Richard Weindruch

Modification of mitochondrial respiration by aging and dietary restriction.

Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice (23-26 months) did not differ from adult mice (9-12 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (beta-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3-7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c ox. activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.

Effect of dietary restriction upon the age-associated decline of lymphocyte DNA repair activity in mice

Effects of dietary restriction (DR) on DNA repair capacity of mouse splenocytes after ultraviolet (UV)-induced damage were assessed. Two mouse cohorts received restricted amounts of purified hypocaloric diets; one was minimally restricted (∼75% of the caloric intake of mice fed a commercial diet ad libitum), the other was severely restricted (∼50% caloric restriction). An inverse correlation between age and DNA repair was present in the two cohorts; however, the regression lines of the two cohorts showed different slopes. DR appears to decelerate the age-associated decline of DNA repair capacity, and this delay might account in part for the improved immune function shown by old mice on DR.

Influence of weaning-initiated dietary restriction on responses to T cell mitogens and on splenic T cell levels in a long-lived F1-hybrid mouse strain

Abstract Previous studies have shown that rodents dietarily restricted (but not malnourished) since weaning (3–4 weeks old) show prolonged maximum survivorship, inhibition of occurrence of late-life diseases and delayed onset of senescent changes in the immune system. Mitogen induced lymphocyte proliferation, known to decline with age, was examined using mice of a long-lived hybrid strain dietarily restricted at weaning. They were fed intermittently on diets enriched in protein, vitamins and salts and providing approximately 50–60% of the calories fed to control mice. In six series of experiments dietarily restricted mice showed a 2–4 fold decrease in the number of nucleated cells per spleen and a 1.3–3.3 fold increase in phytohemagglutin (PHA)-induced tritiated thymidine uptake by spleen cells. However, diet restriction did not influence splenic responses to B cell mitogens ( E. coli lipopolysaccharide and pokeweed mitogen). An increase in splenic concanavalin A (Con A) reactivity was observed in one of three experiments. The higher maximal PHA responses by restricted mice could not be attributed to kinetic differences or to mitogen concentrations. Spleen cells from restricted mice displayed higher PHA responses at both optimal and reduced cell densities. Lymph node cell yields were reduced by underfeeding but PHA responses were not altered by dietary restriction. The immediate fasting vs. feeding state of restricted mice affected both splenic PHA and Con A reactivity as restricted mice studied 48 hours from last feeding revealed lower PHA and Con A responses than were displayed by restricted mice fed 24 hours before being killed. Neither synergy nor suppression was detected in PHA-stimulated mixed cultures of control and restricted spleen cells. The increase in splenic PHA reactivity for restricted mice was associated with a 1.7 fold increase in the proportion of PHA stimulated blast cells (as judged microscopically) and a 1.7–2.0 fold increase in the proportion of splenic T cells (as shown by cytotoxicity and immunofluorescence measurements). This increase in T cell proportions in underfed spleens is not as great as the decrease in total spleen cells yielding a 1.4–2.1 fold fall in absolute T cell numbers in restricted spleens. These findings suggest that dietary restriction of a type which may be described as “undernutrition without malnutrition” raises splenic PHA response capacity at least in part by increasing the proportion of PHA-responsive T cells.

Food intake reduction and immunologic alterations in mice fed dehydroepiandrosterone

A diet containing 0.4% DHEA was fed to male mice of a long-lived strain from 3 weeks until 18 weeks of age. These mice were compared with others fed a control diet ad libitum and with mice pair-fed the control diet in amounts approximating the intake of the DHEA-fed group. Mice fed the DHEA diet failed to eat all of the food presented to them whereas the pair-fed mice ate all of their food. All mice were studied at 18 weeks of age for two age-sensitive immune parameters (spleen lymphocyte proliferation induced by T-cell mitogens [PHA or ConA] and natural killer cell lysis of an allogeneic tumor). DHEA feeding led to: 1) a decrease in food intake (approximately 30% less than for mice fed the control diet ad libitum), 2) a lower body weight at 18 weeks of age (approximately 40% lower than for ad libitum controls) due to a decrease in the body weight gained from 3 weeks through 18 weeks of age (approximately 55% lower than controls), 3) a lower spleen weight (approximately 30% lower than controls) but without lower numbers of nucleated cells per spleen, 4) an increase in PHA-induced proliferation by spleen lymphocytes (approximately 100% higher than for controls) and, 5) no influence on splenic natural killer cell activity. The inhibition of body weight gain for mice fed DHEA appeared due to both a reduction in food intake and a metabolic effect since mice eating DHEA gained less body weight per gram of food eaten than did mice in either group eating the control diet.

INFLUENCE OF LIFE-PROLONGING DIETARY RESTRICTION ON INTESTINAL VITAMIN A ABSORPTION IN MICE

We studied influences of life prolonging dietary restriction on the absorptive capacity of the small intestine for the lipid micronutrient, Vitamin A. Using a well established intestinal perfusion technique, we measured the vitamin absorption rates in vivo in male mice fed regular laboratory chow (LC) ad lib, female mice on 95 kcal/week of a semipurified diet (designated “N” for normally fed), and female mice restricted to a 55 kcal/wk diet fed four days per week (R). The absorption rate of vitamin A was the lowest in LC mice and highest in R mice. Both at 6 and 26 months of age, R mice absorbed significantly more vitamin A than did the N mice. For example, at 6 months, R mice absorbed 10696 pmol/100 cm/hr while N mice absorbed only 7986 pmol/100 cm/hr (p<.01). Thus, dietary restriction increased the absorptive capacity of the small intestine for the lipid micronutrient vitamin A. The mechanisms responsible remain unknown, but could involve changes in the unstirred water layer resistance or in the lipid cell membrane of the enterocytes.

Drug metabolizing capabilities and drug-induced immunosuppression in two strains of H-2 congenic mice

Mice of the C57BL/10.RIII strain (B10.RIII) and C57BL/10.F strains (B10.F) are congenic, differing genetically at the H-2 complex. The former is H-2r and the latter H-2n. The strains differ greatly in mean survival times, with values for females being about 33 months for B10.RIII and 14 months for B10.F. In the present study, we found that B10.F mice had slightly greater uninduced levels of hepatic P-450-mediated monooxygenase activities than their congenic partners. The ratios of B10.F/B10.RIII basal levels of activities of benzo(a)pyrene (BP) hydroxylase, 7-ethoxycoumarin (7-EC) deethylase and p-nitroanisole (p-NA) demethylase were 1.5, 1.7, and 1.2, respectively (p < 0.05 for all comparisons). Cytochrome P-450-independent malathion and phenthoate carboxylesterase basal activities were not different between the strains. Treatment with β-naphthoflavone (β-NF) induced BP hydroxylase 6.0 fold in B10.RIII mice compared with 3.0 fold in B10.F mice. 7-EC deethylase was induced 3.4 fold in B10.RIII mice and 2.6 fold in B10.F mice. Induction of p-NA demethylase was 1.9 fold in both strains. Treatment with phenobarbital induced 7-EC to a greater degree in B10.RIII than in B10.F mice. In parallel studies on these same mice, splenic lymphocytes from the B10.RIII strain displayed a 2.0-fold higher proliferative response to concanavalin A (Con, 6,) stimulation than was seen in B10.F mice. However, after treatment with β-NF, a striking immunosuppressive effect on B10.RIII mice was noted, with a 6.6-fold decrease in Con A response, while B10.F mice were unaffected. Thus, the longer lived strain B10.RIII appears to be more susceptible to immunosuppression and AHH induction by β-NF than does its congenic partner.

DIETARY RESTRICTION IN MICE BEGINNING AT 1 YEAR OF AGE: EFFECTS ON SERUM IMMUNE COMPLEX LEVELS

One year old mice from a long-lived strain were gradually subjected to life-prolonging dietary restriction. Serum immune complex and immunoglobulin G (abbreviated IgG) levels were measured at 13 and 23 months of age. Longitudinal and cross-sectional analyses showed reduced serum immune complex and IgG levels for mice underfed in adulthood.