Josette Alliot

Differential proteome analysis of aging in rat skeletal muscle

To identify the mechanisms underlying muscle aging, we have undertaken a high‐resolution differential proteomic analysis of gastrocnemius muscle in young adults, mature adults, and old LOU/c/jall rats. Two‐dimensional gel electrophoresis and subsequent MALDI‐ToF mass spectrometry analyses led to the identification of 40 differentially expressed proteins. Strikingly, most differences characterized old (30‐month) animals, whereas young (7‐month) and mature (18‐month) adults exhibited similar patterns of expression. Important modifications in contractile (actin, myosin light‐chains, troponins‐T) and cytoskeletal (desmin, tubulin) proteins, and in essential regulatory proteins (gelsolin, myosin binding proteins, CapZ‐β, P23), likely account for dysfunctions in old muscle force generation and speed of contraction. Other features support decreases in cytosolic (triose‐phosphate isomerase, enolase, glycerol‐3‐P dehydrogenase, creatine kinase) and mitochondrial (isocitrate dehydrogenase, cytochrome‐c oxidase) energy metabolisms. Muscle aging is often associated with increased oxidative stress. Accordingly, we observed differential regulation of molecular chaperones (hsp20, hsp27, reticuloplasmin ER60) and of proteins implicated in reactive aldehyde detoxification (aldehyde dehydrogenase, glutathione transferase, glyoxalase). We further noticed up‐regulation of proteins involved in transcriptional elongation (RNA capping protein) and RNA‐editing (Apobec2). Most of these proteins were previously unrecognized as differentially expressed in old muscles, and they represent novel starting points for elucidating the mechanisms of muscle aging.

Changes in Pattern of Macronutrient Intake During Aging in Male and Female Rats

Transversal and longitudinal studies of the natural patterns of food intake were made in male (n = 65) and female (n = 52) Lou rats, from 4 to 34 months of age. The efficiency of body weight control, especially for females, seems to be a main feature of this strain of rat: these rats are small eaters, have lighter body weight, show no development of obesity with age, and have a longer life expectancy than other common strains of rat. A better adaptation to feeding schedules was shown by female groups throughout the experiments. Moreover, an increased consumption for the oldest animals (from 28 months) enables them to keep their body weight constant up to age 34 months, whereas the male rats gradually lost weight with old age (from 28 months). When male and female rats of different age groups were allowed to choose their diet from pure macronutrient sources (protein, fat, and carbohydrate), an age-related shift of preferences from carbohydrate to fat diets was evidenced during daily energy intake. Protein intake also decreased according to age, earlier for males (around 21 months) than for females (34 months).

Reduced serine racemase expression contributes to age-related deficits in hippocampal cognitive function

To gain insight into the contribution of d-serine to impaired cognitive aging, we compared the metabolic pathway and content of the amino acid as well as d-serine-dependent synaptic transmission and plasticity in the hippocampus of young and old rats of the Wistar and Lou/C/Jall strains. Wistar rats display cognitive impairments with aging that are not found in the latter strain, which is therefore considered a model of healthy aging. Both mRNA and protein levels of serine racemase, the d-serine synthesizing enzyme, were decreased in the hippocampus but not in the cerebral cortex or cerebellum of aged Wistar rats, whereas the expression of d-amino acid oxidase, which degrades the amino acid, was not affected. Consequently, hippocampal levels of endogenous d-serine were significantly lower. In contrast, serine racemase expression and d-serine levels were not altered in the hippocampus of aged Lou/C/Jall rats. Ex vivo electrophysiological recordings in hippocampal slices showed a marked reduction in N-methyl-d-aspartate-receptor (NMDA-R)-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) in the CA1 area of aged Wistar rats, which were restored by exogenous d-serine. In contrast, NMDA-R activation, LTP induction and responses to d-serine were not altered in aged Lou/C/Jall rats. These results further strengthen the notion that the serine racemase-dependent pathway is a prime target of hippocampus-dependent cognitive deficits with aging. Understanding the processes that specifically affect serine racemase during aging could thus provide key insights into the treatment of memory deficits in the elderly.

Effects of posttrial vasopressin injections on appetitively motivated learning in rats

Sixty male rats, maintained on 23-hr food deprivation were trained on two types of appetitive tasks: bar pressure responding under a CRF schedule, and under a differentially reinforced (light+, dark-) schedule. Performance of rats treated with lysing vasopressin, injected immediately after each training session, was compared to that of control animals injected with saline. In the CRF stage, treated animals reached learning criterion significantly later than did control rats, and made significantly fewer bar presses. During acquisition and extinction of a light-dark discrimination, learning and retention were not altered by vasopressin, though the number of bar presses was significantly decreased, and a differential effect was found according to previous CRF performance. The results are discussed considering the hypothesis of a facilitatory effect of vasopressin on memory processes.

Age-related changes in nociception and effect of morphine in the Lou rat

Little is known about how the ageing process affects pain sensitivity and a relevant animal model is therefore required. The effect of age on pain reactivity in animals has been investigated by several experimenters but the results are conflicting. Four groups of male and female Lou/C/Jall rats (4–29 months old) were used for our study. Four pain tests based on evaluation of reflex or more integrated behaviours after a thermal (tail immersion test) or mechanical (paw pressure test and von Frey test) stimulation were used. With mechanical stimulus, a significant decrease in the pain threshold across age was observed, females were more sensitive than males. This increase in nociceptive sensitivity to mechanical stimulation was more pronounced on integrated behaviours (struggle reaction) than on withdrawal reflex. An age‐related increase in sensitivity was found on von Frey test. No effect on the latency of reflex induced by thermal stimulation was observed. In addition, a decrease in the spontaneous motor activity during exploration was observed across ageing; this effect was more marked for the females. The effect of morphine at doses of 1, 3 and 9 mg/kg (s.c.) decreased in intensity across ageing. These data demonstrate the need to use (1) various noxious stimuli because differences were observed in the modification of pain reactivity according to the nature of the stimulus; (2) various pain parameters and particularly integrated behaviours; (3) several age groups. In addition, Lou/C/Jall rat could be a useful model for studying of effect of age on pain.

Preserved memory capacities in aged Lou/C/Jall rats.

Although memory impairments are a hallmark of aging, the degree of deficit varies across animal models, and is likely to reflect different states of deterioration in metabolic and endocrinological properties. This study investigated memory-related processes in young (3-4 months) and old (24 months) Sprague-Dawley rats (SD), which develop age-linked pathologies such as obesity or insulin-resistance and Lou/C/Jall rats, which do not develop such impairments. In short- and long-term memory recognition tasks, old Lou/C/Jall rats were never impaired whereas old SD rats were deficient at 1 and 24h latencies. The expression of N-methyl-d-aspartate receptors (NMDAR)-mediated synaptic plasticity in CA1 hippocampal networks shifted towards lower activity values in old Lou/C/Jall rats whereas long-term potentiation was impaired in age-matched SD rats. Age-related decrease in NR2A subunits occurred in both strains, extended to NR2B, NR1 and GluR1 subunits in older animals (28 months) but only in SD rats. Therefore, the Lou/C/Jall rats can be considered as a model of healthy aging, not only in terms of its preserved metabolism, but also in terms of cognition and synaptic plasticity.

Age-related changes in adaptive mechanisms of macronutrient self-selection: evidence for a sexual dimorphism.

The effect of aging on patterns of food intake and nutrient selection was investigated using a longitudinal study. Male (n = 10) and female (n = 10) Wistar-Lou rats from 4 to 28 months of age were repeatedly submitted to a macronutrient self-selection (S-S) regimen while controls were maintained under a standard chow diet (Std). An age-related shift of preferences from CHO to fat diets, and a decrease in protein intake for both males and females were evident. Nevertheless, all these modifications were more pronounced and precocious for males. Physical exercise (45 min/day of swimming, on 6 days) induced a body weight loss and an hypophagia more pronounced for males than for females. S-S regimen results revealed that hypophagia concerned exclusively fat intake and that females significantly increased protein intake during and after the exercise period. Study of longevity curves showed a decrease of the mortality in S-S submitted male rats compared to control rats. This study shows that aging induces a sex-difference in feeding patterns which undoubtedly reveals a sex-difference metabolic requirements. Moreover, these results suggest that allowing rats to select macronutrient intakes could delay the process of senescence.

Delayed age-associated decrease in growth hormone pulsatile secretion and increased orexigenic peptide expression in the Lou C/JaLL rat.

Since modifications in the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis and/or caloric restriction are involved in the ageing process, GH secretory profiles, total IGF-1, ghrelin, and leptin plasma levels and expression of genes implicated in somatotrope axis and food intake regulation in hypothalamus and pituitary were compared in 3-, 12-, and 24-month-old male Lou C/Jall rats and their parent strain, the Wistar rats. The Lou C/Jall strain may appear as a healthy ageing model, since it does not become obese with age and maintains its caloric intake at 2 years of age. The GH pulsatile secretion decreased from 3 months in Wistar, but only after 12 months in Lou C/Jall rats. The IGF-1 levels were lower in Lou C/Jall rats and decreased more steeply with ageing as compared with Wistar rats. The total ghrelin levels were higher in young Lou C/Jall rats than in Wistar rats, but increased similarly with age in both strains. The leptin concentrations increased with ageing only in Wistar rats. By semiquantitative reverse-transcription polymerase chain reaction, pituitary GH secretagogue receptors and GH mRNA levels were more abundant in Lou C/Jall rats, and the latter decreased with ageing in Wistar rats only. Hypothalamic growth-hormone-releasing hormone and GH secretagogue receptor mRNA levels were similar in both strains and transiently increased only in middle-aged Wistar rats. Agouti-related peptide, neuropeptide Y, and orexin mRNA levels were more abundant in the Lou C/Jall rat hypothalamus, and the two former tended to further increase with age only in this strain. Conversely, the hypothalamic pro-opiomelanocortin mRNA levels were higher in old Wistar rats. In conclusion, ageing in Lou C/Jall rats is associated with a delayed decrease in pulsatile GH secretion in the presence of a lower IGF-1 tone and an increase in the expression of orexigenic neuropeptides in the hypothalamus.

Impact of ageing on the antinociceptive effect of reference analgesics in the Lou/c rat

Research on the evolution of experimental pain perception and on the achievement of analgesia with ageing has led so far to contradictory results. This study investigated in the rat the impact of ageing on the antinociceptive effect of reference analgesics, acetaminophen (50, 100, 200, 400 mg kg−1 po), aspirin (50, 100, 200, 400 mg kg−1 sc), clomipramine (5, 10, 20, 40 mg kg−1 sc) and morphine (1.25, 2.5, 5, 10 mg kg−1 sc). Lou/c rats were chosen because they provide a model of healthy ageing and they do not develop obesity with age. Three groups of 40 rats each (mature (4 months), middle‐aged (18 months) and old (26 months)), were treated with each drug at 14 days interval. Two tests were used: a thermal test (tail immersion in 48°C water and measurement of reaction latency) and a mechanical test (paw pressure and measurement of struggle threshold). Results confirm the increased mechanical sensitivity to pain and no change in thermal sensitivity for old rats compared to mature and middle‐aged animals. They show a marked decrease in the effect of morphine with age and no age‐related effect for acetaminophen, aspirin or clomipramine. Plasma levels of morphine and metabolites are not different in the three age groups. It is likely that the influence of age on morphine analgesia is linked mainly to pharmacodynamic rather than pharmacokinetic changes.

Age-related changes in adaptive macronutrient intake in swimming male and female Lou rats.

To evaluate the age-related changes in capacity to adjust the nutrient intake to needs, self-selecting male and female Lou/C/jall rats of 4, 6, 12, 16 and 23 months of age were submitted to a swimming exercise. They were given 6 consecutive days of moderate intensity training (3 x 15 minutes per day). Exercise and postexercise periods were compared with results from the pretraining period. During swimming, a body weight loss and a decrease in both caloric intake and fat selection were observed. This effect was more marked in older groups compared to 4 month-old groups. An increase in protein intake was observed in females, specially in older groups, whereas no effect was seen in males. The ability to increase caloric ingestion and regain weight during the postexercise period decreased with advancing age and was better in females than in males. We also showed an age-related effect on the recovery of initial nutrient intake rate that was more pronounced and more precocious for males. Moreover, males tended to decrease their protein intake, whereas females significantly increased it. The present findings suggest a decrease of capacity of adjusting feeding behavior to metabolic needs in aged rats, may be due to a deterioration of the central control of food intake.