Modulation of cell injury and survival by high glucose and advancing age.

Old age is associated with a higher prevalence of cardiovascular disease and diabetes mellitus. Vascular smooth muscle cells (VSMC) play a role in the pathogenesis of vascular diseases, often a complication of diabetes mellitus. We examined in explanted aortic VSMC from young vs. older rats glucose-related activation of nuclear factor kappaB (NF-kappaB), a transcription factor induced by many oxidants. Data demonstrate that old age is associated with enhanced NF-kappaB activity in unstimulated VSMC that is further increased after exposure to high glucose medium. Furthermore, VSMC from old animals exhibit increased levels of protein carbonyls, an indicator of oxidative stress, and less apoptosis in response to glucose than VSMC isolated from young animals. These changes are accompanied by increased expression of NF-kappaB-related genes, gamma-glutamylcysteine synthetase, inhibitor of apoptosis protein-1 (IAP-1), and inducible nitric oxide synthase (iNOS). Results suggest that high glucose, a putative oxidative stress, causes apoptosis in VSMC from young animals and is associated with greater induction of NF-kappaB in VSMC from older animals. Increases in IAP-1 and decreased apoptosis implicate NF-kappaB as a survival factor in VSMC.

Glutathione-S-Transferase M1 null genotype in autoimmune hepatitis.

Autoimmune hepatitis is associated with genes located in the major histocompatibility complex. The search for genes at other loci that may play a role in disease susceptibility and/or severity is an area of active investigation in autoimmune liver diseases. Genes for glutathione-S-transferases, enzymes that are widely distributed and collectively metabolize carcinogens, pollutants, drugs, and a broad spectrum of harmful, foreign compounds have been associated with liver disease. The objective of this study was to search for a relationship between the glutathione-S-transferase Ml null genotype and autoimmune hepatitis using polymerase chain reaction analysis. The findings indicate that the frequency of the null genotype is not increased in patients with autoimmune hepatitis when compared to control subjects. These results coupled with similar ones in primary biliary cirrhosis do not support a role for this mutation in autoimmune liver disease.

High glucose concentrations induce oxidative damage to mitochondrial DNA in explanted vascular smooth muscle cells.

Oxidative stress is considered to be one of the mechanisms leading to atherosclerosis. It occurs in response to injury or to altered metabolic state. Alterations in cell growth (proliferation or apoptosis) can also contribute to the pathogenesis of atherosclerosis and is influenced by oxidative stress. Smooth muscle cells (SMC) from aortic explants of JCR:LA-cp homozygous cp/cp corpulent rats who are genetically predisposed to develop atherosclerosis exhibit increased SMC proliferation, which can be attenuated by exercise and food restriction. This study was conducted to characterize the effects fo oxidative stress and high glucose media on cell growth and its relationship to mitochondrial DNA integrity and gene expression in explanted aortic SMC from corpulent and lean JCR:LA-cp rats. The results show that SMC from the cp/cp rat appear to be resistant to oxidant-induced cell death and that they accumulate mitochondrial DNA mutations, probably as a result of a reduction in apoptosis. These data suggest that susceptibility to age- and glucose-related atherosclerosis may be related to alterations in redox signaling.

Measurement of intracellular sulfur amino acid metabolism in humans.

Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.

Sex-related differences in methionine metabolism and plasma homocysteine concentrations.

BACKGROUND: Elevated fasting homocysteine concentrations are considered a risk factor for vascular disease. Homocysteine, which is produced by the transmethylation of methionine, can be either remethylated back to methionine or metabolized via transsulfuration to cystathionine. It has been speculated that the lower risk of vascular disease among premenopausal women may be related to lower homocysteine concentrations in women than in men. OBJECTIVE: This study was designed to determine whether sex-related differences exist in methionine cycle kinetics, which may account for the reportedly lower fasting homocysteine concentrations in premenopausal women. DESIGN: Eleven healthy young men and 11 premenopausal women without cardiac risk factors were studied by using stable-isotope-labeled L-[methyl-(2)H(3),1-(13)C]methionine and L-[methyl- (2)H(3)]leucine. After 3 h of tracer infusion, 100 mg unlabeled L-methionine/kg body wt was ingested. Blood and breath samples were obtained at timed intervals. Fat-free mass was estimated by dual-energy X-ray absorptiometry and muscle mass by urinary creatinine excretion. RESULTS: No significant sex-related differences were found in fasting homocysteine concentrations, responses to the oral methionine load, or rates of methionine flux based on carboxyl or methyl labels. However, women had significantly higher remethylation rates than did men (P < 0.005) and a tendency toward higher transmethylation (P < 0.10). Whereas adjustment of remethylation rates for fat-free mass tended to attenuate the sex-related effect (P = 0.08), adjustment for muscle mass did not (P < 0.04). In contrast, significant sex-related differences in leucine flux (P < 0.02) were eliminated after adjustment for either fat-free mass or muscle mass. CONCLUSION: Reported differences between men and women in homocysteine concentrations may be partially explained by differences in rates of homocysteine remethylation.

Oxidation of glutamine by the splanchnic bed in humans.

[1,2-(13)C(2)]glutamine and [ring-(2)H(5)]phenylalanine were infused for 7 h into five postabsorptive healthy subjects on two occasions. On one occasion, the tracers were infused intravenously for 3.5 h and then by a nasogastric tube for 3.5 h. The order of infusion was reversed on the other occasion. From the plasma tracer enrichment measurements at plateau during the intravenous and nasogastric infusion periods, we determined that 27 +/- 2% of the enterally delivered phenylalanine and 64 +/- 2% of the glutamine were removed on the first pass by the splanchnic bed. Glutamine flux was 303 +/- 8 micromol. kg(-1). h(-1). Of the enterally delivered [(13)C]glutamine tracer, 73 +/- 2% was recovered as exhaled CO(2) compared with 58 +/- 1% of the intravenously infused tracer. The fraction of the enterally delivered tracer that was oxidized specifically on the first pass by the splanchnic bed was 53 +/- 2%, comprising 83% of the total tracer extracted. From the appearance of (13)C in plasma glucose, we estimated that 7 and 10% of the intravenously and nasogastrically infused glutamine tracers, respectively, were converted to glucose. The results for glutamine flux and first-pass extraction were similar to our previously reported values when a [2-(15)N]glutamine tracer [Matthews DE, Morano MA, and Campbell RG, Am J Physiol Endocrinol Metab 264: E848-E854, 1993] was used. The results of [(13)C]glutamine tracer disposal demonstrate that the major fate of enteral glutamine extraction is for oxidation and that only a minor portion is used for gluconeogenesis.

L-2-[(13)C]oxothiazolidine-4-carboxylic acid: a probe for precursor mobilization for glutathione synthesis.

L-2-oxothiazolidine-4-carboxylic acid (OTZ), a 5-oxoproline analog, is metabolized by 5-oxoprolinase and converted to cysteine, the rate-limiting amino acid for GSH synthesis, with the release of CO(2). [(13)C]OTZ (1.5 mg/kg) was used in 12 healthy men and women (ages 23-73 yr) to indirectly assess precursor mobilization for GSH synthesis when stores were reduced by 2 g acetaminophen. Expired breath samples were analyzed for (13)CO(2), and results were analyzed using noncompartmental and two-compartment open minimal models. Results show an increase in (13)C excretion (higher OTZ hydrolysis) when GSH stores were reduced and 5-oxoprolinase substrate utilization patterns, consequently, were altered (P < 0. 01). A metabolic rate index (MRI) of the OTZ probe was found to be significantly higher after reduction of GSH content by acetaminophen (P < 0.05). The difference in adaptive capacity (difference between control and postacetaminophen metabolic rate indexes) was two times as large in the young than the old subjects (P < 0.01). These data support the use of [(13)C]OTZ as a probe to identify individuals who may be at risk for low GSH stores or who have an impaired capacity to synthesize GSH.

Strategies for evaluation of signaling pathways and transcription factors altered in aging.

Aging is characterized by an accumulation of oxidative injury to DNA, RNA, proteins, lipids, and carbohydrates. In addition to damage, oxidative stress can initiate cell signaling cascades that modulate cell function, growth, and death. Aging and two common age-related diseases, diabetes mellitus and atherosclerosis, may share common oxidant-related signaling pathways that lead to abnormal transcription factor activation and ultimately to cellular dysfunction, degeneration, or death. This review will focus on approaches to evaluate key redox-sensitive signaling pathways and the transcription factors altered by diabetes, atherosclerosis, and aging.

Plasminogen activator inhibitor type 1 in adults with Down syndrome and protection against macrovascular disease.

In the present study we have shown that adults with Down syndrome have reduced plasminogen activator inhibitor type-1 in blood compared with control subjects matched for age, gender, and body mass. Reduced plasminogen activator inhibitor type-1 may explain the low incidence of atherosclerotic vascular disease and its complications that are attributable to plaque instability, such as myocardial infarction and unstable angina, in subjects with Down syndrome, despite an ever-increasing life expectancy.

Aging: is oxidative stress a marker or is it causal?

Rapid developments in free radical biology and molecular technology have permitted exploration of the free radical theory of aging. Oxidative stress has also been implicated in the pathogenesis of a number of diseases. Studies have found evidence of oxidative damage to macromolecules (DNA, lipids, protein), and data in transgenic Drosophila melanogaster support the hypothesis that oxidative injury might directly cause the aging process. Additional links between oxidative stress and aging focus on mitochondria, leading to development of the mitochondrial theory of aging. However, despite the number of studies describing the association of markers of oxidative damage with advancing age, few, if any definitively link oxidative injury to altered energy production or cellular function. Although a causal role for oxidative stress in the aging process has not been clearly established, this does not preclude attempts to reduce oxidative injury as a means to reduce morbidity and perhaps increase the healthy, useful life span of an individual. This review highlights studies demonstrating enhanced oxidative stress with advancing age and stresses the importance of the balance between oxidants as mediators of disease and important components of signal transduction pathways.

Measurement of homocysteine concentrations and stable isotope tracer enrichments in human plasma.

Elevated levels of plasma homocysteine have been established as an independent risk factor for cardiovascular disease. Homocysteine is in low concentration in plasma (5-15 microM) and is bound to other thiols (e.g., cysteine in plasma proteins) via disulfide bonds. Existing methods for measuring homocysteine have difficulty in reducing and maintaining the reduction of homocysteine for measurement. We describe a GC/MS method that first reduces the disulfides in the physiological sample matrix and then immediately alkylates the free thiols with 4-vinylpyridine to prevent the reformation of the disulfide bonds. We use a deuterated internal standard, [3,3,3',3',4,4,4',4'-2H8]homocystine to account for losses associated with the isolation, derivatization, and measurement of the natural homocysteine. The amino acids are separated and derivatized to form the tert-butyldimethylsilyl derivatives. This method requires only 50 microL of plasma to measure homocysteine concentrations to 5 microM. Total homocysteine concentrations in plasma can be measured routinely from 0.5-mL samples with relative intra- and interday precisions of 1.3 and 4.0%, respectively. This method is sensitive enough to determine tracer enrichments of [1-13C]homocysteine with a detection limit of < 0.3 mol% excess and an average tracer precision of 0.6%.

Aging and high concentrations of glucose potentiate injury to mitochondrial DNA.

Deletions of mitochondrial DNA (mtDNA) are associated with aging and several chronic diseases. We have reported heterogeneous mutations between base pair 8468 and 13446 in mtDNA, the region known as the "common" deletion, in muscle of older humans with impaired glucose tolerance or diabetes mellitus. To further characterize potential effects of age and glycemia on mtDNA integrity, we studied corpulent JCR:LA-cp rats that are characterized by insulin resistance, hyperinsulinemia, and hyperlipidemia, factors strongly associated with both aging and cardiovascular disease. In addition to skeletal muscle, we isolated vascular smooth muscle cells (VSMC) from aortas of 6-, 12-, and 17-month-old rats and exposed them to 5-, 25-, 62-, and 100-mM glucose or a combination of hypoxanthine (100 microM) and xanthine oxidase (0.025 U/ml) to generate reactive oxygen species in separate cultures. Long- and short-fragment and nested polymerase chain reaction was used to detect mutations in the common deletion region. The data demonstrate that aging and the cp genotype confer susceptibility to mtDNA deletions in vivo and that high glucose concentrations can induce mtDNA mutations in vitro. Accordingly, aging and glucose-related oxidative stress and possibly hyperinsulinemia may contribute to alterations in mitochondrial gene integrity and the cp genotype appears to increase the susceptibility of muscle to the age-related accumulation of mtDNA mutations.

Methionine and cysteine kinetics at different intakes of methionine and cysteine in elderly men and women.

Earlier nitrogen balance studies led to the conclusion that requirements for methionine in older individuals are much higher than those in younger adults. Hence, we examined the kinetics of whole-body methionine, cysteine, and leucine metabolism postabsorptively using a continuous intravenous infusion of L-[C2H3, 1-(13)C]methionine, L-[2H3]leucine, and [3,3-2H2]cysteine in 12 elderly men (n = 5) and women (n = 7) given as a 3-h infusion after a 12-h fast (study 1) and in 8 elderly men (n = 4) and women (n = 4) as an 8-h infusion according to a 3-h fasted, 5-h fed protocol (study 2) for 6 d. Before tracer infusion, each of 3 L-amino acid diets supplying the following nominal, but known, amounts (mg x kg(-1) x d(-1)) of methionine and cysteine, respectively, were used in study 2: diet 1: 13 and 0; diet 3: 6.5 and 5.2; and diet 5: 6.5 and 21. Studies 1 and 2 gave values for plasma methionine flux that agreed with the leucine flux data, which, in turn, also appeared to be comparable with findings in healthy younger adults. In study 2, methionine oxidation rates were the same across all diets in the fasted state and the same with diets 1 and 3 in the fed state but lower with diet 5, suggesting a modest sparing effect of dietary cystine on methionine oxidation. Estimated daily methionine balance was at equilibrium for diet 1 and negative (significantly different from zero, P<0.05) with diets 3 and 5. The results were evaluated against our previous findings in younger adults.

Increased prevalence of mitochondrial DNA deletions in skeletal muscle of older individuals with impaired glucose tolerance: possible marker of glycemic stress.

To determine the relationship between mitochondrial DNA (mtDNA) mutations and age-related impaired glucose tolerance (IGT), mtDNA from skeletal muscle of 19 volunteers, ages 55-75 years, with either IGT or diabetes and 17 age- and sex-matched control subjects was analyzed using a long-extension polymerase chain reaction (PCR) combined with a quantitative PCR. We found the common 4,977-bp deletion in 84% of the IGT/diabetes group compared with only 41% in the control group (P < 0.02). Multiple other deletions of different sizes were identified in 13 out of 19 IGT/diabetes patients (68%) compared with 2 out of 17 control subjects (12%) (P < 0.002). Because of the heterogeneity and variation in the mutations identified, we propose that these mtDNA mutations were the result rather than the cause of IGT. The increase in type and frequency of mtDNA deletions in diabetes and IGT patients may be related to oxidative damage by oxygen free radicals. These may be produced in greater amounts as a result of hyperglycemia or may be more abundant because of an abnormality in the scavenging of free radicals by antioxidants.

Diurnal pattern of the interrelationships among leucine oxidation, urea production, and hydrolysis in humans.

We investigated in six healthy adult men, who received an adequate intake of protein (1 g.kg-1.day-1), the relationship among urea production, excretion, and hydrolysis. At the end of a 6-day diet-adjustment period, subjects were studied using a 24-h continuous intravenous [1-13C]leucine and [15N,15N]urea tracer protocol (A. E. El-Khoury, N. K. Fukagawa, M. Sánchez, R. H. Tsay, R. E. Gleason, T. E. Chapman, and V. R. Young. Am. J. Clin. Nutr. 59: 1000-1011, 1994) to determine rates of irreversible protein nitrogen loss and urea kinetics. By combining leucine and urea kinetic data, we found a significant degree of urea hydrolysis over the 24-h period but no evidence to support the thesis that there is a net retention or "salvage" of the urea nitrogen liberated. Our measurements revealed little or no urea hydrolysis during the fed 12-h period of the 24-h tracer protocol but substantial hydrolysis during the 12-h fasting phase. Furthermore, a mass balance model and calculations (APPENDIX) indicated that nitrogen salvage, if any, is quantitatively indistinguishable from insensible nitrogen losses and aggregate estimation errors, accounting for no more than 5% of the nitrogen intake. We conclude that urea hydrolysis, via the intestinal microflora, although representing a component of the overall cycles of nitrogen flow within the body, does not contribute via a net retention of amino nitrogen to the maintenance of body nitrogen homeostasis in healthy adults consuming an adequate diet.

Effect of age on body water and resting metabolic rate.

BACKGROUND: We previously reported that differences in fat-free mass (FFM) estimated by isotope dilution of 18O-labeled water could not fully account for lower resting metabolic rates (RMR) in old men and women compared to RMR in young men. METHODS: Since age-related changes in the distribution of water between extracellular and intracellular spaces could lead to overestimation of FFM in the old, we reanalyzed our data using estimates for total body and intracellular water (TBW and ICW, respectively) derived from published equations and included data from adolescent boys and girls studied under similar conditions. RESULTS: In both sexes, the age-related reduction in RMR remained significant after adjustment for estimated body water compartments (p < .05). While adjusted RMR differed in boys and girls (p < .0001), it did not in old men and women (p = .15). CONCLUSION: We conclude that aging per se reduces RMR in lean tissue, a difference which cannot be fully explained by changes in body water or its distribution. Investigators should be cautious when selecting models and equations to estimate body water compartments.

Plasma methionine and cysteine kinetics in response to an intravenous glutathione infusion in adult humans.

Glutathione (GSH), a tripeptide (gamma-glutamyl-cysteinyl-glycine), is thought to be both a storage and a transport form of cysteine (Cys). In a previous study (T. Hiramatsu, N.K. Fukagawa, J.S. Marchini, J. Cortiella, Y.-M. Yu, T.E. Chapman, and V.R. Young. Am. J. Clin. Nutr. 60: 525-533, 1994), the direct tracer-derived estimate of Cys flux was considerably higher than that predicted from estimates of protein turnover. To further examine the components of plasma Cys flux, seven normal-weight healthy adult men and women (26 +/- 2 yr) received stable isotope tracer infusions of L-[methyl-2H3;1-13C]methionine, L-[3,3-2H2]cysteine, and L-[methyl-2H3]leucine for 460 min. After a 3-h baseline period, GSH was administered at approximately 32 mumol.kg-1.h-1 until the end of the study. Expired breath and blood samples were obtained at timed intervals and analyzed for isotope enrichment using mass spectrometry. Leucine, alpha-ketoisocaproate, and methionine (carboxyl carbon, methyl moiety, remethylation, and transsulfuration) turnover were reduced during GSH administration (P < 0.01). In the final hour of GSH administration, Cys flux increased by 61% from 55.1 +/- 1.7 to 88.7 +/- 5.2 mumol.kg-1.h-1 (P < 0.01), which was essentially equivalent to the rate of exogenous GSH infusion. These data suggest that GSH breakdown accounts for approximately 50% of tracer-derived Cys flux basally and for all of the increase in measured Cys turnover during exogenous GSH infusion.

The 24-h kinetics of leucine oxidation in healthy adults receiving a generous leucine intake via three discrete meals.

The significance of meal size and frequency for the 24-h leucine tracer-balance technique was examined. Continuous measurements of leucine oxidation throughout a 24-h d were performed in six healthy, young adults who were given a weight-maintaining diet (188 kJ.kg-1.d-1; 1 g protein.kg-1.d-1) for 6 d followed by primed, continuous intravenous infusions of L-[1-13C]leucine and [15N-15N]urea. The 24-h study was started at 1800 on day 6 and three equal discrete meals were given at 2000, 0600, and 1200. Leucine oxidation was assessed from plasma [13C]alpha-ketoisocaproate enrichment and 13CO2 excretion. The mean (+/- SD) leucine oxidation after each meal (over 6 h) was not significantly different (P > 0.5) among the three discrete meals: 20.0 +/- 3.9, 20.2 +/- 1.9, and 20.3 +/- 2.4 mg.kg-1.d-1 for the meals given at 2000, 0600, and 1200, respectively. Twenty-four-hour leucine oxidation was 75.0 +/- 7.8 mg.kg-1.d-1 for a leucine dietary intake of 80 mg.kg-1.d-1 (and approximately 9.7 mg tracer.kg-1.d-1). The 24-h pattern in leucine oxidation was paralleled by plasma leucine concentrations. Further, leucine oxidation and urea excretion predicted relatively similar values for 24-h protein oxidation. These data are compared with results from our similar previous studies using a multiple-small-meal feeding protocol.

Estimation of energy requirements in persons with severe central nervous system impairment.

We measured resting metabolic rate (RMR) in 12 persons (aged 10 to 30 years) with severe impairment of the central nervous system requiring gastrostomy feedings, and compared our findings with the RMR predicted from standardized equations. The RMR was 70.6% +/- 15.7% of that predicted from the Mayo Clinic nomogram, 63.7% +/- 18% of that predicted from the Food and Agriculture Organization/World Health Organization/United Nations University equation, and 75.4% +/- 17.3% of that predicted from the Robertson and Reid equation. Energy intake was 756 +/- 225 kcal/day and RMR was 708 +/- 231 kcal/day; RMR adjusted for changes in body energy stores was 729 +/- 231 kcal/day. No significant differences were found between energy intake and RMR, or between energy intake and the sum of RMR and changes in body energy stores. These findings demonstrate that standardized equations overestimate energy needs of individuals with severe CNS impairment. Measures of energy intake adjusted for weight changes represent a valid method to determine energy requirements in a group of individuals with severe CNS impairment. Length was found to be significantly correlated with RMR (r = 0.79; p < 0.01). When the regression equation was tested on another group of subjects from the same residential facility, the equation predicted energy needs reasonably well for those individuals with similar energy intakes but not for all patients with CNS disorders.

Acute effects of fructose and glucose ingestion with and without caffeine in young and old humans.

Aging is associated with a decline in energy expenditure (EE), glucose intolerance, and a reduction in body nitrogen content. In addition, a reduction in the thermic response to glucose but not to fructose or protein has been reported in the elderly. The present study was conducted to further examine nutrient-induced thermogenesis and the effects of specific sugars on amino acid metabolism in relation to age. After 3 days on a weight-maintaining, 250-g carbohydrate diet, 16 healthy non-obese men and women in two age groups (18 to 29 and 66 to 80 years) consumed on 4 different days 500 mL of either a 75-g fructose or 75-g glucose solution, with or without 300 mg caffeine or vitamin C as a placebo. Blood substrate and hormone levels and EE, using indirect calorimetry, were measured at timed intervals for 3 hours after consumption of the drinks. There was no difference in the carbohydrate-induced increase in EE in either young or old even after adjustments for body weight and fat-free mass (FFM). An approximately 20-fold increase in serum caffeine levels increased EE in both groups (P < .003), but had minimal effects on substrate and hormone responses. In contrast to glucose, fructose induced a marked elevation in plasma alanine from combined basal levels of 301 +/- 24 to approximately 500 +/- 18 mumol/L (mean +/- SEM) in both groups (P < .001). However, both fructose and glucose ingestion resulted in a similar decline in branched-chain and aromatic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)