treated with hydrocortisone the aortic glucose oxidation was reduced after diabetes for 4 days. After incubation of normal rat aorta in vitro for 6 hours with cortisol (1 microgram/ml) in the incubation medium a decrease in the aortic glucose oxidation was found. Incubation of aorta with only growth hormone had no effect. These results suggest that cortisol is of importance for the lowered glucose oxidation in diabetic rat aorta.     

Anti-fibrotic cardio protective efficacy of aminoguanidine against streptozotocin induced cardiac fibrosis and high glucose induced collagen up regulation in cardiac fibroblasts

This study mainly focuses on cardio protective anti-fibrotic activity of aminoguanidine against streptozotocin induced cardiac fibrosis and high glucose induced collagen accumulation in cardiac fibroblasts. Dysregulation of matrix metalloproteinase especially 2 and 9 were considered to be responsible for the abnormal collagen deposition, which resulting improper cardiac contractile function in diabetic mice. Mice received a single dose of streptozotocin (100 mg/kg) through tail vein to induce diabetes. Normal and diabetic mice received aminoguanidine orally (100 mg/kg/day) throughout the study period of 8 weeks. Cardiac fibroblasts cultured and exposed to high glucose, aminoguanidine and both for 48 h. Collagen quantitatively estimated in both in vivo and in vitro models. Altered structural changes were studied using the Masson tri-chrome staining, TEM images of cardiac sections. Increased collagen and metalloproteinase activities were confirmed using gelatin zymography, western blotting and gene expression studies. The exact mechanism responsible for high glucose induced collagen up regulation in diabetic heart was incompletely understood. From this above in vivo and in vitro results, we conclude that, the cardio protective anti fibrotic activity of amino guanidine was mainly attributed by exhibiting the inhibitory efficacy against streptozotocin and high glucose induced collagen accumulation probably by inhibiting high glucose altered metalloproteinase-2 and -9 activities.     

Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance

Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846-8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.     

Biosynthesis of collagen and other matrix proteins by articular cartilage in experimental osteoarthrosis.

Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage.     

Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.     

Oxidative stress--mediated alterations in glucose dynamics in a genetic animal model of type II diabetes

Insulin resistance, characterized by an inexorable decline in skeletal muscle glucose utilization and/or an excessive hepatic glucose production, constitutes a major pathogenic importance in a cluster of clinical disorders including diabetes mellitus, hypertension, dyslipidemia, central obesity and coronary artery disease. A novel concept suggests that heightened state of oxidative stress during diabetes contributes, at least in part, to the development of insulin resistance. Several key predictions of this premise were subjected to experimental testing using Goto-Kakizaki (GK) rats as a genetic animal model for non-obese type II diabetes. Euglycemic-hyperinsulinemic clamp studies with an insulin infusion index of 5 mU/kg bw/min were used to measure endogenous glucose production (EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal muscle glucose utilization index (GUI). Moreover, the status of oxidative stress as reflected by the urinary levels of isoprostane and protein carbonyl formation were also assessed as a function of diabetes. Post-absorptive basal EGP and circulating levels of insulin, glucose and free fatty acid (FFA) were elevated in GK rats, compared to their corresponding control values. In contrast, steady state GIR and GDR of the hyperglycemic/hyperinsulinemic animals were reduced, concomitantly with impaired insulin's ability to suppress EGP. Insulin stimulated [3H]-2-deoxyglucose (2-DG) uptake (a measure of glucose transport activity) by various types of skeletal muscle fibers both in vivo and in vitro (isolated muscle, cultured myoblasts) was diminished in diabetic GK rats. This diabetes-related suppression of skeletal muscle glucose utilization was associated with a decrease in insulin's ability to promote the phosphorylation of tyrosine residues of insulin receptor substrate-1 (IRS-1). Similarly, the translocation of GLUT-4 from intracellular compartment to plasma membrane in response to insulin was also reduced in these animals. Oxidative stress-based markers (e.g. urinary isoprostane, carbonyl-bound proteins) were elevated as a function of diabetes. Nullification of the heightened state of oxidative stress in the GK rats with alpha-lipoic acid resulted in a partial amelioration of the diabetes-related impairment of the in vivo and in vitro insulin actions. Collectively, the above data suggest that 1) insulin resistance in GK rats occurs at the hepatic and skeletal muscle levels, 2) muscle cell glucose transport exhibited a blunted response to insulin and it is associated with a major defect in key molecules of both GLUT-4 trafficking and insulin signaling pathways, 3) skeletal muscle insulin resistance in GK rats appears to be of genetic origin and not merely related to a paracrine or autocrine effect, since this phenomenon is also observed in cultured myoblasts over several passages and finally heightened state of oxidative stress may mediate the development of insulin resistance during diabetes.     

Glutathione reverses early effects of glycation on myosin function

Nonenzymatic glycosylation (glycation) has been recognized as an important posttranslational modification underlying alterations of structure and function of extracellular proteins during aging and diabetes. Intracellular proteins may also be affected by this modification, and glycation has been suggested to contribute to aging-related impairment in skeletal muscle function. Glycation is the chemical reaction of reducing sugars with primary amino groups resulting in the formation of irreversible advanced glycation end products. Glutathione is an abundant tripeptide in skeletal muscle. To understand the effect of glutathione on glycated myosin function, we used a single-fiber in vitro motility assay in which myosin is extracted from a single muscle fiber segment to propel fluorescent-labeled actin filaments. Myosin function responded to glucose exposure in a dose-dependent manner, i.e., motility speeds were reduced by 10, 34, and 90% of preincubation values after 30-min exposure to 1, 3, and 6 mM glucose, respectively. The 30-min 6 mM glucose incubation was followed by a 20-min 10 mM glutathione incubation. Glutathione treatment restored motility (0.98 ± 0.06 µm/s, n = 3; P < 0.001) after glucose exposure (0.10 ± 0.07 µm/s, n = 3), close to preincubation levels (1.12 ± 0.06 µm/s, n = 3). It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function. in vitro motility; skeletal muscle fibers; speed of contraction