3-month Rats Research Articles

Age-related changes in satellite cell proliferation by compensatory activation in rat diaphragm muscles

To investigate the age-related changes in satellite cell (SC) proliferation in vivo, we used a compensatory activation (CAC) model of the hemi-diaphragm muscle. Young (2-month), adult (14-month) and old (24-month) rats were randomly divided into control and CAC groups. In the CAC group, denervation surgery in the left hemi-diaphragm was performed to induce CAC of the right hemi-diaphragm. Six days after the surgery, the CAC diaphragm muscle was removed and separated into two blocks for immunohistochemical staining and real time RT-PCR procedures. The number of SCs in type I and IIa fibers were not affected significantly by the CAC in any age groups, but that in type IIx/b fibers was significantly increased in the young and adult groups. As compared to the age-matched control group, the Pax7 mRNA expression level was significantly higher in the young and adult CAC groups, but not in the old CAC group. These results may suggest that the mechanism of SC proliferation in type IIx/b fibers is impaired in aged diaphragm muscles.

MiR-335 and miR-34a Promote Renal Senescence by Suppressing Mitochondrial Antioxidative Enzymes

The molecular basis for aging of the kidney is not well understood. MicroRNAs (miRNAs) contribute to processes such as development, differentiation, and apoptosis, but their contribution to the aging process is unknown. Here, we analyzed the miRNA expression profile of young (3-month) and old (24-month) rat kidneys and identified the biologic pathways and genes regulated by differentially expressed miRNAs. We observed upregulation of 18 miRNAs with aging, mainly regulating the genes associated with energy metabolism, cell proliferation, antioxidative defense, and extracellular matrix degradation; in contrast, we observed downregulation of 7 miRNAs with aging, principally targeting the genes associated with the immune inflammatory response and cell-cycle arrest. Bioinformatics analysis suggested that superoxide dismutase 2 (SOD2) and thioredoxin reductase 2 (Txnrd2), located in the mitochondria, are potential targets of miR-335 and miR-34a, respectively. Aging mesangial cells exhibited significant upregulation of miR-335 and miR-34a and marked downregulation of SOD2 and Txnrd2. miR-335 and miR-34a inhibited expression of SOD2 and Txnrd2 by binding to the 3'-untranslated regions of each gene, respectively. Overexpression of miR-335 and miR-34a induced premature senescence of young mesangial cells via suppression of SOD2 and Txnrd2 with a concomitant increase in reactive oxygen species (ROS). Conversely, antisense miR-335 and miR-34a inhibited senescence of old mesangial cells via upregulation of SOD2 and Txnrd2 with a concomitant decrease in ROS. In conclusion, these results suggest that miRNAs may contribute to renal aging by inhibiting intracellular pathways such as those involving the mitochondrial antioxidative enzymes SOD2 and Txnrd2.

4-Vinylcyclohexene diepoxide (VCD) inhibits mammary epithelial differentiation and induces fibroadenoma formation in female Sprague Dawley rats

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, both doses of VCD accelerated mammary tumor onset by 5 months, increasing incidence to 84% (vs. 38% in controls). Tumor development was independent of time in persistent estrus, 17β-estradiol, androstenedione and prolactin. Delay in VCD administration until after completion of mammary epithelial differentiation (3 months) did not alter tumor formation despite acceleration of ovarian senescence. VCD administration to 1-month rats acutely decreased mammary alveolar bud number and expression of β-casein, suggesting that VCD's tumorigenic effect requires exposure during mammary epithelial differentiation.

Age-Related Increases of Macroautophagy and Chaperone-Mediated Autophagy in Rat Nucleus Pulposus

Objective: Excessive apoptosis plays an important role in the progression of intervertebral disc degeneration. However, the effect of autophagy, another type of programmed cell death, on the pathogenesis of disc degeneration is still unclear. Macroautophagy and chaperone-mediated autophagy (CMA) change and intervertebral disc degeneration aggravates with age. This study aims at examining the expression changes of light chain 3 (LC3), lysosome-associated membrane protein 2A (LAMP-2A), and Hsc70, the indicator substrates of macroautophagy and CMA, in rat nucleus pulposus (NP) to prove that macroautophagy and CMA are both related with age. Methods: Female Sprague–Dawley rats of 3, 12, and 24 months (n = 8 per age) were used in this study. Autophagic vacuoles in NP cells were detected by transmission electron microscopy. In NP, the expressions of LC3-II and LAMP-2A protein and mRNA were examined by immunohistochemistry and reverse transcription polymerase chain reaction, respectively. LC3-II, LC3-I, and LAMP-2A protein were also measured by western blot. The mRNA and protein level of myocyte enhancer factor-2D regulated by LAMP-2A and Hsc70 were detected by reverse transcriptase polymerase chain reaction and western blot, respectively. Results: Transmission electron microscopy showed more autophagic vacuoles in 12- and 24-month groups than in 3-month group. Expression of LC3-II and LC3-II/LC3-I in 24-month group was significantly higher than in 3-month group (p < 0.05). Meanwhile, LAMP-2A expression was significantly higher in 24-month group than in 3-month group (p < 0.05). However, lower expression of Hsc70 and myocyte enhancer factor-2D was found in the 24-month rats than in 3-month group (p < 0.05, p < 0.05, respectively). Conclusion: Macroautophagy and CMA were present and increased with age in rat NP.

Effects of calorie restriction on cardioprotection and cardiovascular health

Multiple health benefits of calorie restriction (CR) and alternate day fasting (ADF) regimens are widely recognized. Experimental data concerning the effects of calorie restriction on cardiac health are more controversial, ranging from evidence that ADF protects heart from ischemic damage but results in developing of diastolic dysfunction, to reports that CR ameliorates the age-associated diastolic dysfunction. Here we investigated the effects of chronic CR on morphology and function of the cardiovascular system of aged rats and cardioprotective effect of CR against ischemic damage in the experimental rat model of MI. Cardiovascular fitness of 24-month old Fisher 344 rats maintained through life on ad libitum (AL) or CR diets was extensively evaluated via echocardiography, dobutamine stress test, pressure–volume loop analyses, pulse wave velocity measurements, and histology. Groups of 2-month old AL and 29-month old CR rats were studied for comparison. Myocardial infarction (MI) was induced by a permanent ligation of the anterior descending coronary artery in 5-month old rats maintained for 3months on CR or AL. MI size was evaluated histologically 24hrs following coronary ligation. Cardiac remodeling was followed-up via echocardiography. Age-associated changes in 24-month old rats consisted of 33% increase of fibrosis in the myocardium and more than 2 fold increase of the collagen in the tunica media of the aorta. There was a significant decrease in the density and total number of cardiomyocytes, while their size was increased. These morphological changes were manifested in a decline of systolic and diastolic cardiac function, increase of left ventricular and aortic stiffness, and arterio-ventricular uncoupling. Tachycardic response to dobutamine challenge was absent in the old rats. Compared to AL rats, 24-month old CR rats had reduced levels of cardiac and aortic fibrosis, increased density of cardiomyocytes that were smaller in size, attenuated diastolic dysfunction, normal systolic function and arterio-ventricular coupling. Tachycardic response to dobutamine was also intact in CR 24-month old rats and aortic stiffness was reduced. Adjustment for body weight differences through ratiometric or allometric scaling did not affect the overall pattern of differences between AL and CR rats. Attenuation of morphological and functional age-associated changes in 24-month old CR rats either was not observed at all or was smaller in 29-month old CR rats. Size of MI induced by a permanent coronary ligation as well as post-MI cardiac remodeling and function were similar in CR and AL rats. CR does not increase tolerance of myocardium to ischemic damage, but attenuates the age-associated changes in the heart and major vessels. The attenuation of age-associated changes by CR cannot be explained by the effect of lower body weight but are attributable to more intimate cellular mechanisms of CR itself. Attenuation of age-associated changes by CR waned with advancing age, and is consistent with the idea that CR postponed senescence.

An Evaluation of Chronic 6- and 12-Month Rat Toxicology Studies as Predictors of 2-Year Tumor Outcome

Chronic 6- and 12-month rat toxicology studies were evaluated for their ability to predict tumor outcome in 2-year rat carcinogenicity studies for 80 pharmaceuticals from commercial and Merck databases. The data consisted of 62 (6-month) and 54 (12-month) studies, which included 30 rat carcinogens and 50 noncarcinogens in 2-year studies. The histopathology findings considered as evidence of potential preneoplasia in the chronic studies were hyperplasia, cellular hypertrophy, and atypical cellular foci. The authors hypothesized that a whole animal-based approach should be taken, wherein (1) evidence of potential preneoplasia in any tissue may serve as a sensitive predictor of tumor outcome in any tissue in the whole animal and not necessarily the same tissue and (2) the absence of evidence for potential preneoplasia in all tissues may serve as a strong negative predictor of tumor outcome in any tissue. Based on this whole animal approach, 25 of 30 rat carcinogens showed histopathologic signals in chronic toxicology studies, yielding a test sensitivity of 83%. The negative predictivity of the absence of histopathology findings in chronic toxicology studies of 50 nontumorigenic compounds was 88%. The value of the extra 6-month treatment was not apparent. The 5 false negatives (negative chronic studies but positive 2-year studies) are for marketed drugs approved for non-life-threatening conditions and associated with rat-specific mechanisms. The absence of preneoplasia in the whole animal is a reliable predictor of negative tumor outcome in 2-year rat studies, and approximately 50% rat carcinogenicity studies could be eliminated for the 80 pharmaceuticals examined, with no risk to humans and with a substantial reduction in animal usage and drug development time.

Expression and significance of Beclin-1 and microtubule-associated protein 1 light chain 3 in nucleus pulpous of rats during the aging process

Objective To evaluate the expression and significance of Beclin-1 and microtubule-associated protein 1 light chain 3 ( MAPLC3 ) in nucleus pulpous of rats during the aging process. Methods The models of youth, middle-aged and old rats were made with the 3-month, 12-month and 24-months SD rats respectively. The grades of intervertebral disc degeneration were measured with HE staining. The expression of autophagosome in nucleus pulpous was detected under a transmission electron microscope ( TEM ). The changes of Beclin-1 and MAPLC3 in nucleus pulpous were assayed by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Results The aging process of rats may reflect the process of intervertebral disc degeneration. There was expression of autophagosome in nucleus pulpous in every group. Beclin-1 and MAPLC3 were expressed in nucleus pulpous cells in each group ( ayerage absorbance values were 0. 42 ± 0. 05, 0. 47 ± 0. 06, 0. 52 ± 0. 05 and 0. 34 ± 0. 06, 0. 39 ± 0. 05,0. 46 ±0. 09 respectively) and both of them were higher in old group (P <0. 01 ). Beclin-1 and MAPLC3 mRNA was expressed in nucleus pulpous in each group (0.86 ±0. 12, 0.93 ±0. 14, 1.01 ±0. 13 and 1.09 ± 0.06,1.15 ± 0. 07, 1.33 ± 0. 11 respectively) and both of them were higher in old group ( P <0. 05 and P < 0. 01 respectively). Conclusion Autophagy resides in the process of intervertebral disc degeneration. The expression of MAPLC3 and Beclinl was up-regulated in nucleus pulpous in different groups and autophagy. Up-regulation of MAPLC3 and Beclinl may be related to the process of the lumbar disc degeneration. Key words: Intervertebral disc degeneration; Aging; Autophagy; Beclin-1; MAPLC3

Decreased Brainstem Function Following Cardiac Arrest and Resuscitation in Aged Rat

There is a high incidence of cardiac arrest and poorer post-resuscitation outcome in the elderly population. Cardiac arrest and resuscitation results in ischemia/reperfusion injury associated with oxidative stress, leading to post-resuscitation mortality and delayed selective neuronal cell loss. In this study we investigated recovery following cardiac arrest and resuscitation in the aged rat brain. Male Fischer 344 rats (6, 12 and 24months old) underwent 7minute cardiac arrest before resuscitation. Overall survival and hippocampal neuronal counts were determined at 4days of recovery. Brainstem function was assessed by hypoxic ventilatory response (HVR). Mitochondria of brainstem, cortex and hippocampus were isolated and assessed for respiratory function. Effect of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) was used as a treatment strategy against oxidative stress in the 6 and 24-month old rats. The time course of mitochondrial function was established using 3-month old Wistar rats with 12-minute cardiac arrest. In the 24-month old rats, overall survival rate, hippocampal CA1 neuronal counts, HVR, and brain mitochondrial respiratory control ratio were significantly reduced following cardiac arrest and resuscitation compared to the younger rats, and PBN treatment improved outcome. The data suggest that (i) there was increased susceptibility to ischemia/reperfusion in aged rat brain; (ii) HVR was decreased in the aged rats; (iii) brain mitochondrial respiratory function related to coupled oxidation was decreased following cardiac arrest and resuscitation in rats, more so in the aged; and (iv) treatment with an antioxidant, such as PBN, reduced the oxidative damage following cardiac arrest and resuscitation.

3α-androstanediol, but not testosterone, attenuates age-related decrements in cognitive, anxiety, and depressive behavior of male rats

Some hippocampally-influenced affective and/or cognitive processes decline with aging. The role of androgens in this process is of interest. Testosterone (T) is aromatized to estrogen, and reduced to dihydrotestosterone (DHT), which is converted to 5α-androstane, 3α, 17α-diol (3α-diol). To determine the extent to which some age-related decline in hippocampally-influenced behaviors may be due to androgens, we examined the effects of variation in androgen levels due to age, gonadectomy, and androgen replacement on cognitive (inhibitory avoidance, Morris water maze) and affective (defensive freezing, forced swim) behavior among young (4 months), middle-aged (13 months), and aged (24 months) male rats. Plasma and hippocampal levels of androgens were determined. In experiment 1, comparisons were made between 4-, 13-, and 24-month-old rats that were intact or gonadectomized (GDX) and administered a T-filled or empty silastic capsule. There was age-related decline in performance of the inhibitory avoidance, water maze, defensive freezing, and forced swim tasks, and hippocampal 3α-diol levels. Chronic, long-term (1–4 weeks) T-replacement reversed the effects of GDX in 4- and 13-month-old, but not 24-month-old, rats in the inhibitory avoidance task. Experiments 2 and 3 assessed whether acute subcutaneous T or 3α-diol, respectively, could reverse age-associated decline in performance. 3α-diol, but not T, compared to vehicle, improved performance in the inhibitory avoidance, water maze, forced swim, and defensive freezing tasks, irrespective of age. Thus, age is associated with a decrease in 3α-diol production and 3α-diol administration reinstates cognitive and affective performance of aged male rats.

Effects of chronic treatment with a low dose of nicorandil on the function of the rat aorta during ageing

1. It is known that ATP-sensitive potassium (K(ATP)) channels regulate the membrane potential of smooth muscle cells and vascular tone. Because their activity is altered during ageing, many pharmacological treatments aimed at improving K(ATP) channel and cardiovascular functions have been evaluated. Nicorandil, a K(ATP) channel opener, nitric oxide (NO) donor and anti-oxidant, induces vasodilation, decreases blood pressure and exhibits cardioprotection in ageing, as well as after ischaemia-reperfusion. 2. In the present study, using tension myography and biochemical and histological techniques, we investigated the effects of chronic (2 months) low-dose nicorandil (0.1 mg/kg per day) treatment on the function of rat aorta during ageing (in 4-, 12- and 24-month old rats). 3. The results showed that chronic nicorandil treatment significantly improves mechanical relaxation and noradrenaline-induced vasoconstriction in aged rats. At all ages, the nicorandil-induced vasodilation was primarily mediated by its NO donor group. Nicorandil treatment resulted in an additional 0.5-1 elastic lamella in the aorta and decreased total protein, collagen and elastin content in the aortic wall at all ages. However, in 4-month-old rats, nicorandil significantly increased the elastin : total protein ratio by 19%. 4. In contrast with results of previous studies that used high doses of nicorandil (i.e. 60 mg/kg per day), low-dose nicorandil treatment in the present study did not lead to a progressive desensitization to nicorandil and may be beneficial in improving arterial function in ageing or cardiovascular diseases.

Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport

Respiratory enzyme complex dysfunction is mechanistically involved in mitochondrial failure leading to neurodegenerative disease, but the pathway is unclear. Here, age-related differences in mitochondrial respiration were measured in both whole and permeabilized neurons from 9-month and 24-month adult rat cortex cultured in common conditions. After permeabilization, respiration increased in both ages of neurons with excess substrates. To dissect specific deficiencies in the respiratory chain, inhibitors for each respiratory chain complex were used to isolate their contributions. Relative to neurons from 9-month rats, in neurons isolated from 24-month rats, complexes I, III, and IV were more sensitive to selective inhibition. Flux control point analysis identified complex I in neurons isolated from 24-month rats as the most sensitive to endogenous substrate availability. The greatest age-related deficit in flux capacity occurred at complex IV with a 29% decrease in neurons isolated from 24-month rats relative to those from 9-month rats. The deficits in complexes I and III may contribute to a redox shift in the quinone pool within the electron transport chain, further extending these age-related deficits. Together these changes could lead to an age-related catastrophic decline in energy production and neuronal death.

The repair of a large duodenal defect by a pedicled gastric seromuscular flap

The aim was to evaluate the efficacy of a pedicled gastric seromuscular flap for the closure of a large duodenal defect. A large defect of the second duodenal part was repaired by a gastric seromuscular flap. Of 35 rats, 9 rats were euthanized at 2 weeks, 12 rats at 2 months, and 14 rats at 4 months for the histopathological evaluation of the patch and normal duodenum (control) adjacent to the patch. All rats survived. The patch was completely covered by neomucosa in all of the 4-month rats, and in 8 of the 12 2-month rats. The villous height of the neomucosa was significantly higher in the 4-month rats in comparison to the other rats (P < 0.001). However, a normal duodenum had higher villi than in that of the patches (P < 0.001). The crypt density of the neomucosa was significantly increased in the 4-month rats in comparison to the 2-week and the 2-month rats (P < 0.001 and P < 0.05 group, respectively). The crypt density was higher in the controls than in the neomucosa covered patch of the 2-week and the 2-month rats (P < 0.001 and P < 0.05, respectively). The crypt depth of the neomucosa increased significantly in the 4-month rats and in the controls versus the 2-week rats (P < 0.05). The new mucosal barrier overlaying the patch appeared to be satisfactory. This technique, which has not been described previously, is likely to be useful for the repair of the large duodenal defect.

Exploration of senescence-associated genes by differential display reverse transcription polymerase chain reaction: Prosaposin as a novel senescence-associated gene

To understand the process and molecular mechanism of cellular aging, we explored novel senescence-associated genes using rat tissues with different age. Using total RNAs from 6 and 24-month old rat tissues, differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) were performed and 12 differentially expressed genes (DEGs) which were down- or up-regulated in aged rat tissues were found. Among these DEGs, the level of prosaposin mRNA was elevated of in senescent human dermal fibroblast (HDF) and human umbilical vein endothelial cells (HUVECs). It was further confirmed that expression of prosaposin was up-regulated in prematurely senescent HUVECs induced by hydrogen peroxide or interferon-gamma treatment. Taken together, we report the up-regulation of prosaposin in the senescent HDF and HUVECs as well as in hydrogen peroxide or interferon-gamma induced prematurely senescent HUVECs, suggesting that prosaposin might be a novel senescence-associated gene.

Cerebral metabolic effects of acetyl- l-carnitine in rats during aging

The aim of the present study was to investigate the neuronal structures that mediate the antiaging properties of acetyl-l-carnitine (ALCAR). The regional cerebral metabolic rates for glucose (rCMRglc) have been determined with the quantitative autoradiographic [ 14C]2-deoxyglucose procedure at different times after i.v. administration of saline or ALCAR 500 mg/kg to naïve, non pretreated 3-, 12- and 24-month-old rats and to 24-month-old rats pretreated with ALCAR (100 mg/kg/day, for 3 months). rCMRglc increased maximally at 30 min after ALCAR in 3-, 12- and 24-month old rats (14, 15 and 15 areas affected, 19, 24 and 22% mean increments). Peak metabolic activations occurred with similar magnitude in motor, visual, limbic and thalamic areas in all age rats and with larger magnitude in hippocampal and thalamic areas in aged rats. Cerebral metabolic activations subsided by 60 min after ALCAR in 3-month rats (3 brain regions affected, 4% decrease) and persisted by that time in 12- and 24-month-old rats (14 and 12 regions affected, 15 and 20% increases). Cerebral activations were enhanced in aged rats after chronic treatment with ALCAR (24 brain regions affected, 20% mean increase). Hence, during aging, metabolic responsivity to ALCAR is maintained in most brain areas and increased in limbic and thalamic regions. Increased responsivity to ALCAR may result from undetermined pharmacokinetic factors and/or from a higher sensitivity and contribute to the aging reversal properties of ALCAR.

Nicorandil protects ATP-sensitive potassium channels against oxidation-induced dysfunction in cardiomyocytes of aging rats

ATP-sensitive potassium channels (K(ATP) channels) regulate vascular tone and cardiac contraction through their action on the membrane potential of smooth muscle cells and cardiomyocytes. Because aging and diseases alter K(ATP) channel activity, many pharmacological treatments aimed at improving their function, therefore cardiovascular function, have been evaluated. Nicorandil, a K(ATP) channel opener, nitric oxide donor and antioxidant, is used as a treatment of angina pectoris and induces vasodilation, blood pressure decrease and cardioprotection in aging as well as after ischemia-reperfusion. Here, using the patch-clamp technique, we have studied the effect a chronic low dose of nicorandil (0.1 mg/kg per day for 2 months), on the activity of cardiomyocyte K(ATP) channels as a function of age, in newborn, 4-, 12- and 24-month old rats. Nicorandil exerted an anti-oxidant and protective action on cardiomyocyte K(ATP) channels, especially in aged animals, leading to restoration of a normal channel activity. These findings could justify further therapeutical applications.

Influence of aging and long-term swimming exercise on parvalbumin distribution in rat hearts

Parvalbumin (PV), which is a small (12kDa) cytoplasmic calcium-binding protein, has been implicated in mediating relaxation in cardiac myocytes. The influence of aging and exercise on the distribution of PV in rat heart was investigated. Male Wistar rats aged 3, 6, 12 and 18-months were divided into sedentary and exercise groups. The exercise group underwent exercise in the form of regular swimming for 6 months. The hearts were processed for immunohistochemistry and Western blotting. The intensity of PV immunoreactivity was strong in the 9 and 12-month hearts and decreased in the 18-month hearts. The smallest amount was detected in the 24-month rat hearts when compared to those of the 9, 12 and 18-month rat hearts. Significantly less PV was detected in the 18 and 24-month hearts compared to the 12-month rat hearts (P<0.05). The intensity of PV immunoreactivity was considerably stronger in hearts of the 9, 12 and 18-months exercised rats than in hearts of age-matched sedentary rats. However, in the hearts of 24-month rats, immunoreactivity was only slightly stronger in the exercised rats in comparison with those of sedentary rats. A significant increase of PV detection in hearts was found in the exercised rats in comparison with sedentary rats in the 9 (P<0.05) and 18-month samples (P<0.01). Our data indicate that PV is down-regulated in the rat heart during aging. In addition, our data indicate that long-term swimming exercise could induce an increase of PV expression.

Age-related alterations of Nestin-immunoreactive neurons in rat basal forebrain with aged memory deficit

Age-related and aged memory deficit changes in Nestin-immunoreactive (Nestin-IR) neurons were studied following recent evidence of distinct Nestin-IR neurons within adult rat basal forebrain. Morris water maze task assessed spatial learning capacity of 3- and 24-month rats (aged-impaired and aged-unimpaired groups). Nestin-IR neuron distributional and morphological features were investigated by immunohistochemistry and positive neuronal number calculation. Nestin-IR neuron number declined with aging, especially aged-impaired. Significant negative correlations existed between average escape latencies and Nestin-IR neuron number in medial septum-diagonal band of Broca (MS-DBB). Correlations of rostral portion [medial septum (MS) and vertical limb diagonal band (vDB)] were higher than caudal portion [horizontal limb diagonal band (hDB)]. Aged-impaired showed reduced complexity of Nestin-IR neuron dendrite arborization and dendritic length. Nestin-IR astrocyte-like cells appeared scattered among Nestin-IR neurons on some aged-impaired slices. In conclusion, aged-impaired rats showed worse cognitive spatial performance and less Nestin-IR neuronal number compared to aged-unimpaired. Nestin-IR neuronal loss and morphological changes are some pathological characteristics of rat aged basal forebrain and may be important in neurobiological mechanisms of brain aging and aged memory deficit.

Aging-induced alterations in gene transcripts and functional activity of mitochondrial oxidative phosphorylation complexes in the heart

Aging is associated with progressive decline in energetic reserves compromising cardiac performance and tolerance to injury. Although deviations in mitochondrial functions have been documented in senescent heart, the molecular bases for the decline in energy metabolism are only partially understood. Here, high-throughput transcription profiles of genes coding for mitochondrial proteins in ventricles from adult (6-months) and aged (24-months) rats were compared using microarrays. Out of 614 genes encoding for mitochondrial proteins, 94 were differentially expressed with 95% downregulated in the aged. The majority of changes affected genes coding for proteins involved in oxidative phosphorylation (39), substrate metabolism (14) and tricarboxylic acid cycle (6). Compared to adult, gene expression changes in aged hearts translated into a reduced mitochondrial functional capacity, with decreased NADH-dehydrogenase and F 0F 1 ATPase complex activities and capacity for oxygen-utilization and ATP synthesis. Expression of genes coding for transcription co-activator factors involved in the regulation of mitochondrial metabolism and biogenesis were downregulated in aged ventricles without reduction in mitochondrial density. Thus, aging induces a selective decline in activities of oxidative phosphorylation complexes I and V within a broader transcriptional downregulation of mitochondrial genes, providing a substrate for reduced energetic efficiency associated with senescence.

Aging impairs insulin-stimulated glucose uptake in rat skeletal muscle via suppressing AMPKα

Insufficient intracellular fat oxidation is an important contributor to aging-related insulin resistance, while the precise mechanism underlying is unclear. AMP-activated protein kinase (AMPK) is an important regulator of intracellular fat oxidation and was evidenced to play a key role in high-glucose and high-fat induced glucose intolerance. In the present study, we investigated whether altered AMPK expression or activity was also involved in aging-related insulin resistance. Insulin sensitivity of rats' skeletal muscles was evaluated using in-vitro glucose uptake assay. Activity of alpha subunit of AMPK (AMPKalpha) was evaluated by measuring the phosphorylation of both AMPKalpha (P-AMPKalpha) and acetyl-CoA carboxylase (P-ACC), while expression of AMPKalpha was assessed by determining the mRNA levels of AMPKalpha1 and AMPKalpha2, and protein contents of AMPKalpha. Compared with 4-month old rats, 24-month old rats exhibited obviously impaired insulin sensitivity. At the same time, AMPKalpha activity significantly decreased, while AMPKalpha expression did not alter during aging. Glucose transporter 4 expression also decreased in old rats. Compared with 24-month old rats, administration of the specific activator of AMPK, 5-aminoimidazole-4-carboxamide riboside (AICAR), significantly elevated AMPKalpha activity and GluT4 expression. Also, aging-related insulin resistance was significantly ameliorated by AICAR treatment. In conclusion, aging-related insulin resistance is associated with impaired AMPKalpha activity and could be ameliorated by AICAR, thus indicating a possible role of AMPK in aging-induced insulin resistance.

Kupffer cells are not responsible for splanchnic sequestration of amino acids in old rats

Background: In the postprandial state, splanchnic extraction of leucine is two-fold higher in elderly humans and in old rats compared with adults. This splanchnic sequestration of Leu (SSLeu) reduces the peripheral availability of dietary Leu, which is a key player in the regulation of muscle protein synthesis. Thus, SSLeu could contribute to sarcopenia, which is a major public health issue. However, the underlying mechanism of SSLeu remains totally unknown. Our working hypothesis is that the low-grade inflammation occurring during aging could lead to Kupffer cell (KC) activation in the liver which, in turn, could lead to SSLeu and the subsequent decrease in peripheral Leu availability. Methods: 24-month old rats received an i.v. injection of either GdCl3 (to destroy KC) or saline (control group). 24 hours later, the anesthetized animals underwent surgery and were studied in fed state. A primed continuous infusion of 1-13C-Leucine was administered in the jugular vein. Nutrition and 2H3-Leucine were continuously infused into the duodenum (id) during 60 min. Blood samples were then collected from the carotid artery. Splanchnic extraction (SPE) = [1 − (Ra 2H3-Leu / Ra 13C-Leu)] x100 where Ra = rate of appearance. Results: Conclusions: In old rats, the age-related activation of KC is not directly responsible for SSLeu. Indeed, invalidation of KC was unable to normalize SSLeu to what the value observed in adult rats (SPE = 25%). To our knowledge, this is the first mechanistic study in the field, and it enables an important hypothesis to be ruled out.