Age-related alterations in transcriptional regulation of hepatic glutathione synthesis : remediation by R-alpha-lipoic acid

Glutathione (GSH) is the predominant low molecular weight thiol antioxidant in liver tissue. GSH plays an important role in maintaining the intracellular thiol redox ratio as well as detoxification of electrophiles and xenobiotics. Aging leads to a significant decline (35%; P ≤ 0.05) in hepatocellular GSH levels. Using young (2-4 months corresponding to an adolescent human) and old (24-28 months corresponding to a 60-75 year old person) male Fischer 344 rats, we determined that the age-related loss of GSH levels were due to lower activity (53 + 6%; P ≤ 0.05) and levels of the rate-limiting enzyme, γ-glutamate cysteine ligase (GCL). GCL is composed of a catalytic subunit (GCLC) and also a modulatory subunit (GCLM) that affects the KM of its substrate. Since GCLC levels are regulated by transcription, we sought to elucidate its precise transcriptional mechanism and whether aging alters the transcriptome of the enzyme subunit. A cis-acting DNA sequence called the antioxidant response element (ARE) has been previously implicated in the transcriptional regulation of Phase II enzymes, including GCLC and GCLM. Computer-based analysis of the promoter region of Gclc revealed the presence of three putative AREs and a single cis element (ARE-like) containing the core but not the flanking nucleotides of the ARE. Results from experiments where H4IIE rat hepatoma cells were transfected with luciferase reporter constructs containing individual Gclc ARE elements revealed that only the ARE element 3.9 kb upstream of the transcriptional start site (ARE3) possessed basal transcriptional activity. Electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments on liver tissue and primary hepatocytes in culture showed that NF-E2-related factor 2 (Nrf2) was the predominant transcription factor bound to ARE3 and was partnered with small maf proteins, c-Jun, c-Fos and the histone acetyltransferase CREB-Binding Protein (CBP). In aging, nuclear steady-state levels of Nrf2 showed a profound 51 + 7% (P ≤ 0.0001) decline leading to lower Nrf2-ARE3 binding (40%) and transcriptional activity (70 + 10%; P ≤ 0.05), consistent with the loss in GCLC levels. Concomitantly, the transcriptional repressor Bach1 was enriched at the ARE3 site and was accompanied by a loss of CBP. These results show that a negative remodeling of the active Gclc transcriptional complex occurs in the liver of old rats. Furthermore, Nrf2 was detected at the ARE-like site, which was not transcriptionally active in hepatocytes from young rats. Thus, a promoter switching mechanism may occur with age. In previously published reports, we demonstrated that administration of the dithiol compound R-alpha-lipoic acid (LA; 40 mg/kg body weight; intraperitoneal injection) to old rats reversed the age-related decline in hepatic glutathione levels. LA admininstration both to old rats and to hepatocytes in primary cell culture (100 μM) replenished nuclear Nrf2 levels lost during aging. Additionally, LA increased Nrf2 enrichment and activity of both the ARE3 and ARE-like promoters albeit, to a greater extent at the ARE-like promoter (60 + 10%; P ≤ 0.05). This was accompanied by reversal of the age-related decline in GCLC expression, protein levels and GCL activity. Thus, LA maintains hepatic GSH status during aging by permitting normal ARE-mediated GCLC expression, suggesting that it would be a good therapeutic agent to restore GSH-dependent detoxification systems during aging

A moderate increase in dietary zinc reduces DNA strand breaks in leukocytes and alters plasma proteins without changing plasma zinc concentrations.

BackgroundFood fortification has been recommended to improve a population's micronutrient status. Biofortification techniques modestly elevate the zinc content of cereals, but few studies have reported a positive impact on functional indicators of zinc status.ObjectiveWe determined the impact of a modest increase in dietary zinc that was similar to that provided by biofortification programs on whole-body and cellular indicators of zinc status.DesignEighteen men participated in a 6-wk controlled consumption study of a low-zinc, rice-based diet. The diet contained 6 mg Zn/d for 2 wk and was followed by 10 mg Zn/d for 4 wk. To reduce zinc absorption, phytate was added to the diet during the initial period. Indicators of zinc homeostasis, including total absorbed zinc (TAZ), the exchangeable zinc pool (EZP), plasma and cellular zinc concentrations, zinc transporter gene expression, and other metabolic indicators (i.e., DNA damage, inflammation, and oxidative stress), were measured before and after each dietary-zinc period.ResultsTAZ increased with increased dietary zinc, but plasma zinc concentrations and EZP size were unchanged. Erythrocyte and leukocyte zinc concentrations and zinc transporter expressions were not altered. However, leukocyte DNA strand breaks decreased with increased dietary zinc, and the level of proteins involved in DNA repair and antioxidant and immune functions were restored after the dietary-zinc increase.ConclusionsA moderate 4-mg/d increase in dietary zinc, similar to that which would be expected from zinc-biofortified crops, improves zinc absorption but does not alter plasma zinc. The repair of DNA strand breaks improves, as do serum protein concentrations that are associated with the DNA repair process. This trial was registered at clinicaltrials.gov as NCT02861352

A multicomponent nutrient bar promotes weight loss and improves dyslipidemia and insulin resistance in the overweight/obese: chronic inflammation blunts these improvements

This study determined if twice-daily consumption of a nutrient-dense bar intended to fill gaps in Western diets, without other dietary/lifestyle requirements, favorably shifted metabolic/anthropometric indicators of dysregulation in a healthy direction. Three 8-wk clinical trials in 43 healthy lean and overweight/obese (OW/OB) adults, who served as their own controls, were pooled for analysis. In less inflamed OW/OB [highsensitivity C-reactive protein (hsCRP) \u3c1.5], statistically significant decreases occurred in weight (-1.1 ± 0.5 kg), waist circumference (-3.1 ± 1.4 cm), diastolic blood pressure (-4.1 ± 1.6 mmHg), heart rate [HR;-4.0 ± 1.7 beats per minute (bpm)], triglycerides (272638.2mg/dl), insulin resistance (homeostatic model of insulin resistance) (-0.72 ± 0.3), and insulin (-2.8 ± 1.3 mU/L); an increase in HDL-2b (+303 ± 116 nM) and realignment of LDL lipid subfractions toward a less atherogenic profile [decreased small LDL IIIb (-44 ± 23.5 nM), LDL IIIa (299643.7nM),andincreased largeLDLI (+66±28.0nM)]. In the more inflamed OW/OB (hsCRP \u3e1.5), inflammation was reduced at 2 wk (20.66 mg/L), and HR at 8 wk (-3.4 ± 1.3 bpm). The large HDL subfraction (10.5–14.5 nm) increased at 8 wk (+346 ± 126 nM). Metabolic improvements were also observed in lean participants. Thus, favorable changes in measures of cardiovascular health, insulin resistance, inflammation, and obesity were initiated within 8 wk in the OW/OB by replacing deficiencies in Western diets without requiring other dietary or lifestyle modifications; chronic inflammation blunted most improvements.—McCann, J. C., Shigenaga, M. K., Mietus-Snyder, M. L., Lal, A., Suh, J. H., Krauss, R. M., Gildengorin, G. L., Goldrich, A. M., Block, D. S., Shenvi, S. V.,McHugh, T. H.,Olson,D. A., Ames, B.N. A multicomponent nutrient bar promotes weight loss and improves dyslipidemia and insulin resistance in the overweight/obese: chronic inflammation blunts these improvements

A moderate increase in dietary zinc reduces DNA strand breaks in leukocytes and alters plasma proteins without changing plasma zinc concentrations

Background: Food fortification has been recommended to improve a population’s micronutrient status. Biofortification techniques modestly elevate the zinc content of cereals, but few studies have reported a positive impact on functional indicators of zinc status. Objective: We determined the impact of a modest increase in dietary zinc that was similar to that provided by biofortification programs on whole-body and cellular indicators of zinc status. Design: Eighteen men participated in a 6-wk controlled consumption study of a low-zinc, rice-based diet. The diet contained 6 mg Zn/d for 2 wk and was followed by 10 mg Zn/d for 4 wk. To reduce zinc absorption, phytate was added to the diet during the initial period. Indicators of zinc homeostasis, including total absorbed zinc (TAZ), the exchangeable zinc pool (EZP), plasma and cellular zinc concentrations, zinc transporter gene expression, and other metabolic indicators (i.e., DNA damage, inflammation, and oxidative stress), were measured before and after each dietary-zinc period. Results: TAZ increased with increased dietary zinc, but plasma zinc concentrations and EZP size were unchanged. Erythrocyte and leukocyte zinc concentrations and zinc transporter expressions were not altered. However, leukocyte DNA strand breaks decreased with increased dietary zinc, and the level of proteins involved in DNA repair and antioxidant and immune functions were restored after the dietary-zinc increase. Conclusions: A moderate 4-mg/d increase in dietary zinc, similar to that which would be expected from zinc-biofortified crops, improves zinc absorption but does not alter plasma zinc. The repair of DNA strand breaks improves, as do serum protein concentrations that are associated with the DNA repair process. This trial was registered at clinicaltrials.gov as NCT02861352

A nutrient-dense, high-fiber, fruit-based supplement bar increases HDL cholesterol, particularly large HDL, lowers homocysteine, and raises glutathione in a 2-wk trial.

Dietary intake modulates disease risk, but little is known how components within food mixtures affect pathophysiology. A low-calorie, high-fiber, fruit-based nutrient-dense bar of defined composition (e.g., vitamins and minerals, fruit polyphenolics, β-glucan, docosahexaenoic acid) appropriate for deconstruction and mechanistic studies is described and evaluated in a pilot trial. The bar was developed in collaboration with the U.S. Department of Agriculture. Changes in cardiovascular disease and diabetes risk biomarkers were measured after 2 wk twice-daily consumption of the bar, and compared against baseline controls in 25 healthy adults. Plasma HDL-cholesterol (HDL-c) increased 6.2% (P=0.001), due primarily to a 28% increase in large HDL (HDL-L; P<0.0001). Total plasma homocysteine (Hcy) decreased 19% (P=0.017), and glutathione (GSH) increased 20% (P=0.011). The changes in HDL and Hcy are in the direction associated with decreased risk of cardiovascular disease and cognitive decline; increased GSH reflects improved antioxidant defense. Changes in biomarkers linked to insulin resistance and inflammation were not observed. A defined food-based supplement can, within 2 wk, positively impact metabolic biomarkers linked to disease risk. These results lay the groundwork for mechanistic/deconstruction experiments to identify critical bar components and putative synergistic combinations responsible for observed effects.—Mietus-Snyder, M. L., Shigenaga, M. K., Suh, J. H., Shenvi, S. V., Lal, A., McHugh, T., Olson, D., Lilienstein, J., Krauss, R. M., Gildengoren, G., McCann, J. C., Ames, B. N. A nutrient-dense, high-fiber, fruit-based supplement bar increases HDL cholesterol, particularly large HDL, lowers homocysteine, and raises glutathione in a 2-wk trial