Effects of non-glycated and glycated glucagon-like peptide-1(7-36) amide on glucose metabolism in isolated mouse abdominal muscle

This study investigated the actions of non-glycated and glycated glucagon-like peptide-1(7-36)amide (tGLP-1) on glucose uptake and metabolism in isolated mouse abdominal muscle. Monoglycated tGLP-1 (Mr 3463.8) was prepared under hyperglycemic reducing conditions and purified by HPLC. Non-glycated tGLP-1 (10(-10)-10(-8) mol/l) stimulated both 2-deoxy-D-[1-3H]glucose uptake (1.3-1.5 fold) and 14C-glucose oxidation (1.4-1.7 fold) in muscle compared to controls without tGLP-1. Glycation reduced these stimulatory effects by 27-33% and 25% (at 10(-9) mol/l), respectively. tGLP-1 (10(-10)-10(-8) mol/l) promoted muscle glycogenesis and lactate production, whereas glycated peptide was ineffective below 10(-9) mol/l. This study demonstrates that tGLP-1 has potent glycogenic effects in mouse abdominal muscle in vitro and that glycation impairs its action.     

Plasma disappearance of glycated and non-glycated albumin in type 1 (insulin-dependent) diabetes mellitus: evidence for charge dependent alterations of the plasma to lymph pathway

The fractional plasma escape rates of glycated and non-glycated albumin have earlier been measured in groups of Type 1 (insulin-dependent) diabetic patients and control subjects. The escape of non-glycated albumin was similar in control subjects and normoalbuminuric patients, but elevated in patients with micro or macroalbuminuria. In all groups the escape rate of glycated albumin was lower than that of non-glycated albumin. Glycation increases the anionic charge of albumin. To assay for charge-dependent alterations of transport a selectivity index (non-glycated albumin/glycated albumin transport ratio) was determined from the disappearance data. The index was high in control subjects (1.021 +/- 0.0057 (SEM)). This reflects a mean difference between the two escape rates of 2.1% per hour (for comparison the mean of the fractional escape rate of non-glycated albumin of the normal control subjects was 4.7% per hour). The index was numerically even higher in normoalbuminuric patients (1.031 +/- 0.0047 (SEM)), but reached significantly lower levels in patients with microalbuminuria (1.013 +/- 0.0030 (SEM), p < 0.02). Patients with clinical nephropathy had very low levels indicating loss of selectivity (1.002 +/- 0.0068 (SEM), p < 0.001). This pattern accords well with measurements of renal clearance selectivity indices, suggesting a general, progressive deterioration of anionic perivascular barrier components in diabetic microangiopathy. The structural target for these changes is likely to be the glycosaminoglycans of the glomerular basal membrane and the interstitial matrix.     

IgG glycation and function during continuous ambulatory peritoneal dialysis

IgG in dialysate may have an important role in anti-infection mechanisms during continuous ambulatory peritoneal dialysis (CAPD). As Fc fragment oligosaccharidic chains are crucial for IgG effector functions, we have tested the hypothesis that IgG glycation might occur during CAPD and modify IgG properties. Purified normal IgG was incubated with glucose solutions of different concentrations and pH. Separation of glycated IgG was performed by affinity chromatography. Complement activation (C3c deposition) and phagocytosis by polymorphonuclear leucocytes (PMN) were studied in vitro using Staphylococcus aureus Wood (STAW) as antigen. In addition, we compared the percentages of glycated IgG in IgG purified from sera and dialysates of 12 CAPD patients. The percentage of glycated IgG after in vitro incubation of normal IgG with glucose solutions was directly proportional to glucose concentrations, incubation time and pH. Glycated IgG anti-STAW induced a higher C3c deposition than non-glycated IgG anti-STAW (C3c/IgG (mean +/- SD) 0.96 +/- 0.06 vs 0.79 +/- 0.08; P = 0.027). PMN phagocytosis was not affected by IgG glycation. The percentages of glycated IgG in dialysates of CAPD patients were greater than those in corresponding sera (5.38 +/- 2.36% vs 4.56 +/- 2.47%; P = 0.006). It is concluded that IgG glycation may take place in the peritoneal cavity during CAPD and lead to enhanced complement activation. This could explain the high degree of complement activation previously described in dialysate of CAPD patients and might theoretically result in a reduction of complement factors available in dialysate for adequate anti-infection mechanisms.     

Demonstration and isolation of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C

The corss-linking reagent p,p'-difluoro-m,m'-dinitrodiphenylsulfone has been used to fix in the tetramer form the various species of hemoglobin present in mixtures of hemoglobin A and hemoglobin C and of hemoglobin S and hemoglobin C. Following reaction, the presence of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C in these hemoglobin mixtures was demonstrated electrophoretically and the hybrids were isolated by ion-exchange chromatography. The identity of the alpha 2 A beta A beta C hybrid was further verified by peptide analysis. The success in cross-linking alpha 2 A beta 2 C, alpha 2 A beta A beta C, and alpha 2 A beta S beta C with p,p'-difluoro-m,m'-dinitrodiphenylsulfone shows that the distance between the alpha chain amino terminals in solution for these hemoglobin species is the same as in normal hemoglobin.     

Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a Kcat of 14.3 s-1 and a kcat/KM of 2.70 x 10(6) s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.     

Glycated proteins modulate tissue-plasminogen activator-catalyzed plasminogen activation

Plasminogen activation by tissue-plasminogen activator (t-PA) is accelerated by the presence of a macromolecular surface, which acts as a template that brings enzyme and substrate in close proximity. Modification of lysine residues, which are important for this template function, occurs in diabetic patients as a consequence of glycation of proteins. In this study, we investigated the effects of glycation of fibrin and other proteins in t-PA-catalyzed plasmin formation. Plasminogen activation on glycated fibrin(ogen) was increased compared to non-glycated fibrin(ogen), which could fully be attributed to an increased affinity of t-PA for glycated fibrin(ogen). Binding of plasminogen to glycated fibrin was increased, but did not contribute to increased plasminogen activation. Both plasminogen activator inhibitor-1 (PAI-1) binding and activity were increased on glycated fibrin. Induction of template function in plasminogen activation was also observed on immobilized glycated bovine serum albumin (BSA) and human gamma-globulins (IgG). Increased plasmin generation at sites of deposition of glycated proteins may lead to increased extracellular matrix breakdown and thereby affect the integrity of the endothelial monolayer. Moreover, soluble glycated BSA and glycated IgG can inhibit t-PA binding to immobilized glycated fibrin and interfere with fibrinolysis in diabetic patients.     

Purity and antigenicity of the new insulin preparations

A short review on new types of insulin preparations purified by chromatography is presented. The purity of these new chromatographed insulins, which have almost completely replaced the conventional types of insulin, has been considerably improved. Our own investigations have revealed significantly lower antigenicity of porcine depot preparations compared with beef or mixed beef-pork insulins. By means of gel filtration and additional anion exchange chromatography the antigenicity of porcine insulin can be significantly reduced. Monocomponent insulins prepared by these methods produce low titers of insulin antibodies in only one third of diabetics treated by insulin for the first time. Mixed beef-pork Lente Insulin purified by single chromatography is also less antigenic than Lente Insulin of conventional purity, but more antigenic than pork insulin (e.g. Monotard). Pork insulin purified by chromatography is indicated in cases of insulin allergy, insulin resistance and lipoatrophy, and also for first insulin treatment in younger and middle-aged diabetics.     

Isolation and characterization of anti morphine analgesic peptide from canine brain

The anti-morphine peptide from canine brain, which is homologous to alpha subunit of hemoglobin of canine, has been isolated and characterized. The canine brain was homogenized and extracted in 0.1 M acetic acid. The supernatant was collected, and purified by chromatography of Sephadex G-50 and ion-exchange and RP-HPLC. A polypeptide was then obtained, which is termed as AMP5. It shows prominent antimorphine analgesic activity. AMP5 has an apparent molecular mass of 10.23kD. The isoelectric point was estimated to be around pH 6.7 by IEF. The amino acid composition of AMP5 and sequence of the N-terminal 50 amino acid residues was determined.     

Experimental benznidazole encephalopathy: II. Electroencephalographic and morphological alterations

Glycated hemoglobin can be degraded by proteolytic enzyme(s) in the erythrocyte. The enzyme(s) co-elutes with glycated hemoglobin when the latter is separated from erythrocyte lysates using the cation-exchanger Bio Rex-70. A further purification of the Bio Rex eluant on DEAE Sephadex A-50 separated the enzyme(s) from glycated hemoglobin. Studies with the Bio Rex eluant showed that degradation of glycated hemoglobin is maximum at 37 degrees C at pH 8.6. Proteolytic degradation is inhibited by 5 mM N-ethylmaleimide (NEM), 5 mM ethylenediamine tetraacetic acid (EDTA) and 0.6 mM n-p-tosyl-L-lysine choromethyl ketone (TLCK) (100-87 and 76% inhibition respectively). This study also examines the possibility that oxidative-damage to glycated hemoglobin increases its susceptibility to proteolytic degradation. When incubated with various anti-oxidants like DTPA, uric acid, mannitol and butylated hydroxy toluene (BHT), proteolytic degradation of glycated hemoglobin decreased by 66.1, 50.7 and 38% respectively.     

The validity of basal blood glucose in the control of non-insulin-dependent diabetic patients

BACKGROUND: To validate basal glucemia as a control method for non-insulin dependent diabetes mellitus, and to determine the cut-off point that best characterizes good control. PATIENTS AND METHODS: A transversal, observational study of 256 patients who participated in a diabetes mellitus follow-up program during 1993. In the study, glucemia validity indicators were evaluated after making 2 X 2 tables and ROC (receiver operating characteristic) curves for the different values. Control values of glycated hemoglobin was used as to define a good (< 6.5%) and moderate (< 8%). RESULTS: The values of glucemia considered to be "good" as regards control (from 80 to 110 mg/dl, 4.4-6.05 mmol/dl) have good sensitivity (from 97.3% to 100%) and negative predictive values (from 85.7% to 100%) but extremely bad specificity (from 3.8% to 22.7%) and only moderate positive predictive values (from 59.5% to 64.1%) in reference to values of glycated hemoglobin of 6.5%. The same occurs for 8% as regards sensitivity (from 98.6% to 100%), negative predictive value (from 96.4% to 100%) and specificity from 2.1% to 14.5%). Positive predictive value worsens (from 27.8% to 30.3%). The most effective and most accurate values of glucemia in the ROC curves are 150 mg/dl (8.25 mmol/l) if the control of glycated hemoglobin is good, and 170 mg/dl (9.35 mmol/l) if it is moderate. CONCLUSIONS: The glucemia control figures recommended by consensus produce false positives when they are compared to glycated hemoglobin. In the analysis of effectiveness and ROC curves greater accuracy is obtained with glucemia values that are slightly higher than those recommended.     

Differential gene expression of angiotensin II receptor subtypes in the epididymides of mature and immature rats

Electrospray Ionization Mass Spectrometry can be applied to detect aberrant proteins using intact molecules. Direct examination of hemolysate might well facilitate rapid ascertainment of a variant hemoglobin (Hb) provided that the mass difference between normal and abnormal chains is larger than the resolution power of standard instruments (i.e. = 10 Da). We propose immunoprecipitation as a preparation method of plasma and cell proteins other than Hb prior to MS. Amino acid sequences of various variants detected by MS were determined by MS/MS. Some of these variants were new. These new variants were; 1: Hb Sagami[beta 139(H17)Asn-->Thr]. 2: Hb Hokusetsu[beta 52(D3)Asp-->Gly]. 3: a variant transthyretin, amyloidogenic, [38Asp-->Ala]. 4: a variant transthyretin, non-amyloidogenic, [101Gly-->Ser]. The abundance of ion peaks showed the approximate ratio of each component, which was in agreement with the ratio obtained by chromatography and by ESIMS in the analyses of glycated hemoglobin. Samples with low kidney function (BUN > 50 mg/dl, creatinine > 2.5 mg/dl) showed higher values of glycated Hb on routine HPLC than the MS method. Samples containing high carbamylated Hb might cause this discrepancy.     

Protein glycation in the aging male Sprague-Dawley rat: effects of antiaging diet restrictions

Protein glycation and accumulation of advanced glycosylated end-products (AGEs) are supposed to play an important role in the process of aging. Dietary restriction increases life span and delays the onset of most age-associated diseases. Age-dependent changes in glucose homeostasis and glycated plasma proteins and hemoglobin were determined, and AGEs formation was measured as fluorescence in skin and aortic collagens in male Sprague-Dawley rats fed ad libitum or subjected to every-other-day feeding or 40% food restriction. In aging control rats, skin and aortic collagen-linked fluorescence increased with a similar exponential curve (aortic value being always higher), whereas glycated plasma protein and hemoglobin decreased slightly. Dietary restrictions decreased glycated plasma proteins and fluorescent products in skin collagen of younger but not older rats, and did not affect glycated hemoglobin or aortic collagen fluorescence. In conclusion, our data indicate that age-related changes in glucose homeostasis do not play a substantial role in aging; and collagen-linked fluorescence increases significantly during aging, but it may not be sensitive to dietary intervention.     

The use of electrophoretic methods for determining modified proteins in diabetic patients

Modified proteins were determined by isoelectric focusing in borate-polyol system with subsequent colorimetry (micromethod) and electrophoresis of blood serum on paper with subsequent TCA-ethanol treatment. Increased levels of glycated hemoglobin and modified albumin and changed light absorbance of glycated albumin were detected. The levels of glycated hemoglobin assessed by the micromethod and colorimetry without calibration did not correlate.     

Surgical treatment of vertebral and posterior inferior cerebellar artery aneurysms

Geochelone carbonaria hemoglobin (Hb) was analyzed by polyacrylamide gel electrophoresis (PAGE) and purified by ion exchange chromatography on CM-cellulose. Seven fractions were obtained using fresh Hb preparations. CM-cellulose chromatography of Hb reacted with iodoacetamide, showed one minor (HbI) and one major band (HbII). Analysis of the molecular masses of recently collected Hb and of aged solutions determined by gel filtration showed that polymerization increased with the duration of storage. The reaction with oxidized glutathione changed the electrophoretic pattern of Hb, and highlighted the bands corresponding to glutathionyl-Hb. The presence of these bands in fresh Hb solutions and in alkylated preparations suggests that they may occur in vivo. PAGE under dissociating conditions showed that the hemolysate contained 3 different polypeptide chains (G1, G2 and G3). Both Hb components shared the G1 globin chain with HbI containing G1 and G2 and HbII, G1 and G3 chains.     

Some properties of purified skeletal muscle alpha-actinin

Highly purified alpha-actinin can be made by using the low ionic strength extraction procedure previously described (Arakawa N., Robson, R. M., and Goll, D. E. (1970) Biochim. Biophys. Acta 200, 284-295) and then subjecting the crude alpha-actinin fraction obtained with this extraction procedure to successive chromatography on DEAE-cellulose and hydroxyapatite. Hydrozyapatite chromatography specifically removes a protein having a subunit molecular weight of 42,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hydroxyapatite-purified alpha-actinin sediments entirely as a 6.21 S boundary in the analytical ultracentrifuge with no trace of the small 9 to 10 S boundary seen in earlier alpha-actinin preparations purified by DEAE-cellulose chromatography. In 100 mM KCl, 20 mM Tris-acetate, pH 7.5, hydroxyapatite-purified alpha-actinin has a diffusion coefficient (D020,w) of 2.71 X 10(-7) cm2-s-1, an intrinsic viscosity of 20.6 ml-g-1, a molecular weight of 201,000 +/- 4,300 (plus or minus least squares standard error) as determined by sedimentation equilibrium, and a molecular weight of 210,000 as determined by sedimentation diffusion. In 6 M guanidine HCl, hydroxyapatite-purified alpha-actinin has a molecular weight of 106,000 +/- 6,300 as determined by sedimentation equilibrium and a molecular weight of 100,000 as determined by a calibrated 4% agarose gel permeation column. SDS-polyacrylamide gel electrophoresis gives a molecular weight of 96,000 to 100,000 for hydroxyapatite-purified alpha-actinin. Rod-shaped particles 44 X 390 to 400 A are seen in electron micrographs of negatively stained alpha-actinin. By assuming 45% hydration and a molecular weight of 206,000, dimensions of approximately 40 X 500 A can be calculated for the alpha-actinin molecule by using either s 020, w, D 020, w, intrinsic viscosity, or a calibrated 6% agarose gel permeation column. Hydroxyapatite-purified alpha-actinin has an alpha-helical content of 74% as measured by circular dichroism at 208 nm.     

Glycated cholecystokinin-8 has an enhanced satiating activity and is protected against enzymatic degradation

Monoglycated cholecystokinin octapeptide (CCK-8) (glucitol-Asp1 adduct) modified at the NH2-terminus was prepared under hyperglycemic conditions, purified by high-performance liquid chromatography, and characterized by mass spectrometry (Mr 1228.4 Da) and peptide sequencing. CCK-8 (100 nmol/kg, i.p.) significantly (P < 0.001) reduced voluntary food intake of fasted mice for up to 30 min after its administration, compared with saline-administered controls. Glycated CCK-8 reduced food intake at 30-120 min (P < 0.01 to P < 0.001) and significantly reduced feeding compared with CCK-8 from 60 to 120 min (P < 0.01). In vitro plasma degradation studies indicated that glycated CCK-8 was resistant to the normal rapid enzymatic conversion to CCK fragments. This study demonstrated that CCK-8 is a potent short-term inhibitor of food intake, and that structural modification of this peptide by amino-terminal glycation leads to enhanced satiating activity, partially due to increased resistance to serum aminopeptidase degradation.     

What is hemoglobin A1c? An analysis of glycated hemoglobins by electrospray ionization mass spectrometry

Hemoglobin A1c (HbA1c) is a stable minor Hb variant formed in vivo by posttranslational modification by glucose, originally identified by using cation exchange chromatography, and containing primarily glycated N-terminal beta-chains. However, the structure(s) of the quantified species has not been elucidated, and the available methods lack a reference standard. We used electrospray ionization mass spectrometry to determine the extent of glycation of samples separated by boronate affinity and/or cation exchange chromatography. Analyses of clinical samples were consistent with the curvilinear relationship of patient glucose and HbA1c. As glycation increased, the ratio of beta-chain to alpha-chain glycation increased, and the number of glycation sites on the beta-chain increased, although these were relatively minor components. We found several glycated species that cochromatographed with HbA1c on cation exchange, including species with both glycated alpha- and beta-chains, nonglycated alpha- and glycated beta-chains, and multiply glycated beta-chains. The combined use of affinity and cation exchange chromatography with structural confirmation by electrospray ionization mass spectrometry was found to be useful in producing samples of sufficient purity for the standardization of glycohemoglobin clinical assays.     

In vitro regulation of an inducible-type NO synthase in the rat seminiferous tubule cells

S-Adenosyl-L-methionine:L-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-L-methionine (SMM) from L-methionine and S-adenosyl-L-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).     

The importance of quaternary structure in the expression of the C1-binding site of IgM

The ability of C1 to bind to the Fc5mu-fragment of a monoclonal IgM was determined by means of a haemolytic C1-inhibition assay. Fc5mu fragments were produced by trypsin digestion at five different temperatures ranging from 54-62 degrees (2 degrees increments) and were purified by immunoadsorption through a column of monospecific anti-Fabmu and by molecular exclusion chromatography. Analytical ultracentrifugation showed the final preparations to be free of aggregates. A plot of mug Fc5mu required to inhibit 50% of available C1 versus temperatures of production of the fragment yielded a curve with a minimum at 58-60 degrees. Upon mild reduction and alkylation of these Fc5mu fragments their C1-fixing capacity became approximately the same irrespective of temperature of production. Fc5mu was also prepared at 25 degrees in the presence of 5 M urea, purified by immunoadsorption as before and aliquots then exposed to temperatures ranging from 40-70 degrees (5 degrees increments) for 15 min. After aggregates had been removed by chromatography a similar minimum in C1-fixation was again observed at 60 degrees. Reduction and alkylation once more abolished these differences. Fc5mu and its reduced and alkylated subunits, produced at 60 degrees and then exposed to various concentrations of urea (0-7 M) for 24 hd did not yield a minimum in C1 fixation. Reduced and alkylated Fcmu incubated at various temperatures (40-70 degrees) also did not fix C1 differentially. Examination in the near and far u.v. region of the circular dichroism spectra of different Fc5mu preparations showed a gradual loss of structure associated with restricted aromatic chromophores and secondary (beta) structure with increased temperature. Urea denaturation had a more pronounced and irreversible effect on Fc5 mu conformation. These changes could not be correlated with the CU-fixation patterns observed. It would therefore appear that elevated temperatures induce a static change in the pentameric FC-part of IgM which in turn directly influences or modulates the availability of the C1-binding site. The importance of disulphide bonds in maintaining these temperature-induced changes in Fc5mu was also indicated.     

Evaluation of assay methods of glycohemoglobin: reference method and parameter

In this paper, the characteristics of the analytical procedures allowing the measurement of glycated hemoglobin products are presented. With respect to their respective performances, the authors recommend the specific measurement of HbA1c and the more reliable procedure, high pressure liquid chromatography (HPLC).