David W. Killilea

Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-l- carnitine and/or R-α-lipoic acid

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-l-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R-α-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.

Oxidative stress and inflammation in iron‐overloaded patients with β‐thalassaemia or sickle cell disease

Blood transfusion therapy is life‐saving for patients with β‐thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1·8‐fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0·001) and specific diagnosis (P = 0·019). A significant 2‐fold elevation of non‐transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2·2‐fold elevation of the inflammatory marker interleukin (IL)‐6 relative to controls, and a 3·6‐ and 1·7‐fold increase in IL‐5 and IL‐10 relative to thalassaemia. Although α‐tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, γ‐tocopherol, a nitric oxide‐selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and γ‐tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimers disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.

Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans

Lithium (Li+) has been used to treat mood affect disorders, including bipolar, for decades. This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the one or more underlying mechanisms of its therapeutic action are not understood. Here we describe a pharmacogenetic study of Li+ in the nematode Caenorhabditis elegans. Exposure to Li+ at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li+ treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference of T08D10.2 is sufficient to extend longevity (∼25% median increase), suggesting Li+ regulates survival by modulating histone methylation and chromatin structure.

Magnesium deficiency accelerates cellular senescence in cultured human fibroblasts

Magnesium inadequacy affects more than half of the U.S. population and is associated with increased risk for many age-related diseases, yet the underlying mechanisms are unknown. Altered cellular physiology has been demonstrated after acute exposure to severe magnesium deficiency, but few reports have addressed the consequences of long-term exposure to moderate magnesium deficiency in human cells. Therefore, IMR-90 human fibroblasts were continuously cultured in magnesium-deficient conditions to determine the long-term effects on the cells. These fibroblasts did not demonstrate differences in cellular viability or plating efficiency but did exhibit a decreased replicative lifespan in populations cultured in magnesium-deficient compared with standard media conditions, both at ambient (20% O2) and physiological (5% O2) oxygen tension. The growth rates for immortalized IMR-90 fibroblasts were not affected under the same conditions. IMR-90 fibroblast populations cultured in magnesium-deficient conditions had increased senescence-associated β-galactosidase activity and increased p16INK4a and p21WAF1 protein expression compared with cultures from standard media conditions. Telomere attrition was also accelerated in cell populations from magnesium-deficient cultures. Thus, the long-term consequence of inadequate magnesium availability in human fibroblast cultures was accelerated cellular senescence, which may be a mechanism through which chronic magnesium inadequacy could promote or exacerbate age-related disease.

Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene

Hypoxia, a stimulus for angiogenesis and vascular remodeling, has been proposed to use reactive oxygen species as second messengers in signal transduction. This contention remains controversial, in part because of vagaries associated with fluorescence‐based methods of free‐radical detection. We took a different approach. Rat main pulmonary artery endothelial cells (PAECs) were cultured in hypoxia for up to 48 h, and, with dichlorofluorescein fluorescence to detect free‐radical production, we used quantitative Southern blot and ligation‐mediated PCR analyses to search for oxidative modifications in the mitochondrial genome and in the nuclear vascular endothelial cell growth factor (VEGF) gene. In accord with previous studies in other cell types, we found that acute hypoxic exposure promoted time‐dependent dichlorofluorescein fluorescence in PAECs. Quantitative Southern blot analysis showed that although hypoxia failed to alter mitochondrial DNA integrity, prominent oxidative lesions occurred in a 5.0‐kb sequence of the VEGF promoter. Using ligation‐mediated PCR to map the modifications at single nucleotide resolution, we found clusters of oxidized bases in a VEGF promoter sequence that included the AP‐1 and HIF‐1 response elements. These actions of hypoxia differed from exogenous xanthine oxidase, which obliterated the mitochondrial genome but failed to erode integrity of the VEGF promoter. Our observations indicate that hypoxia promotes an oxidant stress in main PAECs as detected by oxidative base modifications in the nuclear VEGF gene. The presence of hypoxia‐induced, oxidative base modifications in functionally significant sequences within the VEGF promoter suggests new concepts for mechanisms by which reactive oxygen species participate in hypoxic signal transduction.

Iron Accumulation During Cellular Senescence in Human Fibroblasts In Vitro

Iron accumulates as a function of age in several tissues in vivo and is associated with the pathology of numerous age-related diseases. The molecular basis of this change may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary human fibroblast cells (IMR-90) were studied in vitro as a model of cellular senescence. Total iron content increased exponentially during cellular senescence, resulting in 10-fold higher levels of iron compared with young cells. Low-dose hydrogen peroxide (H2O2) induced early senescence in IMR-90s and concomitantly accelerated iron accumulation. Furthermore, senescence-related and H2O2-stimulated iron accumulation was attenuated by N-tert-butylhydroxylamine (NtBHA), a mitochondrial antioxidant that delays senescence in vitro. However, SV40-transformed, immortalized IMR-90s showed no time-dependent changes in metal content in culture or when treated with H2O2 and/or NtBHA. These data indicate that iron accumulation occurs during normal cellular senescence in vitro. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells.

A Drosophila Model Identifies a Critical Role for Zinc in Mineralization for Kidney Stone Disease

Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall’s plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

Stunting Prevalence, Plasma Zinc Concentrations, and Dietary Zinc Intakes in a Nationally Representative Sample Suggest a High Risk of Zinc Deficiency among Women and Young Children in Cameroon

Before initiating a mass zinc fortification program, this study assessed the prevalence of and risk factors for low zinc status among Cameroonian women and children. In a nationally representative survey, we randomly selected 30 clusters in each of 3 strata (North, South, and Yaoundé/Douala) and 10 households per cluster, each with a woman aged 15-49 y and a child aged 12-59 mo (n = 1002 households). Twenty-four-hour dietary recalls (with duplicates in a subset) and anthropometric measurements were conducted, and non-fasting blood was collected to measure plasma zinc concentration (PZC) and markers of inflammation. PZC was adjusted for methodologic factors (time of collection and processing, and presence of inflammation). The prevalence of stunting was 33% (32% South; 46% North; 13% Yaoundé/Douala). Among women, 82% had low adjusted PZC (<50 μg/dL for pregnant women; <66 μg/dL for others; 79% South, 89% North, 76% Yaoundé/Douala). Among children, 83% had low adjusted PZC (<65 μg/dL; 80% South, 92% North, 74% Yaoundé/Douala). Risk factors for low PZC among women and children and for low height-for-age Z-score among children were similar and included residence in the North region and rural areas and households with low socioeconomic status. Using estimated average requirement values from the International Zinc Nutrition Consultative Group (IZiNCG), 29 and 41% of women had inadequate zinc intakes, assuming moderate and low bioavailability, respectively, but only 8% of children had inadequate zinc intake. Depending on the estimated physiologic zinc requirement applied, 17% (IZiNCG) and 92% (Institute of Medicine) of women had inadequate absorbable zinc intakes. Total zinc intakes were greatest in the North region, possibly because of different dietary patterns in this area. Zinc deficiency is a public health problem among women and children in Cameroon, although PZC and dietary zinc yield different estimates of the prevalence of deficiency. Large-scale programs to improve zinc nutrition, including food fortification, are needed.

Zinc Deficiency Augments Leptin Production and Exacerbates Macrophage Infiltration into Adipose Tissue in Mice Fed a High-Fat Diet

Zinc (Zn) deficiency and obesity are global public health problems. Zn deficiency is associated with obesity and comorbid conditions that include insulin resistance and type 2 diabetes. However, the function of Zn in obesity remains unclear. Using a mouse model of combined high-fat and low-Zn intake (0.5-1.5 mg/kg), we investigated whether Zn deficiency exacerbates the extent of adiposity as well as perturbations in metabolic and immune function. C57BL/6 mice were randomly assigned to receive either a high-fat diet (HFD) or a control (C) diet for 6 wk, followed by further subdivision into 2 additional groups fed Zn-deficient diets (C-Zn, HFD-Zn), along with a C diet and an HFD, for 3 wk (n = 8-9 mice/group). The extent of visceral fat, insulin resistance, or systemic inflammation was unaffected by Zn deficiency. Strikingly, Zn deficiency significantly augmented circulating leptin concentrations (HFD-Zn vs. HFD: 3.15 ± 0.16 vs. 2.59 ± 0.12 μg/L, respectively) and leptin signaling in the liver of obese mice. Furthermore, gene expression of macrophage-specific markers ADAM8 (A disintegrin and metalloproteinase domain-containing protein 8) and CD68 (cluster of differentiation 68) was significantly greater in adipose tissue in the HFD-Zn group than in the HFD group, as confirmed by CD68 protein analysis, indicative of increased macrophage infiltration. Inspection of Zn content and mRNA profiles of all Zn transporters in the adipose tissue revealed alterations of Zn metabolism to obesity and Zn deficiency. Our results demonstrate that Zn deficiency increases leptin production and exacerbates macrophage infiltration into adipose tissue in obese mice, indicating the importance of Zn in metabolic and immune dysregulation in obesity.