Benjamin S. Szwergold

Observation of inositol pentakis- and hexakis-phosphates in mammalian tissues by 31P NMR

In analyzing the 31P NMR spectra of extracts of mammalian tissues and cells we have identified inositol pentakis- and hexakis-phosphates in essentially all of the samples examined. These compounds were present at concentrations of at least 5-15 microM. While the sources and functions of these compounds in mammalian cells are not clear, they may play an important role in phosphoinositol metabolism. For example, one obvious possibility is that these compounds may be sources of or sinks for the Ca++ mobilizing inositol tris- and tetrakis-phosphates.

Glyceraldehyde-3-phosphate dehydrogenase activity as an independent modifier of methylglyoxal levels in diabetes

Methylglyoxal (MG) may be an important cause of diabetic complications. Its primary source is dihydroxyacetone phosphate (DHAP) whose levels are partially controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using a human red blood cell (RBC) culture, we examined the effect of modifying GAPDH activity on MG production. With the inhibitor koningic acid (KA), we showed a linear, concentration-dependent GAPDH inhibition, with 5 microM KA leading to a 79% reduction of GAPDH activity and a sixfold increase in MG. Changes in redox state produced by elevated pH also resulted in a 2.4-fold increase in MG production at pH 7.5 and a 13.4-fold increase at pH 7.8. We found substantial inter-individual variation in DHAP and MG levels and an inverse relationship between GAPDH activity and MG production (R=0.57, P=0.005) in type 2 diabetes. A similar relationship between GAPDH activity and MG was observed in vivo in type 1 diabetes (R=0.29, P=0.0018). Widely varying rates of progression of diabetic complications are seen among individuals. We postulate that modification of GAPDH by environmental factors or genetic dysregulation and the resultant differences in MG production could at least partially account for this observation.

Identification of Two Gene Clusters and a Transcriptional Regulator Required for Pseudomonas aeruginosa Glycine Betaine Catabolism

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-(13)C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and (13)C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.

Structural studies of a phosphocholine substituted β-(1,3); (1,6) macrocyclic glucan from Bradyrhizobium japonicum USDA 110

In our previous in vivo 31P study of intact nitrogen-fixing nodules (Rolin, D.B., Boswell, R.T., Sloger, C., Tu, S.I. and Pfeffer, P.E., 1989 Plant Physiol. 89, 1238-1246), we observed an unknown phosphodiester. The compound was also observed in the spectra of isolated bacteroids as well as extracts of the colonizing Bradyrhizobium japonicum USDA 110. In order to characterize the phosphodiester in the present study, we took advantage of the relatively hydrophobic nature of the material and purified it by elution from a C-18 silica reverse-phase chromatography column followed by final separation on an aminopropyl silica HPLC column. Structural characterization of this compound with a molecular weight of 2271 (FAB mass spectrometry), using 13C-1H and 31P-1H heteronuclear 2D COSY and double quantum 2D phase sensitive homonuclear 1H COSY NMR spectra, demonstrated that the molecule contained beta-(1,3); beta-(1,6); beta-(1,3,6) and beta-linked non-reducing terminal glucose units in the ratio of 5:6:1:1, respectively, as well as one C-6 substituted phosphocholine (PC) moiety associated with one group of (1,3) beta-glucose residues. Carbohydrate degradation analysis indicated that this material was a macrocyclic glucan, (absence of a reducing end group) with two separated units containing three consecutively linked beta-(1,3) glucose residues and 6 beta-(1,6) glucose residues. The sequences of beta-(1,3)-linked glucose units contained a single non-reducing, terminal, unsubstituted glucose linked at the C-6 position and a PC group attached primarily to an unsubstituted C-6 position of a beta-(1,3)-linked glucose.

A novel mouse model of Niemann–Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

Transglycation - a potential new mechanism for deglycation of Schiff's bases.

Abstract: Nonenzymatic glycation is believed to play a major role in the development of diabetic complications. Over the past several years we and others have shown that in cells this nonenzymatic process can be reversed by an ATP‐dependent reaction catalyzed by fructosamine‐3‐kinase (FN3K) and possibly by its isozyme, fructosamine‐3‐kinase‐related protein (FN3KRP). In this study we provide the first evidence that this FN3K‐dependent deglycation, acting on the Amadori products, is complemented by another deglycation process operating on the very first product of nonenzymatic glycation, glucosylamines (Schiffs bases). We postulate that the first step in this Schiffs‐base deglycation process occurs by transfer of the sugar moiety from macromolecule‐bound glucosylamine to one of the low‐molecular weight intracellular nucleophiles—in particular, glutathione. We term this reaction transglycation, and in this study we demonstrate that it occurs readily and spontaneously in vitro. We further propose that one of the spontaneously formed glucose‐glutathione adduct(s) is subsequently removed from cells by a multidrug‐resistance pump (MRP, MDR‐protein, ATP‐binding‐cassette protein), metabolized, and excreted in urine. In support of this latter contention, we show that at least one transglycation product, glucose‐cysteine, is found in human urine and that its concentrations are increased in diabetes.

Some Clues as to the Regulation, Expression, Function, and Distribution of Fructosamine‐3‐Kinase and Fructosamine‐3‐Kinase‐Related Protein

Abstract: Fructosamine‐3‐kinase (FN3K) and the more recently discovered fructosamine‐3‐kinase‐related protein (FN3KRP) appear to protect proteins from nonenzymatic glycation. To gain a better understanding of these enzymes we performed a series of investigations including (1) in silico comparisons of their promoters; (2) real‐time PCR analysis of their expression in human tissues; (3) effects of hyperglycemia, interleukin‐1β (IL‐1β), and nuclear factor kappa‐B (NFκB) activation on their mRNA levels; (4) effects of small interfering RNA (siRNA) suppression of FN3K expression (knockdown) in cultured cells and (5) search of FN3K and FN3KRP homologs in available genomic and EST (expressed sequence tag) databases. Our results indicate that (1) both FN3K and FN3KRP promoters are TATA‐less and CAAT‐less and contain several homologous CpG islands and Sp1 binding sites. (2) Both genes are expressed in all human tissue examined, with FN3K showing significantly higher levels in tissues susceptible to nonenzymatic glycation and diabetic complications. (3) Treatment of fibroblasts with high glucose, IL‐1β, and activation of NFκB does not affect the expression of either FN3K or FN3KRP. (4) Knockdown of FN3K in cultured cells inhibits or arrests their growth. (5) FN3K‐like genes are widely distributed in nature, with the notable exception of insects and yeasts. These data suggest that FN3K and FN3KRP are constitutive “housekeeping” genes and that they play an important role in cell metabolism, possibly as deglycating enzymes.

Quantitation of Resonances in Biological 31P NMR Spectra via Principal Component Analysis: Potential and Limitations

This paper examines the potential and limitations of peak area quantitation of biological NMR spectra using principal component analysis (PCA), including its requirement for prior knowledge. The principles of the method are presented without in‐depth mathematical treatment. PCA is illustrated for simulated data, 31P NMR spectra obtained consecutively over 1–2.5 days from perfused Rat‐2 cells metabolizing the choline analogue phosphoniumcholine (Chop) and in vivo proton‐decoupled, NOE‐enhanced, three‐dimensional CSI localized 31P NMR spectra of the liver of healthy volunteers. The results show that PCA can be used to quantitate strongly overlapping peaks without prior knowledge of the peak shapes or positions and to reconstruct spectra with significantly reduced noise variance. Two major limitations of PCA are presented: (1) PCA cannot separate peaks whose intensities are well correlated; (2) PCA is sensitive to differences in chemical shift and line‐width of peaks between spectra. The discussion focusses on what knowledge of the biological and spectroscopic features of the samples and the principles of PCA is necessary for peak area quantitation via PCA.

Nonenzymatic glycation/enzymatic deglycation: a novel hypothesis on the etiology of diabetic complications

Abstract Nonenzymatic glycation appears to play a major role in the development of diabetic complications. Key early intermediates in the nonenzymatic glycation cascade are glucoselysines (GL) and fructoselysines (FL). In 1997, we proposed that intracellular nonenzymatic glycation is controlled by an enzymatic deglycation process catalyzed by fructosamine-3-kinase (FN3K). FN3K phosphorylates FL to fructoselysine-3-phosphate (FL3P), which then decomposes regenerating an unmodified lysine residue. With the recent purification, sequencing and cloning of FN3K [Diabetes 50 (2001) 2139; Diabetes 49 (2000) 1627] the concept of enzymatic deglycation has received considerable experimental support. In this paper, we provide evidence of enzymatic deglycation activity in vivo involving both FN3K-dependent and FN3K-independent mechanisms. Based on these data, we propose a new theory on the development of diabetic complications: the nonenzymatic glycation/enzymatic deglycation hypothesis. We postulate that enzymatic deglycation is an essential defense system in mammalian cells. In diabetes, this system is stressed and often overwhelmed by episodes of extreme hyperglycemia during which nonenzymatic glycation proceeds unchecked. This results in cumulative damage to essential proteins and leads ultimately to cellular dysfunction. Susceptibility to diabetic complications is thus a consequence of two factors: severity of the hyperglycemic stress and ability of the deglycating system to cope with that stress.