The effects of aging and calorie restriction on plasma nutrient levels in male and female Emory mice.

We examined the effect of diet, age (4.5, 13 and 23 months), and sex on plasma levels of retinol, tocopherol, ascorbate, cholesterol, glucose and glycohemoglobin in male and female Emory mice which were fed control (C) and 50% calorie restricted (R) diets. Results showed that C fed animals tended to have higher levels of plasma ascorbate (50-71%), cholesterol (23-71%), glucose (38-81%) and glycohemoglobin (50%). However, these diet differences varied with the age and sex of the animals. Plasma retinol levels were lower only in R males vs. C males (50%). Novel sex-related differences in levels of plasma retinol (2-fold higher in C male mice than in C or R female mice) are described. Aging was associated with trends towards lower levels of plasma ascorbate (14-25%), glucose (34-36%) and glycohemoglobin (47-57%) from 4.5 to 23 months of age. However, these age differences depended upon the diet and sex of the animals. These data suggest that lower plasma levels of glucose, glycosylated hemoglobin and cholesterol may be causally related to the life extension noted in R animals since elevated levels of these moieties have been related to aging. Since oxidative stress is thought to be causally related to aging it appears unlikely that retinol, tocopherol and ascorbate are causally related to R-induced life-extension.

Regulation of ubiquitin-conjugating enzymes by glutathione following oxidative stress.

Upon oxidative stress cells show an increase in the oxidized glutathione (GSSG) to reduced glutathione (GSH) ratio with a concomitant decrease in activity of the ubiquitinylation pathway. Because most of the enzymes involved in the attachment of ubiquitin to substrate proteins contain active site sulfhydryls that might be covalently modified (thiolated) upon enhancement of GSSG levels (glutathiolation), it appeared plausible that glutathiolation might alter ubiquitinylation rates upon cellular oxidative stress. This hypothesis was explored using intact retina and retinal pigment epithelial (RPE) cell models. Exposure of intact bovine retina and RPE cells to H2O2 (0.1-1.7 micromol/mg) resulted in a dose-dependent increase in the GSSG:GSH ratio and coincident dose-dependent reductions in the levels of endogenous ubiquitin-activating enzyme (E1)-ubiquitin thiol esters and endogenous protein-ubiquitin conjugates and in the ability to form de novo retinal protein-125I-labeled ubiquitin conjugates. Oxidant-induced decrements in ubiquitin conjugates were associated with 60-80% reductions in E1 and ubiquitin-conjugating enzyme (E2) activities as measured by formation of ubiquitin thiol esters. When GSH levels in RPE cells recovered to preoxidation levels following H2O2 removal, endogenous E1 activity and protein-ubiquitin conjugates were restored. Evidence that S thiolation of E1 and E2 enzymes is the biochemical link between cellular redox state and E1/E2 activities includes: (i) 5-fold increases in levels of immunoprecipitable, dithiothreitol-labile 35S-E1 adducts in metabolically labeled, H2O2-treated, RPE cells; (ii) diminished formation of E1- and E2-125I-labeled ubiquitin thiol esters, oligomerization of E225K, and coincident reductions in protein-125I-labeled ubiquitin conjugates in supernatants from nonstressed retinas upon addition of levels of GSSG equivalent to levels measured in oxidatively stressed retinas; and (iii) partial restoration of E1 and E2 activities and levels of protein-125I-labeled ubiquitin conjugates in supernatants from H2O2-treated retinas when GSSG:GSH ratios were restored to preoxidation levels by the addition of physiological levels of GSH. These data suggest that the cellular redox status modulates protein ubiquitinylation via reversible S thiolation of E1 and E2 enzymes, presumably by glutathione.

Antioxidant enzyme activities in lens, liver and kidney of calorie restricted Emory mice.

Dietary calorie restriction extends both mean and maximum life span and retards age-related diseases, including eye lens cataract in Emory mice. The beneficial effects of calorie restriction have been hypothesized to reflect enhanced tissue antioxidant capacity. As a test of this hypothesis, we reared male and female Emory mice on control (C) or 40% calorie-restricted (R) diets. We then determined activities of total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) in eye lens, liver and kidney of young (4.5 or 6 months), mature (11 or 12 months) and old (22 months) animals. Effects of diet, age and sex were evaluated by multi-factor ANOVA. Only kidney GR activities (mean +/- S.E.M.) were significantly enhanced with the R diet (R, 61 +/- 2 vs. C, 54 +/- 3 U/mg protein; P = 0.03). More frequently, we noted reduced antioxidant enzyme activity in R as compared with C animals, including reduced activities of T-SOD in lens, liver and kidney, Cu/Zn-SOD in liver and kidney, liver Mn-SOD and liver CAT (P < 0.05). Effects of age on antioxidant enzyme activity in C mice included age-dependent decreases in lens and kidney CAT and in liver Mn-SOD. There was also an age-dependent increases in liver and kidney Cu/Zn-SOD and liver GR. None of these age-dependent alterations in antioxidant enzyme function were attenuated in tissues of mice fed the R diet. Values for liver CAT were significantly lower in females than in males (P = 0.05). These results indicate that antioxidant enzyme activities in Emory mouse tissues are influenced by diet, age and sex. However, it is unlikely that increased lifespan and attenuation of cataract (and perhaps other age-dependent debilities), which are associated with the R diet in the Emory mouse, are due to enhanced antioxidant enzyme capabilities.

Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt and rhodopsin.

In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pI 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt as a substrate for ubiquitin-dependent proteolysis (Obin, M. S., Nowell, T., and Taylor, A. (1994) Biochem. Biophys. Res. Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to Gt, included (i) the presence in ROS membranes "stripped" of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of 125I-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a "ladder" of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these 125I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of 125I-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (E1); (ii) four ubiquitin carrier proteins (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, an enzyme required for "N-end rule" proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass 125I-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties. Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.

Dietary restriction delays cataract and reduces ascorbate levels in Emory mice.

Dietary restriction can effectively extend lifespan and retard many age-related debilities. One hypothesis to explain the beneficial effects of dietary restriction is that it prolongs maintenance of cellular homeostasis by limiting endogenous oxidative stress and preserves oxidative defense mechanisms during aging. Ascorbate, a primary antioxidant, may play a major role in preventing oxidative damage. Ascorbate levels were determined in dietary restricted (R) and control (C) Emory mice, a strain which develops age-related cataract due in part to oxidative damage to lens proteins. Mice which consumed a diet restricted by 40% in calories had lower ascorbate concentrations in plasma, liver and kidney. Nevertheless, R animals showed significantly delayed progression of cataract which extended over the entire second half of life. The R diet did not result in different ascorbate levels in this lens. Aging was associated with a decrease in ascorbate in all the examined tissues except lens of both the R and C groups. It is not clear from these data that ascorbate is a prominent factor in the delay of cataract formation or other debilities in R Emory mice. However, it also appears unlikely that lens ascorbate is cataractogenic.

Dietary calorie restriction in the Emory mouse: effects on lifespan, eye lens cataract prevalence and progression, levels of ascorbate, glutathione, glucose, and glycohemoglobin, tail collagen breaktime, DNA and RNA oxidation, skin integrity, fecundity, and cancer.

The Emory mouse is the best model for age-related cataract. In this work we compare the effects of feeding a control diet (C) with a diet restricted (R) by 40% relative to C animals. In the R animals, median lifespan was extended by 40%. The proportion of R mice with advanced cataract was lower than C mice as early as 5 months of age. The mean grade of cataract was lower in R animals, beginning at 11 months and continuing until the end of the study. Ascorbate levels in R plasma and liver were 41-56% of C animals. There was no difference between diet groups with respect to lens ascorbate. Aging was associated with a decrease in ascorbate in lenses and kidneys in C and R mice. By 22 months, R animals had 48% higher liver glutathione levels than C mice. Liver glutathione levels were maximal at 12 months. Plasma glucose levels were > 27% lower in R animals at 6.5 and 22 months, and there was a 14% increase in glucose levels upon aging for both diet groups. In R mice, glycohemoglobin levels were 51% lower and tail collagen breaktime was decreased by 40%, even in younger animals. Collagen breaktime increased > 360% upon aging for both diet groups. Rates of production of urinary oxo8dG and oxo8G were higher in R animals compared with C animals, and increased upon aging. C animals exhibited more cancer and dermatological lesions, but less tail tip necrosis and inflamed genitals than R mice. These data allow evaluation of several theories of aging.

Isolation of bovine kidney leucine aminopeptidase cDNA: comparison with the lens enzyme and tissue-specific expression of two mRNAs.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Leucine aminopeptidase (LAP) from bovine lens is the best characterized aminopeptidase and the only LAP for which the amino acid sequence was determined by protein sequencing. Using this sequence information, we isolated a bovine kidney LAP cDNA and compared its deduced amino acid sequence to the published amino acid sequence for bovine lens LAP. Overall, the sequences are highly conserved. However, several differences are observed. The kidney LAP cDNA indicates a 26 amino acid extension at the amino terminus which is not found in the mature purified lens LAP. The cDNA also indicates an additional octapeptide in the C-terminal region which was not indicated in the published lens LAP amino acid sequence but which was required for best fit of crystallographic data regarding bovine lens LAP. Several other single amino acid changes were also noted. Levels of LAP transcripts were examined in bovine lens and kidney tissue as well as in cultured lens cells. Lens epithelial tissue showed only one LAP transcript (2.4 kb) whereas two transcripts (2.0 and 2.4 kb) were observed in cultured lens cells derived from epithelial tissue and in kidney tissue. Using Northern blot analysis, we correlated LAP mRNA levels with previously determined changes of LAP activity in aging lens tissue and in progressively passaged lens epithelial cells which were used to simulate aging in vitro. No differences were found in LAP mRNA levels in epithelial tissue from old and young lenses.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine lens epithelial cells have a ubiquitin-dependent proteolysis system.

Lens cells must remove obsolete or damaged proteins produced during development, maturation and aging to maintain lens transparency. In reticulocytes removal of abnormal or obsolete proteins is thought to involve a ubiquitin-dependent proteolytic pathway. Two hallmarks of ubiquitin (Ub) dependent proteolysis have previously been demonstrated in lens cell or tissue supernatants: (1) the presence of ubiquitin conjugates, and (2) ATP-dependent proteolysis. Nevertheless, conclusive proof was lacking of a requirement for ubiquitination of substrate proteins for proteolysis. Here we show that in bovine lens epithelial cell (BLEC) supernatant, ATP-dependent proteolysis is also ubiquitin-dependent. Ubiquitin-activating enzyme (E1), the first enzyme in the cascade of ubiquitin ligation, was purified over 3000-fold from a rabbit reticulocyte lysate using Ubiquitin-Sepharose, and showed ATP-PPi exchange activity. Antiserum to E1 was prepared in goats and affinity-purified on Protein G-Sepharose. Western blot analysis revealed that both the goat antiserum and purified antibody (anti-E1(IgG)) recognize specifically E1. Anti-E1(IgG) inhibits 86% of the ATP-dependent degradation of labeled histone H2A in reticulocyte lysate and 75% of the ATP-dependent degradation in BLEC. Upon reconstitution with purified E1, 100% and 80% of proteolysis was restored in reticulocytes and BLEC supernatant, respectively. This confirms that there is a ubiquitin-dependent proteolytic system in lens.

Ubiquitin and ubiquitin conjugates in human lens.

Ubiquitin, an 8.5 kDa polypeptide found almost universally in plants and animals, is a normal component in the lens. The best documented function for ubiquitin involves its conjugation to proteins as a signal to initiate degradation. Conjugates for ubiquitin-dependent degradation tend to be of very high molecular mass and are rapidly degraded. Another role of ubiquitin conjugation may be as a stabilizer during stress for protection of constituent proteins, resulting in ubiquitin conjugates that are long-lived. Examination of clear and cataractous human lenses of < 1 to > 50 years revealed the dramatic accumulation of ubiquitin and ubiquitin conjugates with age, beginning at approximately 10 years. Epithelial tissue contained predominantly conjugates of > 250 kDa, although ubiquitin conjugates were found at 98 and 40-60 kDa in tissues from older donors. The water-soluble, urea-soluble and urea-insoluble fractions of lens cortex and core also contain ubiquitin conjugates that accrue with age. High molecular mass conjugates (> 250 kDa) are particularly prominent in older lens tissue. Cataractous lenses, as compared with normal lenses of the same age, show more of these high molecular mass conjugates in the urea-soluble and urea-insoluble fractions of cortex and core. Heterogeneous conjugates in the 20-85 kDa range accumulate in an age-related fashion in all lens cortex and core fractions. While levels of free ubiquitin are significant in the epithelium and the water-soluble cortex and core for all ages, there is no detectable free ubiquitin in the urea-soluble and urea-insoluble fraction under conditions used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for ATP and ubiquitin dependent degradation of proteins in cultured bovine lens epithelial cells.

Degradation of endogenous lens proteins has been difficult to show under physiological conditions using lens tissue preparations. In contrast, active proteolytic systems in cultured bovine lens epithelial (BLE) cells have been demonstrated previously. BLE cells also contain ubiquitin, a 76 amino-acid polypeptide which is conjugated to proteins in an ATP/Mg(2+)-dependent process prior to their cytosolic proteolysis. In this study, we show that histone H2A, alpha-crystallin, and actin are conjugated to ubiquitin, resulting in higher molecular mass species, which are detected by anti-ubiquitin antibodies. These proteins are also degraded in cell-free assays containing BLE cell supernatants under physiological conditions in an ATP/Mg(2+)-dependent manner. Observation of 125I-labeled proteolytic fragments was made after SDS polyacrylamide gel electrophoresis of the assays. Quantitation of trichloroacetic acid-soluble radiolabeled fragments generated in the presence of ATP/Mg2+ revealed that, with BLE cell supernatant, 25% of the histone H2A was degraded in 3 hr. Proteolysis of alpha-crystallin and actin amounted to 2.3% and 2.9%, respectively. The requirement of ATP/Mg2+ for proteolysis and the observation of ubiquitin conjugation to the same proteins is consistent with the presence of a ubiquitin-dependent proteolytic pathway in BLE cells. Additionally, in this study the BLE cell proteases were even more active on some substrates than the reticulocyte ubiquitin/ATP-dependent proteolytic system.