Loss of potentiating effect of hypoglycemia on the glucagon response to hyperaminoacidemia in IDDM.

IDDM subjects lose the ability to release glucagon during hypoglycemia. Because replacement of basal levels of amino acids enhances the glucagon response to hypoglycemia in healthy subjects, we tested whether raising amino acid levels during hypoglycemia could reverse the defective alpha-cell response in IDDM patients. For this purpose, 11 IDDM patients (HbA1 9.4 +/- 0.6%) and 8 healthy, nondiabetic subjects received two hypoglycemic insulin clamp studies (0.8 mU.kg-1 x min-1) in which plasma glucose was clamped at 55 mg/dl (3.08 mM) for 180 min. During one of the studies, an infusion of amino acids was superimposed between 120 and 180 min (0.3 g.kg-1 x h-1). This dose of amino acids had a small effect on plasma glucagon levels during euglycemic hyperinsulinemia that was comparable in normal and IDDM subjects. In healthy control subjects, plasma glucagon rose by 80% during the initial hypoglycemic phase of the study. The addition of amino acids produced a further sharp (200-250 ng/L, P < 0.02) rise in plasma glucagon, such a change did not occur in the absence of amino acids. In contrast, plasma glucagon in IDDM patients failed to increase during hypoglycemia alone and rose by only 40-50 ng/L (P < 0.05 vs. controls) when amino acid infusion was superimposed, even though plasma amino acid levels rose to the same extent in IDDM and control subjects. More importantly, the rise in glucagon produced by amino acids was comparable during hypoglycemic and euglycemic hyperinsulinemia in the IDDM patients, results strikingly different from those observed in nondiabetic control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose transport in human skeletal muscle. The in vivo response to insulin.

Transmembrane glucose transport plays a key role in determining insulin sensitivity. We have measured in vivo WBGU, FGU, and K(in) and K(out) of 3-O-methyl-D-glucose in forearm skeletal muscle by combining the euglycemic clamp technique, the forearm-balance technique, and a novel dual-tracer (1-[3H]-L-glucose and 3-O-[14C]-methyl-D-glucose) technique for measuring in vivo transmembrane transport. Twenty-seven healthy, lean subjects were studied. During saline infusion, insulin concentration, FGU (n = 6), K(in), and K(out) (n = 4) were similar to baseline. During SRIF-induced hypoinsulinemia (insulin < 15 pM, n = 4) WBGU was close to 0, and FGU, K(in), and K(out) were unchanged from basal (insulin = 48 pM) values. During insulin clamps at plasma insulin levels of approximately 180 (n = 4), approximately 420 (n = 5), approximately 3000 (n = 4), and approximately 9500 pM (n = 4), WBGU was 14.2 +/- 1.3, 34.2 +/- 4.1 (P < 0.05 vs. previous step), 55.8 +/- 1.8 (P < 0.05 vs. previous step), and 56.1 +/- 6.3 mumol.min-1.kg-1 of body weight (NS vs. previous step), respectively. Graded hyperinsulinemia concomitantly increased FGU from a basal value of 4.7 +/- 0.5 mumol.min-1.kg-1 up to 10.9 +/- 2.3 (P < 0.05 vs. basal value), 26.6 +/- 4.5 (P < 0.05 vs. previous step), 54.8 +/- 4.3 (P < 0.05 vs. previous step), and 61.1 +/- 10.8 mumol.min-1.kg-1 of forearm tissues (NS vs. previous step), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Mild hypoglycemia and impairment of brain stem and cortical evoked potentials in healthy subjects.

To evaluate the impact of mild hypoglycemia on CNS function in healthy adults, we measured brain stem auditory evoked potentials and P300 potentials (elicited by cognitive processing of auditory stimuli) during hypoglycemic or euglycemic insulin clamps (80 mU.m-2.min-1). In the hypoglycemic clamp study (n = 8), plasma glucose was allowed to fall from 4.6 to 3 mM in hourly approximately 0.5-mM steps and subsequently returned to euglycemic baseline levels. In the euglycemic clamp study (n = 8), plasma glucose was maintained at baseline levels throughout. Neither brain stem nor P300 responses changed during the euglycemic control study; symptoms and counterregulatory hormones were also unaffected. During the hypoglycemia study, epinephrine and growth hormone rose once plasma glucose reached 3.4 +/- 0.1 mM. Brain stem and P300 potentials remained unchanged until the 3-mM glucose step, when neurophysiological changes suddenly developed in conjunction with reported symptoms. At this glucose level, the wave V component of the brain stem potential was selectively altered in 7 of 8 subjects. Furthermore, P300 latency significantly increased, and amplitude diminished. Changes in both brain stem and cortical (P300) responses reversed when euglycemia was restored. We conclude that modest reductions in plasma glucose (to 3 mM) produce marked alterations in both brain stem and cortical responses to auditory stimuli. These changes in neural function appear at the same time as symptoms and follow rather than precede the rise in counterregulatory hormones during hypoglycemia. Our data suggest that the adverse effects of mild hypoglycemia on brain function are not limited to higher centers but also involve the brain stem.

Effect of acute physiological elevations of insulin on circulating androgen levels in nonobese women.

Extreme pharmacological elevation of the circulating insulin level acutely lowers dehydroepiandrosterone sulfate (DHEAS) levels. To assess whether more physiological elevations in plasma insulin (due to exogenous infusion or endogenous secretion) would have similar effects, we examined the levels of DHEAS, androstenedione, testosterone, and free testosterone before and after euglycemic hyperinsulinemic and hyperglycemic hyperinsulinemic clamp studies. Studies were performed in women within 20% of ideal body weight after an overnight fast. Androgen levels were measured before and at the conclusion of studies in which either insulin was infused exogenously at 1 mU/kg.min or endogenous insulin secretion was stimulated for 2 h by elevation of the plasma glucose concentration by 125 mg/dL above basal levels by an exogenous glucose infusion. Basal plasma DHEAS (6.2 +/- 0.5 mumol/L) declined to 5.2 +/- 0.4 mumol/L (P less than 0.001) during the euglycemic insulin clamp, without any significant change in testosterone, free testosterone, or androstenedione. During the hyperglycemic clamp, DHEAS fell from 6.7 +/- 0.5 to 5.1 +/- 0.4 mumol/L (P less than 0.001) in response to endogenous hyperinsulinemia; plasma testosterone, free testosterone, and androstenedione did not change significantly. There was no correlation between the elevation in plasma insulin concentration and the fall in DHEAS during either the euglycemic or hyperglycemic clamps. However, the magnitude of fall of DHEAS was directly correlated with the initial DHEAS level in both the euglycemic (r = 0.51; P less than 0.05) and hyperglycemic (r = 0.75; P less than 0.01) studies. This association of hyperinsulinemia with a reduction of circulating levels of DHEAS, but not other C-19 steroids (e.g. testosterone and androstenedione) may reflect differential mechanisms by which DHEAS levels are regulated and suggests that insulin either inhibits its biosynthesis and/or secretion, or enhances its MCR.

Suppression of counterregulatory hormone response to hypoglycemia by insulin per se.

Although assessment of counterregulatory hormone responses to hypoglycemia relies upon insulin to lower the glucose level, it is not known if the exogenous insulin does used itself influences the magnitude of the hormone response. To assess this, 12 normal subjects randomly received 2 hypoglycemic clamp studies in which the only variable was the insulin dose (0.6 or 5.0 mU/kg-min). Despite 10-fold differences in circulating insulin (265 +/- 29 vs 2576 +/- 222 pmol/L respectively), the hypoglycemic stimulus did not vary. Glucose levels fell over one hour, and then were maintained for two hours at the same hypoglycemic plateau (approximately 3.1 mmol/L for each study) by a variable glucose infusion. Although basal counterregulatory hormone levels in low and high dose studies were indistinguishable, during hypoglycemia the response of epinephrine, growth hormone, and glucagon was significantly suppressed when the degree of hyperinsulinemia was increased. We conclude that raising the magnitude of hyperinsulinemia suppresses the magnitude of the counterregulatory hormone response to hypoglycemia in normal subjects. This modulating effect of insulin per se is yet another variable in the interpretation of hypoglycemic counterregulation.

Characteristics of spontaneously agglutinating Proteus mirabilis strains from bacteriuric patients.

Out of 210 Proteus mirabilis isolates from bacteriuric patients a total of eight spontaneously agglutinating strains were found. SDS-PAGE analysis of their lipopolysaccharides (LPS), the separation of polysaccharide fractions (PS) by gel filtration, and chemical characterization of PSs were performed. Out of the eight strains one S form and one mutant classified as Rc were detected. The remaining six strains were recognized as Ra and intermediate forms. When tested in a hematogenous infection model in mice, the P. mirabilis Rc mutant survived in kidneys for at least two weeks, while the Re mutant used as control was eliminated within 20 h after the challenge. These data indicated that strains of P. mirabilis may be pathogenic even if they express very incomplete LPS.

Structure of the O-specific polysaccharide of Proteus penneri strain 41 from a new proposed serogroup O62.

O-specific polysaccharide of Proteus penneri strain 41 was studied using 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, heteronuclear 13C,1H-correlation (HETCOR) and one-dimensional NOE spectroscopy, and the following structure of a non-stoichiometrically O-acetylated hexasaccharide repeating unit was established:[structure: see text] where RGlcNAc is 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxyglucose. Cross-reactivity of anti-P. penneri 41 O-serum with other P. penneri strains is discussed, and a new, separate O62 serogroup is proposed which is the next Proteus O-serogroup containing P. penneri strains only.

Serological investigations on ribitol-containing lipopolysaccharides from Proteus mirabilis.

Lipopolysaccharides containing or noncontaining ribitol derived from several Proteus mirabilis strains were studied using passive hemagglutination, hemagglutination-inhibition and semi-quantitative precipitin tests. The results indicate that ribitol plays a role in the serological specificity of the respective lipopolysaccharides.

Lipopolysaccharides of flagellated and non-flagellated Proteus vulgaris strains.

Lipopolysaccharides from two strains of Proteus vulgaris were analyzed. One strain (08) was motile, giving swarming growth on solid media and the other (04)--non-flagellated, not able to swarm. Both lipopolysaccharides appeared to be heterogeneous and were separated into two fractions each. Yield, chemical composition and SDS-polyacrylamide gel electrophoresis showed differences between fractions in percentage content of 0-specific and R-core polysaccharides. Relation between ability to swarming growth of Proteus strains and heterogeneity of their lipopolysaccharides is also discussed.

Serological characterization of Proteus penneri species novum.

Serological characterization of the first collection of the 20 Proteus penneri strains is presented. All anti-0 sera were examined in microagglutination, semi-quantitative precipitation and passive hemagglutination tests. Some P. penneri lipopolysaccharides showed strong cross-reactivity in passive hemagglutination additionally confirmed by inhibition in this test. Serological similarity between species within genus Proteus is discussed.

Studies of a receptor for FP3-phage in Salmonella minnesota R595.

The adsorption rate constant of the phage FP3 to sensitive S. minnesota R595 strain was used to evaluate the inactivating capacity towards phage FP3 exhibited by isolated glycolipid and free lipid A. The results suggest that phage FP3 receptor is localized in the glycolipid structure. Phage FP3 is not adsorbed on the other Re mutant cells, Proteus R45, but respective products: glycolipid and free lipid A from R45 exhibitory the inhibition capacity towards phage FP3, comparable to the activity of the products derived from the host strain; the minimal PhI50 concentration of glycolipids is 7.8 and 15.6 microgram/ml respectively.

Structural and immunochemical studies on the O-specific polysaccharide of Proteus penneri strain 15.

On the basis of sugar analysis and 1H- and 13C-NMR spectroscopy, it was shown that the O-specific polysaccharide of Proteus penneri strain 15 has a trisaccharide repeating unit, including an acetal-linked pyruvic acid residue, and is structurally identical to the capsular polysaccharide of Proteus vulgaris strain ATCC 49990. Serological studies supported this conclusion and demonstrated the presence in the homological antiserum of both anti-core and anti-O chain antibodies reacting with a lipopolysaccharide (LPS) epitope containing N-acetylglucosamine and galactose residues.

Structure of the O-specific polysaccharide chain and serological characterization of the Proteus penneri 62 lipopolysaccharide compared with the lipopolysaccharides of the P. penneri strains.

The chemical structure of the O-specific polysaccharide chain of Proteus penneri 62 lipopolysaccharide (LPS) containing N-acetylisomuramic acid was established using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride and 1H and 13C NMR spectroscopy. Cross reactivity of the anti-O-serum P. penneri 62 with a number of other strains of the same species isolated in the USA, Canada, Germany and Poland is discussed.

Lipopolysaccharides of Proteus penneri species novum.

The lipopolysaccharides (LPS), obtained from twenty strains of Proteus penneri, were shown to contain 3-deoxy-D-manno-2-octulosonic acid (KDO), L-glycero-D-manno- and D-glycero-D-manno-heptoses, glucose, 2-amino-2-deoxyglucose, 2-amino-2-deoxygalactose, and galacturonic acid. Galactose was lacking in three LPS and, in some LPS, glucuronic acid, rhamnose, lysine, and two unknown constituents were detected. Chemotypes of the P. penneri are discussed.

Structure of the O-specific polysaccharide of Proteus penneri strain 25 containing N-(L-alanyl) and multiple O-acetyl groups in a tetrasaccharide repeating unit.

Based on sugar and methylation analyses, O-deacetylation, Smith degradation, and 1H and 13C NMR spectroscopy, including 2D COSY, 1H-detected 1H, 13C heteronuclear single-quantum coherence (HSQC), and 1H-detected 1H, 13C heteronuclear multiple-bond connectivity (HMBC) experiments, the following structure of the O-specific polysaccharide of Proteus penneri strain 25 was established: [formula: see text] where D-GlcN(L-Ala) is 2-(L-alanylamido)-2-deoxy-D-glucose.

Structure of a new neutral O-specific polysaccharide of Proteus penneri 34.

The O-specific polysaccharide of Proteus penneri strain 34 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C HMQC experiments. The following structure was established, which is unique among the known structures of Proteus O-antigens:-->4)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-beta- D-GalpNAc-(1-->4)-beta-D-Galp-(1-->. Accordingly, no cross-reaction was observed between P. penneri 34 O-antiserum and O-antigens of other Proteus strains. Therefore, the strain studied should belong to a new Proteus serogroup O65.

Structure of the O-specific polysaccharide of a serologically separate Proteus penneri strain 22.

The O-specific polysaccharide chain (O-antigen) of Proteus penneri strain 22 lipopolysaccharide was studied using chemical methods, including partial acid hydrolysis and Smith degradation, as well as one- and two-dimensional 1H and 13C NMR spectroscopy. The following structure of the pentasaccharide repeating unit was established: [sequence: see text] The O-specific polysaccharide contains a GalNAc residue in the furanose form which has not been hitherto found in bacterial polysaccharides. The O-antigen studied is serologically and structurally unique among Proteus strains and, therefore, a new Proteus serogroup O63 is proposed for P. penneri strain 22.

Structure of a new N-acetylisomuramic acid-containing O-specific polysaccharide of Proteus penneri strains 19 and 35.

O-Specific polysaccharides, together with oligosaccharide products of their degradation, were isolated by GPC after mild acid delipidation of lipopolysaccharides of Proteus penneri strains 19 and 35. The polysaccharides had the same trisaccharide repeating unit containing one residue each of D-galactose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (N-acetylisomuramic acid). On the basis of 1D and 2D 1H and 13C NMR spectroscopy, including 2D correlation spectroscopy (COSY), rotating-frame NOE spectroscopy (ROESY), and H-detected heteronuclear 1H,13C multiple-quantum coherence (HMQC), the following structure of the repeating unit was established: [formula: see text]. The oligosaccharide products formed by cleavage of the glycosidic linkage of GlcNAc represent a chemical trisaccharide repeating unit of the polysaccharide and its oligomer homologs. The ease of hydrolysis of the polysaccharide is associated with the closeness of the glycosyl group and the lactic acid residue in N-acetylisomuramic acid. The polysaccharides studied are structurally related to the O-specific polysaccharides of P. penneri strains 62 and 71 studied by us earlier.

Structure of the O-specific polysaccharide of Proteus mirabilis O16 containing ethanolamine phosphate and ribitol phosphate.

The O-specific polysaccharide of Proteus mirabilis O16 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, HMQC-TOCSY, and 1H,31P HMQC experiments, along with chemical methods. The polysaccharide was found to be a ribitol teichoic acid-like polymer having the following structure [structure: see text].

[Selected properties of strains of a new species of Proteus penneri from the second American collection]. / Wybrane wlasciwosci szczepów nowego gatunku Proteus penneri z drugiej kolekcji amerykanskiej.

The second collection of the novel species Proteus penneri consists of 25 strains from which only two have shown rough from properties in the tests differentiating S and R variants of bacteria. The migration pattern of their lipopolysaccharides in gel electrophoresis was leader-like, typical for smooth organisms. 13 out of 25 lipopolysaccharide preparations showed strong-reactivity with anti-0 sera in semi-quantitative precipitation test. Serological similarity between the strains within species Proteus penneri is discussed.